JH2005

April - June 2005 Volume 39 Number 2
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Kasetsart Journal : Natural Science April - June 2005 Volume 39 Number 2
The Kasetsart Journal

Advisor : Samakkee Boonyawat Rangsit Suwanketnikom Editor-in-Chief : Associate Editors : Ed Sarobol Wanchai Chanprasert, Natural Science Suparp Chatraphorn, Social Science Natural Sciences Amara Thongpan Pornsri Chairatanayuth Onanong Naivikul Praparat Hormchan Korchoke Chantawarangul Aree Thunyakijjanukij Overseas Members G. Baker (Mississippi State University, USA.) A. Bruce Bishop (Utah State University, USA.) John Hampton (Lincoln University, New Zealand) Helen H. Keenan (University of Stathclyde, Scotland) Chitochi Miki (Tokyo Institute of Technology, Japan) Eiji Nawata (Kyoto University, Japan) Manager : Orawan Wongwanich Social Sciences Suwanna Thuvachote Pongpan Trimongkholkul Matrini Ruktanonchai Nongnuch Sriussadaporn Patana Sukprasert
Editorial Board :
Assistant Managers : Surai Suwannarat Business Office : Kasetsart University Research and Development Institute (KURDI) Kasetsart University, Chatuchak, Bangkok 10900. The Kasetsart Journal is a publication of Kasetsart University intended to make available the results of technical work in the natural and the social sciences. Articles are contributed by Kasetsart University faculty members as well as by those from other institutions. The Kasetsart Journal : Natural Sciences edition is issued four times per year in March, June, September and December while The Kasetsart Journal : Social Sciences edition is issued twice a year in June and December. Exchange publications should be addressed to The Librarian, Main Library, Kasetsart University, Bangkok 10900, Thailand.
KASETSART JOURNAL
NATURAL SCIENCE
The publication of Kasetsart University
VOLUME 39 April - June 2005 NUMBER 2
Response of Weeds and Yield of Dry Direct Seeded Rice to Tillage and Weed Management ................................................ Jagat Devi Ranjit and Rungsit Suwanketnikom 165 Screening and Selection for Physiological Characters Contributing to Salinity Tolerance in Rice ................... Duangjai Suriya-arunroj, Nopporn Supapoj, Apichart Vanavichit .................................................................................... and Theerayut Toojinda 174 Weed Control Measures and Moisture Conservation Practices Effects on Seedbank Composition and Vertical Distribution in the Soil ............... Girma Woldetsadik, Sombat Chinawong, Rungsit Suwanketnikom, ............................................................. Sunanta Juntakool and Visoot Verasan 186 Genetic Diversity of Elite and Exotic Oilseed Meadowfoam Germplasm using AFLP Markers ....................................................... Sureeporn Katengam and Steven J. Knapp 194 Effects of Gramma Radiation on Azuki Bean Weevil, Callosobruchus chinensis (L.) ...................... Jakarpong Supawan, Praparat Hormchan, Manon Sutantawong ............................................................................... and Arunee Wongpiyasatid 206 Occurrence and Distribution of Major Seedborne Fungi Associated with Phaseolus Bean Seeds in Ethiopia ....................................................... Mohammed Yesuf and Somsiri Sangchote 216 Short-Term Stressor Effects of Water Deprivation Prior to the Onset of Lay on Subsequent Reproductive Performance of ISA Brown Pullets ............................................. Nirat Gongruttananun and Ratana Chotesangasa 226 Pharmacokinetics and Withdrawal Times of Enrofloxacin in Ducks ................... Natthasit Tansakul, Amnart Poapolathep, Naruamol Klangkaew, .............................................. Napasorn Phaochoosak and Wanida Passudaruk 235 Antimicrobial Resistance of Campylobacter jejuni Isolated from Chicken in Nakhon Pathom Province, Thailand .............. Jananya Sukhapesna, Patamaporn Amavisit, Worawidh Wajjwalku, ....................................... Arinthip Thamchaipenet and Thavajchai Sukpuaram 240 Hematology, Cytochemistry and Ultrastructure of Blood Cells in Asiatic Black Bear (Ursus thibetanus) ......................... Chaleow Salakij, Jarernsak Salakij, Nual-Anong Narkkong, ............................................ Ludda Trongwonsa and Rattapan Pattanarangsan 247
Probiotic Properties of Bacillus pumilus, Bacillus sphaericus and Bacillus subtilis in Black Tiger Shrimp (Penaeus monodon Fabricius) Culture .......... Watchariya Purivirojkul, Monchan Maketon and Nontawith Areechon Extracts of Thai Indigenous Vegetables as Rancid Inhibitor in a Model System ......................................... Plernchai Tangkanakul, Gassinee Trakoontivakorn ....................................................................... and Chansuda Jariyavattanavijit Screening and Characterization of Lactic Acid Bacteria Producing Antimicrobial Substance against Staphylococcus aureus ........ Chatinan Ratanapibulsawat, Pumrussiri Kroujkaew, Ohmomo Sadahiro ................................................................................... and Sunee Nitisinprasert Studies on Nham-Plas Processing by Using Rock Salt and Solar Salt . Mathana Sangjindavong, Pranisa Chuapoehuk and Daungdoen Vareevanich Product Development of Crocodile Jerky ........... Sinee Nongtaodum, Nongnuch Raksakulthai and Mayuree Chaiyawat Utilization of Fish Flour in Canned Concentrated Seasoning Stock for Thai Foods Preparation ......... Plernchai Tangkanakul, Payom Auttaviboonkul, Patcharee Tungtrakul, ....................... Mantana Ruamrux Chidchom Hiraga, Kanjanarat Thaveesook ................................................................................... and Montatip Yunchalad Lightning Surge Response of Concrete Pole due to Effect of the Electrical Properties of Concrete based on the Electromagnetic Field Method ....................................................... Samroeng Hintamai and Jamnarn Hokierti
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Kasetsart J. (Nat. Sci.) 39 : 165 - 173 (2005)
Response of Weeds and Yield of Dry Direct Seeded Rice to Tillage and Weed Management
Jagat Devi Ranjit1 and Rungsit Suwanketnikom2
ABSTRACT The study was initiated to assess the performance of rice (Oryza sativa) under dry direct seeded environment with two tillage systems of conventional tillage and minimum tillage and five weed management treatments namely unweeded control, handweeding twice 25 and 45 days after seeding, anilophos + one handweeding, bispyribac-sodium, and straw mulch + bispyribac-sodium as an alternate method of transplanting in the mid-hill ecology. Both anilophos and bispyribacsodium were found to reduce narrowleaf and broadleaf weeds compared to unweeded control. However, anilophos reduced Cyperus difformis, C. sanguinolentus, and C. iria 4 weeks after seeding (WAS) but not Ammania sp. and Dopatrium junceum 8 WAS. Bispyribacsodium and straw mulch + bispyribac-sodium reduced the population of Alternanthera philoxeroides, Ammania sp., Commelina diffusa, C. difformis, C. iria, and D. junceum 8 WAS. No phytotoxic effect on the rice plants was observed due to both herbicides. Yield and yield attributes were not affected by the tillage systems. The weed managements were found to affect the numbers of tiller per square meter and grain yield. The increasing number of weed did not affect the plant height of rice (Khumal-4). The numbers of tiller and grain yield highly affected the increasing number of weed population. Anilophos plus one handweeding, straw mulch plus bispyribac-sodium, handweeded twice and bispyribacsodium alone gave higher yield compared to unweeded control. Promising grain yield could be achieved with the anilophos or bispyribac-sodium with additional physical or mechanical control methods in dry direct seeded rice. Key words: dry direct seeded rice, bispyribac-sodium, anilophos, tillage, weed flora INTRODUCTION Transplanting is the popular rice establishment practice throughout Nepal with very little in direct seeding in some pocket areas. But with the ascending problem of labor and time, alternate method of rice culture may be beneficial in the future. However, direct seeding will be an alternate option to transplanting. Puddling for rice transplanting also makes land preparation difficult for wheat crop in ricewheat rotation resulting in
1 2
cloddy soil structure, loss of soil moisture, delayed and inadequate seed soil contact (Sharma and De Datta, 1985). Weeds are one of the limiting factors in direct seeded rice in reducing the yield. Weeds account for 50-80% yield reduction in rainfed uplands (Ranjit et al., 1989; Sinha et al., 1996). Yield reduction in rice is even higher (97%) due to competition of Echinochloa crusgalli (Kurchania et al., 1991). However, Echinochloa spp. was reported to be more competitive causing greater loss in growth and yield of rice compared to C.
Agronomy Division, Nepal Agriculture Research Council, GPO 404, Kathmandu, Nepal. Department of Agronomy, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.
Received date : 06/07/04
Accepted date : 01/03/05
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Kasetsart J. (Nat. Sci.) 39 (2)
difformis, Eclipta alba, Marsilia minuta, and Paspalum distichum (Srinivasan and Palaniappon, 1994). The yield losses caused by different weeds depends on the type of rice culture, weed infestation, density and weed species prevalence. Hand weeding is the most popular method of weed management in Nepal as well as in many parts of the world. Besides hand pulling and hand weeding, a number of herbicides have been developed and tested for the direct seeded rice around the world. Herbicides such as butachlor, thiobencarb, pendimethalin, oxyfluorfen, propanil, quinclorac, ioxynil, 2,4-D, piperophos + sulfonylurea, bentazone, molinate and bispyribacsodium have been tested in direct seeded rice in the past research (Biswas et al., 1992; Chin, 1999; Crawford and Jordan, 1995; Im, et al., 1999; Ranjit et al., 1989). Many factors affected cause the change of weed communities. Weed flora in the rainfed ecosystem has been reported to be the most complex compared to irrigated rice, but the weed management is the most important and can fill up at least 15% yield gap in different growing conditions (Moody, 1982). This study aimed to assess the responses of weed and yield attributes of dry direct seeded rice to tillage and weed management with bispyribac-sodium and anilophos herbicide and straw mulch in the midhill ecology. MATERIALS AND METHODS This experiment was conducted in the lowland field at Agronomy farm, Khumaltar, Nepal in a split plot design with RCBD replicated 4 times during the summer season of 2002. The main plots and sub plots were compiled of tillage and weed management respectively. The plot size was 4m 5m (20m2) and row spacing 20 cm. The field was located at an elevation of 1360 m above mean sea level on 27 40N latitude and 85 20E longitude. Land preparation was done with 2 ploughing and 2 harrowing in case of conventional tillage
(CT). But, for minimum tillage (MT), about 5-7 cm deep ploughing (only one time) was undertaken by the Chinese Seed Drill. Rice seeding was done after wheat harvest. Planting was carried out after making a line with hand hoe for both tillage systems. Planting and harvesting were conducted in June and October respectively. The variety used was Khumal-4. Seed rate was 90 kg/ha. Chemical fertilizer was applied at 100 kg nitrogen, 50 kg phosphorus, and 30 kg potash per hectare. Nitrogen was splitted in two halves. The 1st half was given as basal dose during planting and 2nd half during top dressing 45 days after planting. Chopped rice straw at 4 t/ha was used for the mulch treatment one day after rice seeding. Weed count was initiated from 0.50 m2 placing 50 cm by 50 cm quadrat at 2 places in each plot. Weed count was performed 3 times first 4 weeks after rice seeding (WAS), the second one 8 WAS and the 3rd at milking stage of rice (MSR). The first and second weed counts were carried out from the same spots in each plot but the third count was done from the different spot in each plot to see the changes in weed flora during the reproductive stage of rice. Individual weed species was counted. Weeds were pulled during the second and third counts and biomass was recorded after separating and cutting the roots of the narrowleaf and broadleaf weeds. Chopped rice straw @ 4t/ha was used for the mulch treatment one day after rice seeding. There were 5 weed management treatments namely unweeded control (W1), twice hand weeding 25 and 45 days after sowing (DAS) (W 2 ), preemergence application of anilophos [S[N(4chloro-phenyl-)-N-isopropyl-carbamoyl-methyl-]o, o-dimethyl-dithiophosphate, trade name = Arozin 30EC] @ 0.4kg ai/ha (W 3 ), postemergence application of bispyribac-sodium [ 2,6-bis{(4,6-dimethoxypyrimidin-2-yl) oxy}benzoate, trade name = Nominee10 EC] @
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50 g ai (W4), and rice straw mulch @ 4 t/ha + postemergence application of bispyribac-sodium @ 40 g ai /ha 40 days after seeding (DAS) (W5). Bispyribac-sodium was applied after mixing with 1/1 v/v surfactant. Anilophos was applied one day after rice sowing. Aspee backpack sprayer with 4 flat fan nozzles (8002) was used for herbicide spray. The spray volume was 500 l/ha. The weather during herbicide spray was sunny sky with patches of cloud and mild wind. Plant height (cm), tillers per square meter, seeds per panicle, thousand seed weight (g) and grain yield (kg/ha) were recorded. Plant height was recorded from the averages of 5 plant in each plot. Tillers were recorded from one square meter in each plot. Harvesting was done from 9.60 square meter (3m 3.20m). Grain yield was adjusted at 14 percent moisture contents. The mean minimum temperature during the rice crop ranged from 20.3C (June) to 12.8C (October) and the maximum temperature ranged
from 28.5C (June) to 24.7C (October). The total rainfall was 993.5 mm from June to October. The percent soil moisture during the rice crop was 4053. RESULTS AND DISCUSSION Weeds were categorized in narrowleaf (grass and sedge), broadleaf (monocot and dicot) and pteridophyte. The important species were A. philoxeroides, C. diffusa, C. difformis, C. iria, C. sanguinolentus, Ceratopteris thalictroides, E. colona, F. miliacea, Lindernia procumbens, and P. distichum (Table1). Response of weeds and weed biomass to tillage and management Both narrowleaf and broadleaf weeds and their biomass were found not to be different due to tillage in all counts. However, P. distichum was noticed to be more in minimum tillage 8 WAS and
Table 1 Weed species recorded in the experimental field at different stages of direct seeded rice.
Weeds species Narrowleaf weeds : Cynodon dactylon L. Pers Cyperus difformis L. C. dilutus L C. iria L C. sanguinolentus Vahl. Echinochloa colona L. (Link) E. crusgalli (L) P. Beauv. Eriocaulan sp. Eriocaulan sieboldtianum Sieb.et Zucc Fimbristylis miliacea Vahl. Paspalum distichum L. Panium sp. Scirpus juncoides Roxb. Broadleaf weeds (Dicot) : Ageratum conyzoides L. Eclipta prostrata L. Erigeron sp. Asteraceae Asteraceae Asteraceae Poaceae Cyperaceae Cyperaceae Cyperaceae Cyperaceae Poaceae Poaceae Eriocaulaceae Eriocaulaceae Cyperaceae Poaceae Poaceae Cyperaceae Family Weeds species Alternanthera philoxeroides (Mart) Griseb. Ammania baccifera L. Dopatrium junceum Hamilt. Lindernia procumbens Philcox Polygonum hydropiper L. Rotola indica Koehne Rorrippa indica Vandellia angustifolia Benth. Broadleaf weeds (Monocot) : Commelina diffusa Burm.f Murdania sp. Monochoria vaginalis Presl. Sagittaria guayanensis H.B.K Pteridophyte : Ceratopteris thalictroides (L) Brongn Parkeriaceae Commelinaceae Commelinaceae Pontederiaceae Alismataceae Family Amaranthaceae Lythraceae Scrophulariaceae Scrophulariaceae Polygonaceae Lythraceae Brassicaceae Scrophulariaceae
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MSR although the population was not high. But the number of C. sanguinolentus was less in minimum tillage 8 WAS (Figure 1). It has been reported that different tillage systems have different rates of weed suppression. Reduced tillage (one round tillage + leveling) resulted in heavy infestation of F. miliacea. But conventional tillage increased the amount of M. vaginalis. A seeding rate of 100 kg/ha significantly reduced sedges and broadleaf biomass 60 days after planting but not E. crusgalli (Azmi and Mortimer, 1999). However, the tillage did not affect the total populations of weed in the study. It might need a few years to see the change in weed population and species (Table 2). Total number of weed was different due to weed management treatments in all counts except broadleaf weeds 4 WAS. Preemergence application of anilophos + handweeding, straw mulch + bispyribac-sodium and bispyribac-sodium alone
(a) Cyperus sanguinolentus
25
a b
CT MT
20 15 10 5 0
4 WAS
8 WAS
MSR
(b) Paspalum distichum

15
a
10 5 0
4 WAS 8 WAS MSR
a b
CT MT
Figure 1 Weed species response to conventional tillage (CT) and minimum tillage (MT) at 4 WAS, 8 WAS, and MSR. Values in the bars with the same letters above are not significantly different at the 0.05 level. Bars without letter are not significantly different.
reduced the narrowleaf weeds in all counts (Table 2). Among the individual species, anilophos and straw mulch suppressed more amount of C. difformis and C. sanguinolentus than the unweeded control 4 WAS (Figure 2). All weed control treatments were found to suppress both narrowleaf and broadleaf weeds 8 WAS, and MSR (Table 2). Post-emergence application of bispyribacsodium alone and straw mulch + bispyribac-sodium were found to suppress both types of weed namely Ammania sp. and D. junceum, but both of them were not suppressed by anilophos when compared to the unweeded control 8 WAS (Figure 2). The number of these weeds decreased during the maturity stage of rice. Weeds like Cyperus spp., C. diffusa, and D. junceum were suppressed by bispyribac-sodium alone and in combination with straw mulch (Figure 2). Narrowleaf and broadleaf weed biomasses were significantly different due to weed management 8 WAS and MSR. Higher weed biomass was recorded in the unweeded control. The rest of the weed management treatments lowered the weed biomass in the same range. The herbicide application was also equally effective as twice handweeding (Table 3). Earlier researches also reported that bispyribacsodium controlled many narrowleaf and broadleaf weeds such as C. diffusa, C. iria, E. crusgalli, Fimbristyllis spp., Leersia oryzoides, Murdania sp., P. distichum, Polygonum sp., Sagittaria spp., Scirpus spp., and Sphenoclea zeylanica (Han, 2001; Kobayashi et al., 1995; Shinohara et al., 1994; Tachikawa et al., 1997; Yokohama et al., 1993). A. philoxiroides, Aeschenomene indica, Ammania coccinea, and Heteranthera limosa were also controlled by KIH 2023 (bispyribacsoduim) (Braverman and Jordan, 1996). Anilophos + ethoxysulfuron or anilophos alone controlled the most dominant weed Cyperus sp., F. miliacia and also Saccolepis interrupta (Moorthy et al., 1999; Screedevi and Thomas, 1993).
Plants/0.50 m2
Plants/0.50 m2
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Table 2 Narrowleaf and broadleaf weeds as affected by tillage and weed management. Treatment Narrowleaf weeds 4 WAS 8 WAS MSR Broadleaf weeds 4 WAS 8 WAS MSR
------------------------ (Plants/0.50 m2)-----------------------Tillage : Conventional tillage (CT) 921 Minimum tillage (MT) 145 Weed management : Unweeded control (W1) 172 a Handweeding twice (W2) 161 a Anilophos + handweeding one (W3) 63 b Bisbyribac-sodium (W4) 127 ab Straw mulch + bisbyribac-sodium (W5) 70 b Tillage (T) NS2 Weed management (W) ** TxW NS
1
65 79 117 a 90 ab 59 bc 55 c 40 c NS ** NS
31 43 58 a 48 ab 34 bc 21 c 23 c NS ** *
23 36 27 32 28 35 24 NS NS NS
45 45 25 c 56 b 107 a 12 c 25 c NS ** NS
11 11 30 a 7b 7b 5b 7b NS ** NS
Means within the same column and grouping followed by the same letters are not different according to Fishers protected test P=0.05. Treatment effects and interactions were significant at 5% (*), significant at 1% (**) or nonsignificant (NS).
(a) Cyperus difformis 150

Plants/0.50m2
W1
Plants/0.50m2
(b) Cyperus iria 20 15 10 5 0 b b b b 4WAS 8WAS a a b b b MSR a
W1 W2 W3 W4 W5
a a 100 50 0 4WAS ab b b a a
W2 W3 W4 b a b MSR W5
8WAS
(c) Cyperus sanguinolentus 50

Plants/0.502 Plants/0.50m2
W1 25 W1 W2 W3 20 15 10 5 0 b b b b 4WAS 8WAS b b b b a (d) Commelina diffusa a W2 W3 W4 W5
40 30 20 10 0 4WAS 8WAS MSR a a a b b b c bc bc a b b b b
W4 W5
MSR
Figure 2 Weed species responses to different weed managements of W1 (unweeded control), W2 (handweeded twice), W3 (anilophos + one weeding), W4 (bispyribac-sodium), and W5 (straw mulch + bispyribac-sodium) at 4 WAS, 8 WAS, and MSR. Values in the bars with the same letters above are not significantly different at 0.05 level. Bars without letters are not significantly different.
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Table 3 Effects of weed management on dry weed biomass at different stages of rice. 8 Weeks after sowing (WAS) Narrowleaf Broadleaf Maturity stage of rice (MSR) Narrowleaf Broadleaf
Treatments
--------------------------------(g/0.50 m2)-----------------------------Tillage : Conventional tillage (CT) Minimum tillage (MT) Weed management : Unweeded control (W1) Handweeding twice (W2) Anilophos + handweeding one (W3) Bisbyribac-sodium (W4) Straw mulch + bisbyribacsodium (W5) Tillage (T) Weed management (W) TW
1
35.61 48.7 113.1 a 20.9 b 14.5 b 38.4 b 24.0 b NS2 ** NS
5.8 8.2 18.1 a 4.6 b 4.5 b 2.3 b 5.6 b NS ** NS
30.5 43.9 88.3a 11.1 b 24.4 b 35.5 b 26.8 b NS ** NS
13.4 13.7 50.8 a 2.4 b 3.7 b 5.3 b 5.5 b NS ** NS
Response of yield attributes of rice to tillage There were no significant differences on plant height, tillers per square meter, thousand seed weight and grain yield due to tillage. It showed that dry direct seeding rice in conventional and minimum tillage did not affect the yield attributes and could be planted in both tillage systems (Table 4). Hobbs et al. (2002) also reported that rice yield was in the same range in both puddled and unpuddled rice cultures. This might be due to the condition under the unpuddled rice culture where the weeds were more properly controlled, since, in general, weeds in unpuddled rice culture were more serious problem than in puddled rice culture. Response of yields attributes to weed management Tillers per square meter, grain yield and dry straw weight were significantly different due
to weed management, but not plant height, number of seeds per panicle, and thousand seed weight. It showed that higher numbers of weed did not affect plant height because the plant height in other weed management treatments was in the same range of the unweeded control. Number of tillers per square meter ranged from 205 in unweeded control to 335 in straw mulch + bispyribac-sodium. Higher yield (6,708 kg/ha) was recorded in handweeding twice, straw mulch + bispyribac-sodium (6,445 kg/ha), and anilophos + one handweeding (6,416 kg/ha) which were at par to each other. Bispyribac-sodium alone yielded 5,469 kg/ha which was higher than that in unweeded control (2,136 kg/ha) (Table 4). All weed management treatments except unweeded control in this experiment gave promising yields up to 670 kg/ha. In the study, both herbicides did not show any phytotoxic effect on rice plants. However, the phytotoxic effect of these herbicides on different agroecological rice cultivars needs to
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Table 4 Effects of tillage and weed management on plant height, tillers, seeds/panicle, thousand seed weight, grain yield and dry straw weight of dry direct seeded rice. Treatments
Plant height (cm) Tillage : Conventional tillage (CT) Minimum tillage (MT) Weed management : Unweeded control (W1) Handweeding twice (W2) Anilophos + handweeding one (W3) Bisbyribac-sodium (W4) Straw mulch + bisbyribac-sodium (W5) Tillage (T) Weed management (W) TW
1
Tiller
Seed/panicle 1000 Filled Unfilled seed wt. (g) 18.7 19.4 19.1 19.3 18.9 19.2 18.8 NS NS NS
Grain Straw yield biomass (kg/ha) 5630 5239 2136c 6708a 6416a 5469b 6445a NS ** NS (kg/ha) 7395 6432 3989b 7701a 7541a 7195a 8140a NS ** NS
(no./ m2) ----(no./panicle)---281 256 205c 258ab 277ab 270abc 335a NS NS NS 146 126 106 138 170 128 139 NS * NS 12 11 11 11 11 10 15 NS NS NS
127.61 127.8 127.4 127.2 128.5 127.5 128.0 NS2 NS NS
be assessed in the future. The rotational effect of these herbicides to wheat herbicides should be studied in depth in different agroecological environments to find the effect on crop and weed shifts in the future. With the increasing number of narrowleaf weed population, both tillers per square meter and grain yield decreased (Table 2, 4, and Figure 3). In this study Cyperus spp. were the found to be dominant narrowleaf weed. Broadleaf weed like D. junceum did not affect the rice yields (Figure 3). Because the yield in anilophos treatment was higher, even the broadleaf weeds was not suppressed. The yield reduction might be depended on weed species. However, the low yield in bispyribacsodium alone compared to other treatments W2, W3 and W5 was actually not known although it suppressed both narrowleaf and broadleaf weeds (Figure 3). This herbicide might need to be assessed with regard to time, rate and the cultivar in different
agroecological environments for more seasons. CONCLUSION Most common weeds associated with dry direct seeded rice were A. philoxiroides, C. difformis, C. iria, C. sanguinolentus, C. diffusa, D. junceum, E. colona, and Lindernia sp. Both narrowleaf and broadleaf weeds were not significantly different due to tillage, but was significantly different due to weed management. Both narrowleaf and broadleaf weeds were reduced by bispyribac-sodium. Weeds like A. philoxiroides, Cyperus spp., and D. junceum were significantly reduced. However, Ammania sp. and D. junceum were not suppressed by anilophos. No phytotoxic effect on rice plants has been observed due to both herbicides. This study showed that both herbicides could be applied in dry direct seeded rice culture in the mid hill ecology. The weed managements showed significant impact on tillers and grain
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8 WAS
YL NL 8000
LSD= 593 0.05 LSD= 32 0.05

