You are on page 1of 8

Soil Science Society of America Journal

Soil Sci. Soc. Am. J. 78:538545


doi:10.2136/sssaj2013.07.0315
Received 30 July 2013
*Corresponding author zazahir@yahoo.com
Soil Science Society of America, 5585 Guilford Rd., Madison WI 53711 USA
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by
any means, electronic or mechanical, including photocopying, recording, or any information storage
and retrieval system, without permission in writing from the publisher. Permission for printing and for
reprinting the material contained herein has been obtained by the publisher.
Scrutinizing Rhizobia to Rescue Maize
Growth under Reduced Water Conditions
Soil Biology & Biochemistry
D
rought is a major constraint to the productivity of cereals (reducing yield
up to 40.8%) in arid and semiarid areas (Boyer, 1982) like Pakistan.
Change in climate due to global warming is resulting in irregular rainfall,
fash fooding, increased salinity, high temperature, and drought in the country,
which contributes substantially to reduced growth and yield of these crops. Pakistan
has been facing water defcit situations for many years due to reduced river fow, low
rainfall, and high salinity (Economic Survey of Pakistan, 2011). Terefore, several
approaches have been reported in the literature to mitigate the drought-induced im-
pacts on plant growth. Mulching, bed planting, deep tillage, cover cropping (physi-
cal methods), and foliar sprays of chemicals such as plant growth regulators (indole
acetic acid, zeatin, and abscisic acid) and osmo-protectants (silver, silicon, proline,
glycine betaine, salicylic acid, and jasmonic acid) have been adopted to ameliorate
the impact of drought stress (Hussain et al., 2011). However, conventional breeding
and selection of drought tolerant genotypes on the basis of osmoregulation have also
been adopted (Hussain et al., 1986; Ahmad et al., 1987) but this is a slow and lengthy
process. On the other hand, rhizobiaa group of N fxing bacteria (in symbiosis
Muhammad Baqir Hussain
Zahir Ahmad Zahir*
Hafz Naeem Asghar
Sajid Mahmood
Institute of Soil & Environ. Sciences
University of Agriculture
Faisalabad, (38040)
Pakistan
Availability of fresh water for crop production is a major concern these days.
Therefore inoculation biotechnology has been tried as a tool to support crop
growth and yield under water defcit conditions. Thirty rhizobia were isolated
from the nodules of three legumes (Lens culinaris, Vigna radiata, and Cicer
arietinum) cultivated in arid and semiarid regions of Punjab (Pakistan). Nine
fast growing isolates were evaluated for drought tolerance capability with
PEG-6000-induced water stress. These nine isolates were assessed for plant-
growth-promoting-activity on maize under drought in gnotobiotic conditions
by planting inoculated seeds between flter sheets soaked with each respec-
tive inoculum. Drought was imposed by applying PEG solutions of -0.61 and
-1.2 MPa osmotic potential and isolates rhizobial strain (RS)-1, RS-3, RS-8,
and RS-12 were selected as prominent growth promoters. Selected iso-
lates were evaluated in a pot trial under natural conditions where irrigation
was delayed at the vegetative and reproductive growth stages of the maize
crop to develop water defcit stress. Improvement in fresh cob yield, root/
shoot length, shoot fresh weight and drought tolerance index was observed.
The isolates displayed P-solubilization (RS-1 and RS-12), siderophores (RS-3
and RS-8), oxidase (RS-1), indole acetic acid, catalases, and exopolysaccha-
rides production ability (RS-1, RS-3, RS-8, and RS-12). Isolates RS-1 and
RS-3 were identifed as Rhizobium phaseoli whereas RS-8 and RS-12 were
Mesorhizobium ciceri. Results suggested rhizobia as potential inoculants to
maize for improving growth and productivity under drought. However, evalu-
ation in feld conditions is required to confrm their capability.
Abbreviations: RS, rhizobial strain; YEM, yeast extract mannitol.
Published April 8, 2014
Published March 21, 2014
www.soils.org/publications/sssaj 539
Soil Biology & Biochemistry
with legumes) have been reported to improve growth and produc-
tivity of non-legumes through mechanisms other than biological
N
2
fxation (Noel et al., 1996; Hussain et al., 2009). Rhizobia may
perform as bio-fertilizers (improving nutrition and nutrient-use
efciency; Zaidi et al., 2009), bio-stimulators (plant growth regu-
lator production; Etesami et al., 2009) or bio-protectants (antibio-
sis, chitinase, osmolytes, and 1-aminocyclopropane-1-carboxylate
[ACC] deaminase production; Zahir et al., 2008). Plant growth
promotion under normal conditions has been tested in rhizobi-
um-inoculated rice (Oryza sativa L.), maize (Zea mays L.), and
wheat (Triticum aestivum L.; Yanni et al., 1997, 2001; Gutirrez-
Zamora and Martnez-Romero, 2001; Mehboob et al., 2011).
However, information regarding plant growth promotion efects
of rhizobial inoculation on non-legumes exposed to drought
is scarce. Rhizobia are capable of surviving in soils with limiting
moisture levels; however, population densities tend to be lower un-
der desiccated conditions and increase as the moisture stress is re-
lieved (Fuhrmann et al., 1986). Athar and Johnson (1997), Athar
(1998), and Elboutahiri et al. (2010) observed enormous ability of
rhizobia to survive under water potentials up to -1.5 MPa.
Rhizobia can alleviate the efect of water stress in plants by
altering leaf stomatal conductance, transpiration, photosynthetic
capacity, and root morphology in inoculated non-legumes (Chi
et al., 2005). Terefore, Alami et al. (2000) evaluated an exopoly-
saccharide producing strain, YAS34, belonging to Rhizobium
species for ameliorating the impact of drought and improving
water and nutrient-use efciency in sunfower (Helianthus ann-
uus L.). Tey recorded a signifcant increase in N uptake and im-
proved root and shoot dry biomass under normal and water lim-
ited conditions. Similarly, rhizobial strain KYGT207 (isolated
from the drying soil) improved the growth of wheat under water
defcit conditions due to the improved aggregation of rhizo-
sphere soil (Kaci et al., 2005). Following the literature reviewed,
we found a room for rhizobial inoculation to non-legumes under
drought conditions as scanty information is available in this area.