MSR
YL NL 150 8000
LSD= 593 0.05 LSD= 16 0.05
80 60 40 20 0 W1 W2 W3 W4 W5
Plants/0.50 m2
6000 4000 2000 0 W1 W2 W3 W4 W5
100 50 0
6000 4000 2000 0
8 WAS
YL BL 8000
LSD= 593 0.05 LSD= 27 0.05
MSR
YL BL 150 8000
LSD= 593 0.05 LSD= 9 0.05
40 30 20 10 0
Plants/0.50 m2
6000 4000 2000 0 W1 W2 W3 W4 W5
100 50 0
6000 4000 2000 0 W1 W2 W3 W4 W5
Figure 3 Grain yields of rice as affected by narrow leaf (NL) and broadleaf weed (BL) under different weed managements of W1 of (unweeded control), W2 (handweeded twice), W3 (anilophos + 1 weeding), W4 (bispyribac-sodium), and W5 (straw mulch + bispyribac-sodium) 8 WAS and MSR. yield. With the increasing number of weed population, the numbers of tiller and grain yields decreased. All weed management gave comparable yields to twice handweeding. With the proper weed management 150-200 percent rice yield could be increased so that the drudgery operation like seedbed preparation and transplanting could be avoided in dry direct seeded rice culture. ACKNOWLEDGEMENTS The authors would like to give their sincere thanks to Chief and the staffs of the Agronomy Division for providing the field and other facilities to conduct this research. We are thankful to Dr. P. R. Hobbs, former regional agronomist CIMMYT/ Nepal, Rice Wheat Consortium New Delhi and Kumiai Chemical Company, Japan for their cooperation in providing the rice herbicides for this research. Nepal Agricultural Research Council, Nepal supported this program. LITERATURE CITED Azmi, M.and A.M. Mortimer. 1999. Effect of tillage practices, seeding rates and herbicides on weed infestations in direct seeded rice, pp. 199-204. In Proc. 17th Asian Pacific Weed Science Society Conference, Bangkok, Thailand. Biswas, J.C., S.A. Sattar, and S.B. Siddique. 1992. Evaluation of herbicides in direct seeded rice in Bangladesh. Bangladesh Rice Journal. 2(1-2): 40-43. Braverman, M.P. and D.L Jordan. 1996. Efficacy of KIH-2023 in dryand water seeded rice
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Yield kg/ha
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Yield kg/ha
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(Oryza sativa). Weed Tech. 10: 876-882. Chin, D.V. 1999. Bispyribac-sodium, a new selective rice herbicide in direct seeded rice in Vietnam, pp. 443-446. In Proc. 17th Asian Pacific Weed Science Society Conference. Bangkok, Thailand. Crawford, S.H. and D.L. Jordan. 1995. Comparison of single and multiple applications of propanil and residual herbicides in dry seeded rice (Oryza sativa). Weed Tech. 9(1): 153-157. Han, F.C. 2001. Effect of Nominee (Bispyribacsodium) on rice cutgrass (Leersia oryzoides) in water seeded rice in Heilongjiang, China, pp. 787-792. In Proc. of 18th Asian Pacific Weed Science Society Conference, Beijing, China. Hobbs, P.R., Y. Singh, G.S. Giri, J.G. Lauren, and J.M. Duxbury. 2002. Direct seeding and reduced tillage options in the rice-wheat systems of the Indo-Gangetic plains of South Asia, pp. 201-215. In Pandey, S., M. Mortimer, I. Wade, T.P. Tuong, K. Lopez and B. Hardy (eds). Direct seeding: research issues and opportunities. In Proc. of the International Workshop on Direct Seeding in Asian Rice Systems: Strategic Research Issues and Opportunities, Bangkok, Thailand, International Rice Research Institute, Los Banos, Philippines. Im, I.B., C.K. Kang, S.S. Han, and S.Y. Cho, 1999. Weed control by weed emergence types in dry direct seeded rice fields. Korean Journal of Weed Science. 19(1): 7-14. Kobayashi, K., M. Yokohama, O. Watanabe, H. Sadohara, and N. Wada. 1995. KIH 2023 a new post emergence herbicide in rice (Oryza sativa), pp. 221-226. In Proc. 15th Asian Pacific Weed Science Society Conference. Tsukuba, Japan. Kurchania, S.P., J.P. Tiwari, and N.R. Paradkar. 1991. Weed control in rice (Oryza sativa) wheat (Triticum sativum) cropping system. Indian Jour. Agric. Sci. 61(10): 720-725. Moody, K. 1982. The status of weed control in rice
in Asia. FAO Plant Protection Bulletin. 30: 1-10. Moorthy, B.T.S., S. Saha and S. Sanjoy. 1999. Relative efficacy of different herbicides for weed control in direct seeded rice in puddled soil. Indian Jour. of Weed Sci. 31(3-4): 210213. Ranjit, J.D., K.P. Bhurer, K.P. Koirala, Y. Thakur, and D.N. Choudhary. 1989. Screening of herbicides in upland and transplanted rice, pp. 129-139. In Proc. 14th Summer crops workshop. Parwanipur, Nepal. Screedevi, P. and C.G. Thomas. 1993. Control of Saccolepis interrupta (Wild) Stapf in dry seeded rice in Kerela, pp.10-12. In Proc. Indian Society of Weed Science International Symposium. India. Sharma, P.K. and S.K. De Datta. 1985. Effects of puddling on soil physical properties and processes. In Soil Physics and Rice. IRRI publications. Shinohara, T., M Yokoyama, O. Watanabe, K. Kawano, and S. Shigematsu. 1994. KIH 2023, A new post emergence herbicide in rice, p 8. In Proc. Weed Science Society of America, Abstract. Sinha, P.K., C.V. Singh, R.K. Mishra, D. Maiti, V.D. Shukla and M. Variar. 1996. Rainfed upland ricefuture strategies. Indian Farming. 66(9): 25-29. Srinivasan, G. and S. Palaniappon. 1994. Effect of major weed species on growth and yield of rice. Indian Jour. Agron. 69(1): 13-15. Tachikawa, S., T. Miyazawa, and H. Sadohara. 1997. Vegetation management by KIH-2023 in rice levees and highways and railroad rightof-ways, pp. 114-117. In Proc. 16th Asian Pacific Weed Science Society Conference. Kualalumpur, Malaysia. Yokohama, M.,O. Watanabe, K. Kawano, S. Shigematsu and N. Wada. 1993. KIH-2023, A new post-emergence herbicide in rice pp. 61-66. In Brighton Crop Protection Conference-Weeds.
Screening and Selection for Physiological Characters Contributing to Salinity Tolerance in Rice
Duangjai Suriya-arunroj1 ,Nopporn Supapoj1, Apichart Vanavichit2 and Theerayut Toojinda3
ABSTRACT Two screening techniques, one initial screening using nutrient solution culture at young seedling stage and the other using soil in pots at vegetative stage, were used for identification of genotypes for salinity tolerance. Sixteen rice lines and cultivars were screened initially at seedling stage. Germinated seeds were placed on styrofoam plates floated on nutrient solution in a plastic container. After 14 days of sowing, the seedlings were subjected to salinization. The results showed that there were 3 groups of rice with different levels of response to salinity ; tolerant group consisted of Pokkali, FL496 and FL530, moderately tolerant group consisted of FL358, FL367, FL411, FL416, FL434, FL443, FL478, FL523, FL563, KMK and DDG, and susceptible group were KDML105 and RD6. To confirm the reliability of this initial screening technique, the visual salt-injury symptom was compared with the mean performance of salinity damage rating, Na+, K+ content and Na+/K+ absorption ratio in young leaves , old leaves and stem to identify physiological characters contributing to salinity tolerance in rice in the vegetative experiment. Eight lines/cultivars of rice were selected from the seedling screening to further investigate tolerant ability at vegetative stage in soil medium. In this test, 21 day-old seedling were subjected to 3 levels of salinity, 4,8 and 12 dS /m. The results showed visual scores to match with the Na+/K+ ratio, the cultivars with low Na+/K+ ratio had high tolerant ability and the susceptible one had high Na+/K+ ratio. The two selected salt tolerant recombinant inbred lines were FL496 and FL478 and two landrace cultivars selected were DDG and KMK which will be used as donors to introgress salt tolerant QTL into target cultivars (KDML105 and RD6) in salt tolerance breeding program. Key words: rice, salt tolerant rice, salt tolerance screening, Na+/K+ ratio INTRODUCTION Soil salinity is the single most widespread soil toxicity problem in rice growing countries. Thus, an increase in salinity resistance in rice is necessary for further expansion of rice growing area because good agricultural land is limited
1 2 3
(Toenniessen, 1984). Breeding for salinity tolerance in rice requires reliable screening techniques and must be rapid to keep pace with a large amount of breeding materials. Salt sensitivity of rice varies not only among genotypes but also among developmental stages of the plant (Akbar and Yabuno, 1974). According to Pearson and
Ubon Ratchathani Rice Research Center, Ubon Ratchathani 34000, Thailand. Rice Genome Project, Kasetsart University Khampaeng Saen Campus, Nakhon Pathom 74130, Thailand. BIOTEC, National Center for Genetic Engineering and Biotechnology. Kasetsart University, Khampaeng Saen Campus, Nakhon Prathom 74130, Thailand.
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Ayers (1960), rice is very tolerant to salt during germination, but very sensitive during the early seedling stages. Once panicles have developed in the leaf sheaths, subsequent phases of development are not sensitive to salt (Kaddah et al., 1973). Two screening techniques were developed (Gregorio et al., 1997) for use at seedling stage and vegetative and reproductive stages. In this study the second techniques used was only for vegetative stage. The two screening techniques adopted were 1. screening at seedling stage in which salinity was imposed to medium solution 14 days after growing in the solution and 2. screening at vegetative stage where the rice plants in pots were salinized 21 days after sowing. The purpose of the latter was to confirm the result of the first screening and also to identify physiological traits associated with salinity tolerance. Physiological mechanisms confering Na + exclusion and selectivity for K+ and Na+ have been described for salt tolerant ability of plant. Thus the visual saltinjury symptoms were compared with Na+, K+ content and Na+/K+ ratio in young leaf, old leaf and stem of selected lines and cultivars. Rice plants maintain the leaf water content to avoid the injury from drought stress which is the early phase of salt stress (Munns,1993) or osmotic phase. K+ and organic solutes accumulate in the cytoplasm and organelles to balance the osmotic pressure of ions in the vacuoles (Munns and James, 2003). Mechanisms of salt tolerance at the cellular level involve keeping the salt out of cytoplasm and sequestering it in the vacuoles of the cell. The objectives of this study were to elucidate how some rice cultivars had an ability to grow in salinity conditions, with particular emphasis on phenological development, leaf injury (salt tolerant scoring), dry matter growth, plant height, Na+ and K+ concentration in plant parts and relative water content, to identify physiological traits for salinity tolerance and to select salt tolerant cultivars/lines. The information obtained from this work would assist in identifying traits which could be used as
selection criteria for salt tolerance in rice. MATERIALS AND METHODS 1. Tested materials At seedling stage, 16 rice lines/cultivars were used; IR66946-3R-58-1-1(FL358), IR669463R-67-1-1(FL367), IR66946-3R-111-1-1 (FL411), IR66946-3R-116-1-1(FL416), IR669463R-134-1-1(FL434), IR66946-3R-143-1-1 (FL443), IR66946-3R-178-1-1(FL478), IR669463R-196-1-1(FL496), IR66946-3R-223-1-1 (FL523), IR66946-3R-230-1-1(FL530), IR669463R-263-1-1(FL563) (the progenies of the cross Pokkali/IR29 which were selected based on different salinity tolerant abilities classified by Gregorio (1997), Khao Mahk Khaek (KMK), Daeng Dawk Gok (DDG), RD6, Khao Dawk Mali105 (KDML105)(Thai landrace cultivars), and Pokkali(tolerant cultivar). Since KDML105 and RD6 were moderately sensitive to salt (score 7) so these cultivars were used as susceptible checks. The lines and cultivars used for vegetative stage screening were selected from the lines or cultivar found to vary on tolerance to salinity during seedling stage. The 8 lines and cultivars selected were Pokkali (tolerant check), IR29 (susceptible check), KMK, KDML105, FL478, FL496, DDG, and RD6. 2. Screening at seedling stage Seeds of the 16 lines/cultivar were surface sterilized with 10% Clorox (5.25%w/w sodium hypochlorite) for 30 minutes, then rinsed with distilled water. Sterilized seeds were incubated in petridishes at room temperature (38-42C) for 5-7 days to germinate. Germinated seeds were placed at 1 seed per a small hole on a styrofoam plate with a nylon net supported at the bottom. The plates were floated on a nutrient solution recommended by Yoshida et al.(1976). After 14 days of growth, the seedlings were subjected to salinization (EC 6 dS /m) by adding NaCl to the nutrient solution.
176
The nutrient solution was renewed once a week, and its pH was maintained daily at 5.5 (adjusted by adding either 1N NaOH or HCl). The seedlings were grown in screenhouse at Ubon Ratchathani Rice Research Center, Ubon Ratchathani, Thailand. The experimental design was a 16 2 factorial in RCB design with 3 replications. The treatments consisted of 16 rice cultivars and 2 salinity levels of 0 and 6 dS /m. Each experimental unit consisted of 18 plants. The salt tolerant scoring (Table 1) was recorded 16 days after salinization (Gregorio et al.,1997). At the same time, leaf and shoot samples were taken for relative water content and Na + and K+ content determination, respectively. For relative water content, the youngest fully expanded leaf was used. One centimeter long leaf sample cut at 1/3 of the leaf from the leaf tip was taken. Two samples were weighed to determine fresh weight (FW), then soaked in distilled water at 25C for 4 hrs and weighed again to record the turgid weight (TW) and oven-dried at 80C for 24 hrs to determine the dry weight (DW). The Relative Water Content (RWC) was computed as follows %RWC =
Na+ and K + analysis using atomic absorption spectrophotometer. 3. Screening at vegetative stage The purposes of this study were to confirm the reliability of the screening at seedling stage and to determine the differential responses to salinity of the parents and progenies for physiological characters including Na+/K+ ratio in young leaves (1-4 leaves from the top), old leaves(below the 4th leaves from the top) and stems, RWC and salt tolerant scoring. Preparation of pots : Black plastic bags with 15 cm 15 cm surface area and 17 cm height were used as experimental pots. Holes with 3-4 mm in diameter, were made at 2-cm spacing on the side wall of these bags to allow movement of the salinized water into the soil. A cotton bag was placed inside each plastic bag and filled with fertilized soil (45 mg N, 27 mg P2O5 and depending on K treatment 18, 22, or 36 mg K2O /kg of soil used)up to 2 cm below the rim of the bag. The cotton bags were used once only. Then the bags were placed in the plastic container filled up with tap water to the same level as soil in plastic bags. Six pregerminated seeds of each entry were placed on soil surface of each bag. Two weeks after seeding, seedlings were thinned to three per bag. Water level was then raised to 1 cm above the soil surface and maintained daily. Pesticides were
FW - DW 100 TW - DW
Shoot samples for Na+ and K+ content analysis were oven-dried for 3 days at 80C. Dried samples were finely ground, and 0.3 g powder from each sample was taken for
Table 1 Modified standard evaluation score (SES) of visual salt injury (Gregorio et al., 1997). Score 1 3 5 7 9 Observation Normal growth Nearly normal growth; leaf tips or few leaves whitish and rolled Growth severely retarded; most leaves rolled; only a few are elongating Complete cessation of growth ; most leaves dry; some plants dying Almost all plant dead or dying Tolerance level Highly tolerant Tolerant Moderately tolerant Susceptible Highly susceptible
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applied to the plants as necessary. Salinization : when the seedlings were 21 days old, water was siphoned from the contaniers; 3-hours elapsed before all water was drained out of the bags. Salinized water solutions with concentration of 4, 8 and 12 dS /m were prepared by dissolving table salt (NaCl) in water while stirring. The plastic container was filled up with salinized water solution. The solution level was maintained at 1 cm above soil surface by adding tap water and salinity levels were monitored for each treatment. Plants were exposed to different salinity levels for 12 weeks when the experiment was completed. The experiment was laid out in a split plot design: Main plots were arranged as 4 3 factorials ; Factor 1: salinity levels = 4 levels ; 0, 4, 8, and 12 dS /m Factor 2: Potassium(K) application rates = 3 rates ; 31.25, 43.75, and 68.75 kg K2O /ha which represented low, reccommended and high rate, respectively. The purpose of different levels of K application was to determine whether K level affected Na absorption and hence salt tolerant scoring of rice plants. Sub plots were 8 lines/cultivars of rice selected for difference in salinity tolerance from screening at seedling stage. They were Pokkali, IR29, KMK, KDML105, FL478, FL496, DDG and RD. The experiment was conducted in a screenhouse at Ubon Ratchathani Rice Research Center, Ubon Ratchathani, Thailand, during June September 2001. The average surrounding temperature of the screenhouse was (day/night temperature) 37/29C. RESULTS 1. Seedling stage screening There was no significant difference in salt tolerant scoring, RWC and Na+/K+ ratio when seedlings were grown at the normal nutrient
solution(data not shown). Physiological traits contributing to salt tolerance of rice grown in 6 dS/ m solution are shown in Table 2. Visual symptom observation illustrated that the lines which had low scores (salt tolerant) compared to Pokkali(tolerant check) were FL416, FL478, FL496, and FL530(Table 2). The lines identified as moderately susceptible to salinity (with intermediate scores) were FL358, FL367, FL411, FL434, FL443, FL523, and FL563, while the susceptible cultivars were RD6 and KDML105. Genotypic ranking for RWC determined at midday was similar to that determined at predawn. The liness FL478, FL496 and FL530 had high RWC compared to Pokkali (Table 2). The two landrace cultivars, KMK and DDG, and FL416, FL434, FL443, FL523, FL358, FL367, FL411, FL563, and Pokkali were rated as having moderately high RWC. Cultivars with low RWC were RD6 and KDML105. In this experiment, the lines FL496 and FL530 had low Na+/K+ indicating that these lines were tolerant to salinity compared to Pokkali with respect to Na+/K+ ratio. The moderately tolerant lines and cultivars were FL358, FL367, FL411, FL416, FL434, FL443, FL478, FL523, FL563, KMK and DDG, while the cultivars RD6 and KDML105 were susceptible to salinity (with high Na+/K+ ratio, Table 2). 2. Vegetative stage screening 1 week after salinization (WAS), most rice cultivars were scored 1 at salinity level of 4 dS/m (Table 3). This indicated that at this salinity level the salt content has mild effects on rice growth except for IR29 (score 2). At 8 dS/m, IR29 was the most susceptible (score 4) while KDML105 was the second most susceptible (score 3). The tolerant lines/cultivars identified were Pokkali, FL496 and DDG which scored 1. At the salinity level of 12 dS/m, most lines scored 3 except for IR29 and KDML105 which scored 5 and 4, respectively. Similar trends were obtained 2 WAS at 4 dS/m. At
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8 and 12 dS/m, salt affected these lines and cultivars more severely than at 4 ds/m. After 12 weeks of salinization, the rice plants were severely damaged by the salt at these levels. Most susceptible
cultivatrs, such as IR29, RD6 and KDML105 were dead or nearly dead, at 8 dS/m. While young leaves of Pokkali, KMK and DDG were still green, old leaves were dead. All plants were dead after
Table 2 Physiological traits contributing to salinity tolerance in rice grown at salinity level of 6 dS/m.
Salt tolerance scoring* 5.7 b 5.0 bcd 5.7 b 3.3 de 5.3 bc 5.3 bc 3.0 e 3.0 e 4.7 b-e 3.7 cde 5.0 bcd 4.3 b-e 4.3 b-e 7.7 a 7.7 a 3.0 e 32.3 Plant weight (g) At 6 dS/m 0.638 bcd 0.748 a-d 0.607 bcd 0.462 cd 0.565 bcd 0.578 bcd 0.665 a-d 0.523 bcd 0.633 bcd 0.506 cd 0.593 bcd 0.855 abc 1.049 a 0.338 d 0.557 bcd 0.940 ab 29.0 Plant height (cm) At 6 dS/m 71.85 ab 66.76 b-e 70.06 a-d 51.96 fg 78.07 a 64.41 cde 55.72 efg 60.68 c-f 78.73 a 48.53 g 77.39 ab 63.42 cde 65.58 cde 59.83 def 61.53 c-f 80.61 a 8.5 Relative water content (%)1/ Midday 85.80 bc 86.98 abc 87.94 abc 91.96 ab 88.50 ab 88.90 ab 94.16 a 94.28 a 91.06 ab 93.91 a 86.04 bc 91.08 ab 92.00 ab 81.35 c 75.00 d 93.27 a 4.2 Predawn 84.28 de 87.35 b-e 91.61 a-d 94.11 ab 86.73 b-e 85.04 cde 97.93 a 96.68 a 92.51 abc 96.39 a 90.75 a-d 90.34 a-d 90.18 a-d 82.06 e 81.80 e 94.12 ab 4.5 Na+/K+ ratio* 0.246 bc 0.266 bc 0.241 bc 0.169 bc 0.182 bc 0.232 bc 0.183 bc 0.158 c 0.200 bc 0.126 c 0.318 b 0.209 bc 0.233 bc 0.729 a 0.668 a 0.236 bc 71.8
Lines/cultivars FL358 FL367 FL411 FL416 FL434 FL443 FL478 FL496 FL523 FL530 FL563 KMK DDG RD6 KDML105 Pokkali CV(%)
1/
The data were collected 16 days after salinization
Table 3 Salinity tolerant scoring at 4 salinty levels recorded 1,2 and 12 weeks after salinization. Variety
0 dS/m
1 WAS
4 dS/m 8 dS/m 12 dS/m 0 dS/m
2 WAS
4 dS/m 8 dS/m 12 dS/m 0 dS/m
12 WAS
4 dS/m 8 dS/m 12 dS/m
Pokkali FL 496 DDG KMK FL 478 KDML105 RD6 IR 29 Mean CV (%)
1a 1a 1a 1a 1a 1a 1a 1a 1
1b 1b 1b 1b 1b 1b 1b 2a 1
1e 1e 1e 2 cd 2 cd 3b 2 cd 4a 2 37.1
3 cd 3 cd 3 cd 3 cd 3 cd 4b 3 cd 5a 3
1a 1a 1a 1a 1a 1a 1a 1a 1
1 cd 1 cd 2 bc 1 cd 2 bc 2 bc 2 bc 3a 2
2d 3c 3c 3c 4b 4b 4b 5a 4 24.1
3d 4c 4c 4c 5b 5b 5b 7a 5
1a 1a 1a 1a 1a 1a 1a 1a 1
1 bc 2b 1 bc 1 bc 1 bc 1 bc 1 bc 7a 2
3e 7c 6d 5d 7c 8b 9a 9a 7 23.0
6b 8a 9a 9a 9a 9a 9a 9a 9
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0.0239 a 0.0154 b 0.0129 b 0.0116 b 0.0147 b 0.0145 b 0.0130 b 0.0291 a 0.0156 b 0.0197 b 0.0152 b 0.0176 b 0.0183 b 0.0152 b 0.0153 b 0.0149 b 0.0308 ab 1.781 ab 1.855 a cv (%) RD 6 2.007 ab 23.8 1.843 ab 0.0155 a 0.0257 ab 30.1 0.0439 a 0.0236 b 0.0216 bc 25.7
Total dry weight and total Na+ and K+ uptake Pokkali had the highest total dry weight (TDW) and high total Na+ and K+ uptake (Table 4) rendering this cultivar to have balance Na+ and K+ in its cell. This implied that Pokkali had high salt tolerant ability because of dilution effect. On the other hand, some lines/cultivars had high Na+ uptake but low K+ uptake resulting in imbalance Na+ and K+ in their cells. These lines/cultivars included KDML105, RD6 and IR29 which were rated as salt susceptible. Effect of Potassium(K) application rate There was no significant difference in salt tolerant scoring among potassium application rates of 31.25, 43.75, and 68.75 kg K2O ha-1(K1, K2, and K3). This result indicated that the 3 potassium application rates, which represented low, recommended and high rate, had no significant effect on salinity tolerance (Munns et al., 2002). This suggested that some rice genotypes might have some mechanisms, such as Na+ exclusion, which allowed plants to handle high levels of salt accumulation in their leaves to maintain low Na+/ K+ ratio(Gregorio and Senadhira,1993).
Table 4 Total dry weight and total Na+ and K+ uptake of 8 rice lines/cultivars at 4 salinity levels.
Total Na+ uptake (g/plant) 4 dS /m 8 dS /m 12 dS /m Total dry weight (g/plant) 4 dS /m 8 dS /m 12 dS /m 0 dS /m Lines/cultiuars 0 dS /m
Pokkali IR 29 KMK KDML 105 FL 478 FL 496 DDG
2.442 a 1.375 a 1.661 a 1.619 a 1.631 a 1.319 a 1.177 a
2.622 a 1.108 c 1.584 bc 2.411 a 1.625 bc 1.981 ab 1.386 bc
2.519 a 1.696 b 2.028 ab 1.771 ab 1.768 ab 1.761 ab 1.659 b
2.327 a 1.713 ab 1.588 ab 1.530 b 1.601 ab 1.467 b 1.513 b
0.0224 a 0.0111 a 0.0139 a 0.0135 a 0.0138 a 0.0104 a 0.0082 a
0.0291 ab 0.0112 c 0.0201 abc 0.0335 a 0.0185 bc 0,0204 abc 0.0155 bc
0.0393 a 0.0301 ab 0.0334 ab 0.0339 ab 0.0272 ab 0.0243 b 0.0248 ab
0.0399 ab 0.0405 ab 0.0296 abc 0.0389 ab 0.0346 abc 0.0219 c 0.0270 bc
salinization at 12 dS/m for 12 weeks. The results from this experiment indicated that at salinity level of 4 dS/m, there was slight or no effect on rice growth compared to that grown in normal condition. Screening salt tolerant rice at this condition(day/night temperature = 32-37/2329C) should not be at this level. Therefore the screening was based on the data obtained from salinity levels of 8 dS/m and 12 dS/m 2 weeks after salinization. The line and cultivars which were tolerant to these conditions were Pokkali, KMK, FL496 and DDG. Moderately tolerant line and cultivars were KDML105, FL478, and RD6. The susceptible cultivar was IR29.
Total K+ uptake (g/plant) 4 dS /m 8 dS /m 12 dS /m 0 dS /m
0.0324 a 0.0148 c 0.0201 bc 0.0199 bc 0.0196 bc 0.0163 bc 0.0135 c
0.0326 a 0.0109 d 0.0176 bcd 0.0245 b 0.0172 bcd 0.0199 bc 0.0152 cd
180
Old leaves (after the 4 th leaf from the top) 0 dS /m 4 dS /m 8 dS /m 12 dS /m
Table 5 K+ concentration (%) in 3 parts of rice plant at 1 week after salinization.
1.357 a 0.935 d 1.215 abc 0.973 d 1.173 bc 1.327 ab 1.079 cd 1.087 cd 1.062 ab 1.129 a 1.031 abc 0.858 d 0.988 bc 1.028 abc 0.960 bc 0.950 cd Pokkali IR 29 KMK KDML 105 FL 478 FL 496 DDG RD 6 1.078 b 1.125 b 1.295 a 1.133 b 1.153 b 1.265 a 1.142 b 1.263 a cv (%) 1.122 abc 1.038 bcd 1.156 a 0.951 d 1.029 cd 1.070 abc 1.061 abc 1.143 ab 9.5 1.168 b 1.344 a 0.984 de 0.904 e 1.098 bc 1.129 bc 1.072 bcd 1.043 cd 1.458 a 1.215 b 1.304 ab 1.330 ab 1.331 ab 1.349 ab 1.346 ab 1.356 ab 1.443 a 0.945 c 1.186 b 1.070 bc 1.079 bc 1.218 b 1.205 b 1.210 b 15
Sodium concentrations in different parts of rice plant In young leaves, at 8 dS/ m and 12 dS/ m levels of salinity, high Na+ concentration was found in cultivars IR29, KMK, KDML105 and RD6. Moderately high Na+ concentration was found in FL478 and DDG, and low Na + concentration were found in Pokkali and FL496. In old leaves, similar trends were obtained; high Na+ concentration was found in the cultivars IR29, KDML105 and KMK. FL478 and DDG had accumulated medium of Na+ concentration, while Pokkali and FL496 had low Na+ concentration in old leaves. In stems, a high concentration of Na+ was found at high salinity level (12 dS/ m) in IR29, KMK, KDML105 FL478, and RD6. On the other hand, DDG had moderately low Na+ concentration and Pokkali and FL496 had low Na+ concentration in stems (data not shown). Potassium concentrations in different parts of rice plant There were high K+ concentrations in young leaves of KMK, FL496 and RD6 at 0 dS /m of salinity (Table 5). At 4 dS /m salinity level, KMK had the highest K+ concentration, however it was not significantly different from Pokkali, FL496, DDG and RD6. At 8 and 12 dS /m, IR29 had the highest K+ concentration. In most salinity levels, KDML105 had the lowest K+ concentration. In the old leaves, for all salinity levels, Pokkali had the highest K+ concentration except at 12 dS /m where FL496 had the highest K+ concentration. IR29 had the lowest K+ concentration in all cases studied. In stems, the lines and cultivars which had high K+ concentration at 0 dS /m were FL478, Pokkali, KMK, FL496 and DDG, while RD6 had moderately high K+ concentration IR29 and KDML105 had low K+ concentration. Similar trend was observed in stems of rice plant grown at 4, 8 and 12 dS /m where Pokkali had the highest K+ concentration, and KMK, FL478, FL496 and DDG had moderately high K+ concentration. In all
12 dS /m Stem 8 dS /m
0.926 a 0.557 d 0.657 c 0.622 cd 0.751 b 0.742 b 0.627 cd 0.579 cd 1.308 a 1.003 b 1.024 b 1.093 b 1.068 b 1.348 a 0.993 b 1.072 b 1.206 ab 0.970 d 1.182 ab 1.085 c 1.221 a 1.168 abc 1.134 abc 1.123 abc 1.056 a 0.695 e 0.888 b 0.756 de 0.859 bc 0.851 bc 0.834 bcd 0.780 cde 10.8
Lines/cultiuars
Young leaves (1-4 leaves from the top) 0 dS /m 4 dS /m 8 dS /m 12 dS /m
0 dS/ m
4 dS /m
0.888 a 0.482 e 0.610 cd 0.530 de 0.640 c 0.725 b 0.630 c 0.591 cd
181
2.520 d 5.671 a 4.641 bc 5.605 a 4.788 b 3.138 d 4.008 c 4.512 bc 2.284 c 4.077 a 3.504 ab 3.982 a 2.894 bc 2.682 c 3.428 ab 4.086 a 1.550 d 3.069 a 2.230 bc 3.196 a 2.085 cd 1.545 d 2.335 bc 2.742 ab 0.398 de 0.571 bcd 0.619 bc 0.993 a 0.433 de 0.369 e 0.491 cde 0.739 b Pokkali IR 29 KMK KDML 105 FL 478 FL 496 DDG RD 6 0.245 a 0.226 a 0.225 a 0.195 a 0.201 a 0.178 a 0.212 a 0.193 a cv (%) 0.262 b 0.297 ab 0.353 ab 0.472 a 0.336 ab 0.269 b 0.326 ab 0.320 ab 37.5 0.385 f 0.808 cd 1.028 b 1.269 a 0.666 de 0.404 f 0.566 e 0.920 bc 0.822 a 0.912 a 0.870 a 0.868 a 0.763 a 0.835 a 0.871 a 0.830 a 1.093 b 1.702 ab 1.634 ab 1.926 a 1.582 ab 1.212 b 1.369 ab 1.636 ab 34.9 1.704 c 3.058 ab 2.573 b 3.583 a 2.814 b 1.807 c 2.697 b 3.214 ab 1.056 a 1.042 a 1.058 a 1.319 a 0.975 a 0.944 a 0.964 a 1.044 a 1.516 c 2.213 abc 2.200 abc 2.636 a 2.009 abc 1.702 bc 1.911 abc 2.356 ab 26.4
salinity levels tested, IR29, KDML105 and RD6 had low K+ concentration (Table 5). Sodium/Potassium ratios (Na+/K+ ratio) in different parts of rice plant As for Na+/K+ ratios in young leaves, there were no significant genotypic differences in Na+/ K+ ratio at 0 dS /m and 4 dS /m. The different response appeared at salinity levels of 8 and 12 dS /m. Low Na+/K+ ratios were found in the leaves of Pokkali, FL478, FL496 and DDG, while high Na+/K+ ratio was found in KDML105, RD6, KMK and IR29 (Table 6). In the old leaves, at 0 dS /m and 4 dS /m, Na+/K+ ratios of all lines and cultivars did not show significant difference. At 8 and 12 dS /m, there was tendency of low Na+/K+ in Pokkali, FL478, FL496, KMK and DDG, which were tolerant to salinity (low Na+/K+ ratio Table 6), while high ratios were found in KDML105, RD6 and IR29. In stems, the Na+/K+ ratio appeared to be higher than those in young and old leaves. Pokkali expressed the lowest Na+/K+ ratio at 4, 8 and 12 dS /m while KDML105 had the highest Na+/K+ ratio at 4 dS /m. At 8 and 12 dS/m, only Pokkali and FL496 had low Na+/K+ ratio, while FL478, DDG and KMK had intermediate Na+/K+ ratios, while IR29, KDML105 and RD6 had high Na+/K+ ratios compared to Pokkali (Table 6). DISCUSSION The results of the study indicated that there was slight or no effect of salinity on rice growth at salinity level of 4 dS/m . This level might not be suitable for screening salt tolerance in rice. However, at 8 dS /m (day/night temperature = 37/ 29C) most rice plants were dead 12 WAS. Therefore, the screening should be conducted at salinity level of 8 dS /m and 12 dS /m and data should be collected 2 WAS. The shoot Na+/K+ ratio is considered to be a reliable parameter used to evaluate salt tolerance ability of rice cultivars (Gregorio et al., 1997; Chotechuen, 2001; Mishra
Table 6 Na+/K+ ratio in 3 parts of rice plant at 1 week after salinization.
Lines/cultiuars
Young leaves (1-4 leaves from the top) 0 dS /m 4 dS/ m 8 dS /m 12 dS /m
Old leaves (after the 4 th leaf from the top) 0 dS /m 4 dS /m 8 dS /m 12 dS /m
0 dS /m
4 dS /m
Stem 8 dS /m
12 dS /m
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et al., 1998). Cultivars with low Na+/K+ ratio usually have an ability to adjust the Na+ content in parts of the plant to prevent toxicity of the ions. At the same time, the rice plant also has K+ absorption capacity to balance the Na+ in the cell (Gregorio et al., 1993). Therefore, the cultivars which have the ability of Na+/K+ balance (low Na+/K+ ratio) is classified as salt tolerant cultivars. In this work, a strong positive correlation (r = 0.84**) between Na+/K+ ratio and salt tolerant scoring (Figure1a) and negative correlation (r = - 0.71**) between Na+/K+ ratio and relative water content (Figure1b) in rice plant were found. Usually, when rice plants are subjected to stress conditions caused by salinity, the tolerant plants will markedly accumulate a number of solute particles, i.e., proline, glycine betain (Bray et al., 2000) and also K+ which is an essential element in many enzyme activators or cofactors and catalysts in plant mechanism(Evans and Sorgor 1966). In general, many researchers point out that in plants, high affinity K+ uptake transporter correlates with low Na+ uptake. In other words, the lower the Na+ uptake, the higher the K + uptake when rice plants are under stresses(Munns et al., 2002; Amtmann and Sanders, 1999, Blumwald, 2000; Munns et al., 2003). Therefore, the concentrations of proline and glycine betain in plant cells are high. In this situation, the
9
percentages of water in the cells were also high due to the osmotic adjustment. This was demonstrated by high relaltive water content in plant under such conditions. It could be concluded that under stress conditions, plants with low Na+/K+ ratio or high K+/Na+ ratio and high relative water content were salinity tolerant. At 8, and 12 dS /m, there were high external Na+ concentrations which affected rice growth as also reported by Greenway (1972) on its effect to reduce availability of essential elements such as K+ by nutrient deficiency. Sodium can partially substitute K+ in a number of crops, while substitution is minimally effective in others. However, the critical level of K+ in plant tissue is relatively high (about 200 ppm), and nearly all K+ are absorbed during vegetative growth (Gardner et al., 1985). In this experiment, a higher level of K+ application (68.75 kg /ha as compared to 43.75 kg /ha) was not able to compete with the high Na+ concentration (at 8 and 12 dS /m) and had no effect on K+ concentration and salt tolerant ability. Salt tolerant was also defined as genotypic differences in biomass production in saline versus non-saline condition over prolonged period of 3-4 weeks. Short term experiment (1 week) measuring of plant size revealed large decrease in growth rate but little genotypic different (Munns and James,
100 98 96 94 92 90 88 86 84 82 80 78 0.0
Salt tolerance scoring
6 5 4 3 2 0.0 .1 .2 .3 .4 .5 .6 .7 .8
%RWC
r = .84**
r = - 0.71**
.1
.2
.3
.4
.5
.6
.7
.8
Na+/K+ ratio
Na+/K+ ratio
Figure 1a Relationship between Na+/K+ ratio and salt tolerant scoring of 16 rice cultivars/ lines grown in nutrient solution at 6 dS/m.
Figure 1b Relationship between Na+/K+ ratio and %RWC of 16 rice cultivars/lines grown in nutrient solution at 6 dS/m.
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2003). Therefore, additional application of K fertilizer in Northeast Thailand soil which is very low in nutrient contents may not be enough to cope with the high Na+ concentration in salinized soil condition. The relation of salt tolerant lines to plant size (both plant weight and plant height) in this experiment indicated that there were 2 types of salt tolerance ; the first type was associated with the large plant size such as Pokkali (Table 2) with plant weight of 0.940 g/plant, plant height of 80.61 cm, moderately low RWC, Na+/K+ ratio and salt tolerant scoring of 3, 2 WAS. This salt tolerant capability was a dilution effect of the large volume of the vegetative shoot (Yeo and Flower, 1984) and the ability of higher K+ uptake, resulting in low Na+/K+ ratios in shoot and root (Neue, 1991). The second type of salt tolerant mechanism was observed in the lines FL478, FL496, and FL530 which had small plant sizes but high RWC both at predawn and midday. Low Na+/K+ ratio and low salt tolerant scoring indicated that these lines had the mechanism to protect the water loss of stressed plants. This was the type reported by Bolhar-Nor Denkampf and Draxler (1993) in which they clarified that during stomata opening, the starch in chloroplast of guard cell, was degraded. This caused K+ to move from subsidiary cells to enhance osmotic value in vacuoles (osmotic adjustment) and subsequently increased turgor pressure (leaf water potential). The reduction of nutrient uptake and Na+ accumulation in plants grown under high saline medium were also found in long-term response (Munns and Termat, 1986). The longterm effect resulted from the accumulation of salt within expanded leaves (Yeo et al., 1991). Yeo and Flower (1986) also reported that the salinity resistance was not conferred by a single factor, but was indeed the sum of many contributory physiological traits, which were not necessary linked. Pokkali resistant ability, therefore, was contributed not only by dilution effect but also by second type of salt tolerant mechanism i.e. Na+ exclusion. Pokkali plant size itself rendered dilution
effect to contribute predominately, while in RILs derived from Pokkali and IR29, Na+ exclusion mechanism (Munns et al., 2002) was predominant. The selection for salt tolerant parents can be made using nutrient solution for screening at seedling stage. The relationship between salinity tolerant scoring and Na+/K+ ratio was also taken into consideration because Na+/K+ ratio rather than Na+ alone has been used as an index of salinity tolerance for cultivars comparison in rice (Asch et al., 2000; Zhu et al.,2001). The cultivars selected in this manner were confirmed to be also adapted well to salinized soil condition during vegetative stage. The Na+/K+ ratio also indicated that the tolerant cultivar/lines were Pokkali and FL496. Moderately tolerant cultivar/lines were FL478, DDG and KMK and susceptible cultivars were IR29, KDML105 and RD6. CONCLUSION Screening for salt tolerant parent materials was conducted based on physiological characters, visual symptom(salt tolerant scoring), relative water content, Na+ and K+ concentrations and Na+/K+ ratios in shoot and different parts of rice plant. The screening at seedling stage using nutrient solution culture was the most appropriate and reliable technique for a large number of plant materials generated in each year. The screening at vegetative stage confirmed that the lines were salinity tolerant when grown in pots. In this study, the lines identified as salt tolerant donors were FL478, FL496 and FL530. KMK and DDG were moderately tolerant donors (landrace cultivars) and KDML105 and RD6 were salt susceptible cultivars. The lines and cultivars which were salt tolerant and moderately tolerant will be used as salt tolerant parent in breeding program. ACKNOWLEDGEMEMTS We are grateful to Rockefeller Foundation,
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for granting the scholarship and we would like to acknowledge Dr. Shu Fukai (School of Land and Food Science, the University of Queensland) for editing this manuscript.
LITERATURE CITED Akbar, M. and Yabuno, T. 1974. Breeding for saline-resistant variety of rice. II. Comparative performance of some rice varieties to salinity during early development stage. Japan J. Breed. 24 : 176-181. Amtmann, A. and Sanders, D. 1999. Mechanisms of Na+ uptake by plant cells. Adv. Bot. Res. 29 : 76 112. Asch, F., Dingkuhn, M., Dorffing, K. and Meizan, K. 2000. Leaf K/Na ratio predicts salinity induced yield loss in irrigated rice. Euphytica 113: 109-118. Blumwald, E. 2000. Sodium transport and salt tolerance in plant cells. Curr. Opin. Cell Biol. 12 : 431 434. Bolhar-Nor Denkampf, H. R. and Draxler, G. 1993. Functional leaf anatomy In D.O. Hall, J.M. O. SCURLOCK, H.R. Bolhar-Nor Denkampf, R.C. Leegood and S.P. Long (eds.) Photosynthesis and Production in a Changing Environment. A Field and Laboratory Manual. Chapman and Hall, London. pp. 99-112. Bray, A.E., Bailey-Serres, J. and Weretilnyk, E. 2000. Responses to abiotic stresses. In B. Buchanan, W. Gruissem,R. Jones, (eds.) Biochemistry & Molecular Biology of Plants. Rockville, Marryland, pp. 1158 1175. Chotechuen,S. 2001. Responses of rice (Oryza sativa L.) to salt stress. Paper presented to the Rice Research Seminars held by Prachinburi Rice Research Center and Ayutthaya Rice Experiment Station on 5-6 March 2001 at Aran Mermaid Hotel, Sra Kaew, Thailand.
pp.34-77. Evan, H.J. and Sorger, G.J. 1966. Role of mineral elements with emphasis on the univalent cations. Annu. Rev. Plant Physiol.17: 4776. Gardner, F.P., Pearce, R.B. and Michell, R.L. 1985. Physiology of Crop Plants. The Iowa State University Press, Ames, Iowa. pp.98131. Greenway, H. 1972. Salinity plant growth and metabolism. J. Aust. Insti. Agri. Sci. 39 : 24 34. Gregorio, G.B. 1997. Tagging salinity tolerance genes in rice using amplified fragment length polymorphism (AFLP). UPLB, Ph.D Thesis, 118 p. Gregorio, G.B., Dharmawansa, S. and Mendoza, R.D. 1997. Screening rice for salinity tolerance. IRRI Discussion Paper Series NO.22 International Rice Research Institute, Manila, Philippines. pp.2-23. Gregorio, G.B. and Dharmawansa, S.1993. Genetic analysis of salinity tolerance in rice (Oryza sativa L.) Theor. Appl. Gen. 86 : 333-338. Kaddah, M.T., Lehman, W.F. and Robinson, F.E. 1973. Tolerance of rice (Oryza sativa L.) to salt during boot, flowering, and grain filling stages, Agronomy J. 65: 845 847. Mishra, B., Singh, R.K. and Jetly, V.1998. Inheritance pattern of salinity tolerance in rice. J. Genet. Breed. 52: 325-331. Munns, R. and Termat, A. 1986. Whole plant responses to salinity. Aust. J. Plant Physiol. 13: 143 160. Munns, R.1993. Physiological processes limiting plant growth in saline soil : some dogmas and hypothesis. Plant Cell Enviro.16 :15 24. Munns, R., Hussain, S., Rivelli, A.R., James, R.A., Condon, A.G., Linsay, M.P., Lagudah, E.S., Schachtman, D.P. and Hare, R.A. 2002. Avenues for increasing salt tolerance of crops, and the role of physiological based selection traits. Plant and Soil 247: 93-105. Munns, R. and James, R.A. 2003. Screening
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methods for salinity tolerance : a case study with tetraploid wheat. Plant and Soil 253: 201-218. Neue, H. U. 1991. Adverse soil tolerance of rice : Mechanisms and screening techniques. pp. 243-250. In P. Deturck and F.N. Ponnamperuma (eds.) Rice Production on Acid Soil of the Tropics. Institute of Fundamental Studies, Kandy, Sri Lanka. Pearson, G.A. and Ayers, A. D. 1960. Rice as a crop for salt-affected soil in process of reclamation. U.S. Agri. Prod. Rep. 43 : 1-13. Toenniessen, G.A. 1984. Review of the world food situation and the role of salt-tolerant plants. pp.399-413. In R.C. Staples, G.A. Toenniessen (eds.) Salinity Tolerance in Plants Strategies for Crop Improvement. Wiley, New York. Yeo, A.R. and Flowers, T.J.1984. Mechanisms of salinity resistance in rice and their role as physiological criteria in plant breeding. pp.151-170. In R.C. Staples, and G.A.
Toenniessen (eds.) Salinity Tolerance in PlantsStrategies for Crop Improvement. Wiley,New York. Yeo, A. R. and Flowers, T. J. 1986. Salinity resistance in rice (Oryza sativa L.) and a pyramiding approach to breeding varieties for saline soils. Aust. J. Plant Physiol. 13: 161 173. Yeo, A. R., Lee, K. S., Izard, P., Boursier, P. J. and Flowers, T. J. 1991. Short and long term effects of salinity on leaf growth in rice (Oryza sativa L.). J. Exp. Bot. 42: 881 889. Yoshida, S., Forno, O. A., Cock, J. H. and Gomez, K. A. 1976. Laboratory Manual for Physiological Studies of Rice. International Rice Research Institute. Manila. pp.61 66. Zhu, G.Y.,Kinet, J.M. and Lutts, S. 2001. Characterisation of rice (Oryza sativa L.) F3 populations selected for salt resistance. I Physiological behaviour during vegetative growth. Euphytica 121: 25-263.
Weed Control Measures and Moisture Conservation Practices Effects on Seedbank Composition and Vertical Distribution in the Soil
Girma Woldetsadik1, Sombat Chinawong2, Rungsit Suwanketnikom3, Sunanta Juntakool3 and Visoot Verasan4
ABSTRACT Change in the weed seedbank due to crop production practices is an important determination of subsequent weed problems. Research was conducted to compare weed seed bank composition and vertical distribution of weed seed in the soil among four weed control measures and four moisture conservation practices at Dera Sub-Center and Melkassa Agricultural Research Center. Seed numbers at 45 cm depth were lower in pre-emergence of primagram at the rate of 3 L/ha treatment (137 seeds/m-2) and pre- and post-emergence of primagram at the rate of 3 L/ha plus 2, 4-D at the rate of 1 L/ha (105 seeds/m-2) at Dera and Melkassa, respectively. More than 60% of the weed seed bank was concentrated in the upper 15 cm of soil layer in post-emergence treatment at Dera and pre plus post-emergence at Melkassa site. The seed bank of the moisture conservation treatments was more uniformly distributed over depth and greater than the other systems. Chenopodium fasciulosum, Cyprus rotundus, Eragrostis aspera, and Sorghum arundenanceum were the most commonly found in the seed bank. Key words: moisture conservation, flat bed, ridge, vertical distribution, and furrow
INTRODUCTION The weed seed bank, which comprises of viable seeds in the soil and on its surface, is the principal source of annual weeds in the field crops. Size and composition of the seed bank as well as above ground weed flora reflect past and present weed, crop, and soil management (Roberts et al., 1981). Reducing the size of weed seed bank has been a long-term goal of weed management strategies, especially for field cropped continuously (Schweizer et al., 1984). Additions and losses of
seed from the seed bank are affected by physical, biological and management factors that interact over time to result in shifts in weed flora (Cavers and Benoit,1989). Many weed infestations in cropping seasons arise from the weed seed bank, so changes in the seed bank due to agricultural management practices ultimately result in changing in observed weed flora. However, seed bank changes must be of sufficient magnitude to produce detectable changes in weed flora since only a small percentage of seed residing in soil is expressed as flora during any
1 2 3
Bako Agricultural Research Center, P.O. Box-3, Bako, Ethiopia. Faculty of Agriculture at Kamphaeng Saen, Department of Agronomy, Kasetsart University, Nakom Pathom 73140, Thailand. Faculty of Agriculture, Department of Agronomy, Kasetsart University, Bangkok 10900, Thailand.
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given growing season (Roberts, 1963; Harper, 1977). Weed control by herbicides and mulch are two primary practices that have an impact on weed seed banks. Recognizing the importance of herbicide and mulching in altering species composition in the weed seed bank can lead to improved strategies for weed management. Weed seed depth in the soil influences germination and seedling. Seed at or just below the soil surface often germinate more than seed buried deeper in the soil (Chepil, 1946; Herr et al., 1970). Seed placed deep by plowing may remain dormant until further tillage places them where germination may occur. Weed species with long dormancy are favored by plowing. Seed buried deep in the soil also takes longer to emerge and develops seedling characteristics than weed placed shallow (Mester and Buhler, 1990). Weak seedlings are easier to kill by chemical or mechanical methods than more vigorous plants (Herr et al., 1970, Mester and Buhler, 1990). Depth for optimum germination and development varies among species. Thus, it is very important to analyze the seed bank in the soil, because seed density estimates is useful for predicting weed infestation and enables a minimum use of herbicides (Buhler et al., 1992). Effective weed management is critical for successful maize production. The use of herbicides can also influence the species composition of the seed bank, and may increase or decrease it, depending on the chemicals used (Ball et al., 1989), and they can also cause species shifting (Roberts, 1968). In general, it can be said that interactions among herbicides, land preparation and cultural practice have altered the sizes and natures of seed bank (Roberts, 1981). One key factor that can influence herbicide effectiveness is the propertied weed populations in a field. The numbers of weed seed found in the soil seed bank determine the propertied weed population in a field. The seed bank is a continuous fluctuation due to introductions of new weed seed and losses
of seed from germination or decay (Maxwell and Ghersa, 1992). The soil seed bank population declined by more than 90% after five years of continuous maize with excellent control (Schweizer et al., 1984). However, the seed bank rapidly increased when the level of weed management reduced. The level of input required to obtain acceptable weed control is related to the size of the seed bank. Little work has been done quantifying depth distribution of weed seed by herbicide and moisture conservation practices under field conditions. No previous work in herbicide and mulch effects on seed germination was found in Ethiopia. This experiment evaluated effects of herbicide and weed management on numbers, depth distribution, and viability of the soil weed seed bank. The objective of this study was to compare weed seed bank composition and vertical distribution of weed seed in the soil among four weed control measures and four moisture conservation practices. MATERIALS AND METHODS Study site and agronomic practices A field experiment was conducted at Dera sub-Center and Melkassa Agricultural Research Center during the rainy season of 2003. The soil types at Dera and Melkassa are diverse, most of them are shallow and the organic matter content is quite low between 0 and 2% in most areas, resulting in poor waterholding capacity. The soils are generally brown, grayish brown or light brown. The textures of the soil are either clay loam, loam or sandy loam. The experiment was laid out in a split- split - plot design, comprising 3 levels of soil depth [0-15 cm, 15-30 cm, and 30- 45 cm] in main plots, 4 levels of fertilizer [N0, Control, N1, 10, N2, 20, and N3, 30 kg N/ha] in sub-plots and 4 weed control treatments [W 0, weedy check, W 1, primagram Gold 660 SC 3 L/ha (pre-emergence); W2, primagram Gold 660 SC 3 L/ha and 2, 4-D 1
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L/ha (pre and post-emergence); W3, 2, 4-D 1 L/ha (post-emergence)] in sub- sub plots and was replicated thrice. Maize variety Melkassa-1 double top-cross early duration was sown in rows of 25 cm apart on 8 July 2003 and 29 June 2003 at Dera and Melkassa, respectively. Full dose of P2O5 and half dose of N were applied basal and remaining N was top dressed in 2 equal splits. The cropping system was a maize -pulse rotation, except for 2002 to 2003 where maize followed maize. Agronomic practices and herbicide treatments were consistent with those recommended for maize in Melkassa agricultural research center. Weed control treatments representing those most commonly used in the areas included primagram at the rate of 3 L/ha. The herbicide was applied within 3 days after maize planting and before weeds had begun to emerge. Post-emergence of 2, 4-D was used at the rate of 1 L/ha one month after maize emergence. Seed bank analysis Studies on seed bank in cultivated soil have led to the development of many techniques for estimating seed density from soil samples. To determine the numbers of viable seed in the soil, soil samples were taken on 15 May 2003, after seedbed preparation but prior to seeding and herbicide application. To measure the vertical distribution of weed seed to a depth of 45 cm in the soil, the soil was collected at surface of 0-15 cm, 16-30 cm and 31- 45 cm depths and the 3 samples from each depth were pooled. The soil was airdried and sieved through a 2 mm screen to break up large soil peds. The soil was spreaded in 22 cm square trays and watered twice daily in a greenhouse. Soil samples containing seeds were watered as needed and maintained with water at a depth of 1 cm. Weed seedlings that emerged were identified and counted by species and removed every 7 days from the beginning of weed emergence until no weed emergence was observed at the seedleaf stage or 1-leaf stage to avoid the periodic
interaction of weeds, throughout the growing period. Analysis Analyses of variance (ANOVAs) were used to test the effect of weed control and moisture conservation practices on the seed banks and vertical distribution of seeds in the soil. Means were separated by LSD (0.05). Vertical distribution of seed was expressed as a proportion of the total seed bank. RESULTS AND DISCUSSION Seed bank size Sizes of the seed bank differed among weed control measures and moisture conservation practices (Table 1). The highest total seed number was found in the Melkassa soil where 366 and 300 seeds/ m-2 were observed in post herbicide application and ridge and furrow treatments, respectively. Weedy check gave 222 total weed seeds/ m-2 at Melkassa after post herbicide 2, 4-D. Even though the total weed seeds at Dera for weed control measurers were similar to the highest total weed seeds/ 187 m-2 was from weedy check. Primagram at the rate of 3 L/ha caused no difference in seed numbers across weed management due to effective weed control and low seed numbers. In contrast, 2, 4-D at the rate 1 L/ha caused greatest difference for weed management by depth. Primagram plus 2, 4 D had fewer seed in the soil profile than 2, 4-D. Lower seed numbers in pre plus post herbicide application than in control might be due to better weed control in these weed control treatments and to the stimulatory effect of weed control in inducing weed seed germination (Barralis and Chadoeuf, 1980). Seed numbers in ridge and furrow were higher than those in flat bed; flat bed plus straw mulching and ridge and furrow plus straw mulching at the Melkassa site. Differences among sites could be attributed in part to differences in previous vegetative cover in
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addition to differences in soil type. Pre-emergence of primagram plus post-emergence of 2, 4-D at both sites were best to suppress weeds from weed control measures. The effect of mulch to control weeds was found effective only at Melkassa. Vertical distribution The vertical distributions of seed in the soil varied among weed control treatments (Table 2). The top 0-15 cm of soil contained 43.5, 40.2, 40, and 61 % of the seed bank in weedy check, primagram, primagram plus 2, 4-D, and 2, 4-D at Dera, respectively. At Melkasaa, the top 0-15 cm of soil contained 59.1, 56.8, 62.7, and 58.3 % in weedy check, primagram, primagram plus 2, 4-D and 2, 4-D weed control measures, respectively which were greater as compared to those at Dera site. The highest concentrations of weed seed were found in 0-15 cm layer. In 16-30 cm, distribution of weed seeds in the soil declined and were found in the ranges from 18.4 in post-emergence of 2, 4D to 46.5% in pre-emergence of primagram at Dera and from 19.7 in post-emergence of 2, 4-D to 33.5% in pre-emergence of primagram at Melkassa, which were almost similar in weed seeds in the soil among the four weed management systems. Seeds in 31-45 cm soil depth declined steadily and were found in the range from 13.3 in pre-emergence of primagram to 22.7 % in pre plus post-emergence of primagram plus 2, 4-D at Dera in the order of primagram at the rate of 3 L/ha, weedy check, 2, 4D at the rate of 1 L/ha and primagram @ 3 L/ha plus 2, 4-D at the rate of 1 L/ha, respectively. In moisture conservation practice, at Dera flat bed plus straw mulching recorded the lowest percent of weed seed at 0-15 cm soil depth and the highest at 16-30 cm were recorded. Ridge and furrow recorded the highest weed seed at 0-15 cm and the lowest at 31-45 cm, while ridge and furrow plus straw mulching recorded the lowest percent of weed seed at 16-30 cm and the highest at 31-45 cm. At Melkassa flat bed plus straw mulching showed the lowest percent of weed seed at 0-15 cm
and 31- 45 cm and the highest at 16-30 cm where as flat bed without straw mulching treatment recorded the highest percent of weed seed at 16-30 cm and the lowest at 16-30 cm. Ridge and furrow with and without straw mulching expressed medium percents of weed seed at the three depths and which were not different from one another. At both sites, flat bed plus straw mulching recorded the lowest percentage of weed seed at 0-15 cm layer. The highest number of weed seed among the four weed control measures was obtained from weedy check followed by primagram @ 3 L/ha treated plots (Table 3). Ridge and furrow also gave the highest total weed seed followed by flat bed. The control measure with mulch gave less number of weed seed as compared to without mulch. Over 52.7% seeds were concentrated in the top 0-15 cm layer in 2, 4-D @ 1 L/ha herbicide applied treatment and flat bed planting method (Table 3). Weed control measurers ranked 2, 4- D > weedy check > primagram > primagram + 2, 4-D for total seed in the top 45 cm soil depth. Depths by weed control interaction were also significant at both sites. Species composition At four weed control measures, twentyeight weed species were identified in the seed bank or in the field. Almost all of the species observed only in seed bank samples were relatively common at the experimental site; hence it was surprising that these species appeared in the soil samples. Over 90 % of each seed bank among weed control treatments and soils were composed of fewer weed species with 18 species being common to both sites (Table 4). The weeds in different combinations were found at each seed bank. The results showed that the weed seed bank was mainly composed of Chenopodium fasciulosum, Eragrostis aspera, Cyperus rotundus, Sorghum arundenanceum, Flaveria trinerva, Argemone mexicana, Hetilotropium cineraseioen, Amaranthus hybridus, Anagalis arvensis, Nicandra
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phylasoides, Datura stramonium, Galinsoga parviflora, Euphorbia heterophylla, Launea comuta, Portulaca oleracea, Foeum vulgare, etc. at the two sites. Chenopodium fasciulosum was the only species present in each weed control treated plots constituted 21.7 % of the total seed bank
across two locations and consistently made up greater percentages of the seed bank in pre-, pre plus post- and post- emergences than weedy check. Cyprus rotundus, Eragrostis aspera and Sorghum arundenanceum were the next most abundant species at each site. Galinsoga parviflora was
Table 1 Numbers of seed per m2 at a depth of 45 cm in four-weed control measures and averaged moisture conservation practices. Total weed seeds Weed control measures W0 (weedy check) W1 (Primagram) W2 (Primagram + 2, 4-D) W3 (2, 4-D) LSD (0.05) Moisture conservation practices FB (Flat bed) R & F (Ridge and furrow) FB + SM (Flat bed + straw mulch) R & F + SM (Ridge and furrow + straw mulch) LSD (0.05) Dera Melkassa
187 184 137 135 29.92 154 160 167 152 35.22
222 144 105 366 66.51 262 300 168 203 37.08
Table 2 Influences of weed control measure and moisture conservation practices on vertical distributions of total weed seed (%) to a 45 cm soil depth after maize cropping. Dera Depth (cm)Weed control measures W0 W1 W2 0-15 43.5 40.2 40 15-30 39.8 46.5 37.3 30-45 16.8 13.3 22.7 Moisture conservation practices Depth (cm) FB R & F FB + SM 0-15 48.8 51.8 36.5 15-30 31 33.8 46.9 30-45 20.2 10.6 10.8 Melkassa
W3 61 18.4 20.6 R & F + SM 48.6 30.3 21.1
W0 59.1 28.7 12.2 FB 61.3 23.2 15.5
W1 56.8 33.5 9.7 R&F 59.2 28.7 12.1
W2 62.7 22.9 14.4
W3 58.3 19.7 22
FB + SM R and F + SM 58.8 55.9 30.4 26 10.8 18.1
W0 = weedy check, W1= primagram @ 3 L/ha, W2 = primagram @ 3 L/ha + 2, 4-D @ 1 L/ha, and W3 = 2, 4-D @ 1 L/ha. FB = flat bed, R & F = ridge and furrow, FB + SM= flat bed + straw mulch, and R & F + SM = ridge and furrow + straw mulch. L1 = Dera and L2 = Melkassa
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found in all weed control measures at both sites but was not present in post herbicide treatment. Commelina benghalensis, Agenatum spp, Convolvulus spp were very small in number and commonly found in Dera while Euphorbia heterophylla, Leucas martini, Sidaalba spp, Tagets minuta, Tribulus spp, and Portulaca oleracea were found only in Melkassa. CONCLUSION In this study, the herbicides and residues
could influence weed population levels, the rate of population growth and species composition. This might indicate that herbicides and residues were effective enough to prevent excessive weed seed production. This result was obtained from oneyear data, the longterm effects of herbicide and residue on weed populations were not studied. Further studies are needed to examine the interaction effects of herbicide with residue application, allelophaty and shading effects and may result in reduced weed seedling emergence. It also needs further study on farmers field where
Table 3 Distribution of weed seed by soil depth as affected by weed control and moisture conservation practices. Weed seed as affected by depth (cm) Weed control Moisture conservation Dera 16-30 No m-2 Weedy check (control) FB 35 18 3 R&F 24 20 5 FB + SM 28 14 5 R & F +SM 23 7 5 Primagram @ 3 L/ha (pre-emergence) FB 23 15 1 R&F 42 16 12 FB + SM 25 12 4 R & F + SM 12 20 2 Primagram @ 3 L/ha and 2, 4-D @ 1 L/ha (pre and post-emergence) FB 6 87 3 R&F 15 7 2 FB + SM 37 12 6 R & F + SM 16 19 6 2, 4-D @ 1 L/ha (post-emergence) FB 33 4 5 R&F 13 8 6 FB + SM 14 8 2 R & F + SM 21 15 6 42 31 21 29 18 17 15 27 13 19 11 15 85 82 13 45 16 11 10 14 12 10 13 7 7 8 5 5 22 25 8 20 14 6 16 12 8 5 4 8 7 5 4 6 19 25 7 15 Melkassa 16-30
0-15
31-45
0-15
31-45
FB = flat bed, R & F = ridge and furrow, FB + SM= flat bed + straw mulch, and R & F + SM = Ridge and furrow + straw mulch. L1 = Dera and L2 = Melkassa
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herbicides and residues were rarely or not applied. The results and information obtained would expedite the development of management strategies to reduce populations of weed with seed bank
regeneration strategies and to manage the successional dynamics of weed in weed control measures.
Table 4 Seed bank compositions (45 cm deep) in two sites after one year of maize cropping with four weed control measures. Seed bank composition as affected by soil and weed control Dera Melkassa W0 W1 W2 W3 W0 W1 W2 W3 43 12 26 2 19 15 26 1 7 12 3 * 1 2 * 1 * 2 1 0 2 1 1 3 * * * 89 3 19 2 9 9 13 3 13 3 7 * 3 0 * 1 * 3 1 1 0 0 3 0 * * * 56 1 7 4 36 7 3 5 8 2 0 * 1 1 * 1 * 3 1 0 2 0 0 0 * * * 55 1 2 1 24 3 22 3 11 7 0 * 0 2 * 2 * 4 0 0 0 0 1 1 * * * 10 24 12 5 5 16 9 3 5 9 3 4 1 2 1 0 0 0 0 0 1 * * * 0 0 1 30 13 6 14 3 7 2 4 8 3 7 1 3 1 1 0 2 1 1 1 0 * * * 1 0 1 9 7 1 11 6 5 1 12 6 2 0 0 1 1 1 0 0 1 1 0 0 * * * 0 0 1 11 12 27 6 19 3 19 6 2 3 0 2 0 1 0 2 0 0 0 0 0 * * * 1 1 1
Species
Chenopodium fasciulosum Flaveria trinerva Cyperus rotundus Amaranthus hybridus Eragrostis aspera Argemone mexicana Sorghum arundenanceum Nicandra physaloides Heliotropism cineraseioen Anagalis arvensis Datura stramonium Euphorbia heterophylla Galinsoga parviflora Launea comuta Portulaca oleracea Foeum vulgare Tagetes minuta Erucastrum arabicum Setaria pumila Eleusine indica Xanthium strumarium Commelina benghalensis Ageratum conyzoides Convolvulus arvensis Leucas martini Sidaalba spinosa Tribulus terrestris
Values are means of 3 replications at Melkassa and Dera sites.* Species not found at that site;W0 = weedy check (control); W1 = Primagram 660 SC @ 3 L/ha (pre emergence); W2 = Primagram 660 SC @ 3 L/ha and 2, 4-D @ 1 L/ha (pre and post emergence); W3 = 2, 4-D @ 1 L/ha (post emergence). L1 = Dera and L2 = Melkassa
193
LITERATURE CITED Ball, D. A. and S. D. Miller. 1989. A composition of techniques for estimation of arable soil seed banks and relationship to weed flora. Weed Res. 29: 365-373. Barralis, G. and R. Chadoeuf. 1980. Etude de la dynanique d une communaute adventice. 1. Evolution de la flora adventice au course du cycle vegetative dune culture. Weed Res. 20: 231-237. Buhler, D. D., J. L. Gunsolus and D. F. Ralston. 1992. Integrated weed management techniques to reduce herbicide inputs in Soybean. Agron. J. 84: 973-978. Burnside , O. C., R. S. Moomaw, F. W. Roeth, G. A. Wicks and R. G. Wilson. 1986. Weed seed demise in soil in weed free corn (Zea mays L.) production across Nebraska. Weed Sci. 34: 248-251. Cavers, P. B. and D. L. Benoit 1989. Seed banks in arable land. pp 309-328 in M. A. Leck, V. T. Parker and R. L. Simpson, eds. Ecology of Soil Seed Banks. Academic Press New York Chepil, W. S. 1946. Germination of weed seeds: 11. The influence of tillage treatments on germination. Science. Agric 8: 347-357.
Harper, C. A. 1977.The seed bank, pp 83-110. In: Population Biology of plants. Academic Press, London. Herr, D. E. and E. W. Stroube. 1970. Velvetleaf control as influenced by herbicide placement and seed depth. Weed Sci. 18: 459-461. Maxwell, B.D. and C. Ghersa. 1992. The influence of weed seed dispersion versus the effect of competition on crop yield. Weed Techn. 6: 196-204. Mester, J. C. and D. D.Buhler.1990. Effect of seedling depth on Velvetleaf seedling development and response to cynazine. Weed Sci. 38: 34-38. Roberts, H. A. 1963. Studies ion the weeds of vegetable crops. 111. Effect of different t primary cultivation on the w weed seeds in the soil. J. of Ecology 51: 83-95. Roberts, H. A. 1968. The changing population of viable weeds seeds in an arable soil. Weed Res. 8: 253-256. Roberts, H. A. and J. E. Neilson. 1981. Seed banks in soils. Adv. Appl. Biology 6: 1-55. Schweitzer, E. E. and R. L. Zimdahl. 1984. Weed seed decline in irrigated soil after six years of continuous corn (Zea mays L.) and herbicides. Weed Sci. 32: 76-3.
Genetic Diversity of Elite and Exotic Oilseed Meadowfoam Germplasm using AFLP Markers
Sureeporn Katengam1 and Steven J. Knapp2
ABSTRACT Amplified fragment length polymorphism (AFLP) is a PCR-based marker, which is suitable for DNA fingerprinting. The AFLP fingerprinting has not been described in meadowfoam. This powerful method was utilized to access genetic diversity of 41 meadowfoam accessions belonging to the genus Limnanthes. The objectives were to estimate polymorphic information contents (PIC) for AFLP markers and genetic distance among germplasm, and to assess the pattern of genetic diversity in meadowfoam germplasm. One hundred and seventy six polymorphic AFLP markers were produced using 6 primer combinations across 41 accessions. The PIC value ranged from 0.0 to 0.5 and 42 % of germplasm showed high PIC scores in a range between 0.45 and 0.5. Genetic distance ranged from 0.14 to 0.55 with an average of 0.44. The UPGMA clustering phenogram based on the distance matrix was consistent with the known taxonomic classification. The first three principal coordinate analyses accounted for 37 % of total variation of genetic distance estimated. Cluster analysis and principal component analysis clearly separated L. floccosa from L. alba. Within L. alba, subspecies alba and versicolor were distinctly separated into two groups. The results suggested genetic diversity among meadowfoam germplasm was very high. This information is useful to layout framework for meadowfoam improvement thereby enhancing productivity and performance of cultivated meadowfoam. Key words: meadowfoam, Limnanthes sp., genetic diversity, DNA fingerprinting, AFLP
INTRODUCTION Meadowfoam (Limnanthes sp.) is an annual oil seed crop, native to Southern Oregon and California (Mason, 1952). Seed oil of meadowfoam contains unique unsaturated very long chain fatty acids (C20 and C22) with outstanding oxidative stability (Isbell, 1997). Cultivated meadowfoam which belongs to section Inflexae, family Limnanthaceae is based on Limnanthes alba. The section Inflexae comprises of 4 species, namely L.
alba, L. floccosa, L. gracilis and L. montana. The primary gene pool of L. alba is composed of L. alba ssp. alba and L. alba ssp. versicolor, whereas L. floccosa, L. gracilis, and L. montana are identified as a secondary gene pool of L. alba. Meadowfoam has been domesticated since 1973 (Jain, 1986). The L. alba was evaluated as the most promising species in this genus for its lower moisture requirements, adaptation to wide ranges of environments, and high seed yield (Gentry and Miller, 1965). The first non- shattering cultivar,
Department of Agronomy, Faculty of Agriculture, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand. sureeporn.k@ubu.ac.th Department of Crop and Soil Science, Oregon State University, Corvallis, OR, 97331 USA.
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Foamore, developed for commercial production was released in 1974 (Calhoun and Crane, 1975). Breeding and cultivars development is underway at the Oregon State University with the main goal of increasing the productivity of meadowfoam by developing superior cultivars, discovering and developing novel phenotypes, and advancing our understanding of the genetics of economically important traits (Knapp and Crane, 1999). Knowledge of the genetic diversity and relationships among germplasm is essential to the improvement of meadowfoam. Generally, the genetic diversity of germplasm collections can be obtained from pedigree records, morphological traits, isozyme and DNA markers (Smith et al., 1990; Mumm and Dudley, 1994). However, a small number of polymorphic markers obtained from isozyme markers and unfavorable phenotypic expressions of some morphological traits due to the environmental effects are known as limitations of these markers (Smith et al., 1990). The advent of DNA markers opens the ways to solve this problem since DNA markers can reveal tremendous number of genetic loci and they are phenotypic neutral and not subjected to environmental effects. A variety of DNA markers have been applied to cultivar improvement and germplasm management. Owing to its capacity to reveal a large number of marker loci in a short period of time (Vos et al., 1995), AFLP (Amplified fragment length polymorphism) appears to be the leading DNA-based marker systems for DNA fingerprints. AFLP has shown to be a powerful tool for genetic diversity study in many plant species such as soybean (Maughan et al., 1996), barley (Ellis et al., 1997), and rice (Zhu et al., 1998). The high throughout AFLP markers were employed to evaluate the genetic diversity among recent meadowfoam germplasm. The objectives were (1) to estimate polymorphic information contents (PIC) for AFLP markers and estimate genetic distance among inbreds, open-pollinated cultivars, wild population and all genotypes, (2) to
assess the pattern of genetic diversity and relationships of meadowfoam germplasm using UPGMA cluster analysis and principal coordinate analysis. MATERIAL AND METHODS Plant materials A total of 41 meadowfoam accessions representing nine inbred lines, eight openpollinated cultivars, and 24 wild populations were included in this diversity study (Table1). The meadowfoam seeds were germinated and grown as described by Katengam et al. (2002). Leaves from 50 to 55 day-old plants were harvested, immediately frozen, and stored at -80C prior to DNA extraction. AFLP fingerprints Genomic DNA was extracted from frozen tissue using a protocol similar to Lodhi et al. (1994) with minor modification. AFLP analysis was carried out essentially as developed by Keygene (Waeningen, NL) with the minor modification that the selection of a subset of fragments on steptavidin beads was omitted (Vos et al., 1995). AFLP fingerprints were produced using six MseI-EcoRI primer pairs with three selective nucleotides (Table 2). Data analysis Gene diversity was used to describe the relative value of AFLP marker with respect to the degree of polymorphism exhibited for each polymorphic locus. Thus, Gene diversity =
i =1 pi2
k
where pi is the frequency of ith allele and k is the number of alleles (Ott, 1991). Anderson et. al. (1993) indicated that gene diversity was essentially the same as the polymorphic information content (PIC) as used by Botstein et al. (1980). This parameter is sometimes called heterozygosity. Due
196
Table 1 Meadowfoam germplasm (41 accessions) for AFLP fingerprinting. Accessions 1. OMF63 S5 2. OMF64 S5 3. OMF66 S5 4. OMF109-1 5. OMF109-2 6. OMF109-3 7. OMF40-11 (Mermaid S5) 8. LAG109 F4 9. LAG111 F4 10. OMF66 (Redding) 11. OMF 158 12. OMF159 13. OMF160 14. OMF161 15. OMF57 16. OMF52 17. OMF53 18. PI 374793 19. PI 374794 20. PI 374795 21. PI 374796 22. PI 374797 23. PI 374798 24. PI 367900 25. PI 374792 26. Foamore 27. Mermaid 28. Floral 29. Knowles (OMF69) 30. OMF86 31. OMF78 32. OMF87 33. PI 283724 34. PI 420137 35. PI 283720 36. PI 420133 37. PI 283719 38. OSU-LF-4 39. PI 283721 40. PI 283725 41. OMF62-92 Description Self-pollinated inbred line (Selected from OMF159) Self-pollinated inbred line (Selected from OMF160) Self-pollinated inbred line (Selected from OMF66) Self-pollinated inbred line (Selected from Mermaid x OMF62/ OMF64) Self-pollinated inbred line (Selected from Mermaid x OMF62/ OMF64) Self-pollinated inbred line (Selected from Mermaid x OMF62/ OMF64) Insect-pollinated L. alba ssp. alba inbred line (Selected from PI 283703) Self-pollinated Mermaid x L. gracilis ssp. parishii inbred line Self-pollinated Mermaid x L. gracilis ssp. parishii inbred line Wild L. alba ssp. versicolor population Wild L. alba ssp. versicolor population (Recollected PI 283705) Wild L. alba ssp. versicolor population (Recollected PI 374791) Wild L. alba ssp. versicolor population (Recollected PI 374801) Wild L. alba ssp. versicolor population (Recollected PI 374802) Wild L. alba ssp. versicolor population (UC328 or UC457) Wild L. alba ssp. alba population (UC- Calaveras) Wild L. alba ssp. alba population (UC-Sonoma) Wild L. alba ssp. alba population (Placer county) Wild L. alba ssp. alba population (Placer county) Wild L. alba ssp. alba population (Placer county) Wild L. alba ssp. alba population (Butte county) Wild L. alba ssp. alba population (Butte county) Wild L. alba ssp. alba population (Butte county) Wild L. alba ssp. alba population (Sacremento county) Wild L. alba ssp. alba population (Shasta county) Open-pollinated cultivar (Selected from PI 283704) Open-pollinated cultivar (Selected from PI 283703) Open-pollinated cultivar (Mermaid x L. floccosa ssp. grandiflora) Open-pollinated cultivar (Selected from bulk of L. alba ssp. alba) Open-pollinated cultivar (Selected from Knowles) Open-pollinated cultivar (Selected from intermating between L. alba ssp.alba and ssp. versicolor) High oil open-pollinated population (Selected from OMF62) L. gracilis ssp. parishii (Wild species) L. gracilis ssp. gracilis (Wild species) L. floccosa ssp. bellingeriana (Wild species) L. floccosa ssp. grandiflora (Wild species) L. floccosa ssp. floccosa (Wild species) L. floccosa ssp. californica (Wild species) L. floccosa ssp. pumila (Wild species) L. montana (Wild species) High oil open-pollinated population (L. alba ssp. alba)
197
to a bi-allelic feature, the PIC value for AFLP markers therefore ranges from 0.0 (monomorphic) to 0.5 (polymorphic). Binary data representing the presence (1) and absence (0) of specific AFLP marker was generated. Only unambiguous polymorphic bands were scored and entered into a binary matrix as input for the genetic distance analysis. The genetic distance of Roger as modified by Wright (1978) was estimated among all genotypes using NTSYSpc, version 1.8 (Rohlf, 1993). A dendrogram was subsequently generated by cluster analysis based on the unweighted pair group method on the basis of arithmetic averages (UPGMA) using a genetic distance matrix. Goodness of fit of a cluster analysis was tested using cophenetic correlation (r) value from MXCOMP program in NTSYS, which allowed direct comparison between the original dissimilarity matrix that was clustered and the cophenetic value matrix. Principal coordinate analysis based on genetic distance matrix was carried out using the PROC
PRINCOMP procedure of SAS (1996) (SAS Institute, Inc., Cary, NC) to visualize the dispersion of individuals in relation to the first three principal axes of variation. RESULTS AFLP fingerprinting The AFLP fingerprinting of 41 accessions including nine inbred lines, eight open-pollinated cultivars, and 24 wild populations of four species (10 taxa) was performed using six MseI-EcoRI primer combinations (Table 2). These primer combinations were chosen based on previous information of polymorphism level from screening parents for AFLP meadowfoam mapping study (Katengam et al., 2002). A total of 176 AFLP markers were revealed from six primer combinations, which were polymorphic between two or more accessions across the 41 germplasm (Table 3). The polymorphic markers from each primer combination varied from 18 to 40 markers
Table 2 Oligonucleotide adapters and primers used for AFLP fingerprinting. Adaptors: EcoRI adaptors* MseI adaptors* AFLP primers EcoRI +1** MseI +1** EcoRI +3 MseI +3
91M35 91M36 92A18 92A19 92R11 92H20 92SO5 92G23 92G24 92G29 92G30 92F10 92F41
5-CTCGTAGACTGCGTACC-3 3-CTGACGCATGGTTAA-5 5-GACGATGAGTCCTGAG-3 3-TACTCAGGACTCAT-5 5-AGACTGCGTACCAATTC / A-3 5-GACGATGAGTCCTGAGTAA / C-3 5-GACTGCGTACCAATTC / ACA-3 5-GATGAGTCCTGAGTAA / CAG-3 5-GATGAGTCCTGAGTAA / CAT-3 5-GATGAGTCCTGAGTAA / CTG-3 5-GATGAGTCCTGAGTAA / CTC-3 5-GATGAGTCCTGAGTAA / CAC-3 5-GATGAGTCCTGAGTAA / CAA-3
* = EcoRI and MseI adaptors were ligated onto the ends of genomic restriction fragments. ** = EcoRI+1 and MseI+1 primers were used in the preamplification of template DNA. The AFLP markers were generated using pairs of EcoRI+3 and MseI +3 primers.
198
with an average of 29 markers per primer pair. The sizes of these markers ranged from 50 to 250 bp. Out of 176 AFLP markers, 142 and 138 AFLP markers showed polymorphism in at least two inbred lines and two cultivars, respectively, whereas 175 AFLP markers revealed polymorphism in at least two wild populations of meadowfoam. Gene diversity (Polymorphic information content, PIC) Estimation of gene diversity in meadowfoam germplasm was represented by polymorphic information content (PIC) value which showed the probability of polymorphism between two randomly lines. The PIC scores for 176 AFLP markers ranged from 0.0 to 0.5 (Figure1). Mean PIC scores was 0.31 for inbred lines, 0.30 for open-pollinated cultivars, 0.40 for wild populations, and 0.39 for all genotypes based on 142, 138, 175, and 176 polymorphic AFLP markers respectively. The distribution of PIC score was dramatically increased from 0.0 to 0.5. Nearly half of markers (42.61%) showed maximum PIC scores with a range of 0.45 - 0.50, indicating a high genetic diversity in meadowfoam germplasm. Distance analysis Genetic distance among 41 accessions
based on 176 AFLP markers was estimated using Rogers genetic distance as modified by Wright (1978), ranging from 0.14 to 0.55, with an average of 0.44 (Table 4). The distances estimated among nine inbred lines varied from 0.14 (between OMF109-1 and OMF109-3) to 0.47 (between LAG109F4 and OMF109-1, OMF109-2, and OMF109-3) with an average of 0.39. OMF109-1, OMF109-2 and OMF109-3 were related as they were developed from the same cross (Mermaid x OMF62/ OMF64), but were selected for different fatty acid concentration profiles. OMF109-2 was
80 70 60
No. of AFLP markers
50 40 30 20 10 0
0.0
5 0.0
5-0
.1 0.1
-0.
15
0.1
5-0
.2
-0. 0.2
25
0.2
5-0
.3
-0. 0.3
35
5-0 0.3
.4 0.4
-0.
45
0.4
5-0
.5
Polymorphic information content
Figure 1 Distribution of polymorphic information content (PIC ) scored for 176 AFLP markers among 41 meadowfoam accessions.
Table 3 Total numbers of informative AFLP marker detected with six primer combinations (one EcoRI+3 primers (ACA) and six MseI+3 primers) used in diversity study. Primers combinations (EcoRI+3 / MseI+3) ACACTC ACACAG ACACTG ACACAC ACACAA ACACAT Total Average Total polymorphic AFLP markers 28 38 40 35 18 20 176 29.33
Table 4 Genetic distance matrix estimated by Roger-W from AFLP fingerprints of 41 meadowfoam accessions.
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
199
1. OMF63S5 2. OMF64S5 3. OMF66S5 4 OMF109-1 5 OMF109-2 6 OMF109-3 7 OMF40-11 8 LAG109F4 9 LAG111F4 10 OMF66 11 OMF158 12 OMF159 13 OMF160 14 OMF161 15 OMF57 16 OMF52 17 OMF53 18 PI 374793 19 PI 374794 20 PI 374795 21 PI 374796 22 PI 374797 23 PI 374798 24 PI 367900 25 PI 374792 26 Foamore 27 Mermaid 28 Floral 29 Knowles 30 OMF86 31 OMF78 32 OMF87 33 PI 283724 34 PI 420137 35 PI 283720 36 PI 420133 37 PI 283719 38 OSU-LF4 39 PI 283721 40 PI 283725 41 OMF6229 0 0.27 0.43 0.47 0.44 0.43 0.42 0.42 0.42 0.37 0.47 0.48 0.46 0.46 0.44 0.46 0.45 0.44 0.45 0.45 0.43 0.46 0.48 0.43 0.43 0.43 0.41 0.42 0.48 0.47 0.50 0.52 0.51 0.49 0.46 0.45 0.42 0 0.43 0.47 0.44 0.43 0.42 0.42 0.41 0.36 0.49 0.50 0.46 0.45 0.46 0.49 0.48 0.47 0.46 0.47 0.42 0.45 0.49 0.46 0.45 0.42 0.40 0.42 0.50 0.46 0.50 0.51 0.49 0.48 0.46 0.45 0.44 0 0.38 0.42 0.41 0.41 0.40 0.41 0.44 0.43 0.42 0.40 0.33 0.42 0.41 0.41 0.40 0.42 0.43 0.41 0.43 0.37 0.37 0.40 0.38 0.40 0.39 0.47 0.41 0.48 0.47 0.49 0.48 0.46 0.43 0.39 0 0.30 0.45 0.43 0.47 0.46 0.47 0.44 0.47 0.47 0.41 0.47 0.45 0.46 0.45 0.47 0.48 0.46 0.49 0.48 0.41 0.47 0.46 0.49 0.47 0.46 0.45 0.48 0.50 0.46 0.51 0.49 0.45 0.45 0 0.42 0.38 0.41 0.42 0.41 0.44 0.44 0.44 0.38 0.43 0.45 0.42 0.41 0.44 0.45 0.42 0.43 0.42 0.38 0.43 0.45 0.45 0.43 0.44 0.41 0.46 0.46 0.45 0.48 0.45 0.40 0.42 0 0.32 0.37 0.35 0.34 0.47 0.47 0.42 0.41 0.41 0.44 0.40 0.38 0.40 0.44 0.40 0.44 0.46 0.42 0.44 0.41 0.41 0.40 0.52 0.46 0.53 0.51 0.49 0.52 0.47 0.46 0.46 0 0.35 0.37 0.34 0.45 0.47 0.43 0.41 0.43 0.47 0.41 0.40 0.40 0.44 0.38 0.41 0.43 0.42 0.43 0.41 0.40 0.41 0.48 0.43 0.51 0.49 0.49 0.52 0.46 0.39 0.47 0 0.34 0.38 0.46 0.48 0.44 0.43 0.45 0.46 0.43 0.42 0.40 0.46 0.36 0.41 0.44 0.40 0.44 0.42 0.40 0.41 0.49 0.42 0.54 0.52 0.55 0.52 0.49 0.38 0.45 0 0.38 0.47 0.48 0.42 0.38 0.43 0.43 0.41 0.39 0.39 0.44 0.37 0.38 0.42 0.39 0.40 0.37 0.41 0.41 0.50 0.43 0.52 0.50 0.51 0.51 0.47 0.44 0.45 0 0.46 0.48 0.43 0.41 0.43 0.44 0.43 0.43 0.43 0.44 0.39 0.45 0.46 0.45 0.43 0.42 0.40 0.43 0.49 0.43 0.52 0.53 0.52 0.52 0.47 0.40 0.46 0 0.42 0.47 0.44 0.47 0.49 0.45 0.45 0.49 0.47 0.46 0.49 0.46 0.46 0.46 0.47 0.46 0.49 0.45 0.49 0.47 0.48 0.48 0.48 0.47 0.46 0.47 0 0.44 0.44 0.44 0.47 0.48 0.46 0.46 0.43 0.43 0.44 0.45 0.42 0.43 0.47 0.46 0.46 0.48 0.47 0.46 0.48 0.48 0.49 0.45 0.47 0.44 0 0.41 0.44 0.46 0.46 0.40 0.43 0.40 0.43 0.44 0.43 0.42 0.43 0.40 0.42 0.43 0.45 0.44 0.47 0.49 0.50 0.51 0.51 0.41 0.44 0 0.36 0.42 0.34 0.38 0.40 0.40 0.41 0.38 0.40 0.40 0.40 0.40 0.39 0.41 0.47 0.38 0.49 0.51 0.49 0.49 0.48 0.42 0.45 0 0.40 0.38 0.38 0.42 0.41 0.41 0.40 0.41 0.45 0.44 0.46 0.43 0.44 0.47 0.46 0.45 0.47 0.47 0.46 0.45 0.46 0.45
1 0 0.31 0.33 0.37 0.37 0.36 0.35 0.41 0.35 0.31 0.30 0.33 0.32 0.32 0.43 0.43 0.38 0.38 0.39 0.40 0.38 0.37 0.37 0.38 0.38 0.37 0.40 0.38 0.40 0.37 0.35 0.36 0.47 0.38 0.47 0.46 0.46 0.47 0.45 0.38 0.39 0 0.39 0.43 0.42 0.45 0.47 0.42 0.44 0.44 0.42 0.44 0.47 0.44 0.51 0.44 0.49 0.49 0.49 0.51 0.49 0.44 0.47
0 0.44 0.39 0.38 0.39 0.43 0.44 0.42 0.41 0.38 0.41 0.38 0.38 0.49 0.49 0.44 0.46 0.49 0.47 0.46 0.43 0.40 0.47 0.42 0.45 0.46 0.43 0.39 0.39 0.40 0.41 0.51 0.47 0.52 0.52 0.52 0.52 0.49 0.41 0.42
0 0.46 0.46 0.45 0.42 0.45 0.40 0.32 0.35 0.39 0.38 0.38 0.47 0.45 0.43 0.39 0.39 0.45 0.41 0.40 0.42 0.43 0.40 0.40 0.43 0.44 0.44 0.42 0.43 0.43 0.49 0.44 0.49 0.51 0.51 0.50 0.46 0.43 0.44
0 0.25 0.14 0.42 0.47 0.44 0.42 0.42 0.42 0.41 0.37 0.47 0.49 0.46 0.45 0.45 0.50 0.47 0.46 0.46 0.46 0.42 0.46 0.49 0.45 0.44 0.41 0.39 0.42 0.49 0.46 0.49 0.50 0.50 0.48 0.46 0.46 0.43
200
Table 4 Continued.
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
21
22
23
24
21 PI 374796 22 PI 374797 23 PI 374798 24 PI 367900 25 PI 374792 26 Foamore 27 Mermaid 28 Floral 29 Knowles 30 OMF86 31 OMF78 32 OMF87 33 PI 283724 34 PI 420137 35 PI 283720 36 PI 420133 37 PI 283719 38 OSU-LF4 39 PI 283721 40 PI 283725 0 0.40 0.42 0.42 0.38 0.40 0.41 0.40 0.46 0.42 0.49 0.49 0.50 0.48 0.44 0.39 0 0.41 0.41 0.41 0.44 0.40 0.42 0.46 0.38 0.52 0.49 0.51 0.51 0.47 0.41 0 0.41 0.39 0.38 0.41 0.40 0.46 0.41 0.49 0.45 0.48 0.48 0.46 0.40 0 0.38 0.39 0.36 0.41 0.47 0.42 0.51 0.50 0.52 0.53 0.48 0.43 0 0.34 0.40 0.40 0.48 0.40 0.50 0.51 0.51 0.49 0.47 0.40 0 0.34 0.38 0.47 0.43 0.48 0.50 0.50 0.50 0.49 0.40 0 0.37 0.49 0.41 0.52 0.51 0.51 0.52 0.49 0.42 0 0.49 0.40 0.50 0.48 0.49 0.49 0.45 0.38 0 0.47 0.44 0.48 0.48 0.49 0.49 0.46 0 0.49 0.50 0.51 0.48 0.49 0.39 0.40 0.47 0.47 0 0.40 0.32 0.39 0.44 0.52 0.49 0.42 0.43 0 0.41 0.34 0.33 0.51 0.49 0.42 0.41 0 0.41 0.43 0.52 0.49 0.44 0.46 0 0.33 0.49 0.47 0.44 0 0.48 0.40 0 0.43
0 0.35 0.37 0.43 0.42 0.40 0.43 0.43 0.43 0.44 0.43 0.43 0.47 0.43 0.51 0.49 0.50 0.52 0.47 0.44
0 0.39 0.41 0.41 0.40 0.42 0.42 0.42 0.41 0.43 0.40 0.49 0.43 0.49 0.49 0.48 0.52 0.46 0.42
0 0.39 0.40 0.35 0.43 0.41 0.39 0.38 0.39 0.35 0.48 0.39 0.49 0.49 0.51 0.50 0.48 0.41
0 0.40 0.40 0.44 0.42 0.43 0.42 0.43 0.42 0.45 0.42 0.46 0.49 0.51 0.49 0.48 0.44
41 OMF6229
0.49
0.46
0.45
0.46
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selected for L. alba ssp. versicolor fatty acid profile, which had high dienoic (22:2 D5D13) and low erucic acid (22:1D13) content while OMF 109-3 was selected based on L. alba ssp. alba fatty acid profile, which had high erucic acid but low dienoic acid content. OMF109-1 was selected based on heterozygote progeny, which had fatty acid profiles between these two subspecies. In contrast to OMF109 inbred lines, LAG109F4 was derived from an interspecific cross between L. alba (Mermaid) and L. gracilis ssp. parishii, therefore it showed the greatest distance to those derived from intersubspecific (L. alba) crosses. Amongst the eight open-pollinated cultivars, the distance estimated varied from 0.34 to 0.46 with an average of 0.40. The greatest distance was found between Foamore and OMF6229, whereas the least distance or close relationship was found between OMF86 and Knowles and OMF86 and OMF78. Among 24 wild meadowfoam populations, the greatest distance (0.55) was found between OMF159 (L. alba ssp. versicolor) and PI 283719 (L. floccosa ssp. floccosa). The least distance (0.32) was found between two wild populations of L. alba ssp. versicolor. The average genetic distance among wild population was 0.45 indicating high genetic diversity in these wild populations. Cluster analysis Cluster analysis using UPGMA (unweighted pair group method based on arithmetic mean) was performed to examine genetic relationships among meadowfoam germplasms. A phenogram was produced from the UPGMA cluster analysis of genetic distance matrix for 41 accessions based on mean AFLP data from each accession (Figure 2). There were three major diverse clusters. The first cluster (I) was comprised of L. alba ssp. versicolor. Wild populations of L. alba ssp. versicolor and inbred lines derived from them tended to group together in this cluster. The second and largest cluster (II) was primarily
comprised of L. alba ssp. alba with two distinct subclusters. The first subcluster included wild populations of L. alba ssp. alba and the other consisted of all elite germplasm (open-pollinated cultivars) and wild populations of L. alba ssp. alba (PI 374798), L. gracilis spp. gracilis (PI 420137), and L. montana (PI 283725). The third cluster (III) was composed of five taxa of L. floccosa including subspecies bellingeriana, floccosa, grandiflora, california and pumila (PI 283720, PI 283719, PI 420133, OSU-LF4 and PI 283721, respectively). Three inbred lines (OMF62-29, LAG109F4 and LAG111-F4), separately formed a small cluster far from the others (cluster IV). OMF6229 was high oil content enhanced germplasm derived from L. alba ssp. alba, while LAG109-F4 and LAG111-F4 were inbred lines derived from interspecific cross between L. alba ssp. alba (Mermaid) and L. gracilis ssp. parishii. This phenogram showed that these two inbred lines were in between Mermaid and L. gracilis ssp. parishii indicating that they were equally related to their parents. The remaining two small clusters (V and VI) consisted of two wild populations of L. alba ssp. alba, OMF53 and PI 367900 and two wild populations of L. alba alba (OMF52) and L. alba versicolor (OMF57), respectively. The latter was distantly related to their groups (Figure 2). The goodness of fit of this UPGMA cluster analysis was performed based on the cophenetic correlation (r) value between the cophenetic value matrix and the original distance matrix. The cophenetic correlation was high (r = 0.85) indicating a good fit of the UPGMA cluster analysis performed. Principal coordinate analysis (PCA) A two-dimensional presentation of genetic distance produced by principal coordinate analysis is shown in Figure 3. The first three principal coordinates accounted for 37 % of the total variation in AFLP-based genetic distance (the first, second, and the third eigenvalues were 0.22, 0.09, and
202
0.06, respectively). The first and the second as well as the first and the third coordinate clearly separated the wild populations of L. floccosa from the other populations. Within wild populations of L. alba, L. alba ssp. versicolor were clustered and separated from the L. alba ssp. alba. DISCUSSION In this study, 41 accessions of meadowfoam was fingerprinted including nine inbred lines, eight open-pollinated cultivars and 24 wild populations. One hundred seventy six polymorphic AFLP makers were produced from six primer combinations in 41 accessions whereas 141 and
0.5 0.4 0.3
138 AFLP markers were polymorphic among inbred lines and open-pollinated cultivars, respectively). Abundant of AFLP markers providing an efficient mean to UPGMA cluster analysis (Rohlf, 1993) with the cophenetic correlation (r) of 0.84 suggested that a good fit of cluster analysis was performed. The dendrogram produced from UPGMA cluster analysis showed concordance with the taxonomic classification (Mason, 1952) and previous systematic and phylogeny studies using morphological traits and allozyme markers (McNeill and Jain, 1983). The principal component analysis provided three-dimensional presentation of estimated genetic distance and supported the results of the
0.2 0.1
OMF63 S5 OMF158 OMF66 OMF66 S5 OMF161 OMF159 OMF160 PI 374792 OMF64 S5 OMF109-1 OMF109-3 OMF109-2 OMF40-11 PI 374793 PI 374794 PI 374796 PI 374797 PI 374795 PI 374798 OMF87 Foamore PI 420137 PI 283725 Mermaid Floral OMF78 Knowles OMF86 OMF53 PI 367900 LAG109-F4 LAG111-F4 OMF62-29 OMF57 OMF52 PI 283724 PI 283720 PI 283719 PI 420133 OSU-LF4 PI 283721
II
IV V VI
III
Figure 2 A dendrogram produced by UPGMA clustering of Roger-W genetic distance based on AFLP data among 41 meadowfoam accessions.
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UPGMA cluster analysis. L. floccosa subspecies were distinctly separated from the others. Within L. floccosa, two subgroups were clearly distinguished in which L. floccosa subspecies floccosa and bellingeriana were closely related with genetic distance estimated at 0.32 (Table 4), while the other members of this species, grandiflora, californica, and pumila formed a more distantly related groups. This result is not only in agreement with previous studies of allozyme markers and morphological traits (McNeill and Jain, 1983) but agreed with morphological or taxonomical classification of this species as described by Mason (1952) and Arroyo (1973). The subspecies floccosa and bellingeriana were classified as fully autogamous, producing cleistogamous flowers, while the remaining three subspecies, grandiflora, californica, and pumila, were assigned as semi-autogamous due to their relatively more chasmogamous flowers and the presence of a small degree of protandry.
Figure 3 Principal coordinate plots of 41 meadowfoam germplasm for the first, second and third principal coordinates estimated with 176 AFLP markers, using Roger-W distance matrix. , L. alba ssp. alba; , L. alba ssp. versicolor; D, L. floccosa; , L. glacillis and L. montana; , Interspecific hybrid.
Limnanthes species contains a wide range of mating systems from cleistogamy involving full autogamy in L. floccosa through intermediate stage in L. gracilis ssp. parishii, L. graclilis ssp. gracilis, and L. montana, to L. alba with dominantly protandous, showy, insect-pollinated flower and with the lowest autofertility in the section Inflexae (Mason, 1952; Arroyo, 1973). L. alba Benth. has been domesticated since 1971, and several openpollinated cultivars have been developed for commercial production. These results revealed two distinct clusters including wild populations of L. alba ssp. alba and L. alba spp versicolor (Figure 2) which was consistent with taxonomic classification. Commercial open-pollinated cultivars formed a subgroup within L.alba ssp. alba cluster. Foamore was the first meadowfoam cultivar developed (Calhoun and Crane, 1975), followed by Mermaid and Floral (Calhoun and Crane, 1984). All cultivars were developed by mass selection. Knowles and OMF86 were closely related since they were derived from OMF58 by one and two cycles of recurrent half-sib family selection, respectively. OMF78 was developed by one cycle of recurrent half-sib family selection in OMF59. The result from genetic distance showed that all three cultivars were closely related. L. alba was addressed as an outcrossing species and primarily consisted of two subspecies, alba and versicolor (Arroyo, 1973; Brown et al., 1979). The mating systems of this species and the other species in section Inflexae have been widely investigated (Arroyo, 1975; McNeill and Jain, 1983). Several studies reported the presence of self-pollinated progeny in wild populations of L. alba (Arroyo, 1975). Knapp and Crane (1997) screened 26 accessions of L. alba for self-pollinated phenotypes and found that six populations of L. alba ssp. versicolor produced seed in a high percentage of flowers, which indicated that these geographically isolated populations seemed to have allelic diversity for self-pollination. L. alba ssp. versicolor is distributed from ~37 to 41N and ~
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120 to 123 W in central and northern California (Brown et al., 1979; McNeill and Jain, 1983). Self- pollination seems to be concentrated in populations originating near Redding California (40.5 N, 122.4 W), and OMF66 (Redding) was found to be a source of self-pollinated phenotypes (Knapp and Crane, 1997). Self-pollinated inbred lines have been developed from OMF66 and two other wild populations of L. alba ssp. versicolor (OMF159 and OMF160) (Table 1). The selfpollinated inbred lines developed from these species provide useful resources for developing elite meadowfoam cultivars (Table 1). CONCLUSION AFLP fingerprinting was proven to be a promising approach for evaluating genetic diversity in addition to constructing a genetic linkage map in meadowform. AFLPs revealed the great diversity among meadowfoam germplasm, which broadened the opportunity for meadowfoam improvement. Wild species primarily contained sources of many desirable genes underlying important agronomic and quality traits. Interspecific and intersubspecific hybridization among meadowfoam germplasm provided an opportunity to broaden the genetic base of meadowfoam cultivars. Molecular breeding and genome mapping underlying economically important traits, particularly fatty acid concentration (low erucic acid content) and selfpollination traits, are underway in the laboratory, and this information will be used in marker-assisted selection for meadowfoam cultivar improvement. ACKNOWLEDGEMENTS This research was funded by the Paul C. Berger Endowment and USDA (#58-5114-8-1021 and # 58-3620-8-107).
LITERATURE CITED Anderson J.A., G.A. Churchill, J.E. Autrique, S.D. Tankslay, and M.E. Sorrells. 1993. Optimizing parental selection for genetic linkage maps. Genome 36: 181-186. Arroyo M.T.K. 1973. A taximmetric study of intraspecific variation in autogamous Limnanthes floccosa (Limnanthaceae). Brittonia 25:177-191. Arroyo M.T.K. 1975. Electrophoretic studies of genetic variation in natural populations of allogamous Limnanthes alba and autogamous Limnanthes floccosa (Limnanthaceae). Heredity 35(2):153-164. Botstein D., R.L.White, M. Skolnick and R. Davis. 1980. Construction of genetic linkage map in human using restriction fragment length polymorphism. Am. J. Hum. Genet. 32:314331. Brown C.R., H. Hauptli and S.K. Jain. 1979. Variation in Limnanthes alba: A biosystematic survey of germ plasm resources. Eco. Bot. 33:267-274. Calhoun W. and J.M. Crane. 1975. Registration of Foamore meadowfoam. Oregon Agr. Exp. Sta., Corvallis. Calhoun W. and J.M. Crane. 1984. Registration of Mermaid Meadowfoam. Oregon Agr. Exp. Sta., Corvallis. Ellis R.P., J.W. McNiclo, E. Baird, A. Booth, P. Lawrence, B. Thomus and W. Powell. 1997. The use of AFLP to examine genetic relatedness in barley. Mol. breeding 3:359369. Gentry H.S. and R.W Miller. 1965. The search for new industrial crops. IV. Perspectives of Limnanthes. Eco. Bot. 19:25-32. Isbell T.A. 1997. Development of meadowfoam as industrial crop through novel fatty acid derivatives. Lipid Technol. 9:140-144. Jain S. 1986. Domestication of Limnanthes (Meadowfoam) as a new oil crop. In FAO/
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IAEA (ed.) Plant domestication induced mutation. Proceedings of an advisory group meeting on the possible use of mutation breeding for rapid domestication of new crop plants. p 121-134, Vienna, Austria. Katengam S., J.M. Crane and S.J. Knapp. 2002. The development of a genetic map for meadowfoam comprised of amplified fragment length polymorphisms. Theor. Appl. Genet. 104:92-96. Knapp S.J. and J.M. Crane. 1997. The development of self-pollinated inbred lines of meadowfoam by direct selection in open-pollinated. Crop Sci. 37:1770-1775. Knapp S.J. and J.M. Crane. 1999. Breeding advances and germplasm resources in meadowfoam: A novel very long-chain oilseed. In Janick J pp. 222-233. Perspectives on new crops and uses. Ashs Press, Alexandria, VA. Lodhi M.A., G.-N. Ye, N.F. Weeden and B.I. Reisch. 1994. A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Mol. Biol. Rep. 12(1):6-13. Mason C.T. 1952. A systematic study of the genus Limnanthes. Bot. Publ. 25:455-512. Maughan P.J., M.A. Saghai Maroof, G.R. Buss and G.M. Huestis. 1996. Amplified fragment length polymorphism (AFLP) in soybean: species, diversity, inheritance, and nearisogenic line analysis. Theor. Appl. Genet. 93:392-401. McNeill C.I. and S.K. Jain. 1983 Genetic differentiation studies and phylogenetic
inference in the plant genus Limnanthes (section Inflexae). Theor. Appl. Genet. 66:257-269. Mumm R.H. and J.W. Dudley. 1994. A classification of 148 U.S. maize inbreds: I. Cluster analysis based on RFLPs. Crop Sci. 34:842-851. Ott J. 1991. Analysis of human genetic linkage. John Hopkins University Press, Baltimore, Maryland. Rohlf F.J. 1993. NTSYS-pc numerical taxonomy and multivariate analysis system. Version 1.8. Exster Software, Setauket, New York. SAS 6.12. 1996. SAS Users guide. SAS institute Inc. Cary, North Carolina. Smith O.S., J.S.C. Smith, S.L. Bowen, R.A. Tenborg and S.J. Wall, 1990. Similarities among a group of elite inbreds as measured by pedigree, F1 grain yield, grain yield, heterosis, and RFLPs. Theor. Appl. Genet. 80:833840. Vos P., R. Hoger, M. Bleeker, M. Reijans, T. van de Lee, M. Hornes, A. Frijters, J. Pot, J. Peleman, M. Kuiper, and M. Zabeau. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids. Res. 23(21): 4407-4414. Wright S. 1978. Evolutions and the genetics of populations. Vol.4 Variability within and among natural populations. University of Chicago Press, Chicago. Zhu J., M.D.Gale, S. Quarrie, M.T. Jackson and G.J. Bryan, 1998. AFLP markers for the study of rice biodiversity. Theor. Appl. Genet. 96:602-611.
Effects of Gramma Radiation on Azuki Bean Weevil, Callosobruchus chinensis (L.)

Jakarpong Supawan1, Praparat Hormchan1, Manon Sutantawong2 and Arunee Wongpiyasatid3
ABSTRACT Effects of gamma radiation at various doses on eggs, larvae, pupae and adults of azuki bean weevil, Callosobruchus chinensis (L.) were studied. It was found that there were no significant differences between percent egg mortalities at 40 and 80 Gy and among 120, 160 and 180 Gy. As for the larvae, percent mortalities at all doses were significantly different from that of the control. Significant differences were also found among tested doses from that of the control in percent pupal mortality. After 4 and 7 days of irradiation, percent adult mortalities were found to be significantly different from those of the control at every dose. No significant differences were found among percent mortalities at all tested doses between 4 and 7 days. Percent sterilities at 100 and 120 Gy were not significantly different. When untreated male mating with treated female, there were significant differences in fecundity from the control and from among one another. In treated larvae at 100, 300 and 500 Gy, of all doses the degrees of melanization decreased with the increasing dose. The percent phenoloxidase (PO) activities of the treated larvae reduced compared with that of the control. Key words: gamma radiation, azuki bean weevil, Callosobruchus chinensis, melanization, phenoloxidase activity
INTRODUCTION Irradiation of insect has received wide attention in all aspects, from the fundamentals of genetics, through the different approaches of exploratory development to technology and successful application to limited areas or to countrywide insect-pest eradication programs. Radiation studies on specific effects have been carried out on male and female germ cells in Diptera, Hymenoptera, Coleoptera and Hemiptera in order to measure variation in response and sensitivity to
radiation at different stages of oogenesis and spermatogenesis (IAEA, 1963). Many species of stored-product pests are cosmopolitan, but other serious pests such as the khapra beetle (Trogoderma granarium), the larger grain borer (Prostephanus truncatus), and various species of legume weevil (Bruchidae) are not. Irradiation has been proposed as a possible quarantine treatment for various species of fruit fly, mango seed weevil, and codling moth but not for stored-product pests. However, irradiation has good potential for control of these pests, especially
1 2 3
Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand. Biological Science Division, Office of Atoms for Peace, Bangkok 10900, Thailand. Department of Applied Radiation and Isotopes, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
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since the radiosensitivity of many of them is well documented (Brower and Tilton, 1985). The genera Bruchus and Callosobruchus are particularly serious, and moderate to heavy infestations have been reported in the region, because the insects pass a major portion of their life-cycles inside the seeds (Quraishi and Metin, 1963). The most serious of these species in Asia are azuki bean weevil (Callosobruchus chinensis), cowpea weevil (Callobruchus maculatus) and graham bean weevil (Callosobruchus analis). The three bruchid pests have different distribution ranges. C. chinensis occurs in Asia, where it is a pest on azuki bean, chickpea, cowpea, mungbean, peanut, soybean and other grain legumes. The aims of the study were to investigate the effects of gamma radiation on various developmental stages and reproduction of C. chinensis and to determine if change in melanization of irradiated C. chinensis larvae could be used to indicate irradiation exposure including analysis of phenoloxidase activity. MATERIALS AND METHODS Laboratory rearing of C. chinensis The culture was started with eggs of Callosobruchus chinensis (obtained from Stored Pest Division, DOA) on Vigna angularis Sayi seeds kept at room temperature of 27 2C, 70 10 %, relative humidity (RH) and 10 : 14 (light : dark) photoperiod. This included the rearing of larvae and pupae as well as the adults in azuki bean seeds. All life stages were used in various experiments. Effects of gamma radiation on various developmental stages of C. chinensis Eggs: four-day-old eggs from laboratory culture were irradiated with 0, 40, 60, 120, 160 and 180 Gy in a Cobalt 60 gamma irradiator (Gamma Cell 220) at the Office of Atoms for Peace. In each replication, 100 eggs were taken and each dose treatment was replicated three times. After
irradiation, the eggs were reared in petri dishes (1 cm tall 9 cm diameter). The viabilities of irradiated and control egg were recorded at the end of ten days. Wherever hatching occurred, the larvae were provided with azuki bean seeds and allowed to complete development. Larvae: Seeds of 10-day-old egg laying with larvae inside were irradiated with doses of 0, 100, 300, 500 and 800 Gy. For each dose, three replications of 100 larvae each, were used. After irradiation, the larvae were dissected from seeds, reared in petri dishes (1 cm tall 9 cm diameter), released over azuki bean seeds and kept at 27C. Pupae: Seeds of 22-day-old egg laying with pupae inside were irradiated with doses of 0, 100, 300, 500 and 800 Gy. Each group of irradiated pupae consisted of 100 individuals dissected from seeds. Replications were conducted the same way as in the larvae. After hatching, counts of dead and living insects were recorded every day, together with development and reproduction changes. Adults: Approximately two-day-old adults of C. chinensis were irradiated at 0, 300, 600 and 800 Gy. After irradiation, they were mated into their groups. Data on insect emergence, insect deformation and longevity were recorded. Percent mortality was corrected using Abbotts formula. Duncans new multiple range test (DNMRT) was used in statistical analysis. Effects of gamma radiation on reproduction of C. chinensis females Mature females were irradiated with doses of 0, 40, 60 and 80 Gy while males were irradiated with 0, 50, 100 and 120 Gy respectively. Treated and untreated adults were held in the same conditions previously described. Fecundities and percent sterilities of female were recorded from the following crosses for each radiation dose. Three replications were administered. 1. In each replication, 10 untreated males were crossed with 10 untreated females (The
208 control)
UTM X UTF 2. 10 treated males of different doses were crossed with 10 untreated females TM X UTF 3. 10 treated females of different doses were crossed with 10 untreated males TF X UTM Where UT stands for untreated, T stands for treated, M stands for male and F stands for female. The fecundity was determined by counting the eggs laid and the sterility by the percentages of unhatching egg. The numbers of adult emerged in each case were recorded as percentages of the number emerged from the controls. Effects of gamma radiation on melanization of 10 days old C. chinensis Mungbean seeds of 10 day-old egg laying with larvae inside were irradiated with doses of 0, 100, 300 and 500 Gy. After irradiation, the larvae were reared singly in petri dishes. The larvae were cold-killed by placing in freezer (-201C) for 24 hours after irradiation of 1, 2, 3 weeks. They were then removed from the freezer and kept at room temperature. Melanization was evaluated visually. The observations on melanization process were made using stereomicroscope (30X). The color of melanized body portion (black color) was shown by photo. Effects of gamma radiation on phenoloxidase (PO) activity in C. chinensis Control and irradiated 10-day-old larvae with 300 and 500 Gy were killed by placing them in a freezer (-201C) for a few days. Larvae were then removed from the freezer until used in the experiments. PO activity was to be determined by homogenizing each larval instar individually in 150 ml 0.1 M phosphate buffer solution (pH 6.5) in glass apparatus. The tissue homogenate was added with polyvinylpyrrolidone (oxidation protection)
and centrifuged at 12000 x g for 10 min. The supernatant was used as the enzyme source. Enzyme preparations were kept on ice until tested to avoid possible auto oxidation. The substrate was 3 mg/ml of L-dihydroxyphenylalanine (2methyl dopa, Sigma) in 0.1 M phosphate buffer solution (pH 6.5). One hundred microlitre of the supernatant was added to 150 ml substrate solution and 750 ml 0.5 M phosphate buffer, mixed for few seconds and incubated at 25C for 30 min. The intensity of the red color produced was measured by light absorption at 490 nm using a 6400 Spectrophotometer (Jenway). One unit of PO activity was defined as the amount of enzyme, at pH 6.5, producing a change of one absorbance unit a 490 nm. Specific activity was measured as units per milligram of protein (units/mg protein). Data from spectrophotometric analyses were statistically analyzed at 0.05 level of significance and means separated by the Duncans new multiple range test (DNMRT). RESULTS AND DISCUSSION Effects of gamma radiation on various developmental stages of C. chinensis Eggs Results on egg irradiation at different doses after 10 days are shown in Table 1. It was found that the mortality of 4 days old egg increased with the increasing dose. Percentages of mortality at 0, 40, 80, 120, 160 Gy were 0, 40.6, 75.2, 93.7, 98.3 respectively and no egg hatched after treated with 180 Gy. There were no significant differences between percent mortalities of egg irradiated at 40 and 80 Gy and among those at 120, 160 and 180 Gy. Percent egg mortalities at every dose were also found to be significantly different from that of the control. The experiment showed the eggs of C. chinensis with 100 percent mortality at 180 Gy which was in agreement with Sutantawong (1991) who found that a dose of 180 Gy caused 100%
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mortality in C. maculatus eggs. Larvae Results on larval irradiation at different doses are shown in Table 2. The mortalities of 10 days old C. chinensis larvae were also found to vary directly with the radiation doses. Larval mortalities of full-grown larvae following irradiation at 0, 100, 300, 500 and 800 Gy were 0, 27.37, 83.86, 96.14 and 100 % respectively. At every dose, percent larval mortalities were noticed to be significantly different from that of the control. While there was no significant difference between Table 1 Effects of gamma irradiation on percent mortalities of 4-day-old egg of C. chinensis 10 days after irradiation. Dose (Gy) 0 40 80 120 160 180
1/
% mortality1/ 0a 40.6 b 75.2 b 93.7 c 98.3 c 100 c
percent larval mortalities at 500 and 800 Gy, both were significantly different from those at 100 and 300 Gy. In the experiment, a dose of 800 Gy caused 100% larval mortality while OAEP (1968) and Quraishi and Metin (1963) reported 16000 rad (160 Gy) and 200 Gy respectively to cause 100% mortality of 16 days old C. chinensis larvae after hatching from eggs. Yet, the result was similar to that reported by Sutantawong (1991) which found 500 Gy to cause 100% mortality of 7-10 days old C. maculatus. It was possible that the different result from that of Quraishi and Metin (1963) was in the different ages of larvae used. The 8-day-old larvae in their experiment, compared to the 10-day old as in this study, were more sensitive to radiation as suggested by Molin (2001). Hence, lower dose (200 Gy) than the dose in the experiment was required to cause 100% mortality. Pupae The results on the pupal irradiation are shown in Table 3. The mortality of pupal irradiation also increased with increasing dose. Percentages of pupal mortality at 0, 100, 300, 500 and 800 Gy were 0, 44.33, 69.41, 92.09 and 100 % respectively. Some pupae were found dead inside the seeds, hence they were not used in the treatments. Table 3 Effects of gamma irradiation on the mortalities of 4-day-old C. chinensis pupae 10 days after irradiation. Dose (Gy) 0 100 300 500 800
1/
Means in column followed by the same letters are not significantly different at 5% level as determined by DNMRT.
Table 2 Effects of gamma irradiation on the mortalities of 10-day-old C. chinensis larvae 10 days after irradiation. Dose (Gy) 0 100 300 500 800
1/
% larval mortality1/ 0a 27.37 b 83.86 c 96.14 d 100 d
% pupal mortality1/ 0a 44.33 b 69.41 b 92.09 c 100 c
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Significant differences were found among all doses from that of the control in percent pupal mortality. It was also found that between 500 and 800 Gy, the percent pupal mortality did not significantly different from each other. There was no significant difference of percent pupal mortality at 100 and 300 Gy as well. The result was similar to those of Sutantawong (1991) and Quraishi and Metin (1963) which reported 100 percent pupal mortality of C. maculatus and C. chinensis found at 500 and 470 Gy respectively. This study also agreed with Bhuiya et al. (1985) who found 100 percent pupal mortality of C. chinensis at 800 Gy. Adults Table 4 expresses the adult mortality to increase when irradiated at higher doses. Four days after irradiation, the 2-day old adults irradiated at 0, 300, 600 and 800 Gy were noticed to have percentages of mortality of 0, 16, 86.5 and 97 respectively, while after 7 days after irradiation percent mortalities were 0, 81, 100 and 100 respectively. At every dose after 4 and 7 days of irradiation, percent adult mortalities were found to be significantly different from those of the control. While percent mortalities at 600 and 800 Gy were not significantly different from each other, they Table 4 Effects of gamma irradiation on the mortalities of 2-day-old C. chinensis adults 4 and 7 days after irradiation. Dose (Gy) % mortality after irradiation1/ 4 days 7 days 0a 16 b 86 c 97 c 0a 81 b 100 b 100 b
were from that at 300 Gy 4 days after irradiation. No significant differences among tested doses after 7 days of irradiation were noticed. Table 5 shows the percent mortalities of C. chinensis adult between 4 and 7 days after irradiation at various doses. It was revealed that there were significant differences in percent mortality between 4 and 7 days at 300 and 600 but not at 800 Gy according to t-test analysis. According to the results of Table 4 & 5, 800 Gy at 4 and 7 days or 600 Gy at 7 days after irradiation should be recommended as the killing doses and times for complete control of this insect. However, from an economical point of view, 600 Gy at 7 days of irradiation is likely to cause less expense since higher dose means greater cost while longer checking days does not increase as much expense. Similar results were reported by Sutantawong (1991) who revealed a dose of 1000 Gy 7 days after irradiated to give C. maculatus 100% mortality. The study also agreed with Kovacs and Kiss (1985) who reported that with a treatment of 0.8 kGy all imagoes of T. confusum were dead 7 days after irradiation. As stated by Molin (2001), the effect of radiation on insects are many and varied depending primarily on the species, stage, age and physical factor and the younger metamorphic stages of insect are most radiosensitive than the older stages. Table 5 Effects of gamma irradiation on the mortalities of 2-day-old C. chinensis adult between 4 and 7 days after irradiation. Dose (Gy) % mortality after irradiation 4 days 7 days t value 16 86 97 81 100 100 -6.40* -2.96* -1.73
0 300 600 800

1/
Means in each column followed by the same letters are not significantly different at 5% level as determined by DNMRT.
300 600 800

*
Significant at .05 level.
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Thus, eggs, larvae and young pupae are quite easily killed within a reasonable period of time after treatment, the old pupae and young adults are relatively resistant to the lethal effects of irradiation. Eggs are most sensitive to radiation. This was to explain egg mortality occurring at lower doses as in this study compared to the mortalities of other stages at higher doses. Physical factors also have an influence on the effects of radiation on insects. The differences in temperature and humidity, hence, caused different results to even 2-day old difference in age as between larval mortality in the study of Quraishi and Metin (1963) and this experiment. Effects of gamma radiation on fecundity and sterility of C. chinensis females The effects of gamma irradiation with 2day-old C. chinensis males (TM) on the fecundities and sterilities of untreated female (UTF) compared with the control (UTF X UTM) are shown in Table 6. It was found that the numbers of egg/female at each dose of untreated females were not significantly different from the control or from one another. While the percent sterilities at 100 and 120 Gy were not significantly different they differed from that at 50 Gy. Table 6 Effects of gamma irradiated 2-day-old C. chinensis males mated with untreated females (TM UTF) on the fecundities and sterilities of untreated female. Dose (Gy) Fecundity1/ (Eggs / female) 32.93 a 33.23 a 28.63 a 28.53 a
When untreated male (UTM) mating with treated female (TF) (Table 7), it was revealed that at various doses of radiation the fecundity of treated female decreased resulting in increased percent sterilities from 0 to 80 Gy with dose of 80 Gy gave 100% sterility in female. When female exposed to this dose was paired with a normal adult of the opposite sex, eggs were laid on the seeds but the eggs failed to hatch. It was also found that there were significant differences in fecundity from the control and among one another at all doses. Percent sterilities at every tested dose were significantly different from that of the control (0 Gy) but not among one another. In sterility of female, the results agreed with that of Brower and Tilton (1985) who showed the doses required to sterilize stored-product insects to vary widely from 70 Gy for the cowpea weevil, C. chinensis and the female appeared more susceptible than the male in all cases in the same insects (OAEP, 1968). The result also agreed with Sutantawong (1991) who suggested that female of C. maculatus was susceptible than male at 40, 60 and 80 Gy. According to the results in Table 6 & 7, the lower dose in obtaining complete mortality in females than males was required, hence, irradiated Table 7 Effects of gamma irradiated 2-day-old C. chinensis females mated with untreated males (TF UTM) on their fecundities and sterilities. Dose (Gy) Fecundity1/ (Eggs / female) 32.93 a 24.93 b 21.22 c 8.66 d % Sterility2/
Sterility2/ 76.15 a 79.67 b 90.40 c 91.47 c