We hypothesized that the rhizobial isolates capable for surviv-
al under drought conditions and having the characteristics for
improving plant growth of maize under drought could be good
criteria for selecting potential rhizobial inoculants for improving
growth and yield of maize under drought.
MATERIALS AND METHODS
Isolation, Purifcation, and Preservation of Rhizobia
Numerous rhizobia were isolated from the root nodules of
three legumes (Lens culinaris, Vigna radiata, and Cicer arietinum)
cultivated in arid (Chakwal) and semiarid (Faisalabad) areas of
Punjab (Pakistan). Te plants were dug up with non-rhizosphere
soil to avoid damage to the roots and nodules. Tese plants were
transferred to the laboratory in polythene bags. Non-rhizosphere
soil was removed by gentle shaking and further adhering soil was
removed by washing the roots with tap water. Nodules were cut
from the roots with a sterilized razor blade and collected in sepa-
rate Petri dishes. Disinfestation of nodules was done by dipping
in ethanol (95%, v/v) for 10 s and 5% (v/v) sodium hypochlorite
for 10 min followed by thorough rinsing with sterilized water
(Abd-Alla et al., 2012). Sterilized nodules were rolled on the
Petri dishes containing autoclaved yeast extract mannitol (YEM)
agar medium and incubated at 28 1C for 72 h to confrm the
sterilization (Muzzamal et al., 2012). Tese surface-sterilized
nodules were crushed with a sterilized glass rod in separate steril-
ized test tubes containing sterilized distilled water. Homogenate
suspensions were streaked in Petri dishes containing autoclaved
YEM agar medium and incubated at 28 1C. Further streaking
for isolation was performed three to four times to obtain pure
cultures of the diferent isolates. Nine fast growing isolates were
collected, coded as rhizobial strain (RS) with numbers and pre-
served in glycerol at -20 1C. Tese isolates were confrmed as
rhizobia by streaking on congo-red agar plate (Grams test) and
by studying their colony morphology.
Drought Tolerance Assay
Rhizobial isolates (RS-1, RS-2, RS-3, RS-6, RS-7, RS-8, RS-
10, RS-12, and RS-13) were tested for drought/desiccation tol-
erance capability using PEG-6000 in YEM broth medium (Busse
and Bottomley, 1989). Inoculum for each isolate was prepared
in a sterilized 50-mL conical fask containing 20 mL of YEM
broth and incubated for 72 h at 28 1C, shaking at 100 rpm.
Cells were harvested by centrifugation at 4000 g at 4C for
10 min. For inoculation, an inoculum of optical density (OD)
0.5 McFarland units (i.e., 10
8
cells mL
-1
) was developed by us-
ing densitometer (Den-1 Densitometer, McFarland, UK) in ster-
ilized YEM liquid medium (Shakeri et al., 2011). Un-inoculated
control medium was maintained to subtract its OD from the
OD of the inoculum. Test tubes containing 7 mL of YEM broth
with diferent drought levels (No PEG, 15, 25, and 35% PEG
having osmotic potentials -0.04, -0.69, -1.65, and -2.15 MPa, re-
spectively, measured by Cryoscopic Osmometer [OSMOMAT-
030-D, Gonotec, Germany]) were autoclaved. Afer autoclaving,
osmotic potential was measured again to check any change in the
developed potentials, but there was no signifcant change. For
each isolate triplet test tubes at each drought level were inocu-
lated with 0.5 mL inoculum of OD 0.5 (10
8
cell mL
-1
). A trip-
licate set of un-inoculated control test tubes was also maintained.
Rhizobia show maximum growth afer 96-h incubation at 28
1C and 100 rpm which was confrmed in few samples using di-
lution plate technique (data not shown). Terefore, OD afer the
96-h incubation in an orbital shaking incubator at 28 1C and
100 rpm was measured using densitometer and the correspond-
ing population was counted by dilution plating technique (data
not shown; Wollum, 1982). Rhizobial isolates showing high OD
under drought were considered as drought tolerant.
Screening of Rhizobia for Plant Growth
Promoting Activity under Drought
All nine rhizobial isolates used in the drought tolerance as-
say were further tested for plant growth promoting activity un-
der drought using maize (Zea mays L.) cv. Neelum as the test
plant. Inoculum was prepared as described above. Afer achiev-
540 Soil Science Society of America Journal
ing uniform cell density, two sterilized flter paper sheets were
saturated by pouring on the respective inoculum. Tree surface
sterilized (by dipping in 5% sodium hypochlorite for 10 min)
pre-germinated seeds (germinated with hydro-priming on fl-
ter paper sheets) were dipped in the inoculum of 0.5 OD (i.e.,
10
8
cells mL
-1
) for 10 min and placed between the sheets
soaked in the respective inoculum. Te flter paper sheets were
rolled and put in sterilized glass jars as described by Asghar et
al. (2002). Sterilized broth was used for the control treatment.
Each treatment was replicated thrice. Diferent drought poten-
tials (No PEG, 15 and 25% PEG having potentials -0.02, -0.61,
and -1.20 MPa, respectively) were maintained in sterilized
Hoagland solution (1/2 strength) by using PEG-6000. Light
(275 mol m
-2
s
1
) and dark periods were adjusted at 10 and
14 h, respectively, and a suitable temperature (2530C) was
maintained. Five days afer sowing, drought treatments were
applied by irrigating plants with PEG solutions. Te trial was
harvested afer 15 d of drought treatment and data regarding
root/shoot length (longest root/shoot), and dry biomass were
recorded. Isolates showing signifcant improvement in results
were considered as efcient plant growth promoters.
Pot Trial
Selected isolates from the screening trial (RS-1, RS-3, RS-
8, and RS-12) were evaluated for their capability to improve
maize growth and reduce the impact of water defcit stress in
pots (pot dimensions: height of 30 cm, diameter of 23 cm, con-
tained 12 kg sieved sandy loam soil [pH, 7.3; EC
e
, 2.17 dS m
-1
;
OM, 0.84%; Total N, 0.04%; available P, 7.1 mg kg
-1
; extract-
able K, 107 mg kg
-1
; saturation percentage, 37%] collected from
the experimental feld of the Institute of Soil and Environmental
Sciences, University of Agriculture, Faisalabad) in greenhouse.
For the purpose, inoculum was prepared in the laboratory as
described for the screening trial above. Maize seeds were coated
with the inoculum prepared by mixing each bacterial culture
with autoclaved peat: clay (3:1) and a 15% (w/v) table sugar so-
lution. Inoculated seeds were dried under shade. Five seeds per
pot were sown and thinned to one seedling afer 1 wk of ger-
mination. Water stress was applied either at the vegetative (fve
leaf ) or the reproductive (silking) growth stage of the crop by
delaying irrigation. Te irrigation was delayed until the appear-
ance of wilting symptoms in maize leaves (9 d for vegetative or 7
d for reproductive stage water stress). Plants having drought at
the vegetative stage were not water stressed at the reproductive
stage and vice versa. A control treatment with normal irrigation
was also maintained. Recommended doses of NPK (180, 140,
90 kg ha
-1
, calculated according to the weight of soil) were ap-
plied using urea, diammonium phosphate (DAP), and sulfate of
potash (SOP) fertilizers. A completely randomized design was
followed with three replications. Plants were grown to maturity
and at harvest (96 d afer sowing), diferent growth and yield
parameters (fresh cob yield, longest root and shoot length, and
shoot fresh weight) were recorded.
Drought Tolerance Index (DTI)
A drought (stress) tolerance index was calculated by dividing
the shoot or root length of inoculated seedlings under stress (SLI
or RLI and St) with the shoot or root length of un-inoculated and
unstressed seedlings (SLUI or RLUI and USt) (modifed from
Shetty et al., 1995).
(SLI or RLI and St)
DTI
(SLUI or RLUI and USt)
=
where SLI, shoot length of inoculated seeding; RLI, root length
of inoculated seedling; SLUI, shoot length of un-inoculated
seedling; RLUI, root length of un-inoculated seedling; St =
stressed seedling; USt, un-stressed seedling.
Identifcation and Characterization of Selected
Rhizobial Isolates
Te selected rhizobial isolates were identifed by using the
BIOLOG identifcation system (Microlog System release 4.2,
Biolog Inc., USA). Te confrmation of the rhizobial isolates
was performed by isolation of the rhizobia from the nodules of
Vigna radiata and Cicer arietinum, and on re-inoculation, these
isolates nodulated respective legume hosts. Te N fxation activ-
ity of the nodules was confrmed with the pinkish appearance
in the nodules. Te selected isolates of rhizobia were character-
ized for exopolysaccharides synthesis, following the method de-
scribed by Ashraf et al. (2004); catalase, by adopting MacFaddin
(1980); oxidase activity, following Steel (1961); and IAA (auxin)
production was recorded in the presence or absence of its pre-
cursor L-tryptophan by implementing the protocol of Sarwar et
al. (1992). Organic acid production was assessed by growing iso-
lates on YEM with bromothymol blue (orange/yellow growth is
a positive reaction; Vincent, 1970), siderophores by the method
of Schwyn and Neilands (1987). Mehta and Nautiyal (2001)
scheme was adopted to measure the solubilization of inorganic
P in the medium.
Statistical Analysis
Data were analyzed by analysis of variance (ANOVA;
Steel et al., 1997) and means were compared using Tukeys
test. Sofware used for the analysis was Statistix 8.1 (Analytical
Sofware, USA).
RESULTS
Drought Tolerance Assay
Optical density of the isolates decreased with increase in
the PEG concentration (No PEG, -0.04 MPa; 15% PEG, -0.69
MPa; 25% PEG, -1.65 MPa; and 35% PEG, -2.15 MPa) of the
medium (Table 1). Te decrease in cell number with increas-
ing PEG concentration was also evident using dilution plate
technique (data not shown). Under normal conditions (No
PEG), rhizobial isolate RS-6 produced the maximum OD, fol-
lowed by RS-7. However, isolate RS-8 had the lowest OD un-
der normal conditions. Isolates RS-13 and RS-12 were the low
performers at 15% PEG induced drought stress, but RS-7 was
www.soils.org/publications/sssaj 541
prominent among the isolates followed by RS-6. At 25% PEG
induced drought, RS-3 performed best among the isolates fol-
lowed by RS-8 and RS-2, respectively. Isolate RS-13 remained
the last among the isolates at 25% PEG induced drought as was
in 15% PEG induced drought. Te trend changed at 35% PEG
induced drought, where isolate RS-8 became most efcient in
drought tolerance followed by isolates RS-1. Isolate RS-6 which
was prominent among the isolates at 15% PEG induced drought
and in normal conditions, became most sensitive to drought at
35% PEG induced drought. Sensitivity of rhizobial isolates to
drought stress was calculated by dividing their OD at drought
with their OD under normal condition. Isolate RS-6 was most
sensitive at -2.15 MPa osmotic potential, followed by RS-13 at
both -1.65 and -0.69 MPa osmotic potentials. However, lowest
sensitivity was observed by RS-7, RS-3, and RS-8 (-30.4, -52.5,
and -63.5%) at -0.69, -1.65, and -2.15 MPa osmotic potentials,
respectively, as compared with all other isolates.
Screening on the Basis of Plant Growth
Promoting Activity under Drought
Performance of the inoculated maize seedlings was signif-
cantly diferent from the un-inoculated control for some isolates
but not for others (Table 2). Isolates RS-1, RS-3, RS-8, and RS-
12 were the most prominent in plant growth promotion among
the isolates tested.
Inoculation of rhizobial isolates RS-3, RS-8, and RS-12 sig-
nifcantly improved the root length of the maize seedlings as com-
pared with un-inoculated control under normal conditions where-
as other isolates could not show signifcant diferences (Table 2).