1/2/
0 50 100 120
1/2/
0 40 60 80
76.15 a 98.34 b 99.67 b 100 b
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females should be employed in SIT (Sterile Insect Technique) program. However, since the females in the nature are needed to reduce their fecundities, it is more practical to release the irradiated males in order to mate with these wild females even though, in economical point of view, the expense will be higher. In most SIT program, however, both sexes are released and the response of both males and females to the sterilizing doses has to be assessed. In some species the males are the more sensitive sex, e.g., the screw-worm, Cochliomyia hominivorax, in other species the females are, e.g., the medfly, Ceratitis capitata. In case where the male is the more sensitive sex it would be very advantage to have a system for the removal of females so that a lower radiation dose could be given to the male (Rechcigl and Rechcigl, 1998).
As suggested by Molin (2001), low radiation doses cause insect sterilization or genetically deformed gametes, which higher doses required to induce insect death. In these experiments, 600-800 Gy caused complete mortality while only 100-120 Gy induced complete sterility in C. chinensis adults. Higher radiation doses cause multiple breaks in the chromosomes, thus causing death or sterility. Effects of gamma radiation on melanization process of 10 days old C. chinensis larvae Figure 1 presents the color of C. chinensis at different doses. When larvae were treated with various doses (0-500 Gy), the color variation of the larvae ranged from black to creamy white. The degree of melanization in treated larvae decreased with the increasing doses.
Figure 1 Degrees of darkening in the larvae of azuki bean weevil, C. chinensis 3 days after treatment. A. Control (30X) B. Treated with 100 Gy (30X) C. Treated with 300 Gy (30X) D. Treated with 500 Gy (30X)
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The results similarly agreed with Kongratarpon (2002) who reported melanization occurred in untreated young larvae of cigarette beetle larvae after killing by freezing. Some parts of larvae body became dark brown to black while the rest of the body was yellow-white or grayed-yellow. In the treated young larvae, non-melanization to slight melanization occurred at 100, 300 and 500 Gy. The degree of melanization in treated young larvae decreased with the increasing dose. Ignatiwicz and Banasik-Solgala (1997) reported that after the irradiation treatment with doses ranging from 0.1 to 0.5 Gy, the melanization process was significantly inhibited in young larvae cold-killed after irradiation and old larvae of khapra beetle melanized slowly after their death, so that some visible in the body color were noted as late as after 24 hours. With similarity of this study to such report, the change in melanization of the azuki bean weevil, C. chinensis larvae could not be used for indicating previous exposure of these insects to irradiation, because of the great variability in response of the melanization process to the irradiation treatment. Ignatowicz and Ibrahim (1996) found that the melanization in irradiated young larvae of the confused flour beetle, Tribolium confusum DUAVL., was reduced in the first week after treatment and completely inhibited in the second week. Great variation of melanization in the untreated old larvae partially obscured the effects of gamma radiation on this process. However, the melanization was considerably reduced in all experiments involving old larvae. Banasik-solgala and Stanislaw (1997) found that the degree of melanization differed significantly between treated and untreated the Indian meal moth (Plodia interpunctella HBN), the Mediterranean flour moth (Ephestia (Anagasta) kuehniella ZELL) and the almond moth (Cadra cautella WLK).
Effects of gamma radiation on phenoloxidase (PO) activity in C. chinensis Results of the experiment on the effects of radiation on PO in larvae of azuki bean weevil, C. chinensis, indicated variability of the response of enzyme activity to gamma radiation. Three sets of experiment were conducted to determine PO activity spectrophotometrically. In this assay, the frozen larvae were used to increase the activity of the PO system (Mansour and Franz, 1996). The results of PO activity measurement are shown in Table 8. The preliminary analysis of PO in the irradiated larvae at 0, 100, 300 and 500 Gy showed low activities of PO which was difficult to detect because the larvae had small sizes that were not easy in homogenizing. At 0, 100, 300 and 500 Gy, PO activities of larva were found to be 2.500, 1.867, 0.851 and 0.409 units/mg protein respectively. The values obtained for PO activity showed that with the increasing dose from 0 to 100, 300 and 500 Gy, PO activity decreased. The PO activity was found to be significantly different between 0 and 500 Gy, while not differed from those at 100 and 300 Gy. No significant differences were found among PO activities at 100, 300 and 500 Gy. Percent reduction of PO activity also decreased with the increasing dose where the highest (83.67%) was at 500 Gy and lowest (25.33%) at 100 Gy. Irradiation was found to reduce or eliminate melanization after death and reduced phenoloxidase activity in the fourth instar of C. chinensis larvae. The irradiation dose at which melanization was essentially prevented was from 100 Gy up. The result agreed with Lupa and Ignatowicz (1999) who showed the highest doses to inhibit enzyme activities in larvae of Mediterranean flour moth and the confused flour beetle after irradiated. There was a decreasing of PO activity and significant difference when larvae were treated
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Table 8 Phenoloxidase (PO) activity and percent reduction of irradiated 10-day-old larvae of C. chinensis at different radiation doses. Dose (Gy) Mean of PO activity (units/mg protein) 1/2/ (% Reduction) 2.500 a 1.867 ab (25.32) 0.851 ab (65.96) 0.409 b (83.67)
0 (control) 100 300 500
1/
2/
Means followed by the same types of letter in the same columns are not significantly different from one another as determined by DNMRT at 0.05 level. Units of phenoloxidase at 490 nm.
with 0.3 (kGy) or higher dose of gamma radiation. Lupa (2000), also reported that the phenoloxidase activity in fourth instar of kaphra beetle (Trogoderma granarium) larvae was inhibited by 50 and 150 Gy after 1 week and larvae irradiated with 100 and 300 Gy exhibited higher enzyme activity compared to the control. Surisan (2004) reported the changes in the effect of radiation on PO activity on each instar of cotton bollworm. At 75 and 150 Gy the irradiated insects were found to have PO activities significantly less than that of the control and percent reduction of PO activity also increased from 4th to 1st instar. According to Hara et al. (1991) the active units of cuticle-bound phenoloxidase should be measured during metamorphosis along with hemolymphatic phenoloxidase activity in order to better understand the physiological role of phenoloxidase in cuticle tanning and sclerotization. CONCLUSION The egg, larval, pupal and adult mortalities increased with the increasing dose with the egg being the most sensitive. Sterilities of irradiated male mating with untreated female were
significantly different at all doses while those of irradiated female mating with untreated male significantly different between 0 and other doses. Lower dose was used for female than for male in obtaining complete mortality. Melanization in irradiated larvae decreased with increasing dose and could not be used in detecting radiation exposure due to great variability in response of the process to irradiation. Percent PO activity decreased with the increasing dose. LITERATURE CITED Banasik-Solgala, K. and I. Stanislaw. 1997. Melanization process in irradiated larvae of stored product moth. Polish J. Entomal. 66: 125-134. Bhuiya, A.D., M. Ahmed, R. Rezaur, D.R. Seal, G. Nahar, M.M. Islam and M.S. Islam. 1985. Insect disinfestation of pulses by irradiation, pp. 214-221. In J.H. Moy (ed.). Radiation Disinfestation of Food and Agricultural Product. University of Hawaii Press, America. Dobie, P., C.P. Harines, R.J. Hodges and P.F. Prevett. 1991. Insects and Arachinids of
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Tropical Stored Products: Their Biology and Identification (A Training Manual). Storage Department Tropical Development and Research Institute. UK. 273 p. Hara, T., T. Tsukamoto, K. Wantanabe, N. Yamasaki and M. Funatsu. 1991. Properties of cuticular phenoloxidase from pupae of the housefly, Musca domestica L. Agric. Biol. Chem. 55(1): 13-17. IAEA. 1963. Radiation and Radioisotopes Applied to Insects of Agricultural Importance. International Atomic Energy Agency, Vienna, Austria. 508 p. Ignatowicz, S. and H.Z. Ibrahim. 1996. Reduced melanization after death in larvae of the confused flour beetle, Trogoderma confusum, as a result of the irradiation treatment. Polish J. Entomal. 65: 51-59. Ignatowicz, S. and K. Banasik-Solgala. 1997. Reduced melanization after death in larvae of the khapra beetle, Trogoderma granarium, as a result of the irradiation treatment. Radiobiology 42: 801-802. Kongrat-arpon, T. 2002. Effect of Gamma Radiation on the Cigarette Beetle, Lasioderma serricorne (F.). M.S. Thesis, Kasetsart University, Bangkok. Kovacs, E. and I. Kiss. 1985. Disinfestation of wheat germ, wheat, and dried mushrooms by irradiation. pp. 189-198. In J.H. Moy (ed.). Radiation Disinfestation of Food and Agricultural Product. University of Hawaii Press, America. Lupa, D.A. 2000. Reduced activity of phenoloxidase after treatment with gamma radiation in larvae of the kaphra beetle (Trogoderma granarium Everts), as an easy method to detect irradiated and nonirradiated insects. Ann. Warsan Agri. Univ. Hort.
(Landscape Architecture) 21: 9-16. Lupa, D.A. and S. Ignatowicz. 1999. Change of phenoloxidase activity in stored product pest treated with ionization radiation. Progress in Plant Protection 39(2): 458-462. Mansour, M. and G. Franz. 1996. Effect of gamma ray radiation on the biology of the Periplaneta americana. Restaurator 21(1): 41-54. Molin, R.A. 2001. Food Irradiation: Principles and Applications. John Wiley and Sons, Inc., New York. 469 p. OAEP. 1968. Studies on the Use of Gamma Radiation in the Control of Pea Weevil, Callosobruchus chinensis L. Bangkok, Thailand. 18 p. Quraishi, M.S and M. Metin. 1963. Radiosensitivity of various stages of Callosobruchus chinensis L., pp. 479-484. In International Atomic Energy Agency. Radiation and Radioisotopes Applied to Insects of Agricultural Importance. IAEA, Austria. Rechcigl, J.E. and N.A. Rechcigl. 1998. Biological and Biotechnological Control of Insect Pests. Lewis Publishers, America. 374 p. Ress, D.P. 1960. Coleoptera, pp. 1 - 40. In B. Subramanyam and D.W. Hagstrum (eds.). Integrated Management of Insect in Stored Products. Marcel Dekker, Inc., America. Surisan, S. 2004. Gamma Radiation-Induced Changes in Haemocytes, Melanization and Phenoloxidase Activity of Cotton Bollworm, Heliothis armigera Hbner. M.S. Thesis, Kasetsart University, Bangkok. Sutantawong, M. 1991. Disinfestation of the Cowpea Weevil, Callosobruchus maculatus F. in Stored Mungbean by Gamma Irradiation. Office of Atomic Energy for Peace, Bangkok 16 p.
Occurrence and Distribution of Major Seedborne Fungi Associated with Phaseolus Bean Seeds in Ethiopia
Mohammed Yesuf1 and Somsiri Sangchote2
ABSTRACT A total of 245 seed samples of Phaseolus bean; 172 common beans, 51 climbing beans and 22 green beans were collected from various bean growing areas during 2003 crop season. The incidence and severity of seed infection by the major fungal diseases of bean varied between localities, bean types and cropping practices. Thirteen seed-borne fungal pathogens of different genus were identified from seed samples collected from the major bean growing regions of Ethiopia. The incidence of different seedborne fungi ranging between 0.2 to 14.5% was found to vary from location to location and growing conditions. Among them, Colletotrichum lindemuthianum, Phaeoisariopsis griseola, and Ascochyta phaseolorum were the most widespread and damaging seedborne fungal pathogens associated with Phaseolus bean seeds in Ethiopia. From the total seed samples collected, 26.2%, 19.6% and 13.6% of common bean, climbing bean and green bean respectively were infected by C. lindemuthianum, whereas infection by P. griseola was 18.6% and 15.7% on common bean and climbing beans seeds respectively. Green bean seeds were not infected by the latter two fungi. Seeds collected from south, southwest, and western part of Ethiopia showed heavy seed infection by these major fungal pathogens, whereas seeds produced in dry areas with minimum rainfall or under irrigation showed very low seed infection. Phytophthora rot of beans caused by Phytophthora sp. was also detected from green bean pods and immature seeds produced under irrigation in the central rift valley of the country. The geographic distribution of major seed-borne fungi of Phaseolus beans was mapped. Key words: Phaseolus beans, seedborne fungi, distribution, Ethiopia
INTRODUCTION Different types of Phaseolus beans are widely cultivated in various agro-ecological regions of Ethiopia. The most important Phaseolus beans grown in the country are dry common beans (Phaseolus vulgaris L), climbing beans (Phaseolus coccineus) and French/green beans (Phaseolus vulgaris). The area covered by dry common bean production alone in Ethiopia is estimated to be
more than 200,000 hectare annually (CSA, 2000). Among the biotic stresses of Phaseolus bean production in the country, diseases are considered the major production threats. Under optimum crop production management practices of Ethiopia, there is a potential to produce common beans of 2.5 - 3.0 tons/hectare (Amare, 1987). However, the national average of bean yield in Ethiopia is very low (0.6 - 0.7 ton/ha). The major limiting factor for low yield is thought to be
1 2
Melkassa Agricultural Research Center, P. O. Box 436, Nazareth, Ethiopia. Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.
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diseases (Habtu et al., 1996). Seedborne fungi are among the most important plant pathogens that cause direct and indirect losses of the bean crop throughout the world (Schwartz and Galvez, 1980; Hall, 1994; Abdelmonem, 2000). Losses are associated with high disease epidemic in the field consequently causes reduced seed yield and quality attributing to discoloration and blemish of seeds and green pods (Hellene, 1988). Moreover, seedborne pathogens play an important role for the dispersal and dissemination of several economically important diseases in the production field within the country and between countries (Agarwal and Sinclair, 1997). In a bean production system like in Ethiopia where there is no seed health and certification scheme, the risk of seed infection by seedborne fungi could be high. Different types of fungal diseases are reported to cause damage on the bean plant in the field. Among others, bean anthracnose (Colletotrichum lindemuthianum), angular leaf spot (Phaeoisariopsis griseola), aschochyta blight (Ascochyta phaseolorum), ashy stem blight (Macrophomina phaseolina) are reported to be the major fungal pathogens causing foliar diseases of Phaseolus beans under field condition in Ethiopia (Habtu and Abiy, 1995, Habtu, et al., 1996). However, the association of these major fungal pathogens and other fungi with Phaseolus bean seeds has not been adequately investigated. Survey and identification of plant pathogens is important to understand the association of pathogens with a specific host plant and to describe their geographic distribution (Agrios, 1997). Application of GIS on plant disease distribution map is helpful to store and refer spatially referenced point data (Nelson, et al., 1999). Mathur (1995) emphasised the need of surveys of seedborne pathogen by having good amounts of sample unit of different crop species and cultivars so that it enables researchers to set research priorities. Detail understanding of the type, occurrence, association with seed, and geographic distribution of major
pathogens is a prerequisite to formulate rational integrated and sustainable disease management practices in different agro-ecologies. Therefore, the occurrence of major seedborne fungi of Phaseolus beans and their geographic distribution are needed to be investigated. The objective of this study was to investigate the occurrence, and to map the geographic distribution of major seedborne fungal pathogens associated with Phaseolus bean seeds in various bean growing agro-ecological regions of Ethiopia. MATERIALS AND METHODS Survey areas and sample collection Disease survey was carried out in the major bean growing regions of Ethiopia representing the lowlands (central rift valley), mid altitudes (north and southern part) and highlands (north-west and western part of the country) during the 2003 crop season. A total of 245 Phaseolus bean seed samples (172, 51, and 22) of common bean, climbing bean and green bean respectively were collected from the major bean-growing areas of the country. The seed samples were collected from various sources and cropping practices. These included farmers fields, experimental fields, large-scale commercial farms and local markets. Geographic features like latitude, longitude and altitude were recorded from all surveyed areas using handheld Global Positioning System (GPS), Garmen with 12 channels to trace back the specific locations and types of seedborne fungi. All collected seed samples were kept at +5 C in a refrigerator. The seed samples of different Phaseolus bean collected from various agro-ecologies were tested for seedborne fungi by the standard blotter method (International Seed Testing Association, 1993) and agar plate method. Detection of seedborne fungi using standard blotter method Four hundred untreated and 1% sodium
218
hypochlorite pre-treated seeds were plated on four layers of water soaked blotter, 10 seeds per petri dish. The dishes were then incubated for seven days in a growth chamber SGC097.C with programmer, controller and sensor of combined temperature 24 1C and humidity probe under 95% humidity and 12 hours photoperiod of preprogrammed light conditions (Maden et al., 1975). The bottom of the compartment was filled with clean water covered by a glass plate. Slight air current was forced through a plastic pipe in the center at the bottom against the underside of the glass. Such high air humidity enabled the blotters remoistened during incubation and facilitated fungal sporulation. After seven days of incubation samples were examined using stereomicroscope with 20-30x magnification for the growth of fungi. The habit characters of various fungi associated with seeds was recorded. Further identification of fungi was made using compound microscope. Detection of seedborne fungi using agar plate method Seeds of Phaseolus bean treated with 1% sodium hypochlorite were plated on potato dextrose agar (PDA), and then incubated as it was done in the standard blotter method. Microscopic examination was made after seven days of incubation. Comparison on the efficiency of standard blotter and agar plate methods was also made by using naturally infected seed lots of common bean seed by the three major seed borne fungi (C. lindemuthianum, A. phaseolorum, and P. griseola). Geographic distribution of major seedborne fungi of phaseolus beans Important geographic features such as latitude, longitude, and altitude were recorded with the help of handheld Global Positioning System (GPS) Garmen with 12 channels. Satellite signals were received using the GPS and position of the coordinates was recorded. The collected
coordinate information was downloaded into the computer and all necessary descriptive information were incorporated. Consequently, using the latitude and longitude coordinates the survey route and geographic distribution of major seedborne fungi maps were prepared using Geographic Information System (GIS) software (Arc view GIS) version 8.2. RESULTS AND DISCUSSION Detection and identification of seedborne fungi of phaseolus beans Symptoms of seed infection by seedborne fungi varied depending on bean types and severity of infection. Common bean seeds with severe infection by C. lindemuthianum showed dark brown spot associated with characteristic symptoms of depressed sunken lesion both on pod and seed surface (Figure 3 C & D). When seed infection was not severe, it was difficult to differentiate symptoms of different pathogen by the naked eyes. This study showed that Phaseolus bean seeds could be attacked by several economically important seedborne fungal pathogens. A total of thirteen seed-borne fungi of different genus was identified. Seed infection levels of Phaseolus bean with different seedborne fungi ranges between 0.2 - 14.5%. Bean seed infection levels varied between localities, growing conditions, and bean types. Seeds of common bean were more susceptible to all major seedborne fungal pathogens than climbing beans and green beans. High number of fungal microflora was also associated with common bean seeds (Table 1). The severity of seed infection by the major pathogens was very high on seeds collected from fields of small-scale farmer. This was mainly because small-scale farmers did not have the access to improve disease free seeds. Instead, they used to grow their own saved seeds from the previous harvests, without prior knowledge on the primary inoculum present in the seeds.
219
Bean anthracnose was found to be the most widespread with high percent seed infection. C. lindemuthianum was recovered from 26.2%, 19.6, and 13.6% of the seed samples of common bean, climbing bean and green bean respectively; whereas P. griseola was recovered from 18.6% and 15.7% of common bean and climbing bean respectively. P. griseola and A. phaseolorum were not detected from green bean seeds (Figure 1). All these fungal pathogens are also economically important seedborne pathogens of beans in many parts of the world (Richardson, 1979). C. lindemuthianum is found to be the most important seedborne pathogen and is found in high frequency with severe crop damage followed by P. griseola and A. phaseolorum in many bean growing areas of Ethiopia. Chang et al., (2001) reported that several species of fungal microflora were isolated from naturally infected common bean seeds in Canada and C. lindemuthianum was found to be the most predominant fungus associated with bean seeds. In the western part of Ethiopia, small-scale
farmers grow climbing bean (Phaseolus coccineus) in their gardens. All three major seed borne fungi identified from common beans were also isolated from climbing beans and all the isolates proved to
30
26.2 C. lindemuthianum P. griseola A. phaseolorum 19.6 18.6 15.7
25
Infected samples (%)
20
15
13.6
10
9.9
9.8
5
0 0
0
Common beans Climbing beans Green beans
Phaseolus beans
Figure 1 Infected samples (%) of Phaseolus bean seeds by major seedborne fungi during 2003 crop season.
Table 1 Numbers of infected seed sample and incidences (%) of seedborne fungi of common bean (Phaseolus vulgaris) seed obtained from different agro-ecologies of Ethiopia during 2003 crop season. Fungi Numbers of infected sample Seed infection (%) Range Average 1.2-14.5 0.5-12.3 0.6-7.4 0.3-4.5 0.2-2.5 0.3-2.3 1.2-4.5 0.3-2.3 0.4-2.6 0.6 0.2-1.6 0.4-1.2 8.4 5.6 3.7 2.8 0.8 0.9 2.4 0.4 1.0 0.6 0.5 1.3
Colletotrichum lindemuthianum Phaeoisariopsis griseola Ascochyta phaseolorum Macrophomina phaseoli Fusarium moniliforme Fusarium oxysporum Aspergillus sp. Botrytis sp. Phoma exigua Sclerotinia sp. Trichotecium sp. Alternaria alternta
45 32 17 10 3 5 6 1 4 1 3 4
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attack Phaseolus vulgaris. Since climbing bean is a long duration crop and sometimes it can be grown throughout the year as a garden crop, it could serve as a primary source of inoculum of these major seed borne pathogens from season to season. Whereas, in the central rift valley of the country where 60% of the countrys common bean production is grown (Aleligne, 1990), green beans are cultivated in state owned large commercial farms and also private investors under irrigation in continuous planting throughout the year. This type of cropping system also creates favourable condition for the survival and dissemination of important seed borne pathogens such as C. lindemuthianum. Noitalics rot of beans which is caused by Phytophthora sp. was detected from green bean pods and immature seeds collected from commercial bean farms in the central rift valley. This disease is common and causes damage to the bean plant in the South and Central America (Hall, 1994). In this study, Phytophthora rot of beans caused by Phytophthora sp. was reported for the first time in Ethiopia. Detection of major seedborne pathogens
using the standard blotter method gave the higher amount of seed infection (P < 0.01) as compared to the agar plate method (Figure 4). This might be due to the availability of free moisture on the moist blotter papers than the agar plates. The bean seed coat absorbed more water from the wet blotters and enabled the fungi to easily grow and sporulate, whereas on the agar plates, the bean seeds became dry and sporulation of the fungus reduced as compared to the standard blotter method. Blotter method was the easiest, efficient and economical detection technique for Colletotrichum lindemuthianum, Ascochyta phaseolorum, and Phaeoisariopsis griseola from naturally infected Phaseolus bean seeds. Geographic distribution of major seedborne fungi of Phaseolus beans A total of thirteen seedborne fungi of different genera were identified from samples collected from various bean growing agroecologies of Ethiopia (Table 1 & 2). Seed infection of individual samples by different seedborne fungi ranged from 0.2-14.5% and the most widespread
Table 2 Number of infected seed samples and infection percentage of climbing bean and green bean seeds by different seedborne fungi obtained from different agro-ecologies of Ethiopia during 2003 crop season.
Seed infection (%) Number of infected samples Range Average Climbing Green Climbing Green Climbing Green bean bean bean bean bean bean 10 8 5 4 2 4 0 2 0 6 3 0 0 0 1 2 2 0 6 2 2.0 - 8.6 0.8 - 10.4 1.1 - 6.4 0.4 - 2.4 0.4 -1.8 1.6 - 5.8 0 1.2 - 2.6 0 1.8 - 6.5 1.0 - 4.2 0 0 0 0.9 1.1 - 2.6 1.0 - 2.8 0 1.5 - 4.8 4.8 5.2 3.2 1.2 1.0 3.2 0 1.8 0 2.6 0 0 0 0 1.4 1.6 0 3.1
Fungi
Colletotrichum lindemuthianum Phaeoisariopsis griseola Ascochyta phaseolorum Macrophomina phaseolina Fusarium moniliforme Aspergillus sp. Botrytis sp. Tricotecium sp. Phytophthora sp. Alternaria alternata
1.0 - 2.3
3.4
1.7
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seedborne fungi of Phaseolus beans were C. lindemuthianum, P. griseola, and A. phaseolorum. These pathogens attack major Phaseolus beans and widely distributed in many of bean growing areas of Ethiopia. The south, west, and northwestern part of the country is characterized with high rainfall, whereas the central and eastern part get low and erratic rainfall. However, during the 2003 crop season, there was high rainfall in all parts of the country. This created favourable condition for the development of some of the seedborne pathogens even in the semi-arid areas. In the western part of the country, where there is high and frequent rainfall, all these pathogens were found simultaneously in the same area or bean field (Figure. 2). Bean anthracnose was the most widespread and predominant fungal pathogen in almost all bean growing areas of the country
including the lowland arid areas where there was low and erratic rainfall during the crop season. Even though there was field infection of beans by bean anthracnose in the semi arid areas of the central rift valley, seed infection by C. lindemuthianum was very low. This might be due to the late appearance of the disease in the field and unfavourable environmental conditions during the active growth stage of the bean plant. Angular leaf spot and ascochyta blight were more abundant in areas with high rainfall and humidity particularly to the southern and western part of Ethiopia such as Ambo, Bako, Pawe, Areka and Jimma area (Figure 2). These fungi were not detected from seeds collected from the central rift valley where the bulk of dry common bean production is found (Table 3).
Figure 2 Geographic distribution of major seedborne fungi of Phaseolus beans in Ethiopia during the 2003 main crop season (1- Colletotrichum lindemuthianum, 2-Phaeoisariopsis griseola, 3Ascochyta phaseolorum).
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Table 3 Geographic distribution and severity of major seed borne fungi of common bean at different bean growing areas during 2003 crop season. Geographic locations 39 .50' E 8 .60' N 39 .19' E 8 .91' N 38 .15' E 10 .37' N 37 .91' E 10 .58' N 37 .43' E 10 .84' N 37 .58' E 11 .01' N 36 .60' E 11 .50' N 37 .21' E 10 .77' N 36 .81' E 9 .68N 36 .71' E 9 .14' N 37 .23' E 9 .11' N 37 .86' E 8 .64' N 36 .71' E 8 .75' N 36 .40' E 8 .50' N 36 .64' E 7 .89' N 38 .88' E 7 .74' N 37 .96' E 8 .42' N 37 .78' E 7 .08' N 37 .81' E 6 .92' N 38 .52' E 7 .30' N 38 .51' E 7 .33N 38 .79' E 7 .41' N 38 .94' E 8 .06' N 38 .82' E 8 .27' N 40 .76' E 9 .19' N 42 .27' E 9 .43' N 40 .00' E10 .06' N 39 .97' E 10 .14' N 39 .85' E 11 .11' N 39 .70' E 11 .08' N 39 .72' E 12 .30' N Altitude (M.a.s.l) 1650 1940 2476 2470 1875 1665 1126 2150 1404 2130 1650 2200 2526 2027 1684 1750 1845 1768 1850 1730 1940 1960 1660 1675 1350 2050 1260 1780 1900 1950 1450 Severity of major seedborne fungi C. lindemuthianum P. griseola A. phaseolorum ++ ++ + ++ ++ ++ ++ ++ + +++ +++ +++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + +++ ++ ++ ++ ++ ++ + + ++ ++ +++ +++ + +++ +++ +++ + ++ +++ +++ ++ +++ +++ +++ + + + + + + + ++ + + ++ +++ +++ + + + ++ + -
Location Melkassa Debrezeit Dejen Markos Finoteselam Chagni1 Pawe1 Bure1 Guten Nekemt1 Bako1 Ambo1 Arjo1 Bedele1 Agaro1 Jimma1 Wolkite1 Areka1 Sodo1 Awassa1 Shashemene Arssinegelle Ziway Meki Meisso Alemaya Shoarobit Kemissie Kombolcha Dessie Kobbo
1 - Areas with high rainfall - = No disease; + = Slight; ++ = Moderate; +++ = Severe M.a.s.l. - Meters above sea level
223
Figure 3 Symptoms of major seedborne fungal diseases under field condition on pods and seeds of common beans, Paseolus vulgaris L. (A & B- Angular leaf spot, C & D-Anthracnose, E & FAscochyta blight). CONCLUSION Basic information on the occurrences and geographic distribution of major plant disease, and their association with seeds is very important for setting research priorities for further disease management strategies in different agro-ecologies. From this study, it could be concluded that several seed borne fungi were highly associated with bean seeds. The majority of fungi identified in this study were known to be seed transmitted and caused heavy crop loss in different bean growing parts of the world. The presence of different seed borne pathogens in bean seeds warrants for research attention in the area of seed pathology. Seed certification schemes must be strengthen during production, and release of improved bean varieties to the growers. Infection of bean seeds by large number of seedborne fungi also implies the importance of inspecting seed lots of Phaseolus beans for potential seedborne pathogens in local germplasm maintenance, introduction or exchange
224
20 18 16 14 b b 12 10 8 6 4 2 0 b a a a
Blotter method Agar plate method
LITERATURE CITED Abdelmonem, A.M. 2000. Status of seed pathology and seed health testing in Egypt. Seed Sci. & Technol. 28: 533-547. Agrios, G.N. 1997. Plant pathology. 4th ed., Academic Press, San Diego, 635p. Agarwal, V.K. and J.B. Sinclair, 1997. Principles of seed pathology, 2nd ed. CRC Lewis publishers, New York. 539p. Aleligne K. 1990. Farm survey and on-farm research in haricot bean in the middle rift valley of Ethiopia. In: Proceedings of a national workshop on research on haricot bean in Ethiopia, pp. 3-7, Addis Ababa, Ethiopia, 1-3 October, 1990. Amare, 1987. Haricot bean (Phaseolus vulgaris L.) varieties performance and recommended methods of production. pp. 229-251 In: Proceedings of the 19th National Crop Improvement Conference, Institute of Agricultural Research, Addis Ababa, Ethiopia. Central Statistical Authority (CSA). 2000. Area under production of major crops. Statistical Bulletin No. 245, Addis Ababa, Ethiopia. Chang, K.F., Y. yang, R. Conner, S.F. Hwang, and R.J. Hward, 2001. Relationship of bean seed infection with anthracnose to disease development and seed microflora (Abstr.). Can. J. Plant Pathol. 23: 196. Habtu, A. and T. Abiy, 1995. Disease management in lowland pulses: Progress and possibilities for an integrated approach. In Habtu Assefa (ed.). Proceedings of the 25th anniversary of Nazareth Agricultural Research Center: 25 years of experience in lowland crops research, 20-23 September 1995. Nazareth, Ethiopia. Habtu, A., S. Ivan, and J.C. Zadoks, 1996. Survey of cropping practices and foliar disease of common beans in Ethiopia. Crop Protection, 15 (2): 179-186. Hall, R. 1994. Compendium of bean diseases.
Seed infection (%)
C. lindemuthianum
Major seedborne fungal pathogens
Figure 4 Comparison of standard blotter and agar plate methods for the detection of seedborne fungi using naturally infected common bean seeds.
between institutions and countries. Since there is a change or shift in types and occurrence of plant pathogens in different agro-ecologies, routine disease surveys and identification work is important for better understanding of fungal myco-flora associated with Phaseolus bean seeds. Distribution maps along with illustrations of the spread and spatial patterns of diseases across regions should be emphasised. ACKNOWLEDGMENTS The authors would like to acknowledge Mr. Demeke Nigusse for his assistance on mapping of survey routes and geographic distribution of major seed borne fungi using GIS software. This study was financed by the Ethiopian Agricultural Research Organization (EARO) through the Agricultural Research and Training Project (ARTP).
A. phaseolorum
P. griseola
225
2 nd edition. APS Press, the American Phytopathological Society, St. Paul, Minnesota, 73p. Hellene, R.D. 1988. Bean anthracnose fact sheet. Department of Plant pathology, New York State Agricultural Experiment Station, Geneva Cornell University, pp. 729-740. International Seed Testing Association (ISTA) 1993. International rules for seed testing. Seed Sci & Technol., 21, (Supplement), 288p. Maden, S., D. Singh, S.B. Mathur, and P. Neergaard, 1975. Detection and location of seed-borne inoculum of Ascochyta rabiei and its transmission in chickpea (Cicer arietinum). Seed Sci. & Technol. 3: 667-681. Maude, R.B. 1996. Seed-borne diseases and their control: Principles and practices. CAB International, University press, Cambridge, 280p.
Mathur, S.B. 1995. Some aspects of seed pathology that deserve immediate attention. Indian J. Mycol. pl. Pathol. 25: 13-24. Neergaard, P. 1977. Seed Pathology. Vol. I & II. The Macmillan Press Ltd., London and Asingstoke. 1187p. Nelson, M.R., T.V. Orum, and R. Jaime-Garcia 1999. Application of Geographic Information Systems and Geostatistics in Plant Disease Epidemiology and management. Plant Dis. 83: 308-319. Richardson, M.J. 1979. An annotated list of seedborne diseases, Third edition, Commonwealth Mycological Institute, Kew Surrey, 320p. Schwartz, H.F. and G.E. Galvez. 1980. Bean production problems: Diseases, insect, soil and climate constraints of Phaseolus vulgaris, Cali, Colombia, 424 p.
Short-Term Stressor Effects of Water Deprivation Prior to the Onset of Lay on Subsequent Reproductive Performance of ISA Brown Pullets
Nirat Gongruttananun and Ratana Chotesangasa
ABSTRACT The influence of water deprivation prior to the onset of production on sexual maturity and subsequent reproductive performance was investigated in commercial pullets. Three hundred 16-weekold ISA Brown pullets were used in this study. The birds were housed in cages (100 cages of 3 birds/cage) situated in an open sided poultry shed and randomly divided into three treatment groups. The 3 groups were 1) control (received feed with water at all times) 2) dehydrated and 3) dehydrated+NH4Cl. Following an acclimatization period of 4 weeks, the birds in all groups were placed on a commercial layer ration and the treatments began. In the dehydrated and dehydrated+NH4Cl groups, the drinking water was removed completely from the pullets for 48 hours of dehydration but feed available at all times. The water was then returned to the birds following the water withdrawal period, and thereafter until the end of the trial. The pullets in the dehydrated+NH4Cl group were fed on the layer diet supplemented with 1%NH4Cl throughout the experimental period. It was found that feed intake dropped rapidly, by approximately 50%, when the birds were subjected to water deprivation. Neither age at first egg nor at 50-60 % of production was influenced by the interruption of drinking water supply. Little difference in egg weight at first egg was noted between the dehydrated birds and the normally hydrated hens. Pullets deprived of water were slower coming into egg production especially during the first 2 weeks of production period, however, thereafter egg production was similar among all treatment groups with numerical advantages for the normally hydrated hens. There were no carryover effects of water deprivation on subsequent egg weight, albumen weight, Haugh units, yolk weight or yolk color throughout the entire 12 weeks of the test. Body weights were not different either before the treatment or at the end of the study (P>0.05). The results might be interpreted as indicating that an interruption of drinking water supply for 2 days prior to the commencement of egg production induced transient effects of nutrient deficiency resulted in retardation of reproductive development. The acidified layer ration failed to show any positive effects on reproductive performance for the first 12 weeks of lay. The pullets, however, appeared to overcome the detrimental effects as they approached sexual maturity. Key words: water deprivation, sexual maturity, ammonium chloride, onset of lay, egg weight, egg production
Department of Animal Science, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.
227
INTRODUCTION Water is recognized as a vitally important nutrient for chickens. Lacking of drinking water can have a disastrous effect on the performance of the birds. Interruptions of the water supply could happen as a result of failure of watering systems. As cited by Marsden et al. (1965), there was an occurrence of water deprivation at a turkey breeding flock station due to the weekend attendant failing to refill the water in the water supply. A mortality rate of 13% was observed in older poults and 28% in young birds deprived of drinking water for 2 days. Generally, the birds were weak, unsteady on their feet, and some were dead. Symptoms were ataxia and convulsions including violent flopping, head retraction, gasping, falling over backwards, and lying on their side or back, kicking and died. The removal of drinking water in laying hens even for a few days appears to have a long-term effect on subsequent egg production. Sunde (1962) reported that lack of drinking water for 36 hours had a long term detrimental effect on body weight and egg production. This author indicated that the lowest egg production occurred on the fifth day following water deprivation. Bierer et al. (1965) found reduced feed intake and a sudden drop in egg production in White Leghorn hens deprived of water for 48 hours on Days 3 and 4 following water deprivation. Adams (1973) deprived White Leghorn laying hens of water for 48 to 72 hours and found an 8-week period of declined egg production. This finding was consistent with that reported by Sunde (1962) who observed adverse effects on egg production for 10 weeks in waterdeprived hens. Prolonged water deprivation has been reported that severely affected the avian kidneys resulting in renal failure and sudden death (Siller, 1981). Glahn et al. (1988) demonstrated that adding ammonium chloride in the diet could reduce the incidence of kidney damages without deleterious effects on egg production. However, the many works reported have been conducted
with hens that had been in production for some time and, thus, little or no information is available on the influence of the dehydration mentioned on sexual maturity. The objective of the study was to determine the effects of water deprivation for 48 hours, at the point of lay, on sexual maturity and subsequent reproduction performance in commercial pullets. In addition, the use of a diet acidified with NH4Cl in dehydrated birds was also examined, to determine if dietary acidification could be effective in preventing performance damages. MATERIALS AND METHODS Three hundred ISA Brown pullets, 16 weeks of age, were used in this study. The birds were randomly divided and assigned into three treatment groups as follows: 1) control, 2) dehydrated, and 3) dehydrated+NH4Cl. The pullets were allotted to cages (100 cages of 3 birds/cage) placed in an open-sided layer house, and received 16 hours of light per day. An acclimatization period of 4 weeks was allowed. In the control group, the pullets were provided food and water ad libitum throughout the experimental period. In the dehydrated and dehydrated+NH4Cl groups, the drinking water was withdrawn completely for 48 hours of dehydration but feed was available at all times. The water was then returned (rehydration) to the dehydrated and dehydrated+NH4Cl groups following the water withdrawal period. The pullets in both control and dehydrated groups were fed a layer diet, the birds in the dehydrated+NH4Cl group received the layer diet supplemented with 1%NH4Cl throughout the study. The layer ration used in this study was a mash commercial cornsoybean ration, formulated to have a calculated analysis of 3.25% Ca, 0.5% available phosphorous (aP), and an ME value of 2,851 cal/kg. The acidified layer diet was made by adding 0.45 kg of NH4Cl to 44.9 kg of the layer diet (Glahn et al., 1988). Observations of sexual maturity and subsequent
228
production performance of the experimental birds were made and recorded. Daily egg production was recorded and presented as the numbers of egg per hen per 100 days (percentage of hen-day), and feed intakes were determined during the 2 days of water deprival period, and then biweekly thereafter until 32 weeks of age. All eggs obtained from the last day of each two weeks were assessed individually for egg weight, albumen weight, albumen height (Haugh units), yolk weight and yolk color. Mean body weights of the experimental bird were recorded at the beginning and the termination of the trial. The experiment was commenced in July, 2003 and terminated in December, 2003. The average of minimum and maximum ambient temperatures were 26.04 1.98 C and 33.29 0.66 C, respectively. The statistical evaluation of the data was performed by analysis of variance. Repeated measures ANOVA was used where appropriate. Mean values were compared using Duncans multiple range test to determine significance (Snedecor and Cochran, 1980). Significance was assumed if P<0.05. RESULTS Generally, no deaths occurred during the experimental period. The birds remained seemingly in good health. During the water withdrawal period,
aggressive behavior was observed in the birds in the water deprived groups, such as head movement and nonnutritive pecking. On Day 2 of water withdrawal, the dehydrated pullets appeared to increase their alertness. The degree of aggression, however, disappeared gradually as the drinking water was reintroduced. Birds deprived of water drank rapidly and avidly after the water was returned. Table 1 shows mean values of feed intake of all treatment groups during and after water deprivation. Feed intakes of the pullet in the two groups of dehydration reduced sharply when the drinking water was removed for 48 hours (P<0.05). However, feed consumption of the dehydrated hens caught up, and was similar to that of the normally hydrated birds after 2 weeks of replenishment of the drinking water and remained so until the end of the experimental period (P>0.05). Effects of water deprivation on ages at the onset of lay and at 50% of production are illustrated in Figure 1. Although the significant difference was not noticed, the first egg of the control hens (144.7 days) was laid earlier than that of the dehydrated birds (150 days) and dehydrated+NH4Cl hens (148 days). However, the birds in all groups came to 50% of egg production at the same age (P>0.05). The size of the first egg was significantly affected by the treatments as shown in Figure 2. It was apparent that egg weight of the first egg of the
Table 1 Feed consumption during water deprivation and rehydration in the different treatment groups. Group 48 hours-of dehydration Rehydration (weeks) 6 8
10
12
Control Dehydrated Dehydrated +NH4Cl

a-c
Feed intake (g/bird/2days) 155.012.5a 72.74.1 77.08.3b 73.72.9 62.57.9c 72.93.8
Feed intake (g/bird/d) 80.03.3 89.63.8 96.54.4 81.24.1 91.53.9 94.12.0 80.32.4 88.92.7 92.43.5
100.55.0 107.03.3 101.26.1 108.56.2 100.93.6 106.62.7
Means with no common superscript differ significantly among groups (P<0.05).
229
control pullets (43.9 g) was similar to that of the dehydrated birds (42.7 g), which was significantly heavier than that of the dehydrated+NH4Cl hens (39.3 g) (P<0.05). Influences of water deprivation for 48 hours at the point of lay on subsequent egg production are represented in Table 2. Obviously, withdrawal of drinking water adversely affected hen-day egg production during the initial state of the production period. The dehydrated pullets laid at a lower rate than did the normally hydrated birds interval 20-22 weeks of age (P<0.05) whereas egg production of the birds in the
dehydrated+NH4Cl group was not significantly different from that of the birds in the control group. However, thereafter egg production did not significantly differ for all groups until the end of the study. Details of egg quality measurement of the different treatment groups for the first 12 weeks of production are summarized in Table 3. Deprival of drinking water did not significantly affect subsequent egg weight, or any aspect of egg quality parameters throughout the experimental period (P>0.05). Figure 4 presents mean values of body weight of the different treatment hens at the
175 170 165
Age (days)
160 155 150 145 140 135 130 At first egg At 50% of lay control dehydrated dehydrated+ammonium chloride
Group
Figure 1 Averages of age at the onset of lay and 50% of production of the different treatment hens.
46 45 44 control dehydrated dehydrated+ammonium chloride
Egg weight (g)
43 42 41 40 39 38 37 36
Group
Figure 2 Averages of egg weight at first egg of the different treatment hens.
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Table 2 Effects of water deprivation for 2 days during the pullet-laying transition period on subsequent egg production in the different treatment groups. Group 20-22 22-24 Age interval (weeks) 24-26 26-28 Egg production (%) 58.87.8 74.94.2 54.28.9 71.97.3 49.513.5 74.63.8
28-30
30-32
Control Dehydrated Dehydrated+NH4Cl

a-b
11.46.5a 3.81.1b 6.84.8ab
34.88.5 32.310.6 24.414.0
74.66.3 73.06.3 73.77.2
82.83.5 82.23.8 80.26.0
Means with no common superscript differ significantly among groups (P<0.05).
Table 3 Influences of interruption of drinking water supply for 48 hours prior to the onset of lay on subsequent egg quality parameters in the different treatment groups. Group 22 24 Age (weeks) 26 28 Egg weight (g) 54.91.3 56.42.6 53.71.5 55.31.7 52.92.0 56.41.9 Haugh units 91.82.4 88.74.2 94.32.1 90.20.7 94.03.7 92.13.2 Yolk color 11.90.3 12.50.2 12.40.3 12.20.2 12.20.2 12.20.2 Yolk weight (%) 22.70.4 22.90.4 22.20.2 23.40.2 23.00.9 23.00.2 Albumen weight (%) 67.80.7 67.70.8 68.30.7 67.10.6 67.90.7 67.50.3
30
32
Control Dehydrated Dehydrated+NH4Cl Control Dehydrated Dehydrated+NH4Cl Control Dehydrated Dehydrated+NH4Cl Control Dehydrated Dehydrated+NH4Cl Control Dehydrated Dehydrated+NH4Cl
48.72.6 47.43.6 46.42.7 93.64.5 94.36.4 95.24.1 10.10.8 10.40.4 10.30.4 22.51.2 21.31.2 23.31.9 67.81.4 69.41.5 67.22.1
50.81.0 51.91.5 49.92.6 96.85.2 98.02.2 95.92.7 11.80.3 12.20.3 12.30.4 22.50.8 22.10.9 22.60.9 68.10.6 68.20.8 68.50.8
55.61.9 54.81.4 55.42.5 90.02.2 91.32.4 88.74.2 11.80.3 12.20.1 11.80.3 23.21.2 22.60.6 23.10.2 67.50.8 68.10.7 67.40.2
56.13.3 54.41.0 55.63.3 87.16.0 89.01.0 86.54.7 12.10.1 12.10.2 12.10.2 22.60.3 23.10.6 23.61.5 67.30.7 67.10.6 66.61.7
There were no significant differences between groups (P>0.05). All data in the tables are given as mean standard deviation.
231
beginning and the end of the investigation. No significant differences were observed in body weight among groups before the treatment or at the end of the experiment. DISCUSSION No adverse effects of water deprivation for 2 days at the onset of lay were observed on body weights of the bird as illustrated in Figure 4. However, the hens in the study were not weighed during the water removal. The water deprival period of 48 hours caused no apparent behavioral ill effects on the hens, except for an aggressive behavior. The pullets in both dehydration groups showed an increase in aggressive pecks during the water withdrawal period, especially on Day 2 of water deprivation. The increased degree of aggression might arise from frustration (Duncan and Wood-Gush, 1971) resulting from hens expecting the availability of drinking water but not finding it. The finding of aggressive behavior in the dehydrated hens in the study was similar to that observed in White Leghorn laying hens deprived of food (Simonsen, 1979). It is well documented that deprivation of food or water enhances levels of plasma corticosterone in avian species (Freeman et al., 1980; Arnason et al., 1986). Corticosterone hormone is secreted from the adrenal glands, located anterior and medial to the cephalic lobe of the avian kidneys (Ringer and Meyer, 1976). Freeman et al. (1980) indicated that starvation was a strong stimulus for the releasing of corticosterone in domestic birds. The findings of this study, that removal of drinking water for 48 hours prior to the onset of lay reduces feed intake, were consistent with those reported by several workers (Sunde, 1962; Adams, 1973). Korr (1939) reported that the birds responded to water deprivation by reducing glomerular filtration rates (GFR) and urine flow rates, and increasing the osmotic pressure of blood and urine. Later, dehydration was reported to
induce an increase in plasma osmotic concentrations (Koike et al., 1983). These findings were consistent with those reported by Roberts (1991). Sexual maturity of the experimental pullets, as estimated from age at first egg and at 50% production, was not significantly affected by interruption of drinking water which would be a reflection of a similar body weight of the experimental birds (Figure 4). Several reports from previous studies (Summers et al., 1987; Summers and Leeson, 1994) have shown that there is a specific body weight threshold for the onset of production of birds. The investigators suggested that pullets must achieve the certain body weight in order to trigger the onset of production. It was well documented from previous several studies (Brody et al., 1980; Leeson and Caston, 1991) that regardless of feed or management regimen, if the pullet had the chance to consume diets at all times, the animal could quickly catch up in body weight so that a uniform weight was obtained at the onset of lay. The results from the study indicated that, as the drinking water was withheld, feed intake decreased markedly by approximately 50-60 % comparing with that consumed by the normally hydrated hens (Table 1). This might be comparable to the circumstance of feed restriction for 2 days. However, it was of interest to notice that feed intake in the two dehydration groups increased rapidly and returned to the normal value within 2 weeks after the birds were allowed access to the drinking water again. This demonstrated the ability of the pullets to increase feed consumption in an attempt to achieve a mature body weight as they approached sexual maturity. The results from this study revealed that the interruption of drinking water for 48 hours at the point of lay had minor effects on subsequent reproductive performance due to feed restriction. If one compared feed intake of the experimental birds during water deprivation (Table 1) with the
232
average of egg weight at first egg (Figure 2), it was likely that the amount of nutrient intake supported egg size at the initial phase of lay. In addition, the dehydrated pullets were coming into production slowly than the conventionally reared birds as evidenced by hen-day egg production during the first 2 weeks of production period (Table 2), and age at first egg. It did appear to be a trend in the dehydrated birds for requirement of much more times to be ready-to-lay pullets as compared with the control pullets (Figure 1). Likewise, the trend for slightly lower production was evident for the hens in the two groups of water withdrawal as
90 80
depicted in Figure 3, confirming by the experiences of others (Isaacks et al., 1960; Brake et al., 1985; Summers et al., 1991) that feed restriction during the prelaying phase had only a slightly adverse effect on subsequent egg production. Summers et al. (1991) deprived White Leghorn pullets of food for 6 days at 17 weeks of age, they found that the 6-day feed withdrawal period delayed sexual maturity and reduced egg weight only during the first 2 weeks of production. Brake et al. (1985) stated that nutrient intake during the prelay period was a main factor influencing performance of hens. These authors suggested that protein intake
Hen-day production (%)
70 60 50 40 30 20 10 0 20-22 22-24 24-26 26-28 28-30 30-32 control dehydrated dehydrated+ammonium chloride
Age interval (weeks)
Figure 3 Hen-day egg production of hens on the different treatments from 20 to 32 weeks of age.
1.6 1.5
Body weight (kg)
control dehydrated dehydrated+ammonium chloride
1.4 1.3 1.2 1.1 At the beginning At the end
Age
Figure 4 Averages of body weight at the beginning and the end of the experiment of the different treatment birds.
233
was the major factor allowing accumulation of protein reserves in the body, which resulted in increased egg production and egg weight. Presumably, in this study, the dehydrated birds consumed inadequate amount of nutrients especially protein intakes, resulting from a reduction of feed intake during 2 days of water deprivation, in which the degrees of nutrient deficiency would be so much severely that affected reproductive development. This would indicate that such conditions adversely affect carrying reserves of body composition of the pullets at the point of lay, thereby poor production and small egg size at first egg were observed in the laying house. Unfortunately, the data of gonads of the birds in the current experiment were not determined. Additional studies are needed to conduct whether water deprivation influences the development of reproductive organs of the domestic fowl. The results obtained from the study indicated that withdrawal of drinking water supply for 48 hours prior to the onset of egg production had transiently adverse effects on subsequent reproductive performance as evidenced by reducing of egg weight at first egg and egg production during the initial state of lay, suggesting that due to reduced feed intake during the dehydration period resulted in protein and/or nutrient deficiencies, therefore growth and reproductive development was retarded. However, the pullets deprived of water tended to overcome the nutrient restricted stress effect as they approached sexual maturity. This would be a reflection of the ability of the animals to compensate for such circumstance by increasing feed intake and body composition. Acidification diets with NH4Cl did not exhibit any beneficial effects on sexual performance for the 12 weeks of observation period. These results were in agreement with the results reported by Glahn et al. (1988). The workers demonstrated that dietary acidification with NH4Cl at a level of 1% did not affect production performance. They also indicated
that a major advantage of the NH4Cl acidified diet was that it reduced the incidence of kidney lesions. CONCLUSION Lacking of drinking water for 48 hours, prior to the onset of lay, did not affect sexual maturity, body weight, egg weight or subsequent reproductive performance. However, the pullets deprived of water commenced to lay slightly late during the initial period of production. Obviously, feed consumption of the birds dropped abruptly as the birds were subjected to water deprivation. It was possible that dehydration induced a shortterm stressor, leading to a reduction in feed intake. Reduced feed consumption resulted in nutrient deficiencies and retardation of reproductive development. No beneficial effects on reproductive performance of adding 1%NH4Cl to feed were evident. ACKNOWLEDGEMENTS The authors wish to acknowledge the financial support of the Kasetsart University Research and Development Institute (KURDI) in the carrying out of this work. LITERATURE CITED Adams, A.W. 1973. Consequences of depriving laying hens of water a short time. Poultry Sci. 52: 1221-1223. Arnason, S.S., G.E. Rice, A. Chadwick and E. Skadhauge. 1986. Plasma levels of arginine vasotocin, prolactin, aldosterone and corticosterone during prolonged dehydration in the domestic fowl: effect of dietary NaCl. J. Comp. Physiol B156:383-397. Bierer, B.W., T.H. Eleazer and D.E. Roebuck. 1965. Effect of feed and water deprivation on chickens of various ages. Poultry Sci. 44: 1351.
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Brody, T., Y. Eitan, M. Soller, N. Nir and Z. Nitsan. 1980. Compensatory growth and sexual maturity in breeder females reared under severe food restriction from day of hatching. Br. Poultry Sci. 21: 437-446. Brake, J., J.D. Garlich and E.D. Peebles. 1985. Effect of protein and energy intake by broiler breeders during the prebreeder transition period on subsequent reproductive performance. Poultry Sci. 64: 2335-2340. Duncan, I.J.H. and D.G.M. Wood-Gush. 1971. Frustration and aggression in the domestic fowl. Anim. Behav. 19: 500-504. Freeman, B.M., A.C.C. Manning and I.H. Flack. 1980. Short-term stressor effects of food withdrawal on the immature fowl. Comp. Biochem. Physiol. 67A: 569-571. Glahn, R.P., R.F. Wideman, Jr. and B.S. Cowen. 1988. Effect of dietary acidification and alkalinization on urolith formation and renal function in single comb white leghorn laying hens. Poultry Sci. 67: 1694-1701. Isaacks, R.E., B.L. Reid, R.E. Davies, J.H. Quisenberry and J.R. Couch. 1960. Restricted feeding of broiler type replacement stock. Poultry Sci. 39: 339-346. Koike, T.I., L.R. Pryor and H.L. Neldon. 1983. Plasma volume and electrolytes during progressive water deprivation in chickens (Gallus domesticus). Comp. Biochem. Physiol. 74A: 83-87. Korr, I.M. 1939. The osmotic function of the chicken kidney. J. Cell. Comp. Physiol. 13: 175-179. Leeson, S. and L.J. Caston. 1991. Growth and development of Leghorn pullets subjected to abrupt changes in environment temperature
and dietary energy level. Poultry Sci. 70: 1732-1738. Marsden, S.J., G.S. McKee and M.L. Crandall. 1965. Water deprival and replenishment in poults. Poultry Sci. 44: 793-797. Ringer, R.K. and D.C. Meyer. 1976. Parathyroids, Ultimobrachial Bodies, and the Pineal, pp. 359-371. In P.D. Sturkie (ed.). Avian Physiology 3rd ed. Springer-Verlag, New York. Roberts, J.R. 1991. Effects of water deprivation on renal function and plasma arginine vasotocin in the feral chicken Gallus gallus (Phasianidae). Aust. J. Zool. 39:439-446. Siller, W.G. 1981. Renal pathology of the fowl A review. Avian Patho. 10: 187- 262. Simonsen, H.B. 1979. Effect of feed withdrawal on behavior and egg production in white leghorns on litter and wire. Br. Vet. J. 135: 364-369. Snedecor, G.W. and W.G. Cochran. 1980. Statistical Methods 7th ed. The Iowa State University Press, Ames, Iowa, USA. Summers, J.D. and S. Leeson. 1994. Laying hen performance as influenced by protein intake to sixteen weeks of age and body weight at point of lay. Poultry Sci. 73: 495-501. Summers, J.D., S. Leeson and D. Spratt. 1987. Rearing early maturing pullets. Poultry Sci. 66:1750-1757. Summers, J.D., D. Spratt and J.L. Atkinson. 1991. Delaying sexual maturity of pullets by nutrient restriction at the onset of production. Can. J. Anim. Sci. 71: 1215-1221. Sunde, M.L. 1962. Amino acids, proteins, and stuff. Poultry Sci. 41: 1688.
Pharmacokinetics and Withdrawal Times of Enrofloxacin in Ducks

Natthasit Tansakul1, Amnart Poapolathep1, Naruamol Klangkaew1, Napasorn Phaochoosak1 and Wanida Passudaruk2
ABSTRACT The pharmacokinetic properties of enrofloxacin (EFX) were investigated in healthy ducks following a single administration of EFX with a dose of 10 mg/kg of body weight by intravenous (i.v.), intramuscular (i.m.), subcutaneous (s.c.) or oral (p.o.) route. The plasma concentration-time curve was analyzed using a two compartment model. Mean peak plasma concentration of EFX was 11.49 1.17, 5.65 0.36, 4.99 0.87 and 4.87 0.69 mg/ml after i.v., i.m., s.c. and p.o. administration, respectively. After a single i.v. administration, the pharmacokinetic parameters were found as follow; the elimination half-life (t1/2b) = 6.47 2.85 h, the elimination rate constant (Kel ) = 0.70 0.06 h-1,the apparent volume of distribution Vd(area) = 1.30 0.22 L/kg and the total body clearance (ClB) = 0.89 0.07 L/kg/h. Difference enrofloxacin bioavailability following i.m., s.c. and p.o. administration were 98.77 0.05 %, 85.11 2.71 % and 80.35 0.29%, respectively. The results of pharmacokinetic properties of EFX in ducks should be provided with the dosage regimen, preslaughter withdrawal times and maximum residue limits for ducks. Key words: pharmacokinetic, withdrawal time, antibiotic, enrofloxacin, duck species
INTRODUCTION Enrofloxacin (1-cyclopropyl-6-fluoro-1,4dihydro-4-oxo-7-[4-ethyl-1-piperazinyl] -3quinoline carboxylic acid) is an antimicrobial substance which belongs to the fluoroquinolones groups. This agent reportedly has excellent activities against a wide range of aerobic gramnegative bacteria. It is also active against grampositive bacteria and Mycoplasma spp. Therefore, EFX is commercialized for animal use and potential therapeutic application for many types of infection (Garca-ovando et al.,1999). Similar to that of
other quinolones, these compounds act on inhibition of DNA gyrase and exhibit a bactericidal and mycoplasmacidal activity at low concentrations. The efficacy of EFX reportedly inhibits in vivo replication of certain organisms that are resistant to antibacterial substances i.e., beta-lactam antibiotics, aminoglycosides, tetracyclines, folic acid antagonists and macrolides (Anadn et al., 1995). Limited information is available on disposition, metabolism and safety of EFX use in commercial ducks. The objective of the present study was to investigate the fundamental pharmacokinetic value of EFX on ducks following
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Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900. Thailand. Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900. Thailand.
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intravenous (i.v.), intramuscular (i.m.), subcutaneous (s.c.) and oral (p.o.) administration. Thereafter, the proper therapeutic regimen of EFX should be concerned for ducks. MATERIALS AND METHODS Animals Healthy ducks of an average 1.09 0.24 kg body weight, without previous treatment, were used in the study. Ducks were separated into four groups (30 ducks per group). The animals were fed with a commercial standard diet that was free from any chemotherapeutics three times per day. Water supply was provided ad libitum. Throughout the study they were housed in the animal cage at Division of Experimental Animal, Faculty of Veterinary Medicine, Kasetsart University. Drug administration and sample collection Commercial enrofloxacin containing 50 mg/ml (Baytril 5% sterile solution, Bayer AG, Leverkusen,Germany) was prepared for i.v., i.m., s.c. and p.o. administrations at the same dose of 10 mg/kg body weight for each duck. Randomized 2.5 ml of heparinized blood were taken from the brachial vein in the following preset times:0.0, 0.15, 0.30, 1, 2, 3, 4, 5, 6, 8, 10, 12, 20, 24, 30, 48, 54, 72, 78 and 92 h. Blood samples were collected and centrifuged (3000 X g) for 15 min to collect the plasma (Garca-ovando et al.,1999), placed in a 1.5 ml Eppendorf vial (Laboratory Product, Inc., Rochester, NY.) and stored at -20C until analysis. Method of analysis The concentration of EFX was analyzed using a microbiological diffusion method (Bennett et al.,1966; Anhalt,1985; Limpoka,1992). The method used Escherichia coli ATCC 25922 (Scientific and Technology Institute of Thailand) as test organisms. Standard dose-response curves were obtained using buffer EFX solution. The motten agars were prepared by inoculated with the
organisms in broth. Then the medium was poured 32 ml into each 10 15 cm glass plate. After hardened, 10 mm diameter wells were punched 8 holes per plate. Then the plasma samples and standard control (2 holes) were examined. The samples were allowed to diffuse for 45 min at room temperature prior to incubation for 24 h at 37C. Thereafter, the inhibition zone of the standard preparations and samples were measured using a caliper vernia. The concentrations were recorded from plots of log concentration plus zone diameter of plasma. Calculation of pharmacokinetic parameters The pharmacokinetic values of EFX on plasma concentrations after a single i.v. administration were evaluated by a semilogarithm modified standard technique. A bi-exponential equation was selected for all ducks having been given the drug by the i.v. route and consequently the data were described by a two-compartment open model based on the criteria of improvement in the sum square by plotting of residuals. The following pharmacokinetic parameters were obtained according to the conventional equations previously described by Baggot (1977), Limpoka (1992) and Craigmill et al. (1994). The following equations were used to obtain these pharmacokinetic parameters for a twocompartment pharmacokinetic model. t1/2a = ln 2/a t1/2b = ln 2/b K21 = A(b) + B(a)/ A+B Kel = (a)(b)/ K21 K12 = a+b- K21 - Kel Vd(area) = Dose/Cpo AUC = (A/a) + (B/b) F = AUCother / AUCi.v ClB = (Kel) (Vc) The term of Cpo is the extrapolated plasma concentration to determined the zero- time profile. B was calculated from the elimination phase (Bslope). A was calculated by the residual method
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(OFlaherty, 1981). The a and b are hybrid rate constants describing the initial and terminal decline in plasma concentration and are composed of the microrate constants (K12,K21) of the model. The t1/2a (distribution half-life), t 1/2b (elimination halflife), AUC (area under the curve), Vd(area) (apparent volume of distribution during the post-distribution phase), Bioavailability and Cl B (total body clearance) were calculated. Statistic analysis The pharmacokinetic parameters were calculated by CA-Cricket Graph III, version 1.5J, Computer Associates Inc., NY., U.S.A. Statistical analysis of data was performed using Microsoft Excel, Window XP. RESULTS After a single i.v. administration of 10 mg/ kg of body weight of EFX in ducks, the mean SD pharmacokinetic parameters were calculated and described by a two-compartment open model. Distribution half-life (t1/2a ) was 0.60 0.02 h, whereas the elimination half-life(t1/2b) was 6.47 2.85 h. Table 1. presents the pharmacokinetic parameters. Comparison of the mean SD plasma concentration-time profile of EFX at various routes are shown in Table 2. and Figure 1. EFX was absorbed rapidly. Concentrations of EFX peaked within 30 min by i.m. administration while the peak levels of s.c and p.o. administration were found within 1 h. However, these levels were higher than the therapeutic level (Anonymous; 1987). DISCUSSION Pharmacokinetic variables of EFX after the i.v. administration were best described by a two-compartment open model, with a rapid distribution phase (t1/2a = 0.6 h) and a moderately
prolong elimination phase (t1/2b = 6.47 h). Because of limited reports of fluoroquinolones in ducks, the time to maximum concentration (tmax) differed among enrofloxacin, ciprofloxacin and norfloxacin in chicken were applied as reference. The significant differences (p<0.05) were found that the tmax of ciprofloxacin (0.42 0.08 h) (Atta and Sharif,1997) was reached more rapidly than that of enrofloxacin (1.64 0.04 h) (Anadn et al.,1995) and norfloxacin (1.99 0.17 h) (Laczay et al.,1998) after oral administration. In addition, the peak plasma concentration (Cmax) of ciprofloxacin was the highest (4.67 0.33 mg/ml),which was higher than that of the enrofloxacin (2.44 0.64 mg/ml) and norfloxacin (1.46 0.18 mg/ml). Table 1 Pharmacokinetic data (mean SD) for enrofloxacin determined following intravenous administration at a single dose of 10 mg/kg of body weight in ducks. Pharmacokinetic parameters (units) Cpo (mg/ml) A (mg/ml) a (h-1) B (mg/ml) b (h-1) t1/2a (h) t1/2b (h) K12 (h-1) K21 (h-1) Kel (h-1) Vd(area) (L/kg) ClB (L/kg/h) Bioavailabilityi.m. (%) Bioavailabilitys.c. (%) Bioavailabilityp.o. (%) Enrofloxacin
15.73 14.67 1.16 1.35 0.13 0.60 6.47 0.37 0.22 0.70 1.30 0.89 98.77 85.11 80.35
2.60 3.38 0.03 0.90 0.07 0.02 2.85 0.003 0.13 0.06 0.22 0.07 0.05 2.71 0.29
Note: Pharmacokinetic parameters of EFX were determined by a two-compartment pharmacokinetic model.
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A similar kinetic profile was also observed in chickens after the i.v. administration, the biphasic nature of the plasma concentration-time curve has been reported for EFX (Anadn et al.,1995;Garcaovando et al.,1999). In the present study, the elimination half-life (6.47 2.85 h) was also higher than that recorded in healthy dogs (3.4 h), cattle (1.7h), sheep (3.7 h), horses (5.0 h) and pigs
(5.5 h) (Baggot,2001). However, this parameter was lower than that previously reported in chickens (6.99 0.48 h) (Garca-ovando et al.,1999). Fluoroquinolones are lipid-soluble chemical agents, and their typical Vd values are 24 L/kg (Brown,1996). Nevertheless, lower Vd values i.e., 1.94 0.14 L/kg have been reported for EFX in chickens (Garca-ovando et al.,1999)
Table 2 Mean SD plasma concentrations of enrofloxacin in ducks following i.v., i.m., s.c. or p.o. administration at a single dose of 10 mg/kg of body weight. Hours after dosing i.v. 0.15 0.30 1.00 2.00 3.00 4.00 5.00 6.00 8.00 10.00 11.49 1.17 10.46 1.85 5.59 0.28 4.00 0.25 2.08 0.36 1.97 0.15 1.06 0.16 0.86 0.19 0.47 0.07 0.43 0.04 Plasma concentrations (mg/ml) i.m. s.c. 5.65 0.36 8.97 1.33 7.99 0.63 7.60 0.68 6.76 0.99 5.72 0.58 3.90 0.35 3.57 0.41 1.51 0.17 1.43 0.17 4.99 0.87 6.48 1.07 8.29 0.62 7.19 1.12 7.06 0.58 6.67 0.73 4.44 0.68 2.95 1.13 1.46 0.10 1.42 0.25
p.o. 4.87 0.69 5.55 0.70 7.61 1.20 5.99 0.34 5.74 0.60 4.62 0.39 4.43 0.59 3.99 0.73 2.28 0.21 2.06 0.43
14 12 i.v. i.m. s.c. p.o.
Plasma concentrations (ug/ml)
10 8 6 4 2 0 0 1 2 3 4 5 6 7 8
10
Hour after dosing (hr.)
Figure 1 Comparative mean plasma concentration-time profile of enrofloxacin (EFX) following single i.v.,i.m.,s.c.and p.o. administrations of 10 mg/kg b.w. in ducks.
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The mean SD bioavailability of EFX in ducks was 98.77 0.05% after the i.m. administration, therefore it is likely that the dose of EFX was almost completely absorbed. The bioavailability value of the i.m. administration was also higher than those of the s.c. (85.11 2.71%) and p.o. (80.35 0.29%). Moreover, the drug was detected and remained in the plasma up to 20 h after the s.c. and i.m. administrations while it was up to 24h after the i.v. and p.o. administrations. In conclusion, The biphasic nature of plasma concentration-time curve suggested that a twocompartment pharmacokinetic model would provide an accurate description of pharmacokinetic behaviors. The pattern of plasma concentrationtime profiles between EFX and the other fluoroquinolones were identical following i.m., s.c. or p.o. administration . According to the results of this study a dose of 10 mg/kg body weight of enrofloxacin in ducks may be appropriate for the routes investigated. However, the tissue residues should be further determined by an HPLC assay to get insight into the tissue uptake and the proper withdrawal times of EFX in ducks. ACKNOWLEDGEMENTS This study was financially supported by the Kasetsart University Research and Development Institute (KURDI), THAILAND. The researchers wish to thank Prof. Dr. Malinee Limpoka for her advice on the method of analysis. Special thanks are extended to Kanokwan Bangnoi and Sasithorn Limsuwan for their advice on the use of computer program. LITERATURE CITED Anadn, A., M.R. Martinez-Larranaga, M.J. Diaz, P. Bringas, M.A. Martinez, M.L. FernandezCruz, M.C. Fernandez and R. Fernandez.1995. Pharmacokinetics and residues of enrofloxacin in chickens.Amer. J. of Vet. 56: 501-505. Anhalt, J.P. 1985. Antimicrobial assays. In
Laboratory Procedures in Clinical Microbiology. 2nd ed., J.A. Sprinter Verlag, Washington, New York. 691 p. Anonymous .1987. Baytril Broad-spectrum Antiinfective for the treatment of Bacterial Diseases in Animals. Product information. Bayer Veterinary Department, Leverkusen, Germany. 27 p. Atta, A.H. and L. Sharif. 1997. Pharmacokinetics of ciprofloxacin following intravenous and oral administration in broiler chickens. J. vet. Pharmacol. Therap. 20: 326-329. Baggot, J.D.1977.Principles of Drug in Domestic Animals.W.B. Saunders, Philadelphia. 238 p. Baggot, J.D.2001. The Physiological Basis of Veterinary Clinical Pharmacology. Iowa State University Press. 283p. Bennett, J.V., J.L. Brodie, E.Benner and W.M.M. Kirby. 1966. Simplified, accurate method for antibiotic assay of clinical specimens. Applied Microbiology. 14: 170-175. Brown, S.A. 1996. Fluoroquinolones in animal health. J.Vet.Pharmacol.Therap.19: 1-14. Craigmill, A.L., S.F. Sundlof and J.E. Riviere. 1994. Handbook of Comparative Pharmacokinetics and Residues of Veterinary Therapeutic Drugs, CRC Press, Inc., Boca Raton, Fla. 665 p. Garca- ovando, H., N. Gorla, C. Luders, G. Poloni, C. Errecalde, G. Prieto and I. Puelles. 1999. Comparative pharmacokinetics of enrofloxacin and ciprofloxacin in chickens. J. Vet. Pharmacol. Therap. 22: 209-212. Laczay, P., G. Semjen and J. Lehel .1998. Comparative studies on the pharmacokinetics of norfloxacin in chickens,turkeys and geese after a single oral administration. J. Vet. Pharmacol. Therap. 21: 161-164. Limpoka, M. 1992. Principle Pharmacokinetic in Animals. Charulsanitwong, Bangkok. 195 p. O Flaherty, E. 1981.Toxicants and Drug: Kinetics and Dynamics.A Wiley Interscience Publication, New York. 398 p.
Antimicrobial Resistance of Campylobacter jejuni Isolated from Chicken in Nakhon Pathom Province, Thailand
Jananya Sukhapesna1, Patamaporn Amavisit2, Worawidh Wajjwalku3, Arinthip Thamchaipenet4 and Thavajchai Sukpuaram5
ABSTRACT Campylobacter jejuni isolated from retail market-chicken in Nakhon Pathom province were determined for resistance to quinolone and other antimicrobial agents by broth microdilution method. Sixty-eight C. jejuni strains were resistant to quinolone drugs including nalidixic acid (69.12%), norfloxacin (69.12%), ciprofloxacin (58.82%) and marbofloxacin (25.00%). High proportions of the isolates were resistant to tetracycline (77.94%), sulfamethoxazole (72.06%), kanamycin (51.47%), ampicillin 47.06% and streptomycin (42.65%). Low proportions of the isolates were found resistance to gentamycin (16.18%) and erythromycin (13.23%). Nearly 97% of the isolates were multiple resistances to more than 4 antimicrobial agents tested. Key words: Campylobacter jejuni, broth microdilution method, MICs INTRODUCTION Infection with Campylobacter species has emerged worldwide as one of the leading causes of diarrhea (Rautelin et al., 2003). C. jejuni is one of the main species involved in human infection (Saenz et al., 2000). The genotyping and serotyping analysis revealed that poultry can be a source of Campylobacter infection and the contamination occurred by direct ingestion of undercooked food or cross contamination of raw poultry to other foods (Engberg et al.,2001). Fluoroquinolones and macrolides have been widely used for treatment of Campylobacter
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infections (Aquino et al., 2002). The increasing proportions of Campylobacter isolates have been reported to be resistant to these drugs (Endtz et al., 1991). The over uses of antimicrobial agents in veterinary medicine or as feed additives might result in the emergence and spread of resistance among Campylobacter strains. This caused potentially serious effects on food safety and affected to both veterinary and human health (Piddock et al., 2000). In this study we determined the antimicrobial resistance patterns of sixty-eight C. jejuni isolated from chicken in Nakhon Pathom province and evaluated the range of their minimum
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Center for Agricultural Biotechnology, Kasetsart University, Kamphaengsean Campus, Nakhon Pathom 73140, Thailand. Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand. Department of Pathology, Faculty of Veterinary Medicine, Kasetsart University, Kamphaengsean Campus, Nakhon Pathom 73140, Thailand. Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand. Department of Public Health, Faculty of Veterinary Medicine, Kasetsart University, Kamphaengsean Campus, Nakhon Pathom 73140, Thailand.
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inhibitory concentrations (MICs) using broth microdilution method. MATERIALS AND METHODS Bacterial strains Sixty-eight C. jejuni were isolated from retail market-chicken in Nakhon Pathom province. They were identified at Kamphaengsean Animal Diagnostic Laboratory, Faculty of Veterinary Medicine, Kasetsart University. These isolates were confirmed to be C. jejuni by using PCR assay following the method of Stucki et al. (1995). The isolates were cultured on Columbia agar plate with 5% lysed horse blood and were incubated at 42C for 48 h in microaerophilic atmosphere. C. jejuni colonies were transferred into 5 ml of MuellerHinton broth and then incubated at 37C for 24 h in microaerophilic atmosphere to produce a suspension of 6 to 7 log CFU/ml. Antimicrobial agents Six groups of antimicrobial agents including aminoglycoside (gentamycin, kanamycin and streptomycin), macrolide (erythromycin), penicillin (ampicillin), quinolone (ciprofloxacin, marbofloxacin, nalidixic acid and norfloxacin), sulfanamide (sulfamethoxazole) and tetracycline, were used for this study. Test procedure One hundred microliters of two-fold dilution of each antimicrobial agent ranging from 0.007 to 128 ml/ml was filled in each well of 96 wells sterile microtiter plates (Luber et al., 2003). Each well for susceptibility testing was filled with 100 ml of 6 to 7 log CFU/ml of bacterial suspension, and mixed gently. The plates were incubated at 37C under microaerophilic condition. The MICs was evaluated at 24 hours later.
RESULTS AND DISCUSSION Minimum Inhibitory Concentrations (MICs) were defined as the lowest concentration that exhibits no growth of C. jejuni by visible reading. The MICs of antimicrobial agents for C. jejuni is presented in Table 1. Each of isolates had different MICs in each antimicrobial agents and different range of MICs. The MICs range of gentamycin, kanamycin, streptomycin, erythromycin, ampicillin, ciprofloxacin, marbofloxacin, norfloxacin and sulfamethoxazole were 0.03-2, 1-128, 0.12-16, 0.5-64, 0.25-64, 0.12-16, 0.007-8, 0.06-8 and 0.5-128 mg/ml, respectively. Nalidixic acid and tetracycline had the same MICs range at 0.25-128 mg/ml. The MICs values of tetracycline, nalidixic acid, kanamycin and sulfamethoxazole were higher than other antimicrobial agents. At present, there is no internationally accepted criterion for susceptibility testing of Campylobacter. Resistant breakpoint following EUCAST (2000) method was used in this study. It was classified as susceptibility or resistance according to their individual MICs distribution of each agent. When two or more sub-populations were found, isolates with lower MICs were classified as susceptibility, whereas those from sub-populations with higher MICs were classified as resistance. The distribution of MICs of antimicrobial agents against sixty-eight isolates of C. jejuni is shown in Figure 1. The MICs of these antimicrobial agents was a bimodal distribution. C. jejuni isolates were categorized as resistance when MICs of erythromycin, kanamycin, and sulfamethoxazole exceeded 32 mg/ml; ampicillin and tetracycline exceeded 16 mg/ml; nalidixic acid and streptomycin exceeded 8 mg/ml; ciprofloxacin and marbofloxacin exceeded 4 mg/ml, whereas C. jejuni isolates were found as low resistance, when MICs of gentamycin and norfloxacin exceeded 1 and 2 mg/ml, respectively.
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Antimicrobial concentration ranges, MIC50, MIC90, breakpoints of resistance and percentage of resistance for C. jejuni are presented in Table 2. MIC50 and MIC90 were MIC at which
50% and 90% of the isolates were inhibited, respectively. The data indicated that sulfamethoxazole had MIC50 and MIC90 higher than other agents at 64 and 128 mg/ml, respectively,
Table 1 MICs of 11 antimicrobial agents against sixty-eight isolates of C. jejuni by broth microdilution method. MICs (mg/ml) 2 1 0.5 0.25 0.12 0.06 2 27 8 3 4 6 8 20 8 21 5 11 3 8 1 21 14 4 9 9 37 4 3 1 2 16 10 14 33 7 35 9 6 35 1 23 45 4 9
Agent1 GEN KAN STR ERY AMP CIP MAR NAL NOR SMX TET
1
128
64
32
16
0.03 0.015 0.007 3
25
5 7 33 10 1
1 12 8
9 35 7
7 2 32
22
2 13 4 8
GEN = gentamycin, KAN = kanamycin, STR = streptomycin, ERY = erythromycin, AMP = ampicillin, CIP = ciprofloxacin, MAR = marbofloxacin, NAL = nalidixic acid, NOR = norfloxacin, SMX = sulfamethoxazole, TET = tetracycline MICs = Minimum Inhibitory Concentrations
Table 2 Antimicrobial agents, range of MICs, MIC50, MIC90, breakpoint of resistance and percentage of resistance from sixty-eight C. jejuni isolates as determination by broth microdilution. MIC (mg/ml) MIC50 0.12 32 0.25 1 0.5 4 0.03 16 2 64 32 Break point of resistance (mg/ml) 1 32 8 32 16 4 4 8 2 32 16 Percentage of resistance 16.18% 51.47% 42.65% 13.23% 47.06% 58.82% 25.00% 69.12% 69.12% 72.06% 77.94%
Agent Gentamycin Kanamycin Streptomycin Erythromycin Ampicillin Ciprofloxacin Marbofloxacin Nalidixic acid Norfloxacin Sulfamethoxazole Tetracycline
Range 0.03-2 1-128 0.12-16 0.5-64 0.25-64 0.12-16 0.007-8 0.25-128 0.06-8 0.5-128 0.25-128
MIC90 1 64 16 32 32 8 4 64 8 128 128
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while gentamycin had lowest values at 0.12 and 1 mg/ml, respectively. Resistance to antimicrobial agents was commonly detected among sixty eight C. jejuni isolates. Resistance to four quinolones including ciprofloxacin, marbofloxacin, nalidixic acid and norfloxacin was found at 58.82, 25.00, 69.12 and 69.12%, respectively. The high prevalence of
Erythromycin
100 100
quinolone resistant C. jejuni isolates was similar to the finding of Engberg et al. (2001). However, the prevalence of marbofloxacin resistant isolates of C. jejuni in this study was relatively low (25.00%). The reason was possibly due to marbofloxacin is not commonly used as the other drugs in this group. Endtz et al. (1991) reported that certain isolates of C. jejuni were cross-resistant to various
Sulfamethoxazole
100
Kanamycin
% isolates
% isolates
80 60 40 20 0 0.03 0.25 2 16 128
80 60 40 20 0 0.03 0.25 2 16 128
% isolates
80 60 40 20 0 0.03 0.25 2 16 128
MIC (ug/ml) Ampicillin