At 15% PEG induced drought, isolates RS-3, RS-7, RS-8, and RS-
13 inoculation gave signifcantly high root length with respect to
un-inoculated control. Isolates RS-3 and RS-13 inoculation gave
signifcant improvement in the root length of the seedlings over
control at 25% PEG induced drought. However, isolate RS-3 sub-
stantially improved root length at all water defcit conditions (No
PEG, 15% PEG, and 25% PEG) compared with the
other isolates and the un-inoculated control.
Rhizobial inoculation gave signifcant increase
in the shoot length of the maize seedlings as com-
pared with un-inoculated control under normal
conditions (Table 2). Similarly, inoculation pro-
duced signifcant improvement in the shoot length
over un-inoculated control at 15 and 25% PEG in-
duced drought except RS-10 inoculated seedlings
which showed statistically similar shoot length as
compared with un-inoculated control. Isolate RS-8
showed highest shoot length among the isolates un-
der normal and 15% PEG induced drought. But iso-
late RS-1 gave highest shoot length at 25% PEG in-
duced drought as compared with other inoculants.
Root dry weight was signifcantly improved
with the inoculation of rhizobia over un-inocu-
lated control except RS-13 inoculated seedlings
under normal conditions (Table 2). At 15% PEG
induced drought, rhizobial inoculation signif-
cantly increased the root dry weight with respect
to un-inoculated control. Inoculation of rhizobia
also signifcantly improved root dry weight at 25%
PEG induced drought over un-inoculated control
except RS-2 which showed statistically similar root
dry weight with un-inoculated control. Under nor-
mal and 15% PEG induced drought conditions,
isolate RS-8 showed highest increase in the root
Table 1. Optical density (OD) of rhizobia at densitometer
under normal (No PEG), 15, 25 or 35% PEG-induced drought
in yeast extract mannitol (YEM) broth after a 96-h incubation
at 28 1C and 100 rpm.
Isolate No PEG 15% PEG 25% PEG 35% PEG
RS-1 0.94 c 0.37 d 0.21 b 0.17 ab
RS-2 0.73 e 0.30 f 0.24 b 0.12 ef
RS-3 0.61 f 0.40 c 0.29 a 0.13 d-f
RS-6 1.11 a 0.47 b 0.22 b 0.07 g
RS-7 1.02 b 0.70 a 0.23 b 0.14 c-e
RS-8 0.52 g 0.33 e 0.24 b 0.19 a
RS-10 0.87 d 0.31 ef 0.20 bc 0.16 bc
RS-12 0.73 e 0.25 g 0.17 c 0.15 b-d
RS-13 0.86 d 0.25 g 0.13 d 0.11 f
HSD 0.008 0.007 0.011 0.007
Means sharing similar letters are statistically similar to each other at
p = 0.05. HSD shows honestly signifcance difference. The osmotic
potential of the growth media were as No PEG, -0.04 MPa; 15% PEG,
-0.69 MPa; 25% PEG, -1.65 MPa; and 35% PEG, -2.15 MPa.
Table 2. Screening of rhizobia for plant growth promotion of maize under nor-
mal, 15 or 25% PEG-induced drought in gnotobiotic conditions.
Root length (cm) (n = 3) Shoot length (cm) (n = 3)
Isolates No PEG 15% PEG 25% PEG No PEG 15% PEG 25% PEG
Un-inoculated 13.9 bc 12.7 d 10.7 c 33.2 d 29.2 c 26.4 c
RS-1 17.8 ab 15.0 a-d 14.3 a-c 46.0 ab 44.0 ab 40.5 a
RS-2 15.0 bc 15.0 a-d 11.7 bc 41.2 bc 40.2 b 37.8 ab
RS-3 20.4 a 17.6 a 17.3 a 46.8 a 40.0 ab 39.4 a
RS-6 17.5 a-c 14.2 b-d 13.2 a-c 44.6 a-c 40.0 b 38.1 ab
RS-7 17.2 a-c 16.5 ab 15.6 a-c 47.3 a 40.3 b 38.7 ab
RS-8 19.5 a 16.2 a-c 15.2 a-c 48.0 a 46.3 a 39.5 a
RS-10 13.3 c 13.2 cd 12.5 a-c 41.3 bc 29.8 c 28.8 c
RS-12 19.3 a 15.6 a-d 12.6 a-c 46.3 a 44.9 ab 39.8 a
RS-13 17.0 a-c 16.7 ab 16.3 ab 40.7 c 40.8 ab 34.0 b
HSD 1.19 0.94 1.42 1.41 1.58 1.41
Root dry weight (g plant
-1
) (n = 3) Shoot dry weight (g plant
-1
) (n = 3)
Un-inoculated 0.33 d 0.16 d 0.06 e 0.20 e 0.07 f 0.04 e
RS-1 0.66 b 0.55 a 0.49 a 0.42 a-c 0.40 a 0.17 b-d
RS-2 0.50 c 0.42 b 0.17 de 0.44 ab 0.35 a-c 0.25 ab
RS-3 0.44 c 0.40 bc 0.23 d 0.47 a 0.30 bc 0.26 a
RS-6 0.47 c 0.38 bc 0.25 cd 0.40 a-c 0.15 ef 0.12 de
RS-7 0.49 c 0.41 bc 0.36 bc 0.46 ab 0.43 a 0.23 a-c
RS-8 0.82 a 0.56 a 0.42 ab 0.39 bc 0.36 ab 0.27 a
RS-10 0.48 c 0.29 c 0.25 cd 0.35 cd 0.21 de 0.11 de
RS-12 0.50 c 0.42 b 0.29 cd 0.47 a 0.38 ab 0.16 cd
RS-13 0.43 cd 0.32 bc 0.28 cd 0.29 d 0.27 cd 0.26 a
HSD 0.03 0.04 0.04 0.02 0.02 0.02
Means sharing similar letters are statistically similar to each other at p = 0.05. HSD shows
honestly signifcance difference. No PEG, 15 and 25% PEG solutions were having potentials
-0.02, -0.61 and -1.20 MPa, respectively.
542 Soil Science Society of America Journal
dry weight whereas, at 25% PEG in-
duced drought, RS-1 showed high-
est increase in the root dry weight.