100 100
MIC (ug/ml) Tetracycline

100
MIC (ug/ml) Nalidixic acid
% isolates
80 60 40 20 0 0.03 0.25 2 16 128
% isolates
% isolates
0.25 2 16 128
80 60 40 20 0 0.03
80 60 40 20 0 0.03 0.25 2 16 128
MIC (ug/ml) Streptomycin

100 100
MIC (ug/ml) Ciprofloxacin

100
MIC (ug/ml) Marbofloxacin
% isolates
% isolates
80 60 40 20 0 0.03 0.25 2 16 128
80 60 40 20 0 0.03 0.25 2 16 128
% isolates
80 60 40 20 0 0.01 0.06 0.5 4 32
MIC (ug/ml) Gentamycin

100 100
MIC (ug/ml) Norfloxacin
MIC (ug/ml)
% isolates
80 60 40 20 0 0.03 0.25 2 16 128
% isolates
80 60 40 20 0 0.03 0.25 2 16 128
MIC (ug/ml)
MIC (ug/ml)
Figure 1 Distribution of MICs of 11 antimicrobial agents against sixty-eight isolates of C. jejuni from chicken. Arrow indicated break point of resistance.
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quinolones. Most nalidixic acid-resistant isolates have been reported to be cross-resistant to one, two or three drugs in this group (Reina et al., 1995). In Thailand, prevalence of quinolone resistant Campylobacter species in broiler farms was found to increase from 0% in 1987 to 84% in 1995 (Hoge et al., 1998). In addition, Daniel et al. (2002) found that C. jejuni isolated from Thai population was resistant to ciprofloxacin in high proportion (77%). Quinolone-resistant Campylobacter isolates in human and poultry meat were also found in other countries, including Canada (Gaunt and Piddock, 1996), Spain (Prats et al., 2000), Senegal (Cardinale, 2003) and Taiwan (Engberg et al., 2001). A fluoroquinolone-resistant C. jejuni infection has also been associated with foreign travel to many countries (Cardinale, 2003). It was reported that most clinical isolates of C. jejuni from U.S. troops in Thailand were resistant to ciprofloxacin (Murphy et al., 1996). Regarding the proportion of aminoglycoside resistance, the percentage of isolate resistance in kanamycin was 51.47%, gentamycin was 16.18% and streptomycin was 42.65%. There was a report previously indicated that aminoglycosides resistance was less common in C. jejuni (Trieber and Taylor, 2000). Campylobacter species isolated from humans, pigs, cattle, and broilers were very low in resistance to streptomycin (1%) (Fallon et al., 2003). Moreover, it was demonstrated that there was no Campylobacter from chicken isolates
that was resistant to aminoglycosides (Cabrita et al., 1992). The contrary of aminoglycosides resistance of C. jejuni from this work to the others was possibly due to the administration of these antimicrobial agents in veterinary medicine under certain conditions in Thailand. Therefore, resistances of these agents were found to increase in certain isolates of C. jejuni. In this study, the high proportions of C. jejuni isolates were also resistant to other antimicrobial agents including ampicillin (47.06%), sulfamethozaxole (72.06%) and tetracycline (77.94%). These results are in accordance with the previous report that resistances to ampicillin, chloramphenicol, sulfamethozaxole and tetracycline of C. jejuni were commonly found in Thailand (Daniel et al., 2002). Low resistant level of erythromycin and gentamycin was found in 9 and 11 isolates of C. jejuni, respectively. However, MIC values of these agents were different (Table 2). Since gentamycin showed a very low MIC90, it seems reasonable to consider this drug as an alternative for treatment of C. jejuni infections under certain conditions in Nakhon Pathom province, Thailand. Therefore, it is necessary to select the antimicrobial wisely for its effectiveness. Resistance of C. jejuni isolates to a number of antimicrobial agents is presented in Table 3. Antimicrobial resistance (4 to 8 antimicrobial agents) was detected in sixty-six isolates (97.06%). Resistance to four or more of the drugs tested was
Table 3 Multi-antimicrobial resistant profiles of C. jejuni. Number of antimicrobial agents 0 4 5 6 7 8 Number of resistance isolates (%) 2 (2.94%) 10 (14.70%) 19 (27.94%) 26 (38.24%) 9 (13.24%) 2 (2.94%)
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defined as multiple resistances. Only two isolates of C. jejuni (2.94%) were susceptible to all antimicrobial agents. All of multiple resistance of C. jejuni was consisted of quinolone resistance. One common resistance of this group was nalidixic acid. Most C. jejuni strains were resistant to multi-antimicrobial agents tested. This was possibly due to some development by genetic change or physiological adaptation of the organisms to increase antimicrobial tolerances in consequence of previous exposure to antimicrobials. Therefore, discontinuing the practice of routinely adding growth promoters to animal feeds would reduce the resistant strains of C. jejuni in animals in Thailand. Long-term surveillance data are needed to further evaluate the impact of any intervention in antimicrobial usages. Moreover, antimicrobial susceptibility testing methods for Campylobacter species are needed to be harmonized and standardized for predication that bacteria will respond to treatment from an appropriate agent. CONCLUSION Quinoloneresistant Campylobactor is a concerning issue in human medicine because of failure in treatment of diarrhea cases with quinolone. Scientists are monitoring the import meat products to protect the consumers from this organism. Most, C. jejuni of Thaiisolates have shown high proportion of multiple resistance to many kinds of antimicrobial agent used in the test including quinolone. Erythromycin and gentamycin are alternative drugs for treatment of these cases. Good farming practice should be implemented to reduce the usage of antimicrobials. ACKNOWLEDGEMENTS This work was supported by funding from Center for Agricultural Biotechnology, Kasetsart University, Kamphaengsean Campus. Gratitude
is expressed to Srisamai Viriyarampa for her advices in microbiological techniques. We also acknowledge the Faculty of Veterinary Medicine, Kasetsart University for equipment supporting. LITERATURE CITED Aquino, M.H.C., A.L.L. Filgueiras, M.C.S. Ferreira and S.S. Oliveinia. 2002. Antimicrobial resistance and plasmid profiles of C. jejuni and C. coli from human and animal sources. Lett. Appl. Microbiol. 34: 149-153. Cabrita, J.J., J. Rodrigues, F. Bragance, C. Morgado and A. Goncalves. 1992. Prevalence biotypes, plasmid profiles and antimicrobial resistance of Campylobacter isolated from wild and domestic animals from Northeast Portugal. J. Appl. Bacteriol. 73: 279-285. Cardinale, E.J., A. Dromigny, F. Tall, M. Ndiaye, M. Konte and J.D. Perrier. 2003. Fluoroquinolone susceptibility of Campylobacter strains, Senegal. Antimicrob. Agents Chemother. 58: 178-190. Daniel, W.I., C.W. Hoge, P. Chittima, L. Bodhidatta and K.W. Hickey. 2002. Competitive antibiotic resistance of diarrheal pathogens from Vietnam and Thailand, 1996-99. Emerg. Infect. Dis. 8: 175-201. Endtz, H.P., G.J. Rujis, B. Klingeren, W. H. Jansen and. R.P.Mouton. 1991. Quinolone resistance in Campylobacter isolated from man and poultry following the introduction of fluoroquinolone in veterinary medicine. J. Antimicrob. Chemother. 27: 199-208. Engberg, J., F.M. Aurestrup, D.E. Taylor and I. Nachamkin. 2001. Quinolone and macrolide resistance in C. jejuni and C. coli resistance mechanisms and trend in human isolates. Emerg. Infect. Dis. 7: 24-34. EUCAST. 2000. Terminology relating to methods for the determination of susceptibility of bacteria to antimicrobial agents. Clin. Microbiol. Infect. 6: 503-508.
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Fallon, R., N. Sullivan, M. Maher and C. Carroll. 2003. Antimicrobial resistance of C. jejuni and C. coli isolates from broiler chickens isolated at an Irish poultry processing plant. Lett. Appl. Microbiol. 36: 277-281. Gaunt, P.N. and L.J.V. Piddock. 1996. Ciprofloxacin resistant Campylobacter spp. In humans: an epidemiological and laboratory study. J. Antimicrob. Chemother. 37: 747-757. Hoge, C.W., J.M. Gambel, C. Pitarangsri and P. Echeverria. 1998. Trends in antibiotic resistance diarrhea pathogens isolated in Thailand over 15 years. Clin. Infect. Dis. 26: 341-345. Luber, P., E. Bartelt, E. Genschow, J. Wagner and H. Hahn. 2003. Comparison of broth microdilution, E Test, and agar dilution methods for antibiotic susceptibility testing of Campylobacter jejuni and Campylobacter coli. J. Clin. Microbiol. 41: 1062-8. Murphy, G., P. Echeverria, L. Jackon, M. Arness and C. Leboon. 1996. Ciprofloxacin and azithromycin resistant Campylobacter causing travelers s diarrhea in US troops deployed to Thai in 1994. Clin. Infect. Dis. 22: 864-869. Piddock, L.J.V., V. Ricci, K. Stanley and K.Jones. 2000. Activity of antibiotics used in human medicine for C. jejuni isolated from farm animals and their environment in Lancashire, U.K. J. Antimicrob. Chemother. 46: 303306.
Prats, G., B. Mirelis, T. Lovet, C. Munoz, E. Miro and F. Navarro. 2000. Antibiotic resistance trends in enteropathogenic bacteria isolated in 1985-1987 and 1995-1998 in Barcelona. Antimicrob. Agents Chemother. 44: 11401145. Rautelin, H., A. Vierikko, M.L. Hanninen and M. Vaara. 2003. Antimicrobial susceptibility of Campylobacter strains isolated from Finnish subjects infected domestically or from those infected abroad. Antimicrob. Agents Chemother. 47: 102-105. Reina, J., M.J. Ros and V. Fernandez-Baca. 1995. Resistance to erythromycin in fluoroquinolone-resistant Campylobacter jejuni strains isolated from human feces. J. Antimicrob. Chemother. 35: 351-352. Saenz, Y., M. Zarazaga, M. Lantero and F. Baquero. 2000. Antibiotic resistance in Campylobacter strains isolated from animals, foods, and human in Spain in 1997-1998. Antimicrob. Agents Chemother. 44: 267-271. Stucki, U., J. Frey, J. Nicolet and A.P. Burnens. 1995. Identification of Campylobacter jejuni on the basis of a species-specific gene that encodes a membrane protein. J. Clin. Microbiol. 33: 855859. Trieber, C.A. and D.E.Taylor. 2000. Mechanisms of antibiotic resistance in Campylobacter. Am. Soc. Microbiol. 54: 441-454.
Hematology, Cytochemistry and Ultrastructure of Blood Cells in Asiatic Black Bear (Ursus thibetanus)
Chaleow Salakij1, Jarernsak Salakij1, Nual-Anong Narkkong2, Ludda Trongwonsa3 and Rattapan Pattanarangsan4
ABSTRACT Blood cells from adult Asiatic black bear (Ursus thibetanus) were examined and measured after stainning with modified Wright stain and cytochemical stains, including Sudan Black B (SBB), Periodic acid Schiffs reaction (PAS), a-naphthyl acetate esterase (ANAE), acid phospatase (AcP) and bglucuronidase (b-glu). Red blood cells were uniform in shape, with 7.3 mm mean diameter in size and easy to form rouleaux. Using scanning electron microscopic (SEM) examinations revealed normal and abnormal red blood cell surfaces. Neutrophils contained several vacuoles as detected by light microscope and revealed themselves as large granules under transmission electron microscope. Neutrophils stained strongly positive with SBB, ANAE; weak positive with PAS and negative with AcP and b-glu. Using SEM, neutrophil surfaces revealed several microvilli and some micropores. Eosinophils contained numerous small round red refractive granules with some vacuoles. Eosinophils stained strongly positive with SBB and ANAE but negative with PAS and b-glu. Basophils had variable numbers of intense basophilic granules that obscured the very long lobulated nucleus. Basophils stained moderately positive with SBB but strongly positive with ANAE. Lymphocytes were negative with SBB but have 3 patterns of reactivity with ANAE, AcP and b-glu, including negative, focal dot and fine granular stainings. Monocytes stained moderately with SBB and moderately to strongly with for ANAE and b-glu. The SEM examinations could differentiate white blood cells by their surface contours. Transmission electron microscopic examinations revealed organelles within all blood cells. Key words: Asiatic black bear, blood cell, cytochemistry, morphology, ultrastucture INTRODUCTION Asiatic black bear (Ursus thibetanus) has shaggy black fur with white crescent on the chest, considerably larger than Malayan sun bear (Ursus malayanus). Asiatic black bear has suffered from habitat loss and is now rare in many areas (Francis, 2001). So this endangered species has been studied
1 2 3 4
intensively to determine the health status of the individuals. Veterinary hematology serves as a screening procedure to assess general health, the bodys ability to fight infection in adjunct to patient evaluation or diagnosis (Jain, 1993). Differential white blood cell count is very useful not only in numbering the white blood cells but also provide evidence of anemic condition or
Faculty of Veterinary Medicine, Kasetsart University, Kamphaengsaen, Nakorn Pathom 73140, Thailand. Central Instruments Center, Faculty of Science, Mahasarakarm University, Mahasarskarm 44150, Thailand. National Institute of Animal Health, Kasetklang, Jatujuk, Bangkok 10900, Thailand. Faculty of Veterinary Science, Mahidol University, Salaya, Nakorn Pathom 73170, Thailand.
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reveal the pathogenesis. Blood smear examinations provide more information on morphology of red blood cell, white blood cell and platelets (Mills, 1998). Cytochemical method is useful in diagnosis of acute leukemia in human (Apibal, 1987; Khemtonglang et al., 1997). The purpose of the present study was to characterize the morphology, cytochemical reaction and ultrastrucrure of blood cells in Asiatic black bear. MATERIALS AND METHODS From February to June 2003, five clinically healthy Asiatic black bears in Khao Kheaw Open Zoo were chemical restrained with xylaxine (Rompun) and ketamine. Two millilitres of blood samples were collected from the jugular vein and transferred to tubes containing ethylenediamine tetraacetic acid (EDTA). Some of the blood without anticoagulant was directly smeared on the slides. Two Asiatic black bears were adult males and three were adult females aging between 3-5 years old. Hematology, plasma protein and fibrinogen were determined by manual technique (Schalm et al., 1975) within two hours after blood collection. Two direct blood smears from each bear were stained with a modified Wright and Wrights stains. A minimum of 200 leukocytes were counted for differential leukocyte determinations. The anticoagulated blood was used for reticulocyte count by staining with new methylene blue stain (Schalm et al., 1975). The percentage of reticulocyte presented in 1,000 red blood cells (RBC) was determined. For each hematologic parameter, means, variances and standard errors were calculated using SPSS for Window (Norusis, 1993). Cytochemical staining characteristics of blood cells were evaluated using air-dried blood smears from three Asiatic black bears. Cells were stained with periodic acid Schiffs reaction (PAS), Sudan black B (SBB), anaphthyl acetate esterase (ANAE), acid phosphatase (AcP) and b
glucuronidase (bglu). Cytochemical procedures used were the same as those previously described (Salakij et al., 2002). Positive and negativestained cells were differentiated by counting 500 cells on each of the cytochemically stained smears. For scanning electron microscopy (SEM) and transmission electron microscopy (TEM), blood cells from three Asiatic black bears were processed as described by Salakij et al. (2002). Identification of blood cells by SEM and TEM was based on the relative number, size, shape and distribution of granules and on nuclear appearance. RESULTS There was no blood parasite detected in all bears. Hematological data of Asiatic black bear was tabulated (Table 1). White blood cell differential counts were shown in Table 2. Blood cell diameters were observed and calculated (Table 3). Cytochemical staining patterns of blood cells were summarized (Table 4). The morphology under light microscope, SEM, TEM and cytochemical characteristics of individual blood cells were evaluated, as described below. Erythrocytes Red blood cells (RBCs) or erythrocytes under light microscope showed uniform in shape (Figure 1), slightly biconcave and slightly central pallor as observed by SEM (Figure 4a, 4b) with 7.3 mm mean diameter in size (Table 3) and easy to forming rouleaux. Some defected RBCs (Figure 4c), crenated RBCs (Figure 4d), echinocyte (Figure 5a, 5b), rubricyte (Figure 8b) and metarubricyte (Figure 8c) were also observed. Mature red blood cells were negative for all cytochemical stainings. Ultrastructurally, mature RBC showed only hemoglobin (Figure 8a) while the metarubricytes showed some organelles (Figure 8c) and the rubricytes showed more mitochondria (Figure 8b).
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Table 1 Hematology of Asiatic black bear. Hematology PCV (%) Hemoglobin (g/dL) RBC (1012/L) MCV (fL) MCHC (g/dL) WBC (1011/L) Band neutrophils (109/L) Segmented neutrophils (109/L) Lymphocytes (109/L) Monocytes (109/L) Eosinophils (109/L) Basophils (109/L) Band neutrophils (%) Segmented neutrophils (%) Lymphocytes (%) Monocytes (%) Eosinophils (%) Basophils (%) Plasma protein (g/dL) Fibrinogen (mg/dL) Reticulocyte (%) Asiatic black bear (n = 5) 41.4 1.8 13.3 0.6 6.08 0.39 68.5 2.1 32.1 0.6 6.99 0.59 0.11 0.09 4.15 0.19 1.84 0.28 0.13 0.04 0.73 0.18 0.03 0.02 1.7 1.4 60.3 3.4 25.8 1.7 1.8 0.5 10.0 1.5 0.4 0.2 8.1 0.3 220 49 0.0 0.0
Platelets Bear platelets were approximately 1/5 to 1/ 2 of RBC and had prominent reddish-purple granules which were easily seen in modified Wright stain (Figure 1d, f). Plateletes were not stained with SBB but were moderately to strongly positive with ANAE (Figure 3m). These platelets were seldom seen on RBCs (Figure 5b), but gave rosette formation on monocyte (Figure 5c) and aggregation (Figure 5d). Ultrastructurally, platelets showed dense granules, alpha-granules, glycogen granules and microtubule (Figure 8d). Neutrophils Neutrophils were the most prevalent leukocyte in Asiatic black bear (Table 2) with neutrophil : lymphocyte ratio equal to 62 : 26.
Table 2 White blood cell differential count in Asiatic black bear. Hematology Asiatic black bear (n = 5) 1.7 1.4 60.3 3.4 25.8 1.7 1.8 0.5 10.0 1.5 0.4 0.2
Band neutrophils (%) Segmented neutrophils (%) Lymphocytes (%) Monocytes (%) Eosinophils (%) Basophils (%)
With modified Wright stain, neutrophils showed the same size as basophil (Table 3). Neutrophils showed faintly stained cytoplasm which contained indistinct pale granules and several vacuoles
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Table 3 Mean SD of blood cell diameters (mm) in Asiatic black bear. Cell type Red blood cells Segmented Neutrophils Eosinophils Basophils Lymphocytes small medium large Monocytes No. 50 50 50 18 50 50 17 50 Asiatic black bear 7.30 0.84 13.42 1.10 14.09 1.66 13.06 1.95 8.28 0.78 11.02 1.11 14.40 0.48 15.28 2.14
Table 4 Cytochemical staining patterns of blood cells from Asiatic black bear.
Cell type Neutrophils Eosinophils Basophils Lymphocytes Monocytes Platelets SBB ++ + ++ + PAS ANAE Acid phosphatase b-glucuronidase
+ ++ +++ + ++ - - / focal dot / fine granular - / focal dot / fine granular - / focal dot / fine granular ++ + ++ -
SBB indicates sudan black B; PAS, periodic acid-Schiff; and ANAE, a-naphthyl acetate esterase. Staining was scored as negative (-), weak (, few positive cells), moderate (+), moderate to strong (++), or strong (+++).
(Figure 1a, 2a). Neutrophils have tight constricted and multilobulated nuclei (Figure 1a, 2a). These vacuoles shown in light microscope were revealed as large granules in TEM (Figure 9a-d). Some neutrophils (2-5%) of the female revealed sex chromatin lobe. Neutrophils stained strongly positive with SBB (Figure 3a), moderately stained with ANAE (Figure 3i) and negative with b-glu (Figure 3o). Using SEM, neutrophil surfaces revealed several microvilli and some micropores (Figure 6a, 6b, 6d). Ultrastructurally, neutrophils showed lobed nuclei, small specific granules (Figure 11a, b), large electron-dense granules (Figure 9a-c) and glycogen granules (Figure 9d) were also detected.
Eosinophils Eosinophils varied from 10 to 16 mm (average 14 mm) in diameter (Table 3). Eosinophils contained numerous small round red refractive granules with some vacuoles (Figure 1b, 2b). Eosinopil nuclei were less lobulated than those of neutrophils, and tetralobed, trilobed or bandshaped. Eosinophils stained moderately positive with SBB (Figure 3b) and ANAE (Figure 3j) but weak positive with b-glu (Table 4). Under SEM, eosinophil surfaces revealed larger granule contour (Figure 6c, 6d) than those of basophils (Figure 6e, 6f). Ultrastructurally, eosinophils showed lobed nuclei, large pleomorphic granules with bar-shape structures in some granules, Golgi apparatus, RER and ribosomes (Figure 10a, 10b).
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Figure 1 Light micrographs of blood cells in Asiatic black bear stained with modified Wright stain. a. A segmented neutrophil with many cytoplasmic vacuoles. b. An eosinophil. c. A basophil (left) and a segmented neutrophil. d. A lymphocyte (right) and a segmented neutrophil. e. A monocyte. f. A basophil (right) and a segmented neutrophil.
Figure 2 Light micrographs of blood cells in Asiatic black bear stained with Wrights stain (a-d). a. A segmented neutrophil with some cytoplasmic vacuoles. b. An eosinophil. c. A basophil. d. A lymphocyte (right) and a basophil. e. A leukocyte stained with new methylene blue whilst there was no reticulocyte.
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Figure 3 Cytochemical staining of blood cells in Asiatic black bear a. Sudan black-B (SBB) positive segmented neutrophil. b. SBB positive in the periphery of the granules of the eosinophil. c. SBB positive in basophil. d. Some small dots of SBB positive in a 14 mm monocyte. e. PAS positive in segmented neutrophil. f. PAS positive in two segmented neutrophils and negative in an eosinophil. g. PAS negative in a basophil comparing with PAS positive in two segmented neutrophils. h. PAS negative in lymphocyte (lower right) and monocyte (upper left). i.-m. ANAE reactivity in a segmented neutrophil (i), eosinophil (j), basophil (right) (k), monocyte (upper right) (l) and in platelets (m). n. Negative AcP in a segmented neutrophil and a monocyte. o. Negative b-glu in a segmented neutrophil and a basophil. p. Focal dot positive of b-glu in a lymphocyte.
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Figure 4 Scanning electron micrographs (SEM) of red blood cells (RBCs) in Asiatic black bear. a. A cluster of RBCs showing bicocave disk and crenations. b. Higer magnification of crenated RBCs. c. Defective RBCs. d. A cluster of four echinocytes.
Figure 5 SEM of RBCs and platelets in Asiatic black bear. a. An echinocyte. b. A platelet on an echinocyte. c. Plateletes rosetting on a monocyte. d. Platelets aggregation next to neutrophil.
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Figure 6 SEM of granulocytels in Asiatic black bear. a. A neutrophil showing short microvilli and some micropores. b. A neutrophil with three micropores. c. An eosinophil. d. An eosinophil (right) and a monocyte (left). e. A basophil showing small granule contour. f. A basophil.
Figure 7 SEM of agranulocytes in Asiatic black bear. a. A lymphocyte with several cytoplasmic blebs. b. A monocyte with deep surface fissures.
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Figure 8 Transmission electron micrographs (TEM) of blood cells in Asiatic black bear. a. Mature erythrocytes. b. A rubricyte with some mitochondria (arrows). c. A metarubricyte. d. Platelets containing mitochondria, vacuoles (v) and dense granules (*).
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Figure 9 TEM of neutrophils in Asiatic black bear. a. A segmented neutrophil showing nucleus (N) many fine granules and large granules (L). b. A segmented neutrophil showing two lobed neucleus (N) with heterogeneous granule density of large granules (L). c. A segmented neutrophil (N) with homogeneous granule density of large granules (L). d. A segmented neutrophil (N) with many glycogen granules (arrows).
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Figure 10 TEM of eosinophils and basophils in Asiatic black bear. a. An eosinophil showing neucleus (N) and many granules. b. Higher magnification of eosinophil in (a) showing bar-shape structures (arrows) in their granules. c., d. Basophils showing bilobed neuclei (N) and many small heterogenous granules.
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Figure 11 TEM of lymphocytes and monocytes in Asiatic black bear. a. A lymphocyte showing round neucleus (N) and three mitochondria (arrows). b. A lymphocyte with round nucleus (N). c. A monocyte with kidney-shaped nucleus (N). d. A monocyte with nucleus (N), many mitochondria (MT), ribosomes, vacuoles (v) and pseudopodia (arrows).
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Basophils Basophils in were not frequently observed. They varied from 11 to 15 mm (average 13 mm). Basophils contained variable numbers of intensely basophilic granules (Figure 1c, 1f, 2c, 2d) which obscured the very long lobe nucleus. Basophil granules stained with SBB more faintly than granules of neutrophil (Figure 3c). Basophil granules appeared red-brown when stained with ANAE (Figure 3k) but weak positive or negative with b-glu (Figure 3o). Under SEM, basophil surfaces revealed smaller granule contour (Figure 6e, 6f) than those of eosinophils (Figure 6c, 6d). Ultrastructurally, basophils showed lobed nuclei, small heterogenous electron density granules, some dense granules (Figure 10c) and some mitochondria (Figure 10d). Lymphocytes Lymphocytes in bears were variable in size (7 to 16 mm diameter). Most lymphocytes were small (Figure 1d, 2d) and medium size. Some lymphocytes contained small azurophilic granules in their cytoplasm. Lymphocytes were negative for SBB but had 3 patterns of reactivity for ANAE and b-glu, including negative, focal dot and fine granular stainings. Under SEM, lymphocyte surfaces revealed smooth bulging contour of round nuclei (Figure 7a) with variable numbers of cell membrane blebs. Ultrastructurally, lymphocytes showed round nuclei with peripheral clumps of heterochromatin and some mitochodria (Figure 11a, 11b). Monocytes Monocytes in bears varied from 13 to 17 mm in diameter (Table 4). They are the largest white blood cells with variable shape. The nuclei were extremely variable but usually have lacy chromatin (Figure 1e). The cytoplasm was bluegray and contained variable size of vacuoles (Figure 1e). Monocytes stained moderately positive with SBB with faintly black, small granules scattered in
the cytoplasm (Figure 3d). They were moderately to strongly positive with ANAE showing redbrown fine granular pattern (Figure 3l), but they were negative for PAS (Figure 3h) and b-glu. Under SEM, monocyte surfaces revealed more smooth membrane than those of neutrophil (Figure 7b) with deep fissure and micropores. Ultrastructurally, monocytes showed variable shape of nuclei with several mitochodria and pseudopodia (Figure 11c, 11d). DISCUSSION In this report, we described the light microscope, cytochemical features and ultrastructure of blood cells in Asiatic black bear. Although the erythrocytes in bears has special features such as uniform in shape with central pallor similar to those of dog, they are larger in mean diameter (Jain, 1993) and easy to form rouleaux. The RBC parameters of Asiatic black bear in this study was similar to but had lower number of leukocytes than those of Asiatic black bear in the zoo of Czechoslovakia (Pospisil et al., 1987). There was no blood parasite found in Asiatic black bear of this study while there was a report of filarid worm (Dirofilaria ursi) found in the esophageal and tracheal connective tissue of the male Asiatic black bear on Kyushu island (Yokohata et al., 1990). Ultrastructure of platelets in Asiatic black bear were similar to those in bovine (Fern, 2000) and African elephant (Du Plessis and Stevens, 2002). Platelets in Asiatic black bear were negative for SBB which were the same as those in bovine, cat, dog, horse and green sea turtle (Raskin and Valenciano, 2000). Platelets in Asiatic black bear positively stained with ANAE which were similar to those of dog that were also positively stained with non-specific esterase (Raskin and Valenciano, 2000). And they were similar to platelets in Asian wild dog that were positive with ANAE and b-glu (Salakij et al., 2000). So the ANAE stain would be
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useful to differentiate megakaryocytic leukemia in the bears when using with the other non-specific esterase stains like those in human (Apibal, 1987). The neutrophils in Asiatic black bear contained large granules that were unstained similar to vacuoles under light microscopy that were characteristic of neutrophils in Asiatic black bear. These vacuoles were large and variable in electron density as shown by TEM. This characteristic was not found in goat (Kramer, 2000) or in reindeer (Henkel et al., 1999). Characteristic features of basophils in Asiatic black bear were similar to those in dog; including having a long polymorphonuclearshaped nucleus that is longer than those of neutrophil and thinner than those of most monocyte (Willard et al., 1994). Basophils of Asiatic black bear are the same size as neutrophils but smaller than eosinophils (Table 2). These findings may be useful in differentiation of degranulated basophils from netrophils in Asiatic black bear. The strongest reactivity of ANAE was found in eosinophils which was different from eosinophils in Asian wild dogs that were negative for ANAE (Salakij et al., 2000). The bar-shape structures found in some granules of the eosinophil in Asiatic black bear by TEM were different from eosinophils of dog, cat (Young, 2000), goat (Kramer, 2000) and reindeer (Henkel et al., 1999). In the Asiatic black bear, granulocytes and monocytes stained with SBB. These findings are useful in differentiating acute myelogenous leukemia from acute lymphoblastic leukemia like those in human (Apibal, 1987). ANAE, AcP and b-glu staining characteristics of lymphocytes were similar to those reported in human that can differentiate T-lymphocytes (dot staining) from non T-lymphocytes (negative or fine granular staining) (Apibal, 1987; Khemtonglang et al., 1997). The basophils in Asiatic black bear were strongly positive with SBB and ANAE which is similar to reindeer basophils (Henkel et al., 1999).
The basophils in Asian wild dog were stained strongly positive with SBB, ANAE and b-glu (Salakij et al., 2000). So in the Asiatic black bear, the SBB and ANAE are useful in the diagnosis between basophilic leukemia and megakaryocytic leukemia. The ultrastructure of basophils in Asiatic black bear showed smaller granules than those in goat (Kramer, 2000) and in reindeer (Henkel et al., 1999). The ultrastructure of lymphocytes and monocytes in Asiatic black bear were not different from those in pig (Steffens III, 2000) and in reindeer (Henkel et al., 1999). Under SEM, the eosinophils revealed large granule contour which were easy to differentiate from basophils and neutrophils. This is the first report on the surfaces of blood cells in Asiatic black bear that could demonstrate abnormal shape of red blood cells and differentiation of white blood cells. CONCLUSIONS The neutrophils in Asiatic black bear contained large granules that were unstained and similar to vacuoles under light microscopy. This is characteristic of neutrophils in Asiatic black bear. The basophils in Asiatic black bear contained many small granules that not stained metachromatic with modified Wright stain so their nuclear outline were clearly defied. The results of this study provide more information on the morphology, cytochemical staining and ultrastructural characteristics of blood cells in Asiatic black bear. This information adds to our understanding of blood cells in healthy Asiatic black bears. ACKNOWLEDGEMENTS This research was supported in part by the Kasetsart University Research and Development Institute.
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LlTERATURE CITED Apibal, S. 1987. Laboratory diagnosis of acute leukemia. J. Med. Tech. Assoc. Thailand 15: 69-75. du Plessis, L. and K. Stevens. 2002. Blood platelets of the African elephant. J. Comp. Patho. 127: 208-210. Francis, C. M. 2001. A Photographic Guide to Mammals of Thailand & South-East Asia. ASIABOOKS. Bangkok. 127 p. Khemtonglang, N., C. Kitpetcharatana, B. Petsuriya, K. Rukseree and A. Sriinsut. 1997. Pattern of b-glucuronidase staining in acute leukemias. J. Med. Tech. Assoc. Thailand 25:66-78. Kramer, J. W. 2000. Normal hematology of cattle, sheep and goat, pp. 1075-1084. In B. F. Feldman, J. G. Zinkl and N. C. Jain (eds.). Schalms Veterinary Hematology 5thed. Lippincott Williams and Wilkins, Philadelphia. Henkel, K. A., C. L. Swenson, B. Richardson and R. Common. 1999. Morphology, cytochemical staining and ultrastructure characteristics of reindeer (Rangifer tarandus) leukocytes. Vet. Clin. Path. 28: 8-15. Jain, N.C. 1993. Essentials of Veterinary Hematology. Lea and Febiger. Philadelphia, 417 p. Mills, J. 1998. Interpreting blood smears (or What blood smears are trying to tell you !).Aust. Vet. J. 76 : 596-600. Norusis, M. J. 1993. SPSS for Window Base System Users Guide Release 6.0. SPSS Inc., Chicago, Illinois. 828 p. Pospisil, J., F. Kaset and J. Vahara. 1987. Basic
haematological values in carnivores-I. The Canidae, the Hyaenidae and the Ursidae. Comp. Bioche. Physio. 86: 649-652. Raskin, R. E. and A Valemciano. 2000. Cytochemistry of normal leukocytes. pp. 327346. In B. F. Feldman, J. G. Zinkl and N. C. Jain (eds.). Schalms Veterinary Hematology. 5 thed. Lippincott Williams and Wilkins, Philadelphia. Salakij, C., J. Salakij, J. Rattanakunuprakarn, N. Tengchaisri, W. Tunwattana and S. Apibal. 2000. Morphology and cytochemistry of blood cells from Asian wild dog (Cuon alpinus). Kasetsat J. (Nat. Sci.). 34:518-525. Salakij, C., J. Salakij, S. Apibal, N. Narkkong, L. Chanhome and N. Rochanapat. 2002. Hematology, morphology, cytochemical staining, and ultrastructural characteristics of blood cells in King cobras (Ophiophagus hannah). Vet. Clin. Path. 31 : 116-126. Schalm, O. W., N. C. Jain and D. J. Carrol. 1975. Schalms Veterinary Hematology. 3rded. Lea and Febiger. Philadelphia. 807 p. Steffens III WL. 2000. Ultrastructural features of leukocytes, pp. 326-336. In B. F. Feldman, J. G. Zinkl and N. C. Jain (eds.). Schalms Veterinary Hematology. 5thed. Lippincott Williams and Wilkins, Philadelphia. Willard, M. D., H. Tvedten and G. H. Turnwald. 1994. Small Animal Clinical Diagnosis by Laboratory Methods. 2nded. W. B. Saunders Company. Philadelphia. 377 p. Yokohata, Y., O., Fujita, M. Kamiya, T. Fujita, K. Kaneko and M. Ohbayashi. 1990. Parasites from the Asiatic black bear (Ursus thibetanus) on Kyushu island, Japan. Journal of Wildlife Diseases. 26: 137-138.
Probiotic Properties of Bacillus pumilus, Bacillus sphaericus and Bacillus subtilis in Black Tiger Shrimp (Penaeus monodon Fabricius) Culture
Watchariya Purivirojkul1, Monchan Maketon1 and Nontawith Areechon2
ABSTRACT Three species of the genus Bacillus namely B. pumilus,B. sphaericus, and B. subtilis were isolated from the intestine of Penaeus monodon and tested for possible potential as a probiotic in black tiger shrimp rearing. The competition, colonization and inhibition activities of Bacillus spp. on Vibrio harveyi, a known pathogen in black tiger shrimp aquaculture were conducted. Transmission electron microscope observations showed the size of V. harveyis cell colonized by B. pumilus, B. sphaericus and B. subtilis to be smaller compare with its normal cell. This morphological deviation was permanently changed in every generation of V. harveyi. In addition, three species of the Bacillus could be grown in a wild range of conditions, these include salinity between 0-8 % NaCl, pH from 4 to 11 and temperature ranging of 25-50 C except B. sphaericus which was not amenable to grow at 50 C. Therefore, B. pumilus, B. sphaericus and B. subtilis showed promising potential to be used as a probiotic in black tiger shrimp. As for probiotic properties in black tiger shrimp, the results shown that number of bacteria in intestinal tracts of the shrimps were increased in all treatments from 216 to 803% of the control group, and the amount of V. harveyi, were found reduced from 87.53 to 99.76% of the control, as also confirmed by scanning electron microscope observations. B. subtilis, the mixture of B. sphaericus + B. subtilis, and B. pumilus + B. sphaericus + B. subtilis in culture media showed immunostimulatory features measured by total hemocytes, phenol oxidase, superoxide anion, clearance ability and bactericidal activity which increased by 20.02-23.10, 26.02-39.43, 53.45-66.04, 44.68-59.57 and 50.00%, respectively. Key words: Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis, probiotic, Penaeus monodon
INTRODUCTION Black tiger shrimp (Penaeus monodon Fabricius) has been one of the important export products of Thailand for more than a decade. However, in 2002, Thai shrimp production fell about 40 percent from 2001 to approximately 160,000 tons due to several diseases outbreak at the beginning of the year. Farmers utilized a large
quantities of antibiotics in trying to solve this problem. Some antibiotic such as chloramphenicol was found as a residue in shrimps exported from China, Viet Nam, Indonesia and Thailand. Recently, the European communities are focusing in antibiotic residues in shrimp products by strictly inspection for all shrimp products imported from Asian countries. The use of probiotic bacteria and some
1 2
Department of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand. Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand.
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immunostimulant substances such as glucan and peptidoglycan have become popular methods developed for fighting againt diseases since the past decade (Fuller, 1992). Many genus of bacteria were used as probiotic such as Vibrio (Gullian et al., 2004) Bacillus spp. (Moriarty, 1998; Rengpipat et al., 2000; Gullian et al., 2004), especially those bacteria isolated from the intestine of Penaeus monodon (Rengpipat et al., 2000). There are several mechanisms of probiotic, these include production of inhibitory compounds, competition for chemicals or available energy, competition for adhesion sites, enhancement of the immune response and improvement of water quality (Verschuere et al., 2000) In our studies, we conducted both in vitro and in vivo experiments by focusing on competitive and inhibitive capabilities of bacteria in the genus Bacillus in term of probiotic against Vibrio harveyi. Moreover, we continued study these bacteria on their effectiveness as probiotic properties in P. monodon in terms of growth and immune response indicated by total hemocytes, phenoloxidase activity, superoxide anion, bactericidal activity and clearance ability. Changing of bacterial community in shrimp intestines when feed with Bacillus spp. were also investigated by counting the number of Bacillus spp. and Vibrio spp., scanning electron microscope (SEM) was used for confirmation. MATERIALS AND METHODS This research was performed in both in vitro and in vivo systems. Isolation and identification of Bacillus spp. Bacillus spp. were isolated from the intestine of Penaeus monodon harvested from shrimp farms in Chachoengsao province. Two hundred samples of shrimp were investigated. Intestine were dissolved in 5 ml of 1.5% NaCl per animal and heat shock on water bath at 80 C for
20 min followed by cold shock with normal tap water. Then the solution was spreaded on plates using spread plate technique on Nutrient agar (NA) supplemented with 1.5% NaCl (w/v). These plates were incubated at 35 C for 24 h. Isolates were purified by streaking on NA supplemented with 1.5% NaCl (w/v). Catalase test were used for identifying Bacillus species. Species identification were done by VITEK 32 Bacillus (Biomrieux). 1. Colonization and inhibition activities of three bacteria on Vibrio harveyi in vitro V. harveyi was isolated from black tiger shrimp and streaked on TCBS (Thiosulfate Citrate Bile Sucrose) agar. Bacillus spp. were cultured on nutrient agar supplemented with 1.5% NaCl (w/ v), and both were incubated at 35 C for 24 h. Colonization activities tests were done on NA supplemented with 1.5% NaCl (w/v) by cross streak method. V. harveyi was streaked in the first line and then Bacillus spp. was streaked perpendicular to it. Each type of bacterium streaking was done in triplicate and they were incubated at room temperature for 24 h. 1.1 Morphological change of V. harveyi after colonization V. harveyi was isolated from the colonization area, especially from the cross streaking point as well as from the control. All samples were cultured on TCBS agar and incubated at 35 C for 24 h. Growth rates of the experimental V. harveyi were compared with the normal one. Single colony was used to determine morphological deviation by transmission electron microscope (TEM) 1.2 Investigation for the possibility of V. harveyi to return to its normal shape All V. harveyi samples isolated from the cross streaking point were cultured on TCBS agar. These plates were incubated at 35 C for 24 h and morphological change was determined. The samples were then subcultured every 24 h for three consecutive times. TEM was used to observe the
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change of shapes during the subculture times for investigation o the recovery. 1.3 Bacillus spp. growth in different conditions Temperature, salinity and pH were tested for the effect on the growth of Bacillus spp. The bacteria were prepared on NA supplemented with 1.5% NaCl (w/v) and incubated at 35 C for 24 hours. For temperature study, a single colony was selected from the plate and streaked on the NA supplemented with 1.5% NaCl (w/v) and incubated at 25 C, 35 C and 50 C for 24 h. The single colony of Bacillus spp. from the plate was inoculated in NB supplemented with 1.5% NaCl (w/v). 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10% NaCl were added to the media for salinity study and the pH to 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 for pH study. Test materials were incubated at 35 C for 24 hours. 2. Probiotic and immunology properties of three bacteria against V. harveyi in vivo Penaeus monodon was obtained from shrimp farm in Chachoengsao Province, Thailand. Shrimp with a mean fresh weight of approximately 8-12 g per animal were used. They were acclimatised in an aerated aquarium system at 25 ppt and change water every week before testing. 2.1 Probiotic properties study Three strains of Bacillus, they were B. pumilus, B. sphaericus, B. subtilis. Each bacterium
was formulated at the concentration of 1011-1012 cfu/g powder using clay as a filter. The experiment was designed as a CRD with 8 treatments and 3 replications each as shown in Table 1. Each treatment was blended with the shrimps feed, at the ratio of 5 g : 1 kg. feed and then fed at 3% of the body weight, three times daily. The growth rate were observed at 4 weeks after feeding. Scanning electron microscope (SEM) was used to confirm the present of Bacillus in shrimp intestine. 2.2 Immunology study Preparation of hemolymph samples 0.5 ml of hemolymph from each sample was withdrawn from base of the third walking leg of the shrimp using a syrynge containing 1.5 ml anticoagulant (K-199 + 5% L-cysteine) at 4 weeks after feeding. 2.2.1 Total hemocytes After collected hemolymph, hemocytes were counted using a hemocytometer and calculated as number of blood cells (total hemocytes per cubic millimeter) 2.2.2 Phenoloxidase activity assay The method was modified from Supamattaya et al. (2000).After the blood was withdrawn, the hemocytes were washed three times with shrimp saline (1000 rpm. 4 C 10 min). Hemocyte lysate (HLS) was prepared from hemocytes in a cacodylate buffer pH 7.4 by using
Table 1 Eight treatments of probiotic properties study. Treatment 1 2 3 4 5 6 7 8 Species of Bacillus spp. B. pumilus B. sphaericus B. subtilis B. pumilus + B. sphaericus (1:1) B. pumilus + B. subtilis (1:1) B. sphaericus + B. subtilis (1:1) B. pumilus + B. sphaericus + B. subtilis (1:1:1) No Bacillus (control)
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the sonicator at 30 amplitute for 5 second and the suspension was then centrifuged at 10,000 rpm., 4C for 20 min. The supernatant was collected as HLS. Then 200 ml of trypsin 0.1% in cacodylate buffer was mixed to the 200 ml HLS followed by 200 ml of L-dihydroxyphenylalanine (L-DOPA) 4 mg/ml as the substrate. Enzyme activity was measured as the absorbance of dopachrome at 490 nm wavelength. Measurement of protein content in HLS was made by using the method of Lowry et al. (1951). The phenoloxidase activity was calculated as the increasing of optimum density (OD) per minute per mg of protein as : 1 unit of phenoloxidase = D OD490 / min/ mg protein 2.2.3 Superoxide anion (O2-) The method was modified from Supamattaya et al. (2000). The O2- was detected by reduction of redox dye, nitroblue tetrazolium (NBT). By this method, hemocytes were washed 3 times in K-199 solution. Living cells were separated by using trypan blue solution and adjusted to 1107 cell/ml suspension. 200 ml of the cell suspension of each sample was dropped into well of a 96 microwell sterile plate. The plate was left for 45 min at room temperature for incubation period. Unattached cells were washed out by K199 solution. 100 ml of the reaction mixture (0.5 mg zymozan in 0.5 ml serum + 20 mg NBT in 1 ml DMSO + K-199) was added to each well and the reaction mixture incubated at 25 C 60 min. NBT was reduced by O2- during incubation period into a water insoluable blue formozan. The reaction was inhibited by putting 70% methanol into the samples for 3 min and the samples were then allowed to air dry. 120 ml of 2M NaOH and 140 ml of dimethyl sulfoxide (DMSO) were added to each well in order to dissolve the formozan. The concentration of the prussian-blue-colored solution was measured at 620 nm, KOH/DMSO was used as a blank control. The amount of O2- was indicated by the increasing in absorbance at 620 nm of 0.001 from control.
2.2.4 Clearance ability The method of study the clearance ability was modified from Martin et al. (1993). The bacterial pathogen V. harveyi was subcultured in TSA (Tryptic Soy Agar) with 1.5% NaCl and incubated at 35C for 24 hours. A single colony of V. harveyi was solved in the 1.5% NaCl sterile water. The suspension with the OD of 0.13 (2.45 108 cfu/ml) measured by absorbance value at 640 nm was used to count for the number of bacteria after cultured on TCBS agar by spread plate technique. Then 0.1 ml of the bacterial suspension with the counted number was injected to each tested shrimp while the control was injected with saline water. Three hours after injections, 0.5 ml of the blood from each shrimp was withdrawn for counting the number of bacteria by spread plate technique and calculated for the difference. 2.2.5 Bactericidal activity Serum was separated from hemolymph of each shrimp sample and diluted by 2.6% NaCl in the proportion of 1:2, 1:4, 1:8, 1:16 and 1:32 serum to NaCl. The 0.5 ml of each serum dilution and 0.5 ml of NaCl as the control were used in the study. 0.5 ml of bacterial suspension, V. harveyi, prepared from the method as in number 2.2.4 was put into each serum dilution including the control. The treatments were incubated at room temperature for 3 hours before counting the number of bacteria made by a spread plate technique. Recording of the results were made for the dilution that could decrease 50% V. harveyi compared to the control. 2.3 Study for the bacterial concentration in shrimps intestine The average concentration of both probiotic bacteria and V. harveyi were determined after 4 weeks of feeding. Scanning Electron Microscope (SEM) was used for confirmation the results. RESULTS Isolation and identification of Bacillus spp. Out of twenty isolates from shrimp
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intestines, there were only three species belong to the genus Bacillus and were identified as B. pumilus, B. sphaericus and B. subtilis. The percentage of each species were shown in Table 2 1. Study on the colonization and inhibition activities of three bacteria on Vibrio harveyi in vitro The results showed that only 12 hours after the tested, B. pumilus had inhibition effect against V. harveyi as shown in Figure 1.1. On the test plate, some clear zone area were existed and more colonization areas were observed after 24 hours as shown in Figure 1.2. On the other hand, the clear zone did not appear in the colonized plates of both B. sphaericus and B. subtilis. 1.1 Study on the morphological changed of V. harveyi after colonization The cross streaking point between V. harveyi and each colonizing Bacillus spp. on the TCBS agar was isolated for V. harveyi. Results showed that V. harveyi colonized by B. pumilus had slower growth compare with the control but the others two V. harveyi colonized by B. sphaericus and B. subtilis had normal growth with slightly change as shown by TEM (Figure 2) In addition, the cell of V. harveyi colonized by B. pumilus, was almost destroyed as shown in Figure 2.2, this might occurred from some metabolites produced from B. pumilus. The size of normal V. harveyi and those colonized by B. pumilus, B. sphaericus and B. subtilis were about 0.71 1.54 mm, 0.50 0.78 mm, 0.68 0.96 mm
and 0.68 1.07 mm as shown in Figure 2.1, 2.2, 2.3 and 2.5 respectively. 1.2 Investigation for the possibility of V. harveyi to return to its normal shape After three consecutive subcultures those deviated V. harveyi colonized from each bacterium on the TCBS agar at every 24 hours confirmed that their morphologies were permanently changed
Figure 1 Colonization activities of Bacillus spp. 3 species on V. harveyi in vitro. 1.1 inhibition effect of B. pumilus against Vibrio harveyi after 12 hours. 1.2 inhibition effect of B. pumilus against Vibrio harveyi after 24 hours. 1.3 colonization activities of B. sphaericus against Vibrio harveyi after 24 hours. 1.4 colonization activities of B. subtilis against Vibrio harveyi after 24 hours.
Table 2 The result of isolate Bacillus spp. from shrimp intestine by VITEK 32. Species of Bacillus spp. Bacillus pumilus Bacillus sphaericus Bacillus subtilis Non Bacillus species Total Number of isolated 1 2 3 14 20 Percent 5 10 15 70 100
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compare with the control. Thus, it might not possible for the colonized V. harveyi to return to its regular size and shape again as shown in Figure 2.3, 2.5 and 2.7 1.3 Bacillus spp. growth in different conditions Table 3 showed that B. pumilus and B. subtilis could grow at 25-50 C but B. sphaericus could not grow at 50 C. All three Bacillus species could grow in the salinity ranging between 0-10% and pH ranging from 4 to 11. 2. Study on the probiotic and immunology properties of three bacteria against V. harveyi in vivo 2.1 Probiotic properties study
Percent mean weight increasing of shrimps after 4 weeks fed by the mixture of B. sphaericus + B. subtilis (55.72 24.43) is significantly higher (P<0.05) than the single formulation of B. pumilus, B. sphaericus, B. subtilis and the mixture of B. pumilus + B. sphaericus, the mixture of B. pumilus + B. subtilis, the mixture of B. pumilus + B. sphaericus + B. subtilis and control which percent mean weight increasing were 9.99 5.71, 19.44 9.62, 24.17 23.46, 19.94 4.25, 13.63 7.27, 18.77 7.64 and 12.46 3.22, respectively as shown in Figure 3. 2.2 Immunology study 2.2.1 Total hemocytes Mean of total hemocytes from shrimp hemolymph after cultured with 8 feeds for 4 weeks
Figure 2 Morphological structure of normal V. harveyi compared with those deviated from colonization by TEM. 2.1 normal V. harveyi 2.2 V. harveyi colonized by B. pumilus 2.3 V. harveyi colonized by B. pumilus after three consecutive subcultures 2.4 V. harveyi colonized by B. sphaericus 2.5 V. harveyi colonized by B. sphaericus after three consecutive subcultures 2.6 V. harveyi colonized by B. subtilis 2.7 V. harveyi colonized by B. subtilis after three consecutive subcultures
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showed that B. subtilis (11.03 1.51 106), the mixture of B. sphaericus + B. subtilis (11.28 1.88 106), and the mixture of B. pumilus + B. sphaericus + B. subtilis (11.31 1.46 106) were significantly higher (P<0.05) than those fed with B. pumilus, B. sphaericus, the mixture of B. pumilus + B. sphaericus, the mixture of B. pumilus + B. subtilis and control which total hemocytes were 9.78 0.82, 10.02 1.35, 10.70 1.74, 10.41 1.65 106 and 9.19 0.81 106 cell/ml., respectively, as shown in Figure 4.
2.2.2 Phenoloxidase Mean of phenoloxidase from shrimp hemocytes after fed with B. subtilis (297.04 20.69), the mixture of B. pumilus + B. subtilis (307.56 15.31) and the mixture of B. pumilus + B. sphaericus + B. subtilis (310.58 29.58) for 4 weeks were significantly higher (P<0.05) than fed with B. pumilus, B. sphaericus, the mixture of B. pumilus + B. sphaericus, the mixture of B. sphaericus + B. subtilis and control which phenoloxidase were 278.53 56.47, 253.79
Table 3 Growth of Bacillus spp. in NB at 25, 35 and 50 C; NaCl 0-10% and pH ranging from 3-12. B. pumilus Temperature 25 C 35 C 50 C Salinity 0% NaCl 1% NaCl 2% NaCl 3% NaCl 4% NaCl 5% NaCl 6% NaCl 7% NaCl 8% NaCl 9% NaCl 10% NaCl pH 3 4 5 6 7 8 9 10 11 12 B. sphaericus B. subtilis
+ + + + + + + + + + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + + + + + + + -
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28.92, 234.52 26.77, 280.70 30.64 and 222.75 15.34, respectively, as shown in Figure 5. 2.2.3 Superoxide anion Mean of superoxide anion from shrimp hemocytes after fed with B. subtilis (8.13 2.16), the mixture of B. pumilus + B. sphaericus (8.33 1.66), the mixture of B. sphaericus + B. subtilis (8.40 1.41) and the mixture of B. pumilus + B. sphaericus + B. subtilis (8.80 1.31) for 4 weeks were significantly higher (P<0.05) from B. pumilus, B. sphaericus, the mixture of B. pumilus + B. subtilis and control which superoxide anion were 5.63 1.22, 4.60 1.35, 7.63 0.91 and 5.30 0.92, respectively, as shown in Figure 6. 2.2.4 Clearance ability Clearance ability of shrimp blood circulation system after fed with 8 feeds for 4 weeks and then injected with V. harveyi found that shrimp fed with B. subtilis (346.67 70.95), the
Weight increasing (%)
100 80 60 40 20 0 control 1 2 3 4 5 6
mixture of B. sphaericus + B. subtilis (253.33 122.20) and the mixture of B. pumilus + B. sphaericus + B. subtilis (320.00 98.49) could reduce V. harveyi in hemolymph significantly different (P<0.05) from shrimp fed with B. pumilus, B. sphaericus, the mixture of B. pumilus + B. sphaericus, the mixture of B. pumilus + B. subtilis and control which amount of V. harveyi cells were 456.67 85.05, 660.00 105.36, 483.33 201.08, 420.00 40.00 and 626.67 192.96 cfu/ml, respectively, as shown in Figure 7. 2.2.5 Bactericidal activity Bactericidal activity from shrimp serum at 4 weeks after fed with B. sphaericus, the mixture of B. pumilus + B. subtilis , the mixture of B. sphaericus + B. subtilis and the mixture of B. pumilus + B. sphaericus + B. subtilis (8.80 1.31) at the dilution ratio of 1:8 serum to brine could killed 50 percent of V. harveyi. While, shrimp fed
b b b b b b b feeds
7
1 = B.pumilus 2 = B.sphaericus 3 = B.subtilis, 4 = B.pumilus + B. sphaericus 5 = B.pumilus + B. subtilis 6 = B.sphaericus + B. subtilis 7 = B.pumilus + B. sphaericus + B.subtilis
Figure 3 Percent weight increase of the P. monodon after 4 weeks cultured with 8 feeds.
x 106 cells/ml
15
a
10
unit/min/mg.protein
a
400 a 300 b 200 100 a a b a a a
0 control 1 2 3 4 5 6 7
feeds
0 control 1 2 3 4 5 6 7
feeds
Figure 4 Average of the P. monodon hemocytes after 4 weeks cultured with 8 feeds.
Figure 5 Average of the P. monodon phenoloxidase after 4 weeks cultured with 8 feeds.
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with B. pumilus, B. subtilis, the mixture of B. pumilus + B. sphaericus and control killed 50 percent of V. harveyi at the dilution ratio of 1:4, as shown in Figure 8. 2.3 Study for the bacterial concentration in shrimps intestine 2.3.1 Bacillus spp. The number of Bacillus spp. in shrimp intestine after fed with B. pumilus (803.33 395.01), B. sphaericus (536.67 + 310.86), B. subtilis (359.00 270.63), the mixture of B. pumilus + B. sphaericus (373.33 156.31), the mixture of
unit 12 10 8 6 4 2 0 control 1 2 3 4 5 6 7 feeds cd bcd d
B. pumilus + B. subtilis (216.33 94.87), the mixture of B. sphaericus + B. subtilis (493.33 187.17) and the mixture of B. pumilus + B. sphaericus + B. subtilis (476.00 234.03) for 4 weeks were significantly different (P<0.05) from control which number of Bacillus spp. average was 1.00 1.73 104 cfu/g, as shown in Figure 9. 2.3.2 Vibrio spp. The number of Vibrio spp. in shrimp intestine at 4 weeks after fed with normal feed (control) (970.00 285.13 cfu/g) was significantly different (P<0.05) from shrimp fed with B. pumilus,
cfu/ml.
ab
a abc
1000
a
800 600 400 200 0
b a
b a a a a
feeds
control 1 2 3 4 5 6 7
Figure 6 Average of the P. monodon superoxide anion after 4 weeks cultured with 8 feeds.
Figure 7 Cells of V. harveyi from P. monodon hemolymph after infected with V. harveyi 3 hours after 4 weeks cultured with 8 feeds.
% of V. harveyi decreased
100
50
1 :2 1 :4 1 :8 1 :16 1 :32 1 :2 1 :4 1 :8 1 :16 1 :32 1 :2 1 :4 1 :8 1 :16 1 :32 1 :2 1 :4 1 :8 1 :16 1 :32 1 :2 1 :4 1 :8 1 :16 1 :32 1 :2 1 :4 1 :8 1 :16 1 :32 1 :2 1 :4 1 :8 1 :16 1 :32 1 :2 1 :4 1 :8 1 :16 1 :32
control
feeds
Figure 8 Value of diluted serum to kill V. harveyi 50% (*) of P. monodon after 4 weeks of culture when provided with 8 feeds (1 = B. pumilus, 2 = B. sphaericus, 3 = B. subtilis, 4 = B. pumilus + B. sphaericus, 5 = B. pumilus + B. subtilis, 6 = B. sphaericus + B. subtilis, 7 = B. pumilus + B. sphaericus + B. subtilis).
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B. sphaericus, B. subtilis, the mixture of B. pumilus + B. sphaericus, the mixture of B. pumilus + B. subtilis, the mixture of B. sphaericus + B. subtilis and the mixture of B. pumilus + B. sphaericus + B. subtilis which the number of Vibrio spp. in shrimp intestine were 11.33 1.16, 121.67 11.50, 8.00 1.00, 26.00 3.00, 32.00 3.61, 10.00 3.00, 2.33 1.53 cfu/g, respectively, as shown in Figure 10. 2.3.3 Scanning electron microscope SEM revealed that Bacillus spp. could survived in shrimp intestine after treated with either individual isolation or mixtures of two and three of B. pumilus, B. sphaericus and B. subtilis
x 10 cfu/g
1400 1200 1000 800 600 400 200 0
4
as shown in Figure 11.1. While, in control group, only Vibrio spp. was observed in shrimp intestine as shown in Figure 11.2. DISCUSSION Isolation of Bacillus spp. from shrimp intestine found three species namely B. pumilus, B. sphaericus and B. subtilis. Original of those bacteria might came from water, soil, food or normal flora in the intestine. Bonde (1981) reported that in seawater dominated by B. licheniformis followed by B. subtilis and B. pumilus. Other species encountered in low numbers include B.
cfu/g 1500
a ab ab ab ab b c Control 1 2 3 4 5 6 7 ab
b 1000 500
feeds
a Control 1
a 2
aa 3 4
a 5
a 6
a 7
feeds
Figure 9 The number of Bacillus spp. in P. monodon intestine after 4 weeks of culture when provided with 8 feeds.
Figure 10 The number of Vibrio spp. in P. monodon intestine after 4 weeks of culture when provided with 8 feeds.
Figure 11 Scanning Electron Microscope picture. 11.1 Scanning Electron Microscope picture of Bacillus spp. in shrimp intestine after fed with mixture of B. pumilus, B. sphaericus and B. subtilis ( 10,000) 11.2 Scanning Electron Microscope picture of Vibrio spp. in shrimp intestine (control group) ( 15,000)
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brevis, B. firmus and B. sphaericus, largely in nonpolluted areas. In a numerical study of North Sea sediments, Boey and Herts (1976) found that B. subtilis, B. licheniformis and B. firmus strains predominated. So, it was possible for B. pumilus, B. sphaericus and B. subtilis to contaminate in intestinal of shrimp by sea water. In vitro production of inhibitory compounds toward known pathogens for the considered species has often been used in the selection of putative probiotic strains (Verschuere et al., 2000). In this study we demonstrated that the isolated Bacillus spp. from intestinal of black tiger shrimp are potential competitors for V. harveyi, the results showed colonization effect of each Bacillus spp. to V. harveyi in 24 hours. Special results we received from this experiment, the effective of B. pumilus could produce some substance and effective to destroy V. harveyi confirm this result by clear zone area in the test plates. The inhibition of B. pumilus to V. harveyi observed clear zone of V. harveyi colony in 12 hours and eradicate all of V. harveyi colony in 48-72 hours. Although B. sphaericus and B. subtilis did not show colonization effect but they showed some inhibition effect to V. harveyi, which confirmed by distorted shape of V. harveyi by TEM the shape of V. harveyi had smaller size and some area of cell wall was destroyed. The abnormal shape of V. harveyi were permanently changes confirmed with three consecutive subcultures. Bacillus spp. might produced some metabolites for instance antibiotic (Williams and Vickers, 1986) or enzymes for inhibition and/or digestion (Bruno and Montville, 1993) Regarding to the growth of Bacillus spp., this experiment might confirmed that Bacillus spp. could grow in a wide range of environment, such as salinity up to 8% NaCl, pH 4-11 and temperature up to 50 C, althought B. sphaericus could not grow at 50 C but in the real culture, water temperature never raise so high as 50 C. Using B. subtilis mixed with B. sphaericus
could increase weight of shrimp significantly difference (P<0.05) from control and other experiment groups, while others treatments had no significant different from the control (P>0.05). About immunoenhancement ability of Bacillus spp., Bacillus had peptidoglycan in its cell wall which could increase immune of shrimp (Boonyaratpalin et al., 2000). In this research, B. subtilis had highest efficiency to improve immune parameters of black tiger shrimp consists of total hemocyte, phenol oxidase, superoxide anion and clearance ability. Others treatments containing B. subtilis in the mixtures also increased immune parameters significantly difference from the control as well. These results were similar to Rengpipat et al. (2000). But in our research we measured superoxide anion instead of phagocytic activity because superoxide anion produced in phagocytosis process (Bell and Smith, 1993). Furthermore, after fed shrimp with Bacillus spp. for 4 weeks revealed that Bacillus spp. showed superiority in competition and colonization to Vibrio spp. in shrimp intestine. CONCLUSIONS In summary, it had been demonstrated that B. pumilus, B. sphaericus and B. subtilis had good properties to be used as probiotic for black tiger shrimp. B. pumilus showed colonization activity and produce inhibitory compounds while B. sphaericus and B. subtilis showed only inhibitory effects to V. harveyi. TEM studied found that cell morphology of V. harveyi colonized by B. pumilus, B. sphaericus and B. subtilis changed to smaller sizes compared with normal cell. Moreover, three species of Bacillus spp. could grow in a different environment, including salinity 0-8% NaCl, pH 411 and temperature 25-50 C except B. sphaericus which could not grow at 50 C. When fed shrimp with these Bacillus spp. showed immunoenhancement capability and also colonization and inhibition on V. harveyi in shrimp
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intestine. Therefore, these Bacillus spp. might be applied as good probiotic in shrimp aquaculture. ACKNOWLEDGEMENTS This research was supported by Kasetsart University Research and Development Institute (KURDI). We thank Professor Dr. Toshiaki Itami, Miyazaki University for immune techniques. Assoc. Prof. Kidchakarn Supamattaya, Prince of Songkla University, Thailand for chemical formula. Ms.Kanokphan Srimanopach, Department of Fisheries for VITEK 32 instrument, Ms. Patcharee Umrung, Central lab KU for TEM and Ms. Yuppade Paowpan, Central lab KU for SEM. LITERATURE CITED Bell, K. L. and V. J. Smith. 1993. In vitro superoxide production by hyaline cells of the shore crab Carcinus maenas Dev. Comp. Immunol. 17: 211-219. Boey, A. and M. Herts. 1976. Numerical taxonomy of Bacillus isolates from North Sea sediments. Int. J. Syst. Bacteriol. 26:427441. Bonde, G. J. 1981. Bacillus from marine habitats : Allocation to phena established by numerical Techniques, pp. 181-215. In R. C. W. Berkeley and M. Goodfellow (eds.). The Aerobic Endospore-Forming Bacteria : Classification and Identification, Academic Press, London. Boonyaratpalin, M., K. Supamattaya, J. Pongmaneerat, S. Boonyaratpalin and Y. Toride. 2000. The stimulatory effect of high and low molecule peptidoglycan (PG) on immune responses in black tiger shrimp, Penaeus monodon Fabricius. Songklanakarin J. Sci. Technol. 22 (Suppl.) : 689-696.
Bruno, M. E. C. and T. J. Montville. 1993. Common mechanistic action of bacteriocins from lactic acidbacteria. Appl. Environ. Microbiol. 59:3003 3010. Fuller R. 1992. Probiotics. The Scientific Basis. Chapman and Hall, London. 398 p. Gullian, M., F. Thompson and J. Rodriguez. 2004. Selection of probiotic bacteria and study on their immunostimulatory effect in Penaeus vannamei. Aquaculture 233: 1-14. Lowry, O.H., N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275. Martin, G. G., D. Poole, C. Poole, J. E. Hose, M. Arias, L. Reynolds, N. Mckrell and A. Whang. 1993. Clearance of bacteria injected into the hemolymph of the Penaeid shrimp, Sicyonia ingentis. J. Inver. Pathol. 62: 308-315. Moriarty, D.J.W. 1998. Control of luminous Vibrio species in penaeid aquaculture ponds. Aquaculture 164: 351-358. Rengpipat, S., S. Rukpratanporn, S. Piyatirativarakul and P. Menasveta. 2000. Immunity enhancement in black tiger shrimp (Penaeus monodon) by a probiont bacterium (Bacillus S11). Aquaculture 191: 271-288. Supamattaya, K., J. Pongmaneerat and T. Klowklieng. 2000. The effect of bglucan (MacroGard ) on growth performance, immune response and disease resistance in black tiger shrimp, Penaeus monodon Fabricius. Songklanakarin J. Sci. Technol. 22 : 677-688. Verschuere, L., G. Rombaut, P. Sorgeloos and W. Verstraete. 2000. Probiotic Bacteria as Biological Control Agents in Aquaculture. Microbiol. Mol. Biol. R. 64: 655671. Williams, S. T., and J. C. Vickers. 1986. The ecology of antibiotic production. Microb. Ecol. 12:4352.
Extracts of Thai Indigenous Vegetables as Rancid Inhibitor in a Model System