But variable results were observed
in shoot dry weight, where most of
the inoculated treatments remained
statistically similar to each other, yet
signifcantly diferent from respec-
tive un-inoculated control except
RS-6 (at 15 and 25% PEG induced
drought), and RS-10 (at 25% PEG
induced drought), which were at par
with their respective un-inoculated
controls (Table 2). However, RS-3
and RS-12 gave highest increase in
shoot dry weight over control under
normal conditions. At 15% PEG in-
duced drought, RS-7 gave highest signifcant increase over con-
trol and at 25% PEG induced drought, RS-8 inoculated seed-
lings gave highest shoot dry weight.
Among the plants inoculated with diferent rhizobial iso-
lates, a decrease in drought bearing capability was observed with
an increase in PEG concentration (Fig. 1). However, isolates RS-
1, RS-3, RS-8, and RS-12 improved tolerance index up to 51, 57,
59, and 53%, respectively, over the un-inoculated control at 15%
PEG induced drought (-0.61 MPa, osmotic potential). Whereas,
at 25% PEG level (osmotic potential, -1.20 MPa), isolates RS-1,
RS-3, RS-8, and RS-12 showed 53, 49, 49, and 50% increase in
drought tolerance index, respectively, over un-inoculated con-
trol. Te un-inoculated seedlings showed 12 and 20% reduction
in drought tolerance index (at 15 and 25% PEG, respectively)
with respect to the un-inoculated and unstressed control.
Pot Trial
Rhizobial isolates RS-1, RS-3, RS-8, and RS-12 were evalu-
ated for their ability to promote the growth of
maize under drought stress in pots under green-
house conditions. Data presented in Table 3 dem-
onstrate the signifcant ability of these rhizobia to
enhance the fresh cob yield, root length and shoot
fresh weight of maize. Inoculation and drought
could not make signifcant diferences for the
shoot length of plants. Inoculation with RS-3,
RS-8, and RS-12 gave signifcant improvement
in the root length of the maize plants compared
with un-inoculated control in normal conditions.
However, RS-3 was the only inoculant to show
signifcant improvement for root length among
the treatments when drought was applied at vege-
tative growth stage of the crop. Whereas, drought
at reproductive growth stage was signifcantly
relieved by RS-1 and RS-12 inoculation through
signifcantly improving root length as compared
with un-inoculated control. As far as, shoot fresh
weight is concerned, RS-12 inoculation signifcantly improved
it over control in normal conditions. Inoculation of RS-1, RS-8,
and RS-12 showed signifcant improvement in the shoot fresh
weight as compared with un-inoculated control where drought
was created at reproductive growth stage of the crop. Under
drought at vegetative growth stage of the crop, isolate RS-1 in-
oculation gave signifcant improvement in the shoot fresh weight
with respect to un-inoculated control. Among the isolates, RS-3
showed the highest fresh cob yield compared with all other iso-
lates with 37% increase over the un-inoculated control. Isolate
RS-1 was least efcient among the isolates tested with 27% in-
crease in fresh cob yield over un-inoculated control. Drought
at reproductive stage was more injurious than that at vegetative
growth stage.
Te drought tolerance index (Fig. 2) demonstrated that rhi-
zobial inoculation was a good strategy for reducing the deleteri-
ous impact of drought on maize growth and productivity. Water
defcit stress imposed at the vegetative or reproductive stages
resulted in 7 or 20% reductions in the drought tolerance index,
Table 3. Growth and yield parameters of maize infuenced by rhizobial inocula-
tion in pot conditions where irrigation was skipped at vegetative (D1) or repro-
ductive (D2) growth stages of the crop or irrigated normally (D0).
Shoot length (cm) (n = 3) nonsignifcant Root length (cm) (n = 3)
Isolates D0 D1 D2 D0 D1 D2
Un-inoculated 111.7 102.0 98.7 68.7 b 64.0 b 55.0 c
RS-1 120.0 118.3 115.0 71.7 b 67.0 b 65.7 a
RS-3 117.7 110.3 105.7 80.3 a 78.0 a 58.0 bc
RS-8 129.0 126.7 115.7 79.0 a 68.0 b 61.0 b
RS-12 123.3 112.7 101.0 82.0 a 67.3 b 66.7 a
HSD 19.3 25.9 18.4 4.0 4.0 4.6
Shoot fresh weight (g pot
-1
) (n = 3) Fresh cob yield (g pot
-1
) (n = 3)
Un-inoculated 136.3 b 127.3 b 103.7 b 76.3 d 66.7 d 57.7 c
RS-1 144.3 ab 143.0 a 120.0 a 93.0 b 85.3 b 77.0 a
RS-3 141.3 ab 129.0 b 116.3 ab 100.7 a 92.0 a 82.3 a
RS-8 144.7 ab 140.7 ab 124.3 a 95.7 b 88.7 ab 79.0 a
RS-12 153.7 a 136.3 ab 117.7 a 85.3 c 76.0 c 69.7 b
HSD 15.0 13.9 12.9 4.5 4.2 5.4
Means sharing similar letters are statistically similar to each other at p = 0.05. HSD
shows honestly signifcance difference.
Fig. 1. Drought tolerance index of maize seedlings (on the basis of shoot length) due to rhizobial
inoculation at 15 or 25% PEG-induced drought in gnotobiotic conditions.
www.soils.org/publications/sssaj 543
respectively. Inoculation increased the drought tolerance index of
maize plants; RS-3 was the most promising isolate for increasing
the drought tolerance index in maize plants under pot conditions.
Identifcation and Benefcial Characteristics of
Selected Rhizobial Isolate
Selected isolates were identifed as Rhizobium phaseoli
(RS-1 and RS-3), and Mesorhizobium ciceri (RS-8 and RS-
12), respectively, by use of the BIOLOG identifcation system.
Tese identifcations were confrmed when their respective hosts
produced efective nodules (pink colored) afer re-inoculation.