Plernchai Tangkanakul, Gassinee Trakoontivakorn and Chansuda Jariyavattanavijit
ABSTRACT The antioxidant property of twenty five vegetables extracted with ethanolic and water was determined by monitoring their capacities to scavenge the stable free- radicals DPPH. Oxidative rancidity in oil-in-water emulsion model was evaluated by ferric thiocyanate (FTC) method. Total phenolic content was also determined by Folin-Ciocalteu method. The ethanolic extracts were found to exhibit a higher phenolic content as well as DPPH radical scavenging activities than the water extracts. However, the data indicated that both ethanolic and water extracts had dramatically antioxidant activity determined by FTC method. Sixteen and eighteen plants of ethanolic and water extracts, respectively, performed greater rancid inhibition than synthetic antioxidant (BHA, 10 ppm). This study found no relationship between antioxidant activities through the DPPH radicals scavenging or through lipid radicals scavenging. Key words: vegetables, antioxidant capacity, free radical scavenging activity, total phenolics, rancidity
INTRODUCTION Synthetic antioxidants have been applied for decreasing lipid oxidation during storage of processed food products. The use of chemical additives has raised questions regarding food safety and toxicity (Chang et al., 1977). Many research works have been directed toward safe antioxidants with high antioxidative activity from natural sources. The antioxidant properties of herbs and spices, cinnamon, turmeric, clove, black pepper, nutmeg, dry ginger, rosemary, sage and paprika were continuously reported (Chang et al., 1977; Nakatani et al., 1986; Kikuzaki and Nakatani., 1993; Tomaino et al., 2005). Many plant extracts were revealed on antioxidation efficiency when applied in oils, fats and fat containing foods, meat
products (Karpinska et al., 2001). For example, rosemary and sage prolong the induction period in chicken fat and show antioxidant activity comparable with butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) (Bracco et al., 1981). For Thai local vegetables, study on antioxidant activity was reported regarding to the potency of over 100 varieties by using b-carotene bleaching method ((Nakahara and Trakoontivakorn, 1999; Trakoontivakorn and Saksitpitak, 2000; Na Thalang et al., 2001). More than fifty varieties were reported to contain antioxidant more than 100 mg BHA equivalent in 100g fresh weight. With their high potential and lack in information for rancid inhibition, the present study was aimed to evaluate 25 Thai vegetables for
Institute of Food Research and Product Development, Kasetsart University, Bangkok 10900, Thailand.
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anti-rancidity in a model system. Extract solvents, water and ethanol, were taken into a consideration. Total phenolic content was examined to find a relationship between phenolic content and antioxidant activity of crude extracts from both solvents. Presenting antioxidant activity of plant crude extract was expressed in many aspects. These methods revealed differently on mechanisms of antioxidant defense system, i.e., inhibition of lipid peroxidation, reduction of lipid peroxyl radicals or scavenging of oxygen and hydroxyl radicals (Tsushida et al., 1994; Velioglu et al., 1998; Khknen et al., 1999; Pulido et al., 2000). Stable free radicals, frequently applied to study natural antioxidant efficiency were 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), DPPH or N,N-Dimethyl-pphenylenediamine dihydrochloride (DMPD). Koleva et al. (2002) recommended using DPPH due to being simple, rapid, convenient and independent of sample polarity. Antioxidant capacity was another perspective to present antioxidant activity, applying different methodologies such as TEAC (Trolox equivalent antioxidant capacity, Cook et al., 1998), b-carotene bleaching method expressed as BHA content (Tsushida et al., 1994), stable radical ABTS or DPPH expressed as vitamin C (Kim et al, 2002). These antioxidant capacity methods were developed in an attempt to have a meaningful interpretation relating to health. In this study, rancid inhibition was concerned, therefore, scavengers of DPPH were stated in equivalent to BHA content. MATERIALS AND METHODS Sample preparation Twenty five Thai indigenous vegetables were either purchased from local markets or collected from nature (Table 1). Edible portions of vegetable were weighed in 10 g/plastic bags and
kept at -20 C until extracting. Vegetable extraction Individual vegetable was extracted with 2 different solvents, 95% aqueous ethanol and distilled water. The 10 g of frozen vegetables were homogenized (Ultra Turrax) in 40 ml 95% aqueous ethanol or distilled water at room temperature for 1 min. and centrifuged at 10000 g for 10 min. The residue was re-extracted with either 95% aqueous ethanol or distilled water and extracts were pooled and made to 100 ml. The extracts were stored in capped bottles and kept at -20C until further use for antioxidant capacity and total phenolics determinations and rancid inhibition test. DPPH Radical scavenging activity and antioxidant capacity DPPH scavenging activity was determined using a modified method of Onichi et al. (1994). The free radical scavenging activity of vegetable extracts were tested, indicated as bleaching of the stable 1,1 diphenyl-2-picrylhydrazyl radical (DPPH). A diluted extract of the right concentration to posses not more than 60% scavenging activity (%SA), 0.15 ml, was added to 0.9 ml of 0.1 mM DPPH dissolved in 95% ethanolic solution. The mixture was vertexed and allowed to stand at room temperature. After 20 min., the absorbance was recorded at 517 nm. 95% aqueous ethanol was used as a control. Percentage of DPPH scavenging activity (%SA) was calculated from this equation (C-X)100/C, where C = absorbance of control and X =absorbance of extract. In order to express antioxidant activity of plant extract to an easily understood manner, antioxidant capacity as mg t-butylated hydroxyanisole equivalent (BHAE) /g fresh vegetable was introduced. A standard curve of tbutylated hydroxyanisole (BHA) was obtained from DPPH %SA (x) plotted against various BHA concentrations (y). Prepared concentrations of BHA solution were 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0
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Table 1 Tested part Place and time of collection Dry matter (%) Ethanolic extract AC TP (mg BHAE (mg GAE/ /g FW) g FW) Water extract Ratio of AC TP AC, (mg BHAE (mg GAE/ EtOH/water /g FW) g FW) 17.733.15 18.460.06 30.0810.67 3.531.37 7.120.64 17.681.14 0.460.05 3.860.54 16.351.95 0.510.26 1.560.73 46.779.91 4.780.59 0.620.03 49.067.32 1.760.10 3.610.15 5.740.66 1.110.08 12.820.15 10.480.97 22.47 25.18 26.26 19.19 3.90 114.368.39 24.762.13 23.443.75 7.822.80 14.712.17 7.280.37 6.510.29 40.224.27 12.640.49 11.730.30 5.020.47 4.080.06 0.030.03 33.864.15 0.180.06 1.510.88 1.560.05 0.450.02 4.601.83 0.340.15 37.518.06 6.130.56 15.083.11 2.550.59 6.740.09 0.360.02 1.950.05 11.270.91 12.872.20 0.380.00 11.430.11 1.740.27 3.600.42 1.910.08 0.730.09 13.130.30 0.970.05 12.511.89 12.553.26 5.950.34 2.590.50 1.700.11 68.259.86 4.783.50 19.751.76 34.637.43 0.140.01 1.050.23 31.046.71 7.4 2.6 3.2 1.6 5.0 8.7 3.9 1.7 3.8 2.2 1.9 5.3 1.0 4.9 6.7 1.2 6.1 3.7 2.8 31.2 3.0 4.0 1.6 3.1 2.2 131.7214.38 42.165.49 51.334.42 6.713.84 11.860.11 54.029.45 1.180.22 4.940.69 33.773.54 1.250.04 3.390.08 25.344.96 34.511.14 176.4832.07 15.097.64 31.850.81 174.1814.67 1.240.15 4.060.09 53.910.97 1.950.03 3.390.07 88.720.95 25.340.34 0.030.00 167.2219.06 1.200.07 1.770.08 9.560.02 1.650.07 31.16 23.06 28.86 19.61 28.15 8.72 21.94 18.74 17.18 14.26 17.60 24.32 8.38 25.48 20.14 16.76 8.22 8.16 16.33 9.76
Antioxidant capacity (AC), reported as BHA equivalent (BHAE), and total phenolic (TP) content, reported as gallic acid equivalent (GAE), of vegetables.
Botanical name
Local name
Aganosma marginata Leaf Leaf Fruit Leaf Leaf Leaf Leaf Flower Stem Leaf Leaf Flower Whole Leaf Leaf Leaf Leaf, stem Leaf, stem Leaf, stem Leaf, stem Leaf Leaf Leaf Leaf Filaments Ubon Ratchathani, March Surat Thani, March
Saton
Mamuang himaphan Archidendron jiringa Nieng Barringtonea acutangula Kradon nam
Anacardium occidentale
Careya sphaerica
Kradon bog
Centella asiatica Colubrina asiatica Cratoxylum formosum
Baibua bog Gan tong Tew
Cymbopogon citratus. Dregea volubilis Eugenia grata
Ta kai Huan Sa meg
Feroniella lucida
Ma sung
Glinus oppositifolius
Khee khom
Glochidion wallichianum Gnetum gnemon Gymnema inodorum Ipomoea aquatica Lasia spinosa
Mun pu Lieng Chiengda Phak bung thai Phak nham
Leucaena leucocephala Limonophila aromatica
Kratin Phak ka yheng
Mangifera indica Micromelum minutum Parkia speciosa Passiflora foetida
Ma muang Mhui Sator Ka tok rok
Spirogyra sp.
Tao
Surat Thani, March Ubon Ratchathani, March Sakon Nakorn, February Surat Thani, March Lampang, April Sakon Nakorn, February Bangkok, May Lampang, April Sakon Nakorn, February Sakon Nakorn, February Sakon Nakorn, February Surat Thani, March Surat Thani, March Lampang, April Bangkok, May Sakon Nakorn, February Bangkok, May Ubon Ratchathani, March Lampang, April Surat Thani, March Surat Thani, March Ubon Ratchathani, March Sakon Nakorn, February
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mg/100 ml 95% ethanol. The regression line was y = 0.0832x - 0.0469. Determination of total phenolic contents Total phenolics were determined using the Folin-Ciocalteau reagent, adopted from Singleton and Rossi (1965). Two millilitres of suitable diluted vegetable extracts was transferred and reacted with 10 ml of Folin-Ciocalteau reagent (previously diluted 10 fold with distilled water) in 25 ml volumetric flask. After 30 sec. and before 8 min., 8 ml of 7.5% of sodium carbonate was added and mixed, and the contents of the flask made to volume with distilled water. Solutions were heated in a 40C water bath for 30 min. The color was developed and absorbance measured at 765 nm. The standard curve was prepared using 0, 0.5, 1.0 and 1.5 ml of gallate stock solution (8 mg/100ml) in 25 ml volumetric flask. The regression line between absorbance (y) and gallic acid content (x) was y = 0.0046x + 0.0163. The results were expressed as mg gallic acid equivalent / g of fresh vegetable. Antioxidative assay in model system The lipid oxidation was monitored by ferric thiocyanate (FTC) method described by Kikuzaki and Nakatani (1993). A 20 ml experimental mixture made of 4 ml of an ethanolic extract (0.4 g fresh vegetable), 4.1 ml of 2.51% linoleic acid in 99.5% ethanol, 8 ml of 0.05 M phosphate buffer (pH 7.0) and 3.9 ml of water was placed in a plastic bottle (25 mm diameter, 60 mm height) with a screw cap. For water extract, an assay mixture was prepared the same as above except 3.9 ml of water was replaced by 99.5% ethanol. The mixtures were placed in an oven at 40C in the dark. FTC was carried out by adding 0.1 ml of incubated mixture, 9.7 ml of 75% ethanol and 0.1 ml of 30% ammonium thiocyanate into a test tube. Precisely 3 min after addition of 0.1 ml of 0.02M ferrous chloride in 3.5% hydrochloric acid to the reaction mixture, the absorbance of red color was measured
at 500 nm. Antioxidative assay was carried out with 7 days interval until the absorbance of the tested mixture reached maximum or 142 days. BHA at concentration of 1.0 mg/100 ml and 5.0 mg/100 ml were used as a control. RESULTS AND DISCUSSION Antioxidant capacity Within twenty five vegetables, ethanolic extract was found to obtain more antioxidant capacity than water extract (Table 1). The result agreed with Kaur and Kapoor (2000) who applied b-carotene bleaching test. Ratio of antioxidant capacity of components soluble in ethanol to water of 25 tested vegetables varied in a range of 31.2 to 1.0, with mean 4.7 and median of 3.2. Limonophila aromatica was the one that contained antioxidants most susceptible to dissolve in ethanol. And it was also revealed that antioxidant capacity of ethanolic extracts was in different order from that of water extracts, however, not dramatically. A large variation in the antioxidant capacity was observed in ethanolic extracts, ranging from as high as 176.48 mg BHAE /g fresh weight of Anacardium occidentale to as low as 0.03 mg BHAE /g fresh weight of Glinus oppositifolius. The ranking of five vegetables possessing high antioxidant capacity through ethanol extraction was Anacardium occidentale, Careya sphaerica, Glochidion wallichianum, Aganosma marginata and Mangifera indica. The result agreed to Trakoontivakorn and Saksitpitak (2000) who reported that methanolic extracts of Anacardium occidentale, Careya sphaerica, Glochidion wallichianum and Mangifera indica contained great amount of antioxidants analyzed by b-carotene bleaching method. The results of water extract were found to have antioxidant capacities between 0.03 - 68.25 mg BHAE /g fresh vegetable. The five greatest antioxidant capacities were Anacardium occidentale, Eugenia grata, Mangifera indica, Careya sphaerica and Glochidion wallichianum.
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Total phenolic content The results of phenolic analysis of twenty five vegetables are exhibited in Table 1. The phenolic contents of the vegetable in ethanol extraction varied from 0.62 mg GAE/g fresh weight of Glinus oppositifolius to 54.02 mg GAE/g fresh weight of Careya sphaerica. The ethanolic extractable phenolic compounds greater than 25 mg GAE/g fresh vegetable were found in Anacardium occidentale, Glochidion wallichianum, Aganosma marginata, Mangifera indica, Feroniella lucida, Cratoxylum formosum and Eugenia grata. In water extraction, the total phenolic contents was found highest in Anacardium occidentale, 30.08 mg GAE/g fresh weight, followed by Aganosma marginata, Careya sphaerica, Cratoxylum formosum and Leucaena leucocephala as 18.46, 17.68, 16.35 and 13.13 mg GAE/g fresh weight, respectively. The present study demonstrated that total phenolics in tested Thai vegetables generally were greater than those of Western herbs, 0.23 -17.51 mg GAE/g fresh weight, reported by Zheng and Wang (2001). Phenolic compounds were found to be generally susceptible to dissolve more in ethanol than in water. An exception was observed in Leucaena leucocephala where extractable phenolic compounds were almost double in water extract than in ethanolic extract. Micromelum minutum, Gymnema inodorum and Gnetum gnemon possessed similar phenolic contents in both extract media. A study reported on phenolic components extracting ability that ethanol was less effective than acetone when applied in Greek oregano and summer savory. However, acetone extracts exhibited incredible low in DPPH scavenging activity (Exarchou et al., 2002). With this detection, therefore, an extract medium should be taking into account in order to obtain antioxidative compounds. Phenolic contents existed in these twenty five Thai indigenous vegetables were considered as moderate to high. Converting vegetable weight into dry weight basis, ethanolic extractable phenolic
compounds ranged from 7.28 mg GAE/g dry weight in Cymbopogon citratus to 222.59 mg GAE/g dry weight in Anacardium occidentale. Comparing to the result of Khknen et al. (1999), the selected cereals and vegetables contained greatly lower amount of phenolics, 0.2 6.6 mg GAE/ g dry weight and in moderate level in herb extracts, 9.1 23.1 mg GAE/g dry weight. A relationship between phenolic content and antioxidant activity was extensively investigated, and both positive and negative correlationships were demonstrated. Velioglu et al. (1998), Rapisarda et al. (1999), Zheng and Wang (2001), and many other research groups stated that there was a positive correlation. In the mean time, a few evidences of no significant correlation were confronted (Heinonen et al., 1998; Khknen et al., 1999). In this study, the regression analysis was done separately on the extracting medium, ethanol and water. The results revealed that both ethanolic (R2 = 0.8893) and water extracts (R2 = 0.6601) held a positive linear relationship between phenolic content and antioxidant capacity as displayed in Figure 1. The degree of correlation coefficient pronounced greatly in ethanolic extracts (R = 0.9430) and lesser in water extracts (R = 0.8125). Antioxidative effect in model system The oxidation of oil-in-water emulsion was monitored by the ferric thiocyanate (FTC) method. The FTC method was used to measure the amount of peroxide in initial stages of lipid oxidation. Peroxide oxidizes ferrous iron to the ferric state resulted in the formation of a red thiocyanate complex. The determined values at 500 nm with low absorbance indicated a high ability to delay rancidity. On the contrary, if high absorbance values and sharp increasing curve were noted, an uncontrollable lipid oxidation stage was confronted. In this study, extracts from 25 vegetables were analyzed and illustrated separately by the extracting media (Figure 2).

60.00
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Ethanolic extracts Water extracts
50.00
Total phenolics (mg GAE/g FW)
40.00
R2 = 0.8893
30.00 20.00
R2 = 0.6601
10.00
0.00 0.00 50.00 100.00 150.00 200.00
Antioxidant capacity (mg BHAE/g FW)
Figure 1 Relationship between total phenolic content and antioxidant capacity of 25 vegetables, extracted by ethanol and water. Within 142 days of rancid inhibiting evaluation, only 9 vegetable ethanol extracts were lost or deteriorated in their abilities indicated by reaching an absorbance of 0.4 (Figure 2a). They were Cratoxylum formosum, Aganosma marginata, Anacardium occidentale, Glinus oppositifolius, Feroniella lucida, Micromelum minutum, Careya sphaerica, Spirogyra sp. and Gnetum gnemon. Absorbance of the control, no antioxidant in the system, reached absorbance of 0.4 after day 7. Seven out of 9 vegetables and 0.04 mg BHA reached absorbance of 0.4 within 60 days. From the whole experiment, only the extracts from Glinus oppositifolius, Spirogyra sp., Gnetum gnemon, 0.04 mg BHA and 0.2 mg BHA displayed a sharp raise curve similar to the control. A sharp changing in absorbance is a common oxidation pattern monitored by FTC method as shown in other studies (Kikuzaki and Nakatani, 1993; Chen and Ho, 1997). The indicator used to monitor the generation of rancid odor from oil oxidation during incubation was the absorbance of 0.4 by FTC as reported by Chen and Ho (1997). Extract of Anacardium occidentale and Careya sphaerica could retain absorbance values around 0.4 indicating potential antioxidant plants, and there were other 16 vegetables performed better than 0.2 mg BHA. The plant extracts that exhibited substantial ability in rancid inhibition were Gymnema inodorum, Dregea volubilis, Cymbopogon citratus, Colubrina asiatica, Passiflora foetida, Lasia spinosa and Centella asiatica. Water extracts illustrated a good result as rancid inhibitors as well (Figure 2b). The plants that possessed this property were Leucaena leucocephala, Gymnema inodorum, Dregea volubilis, Archidendron jiringa, Ipomoea aquatica, Passiflora foetida and Spirogyra sp.. Water extracts of plant that could not inhibit rancidity were Glinus oppositifolius, Cratoxylum formosum, Aganosma marginata, Gnetum gnemon, Lasia spinosa, Cymbopogon citratus, Limonophila aromatica and Centella asiatica. It was interesting to point out that some plants performed oppositely in controlling rancidity in this model system. Water extract of Spirogyra sp. contained effective antioxidants but not in ethanolic extract. Whiles, ethanolic extracts of Centella asiatica, Lasia spinosa and Cymbopogon citratus were effective antioxidant, they became ineffective when extracted with water. The plants
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0.8
AM AO AJ
0.7
BA CS CAU
0.6
CAB CF CC DV
Absorbance 500 nm
0.5
EG FL GO
0.4
GW GG GI IA
0.3
LS LL LA
0.2
MI MM PS PF
0.1
S Control BHA 0.04 mg
0 0 20 40 60 Time (days) 80 100 120 140
BHA 0.2 mg
0.8 0.7 0.6
AM AO AJ BA CS CAU CAB CF CC
Absorbance 500 nm
0.5 0.4 0.3 0.2 0.1 0 0 20 40 60 80 100 120 140 Time (days)
DV EG FL GO GW GG GI IA LS LL LA MI MM PS PF S Control BHA 0.04 mg BHA 0.2 mg
Figure 2 Oxidation of oil-in-water emulsion monitored by the ferric thiocyanate method (a) ethanol extraction (b) water extraction. Aganosma marginata (AM), Anacardium occidentale (AO), Archidendron jiringa (AJ), Barringtonea acutangula (BA), Careya sphaerica (CS), Centella asiatica (CAU), Colubrina asiatica (CAB), Cratoxylum formosum (CF), Cymbopogon citrates (CC), Dregea volubilis (DV), Eugenia grata (EG), Feroniella lucida (FL), Glinus oppositifolius (GO), Glochidion wallichianum (GW), Gnetum gnemon (GG), Gymnema inodorum (GI), Ipomoea aquatica (IA), Lasia spinosa (LS), Leucaena leucocephala (LL), Limonophila aromatica (LA), Mangifera indica (MI), Micromelum minutum (MM), Parkia speciosa (PS), Passiflora foetida (PF), Spirogyra sp.(S)

Ethanolic extracts Water extracts
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80
70
60
BHAE content (mg)
50 40 30
20 10 0 0 20 40 60 80 100 120 140 160 180 200
Incubation time (days)
Figure 3 Relationship between BHAE content obtained from antioxidant capacity and incubation time to reach an absorbance of 0.4 by ferric thiocyanate method of 25 vegetables, extracted by ethanol and water.
that displayed as a good antioxidant sources when either extracted with water or ethanol were Passiflora foetida, Dregea volubilis and Gymnema inodorum. Results from the model system experiment revealed that plant extracts good in scavenging DPPH radical was not always good in scavenging lipid radicals. A relationship between BHAE content and incubation time (reached absorbance of 0.4) was investigated. BHAE content of tested vegetables calculated from antioxidant capacity which used DPPH scavenging method as a tool. The regression values of ethanolic extract and water extract were 0.093 and 0.0122, respectively (Figure 3). CONCLUSION It was apparent from the study that plant extracted by different solvents scavenged DPPH radical and lipid radicals differently. Water extracts of some vegetables were efficient comparable to their ethanolic extracts for oxidative rancidity inhibition. And the model of scavenging DPPH
radical could not be used to predict lipid oxidative inhibition. This study also found that extractable antioxidants from 0.4 g of some vegetables demonstrated stronger rancid inhibition than the synthetic antioxidant, 10 ppm BHA. It thus constitutes an interesting source for use as natural protecting agent to prevent oxidative deterioration of food. ACKNOWLEDGMENTS Kasetsart University Research and Development Institute (KURDI) financially supported this research. LITERATURE CITED Bracco, U., J. Loliger and Viret J.L. 1981. Production and use of natural antioxidans. J. Am. Oil Chem. Soc. 58:686-690. Chang, S.S., B. Ostric-Matijasevic, O.A. Hsieh and C.L. Chang. 1977. Natural antioxidants from rosemary and sage. J. Food Sci. 42:11021106.
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Chen, J.H. and C-T Ho. 1997. Antioxidant activities of caffeic acid and its related hydroxycinnamic acid compounds. J. Agric. Food Chem. 45:2374-2378. Cook, J.A., D.J. Vanderjagt, A. Dasgupta, G. Mounkaila, R.S. Glew, W. Blackwell and R.H. Glew. 1998. Use of the Trolox assay to estimate the antioxidant content of seventeen edible wild plants of Niger. Life Sci. 63:105110. Exarchou, V., N. Nenadis, M. Tsimidou, I.P. Gerothanassis, A. Troganis and D. Boskou. 2002. Antioxidant activities and phenolic composition of extracts from Greek oregano, Greek sage and summer savory. J. Agric. Food Chem. 50:5294-5299. Heinonen, M., P.J. Lehtonen and A. Hopia. 1998. Antioxidative activity of berry and fruit wines and liquor. J. Agric. Food Chem. 46:25-31. Karpinska, M., J. Borowski and M. DanowskaOziewicz. 2001. The use of natural antioxidants in ready-to-serve food. Food Chemistry 72:5-9. Kaur, C. and H.C. Kapoor. 2002. Anti-oxidant activity and total phenolic content of some Asian vegetables. Inter. J. Food Sci. Tech. 37:153-161. Khknen, M.P., A.L. Hopia, H.J. Vuorela, J-P. Rauha, K. Pihlaja, T.S. Kujala and M. Heinonen. 1999. Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem. 47:3954-3962. Kikuzaki, H. and N. Nakatani. 1993. Antioxidant effects of some ginger constituents. J. Food Sci. 58:1407-1410. Kim, D-O., W.L. Ki, H.J. Lee and C.Y. Lee. 2002. Vitamin Cequivalent antioxidant capacity (VCEAC) of phenolic phytochemicals. J. Agric. Food Chem. 50:3713-3717. Koleva, I.I., T.A. van Beek, J.P.H. Linssen, A. de Groot and L.N. Evstatieva. 2002. Screening of plant extracts for antioxidant activity: a comparative study on three testing methods.
Phytochemical Analysis 13:8-17. Na Thalang, V., G. Trakoontivakorn and K.Nakahara. 2001. Determination of antioxidant activity of some commonly consumed leafy vegetables in Thailand. JIRCAS J. 9:39-46. Nakahara, K. and G.Trakoontivakorn. 1999. Antioxidative and autimutagenic properties of some local agricultural products in Thailand. In Highlight of Collaborative Research Acfivities between Thai Research Organizations and JIRCAS. Provided for JIRCAS Seminar in Bangkok 1999, Bangkok, Thailand 3 March 1999. pp.141 Nakatani, N., R. Inatani, H. Ohta and A. Nishioka. 1986. Chemical constitutents of pepper and application of food preservation, naturally occurring antioxidative compounds. Environmental Health Perspectives. 67:135-141. Ohnishi, M., H. Morishita, H. Iwahashi, S. Toda, Y. Shirataki, M. Kimura and R. Kido. 1994. Inhibitory effects of chlorogenic acids on linoleic acid peroxidation and haemolysis. Phytochemistry 36 (3):579-583. Pulido, R., L. Bravo and F. Saura-Calixto. 2000. Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/ antioxidant power assay. J. Agric. Food Chem. 48:3396-3402. Rapisarda, P., A. Tomaino, R.L. Cascio, F. Bonina, A. De Pasquale and A. Saija. 1999. Antioxidant effectiveness as influenced by phenolic content of fresh orange juices. J. Agric. Food Chem. 47:4718-4723. Singleton, V.L. and J.A. Rossi. 1965. Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. Am. J. Enol. Vitic. 16:144-158. Tomaino, A., F. Cimino, V. Zimbalatti, V. Venuti, V. Sulfaro, A. De Pasquale and A. Saija. 2005. Influence of heating on antioxidant activity and the chemical composition of some
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41:611-618. Velioglu, Y.S., G. Mazza, L. Gao and B.D. Oomah. 1998. Antioxidant activity and total phenolics in selected fruits, vegetables and grain products. J. Agric. Food Chem 46:41134117. Zheng, W. and S.Y. Wang. 2001. Antioxidant activity and phenolic compounds in selected herbs. J. Agric. Food Chem. 49:5165-5170.
Screening and Characterization of Lactic Acid Bacteria Producing Antimicrobial Substance against Staphylococcus aureus
Chatinan Ratanapibulsawat1, Pumrussiri Kroujkaew2, Ohmomo Sadahiro2 and Sunee Nitisinprasert1
ABSTRACT One isolate of lactic acid bacteria (LAB) producing antimicrobial substance (AMS) against Staphylococcus aureus was selected and designed as LS2-30. Morphological, physical and biochemical tests, and 16S rRNA analysis were performed and revealed that it was Pediococcus acidilactici. Optimal conditions for growth and AMS production were determined. The optimum temperature and initial pH for both growth and AMS production were 45 C and pH 6, respectively. Cell-free culture fluid of LS230 showed an inhibitory action against Staphylococcus aureus, Salmonella sp., Bacillus sp., and E. coli but no activity against all LAB tested. When mode of action was studied, the cell-free culture fluid exhibited a bactericidal mode of action but did not lyse its cells. After removing organic acids, partial purified antimicrobial substance at pH 3 showed inhibitory activity as bactericidal action. Hydrolytic enzyme treatment suggested that AMS structure was neither proteinaceous, starch nor lipid moiety in nature. Therefore, the nature of inhibitory compound secreted by P. acidilactici LS2-30 was still unclear. Key words: antimicrobial substance, lactic acid bacteria, Staphylococcus aureus INTRODUCTION Staphylococcus aureus is an important opportunistic pathogen that causes a variety of diseases in humans and animals. It is widely recognized that antibiotic therapies formulated for intramammary use are generally unsuccessful in eliminating existing S. aureus infections or in preventing the establishment of chronic udder disease (Leitnera et al., 2003). Currently, more than 95% of infectant with S. aureus infections worldwide do not respond to first-line antibiotics such as penicillin or ampicillin (Rubin et al., 1999). Moreover, with increasing public concern over food safety, there is regulatory pressure to justify the use of therapeutic drugs in dairy cattle and to reduce the incidence of residues in milk. The emergence and spread of drug-resistant staphylococci underscores the need to find new modes of prevention and alternative non-antibiotic treatment. In a recent in vitro study, various substances have been found and shown antimicrobial activity against S. aureus, like, lactose chitosan derivative against S. aureus CCRC 12657 (Chen and Chou, 2005), the extract of Panax ginseng root (ginsenoside) (Hu et al., 2003) and lactoferrin with penicillin G (Diarra et al., 2002). In addition, many anti-S. aureus substances producing LAB have been a great interest because most of the LAB are
1 2
Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand. Department of Animal Products, National Institute of Animal Industry (NIAI), Tsukuba, Ibaraki, 305-0901 Japan.
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considered as Generally Recognized as Safe microorganism (Holzapfel et al., 1995). Several LAB species were found to be a potent anti-S. aureus substance producer such as Lactobacillus plantarum BS, Lb. salivarius, Lb. plantarum 35d (Messi et al., 2001), P. acidilactici F (Osmanagaoglu et al., 1998) and Lb. acidophilus LA1 (Bernet-camard et al., 1997). However, most of them produced the substances that inhibited themselves as well. Therefore, it is still important to isolate a novel anti-S. aureus substance with new properties and without significant inhibitory activity against its host. The objective of this study was to characterize LAB producing antimicrobial substance against S. aureus. The antibacterial spectrum and their mode of action were described as well. MATERIALS AND METHODS Bacterial strains and growth conditions Antimicrobial substance (AMS) producing lactic acid bacteria isolated from silage and fermented pork (Nham) were obtained from the culture collection of Department of Biotechnology, Kasetsart University (Thailand). They were anaerobically grown in De Man Rogosa and Shape (MRS, Merck) at 45 C. All strains of S. aureus isolated from mastitis cow milk from the National Institute of Animal Health (Thailand) and pathogenic bacteria used as indicator strains were grown in Nutrient broth at 37 C while LAB was done in MRS at 37 C. E. coli DH5a used as a host for cloning PCR-amplified 16S rDNA was grown in Luria broth at 37 C. All strains were stored at -80 C in desirable media containing 20% (v/v) glycerol, and propagated twice before experimental use. Screening of antimicrobial substance producing LAB Bacterial cells were removed from the
overnight cultures by centrifugation at 10,956 g for 15 min at 4C. Cell-free culture supernatant (CFS) obtained was filter-sterilized with a 0.2 mm pore size filter membrane and further assayed for antimicrobial activity. Determination of antimicrobial activity The spot-on-lawn method was used to determine antimicrobial activity. The desired medium agar plates were overlaid with 5 ml of soft medium agar containing 50 ml of an overnight culture of the indicator strain. 10 ml of sample was spotted onto the overlaid surface. The plates were incubated for 6 and 12 h at 37C and determined for antimicrobial substance (AMS) production. The AMS titre was determined by the serial twofold dilution method previously described by MayrHarting et al. (1972). The activity was defined as the reciprocal of the dilution after the last serial dilution giving a zone of inhibition and expressed as activity units (AU) per milliliter. Identification of the LAB Sixteen-hour culture solution was determined for morphology, gram stain, catalase reaction and motility according to Axelsson (1998). Growth of LAB in MRS broth at the temperatures of 10, 45, 50 and 55 C and NaCl concentration of 6% and 18% were determined by turbidity measurement at 600 nm. CO2 production from glucose was examined by the method of Gerhardt et al. (1981). Carbohydrate fermentation patterns were determined by API CHL50 tests (API, BioMeriuex, France) as described by the manufacturer. 16S rRNA analysis was done by the modified method of Nitisinprasert et al. (2000). Primer 1407B (5-GACGGGCGGTGTGTAC-3) and 8UA (5-AGAGTTTGATCCTGGCTC AG3) were used as forward and reverse primers, respectively. Amplification was carried out in a thermo-cycler (Hybaid, UK) with annealing for 2 min at 55 C. The purified DNA fragment was ligated into pGEM-T Easy vector (Promega,
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USA) and then transformed into E. coli DH5a competent cells. The presence of the PCR product ligated to the plasmid was confirmed by whole cell PCR reaction (Nitisinprasert et al., 2000) and restriction analysis with EcoRI under standard condition. Nucleotide sequences were determined by Bioservice unit (BSU, Thailand). The sequences of 16S rDNA clone were aligned with data from GenBank by BLAST analysis. Optimization of growth and AMS production To determine the effect of temperature and pH on growth and AMS production, the culture solution in MRS medium was incubated at the temperatures of 30, 37, 45, 50 and 55C, and the pH of 4, 5, 6, 7 and 8. Culture solution was sampled every 2 h until 24 h and then determined the values of turbidity at 600 nm, viable cell numbers, pH and AMS activity against S. aureus TM67. Preparation of partial purified AMS (ppAMS) Partially purified AMS (ppAMS) was prepared by vacuum evaporator and cation exchange chromatography. The CFS was concentrated to 10-fold by vacuum evaporator. One mililiter of 10-fold concentrated CFS was loaded onto a Sephadex fast flow column equilibrated with 20 mM sodium citrate buffer (pH 3.0). After initial washing with 20 mM sodium citrate buffer (pH 3.0), AMS was eluted with 1 M NaCl in the same buffer at a flow rate of 1 ml/min. The ppAMS obtained was free from lactic acid and acetic acid tested by HPLC with an Aminex HPX87H cation exchanger. The acids were eluted with 8 mM sulphuric acid at the flow rate of 0.5 ml/min and tartaric acid was used as internal standard. Mode of action To determine the mode of action of CFS and ppAMS, 1% (v/v) of the overnight culture solution of S. aureus was inoculated into NB and cultivated at 37C until the cells reached logarithmic phase. Then, CFS at the concentrations
of 100, 200 and 300 AU/ml was added into the culture broth and further cultivated at 37C. For control, equal amount of sterile distilled water was added instead of CFS. Viable cell numbers and turbidity at 600 nm were monitored at 1-h intervals. For ppAMS, 100 AU/ml of ppAMS was directly added into 102 CFU/ml of S. aureus and the number of viable cell was determined at 1-h intervals. twenty mM sodium citrate buffer (pH 3.0) used instead of ppAMS was performed as a control. Structural determination by enzyme treatment A ppAMS was treated with various enzymes including pepsin, trypsin, proteinase K, protease, a-chymotrypsin, papain, a-amylase and lipase. Each enzyme was added to the ppAMS (100 AU/ ml) to obtain a final concentration of 1 mg/ml. The mixture was incubated at the optimum temperature of each enzyme for 18 h. At the end of incubation, the residual activity of AMS was assayed. RESULTS Screening of AMS-producing lactic acid bacteria Fifteen LAB strains isolated from silage and fermented pork were screened for AMS production as shown in Table 1. All CFS exhibited higher inhibition number activity than CFS adjusted pH to 5 and 6. It indicated a synergistic effect between antimicrobial substance and organic acids. The isolate designed as LS2-30 displayed the highest inhibition number against 9 S. aureus strains at different conditions of CFS, CFS adjusted to pH 5 (CFS-5) and CFS adjusted to pH 6 (CFS6). Moreover, the AMS of LS2-30 was active against food-borne and pathogenic bacteria not only gram positive, S. aureus and Bacillus sp., but also gram negative such as E. coli as shown in Table 2. The inhibition activity against those strains appeared at the range of 100 300 AU/ml (data not shown). The highest activity obtained
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Table 1 The antimicrobial activity of LAB 15 isolates against S. aureus 9 strains. LAB %Inhibition number1/ CFS CFS-5 CFS-6 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 0 55.5 66.7 22.2 11.1 55.5 0 77.8 22.2 44.4 44.4 22.2 11.1 55.6 11.1 22.2 0 0 11.1 0 0 0 55.6 0 11.1 0 0 0 11.1 0
CS1-10 CS1-11 CS4-10 CS4-11 LG1-1 LG1-2 LG2-1 LS2-30 LS2-31 LS2-32 N1-9 SG1-1 SG1-2 SK1-3 SK1-4
1/
performed and analyzed by APILAB program. The results showed that LS2-30 was similar to P. pentosaceus at 92.8%. However, the difference in maltose fermentation was found as shown in Table 3. Therefore, 1,500 bp of the 16S rRNA of LS2-30 was analyzed and the result showed 97% identity to P. acidilactici B1104 (accession no. AJ 305322.1). Optimization of growth and AMS production The effects of temperature and the initial pH of medium on growth and AMS production were monitored at various conditions. The temperature effect was performed at 30, 37, 45, 50 and 55C as shown in Figure 1. Similar growth profile was observed at 37 and 45C, however, the maximum activity of 300 AU/ml obtained at 45 C was greater and even maintained for longer time of 24 h. At high temperature of 50 C, both growth and AMS activity reached the maximum after 18 h and then dropped dramatically while the one of 55 C was clearly inhibited after 6 h. It seems that AMS produced after 6 h actively killed all viable target cells at high temperature of 55 C. The initial pH of 4, 5, 6, 7 and 8 displayed a significant effect on AMS production but not on its growth as shown in Figure 2. At initial pH of 4, 5 and 6, AMS production started immediately after growth began and reached a maximum of 200-300 AU/ml during stationary phase. The maximum of AMS activity was observed after 8 h of cultivation at initial pH of 6. At the higher pH of 7, only low activity of 100 AU/ml was detected after 12 h while there was no AMS production found at the initial pH of 8. Mode of action To determine whether the AMS had bactericidal or bacteriostatic effect on the sensitive strains, the AMS treated condition of 100, 200 and 300 AU/ml and a control (without AMS) were performed as shown in Figure 3. Comparing to the control, the number of viable indicator cell from
% Inhibition number = the number of inhibited S. aureus / total S. aureus tested
was against S. aureus, Salmonella sp., Sal. Typhimurium, B. cereus, and B. subtilis. Interestingly, its AMS was not active against all LAB, M. luteus, and L. innocua. Identification of the strain LS2-30 Morphological and biochemical tests were used to identify this isolate. LS2-30 was grampositive, coccus-shaped, tetrad formed, without catalase activity, non-motile and no spore forming. It grew at high temperatures of 45, 50 and 55C, and did not produce gas indicated as homofermentative LAB. It also grew in MRS broth with the pH adjusted to 4.5 and 9.6, and NaCl concentration to 6% but not to 18%. Based on Bergeys Manual of systematic of bacteriology, this isolate was tentatively assigned to the genus Pediococcus. For species determination, carbohydrate fermentation using API 50 CHL was
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Table 2 The antibacterial spectrum of LS2-30. Indicator strain Staphylococcus aureus Escherichia coli Salmonella sp. Salmonella Typhimurium TISTR 292 (ATCC 13311) Bacillus cereus (isolated from silage) Bacillus subtilis TISTR 025 Bacillus coagulans JCM2257 Bacillus subtilis JCM1465 Bacillus circulans JCM2504 Micrococcus luteus IFO12708 Listeria innocua (ATCC33090) Pediococcus pentosaceus JCM5885 Enterococcus faecalis JCM5803 Lactococcus lactis ssp. lactis (ATCC19435) Lactococcus lactis ssp. cremoris TUA1344L Lactobacillus plantarum (ATCC14917) Lactobacillus sakei subsp. sakei JCM1157 Leuconostoc mesenteroides ssp. mesenteroides JCM6124 Strain tested/inhibited 9/9 13/13 3/3 1/1 1/1 1/1 1/1 1/1 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0 1/0
the treated condition decreased immediately while the optical density of cell suspension still remained. This indicated that CFS of LS2-30 was bactericidal without cell lysis. For ppAMS (Figure 4), the viable cell of 2.5 log CFU/ml decreased to less than 10 cfu/ml after 1 h incubation while the control declined slowly. This confirmed bactericidal effect against S. aureus TM67 while low pH of 3 displayed slight inhibition of its growth. Determination of compound structure by enzyme treatment The structure of ppAMS produced from LS2-30 was examined by various enzymes as shown in Table 4. The antimicrobial activity of ppAMS was not affected after treatment with proteinase K, protease type XIII, papain, pepsin A, trypsin type I, a-chymotrypsin type II, a-amylase type X-A and lipase indicating that the ppAMS
was neither proteinaceous, starch nor lipid moiety in nature. DISCUSSION Isolate LS2-30, obtained from silage and identified as P. acidilactici, was selected to study as an AMS producing strain. Recently, a number of antagonistic substances from P. acidilactici were reported, for example, lactic acid (Stiles, 1996), pediocin PA-1, pediocin AcH, pediocin JD, pediocin SJ-1 (Schved et al., 1994), pediocin 5, pediocin L50, pediocin F (Osmanagaoglu et al., 1998), pediocin AcM (Elegado et al., 1997). They all, except lactic acid, had proteinaceous structure. However, the AMS of LS2-30 seems to be different from the results of various enzyme treatments, it didnt show the structure of either protein or complex compounds of protein, starch or lipid belonging to bacteriocin group IV. Therefore, it
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Table 3 Carbohydrate fermentation pattern of LS2-30 compared with P. pentosaceus. Carbohydrate Glycerol Arabinose Ribose Xylose Galactose Glucose Fructose Mannose Rhamnose Mannitol Sorbitol Amygdalin Salicin Cellubiose Maltose Lactose Melibiose Trehalose Inulin Melezitose Raffinose P. pentosaceus + + + + + + + + + + + + LS2-30 + + + + + + + ? + + + + ? + -
Table 4 Factors affecting antimicrobial activity of ppAMS. Enzyme Proteinase K Protease type XIII Papain Pepsin A trypsin type I a-Chymotrypsin type II a-Amylase type X-A Lipase Control Activity (AU/ml) 100 100 100 100 100 100 100 100 100
+ = positive; - = negative; ? = doubtful
could be either tolerate to enzyme or new compound structure. Considering the inhibition ability, LS2-30 has shown the ability to secrete broad spectrum AMS against different non-related genera, including food-borne pathogens such as S. aureus, Bacillus sp., Salmonella sp., and E. coli. Interestingly, no inhibitory activity of LS2-30 was observed against LAB tested. Most bacteriocins produced by P. acidilactici displayed inhibitory effect to both LAB and some pathogenic microorganisms for example nisin, pediocin AcH (Kalchayanand et al., 1992), salivacin 140 and plantaricin 35d (Messi et al., 2001). Since the definition of bacteriocin indicated that it will inhibit
closely related species (Klaenhammer, 1993), therefore, it was possible that the AMS found was not bacteriocin which correspond to above result of structure determination. This was the first report of non-bacteriocin substance excluding organic acid produced by homofermentative lactic acid bacteria. This property is an advantage to use this compound as a biological preservative in the dairy product and fermented food without any effect to LAB used as a starter culture. The AMS from this isolate were secreted into culture medium at the beginning of the logarithmic growth phase and reached the maximum level during the early stationary phase at 45C. Thus, the production of AMS from LS230 was growth-associated. The same pattern occurred with most of active compound produced by other LAB, such as carnocin U149, leucocin S and plancitaricin S. The initial optimum pH of AMS production was pH 6 which was similar to the previous reports (Mortvedt-Abildgaard et al., 1995). In addition, this strain clearly showed that the inhibitory activity remained stable although the pH was down to pH 4 after prolonging incubation, suggesting that the antimicrobial substance was unaffected under acidic conditions. This could be an important asset allowing its use under acidic conditions such as fermented food.
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Both AMS production and cell growth were observed at the same pH of 6.0 which was similar pattern to pediocin PD-1 production from P. damnosus NCFB 1832 (Nel et al., 2001). This should be beneficial to gain the high cell number and its products during fermentation. The decline in number of living cells of S. aureus recorded after the addition of antimicrobial substance suggested that the mode of action was bactericidal activity without cell lysis. Its action was similar to plantaricin 35d properties as reported by Messi et al. (2001). When high cell concentration
of target strain ( 108 cfu/ml ) were applied, the CFC of 100, 200 and 300 AU/ml led to a decrease in viability of only ca. 50% in 7, 4 and 4 h, respectively. However, when low cell concentration of target strain 102 cfu/ml was applied, only low dose of ppAMS (100 AU/ml) could inhibit its growth. This corresponded to the work done by Boucabeille et al. (1997). It was found that linenscin OC2 of 10,520 AU/ml exhibited bactericidal against 108 cfu/ml of L. innocua while low dose of 850 AU/ml did only bacteriostatic action. Therefore, the inhibition efficiency would depend on the
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Figure 1 Influence of temperature on the cell growth (), pH () and antimicrobial substance production () by P. acidilactici LS2-30 at 30 C (a), 37 C (b), 45 C (c), 50 C (d) and 55 C (e).
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291 CONCLUSION
concentration of antimicrobial substance and the cell concentration of the indicator strain as also supported by de Vuyst and Vandamme (1994). The ambiguous structure of ppAMS still remained. Further study on purification and characterization will explain the inhibition mechanism for application uses in food, feed and pharmaceutical industries.
Fifteen LAB isolates have been screened according to AMS production against 9 Staphylococcus sp. strain. One of them designed as LS2-30 was classified as Pediococcus acidilactici by morphological, physical and biochemical tests, and 16S rRNA. The optimum growth temperature and initial pH were 45 C and pH 6, respectively. CFC of LS2-30 showed an inhibitory action against some pathogens and
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10
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Figure 3 Effect of LS2-30 CFS on the viability (a) and absorbance at 600 nm (b) of S. aureus. CFS was added (arrow) at a final dose of 100 AU/ml (), 200 AU/ml (), 300 AU/ml (X) and control (). foodborne bacteria such as S. aureus, Salmonella sp., Bacillus sp., and E. coli . However, it didnt exhibit activity against all LAB tested. When mode of action was studied, the CFS displayed a bactericidal mode of action without cell lysis. The ppAMS at pH 3.0 showed inhibitory activity as bactericidal action at low concentration of 100 AU/ml against 102 CFU/ml of target strain. By various hydrolytic enzymes treatment, it suggested that the AMS was neither proteinaceous, starch nor lipid moiety in nature. Therefore, the nature of inhibitory compound secreted by P. acidilactici LS2-30 is still unclear. ACKNOWLEDGEMENTS This work was supported in part by a research grant from Graduated School, Kasetsart University. The authors gratefully acknowledge National Institutes of Animal Health, Ministry of Agriculture and Cooperatives, for S. aureus used as indicator strains. LITERATURE CITED Axelsson, L. 1998. Lactic acid bacteria: classification and physiology, pp. 1-72. In S. Salminen and A Von Wright (eds). Lactic Acid Bacteria: Microbiology and Functional Aspect. Marcel Dekker, Inc.,
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Figure 4 Effect of ppAMS of LS2-30 on the viability of S. aureus at 102 cfu/ml. ppAMS was added at 100 AU/ml () and 20 mM sodium citrate buffer (pH 3.0) as control ().
New York. Bernet-Camard, M.F, V. Lievin, D. Brassart, J.R. Neeser, A.L. Servin and S. Hudault. 1997. The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antimicrobial substance(s) active in vitro and in vivo. Appl. Environ. Microbiol. 63: 2747-2753. Boucabeille, C., D. Mengin-Lecreulx, G. Henkes, J.M. Simonet and J. van Heijenoort. 1997. Antibacterial and hemolytic activities of linenscin OC2, a hydrophobic substance produced by Brevibacterium linens OC2. FEMS Microbiol. Lett. 153: 295-301. Chen, Y.L. and C.C. Chou. 2005. Factors affecting
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the susceptibility of Staphylococcus aureus CCRC 12657 to water soluble lactose chitosan derivative. Food Microbiol. 22: 2935. Diarra, M.S., D. Petitclerc and P. Lacasse. 2002. Effect of lactoferrin in combination with penicillin on the morphology and the physiology of Staphylococcus aureus isolated from bovine mastitis. J. Dairy Sci. 85: 11411149. Elegado, F.B., W.J. Kim and D.Y. Kwon. 1997. Rapid purification, partial characterization, and antimicrobial spectrum of the bacteriocin, Pediocin ACM, from Pediococcus acidilactici M. Int. J. Food Microbiol. 37: 1-11. Gerhardt, P., R.G.E. Murray, R.N. Costilow, E.W. Nester, W.A. Wood, N.R. Krieg, and G.B. Phillips. 1981. Manual of Methods for General Bacteriology. Washington: American Society for Microbiology. P. 415. Holzapfel, W.H., R. Geisen and U. Schillinger. 1995. Biological preservation of foods with reference to protective cultures, bacteriocins and food-grade enzymes. Int. J. Food Microbiol. 24: 34362. Hu, S., C. Concha, F. Lin and K.P. Waller. 2003. Adjuvant effect of ginseng extract on the immune responses to immunization against Staphylococcus aureus in dairy cattle. Vet. Immunol. 91: 29-37. Kalchayanand, N., M.B. Hanilin and B. Ray. 1992. Sublethal injury makes Gram-negative bacteria sensitive to the bacteriocin, pediocin AcH and nisin. Lett. Appl. Microbiol. 15: 239-243. Klaenhammer, T.R. 1993. Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol. Rev. 12: 39-86. Leitnera, G., E. Lubashevskyb and Z. Traininb. 2003. Staphylococcus aureus vaccine against mastitis in dairy cows, composition and evaluation of its immunogenicity in a mouse model. Vet. Immunol. Immunopathol. 93: 159167. Mayr-Harting, A., A.J. Hedges and R.C.W. Berkeley. 1972. Methods for studying bacteriocins, pp. 313-342. In J.R. Norris and D.W. Ribbon (eds.) Methods in Microbiology (7a). Academic Press, New York.
Messi, P., M. Bondi, C. Sabia, R. Battini and G. Manicardi. 2001. Detection and preliminary characterization of a bacteriocin (plantaricin 35d) produced by a Lactobacillus plantarum strain. Int. J. Food Microbiol. 64: 193-198. Mortvedt-Abildgaard, C.I., J. Nissen-Meyer, B. Jelle, B. Grenov, M. Skaugen and I.F Nes. 1995. Production and pH-dependent bactericidal activity of lactocin S, a lantibiotic from Lactobacillus sake L45. Appl. Environ. Microbiol. 61: 175-179. Nitisinprasert, S., V. Nilphai, P. Bunyun, P. Sukyai, K. Doi and K. Sonomoto. 2000. Screening and identification of effective thermotolerant lactic acid bacteria producing antimicrobial activity against Escherichia coli and Salmonella sp. resistant to antibiotics. Kasetsart J. 34: 387-400. Nel, H.A., R. Bauer, E.J. Vandamme and L.M.T. Dicks. 2001. Growth optimization of Pediococcus damnosus NCFB 1832 and the influence of pH and nutrients on the production of pediocin PD-1. J. Appl. Microbiol. 91: 1131-1138. Osmanagaoglu, O., U. Gunduz, Y. Beyatli and C. Cokmus. 1998. Purification and characterization of pediocin F, a bacteriocin produced by Pediococcus acidilactici F. Tr. J. Biol. 22: 217-228. Rubin, R.J., C.A. Harrington, A. Poon, K. Dietrich, J.A. Greene and A. Moiduddin. 1999. The economic impact of Staphylococcus aureus infection in New York City hospitals. Emerg. Infect. Dis. 5: 9 17. Schved, F., Y. Henis and B.J. Juven. 1994. Response of spheroplasts and chelatorpermeabilized cells of Gram-negative bacteria to the action of the bacteriocins pediocin SJ1 and nisin. Inter. J. Food Microbiol. 21: 305314. Stiles, M. E. 1996. Biopreservation by lactic acid bacteria. Antonie Leeuwenhoek. 70: 331345. de Vuyst, L. and E.J. Vandamme. 1994. Bacteriocin of Lactic Acid Bacteria: Microbiology, Genetics and Applications Blackie Academic and Professional, London, 536 p.
Studies on Nham-Plas Processing by Using Rock Salt and Solar Salt

Mathana Sangjindavong, Pranisa Chuapoehuk and Daungdoen Vareevanich
ABSTRACT Nham-Pla (fermented fish cakes) were made from striped snake-head fish (Channa striata), Nile tilapia (Oreochromis niloticus), striped catfish (Pangasius hypophthalmus), and hybrid catfish (Clarias macrocephalus x Clarias gariepinus). The results of the organoleptic test of uncooked Nham-Pla prepared from rock salt showed differences (p<0.05) in appearance, texture, odor and average acceptability scores with the exception for dark meat flesh from hybrid catfish which received the lowest acceptability score of color. The organoleptic tests of cooked Nham-Pla prepared by using rock salt revealed that Nham-Pla made from striped catfish received the highest acceptability score for the appearance characteristic and there were no differences in odor, taste and average acceptability score for both samples prepared from rock salt and solar salt. Nham-Pla made from Nile tilapia with solar salt received the lowest acceptability score, but they received the highest acceptability score for texture. Nham-Pla made from snake-head fish using solar salt received better acceptability scores than Nham-Pla prepared by using rock salt. According to the results, rock salt and solar salt were important for Nham-Pla preparation based on species of fresh water fish and some of their characteristics. Key words: Nham-Pla, rock salt, solar salt as Oreochromis niloticus, Pangasius hypophthalmus, Channa striata, Clarias spp., Notopterus chitala and Notopterus notopterus for Nham-Plas processing. Sangjindavong et al. (2000) made a preliminary study on Nham-Pla made from fresh water fish and marine fish. Sensory evaluation tests showed that Nham-Pla made from the fresh water fish had a higher acceptability score than Nham-Pla made from marine fish. Fermented fish products are divided into three main categories (Amano, 1962): 1) Traditional products with high salt content which involve enzymes from fish muscle and intestinal organ. Fish sauce and fish paste are categorized in this group. 2) Traditional products with two steps
INTRODUCTION Nham-Pla or sour fish cake is one of fermented fishery products processed from mostly fresh-water fish. Nham-Pla made from Notopterus chitala are found in the market but the shapes of the product and the ingredients were the same as Som-Fug. Usually Som-Fug is made from minced fresh water fish and the ingredients are salt, minced garlic and cooked rice, but for Nham-Pla some sliced pork skin are added the same as Nham-Moo and carrot is used for decorating the products. Because there were a lot of reservoirs in the country, the people who live around that area have the great chance to use some common fresh-water fish such
Department of Fishery Products, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand.
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reaction: the first step involves enzymes from fish muscle and the second step mainly adds carbohydrate or microbial to start the final reaction. Pla-Ra and Pickled fish are samples of this group. 3) Non-traditional products: acid such as hydrochloric acid (HCI) is added in the processing of fish sauce products. Rock salt as a mineral occurs naturally in the ground. It contains 98-99% sodium chloride and it has a water insolubility level of about 0.51.5%, being mainly calcium sulphate. Solar salt as a natural product is obtained mainly through evaporation of seawater. It contains 85% sodium chloride and has a water insolubility level of less than 0.03%. (http://www.lentech. Com/watersoftener-FAQ.htm) The objective of this study was therefore to assess the quality characteristics of fermented fish cake prepared from four common varieties of freshwater fish by comparing Nham-Pla which using rock salt and solar salt. MATERIALS AND METHODS Completely randomized design (CRD) method with four treatments was adopted for this research. Four kilograms of striped snake-head fish (Channa striata), Nile tilapia (Oreochromis niloticus), striped catfish (Pangasius hypophthalmus), and hybrid catfish (Clarias macrocephalus x C. gariepinus) were bought from the market, cleaned, gutted and filleted, followed by grinding for 10 minutes using Kitchen Aid. Small amount of rock salt or solar salt, up to 30 grams, was added to 1,000 grams of fish minced during the grinding process. All other ingredients (Table 1) were added after grinding and then the samples, weighing about 130-150 grams each, were put into 69 plastic tubes, wrapped with rubber bands on both ends, placed on trays and kept at ambient temperature (28C-30C) for 4 days. Sensory evaluation, chemical analysis and microbiological tests were conducted at
fermentation period of 4 days. Flow diagram of Nham-Pla processing is shown in Figure 1. Quality examination Sensory evaluation A sensory panel consisting of 10 persons from the department of Fishery Products was established. Hedonic scale ranged from 1 to 5 was used to indicate the degree of acceptability of each sample (1 = most unacceptable, 5 = most acceptable). The evaluation criteria was manily focused on appearance, color, odor, flavor and texture (Watt et al., 1989). Statistical analyses Table 1 Ingredients processing. Ingredients used in Nham-Pla
Total weight (gms)
Fish minced Pork skin, sliced Rice, cooked Carrot, sliced Garlic, minced Salt (Rock salt, Solar salt) Chilli
1,000 300 100 150 150 30 10-20
Raw material
Filleting Addition of salt while grinding for 10-15 minutes
Addition of other ingredients and mixed
Filling in plastic tubes (size 6x9) about 130-150 gms each
Fermentation
at ambient temperature (28 oC - 30 oC) for 4 days
Analysis
Figure 1 Flow diagram of Nham-Pla.
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were carried out using Randomized completed block design (RCBD) method. The mean from 3 replicates was compared by the methods of Duncans new multiple range test (DMRT) (Khuantham, 1996). Chemical analysis The value of pH was measured from 1:10 diluted samples (Metrohm 744 pH meter). Lactic acid, expressed as total acid, was examined from titrating diluted samples with 0.1 N standard NaOH, using phenolphthaline as pH indicator and calculating the lactic acid concentration using the equation % lactic acid = (ml alkali x normality alkali 9)/ weight of samples in grams (Frazier et al., 1968). NaCl content was determined according to Volhard (AOAC, 1990). Protein, fat and moisture content were determined according to AOAC (1984). Microbiological analysis Total viable aerobic counts (TVC) were performed on Plate count agar (Merck KGaA), and lactic acid bacteria counted on MRS agar (Merck KGaA). Plates were incubated at 35-37 C for 3 days (TVC count) and incubated at 25C for 3 days (lactic acid bacteria counted). Strains of lactic acid bacteria were identified according to Sharpe et al. (1966). RESULTS Chemical analysis
Proximate compositions of Nham-Pla are shown in Table 2. and 3. Sensory evaluation are shown in Table 7 and 8. Microbiological analysis Microbiological studied are shown in Table 4, 5 and 6 DISCUSSION The effect of rock salt and solar salt on Nham-Pla depended on the species of fish and salt characteristics. Flavor of Nham-Pla related to some lactic acid bacteria which might be found in salt. Sangjindavong (1982) reported that 4 genera of bacteria such as Staphylococcus sp., Bacillus sp. Arthrobacter sp. and Corynebacterium sp. were isolated from solar salt. Sangjindavong et al. (2000) studied on Nham-Pla prepared from striped catfish and Nile tilapia and lactic acid bacteria were isolated namely :- Leuconostoc mesenteroides, Lactobacillus plantarum, Pediococcus damnosus, Lactobacillus leichmanii, Lactobacillus delbruekii, Lactobacillus brevis and Lactobacillus acidophilus. For this study, lactic acid bacteria isolated from Nham-Pla were Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus acidophilus and Pediococcus pentosaceus. Some pathogenic bacteria such as Staphylococcus aureus, Clostridium perfringens, Salmonella spp., and fungi were not detected from Nham-Pla.
Table 2 Chemical composition of Nham-Pla using rock salt. Kinds of Nham-Pla Moisture (%) 75.11 68.02 72.73 71.26 pH Lactic acid (%) 2.37 1.78 2.81 2.17 Salt (%) 1.63 1.72 1.76 1.73 Protein (%) 15.58 17.23 18.17 16.97 Fat (%) 1.06 2.15 0.94 3.25 Ash (%) 2.14 2.49 2.06 2.24
Nile tilapia Hybrid catfish Striped snake-head fish Striped catfish
3.81 4.03 4.06 4.05
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Table 3 Chemical composition of Nham-Pla using solar salt. Kinds of Nham-Pla Moisture (%) 75.58 73.49 73.67 73.16 pH Lactic acid (%) 2.48 2.58 2.68 2.32 Salt (%) 1.59 1.70 1.67 1.8 Protein (%) 17.12 16.62 17.22 17.52 Fat (%) 1.07 2.19 1.03 3.01 Ash (%) 2.33 2.56 2.26 2.35
4.06 4.09 4.10 4.09
Table 4 Total lactic acid bacteria in Nham-Pla. Kinds of Nham-Pla Total lactic acid bacteria (CFU/g) Rock salt Solar salt >300 >300 >300 >300 >300 >300 >300 >300
Table 5 Total viable aerobic counts. Kinds of Nham-Pla Total viable aerobic counts (CFU/g) Rock salt Solar salt 4.65 108 5.65 108 3.95 108 1.64 109 2.90 109 9.25 108 6.10 108 3.40 108
Table 6 Coliforms, faecal coliforms and Escherichia coli in Nham-Pla. Kinds of Nham-Pla Coliforms (MPN/gm) Rock salt Solar salt >1100 >1100 >1100 >1100 >1100 >1100 >1100 Faecal coliforms (MPN/gm) Rock salt Solar salt <3 <3 <3 <3 <3 <3 <3 <3 Escherichia coli (MPN/gm) Rock salt Solar salt <3 <3 <3 <3 <3 <3 <3 <3
Nile tilapia Hybrid catfish Striped snake-head fish Striped catfish>1100
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Table 7 Comparative sensory scores of uncooked Nham-Pla using rock salt and solar salt, fermented at ambient temperature for 4 days.
Sensory properties Kinds of Nham-Pla Appearance Texture Color Odor Average acceptability 3.57abc 1.22 4.14a 0.53 3.50abc 0.52 3.71b 0.61 3.21bc 1.05 2.93c 0.62 3.57abc 0.85 3.64ab 0.93
1. Striped snake-head fish 2. Striped catfish 3. Nile tilapia 4. Hybrid catfish 5. Striped snake-head fish 6. Striped catfish 7. Nile tilapia 8. Hybrid catfish
4.21a 0.11 4.04a 0.50 3.43ab 0.65 3.54ab 1.04 3.71ab 0.83 3.15b 0.86 3.79ab 1.05 3.86a 0.85
3.36 ab 0.84
3.00b 1.18 3.71a 0.83 3.57ab 0.65 4.07a 0.73 3.43ab 0.65 3.46ab 0.63 3.43ab 0.76
4.36a 0.11 3.93a 0.83 3.82ab 0.54 3.68a 0.77 3.82ab 0.88 2.96b 0.97 3.54b 1.05 3.43ab 1.16 3.57b 0.94 3.00b 1.11 2.71c 1.00 2.86b 0.86 3.85ab 0.95 3.36 ab 0.84 3.79ab 0.89 3.21ab 0.89
Means followed by different letter are significantly different (P<0.05) 1-4 Nham-Pla; Using Rock salt 5-8 Nham-Pla; Using Solar salt Table 8 Comparative sensory scores of cooked Nham-Pla using rock salt and solar salt, fermented at ambient temperature for 4 days.
Sensory properties Kinds of Nham-Pla Appearance Texture Color Odor Flavor Average acceptability
1. Striped snake-head fish 2. Striped catfish 3. Nile tilapia 4. Hybrid catfish 5. Striped snake-head fish 6. Striped catfish 7. Nile tilapia 8. Hybrid catfish
3.93ab 0.96 4.20a 0.41 3.90ab 0.84 3.47b 0.83 4.00ab 0.76 3.73ab 0.70 2.66c 0.82 4.00ab 0.65
2.87c 1.25 3.67ab 0.90 4.00a 0.85 3.67ab 0.90 3.53b 0.92 3.20ac 0.85 3.33bc 0.90 3.27bc 0.80
4.00a 0.85 4.00a 0.53 3.90a 0.93 3.13b 0.74 3.87a 0.83 3.40ab 0.83 2.53c 0.74 3.67ab 0.72
3.30ab 1.19 3.60a 0.83 3.40ab 0.91 3.60a 0.74 3.32ab 1.14 3.50ab 1.12 2.73b 0.59 3.00ab 1.13
3.13ab 1.25 3.30ab 1.10 3.80a 0.94 3.63a 0.80 3.23ab 1.24 3.63a 1.01 3.93a 0.70 3.53a 0.64 3.47 ab 0.99 3.57a 0.90 3.33 ab 0.98 3.30ab 0.88 2.80b 0.86 2.67b 0.72 3.27ab 0.80 3.40a 0.74
Means followed by different letter are significantly different (P<0.05)

1-4 Nham-Pla; Using Rock salt 5-8 Nham-Pla; Using Solar salt
CONCLUSIONS Sensory properties of uncooked Nham-Pla prepared from striped snake-head fish and striped catfish using rock salt were highest based on the criteria of appearance, color and odor at
fermentation period of 4 days while Nham-Pla made from striped catfish using rock salt gave the highest average acceptability. Sensory evaluation of cooked Nham-Pla prepared from striped catfish and Nile tilapia both using rock salt showed the highest average acceptability. Nham-Pla made
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from hybrid catfish using rock salt and Nham-Pla made from Nile tilapia using solar salt gave the lowest acceptability. Nham-Pla made from striped snaked-head fish using rock salt had better texure than using rock salt. LITERATURE CITED Amano, K. 1962. The influence of fermentation on the nutritive value of fish with special reference to fermented fish products of Southeast Asia, pp. 180-200. In E. Heen and R. Kruezer (eds.). Fish in Nutrition. Fishing News (Books), London. AOAC. 1984. Official Methods of Analysis. 14th edition. Association of Official Analytical Chemistry. Washington. D.C. 1141 p. AOAC. 1990. Official Methods of Analysis. 15th edition. Association of Official Analytical Chemistry. Washington. D.C. 1296 p. Frazier, W. C., E. H. Marth and R. H. Diebel. 1968. Laboratory Manual for Food Microbiology. 4th edition. Burgess Publishing Companys Minneapolis, MN. 122 p. Horie, S. and N. Hinago. 1924. Bacterial flora in
imported solar salt. Bulletin of the Japanese Society of Scientific Fisheries 40(10) : 10591062. Khuantham, A. 1996. Principles of Experimental Designs. Department of Statistics, Faculty of Science, Kasetsart University, Bangkok. 227 p. Saisithi, P. 1987. Traditional fermented fish products with special reference to Thai Products. Asean Food Journal 3(1) : 3-10. Sangjindavong, M, P. Chuapoehuk and N. Raksakulthai. 2000. Quality characteristics of fermented sour fish cake (Nham-pla). International Journal of Food Properties 3(3) : 407-419. Sangjindavong, M. 1982. Studies on bacteria isolated from solar salt selling at Bangkoks market. Food. Jan-March : 56-63. Watt, B. M., G. I. Ylimalei, L. E. Jefferg and L. G. Elias. 1989. Basic Sensory Methods for Food Evaluation. The International Development Research Center, Ottawa, Canada. 160 p. Water softener FAQ. http://www.lentech.com/ water-softener-FAQ.htm. (2/14/2005)
Product Development of Crocodile Jerky