Characteristics such as catalase and exopolysaccharides pro-
duction were observed for all four isolates (Table 4). Rhizobial
strain-1 (RS-1) was the only isolate positive for oxidase produc-
tion. Further, phosphate solubilization activity and organic acid
production were found in RS-1 and RS-12, but RS-3 and RS-8
did not have these properties. Results for siderophores produc-
tion showed that rhizobial isolates RS-3 and RS-8 had the ability
to synthesize siderophores however, RS-1 and RS-12 were non-
producers. Indole-acetic acid (IAA) production was recorded for
all the isolates in the presence or absence of L-tryptophan. Isolate
RS-1 produced highest IAA, with or without L-tryptophan
(3.95, 30.58 mg L
-1
), respectively, followed by isolate RS-12 and
RS-8. Lowest IAA among the isolates was pro-
duced by isolate RS-3.
DISCUSSION
Survival of inoculated rhizobia in soil is
a major concern and depends on the severity
of the environment. Ofen, inoculant bacte-
ria added to soil sufer from desiccation and
high temperature stresses. Rhizobia have a re-
markable ability to survive under severe water
stress, as indicated by the drought tolerance
test of the isolates presented here (Table 1).
However, drought decreases the rhizobial cell
numbers with increase in the PEG concentra-
tion. Findings of our study are in line with the
results of Athar and Johnson (1997), Athar
(1998), and Elboutahiri et al. (2010) who re-
ported survival capability of rhizobia up to -1.5
MPa water potential. Teir ability to live under severe water def-
cit conditions might be due to exopolysaccharides production
and catalase or oxidase activity (Goyal et al., 1986; Fox, 2005).
As bacterially produced exopolysaccharides enhance the nutrient
and water holding capacity in the micro-environment to prolong
their existence. Catalases are involved in scavenging the stress in-
duced H
2
O
2
to reduce the cell damage and oxidases are helpful
in maintaining cell metabolism under stress. About 82.2% of 157
Sinorhizobium melilotii (isolated from arid and semiarid areas of
Morocco) isolates showed tolerance up to -1.5 MPa (Tami-
Alami et al., 2010). Drought tolerant rhizobia accumulated
osmolytes, and changed their cell morphology to survive under
water defcit stress condition (Busse and Bottomley, 1989). One
most important adaptation of exopolysaccharides producing
Rhizobium leguminosarum bv. viciae 3841 is the development of
bioflm for survival under desiccated conditions (Vanderlinde et
al., 2010). Tose drought tolerant rhizobia could be helpful in
rescuing the plant growth under drought conditions due to pro-
longed existence in the rhizosphere.
Drought tolerance is the ability of a plant to grow; fower and
yield under suboptimal water supply (Farooq et al., 2009). Root
length is a very critical plant growth parameter especially under
reduced water conditions. In our study, inoculation with drought
tolerant rhizobial isolates resulted in remarkable improvement
in maize growth as shown by improved root or shoots length,
root or shoots dry weight (Table 2) and drought tolerance
index (Fig. 1) in pot trial in axenic condition. Aferward, in-
oculation of selected rhizobia RS-1, RS-3, RS-8, and RS-12
improved the cob yield whether drought was applied (at vege-
tative or reproductive stage) or not (Table 3). Similar improve-
ments in the sorghum root or shoots dry weight was observed
with the inoculation of Rhizobium or Bradyrhizobium under
drought (Rashad et al., 2002). Tese improvements in the
growth of sorghum might be due to the ability of Rhizobium
or Bradyrhizobium to produce phytohormones, particularly
auxin or cytokinin which are more important during drought
Fig. 2. Drought tolerance index of maize plants (on the basis of root length) due to rhizobial
inoculation under drought at vegetative or reproductive growth stage of the crop in pots.
Table 4. Identifcation and plant growth promoting characteristics of
selected rhizobial isolates.
Rhizobium phaseoli Mesorhizobium ciceri
RS-1 RS-3 RS-8
RS-12
Oxidase +
Catalase + + + +
Exopolysaccharides + + + +
P-solubilization + +
Siderophores + +
Organic acid + +
IAA production,
mg L
-1
Without L-TRP 3.95 0.92 1.35 3.27
With L-TRP 30.58 12.68 19.98 28.30
Plus sign represents the presence and minus sign shows the absence of the
characteristics.
544 Soil Science Society of America Journal
stress. Nitrogen nutrition, water uptake, and dry biomass of sun-
fower were also enhanced due to the inoculation of exopolysac-
charide producing Rhizobium strain, YAS34, under water stress
(Alami et al., 2000). Moreover, exopolysaccharides producing
rhizobial strain KYGT207 improved the rhizosphere soil aggrega-
tion (Kaci et al., 2005). Fitouri et al. (2012) measured signifcant
improvement in shoot dry weight of Hedysarum when inoculated
with a moderately drought tolerant Rhizobium sullae strain iso-
lated from a semiarid region of Tunisia.
In our study, inoculation with rhizobia improved the
drought tolerance index of maize, which might be due to their
ability to produce exopolysaccharides, antioxidants (catalase),
or phytohormones (IAA; Table 4). Rhizobial production of
exopolysaccharides might have altered the maize rhizosphere by
creating a bioflm on the root surface that might have enhanced
water and nutrient availability to the host. Te increased root
growth observed in inoculated maize could be due to indole-
3-acetic acid production by rhizobial isolates. Te better maize
shoot growth observed, also suggests the involvement of mi-
crobial assisted nutrition with their phosphate solubilization
and siderophores activity (enhanced the iron availability and
uptake). Whereas, oxidase and catalase production might have
assisted rhizobial survival under water stress.
It has been recognized that the plants show variable re-
sponses to water defcit stress at diferent developmental stages
(Mogensen et al., 1985; Gupta et al., 2001). Tese unfavorable
conditions also adversely impact the development of reproduc-
tive organs and grain yields (Mogensen et al., 1985; Gupta et al.,
2001; Kettlewell et al., 2010). In our study, the drought impact
was more severe at reproductive stage of maize as compared with
drought at vegetative growth stage. However, rhizobial inocula-
tion might have improved the rate of photosynthesis and ulti-
mately the cob yield under drought. Similar, fndings have been
reported by diferent researchers due to the inoculation of non-
legumes with benefcial bacteria under drought (Vardharajula et
al., 2011; Yandigeri et al., 2012).