Sinee Nongtaodum1, Nongnuch Raksakulthai2 and Mayuree Chaiyawat2
ABSTRACT Crocodile jerky was developed from freshwater crocodile (Crocodylus siamensis) tail meat. The Ratio Profile Test (RPT) was used to find the most acceptable product. Seasonings (soy sauce, sugar, and pepper) and processing conditions (drying time and temperature, frying time and temperature) were varied. The jerky sample with 8 % soy sauce, 6.5 % sugar, 2.5 % pepper and 3 % white sesame seed, dried at 60C for 2 hours and fried at 160C for 1 minute received the highest acceptability score (P 0.05). The shear force, L*, a*, b* values and aw of the prepared product were 25.8 N., 41.5, 5.3, 9.4 and 0.63, respectively. The proximate composition of the fresh meat was 72.3 % moisture, 20.2 % protein, 5.5 % fat, 1.0 % ash and 0.9 % carbohydrates, and the total viable bacterial count was 1.48 106 CFU/g. The proximate composition of the jerky was 13.9 % moisture, 48.2 % protein, 14.7 % fat, 5.3 % ash and 17.9 % carbohydrates. No microorganism was found in the jerky. The product shelf life was determined according to the thiobarbituric acid number set at 2.5 mg malonaldehyde/Kg by accelerated temperature test. The product was stored in aluminum foil laminated plastic (OPP/LDPE/Al/LDPE/OPP) bags at ambient temperature (303C) under different conditions. The results showed that shelf life was 7 weeks when packed under air, 9 weeks under air with moisture absorber, 14 weeks under air with oxygen absorber and 13 weeks under modified atmosphere of 100 % nitrogen. Key words: crocodile meat, crocodile jerky
INTRODUCTION Freshwater, marine and hybrid crocodiles are farm-raised successfully in Thailand. The most commonly bred species is the freshwater crocodile (Crocodylus siamensis). At present, the number of crocodile farms is increasing. The main purpose of farming is to produce and export crocodile leather products. Crocodile meat, especially from the tail part, is believed to be a nutraceutical and can be cooked with herbs to cure asthma (Maneenopphol, 1998). It is popular among Chinese, Taiwanese, and Korean tourists. Furthermore, canned stew,
1
soup and dried crocodile meat have been processed. In Australia, there are many kinds of crocodile products such as burgers and jerky (Rattanakorn, 1994). Jerky is a product rather similar to Thai semi-dried beef or pork. Since it is light-weight and shelf-stable, making crocodile jerky with a Thai-style taste and flavor might expand the uses of crocodile meat. Therefore, the objectives of this study are as follows: 1. To determine the proximate composition of fresh crocodile meat and prepared crocodile jerky. 2. To develop a process for Thai-style
Department of Food Technology, Faculty of Engineering and Industrial Technology, Silpakorn University, Sanamchandra Palace Campus, Nakorn Pathom 73000, Thailand. Department of Fishery Products, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand.
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crocodile jerky. 3. To set up the thiobarbituric acid (TBA) number as a quality index for the prepared crocodile jerky. 4. To find the shelf life of the prepared crocodile jerky under different storage conditions at ambient temperature. MATERIALS AND METHODS Process development of crocodile jerky Frozen crocodile meat was sliced into thin sheets of approximately 3 6 0.5 cm and marinated with seasonings at 4-6C for 16 hours before drying. The dried product was fried at 160 C for 1 minute. The Ratio Profile Test (RPT) of product using soy sauce (6.0 %), sugar (4.5 %), ground pepper (1.5 %) and sesame seeds (3.0 %) as in semi-dried catfish sticks (Department of Fishery Products, 2000) was conducted. The marinated crocodile meat was dried at 60C for 3 hours then fried at 160C for 1 minute. Sensory evaluation by 10-trained panelists for color, spice odor, hardness, toughness, sweetness, saltiness and aftertaste was carried out. To develop the most acceptable product, appropriate amounts of seasoning and spice were studied by varying one factor at a time. The variables were soy sauce (8, 8.5 and 9.0 % w/w of crocodile meat); sugar (5.0, 6.5 and 8.0 % w/w) and pepper (1.5, 2.0 and 2.5 % w/w). The prepared samples were sensory evaluated by 30 panelists for appearance, color, odor, taste, texture and overall acceptability using a 9-point hedonic scale (1 = dislike extremely and 9 = like extremely). The experimental design used was a randomized completely block design. The scores were statistically analyzed for ANOVA, and Duncans new multiple range tests were employed for comparison among sample means. Optimal drying temperature and time of product with the highest acceptability score were
determined at 50 and 60C for 1.5 and 2 hours. The experimental design was 22 factorial. The samples were sensory evaluated as above. Quality of crocodile jerky Shear force of the prepared crocodile jerky with the highest acceptability score was measured with the TA-HD Texture Analyzer. L*, a* and b* were measured by the Minolta CM-3500. aw was measured with a Thermoconstanter Novasina TH 200. Proximate compositions of fresh crocodile meat and crocodile jerky were analyzed according to AOAC (1984). Total viable bacterial and Salmonella counts were determined according to FDA (1984). Deterioration at an accelerated temperature The prepared crocodile jerky was placed on a glass plate with a cover and stored at 50C. Samples were taken for the TBA number analysis (Woods and Aurand, 1977; Shibata and Kinumaki, 1979) and the rancid odor was determined by 5 trained panelists every day. The TBA number at the point that rancidity could be detected by the panelists was considered as the end of storage time. Shelflife Samples (20 g each) of prepared crocodile jerky were packed in 12 18 cm aluminum foil laminated plastic bags (OPP/PE/Al/PE/OPP of 20/25/7/20/30 microns thickness) and stored at ambient temperature (303C) under different conditions namely: air, air with a moisture absorber bag (silica gel), air with an oxygen absorber bag (ferric) and modified atmosphere of 100 % nitrogen. Samples were taken once a week for the TBA number analysis until TBA number exceeded the predetermined value. The experimental design was a split plot with packaging conditions as a main plot and storage time as a subplot.
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RESULTS AND DISCUSSION Process development of crocodile jerky RPT results are shown in Table 1. The ratios of ideal and sample scores on toughness and hardness were higher than 1.0, but those of spice odor, sweetness and saltiness were lower than 1.0. Therefore, drying time and temperature, amount of spice and seasonings were adjusted in the next step. Sensory evaluation of samples prepared with varying amounts of soy sauce, sugar and pepper contents are shown in Tables 2-4. It may be concluded that the appropriate amounts of soy sauce, sugar and pepper were 8, 6.5 and 2.5% by weight of the crocodile meat, respectively. It was found that drying time and temperature significantly affected the texture of the product (P 0.05). Shear force of products dried at 50 and 60C is shown in Table 5. Sensory evaluation of samples is shown in Table 6. The sample dried at 60C for 2 hours received the highest sensory score for texture with the highest shear force. The appearance, color and overall acceptability scores were the highest as well. It was recommended that jerky with the size of 12.5 5 0.6 cm should be dried at a temperature between 60 and 65C for at least 4 h to keep it from spoiling (http://www.alljerky.com/ wwwboard/wwwboard.html). The size of samples in this study were smaller, thus the drying time required at the same drying temperature was only 2 h. Quality of crocodile jerky Proximate compositions, total bacterial, Salmonella counts of fresh crocodile meat, and prepared crocodile jerky are shown in Table 7. Mitchell et al. (1995) reported that the average proximate compositions of fresh meat of Crocodylus porosus and C. johnstoni were 75.9 % moisture content, 21.1 % protein, 1.9 % fat and
0.95 % ash while Baek and Cadwallader (1997) reported the proximate composition of the tail meat of Alligator mississippiensis to be 29.1 % protein and 2.9 % fat. The fat content of Crocodylus siamensis from our study was as high as 5.5 % which might be due to its feed. However, it could be observed that the fat layer was separated from the muscle. It was found that the process of crocodile jerky was effectively in reducing the number of bacteria. The product had a shear force, L*, a* and b* and aw of 25.8 N, 41.5, 5.3, 9.4 and 0.63, respectively. Deterioration at an accelerated temperature The TBA number was chosen as a quality index in this study since it has been generally used to test rancidity in meat (Green and Cumeze, 1982). In general, the acceptable TBA value in food is less than 20 mg malonaldehyde/Kg sample (Shamberger et al., 1977). However, different types of food have different TBA values for a threshold of rancid odor, e.g., in cooked ground pork and beef, the values are 0.5-1.0 and 0.6-2.0 mg malonaldehyde/Kg, respectively (Tarladgis et al. 1960). For frozen and canned fish, it was reported as good quality when the TBA number was less than 3.0, however they were still acceptable when the number increased to 4-27 mg malonaldehyde/Kg (Shamberger et al. 1977). For fishmeal, the initial TBA number was reported at 21, and a strong rancid odor was found at around 300 mg malonaldehyde/Kg (Green and Cumeze, 1982). Rancid odor in semi-dried catfish sticks could be detected when the TBA number exceeded 2.1 mg malonaldehyde/Kg (Pongchawee, 1994). The initial TBA number of dried fish was 3.1 (Pigott and Tucker, 1990). The panelists accepted fried pork sticks if the TBA number was lower than 3.54 mg malonaldehyde/Kg (Niyomkiatkul, 1986). It was found that panelists could detect a rancid odor when the TBA number of the prepared
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jerky exceeded 2.5 mg malonaldehyde/Kg. Therefore, this value was used as an index of quality during the shelf life study. Shelflife TBA numbers of crocodile jerky stored under different conditions are shown in Figure 1. Packaging conditions and storage time significantly affected the TBA number (P 0.05). The TBA values of sample stored under air showed a trend to be higher than those stored under air with moisture absorber, under modified atmosphere with nitrogen or under air with oxygen absorber. It was also found that the TBA values of samples stored under air with oxygen absorber during the 12, 13 and 14th week were not significantly different (P > 0.05). According to the predetermined value Table 1 Ratio Profile Test of crocodile jerky. Attribute Color Spice odor Toughness Hardness Sweetness Saltiness Aftertaste Ideal score (I) 5.70 6.57 4.40 4.12 5.51 5.21 0.50
of TBA from the accelerated storage condition, the shelf life of the products packed under air, air with a moisture absorber, air with an oxygen absorber and 100 % nitrogen were 7, 9, 14 and 13 weeks, with the TBA numbers of 2.53, 2.63, 2.52 and 2.55 mg malonaldehyde/Kg samples, respectively. The crocodile jerky product of Australia with 2 % fat was reported to have a shelflife of at least 1 year (www.alljerky.com/jerky_product.html). The shorter shelflife of our products was resulted from the higher fat content (14.7 %). CONCLUSION Thai-style crocodile jerky can be prepared from the tail meat of cultured freshwater crocodile with the acceptability scores of 7-8 (like moderately
Sample score (S) 6.12 5.18 5.00 5.73 4.32 4.73 0.52
S/I 1.08 0.78 1.14 1.43 0.79 0.91 1.04
Table 2 Sensory evaluation scores of crocodile jerky with different soy sauce contents. Sensory attribute 8.0 % Appearance Color Odor Flavor Texture Overall acceptability
NS
Sensory evaluation score NS Soy sauce content 8.5 % 6.8 6.9 6.8 6.9 6.8 6.9
9.0 % 6.8 6.8 6.8 6.8 7.0 6.8
6.9 7.0 6.9 7.0 6.8 7.1
Not significantly different.
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to like very much). TBA number can be used effectively as a quality index. Product packed in OPP/PE/Al/PE/OPP bag with oxygen absorber could be kept for 14 weeks. However, since
deterioration of crocodile jerky is found to result from rancidity, the use of vacuum packaging might be able to extend shelf life of this product for longer than 14 weeks.
Table 3 Sensory evaluation scores of crocodile jerky with different sugar contents. Sensory attribute 5.0 % Appearance Color Odor Flavor Texture Overall acceptability
1Values
Sensory evaluation score1 Sugar content 6.5 % 7.2 a 7.2 a 7.2 a 7.5 a 7.3 a 7.5 a
8.0 % 7.3 a 7.2 a 7.2 a 7.4 a 7.2 a 7.4 a
7.1 a 7.1 a 7.0 a 7.2 b 7.2 a 7.2 b
in the same row followed by different letters are significantly different (P 0.05).
Table 4 Sensory evaluation scores of crocodile jerky with different pepper contents. Sensory attribute 1.5 % Appearance Color Odor Flavor Texture Overall acceptability
1
Sensory evaluation score1 Pepper content 2.0 % 7.4 a 7.3 a 7.3 b 7.4 b 7.1 a 7.4 b
2.5 % 7.4 a 7.4 a 7.6 a 7.8 a 7.2 a 7.7 a
7.4 a 7.3 a 7.3 b 7.1 c 7.1 a 7.1 c
Values in the same row followed by different letters are significantly different (P 0.05).
Table 5 Shear force of fried crocodile jerky dried at 50 or 60C for 1.5 or 2.0 hours. Shear force (Newton)1 Drying time (hours) 50 C 1.5 2.0
1
Drying temperature
60 C 14.1 c 20.4 a
12.4 d 16.0 b
Values followed by different letters are significantly different (P 0.05).
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Table 6 Sensory evaluation scores of fried crocodile jerky dried at different times and temperatures. Sensory attribute 50 C Appearance Drying time 1.5 h 2.0 h Means Color Drying time 1.5 h 2.0 h Means Odor Drying time 1.5 h 2.0 h Means Flavor Drying time 1.5 h 2.0 h Means Texture Drying time 1.5 h 2.0 h Means Overall acceptability Drying time 1.5 h 2.0 h Means 6.7 7.0 6.9 B 6.2 6.8 6.5 B 7.2 7.2 7.2 7.3 7.3 7.3 6.5 6.9 6.7 B 6.9 7.0 7.0 B Sensory evaluation score Drying temperature 60 C 6.9 7.5 7.2 A 6.7 7.6 7.1 A 7.2 7.3 7.2 7.4 7.5 7.4 6.8 7.4 7.1 A 7.1 7.3 7.2 A
Means 6.8 b 7.3 a
6.5 b 7.2 a
7.2 7.2
7.4 7.4
6.6 b 7.2 a
7.0 b 7.2 a
Means in the same row followed by different letters (A, B) are significantly different (P 0.05). Means in the same column followed by different letters (a, b) are significantly different (P 0.05).
Table 7 Proximate compositions and microbiological quality of crocodile meat and jerky. Composition and microbiological quality Moisture (%) Protein (%) Fat (%) Ash (%) Carbohydrate (%) Total bacterial count (CFU/g) Salmonella (CFU/g)
N.D. not detected.
Crocodile meat 72.3 20.2 5.5 1.0 0.9 1.48 106 N.D.
Crocodile jerky 13.9 48.2 14.7 5.3 17.9 N.D. N.D.
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TBA Number (mg malonaldehyde/kg)
air air + moisture absorber 100 % nitrogen air + oxygen absorber
10 11 12 13 14 15 16 17
Storage time (week)
Figure 1 Change of TBA number of crocodile jerky stored under different conditions at ambient temperature.
LITERATURE CITED AOAC. 1984. Official Methods of Analysis. 14th ed.; Association of Analytical Chemists, Washington D.C. 1141 p. Baek, H.H., and K.R. Cadwallader. 1997. Aroma volatile in cooked alligator meat. J. Food Sci. 62(2): 321-325 Department of Fishery Products. 2000. Freshwater Fish Processing. Faculty of Fisheries, Kasetsart University 48 p. FDA. 1984. Bacteriological Analytical Manual for Food. 16th ed. Bureau of Food, Division of Microbiology. Washington D.C. 224 p. Green, B. E. and T. H. Cumeze. 1982. Relationship between TBA numbers and inexperienced panelists assessment of oxidized flavor in cooked beef. J. Food Sci. 47: 52-54, 58. http://www.alljerky.com/jerky_product.html. 11 Feb. 2005. http://www.alljerky.com/wwwboard/ wwwboard.html. 11 Feb. 2005. Maneenopphol, P. 1998. Basic knowledge in
crocodile farming. Modern Kaset. 8(3):110. Mitchell, G.E., A.W. Reed and D.B. Houlihan. 1995. Composition of crocodile meat (Crocodylus porosus and C. johnstoni). Food Australia 47:321-325. Niyomkiatkul, J. 1986. Effect of Butylated Hydroxy Anisole, Butylated Hydroxy Toluene and Sorbate on Quality of Pork Stick. M.S. Thesis. Kasetsart University. Bangkok. Pigott, G. M. and B. W. Tucker. 1990. Seafood: Effects of Technology on Nutrition. Marcel Dekker, Inc. New York. 362 p. Pongchawee, K. 1994. Production of Semi-dried Catfish Stick and Shelf Life under Modified Atmosphere Packaging. M.S. Thesis. Kasetsart University. Bangkok. Rattanakorn, P. 1994. Utilization of crocodile. Crocodile News 3(2): 5. Shamberger, R.G., B. A. Shamberger and C. E. Willis. 1977. Malonaldehyde content of food. J. Nutr. 107: 1404-1409.
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Shibata, N. and T. Kinumaki. 1979. An improvement of TBA procedure as the measure of the oxidative determination occurring in fish oil: II- Intactsample procedure. Bull. Jap. Soc. Sci. Fish. 45: 505509. Tarladgis, B.G., B.M. Watt, M.T. Younathan and L.R. Dugan, Jr. 1960. A distillation method
for quantitative determination of malonaldehyde in rancid foods. J. Am. Chem. Soc. 37: 44-48. Woods, A.E. and L.W. Aurand. 1977. Laboratory Manual in Food Chemistry. TheAVI Publishing Co., Inc., Westport, Connecticut. 72 p.
Utilization of Fish Flour in Canned Concentrated Seasoning Stock for Thai Foods Preparation
Plernchai Tangkanakul, Payom Auttaviboonkul, Patcharee Tungtrakul, Mantana Ruamrux Chidchom Hiraga, Kanjanarat Thaveesook and Montatip Yunchalad
ABSTRACT Herring fish flour possessed high contents of protein, calcium and phosphorus at 64.70 g/100 g, 2,576 mg/100 g and 1,531 mg/100 g, respectively. Herring fish flour was used to replace fresh or dehydrated fish meat in developing five canned concentrated seasoning stocks for Thai food; Nam Ya Pla (fish curry sauce), Kaeng Som (spicy sour mixed vegetable), Kaeng Tai Pla (southern hot curry), Kaeng Kua Fag (red curry with wax gourd), and Kaeng Tae Po (red curry with swamp morning glory). Sensory test exhibited that a suitable amount of herring fish flour incorporated was 15% or 18% of curry paste, that accounted for 2.6-5.1% in the recipe. It was determined that 100 g canned stocks provided protein, calcium and phosphorus as 4.32 6.00 g, 126.5 136.4 mg and 83.9 108.6 mg, respectively. Calcium content of fish flour incorporated dishes was high, supplying 10-25% of the adult Recommended Daily Dietary Allowance in a 100 g portion. Key words: fish powder, Thai curry, Thai dishes, processed foods, calcium, canned concentrated seasoning stock
INTRODUCTION Fish and fish products attracted food technologists in development of varieties of food due to their marvellous potential and health benefits (Kinsella, 1986; Kinsella et al., 1990). Fish is an excellent source of protein and rich in vitamins and minerals (Guthrie, 1983). In Thailand, it was reported that over 40 % of animal proteins consumed were derived from fish, since a wide spreading in rivers, lakes and rice fields (FAO, 1982). Besides its wide distribution, the price is relatively lower than other animal proteins. Surprisingly, in 58 developing countries, more than 20% of animal protein supply came from fish (FAO, 1982). It implied that there must be
thousands of recipes prepared from fishes available around the world. Micronutrients in fish are renowned, however, with different quantities depending on the type of fishes. For example, cod, a white meat fish, contains 60 mg sodium, 9 mg calcium and 0.1 mg iron, where as 123 mg, 60 mg and 1.2 mg, respectively, were found in herring, which determined from 100 g raw edible portion (Holland et al., 1993). A gutted herring contained calcium as high as 231-597 mg per 100 g fresh weight (Tahvonen et al., 2000). Common fishes used in Thai cooking are fresh water fishes, such as snakehead fish, catfish and common silver barb. These fishes contained calcium in a range of 1832 mg, 0.61.0 mg iron and 189287 mg phosphorus per
Institute of Food Research and Product Development, Kasetsart University, Bangkok 10900, Thailand.
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100 g raw edible portion (Institute of Nutrition, 1999). In Thailand, the sources of calcium could come from dark green vegetables, dried shrimp and small fishes. A national survey on dietary intake of mineral in 1995 reported that calcium consumption per day per person for Thai people was 344 mg (Department of Health, 1995), while the recommendation amount is 800 mg (Committee on Recommended Daily Dietary Allowances, 1989). Thus, if there is herring in a convenient form available in a market, it could supply as a good source of calcium in fish-containing Thai dishes. From health information, it is well-known that calcium is related to health due to a property on reducing the risk of developing osteoporosis. At present, Thai people are concerned more about food safety and health foods. And food producers trend to produce easy-to-cook/ convenience products, serving a modern life style. Hence, the objectives of this study were to evaluate potential of utilizing herring fish flour into ten different Thai dishes and to develop five most accepted dishes into canned concentrated seasoning stocks. MATERIALS AND METHODS Herring fish flour was obtained from the Norwegian Herring Oil and Meal Industry Research Institute and kept at 18C until used. Ten popular dishes of Thai foods were selected; Kaeng Luang (yellow curry), Pad Thai (stir fried rice noodles), Kaeng Pa (spicy curry without coconut milk), Pad Khee Mao (spicy stir fried noodles), Kaeng Tai Pla (southern hot curry), Kaeng Kua Fag (red curry with wax gourd), Nam Ya Pla (fish curry sauce), Kaeng Liang (spicy vegetable soup), Kaeng Som (spicy sour mixed vegetable) and Kaeng Tae Po (red curry with swamp morning glory). The nature of these ten dishes was possible to add a fish ingredient. Dish description and their major ingredients were presented as followed:
Kaeng Luang
Soup-type dish; dried chilli, turmeric root, garlic, shallot, shrimp paste, tamarind juice, palm sugar, fish sauce, raw papaya, fish. Pad Thai Stirred-fried dish; rice noodle, fish flour, hard tofu, fresh bean sprouts, Chinese chive. Kaeng Pa Soup-type dish; chilli, shallot, garlic, shrimp paste, lemon grass, galanga, coriander root, kaffir lime peel, coriander seed, cumin, pepper, fish, bamboo shoot, long-yard bean, baby corn, brinjal, fingerroot (krachai), holy basil leaves. Pad Khee Mao Stirred-fried dish; rice noodle, pepper, garlic, fish flour, onion, tomato, baby corn, cabbage, holy basil leaves. Kaeng Tai Pla Soup-type dish; chilli, shallot, garlic, lemon grass, galanga, kaffir lime peel, turmeric, shrimp paste, fish, fermented fish viscera, bamboo shoot, brinjal, plate brush eggplant, long-yard bean. Kaeng Kua Fag Soup-type dish with coconut milk; chilli, shallot, garlic, galanga, lemon grass, shrimp paste, coconut milk, dried fish, wax gourd, kaffir lime leaves. Nam Ya Pla Sauce with coconut milk; chilli, shallot, fingerroot, garlic, galanga, lemon grass, shrimp paste, coconut milk, fish. Kaeng Liang Soup-type dish; shallot, pepper, shrimp paste, fingerroot, angled gourd, straw mushroom, pumpkin, baby corn, ivy gourd leaves, hairy basil leaves, fish. Kaeng Som Soup-type dish; dried red chilli, shallot, fingerroot, shrimp paste,
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Kaeng Tae Po
fish, tamarind juice, Chinese radish, long-yard bean, Chinese cabbage. Soup-type dish with coconut milk; dried red chilli, shallot, garlic, galanga, lemon grass, pepper, kaffir lime peel, coriander root, shrimp paste, coconut milk, dried fish, tamarind juice, kaffir lime juice, swamp morning glory, kaffir lime leaves.
texture and overall acceptance. The first test was examined when ten dishes at any four different percentages of fish flour; 6, 8, 10, 12, 15 or 18% of curry paste were added. The result was used to indicate five most acceptant dishes at the best incorporated amount of fish flour. The second test was run to evaluate taste preference of five selected dishes prepared from canned concentrated seasoning stock and their controls, the traditional dishes. Five dishes which were prepared from canned concentrated seasoning stock were mixed with vegetables and water as shown in Table 1. Processing concentrated seasoning stocks Concentrated seasoning stocks of five most satisfied dishes, Nam Ya Pla, Kaeng Som, Kaeng Tai Pla, Kaeng Kua Fag and Kaeng Tae Po, were selected for canning. The pHs of the five dishes were 5.97, 4.37, 5.73, 5.99 and 5.07, respectively. The ingredients of five fish flour incorporated concentrated seasoning stock are shown in Table 2. All concentrated stocks were packed in lacquered tinplate can size No. 2 (307409) and processed at
Sensory evaluation of fish flour incorporated Thai dishes Sensory evaluation was carried out using 25 panellists with a 9-hedonic scale method (9 = like extremely, 8 = like very much, 7 = like moderately, 6 = like slightly, 5 = neither like nor dislike, 4 = dislike slightly, 3 = dislike moderately, 2 = dislike very much, 1 = dislike extremely). Two sensory tests were carried out. The interested attributes were colour, consistency, odour, taste,
Table 1 Preparation of five Thai dishes from canned concentrated seasoning stocks. Product Nam Ya Pla Kaeng Som Kaeng Tai Pla Kaeng Kua Fag Kaeng Tae Po Seasoning stock,1 can (g) 560 580 570 565 580 Water (g) 100 120 150 150 100 Vegetables (g) 520 515 400 275
Table 2 Ingredients of five canned concentrated seasoning stocks (g/100g total weight). Product Nam Ya Pla Kaeng Som Kaeng Tai Pla Kaeng Kua Fag Kaeng Tae Po Fish flour 4.5 4.6 5.1 4.0 2.6 Spices 10.0 10.0 11.6 8.2 7.1 Condiment 4.8 20.0 17.4 8.5 18.7 Coconut milk 80.7 79.3 71.5 Chicken stock 65.4 65.9 -
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121C for 60 min, except Kaeng Som which was processed for 30 min. Chemical analysis Herring fish flour, canned concentrated seasoning stock, foods prepared from concentrated seasoning stock and its control recipe of five Thai dishes were analyzed for nutritional compositions. Proximate compositions; protein, fat, ash and moisture content were determined by methods of AOAC (1984). Total dietary fibre was carried out by enzymaticgravimetric method (Faulks and Timms, 1985; Prosky et al., 1985). The level of potassium, sodium, iron, copper, zinc, cadmium, lead, calcium and magnesium were analyzed through Atomic Absorption Spectrophotometric method followed AOAC (2000). The phosphorus content was determined by means of colourimetric method (AOAC, 1998). Consumer test Two hundred and eighty test subjects were randomly selected for an acceptance test. Three groups of people: 150 students, 80 teachers and 50 workers, were recruited. Each food prepared from canned fish flour concentrated seasoning stock (Table 1) was evaluated for its attributes of colour, odour, taste and overall acceptability. Verbal scale with 5 categories as follows: like very much, like, neither like nor dislike, dislike, dislike very much, was used for colour, odour and taste. For overall acceptability, only 3 categories scale was used, i.e., accepted, neither accepted nor unaccepted and unaccepted. Statistical analysis One way analysis of variance and Duncans multiple range test were applied to the results of sensory data by using IRRISTAT software package (IRRISTAT version 90-1). Significance of differences of five dishes between control and its herring fish flour incorporated dish was evaluated
using students t-test at levels of 0.01 and 0.05. RESULTS AND DISCUSSION Nutrients of herring fish flour Proximate analysis of herring fish flour is shown in Table 3. Herring fish flour contained 64.70 g protein/100 g which exhibits a good protein source. However, not only the protein content but also the quality of protein is important to health. Essential amino acid score is generally considered the most logical method to assess protein quality. The pattern of essential amino acids in herring fish flour protein compared to the reference protein according to FAO/WHO (1973) revealed that all of essential amino acid contents are greater than 80% of FAO/WHO suggestion (data not shown). It could be implied that incorporating fish flour into food products would help improving nutritional status to combat malnutrition problem. Fat content was 10.52 g/100 g which was higher than fresh water fish such as dried snake-head fish, 7.0 g/100 g (Ministry of Public Health, 1987). Ash content was substantially high which had a direct correlation with the quantities of the minerals present (Table 4). Among them calcium and phosphorus level were substantially high. Thus, herring fish flour would be useful as a source of many important minerals. Phithakpol (1984) studied on calcium and phosphorus contents of roller dried fish from sardine and threadfin bream. The results showed that both minerals were noticeably lower than those in the herring fish flour (Table 4). The level of harmful heavy metals, cadmium and lead, in tested herring fish flour was low. Sensory evaluation of ten Thai foods utilizing herring fish flour Ten different Thai dishes which incorporated with four various percentages of herring fish flour were studied for sensory evaluation on six attributes; colour, consistency,
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odour, taste, texture and acceptability (Table 5). Percentages of added herring fish flour in the selected dishes were varied depending on the nature of each dish. It was found that there were many limitations for amount of herring fish flour adding. Foods with small amount of herbs and spices could incorporate less fish flour than foods with high amount of spices, which could be observed in Pad Thai and Pad Khee Mao. Colour of most dishes was darkened when greater amount of herring fish flour was added, lead to lower acceptability, for example, Pad Thai, Pad Khee
Mao, Nam Ya Pla and Kaeng Som. From the results, herring fish flour odour was detected in two dishes of stirred fried noodle, Pad Thai and Pad Khee Mao. Fish flour odour in soup-type dishes were not substantially detected as indicated by less score variation. Texture of both stirred fried noodle dishes was influenced by high percentage of fish flour. The effect was mouthcoating of fish flour particles on the tongue. For souptype dishes, sandy mouth feel was reported when high amount of fish flour was applied. This effect was less pronounced in the
Table 3 Proximate analysis of fish flours, processed foods and foods (per 100 g sample). Raw material Moisture (g) Protein (g) Fat (g) Ash (g) Dietary fibre (g) CHO* (g) Energy (Kcal)
Fish flour Herring 9.31 64.70 10.52 10.81 Sardinea 5.55 75.20 5.26 5.19 a Threadfin bream 6.59 74.51 7.41 5.15 Canned concentrated seasoning stocks Nam Ya Pla 77.92 4.97 11.23 2.25 Kaeng Som 84.15 4.59 1.41 3.29 Kaeng Tai Pla 83.70 6.00 1.67 4.20 Kaeng Kua Fag 75.36 5.09 12.46 3.10 Kaeng Tae Po 65.65 4.32 13.85 2.70 Foods prepared from canned concentrated seasoning stocks Nam Ya Pla 76.86 5.49 11.04 2.31 Kaeng Som 87.91 3.13 0.79 2.10 Kaeng Tai Pla 86.77 4.05 1.30 2.24 Kaeng Kua Fag 82.92 3.56 6.05 1.78 Kaeng Tae Po 76.20 3.74 7.47 2.42 Traditional foods (control) Nam Ya Pla 80.14 3.74 11.23 1.86 Kaeng Som 89.11 2.58 0.54 1.83 Kaeng Tai Pla 87.71 3.68 1.09 2.01 Kaeng Kua Fag 85.34 2.88 5.38 1.62 Kaeng Tae Po 75.92 3.16 7.24 2.29
* carbohydrate, expressed as : 100 (protein + fat + ash + dietary fibre + moisture) ** crude fibre a source: Phithakpol, 1984
4.13 0.72** 0.10** 3.34 1.78 1.52 2.25 2.47 3.89 1.66 1.44 2.31 2.22 2.76 1.99 1.39 2.14 2.33
0.53 8.80 6.34 0.29 4.78 2.91 1.74 11.01 0.41 4.41 4.20 3.38 7.95 0.27 3.95 4.12 2.64 9.06
355.60 122.11 40.40 50.67 139.46 185.97 122.96 37.25 44.70 82.21 113.99 117.11 30.98 41.01 70.50 114.04
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dishes with coconut milk. Overall acceptability was evaluated. And dishes from top five at the highest incorporated percentage (Table 5) were selected for further canning process of concentrated seasoning stock. The result of selected dishes with fish flour content were Nam Ya Pla, 18% fish flour; Kaeng Tae Po, 15% fish flour; Kaeng Tai Pla, 15% fish flour; Kaeng Kua Fag, 15% fish flour and Kaeng Som, 15% fish flour. It was found that herring flour applied in this study did not have any critical characteristic on taste, odour or consistency of the dishes. A
similar research work was done in 1981 by Phithakpol et al., demonstrated that the incorporated roller-dried sardine and threadfin bream had more effect on taste and odour than on colour and consistency. Chemical compositions Canned concentrated seasoning stock Nutrient compositions of the five canned concentrated seasoning stock at zero month are exhibited in Table 3. Protein content of all products fell in the range of 4.32 6.00 g/100 g. Kaeng Tai Pla contained the highest protein content. When
Table 4 Minerals contents in fish flours, processed foods and foods (mg per 100 g sample). Raw material Na Ca P Fe Cu Pb K Mg Zn Cd
Fish flour Herring 706.9 2,576 1,531 10.66 1.69 0.0064 904.4 140.25 11.36 0.03 a Sardine n/d 970 708 5.66 0.80 0.20 n/d n/d 4.52 0.05 Threadfin bream a n/d 514 692 1.59 0.40 0.21 n/d n/d 3.50 0.02 Canned concentrated seasoning stocks Nam Ya Pla 506.6 126.5 102.4 1.21 0.11 n/d n/d n/d n/d n/d Kaeng Som 861.2 135.1 83.9 0.86 0.08 n/d n/d n/d n/d n/d Kaeng Tai Pla 1,000.6 136.4 92.9 1.82 0.10 n/d n/d n/d n/d n/d Kaeng Kua Fag 734.6 129.8 108.6 1.04 0.17 n/d n/d n/d n/d n/d Kaeng Tae Po 591.8 135.3 87.1 1.00 0.21 n/d n/d n/d n/d n/d Foods prepared from canned concentrated seasoning stocks Nam Ya Pla 1,055.0 204.8 175.8 1.83 0.20 n/d n/d n/d n/d n/d Kaeng Som 971.0 122.3 123.4 1.26 0.10 n/d n/d n/d n/d n/d Kaeng Tai Pla 1,144.0 142.5 111.8 1.28 0.23 n/d n/d n/d n/d n/d Kaeng Kua Fag 754.0 90.9 109.9 1.10 0.19 n/d n/d n/d n/d n/d Kaeng Tae Po 1,112.0 119.3 144.2 2.31 0.34 n/d n/d n/d n/d n/d Traditional foods (control) Nam Ya Pla 1,008.6 106.6 138.3 1.46 0.14 n/d n/d n/d n/d n/d Kaeng Som 822.3 70.2 75.4 1.03 0.13 n/d n/d n/d n/d n/d Kaeng Tai Pla 1,037.3 104.0 109.0 1.32 0.11 n/d n/d n/d n/d n/d Kaeng Kua Fag 736.9 63.3 83.9 0.89 0.18 n/d n/d n/d n/d n/d Kaeng Tae Po 996.6 47.6 68.7 1.44 0.31 n/d n/d n/d n/d n/d
source: Phithakpol, 1984 n/d = not determined
a
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Table 5 Sensory evaluation of fish flour incorporated in 10 kinds of Thai dishes. % Fish flour in curry paste Colour Consistency Odour Taste Texture Kaeng Luang 8% 7.34a 6.86a 7.05a 7.02a 6.86a 10% 7.14ab 6.50a 6.79ab 6.64a 6.76a 12% 7.24ab 6.86a 6.98ab 6.79a 6.88a b a b a 15% 6.95 6.71 6.68 6.62 6.52a Pad Thai 6% 7.53a 6.78a 7.20a 7.32a 7.22a 8% 7.40a 6.70a 7.05a 7.10a 6.85a b b b b 10% 6.32 5.88 6.38 6.65 6.07b c b b b 12% 5.60 5.55 6.00 6.30 5.65b Kaeng Pa 8% 7.35a 7.10a 7.28a 7.35a 7.20a 10% 7.40a 6.95a 7.03a 7.20ab 6.90a a a a a 12% 7.38 7.07 7.03 7.35 7.00a a a a b 15% 7.25 6.75 6.60 6.93 6.20b Pad Khee Mao 7.34a 7.26a 7.24a 7.34a 6% 7.55a a a a a 8% 7.24 7.03 6.97 7.16 6.71b 10% 6.68b 6.03b 6.42b 6.63b 5.66c 12% 6.13c 5.68b 5.95b 6.24b 5.18c Kaeng Tai Pla 8% 7.58ab 7.53a 7.53a 7.74a 7.63ab a a a a 10% 7.76 7.71 7.66 7.82 7.74a ab a a a 12% 7.45 7.68 7.45 7.63 7.26bc 15% 7.37b 7.58a 7.37a 7.55a 6.95c Kaeng Kua Fag 8% 7.35bc 7.53a 7.57a 7.63a 7.55a a a a ab 10% 7.72 7.53 7.55 7.43 7.47a 12% 7.50ab 7.47a 7.47a 7.53ab 7.45a 15% 7.13c 7.32a 7.22a 7.22b 6.85b Nam Ya Pla 10% 8.02a 8.00a 8.20a 8.24a 8.11a b a b b 12% 7.70 7.91 7.87 7.98 7.65b 15% 7.41b 7.96a 7.80bc 7.91b 7.35b 18% 7.07c 7.89a 7.57c 7.72b 6.98c Kaeng Liang 8% 7.40a 7.02a 7.43a 7.31a 7.43a 10% 7.31a 7.14a 7.14ab 7.24a 7.05b 12% 7.45a 7.05a 7.02bc 6.76b 6.36c a a c b 15% 7.43 7.05 6.79 6.79 6.38c Kaeng Som 8% 7.97a 7.22a 7.56a 7.72a 7.61a 10% 7.61b 7.06ab 7.25b 7.58ab 7.56a 12% 7.03c 6.86b 7.25b 7.33bc 6.92b c b c c 15% 6.89 6.83 6.86 7.28 6.47b Kaeng Tae Po 8% 8.07a 7.52a 7.80a 7.75a 7.91a 10% 7.73b 7.45a 7.55ab 7.45b 7.52b b a ab ab 12% 7.66 7.52 7.52 7.50 7.50b b a b b 15% 7.57 7.48 7.30 7.41 7.07c
Acceptability 6.95a 6.64a 6.81a 6.57a 7.32a 7.03a 6.30b 5.50c 7.34a 7.05a 7.20a 6.43b 7.24a 6.76a 5.74b 5.32b 7.61a 7.76a 7.42ab 7.11b 7.60a 7.55a 7.35a 6.88b 8.39a 7.78b 7.67b 7.35c 7.25a 7.17b 6.74c 6.57c 7.72a 7.56a 6.94b 6.58b 8.02a 7.50b 7.45b 7.25b
In a column, means for each attribute followed by a same letter are not significantly different at the 5 % level by DMRT
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using coconut milk as an ingredient, fat content in the products was markedly increased. Coconut milk contained dishes, namely Kaeng Tae Po, Kaeng Kua Fag and Nam Ya Pla, exhibited 11.2313.85 g fat in 100 g. Non coconut milk dishes, Kaeng Som and Kaeng Tai Pla contained fat dramatically lower of 1.41 and 1.67 g/100g, respectively. Thus, the products without coconut milk provided low calories which were 40.40 Kcal for Kaeng Som and 50.67 Kcal for Kaeng Tai Pla. As shown in Table 4, calcium and phosphorus contents of all products fall in the range of 126.5 136.4 mg/100 g and 83.9 108.6 mg/100 g, respectively. The ratio of calcium and phosphorus in the products was 1.2-1.6:1. The relationship between calcium and phosphorus in the diet plays an important role in the absorption of both minerals. A dietary ratio of 2 parts calcium to 1 to 2 parts phosphorus was known to promote the highest level of calcium absorption (Guthrie, 1983). Sodium content appeared to be high as expected in canned concentrated seasoning stock. The sodium level in Kaeng Tai Pla was found to be the highest as 1000.6 mg in 100 g. The reason might come from applying taipla (fermented fish viscera), which contained 15,000 mg sodium in 100g (Institute of Nutrition, 1999), as an ingredient in the dish. Iron contents ranged from 0.86-1.82 mg/100 g while copper level fell in the range of 0.08-0.21 mg/100 g. Fish flour incorporated foods and controls The contents of protein and ash were greater in foods prepared from canned concentrated seasoning stocks than their controls (Table 3). Fat, dietary fibre and carbohydrate of five dishes incorporating fish flour were comparable to their controls recipes. The determined minerals in this section were sodium, calcium, phosphorus, iron and copper. In general, minerals level was higher in fish flour incorporated foods than their control. And there was a distinction in case of calcium which the contents appeared to be 1.4 - 2.5 times
higher in all dishes using fish flour than those in the controls (Table 4). The present calcium contents were 90.9, 119.3, 122.3, 142.5 and 204.8 mg in 100g of Kaeng Kua Fag, Kaeng Tae Po, Kaeng Som, Kaeng Tai Pla and Nam Ya Pla, respectively. The Thai Recommended Daily Dietary Allowances for adult on calcium is 800 mg (Committee on Recommended Daily Dietary Allowances, 1989). Therefore, consuming 100g portion of the above dishes obtained calcium about 10-25% of RDA. Sensory evaluation and nutrient of five selected herring fish flour incorporated foods and their controls Dishes prepared from seasoning stock contained herring fish flour were tested along with their controls (Table 6). There was no significant differences found in all aspects on Kaeng Tai Pla at P>0.01. While the control dishes of Nam Ya Pla and Kaeng Som received significantly better scores of all attributes (P<0.01). The results demonstrated that colour was the renowned deteriorate factors. Attributes on taste of the control did not alter much from the fish flour incorporated seasoning stock in Kaeng Tae Po and Kaeng Kua Fag. Even though many attributes were not as good as the control, the verbal explanation of foods contained herring fish flour was positive expression as better than like slightly. The overall acceptability scores of control and fish flour incorporated food were relatively good ranging from 7.43-7.91 and 6.61 7.64, respectively. Consumer test Foods prepared from five different concentrate seasoning stocks were evaluated by 280 panelists (Figure 1). The ratings of colour for the top 2 boxes (combined percentages of like very much and like) for Kaeng Tae Po, Kaeng Tai Pla, Kaeng Kua Fag, Keang Som and Nam Ya Pla were 75.7, 69.2, 64.6, 60.3 and 58.5%, respectively. The result of the bottom 2 boxes scores corresponded to dislike and dislike very much due
316
Table 6 Sensory evaluation of five herring fish flour incorporated dishes and their controls.
Food Colour Nam Ya Pla Control 18% fish flour Kaeng Som Control 15% fish flour Kaeng Tai Pla Control 15% fish flour Kagng Kua Fag Control 15% fish flour Kaeng Tae Po Control 15% fish flour Consistency MeanS.D. Odour Taste
Texture
Acceptability
7.950.58 6.230.83** 8.020.53 6.330.83** 7.820.52 7.520.64* 7.980.49 7.040.66** 7.680.73 6.820.78**
7.680.48 7.230.75** 7.830.49 7.280.65** 7.820.39 7.750.43 7.720.45 7.540.56* 7.680.48 7.410.57**
7.930.47 6.640.68** 7.560.77 6.651.00** 7.640.80 7.540.58 7.630.80 7.110.81** 7.480.63 6.910.70**
7.610.72 6.700.96** 7.410.83 6.561.04** 7.700.61 7.700.70 7.760.56 7.280.77* 7.300.84 6.980.97
7.860.56 6.860.85** 7.720.54 6.740.80** 7.680.65 7.340.50* 7.670.56 6.871.02** 7.640.49 7.040.72**
7.910.59 6.611.00** 7.430.86 6.371.00** 7.800.65 7.640.54 7.830.70 7.20060** 7.540.69 6.931.04*
** Significantly different at = 0.01 by students t-test * Significantly different at = 0.05 by students t-test
to off colour (Figure 1). For traditional recipe of Nam Ya Pla, steamed snake-head fish meat which is white in colour was used. Therefore, applying fish flour in Nam Ya Pla was strongly affected the colour. On the contrary, Kaeng Tai Pla had the least effect since it contained fermented fish viscera, which naturally dark in colour. Odour of fish flour did not affect all five recipes. The panelists responded to the odour of products as like and like very much in a range of 62.3 76.8 %. Spices incorporated in curry pastes probably masked the fish flour odour in addition to a fish sensation that supposed to exist in the dishes. The taste rating for the top 2 boxes was great in Kaeng Tae Po, Nam Ya Pla, Kaeng Kua Fag and Kaeng Som, while, Kaeng Tai Pla was verbally indicated as most dislike among these five dishes. However, the dislike reasons came from chilli hotness or saltiness of product, not from the taste of fish flour. Considering the overall acceptability, Kaeng Tae Po, Nam Ya Pla, Kaeng Som and
Kaeng Kua Fag obtained high percentage of 80.5, 78.3, 74.0 and 68.8, respectively, in the top 2 boxes. The sandy mouth feel was mentioned to cause unacceptability in dishes without coconut milk. CONCLUSION It was possible to use herring fish flour for Thai food preparations. Applying amount was varied due to the nature of foods. Some limitations could be concluded as darken colour, fish flour odour, cooking style, liquid available, coconut milk present or sandy mouth feel. Nutritious wise, herring fish flour possessed good quality protein and high calcium content, in addition to many minerals. The five selected Thai dishes prepared by incorporating fish flour provided 90.9 204.8 mg of calcium per 100g, which were 1.4-2.5 times of the traditional dishes and contributed 10-25% of RDA for calcium.
100 100
colour odour
80 60 40 20 0
Nam Ya Pla Kaeng Som Kaeng Tai Pla Kaeng Kua Fag Kaeng Tae Po
80
60
Like very much + like Neither like nor dislike Dislike + dislike very much
% Consumers
20
0
Kaeng Tae Po
Nam Ya Pla
Kaeng Som
Kaeng Tai Pla
Kaeng Kua Fag
100 100
%Consumers
40
taste
80 60 40 20 0
Kaeng Tae Po Nam Ya Pla Kaeng Som Kaeng Tai Pla
overall acceptability
80
60
Accept Neither accept nor unaccept Unaccept
% Consumers
20
Nam Ya Pla
Kaeng Som
Kaeng Tai Pla
Kaeng Kua Fag
%Consumers
40
Kaeng Kua Fag
Kaeng Tae Po
Figure 1 Percentage of consumers on preference and acceptance toward five Thai dishes prepared from canned concentrated seasoning stocks.
317
318 ACKNOWLEDGEMENTS
This study was carried out under the research project Utilization of Fish Flour in Some Food Products funded by Norwegian Herring Oil and Meal Industry Research Institute. LITERATURE CITED Association of Official Analytical Chemists. 1984. Official method of analysis. (14th ed.) Arlington, Virginia. Association of Official Analytical Chemists. 1998. Official method of analysis. (16th ed.) (P) method 995.11. Arlington, Virginia. Association of Official Analytical Chemists. 2000. Official method of analysis. (17th ed.) (K) method 973.53, (Na) method 973.54, (Fe,Cu, Zn, Cd, Pb) method 999.11 (Ca, Mg) method. 991.25. Gaithersburg, Maryland. Committee on Recommended Daily Dietary Allowances, Department of Health, Ministry of Public Health. 1989. Recommended Daily Dietary Allowances for Healthy Thais. The War Veterans Association of Thailand Publishing Co., Bangkok. Department of Health, 1995. The 4th National food and nutrition survey. Ministry of Public Health, Thailand. FAO 1982. Fishery products and the consumer in developing countries. Report of the FAO/ NORAD round table discussion, Frazers Hill,Malaysia 11-16 June 1982. FAO Fish. Rep. 271, 37 p. FAO/WHO 1973. Energy and protein requirements. Report of a joint FAO/WHO Ad Hoc Expert Committee. WHO Technical report series no. 522; FAO Nutrition Meetings Report Series 52. World Health Organization, Geneva. Faulks, R.M. and S.B. Timms, 1985. A rapid method for determining the carbohydrate component of dietary fibre. Food Chemistry 17:273-287. Guthrie, H.A.1983. Introductory Nutrition, 5th ed. St.Louis, Missouri, The C.V. Mosby Company. pp.135, 614.
Holland B., J. Brown and D.H. Buss. 1993. Fish and Fish Products. Third supplement to 5th edition of McCance and Widdowsons The Composition of Foods. Royal Society of Chemistry, Cambridge. Institute of Nutrition. 1999. Thai Food Composition Tables. 1st ed. Bangkok, Thailand: Paluk Tai Co., Ltd.150 p. Kinsella,J.A. 1986. Food components with potential therapeutic benefits: The n-3 polyunsaturated fatty acids of fish oils. Food Technology 4:89-97. Kinsella, J.E., B. Lokesh and R.A. Stone. 1990. Dietary n 3 polyunsaturated fatty acids and amelioration of cardiovascular disease: possible mechanisms. The American Journal of Clinical Nutrition 52:1 28. Ministry of Public Health, Thailand. 1987. Food Composition Table for Use in Thailand. Nutrition Division, Department of Health, Ministry of Public Health. 48 p. Phithakpol, B., S. Sringam, N. Sarikaputi and T. Panayotou. 1981. Report on the acceptability testing of fish protein concentrate type B and roller dried fish in Thailand. Institute of Food Research and Product Development, Kasetsart University, Bangkok. Report for the Food and Agricultural Organization of the United Nations Rome. 123 p. Phithakpol, B. 1984. Marketability and feasibility of pilot plant production of roller dried fish in Thailand. Institute of Food Research and Product Development, Kasetsart University, Bangkok. Report for the Food and Agricultural Organization of the United Nations Rome. 74 p. Prosky, L., N. G. Asp, L. Furda, J.W. De Vries, T. F. Schweizer and B.F. Harland, 1985. Determination of total dietary fibre in foods and food products: Collaborative study. Journal of the Association of Official Analytical Chemists 68:677-679. Tahvonen, R., T. Aro, J. Nurmi and H. Kallio. 2000. Mineral content in Baltic herring and Baltic herring products. Journal of Food Composition and Analysis 13:893-903.
Lightning Surge Response of Concrete Pole due to Effect of the Electrical Properties of Concrete based on the Electromagnetic Field Method
Samroeng Hintamai and Jamnarn Hokierti
ABSTRACT Lightning performance of overhead distribution line affects the cost of line construction. For economical insulation coordination in distribution line design, it is necessary to accurately predict the lightning surge overvoltage that occurs in the electric power system. In particularly, tower or pole surge impedance is one of the most important parameters for lightning surge analysis of distribution lines. This paper presents the lightning surge response of reinforced concrete pole due to the electrical properties of concrete based on the electromagnetic field theory, which has never been considered in the previous lightning surge analysis. The electrical properties of concrete were measured over the frequency of range from 100 Hz to 40 MHz during 86 days after pouring. The concrete sample was mixed according to a construction standard of the electrical distribution pole of Provincial Electricity Authority (PEA). The cement/sand/aggregate ratio was about 1:1.5:3 and water/cement ratio was approximately 0.3. It was found that the electrical properties of concrete varied significantly over the frequencies and time after pouring. Therefore, lightning surge response of reinforced concrete pole depended on the electrical properties of concrete. The results showed that surge impedance calculated by the proposed formula agreed well with the other measured value obtained from reduced- scale test. Key words: concrete pole, electromagnetic field method, electrical properties of concrete, lightning surge response, surge impedance
INTRODUCTION Thailand is in a tropical zone and has the highest number of thunderstorm days in this zone, about 50-120 days per year. For protection of equipments in the electric power system, a lightning overvoltage is a significant factor. Thus, lightning surge analysis is essential for insulation design of the electric power system. Particularly, tower surge impedance is an important factor in the insulation
coordination design for transmission/distribution lines. A number of experimental and theoretical studies on tower surge impedance have been carried out (Motoyama and Matsubara, 2000). However, surge impedance of concrete pole has not been studied enough for lightning surge analysis. Representative methods to investigate the tower surge characteristics include measurement on real towers, measurement on reduced-scale models, analytical study on simplified geometry
1 2 3
Bako Agricultural Research Center, Ethiopia. Department of Agronomy, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Nakhon Pathom 73140, Thailand. Department of Agronomy, Faculty of Agriculture, Kasetsart University,Bangkok 10900, Thailand.
320
and numerical analysis based on the electromagnetic theory. The first theoretical formulation of tower surge impedance was proposed by Jordan (1934). In this formulation, based on Neumanns inductance formula, it was assumed that the current distribution inside the tower was uniform from the tower bottom. Theoretical formulations of tower surge impedance based on the electromagnetic field theory were proposed by Lundholm et al.(1958), Wagner and Hileman(1959), Sargent and Darveniza (1969) and Okumura and Kijima(1985), considering effects of the vector potential generated by the injection current into the tower only. Propagation velocity inside the tower was assumed at the speed of light to the top of the tower. However, the effect of return stroke current was neglected. The tower was approximated as a vertical cylinder having a height equal to the tower, and a radius equal to the mean equivalent radius of the tower. Propagation velocity inside the tower was assumed at the speed of light. Measurements of the surge impedance of actual transmission towers were reported by Breuer et al.(1958) and Caswell et al. (1958). In both cases, a reflection method was used, and similar impedance values of 165 ohms were obtained at the tower top. Measured propagation velocity inside the tower was almost the speed of light. Another experimental value for actual transmission towers was reported by Kawai (1964). He used a direct method to measure tower surge impedance, and obtained an impedance value of 100 ohms at the tower top. His experimental results showed that the tower response to a vertical current is different from the response to a horizontal current. Measured propagation velocity inside the tower was 7080% of the speed of light. Scale-model measurements were reported by Chisholm (1983) and Wahab et al.(1987). Chisholm used the time-domain reflectometry (TDR) method to measure tower surge impedance.
These measurements were performed using both horizontal and vertical current injection. Measured propagation velocity inside the tower was the speed of light. Wahab et al.(1987) used the direct method to measure tower surge impedance for various angles of current injection. Measured propagation velocity inside the tower was 8090% of the speed of light. These results showed that the tower surge impedance was strongly influenced by the angle of current injection. Field measurements of full-scale tower impedance using the direct method were reported by Ishii et al.(1991) and Yamada et al.(1995). These measurements were performed using inclined and horizontal current injection. Both papers proposed a surge impedance of the tower based on the Electromagnetic Transient Program(EMTP). Propagation velocity inside the tower was assumed at the speed of light. Measurements of surge response of a transmission tower to actual lightning were reported by Matsumoto et al.(1995), Shinjo et al.(1997) and Motoyama et al.(1997). All of them estimated the surge impedance of the tower based on the measurements, and proposed a model of the tower based on the EMTP. The results showed that surge response and surge impedance of the tower depended on the lightning discharge path direction. Theoretical work was reported by Ishii and Baba(1997). They estimated the surge response of a tower by numerical electromagnetic field analysis. The calculated results were compared with field test results (Yamada et al., 1995). The analysis showed that surge response and surge impedance of the tower depended on the arrangement of the current lead. Theoretical formulation of tower surge impedance based on the electromagnetic field theory was proposed by Motoyama and Matsubara(2000). The analysis showed that the tower surge impedance depended on the direction and velocity of return stroke current. Recently, theoretical formulation of pole
321
surge impedance of concrete pole based on the electromagnetic field theory, including the effect of direction and velocity of the return stroke current and the electrical properties of concrete, was proposed by Hintamai and Hokierti(2003). The calculated result showed that surge impedance of concrete pole depended on the electrical properties of concrete. In this paper, a new formula of surge impedance of reinforced concrete pole based on the electromagnetic field theory by taking the effect of the electrical properties of concrete is proposed. The electrical properties of concrete were measured over the frequency range from 100 Hz to 40 MHz during 86 days after pouring. MATERIALS AND METHODS 1. Model of lightning return stroke current In this model, as the downward leader nears the earth, an upward leader (or the return stroke) is initiated progresses upwards with a velocity vR neutralizing the charge lowered by the preceding steeped leader (Chowdhuri et al., 2001). The lightning channel then consists of a vertical column; the lower part, containing current, is rapidly expanding upwards, and the upper part, containing the residual charge of the proceeding steeped leader, is diminishing rapidly, as shown in Figure 1. The surge impedance of the return stroke is a function of the height and the velocity of the return stroke. However, the conservative
assumption that the stroke is a constant current source is almost universally used, i.e., the surge impedance of the stroke is infinite. 2. Electric field produced by a step current The geometry adopted for the calculation of electromagnetic filed is shown in Figure 2. This configuration is a crude approximation to the lightning return stroke which travel up with a propagation velocity vR in lossy dielectric medium from the earth while removing negative charge from the channel previously formed by a downward moving, negative charged, cloud-to-ground leader. From the electromagnetic field theory, the general solution for electric fields in cylindrical coordination at any point (r,f,z) is defined as (Rubinstein and Uman, 1989),
E = -f A , t
(1)
where
E : electric field intensity, V/m, f : scalar electric potential, V and
A : vector magnetic potential, Wb/m . From the Lorentz condition,
R dz' i(z',t)
(r, , z)
h z'
Rl
Perfectly Conducting ground
cloud residual charge vR i earth return stroke

-z'
az
a rs
a a
rs
az
ay a ar
image
ax
Figure 1 Model of lightning return stroke.
Figure 2 Geometry of a vertical conductor above a perfectly conducting ground.
322
.A + me
where
f =0 , t
(2)
m : permeability of medium, H/m and e : complex permittivity of medium, F/m. It can be shown that the inhomogeneous solutions are
A (r, t ) =
m 4p
1 4pe
0 z
I (r , t - R / c) dz , R
(3)
where f(t=) since the current and charge distributions are zeros for times less than a certain time t0 . In this analysis, a step current of magnitude I0 is traveling up in the positive direction inside the concrete pole at velocity vR. It is convenient to use a mathematical expression describing both the real current distribution and its image at the same time as (Rubinstein and Uman, 1989),
f (r, t ) =
where
r(r , t - R / c) dz , R
(4)
The function u(x) is called the Heaviside function and is defined as,
i( z , t ) = I0 u(t - z / v R ) .
(8)
z : traveling distance of the current, m, r : observation coordinates, r : source coordinates,
0, x < 0 u(x ) = 1, x 1
We allow for the presence of the conducting plane by using the method of images. The vector potential Ar in cylindrical coordination at any point (r,f, z) can be integrated to yield,
R = r - r : distance between and , m,

c : propagation velocity of the current in medium, m/s, r : line charge density, C/m and I : current distribution, A. The medium of the vertical structure being considered here have conductivity s, a dielectric constant eR and permeability m. Thus, the complex permittivity is defined as (Plonus, 1988),
A=
2 2 mI0 (h - z ) + (h - z ) + r ln . (9) 4p -z + z 2 + r 2
The heightcan be found by setting the argument of the Heaviside function to zero. Solving for and inserting into (9), the vector potential Ar can be obtained as,
2 mI0 (v Rt - z ) + (v Rt - z ) + r Ar = ln . (10) 4p -z + z 2 + r 2 2
e = e 0 e R - js / w .
(5)
The dipole technique uses infinitesimal time-varying as the source of the electric and magnetic fields. Since the vector potential A r can be found from the current alone, expression f in terms of A allows us to write (1) in terms of the current distribution alone. By solving the Lorentz condition, we obtain,
f ( r, t ) = -
1 . Adt + f (t = -) . me 0
(6)
Meanwhile, we can calculate vector potential at the same point from the image channel. Since to change the actual channel to the image channel all we can change z to z, it is readily seen that
Substituting (6) into (1), we obtain

A 1 E ( r, t ) = . A dt - t . me 0
t
(7)
(v Rt + z ) + (v Rt + z ) + r 2 mI Ai = 0 ln . 4p z + z2 + r2
2
(11)
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The sign of the vector potential from image is the same as from the source because the directions of current are the same for both. The total vector potential is then given by,
A ( z ) = Ar ( z ) + Ai ( - z ).
Z pole =
Vtop I0
(12)
Substituting (12) into (7), we obtain the total electric field intensity in cylindrical coordination at any (r,f, z) point as,
Ez = 60 I0 1 2 . (13) e R - js / e 0w z + r 2
0 t < (r / c) 0, h + h2 + r 2 60 In , r = e R - js / e 0w (r / c) t < (h / n R + r / c) t < (h / n R + r / c) Vtop (h / n R + r / c),
(18) Finally, the surge impedance of concrete pole Zpole can be obtained as,
3. Surge impedance of concrete pole 3.1 Single conductor model If the lightning stroke starts from the top of the pole at, t=0 the pole top voltage Vtop is obtained by,
Vtop = - Ez dz .
0 h
Z pole =
h + h2 + r 2 60 ln , (19) r e R - js / e 0w
where h is the height of the pole. 3.2 Multiconductor model An actual concrete pole is composed of multiconductor as shown in Figure 3. The five conductors being short-circuited at its boundary (top and/or bottom), the total impedance seen from the top is given considering the mutual impedances between the conductors by
Z pole = ( Z11 + 2Z12 + Z13 + Z15 ) d d ln ln 2rg 4 2rGMR, st 60 5 (e r - js / e 0w ) ln d 4 16 2rGMR, st rg 2 2h ln (e r - js / e 0w ) d 60
(14)
For 0 t<(r/c), the wave front of the electromagnetic wave does not pass the point. Therefore,
Vtop = 0 .
(15)
For (r/c) t <(h/vR + r/c), the wave front of electromagnetic wave passes through the point (r,f,z). Therefore,
Vtop =
h + h2 + r 2 60 I0 ln . (16) r e R - js / e 0w
For, t > h/vR + r/c the electromagnetic wave reflected on the ground surface reaches the pole top. Therefore,
(21) where rGMR, st = 4 rst dst1 dst 2

2 (dst
1
2 + dst 2 .
Vtop (t ) = Vtop (h / v R + r / c) .
(17)
Since the voltage Vtop is produced by the vertical return stroke current I0, the pole surge impedance Zpole is defined as,
If it is tedious to calculate each component of the above equation, the total surge impedance is easily evaluated as the impedance of an equivalent circular single conductor with the following geometrical mean radius
324
y x
a) structure inside of concrete pole
Figure 4 Measurement of the electrical properties of concrete. in Figure 3. The concrete sample was formed by placing aluminum plate electrode about 1 cm in thickness. The cement/sand/aggregate ratio in preparing the concrete specimen was about 1:1.5:3 and the water/cement ratio was near 0.3. Capacitance and dissipation factor were measured by impedance analyzer. Dielectric constant and electrical conductivity were calculated from the measured values. Change of the dielectric constant and the electrical conductivity with the curing time and frequency are shown in Figures 5 and 6. In the first 3 days, dielectric constant and electrical conductivity decreased rapidly. After 10 days, their changes become very slow. These changes show good consistency with the chemical change and water content in the hardening period of concrete. Therefore, frequency of lightning current of the first strokes is 25 kHz (10/350 ms), dielectric constant of concrete is about 16 and electrical conductivity is about 0.01 mS/m. Another frequency of lightning current of the subsequent strokes is 1 MHz (0.25/100 ms), dielectric constant of concrete is about 8 and electrical conductivity is about 0.122 mS/m. 2. Propagation velocity of wave The propagation velocity of return stroke current inside a medium of complex permittivity e is varied inversely to the square root of the complex permittivity as (Morshedy, 2000),
b) cross section area Figure 3 Multiconductor model of concrete pole.

h + h 2 + R2 60 GMR , ln RGMR e R - js / e 0w
Z pole =
(22) where RGMR = 5 rg

1/ 5 ( ) (rGMR,st )1/ 5 d 4 .
RESULTS AND DISCUSSION 1. Electrical properties of concrete The electrical properties of concrete were measured over the frequency range from 100 Hz to 40 MHz during 86 days after pouring, using Impedance/Gain-Phase Analyzer Hewlett Packard 4164A with an accuracy of 0.17 percent as shown

1st day
325
180 160 140

6th day 3rd day
dielectric constant
120 100 80 60 40 20 0
20th day 10th day
15th day
28th day
freq.(kHz)
0.1
0.6
4.0
25.1
158.5
1000.0
6309.6
39810.7
55th day
tf (s) 2500
416.6
62.5
10
1.57
0.25
0.04
0.006
86th day
Figure 5 Change of dielectric constant of concrete.

1st day
0.025
3rd day
0.020
6th day
conductivity(S/m)
0.015
10th day
15th day
0.010
20th day
0.005
28th day
0.000 freq.(kHz) 0.1 t f (s) 2500
55th day
0.6 416
4.0 63
25.1 10
158.5 1.5
1000.0 0.25
6309.6 39810.7 0.04 0.006

86th day
Figure 6 Change of electrical conductivity of concrete.
velocity =
3 10 8 . e R - js / e 0w
(23)
The propagation velocity of return stroke current inside the concrete pole is shown in Figure 7. In Figure 7, the propagation velocity of wave inside the concrete pole is about 70 to 103.6 m/ms as the frequency of lightning current is between 25 kHz to 1 MHz.
3. Comparison between calculated result and measured result To clarify the effectiveness of the proposed formula, we show the comparison of surge impedance of reinforced concrete pole calculated by the proposed formula with the measured result (Yamamoto et al., 1997). The surge impedance of reinforced concrete pole was measured by scale model technique. The height of reinforced concrete pole is 14 m and a radius of 0.377 m. The hollow
326

1st day
140
3rd day
120
propagation velocity(m/s)
6th day
100 80 60 40 20 0
10th day
15th day
20th day
28th day
freq.(kHz) tf (s)
0.1 2500
0.6 416.6
4.0 62.5
25.1 10
158.5 1.57
1000.0 0.25
6309.6 0.04
39810.7 0.006
55th day
86th day
Figure 7 Propagation velocity of return stroke current inside the concrete pole. steel reinforced concrete pole was composed of two parts; iron cage and concrete part of about 2 mm in thickness. The surge impedance was measured about 242 ohms, whereas calculated result from this configuration by the proposed formula is about 258 ohms. Therefore, it showed that calculated value is different from measured value about 6.8%. 4. Model of concrete pole This study deals with 22 m in height of concrete pole that imbedded a grounding lead wire of 35 mm2 at the center of the pole from top to bottom and supplemental steel reinforced at square inside of solid taper pole, as shown in Figure 8. 5. Surge impedance of concrete pole Figure 9 shows the calculation of surge impedance of concrete pole by equation (22) due to the effect of the electrical properties of concrete. The frequency of lightning current is between 25 kHz to 1 MHz, surge impedance of concrete pole is about 80 to 119 ohms. CONCLUSIONS In this paper, the formulation of lightning
Figure 8 Configuration of the reinforced concrete pole. surge response of reinforced concrete pole based on the electromagnetic field by taking the effect of the electrical properties of concrete was obtained. The results showed that surge impedance of reinforce concrete pole depended on the geometry of pole, the dielectric constant and the electrical conductivity of concrete. ACKNOWLEDGEMENTS The authors wish to thank the Electrical Properties Measurement Laboratory of the National Metal and Material Technology Center for service

1st day
327
160 140
3rd day
surge impedance(ohms)
120 100
6th day
10th day
80 60 40 20
28th day 15th day
20th day
0 freq.(kHz) t f (s) 0.1 2500 0.6 416.6 4.0 62.5 25.1 10 158.5 1.57 1000.0 0.25 6309.6 39810.7 0.04 0.006
55th day
86th day
Figure 9 Surge impedance of concrete pole. of the concrete electrical properties of measurement. LITERATURE CITED Jordan, C.A. 1934. Lightning computations for transmission lines with overhead ground wires Part II. General Electric Review 34: 180185. Lundholm, R., R.B. Finn, Jr. and W.S. Price. 1958. Calculation of transmission line lightning voltage by field concepts. AIEE Trans. PT. 77: 1271-1283. Wagner, C.F. and A.R. Hileman. 1960. A new approach to calculation of lightning performance of transmission lines. AIEE Trans. PT. 79: 589-603. Sargent, A. and M. Darveniza. 1969. Tower surge impedance. IEEE Trans. PAS. 88: 680-687 Okumura, K. and A. Kijima. 1985. A method for computing surge impedance of transmission line tower by electromagnetic field theory. IEE of Japan Trans. B. 105: 733-740. Breuer,G.D., A.J. Schultz, R.H. Schlomann and W. S. Price. 1958. Field studies of the surge response of a 345-kV transmission tower and ground wire. AIEE Trans. PT. 77: 13921396. Caswell, R.W., I.B. Johnson, E.F. Koncel, Jr. and N. R. Schultz. 1958. Lightning performance of 138-kV twin-circuit transmission lines of Commonwealth Edison companyoperating experience and field studies. IEEE Trans. PT. 77: 14801491. Kawai, M. 1964. Studies of the surge response on a transmission line tower. IEEE Trans. PAS. 83: 3034. Chisholm,W.A., Y.L. Chow and K.D. Srivastava. 1983. Lightning surge response of transmission towers. IEEE Trans. PAS. 102: 32323242. Chisholm W.A. and Y.L. Chow. 1985. Travel time of transmission towers. IEEE Trans. PAS. 104: 29222928. Wahab, M.A.A., I. Matsubara and H. Kinoshita. 1987. An experimental evaluation of some factors affecting tower surge impedance. IEE of Japan Trans. 107: 171177. Ishii M., T. Kawamura, T. Kouno, E. Ohsaki, K. Murotani and T. Higuchi. 1991. Multistory transmission tower model for lightning surge analysis. IEEE Trans. PWRD. 6: 13271335. Yamada T., A. Mochizuki, J. Sawada, E. Zaima, T. Kawamura, A. Ametani, M. Ishii and S. Kato. 1995. Experimental evaluation of a UHV tower model for lightning surge analysis.
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IEEE Trans. PWRD. 10: 393402. Matsumoto Y., O. Sakuma, K. Shinjo, M. Saiki, T. Wakai, T. Sakai, N.Nagasaka, H. Motoyama and M. Ishii. 1995. Measuring of lightning surges on test transmission line equipped with arresters struck by natural and triggered lightning. IEEE Trans. PWRD. 11: 9961002. Shinjo K., Y. Matsumoto, O. Sakuma, T. Wakai, T. Sakai, M. Ishii and H. Motoyama. 1997. Characteristics of transient response of okushishiku test transmission line struck by natural and triggered lightning. IEE of Japan Trans. 117-B: 478487. Motoyama H., K. Shinjo, Y. Matsumoto and N. Itamoto. 1998. Observation and analysis of multiphase back flashover on the okushishiku test transmission line caused by winter lightning. IEEE Trans. PWRD. 13: 13191398. Ishii M. and Y. Baba. 1997. Numerical electromagnetic field analysis of tower surge response. IEEE Trans. PWRD. 12: 483488. Motoyama, H. and H.Matsubara. 2000. Analytical and experimental study on surge response of transmission tower. IEEE Trans. Power Delivery 15: 812-819.
Hintamai, S. and J. Hokierti. 2003. Lightning surge response analysis of concrete pole using the electromagnetic field method, pp. 568573. In Transmission and Distribution Conference and Exposition. Dallas Texas. Chowdhuri, P. S. Li and P. Yan. 2001. Review of research on lightning-induced voltages on an overhead line. IEE Proc.-Gener. Transm. Distrib. 148: 91-95. Rubinstein, M. and M.A.Uman. 1989. Methods for calculating the electromagnetic fields from a known source distribution: application to lightning. IEEE Trans. Electromag. Compat. 31: 183-189. Plonus, M.A. 1988. Applied Electromagnetics. International Editions. New York: McGrawHill. Morshedy, A.El. 2000. High-Voltage Engineerng Theory and Practice. New York: Marcel Dekker, Inc. Yamamoto, K., Z. Kawasaki, K. Matsuura, S. Sekioka and S. Yokoyama. 1997. Study on surge impedance of reinforced concrete pole and grounding lead wire on distribution line by experimental on reduced scale model, pp. 209-212. In Proc. 10th Int. Symp. High Voltage Eng.

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April - June 2005 Volume 39 Number 2

The Kasetsart Journal

284 294 300

Kasetsart J. (Nat. Sci.) 39 : 165 - 173 (2005)

Received date : 06/07/04

Accepted date : 01/03/05

Kasetsart J. (Nat. Sci.) 39 (2)

Kasetsart J. (Nat. Sci.) 39 (2)

Kasetsart J. (Nat. Sci.) 39 (2)

(b) Paspalum distichum

Kasetsart J. (Nat. Sci.) 39 (2)

(a) Cyperus difformis 150

(b) Cyperus iria 20 15 10 5 0 b b b b 4WAS 8WAS a a b b b MSR a

(c) Cyperus sanguinolentus 50

W1 25 W1 W2 W3 20 15 10 5 0 b b b b 4WAS 8WAS b b b b a (d) Commelina diffusa a W2 W3 W4 W5

40 30 20 10 0 4WAS 8WAS MSR a a a b b b c bc bc a b b b b

Kasetsart J. (Nat. Sci.) 39 (2)

35.61 48.7 113.1 a 20.9 b 14.5 b 38.4 b 24.0 b NS2 ** NS

5.8 8.2 18.1 a 4.6 b 4.5 b 2.3 b 5.6 b NS ** NS

30.5 43.9 88.3a 11.1 b 24.4 b 35.5 b 26.8 b NS ** NS

13.4 13.7 50.8 a 2.4 b 3.7 b 5.3 b 5.5 b NS ** NS

Kasetsart J. (Nat. Sci.) 39 (2)

127.61 127.8 127.4 127.2 128.5 127.5 128.0 NS2 NS NS

Kasetsart J. (Nat. Sci.) 39 (2)

6000 4000 2000 0 W1 W2 W3 W4 W5

6000 4000 2000 0

6000 4000 2000 0 W1 W2 W3 W4 W5

6000 4000 2000 0 W1 W2 W3 W4 W5

Kasetsart J. (Nat. Sci.) 39 (2)

Kasetsart J. (Nat. Sci.) 39 : 174 - 185 (2005)

Received date : 21/09/04

Accepted date : 21/02/05

Kasetsart J. (Nat. Sci.) 39 (2)

Kasetsart J. (Nat. Sci.) 39 (2)

Kasetsart J. (Nat. Sci.) 39 (2)

Kasetsart J. (Nat. Sci.) 39 (2)

The data were collected 16 days after salinization

Pokkali FL 496 DDG KMK FL 478 KDML105 RD6 IR 29 Mean CV (%)

Kasetsart J. (Nat. Sci.) 39 (2)

Total Na+ uptake (g/plant) 4 dS /m 8 dS /m 12 dS /m Total dry weight (g/plant) 4 dS /m 8 dS /m 12 dS /m 0 dS /m Lines/cultiuars 0 dS /m

Pokkali IR 29 KMK KDML 105 FL 478 FL 496 DDG

2.442 a 1.375 a 1.661 a 1.619 a 1.631 a 1.319 a 1.177 a

2.622 a 1.108 c 1.584 bc 2.411 a 1.625 bc 1.981 ab 1.386 bc

2.519 a 1.696 b 2.028 ab 1.771 ab 1.768 ab 1.761 ab 1.659 b

2.327 a 1.713 ab 1.588 ab 1.530 b 1.601 ab 1.467 b 1.513 b

0.0224 a 0.0111 a 0.0139 a 0.0135 a 0.0138 a 0.0104 a 0.0082 a

0.0291 ab 0.0112 c 0.0201 abc 0.0335 a 0.0185 bc 0,0204 abc 0.0155 bc

0.0393 a 0.0301 ab 0.0334 ab 0.0339 ab 0.0272 ab 0.0243 b 0.0248 ab

0.0399 ab 0.0405 ab 0.0296 abc 0.0389 ab 0.0346 abc 0.0219 c 0.0270 bc

Total K+ uptake (g/plant) 4 dS /m 8 dS /m 12 dS /m 0 dS /m

0.0324 a 0.0148 c 0.0201 bc 0.0199 bc 0.0196 bc 0.0163 bc 0.0135 c

0.0326 a 0.0109 d 0.0176 bcd 0.0245 b 0.0172 bcd 0.0199 bc 0.0152 cd

Kasetsart J. (Nat. Sci.) 39 (2)

Old leaves (after the 4 th leaf from the top) 0 dS /m 4 dS /m 8 dS /m 12 dS /m

Table 5 K+ concentration (%) in 3 parts of rice plant at 1 week after salinization.

Young leaves (1-4 leaves from the top) 0 dS /m 4 dS /m 8 dS /m 12 dS /m

0.888 a 0.482 e 0.610 cd 0.530 de 0.640 c 0.725 b 0.630 c 0.591 cd

Kasetsart J. (Nat. Sci.) 39 (2)

Table 6 Na+/K+ ratio in 3 parts of rice plant at 1 week after salinization.

Young leaves (1-4 leaves from the top) 0 dS /m 4 dS/ m 8 dS /m 12 dS /m

Old leaves (after the 4 th leaf from the top) 0 dS /m 4 dS /m 8 dS /m 12 dS /m

Kasetsart J. (Nat. Sci.) 39 (2)

Salt tolerance scoring