Afer the identifcation and confrmation of the rhizobial
isolates it can be argued that the rhizobial isolates were selec-
tive in host type and environmental conditions. Rhizobium and
Mesorhizobium are those bacteria which nodulate mung bean
and chickpea (less water consuming crops), respectively. Tey are
also good producers of plant growth regulators such as sidero-
phores, exopolysaccharides, and organic acids (Table 4).
From the fndings of these experiments, it can be inferred
that rhizobial inoculation to maize under reduced water condi-
tions could be an opportunity to reduce drought stress impact
on maize growth. However, isolates of Rhizobium phaseoli (RS-
1, RS-3), and Mesorhizobium ciceri (RS-8, RS-12) were most
profcient. Inoculation of these selected rhizobial isolates could
be helpful for improving maize growth and yield under reduced
water conditions. Increases in the yield of maize with the inoc-
ulation of indigenous rhizobial isolates in arid (Chakwal) and
semiarid (Faisalabad) regions of Punjab (Pakistan) could be sup-
portive in poverty alleviation. Adoption of inoculation biotech-
nology of efcient rhizobia for non-legumes may reduce the pro-
duction costs by reducing the use of chemicals such as fertilizers
and pesticides, with environmental safety as an additional ben-
eft. Moreover, selected isolates need to be evaluated in natural
feld conditions for improving growth and yield of maize under
reduced water conditions before their recommendation for large
scale application.
ACKNOWLEDGMENTS
We are grateful to the Higher Education Commission (HEC) of
Pakistan for fnancial assistance. Funding was provided by HEC under
Indigenous 5000 PhD Fellowship Program (Batch V).
REFERENCES
Abd-Alla, M.H., F.M. Morsy, A.E. El-Enany, and T. Ohyama. 2012. Isolation
and characterization of a heavy-metal-resistant isolate of Rhizobium
leguminosarum bv. viciae potentially applicable for biosorption of Cd
+2
and
Co
+2
. Intl. Biodeterioration. Biodegradation. 67: 48-55. doi:10.1016/j.
ibiod.2011.10.008
Ahmad, N., R.H. Qureshi, M. Sarwar, and T. Mahmood. 1987. Drought
tolerance of wheat genotypes. Pak. J. Agric. Sci. 24: 231-234.
Alami, Y., W.A. Achouak, C. Marol, and T. Heulin. 2000. Rhizosphere soil
aggregation and plant growth promotion of sunfower by an exopolysaccharides
producing Rhizobium sp. strain isolated from sunfower roots. Appl. Environ.
Microbiol. 66: 3393-3398. doi:10.1128/AEM.66.8.3393-3398.2000
Asghar, H.N., Z.A. Zahir, M. Arshad, and A. Khaliq. 2002. Relationship
between in vitro production of auxins by rhizobacteria and their growth-
promoting activities in Brassica juncea L. Biol. Fertil. Soils. 35: 231-237.
doi:10.1007/s00374-002-0462-8
Ashraf, M., S.H. Berge, and O.T. Mahmood. 2004. Inoculating wheat seedlings
with exopolysaccharides producing bacteria restricts sodium uptake and
stimulates plant growth under salt stress. Biol. Fertil. Soils. 40: 57-162.
Athar, M. 1998. Drought tolerance by lentil rhizobia (Rhizobium leguminosarum)
from arid and semiarid areas of Pakistan. Lett. Appl. Microbiol. 26: 38-42.
doi:10.1046/j.1472-765X.1998.00272.x
Athar, M. and D.A. Johnson. 1997. Efect of drought on the growth and survival
of Rhizobium meliloti strains from Pakistan and Nepal. J. Arid Environ. 35:
335-340. doi:10.1006/jare.1996.0163
Boyer, J.S. 1982. Plant productivity and environment. Science 218: 443-448.
doi:10.1126/science.218.4571.443
Busse, M.D., and P.J. Bottomley. 1989. Growth and nodulation responses
of Rhizobium meliloti to water stress induced by permeating and non-
permeating solutes. Appl. Environ. Microbiol. 55: 2431-2436.
Chi, F., S.H. Shen, H.P. Cheng, Y.X. Jing, Y.G. Yanni, and F.B. Dazzo. 2005.
Ascending migration of endophytic rhizobia, from roots to leaves, inside rice
plants and assessment of benefts to rice growth physiology. Appl. Environ.
Microbiol. 71: 7271-7278. doi:10.1128/AEM.71.11.7271-7278.2005
Economic Survey of Pakistan. 2011. Government of Pakistan, Finance Division.
Economic Adviser Wing, Islamabad.
Elboutahiri, N., I. Tami-Alami, and S.M. Udupa. 2010. Phenotypic and genetic
diversity in Sinorhizobium meliloti and S. medicae from drought and salt
afected regions of Morocco. BMC Microbiol. 10: 15. doi:10.1186/1471-
2180-10-15
Etesami, H., H.A. Alikhani, M. Jadidi, and A. Aliakbari. 2009. Efect of
superior IAA producing rhizobia on N, P, K uptake by wheat grown under
greenhouse condition. World Appl. Sci. J. 6: 1629-1633.
Farooq, M., A. Wahid, N. Kobayashi, D. Fujita, and S.M.A. Basra. 2009. Plant
drought stress: efects, mechanisms and management. Agron. Sustain. Dev.
29: 185-212. doi: 10.1051/agro:2008021
Fitouri, S.D., D. Trabelsi, S. Sadi, K. Zribi, F.B. Jeddi and R. Mhamdi. 2012.
Diversity of rhizobia nodulating sulla (Hedysarum coronarium L.) and
selection of inoculant strains for semi-arid Tunisia. Ann. Microbiol. 62:
77-84. doi:10.1007/s13213-011-0229-2
Fox, M.A. 2005. Adaptation of Rhizobium to environmental stress. Ph.D. Tesis.
Univ. of Reading, UK.
Fuhrmann, J., C.B. Davey, and A.G. Wollum. 1986. Desiccation tolerance
of clover rhizobia in sterile soils. Soil Sci. Soc. Am. J. 50: 639-644.
www.soils.org/publications/sssaj 545
doi:10.2136/sssaj1986.03615995005000030019x
Goyal, V., S. Chetal, and H.S. Nainawatee. 1986. Alteration in Rhizobium trifolii
catalase under water stress. Folia Microbiol. 31: 164-166. doi:10.1007/
BF02926835
Gupta, N.K., S. Gupta, and S. Kumar. 2001. Efect of water stress on physiological
attributes and their relationship with growth and yield of wheat cultivars
at diferent stages. J. Agron. Crop Sci. 186: 55-62. doi:10.1046/j.1439-
037x.2001.00457.x
Gutirrez-Zamora, M.L., and E. Martnez-Romero. 2001. Natural endophytic
association between Rhizobium etli and maize (Zea mays L.). J. Biotechnol.
91: 117-126. doi:10.1016/S0168-1656(01)00332-7
Hussain, M.B., I. Mehboob, Z.A. Zahir, M. Naveed, and H.N. Asghar. 2009.
Potential of Rhizobium spp. for improving growth and yield of rice (Oryza
sativa L.). Soil Environ. 28: 49-55.
Hussain, M.B., Z.A. Zahir, and M. Arshad. 2011. Microbial strategies for
inducing drought tolerance in crops. Farming Outlook 10: 24-30.
Hussain, S., K. Alam, and A.R. Chowdhry. 1986. Some genetic studies on characters
involved in drought resistance in wheat. Pak. J. Agric. Sci. 23: 251-260.
Kaci, Y., A. Heyraud, M. Barakat, and T. Heulin. 2005. Isolation and
identifcation of an EPS-producing Rhizobium strain from arid soil
(Algeria): Characterization of its EPS and the efect of inoculation
on wheat rhizosphere soil structure. Res. Microbiol. 156: 522-531.
doi:10.1016/j.resmic.2005.01.012
Kettlewell, P.S., W.L. Heath, and I.M. Haigh. 2010. Yield enhancement of
droughted wheat by flm antitranspirant application: Rationale and
evidence. Agric. Sci. 1: 143147.
MacFaddin, J.F. 1980. Biochemical tests for identifcation of medical bacteria.
Williams and Wilkins, Baltimore, MD.
Mehboob, I., Z.A. Zahir, M. Arshad, A. Tanveer, and Farooq-e-Azam. 2011.
Growth promoting activities of diferent Rhizobium spp., in wheat. Pak.
J. Bot. 43: 1643-1650.
Mehta, S., and C.S. Nautiyal. 2001. An efcient method for qualitative
screening of phosphate solubilizing bacteria. Curr. Microbiol. 43: 51-58.
doi:10.1007/s002840010259
Mogensen, V.O., H.E. Jensen, and M.A. Rab. 1985. Grain yield, yield
components, drought sensitivity and water use efciency of spring wheat
subjected to water stress at various growth stages. Irrigation Sci. 6: 131
140. doi:10.1007/BF00251561
Muzzamal, H., R. Sarwar, I. Sajid, and S. Hasnain. 2012. Isolation, identifcation
and screening of endophytic bacteria antagonistic to bioflm formers. Pak.
J. Zool. 44: 249-257.
Noel, T.C., C. Cheng, C.K. Yost, R.P. Pharis, and M.F. Hynes. 1996. Rhizobium
leguminosarum as a plant growth promoting rhizobacterium: Direct
growth promotion of canola and lettuce. Can. J. Microbiol. 42: 279-283.
doi:10.1139/m96-040
Rashad, M., A. Ragab, and S. Salem. 2002. Te infuence of some Bradyrhizobium
and Rhizobium strains as plant growth promoting rhizobacteria on the
growth and yield of sorghum (Sorghum bicolor L.) plants under drought
stress. J. Plant Nutr. 92: 664-665. doi:10.1007/0-306-47624-X_322
Sarwar, M., M. Arshad, D.A. Martens, and W.T. Frankenberger, Jr. 1992.
Tryptophan dependent biosynthesis of auxins in soil. Plant Soil 147: 207-
215. doi:10.1007/BF00029072
Schwyn, B. and J.B. Neilands. 1987. Universal chemical assay for the detection
and determination of siderophores. Anal. Biochem. 160: 47-56.
doi:10.1016/0003-2697(87)90612-9
Shakeri, S., R. Roghanian, and G. Emtiazi. 2011. Comparison of intracellular
polyhydroxybutyrate granules formation between diferent bacterial cell
subpopulations by fow cytometry. Jundishapur J. Microbiol. 4: 229-238.
Shetty, G., D. Hetrick, and P. Schwat. 1995. Efects of mycorrhizal fertilizers
amendments on zinc tolerance of plants. Environ. Pollution 88: 308-314.
doi:10.1016/0269-7491(95)93444-5 Steel, K.J. 1961. Te oxidase reaction as
a toxic tool. J. Gen. Microbiol. 25: 297-306. doi:10.1099/00221287-25-2-297
Steel, R.G.D., J.H. Torrie, and D.A. Dicky. 1997. Principles and procedures of
statisticsA biometrical approach. 3rd ed. McGraw-Hill, New York, NY.
Tami-Alami, I., N. Elboutahiri, and S.M. Udupa. 2010. Variability in
natural populations of Sinorhizobium meliloti in Morocco. Options
Mditerranennes 92: 265-269.
Vanderlinde, E.M., J.J. Harrison, A. Muszynski, R.W. Carlson, R.J. Turner, and
C.K. Yost. 2010. Identifcation of a novel ABC-transporter required for
desiccation tolerance, and bioflm formation in Rhizobium leguminosarum
bv. viciae 3841. FEMS Microbiol. Ecol. 71: 327-340. doi:10.1111/j.1574-
6941.2009.00824.x
Vardharajula, S., S.Z. Ali, M. Grover, G. Reddy, and V. Bandi. 2011. Drought-
tolerant plant growth promoting Bacillus spp.: Efect on growth, osmolytes,
and antioxidant status of maize under drought stress. J. Plant Interact. 6:
114. doi:10.1080/17429145.2010.535178
Vincent, J.M. 1970. A Manual for the practical study of root nodule bacteria.
1st ed. International Biological Program, Blackwell Scientifc Publications,
Oxford, UK.
Wollum, A.G., II. 1982. Cultural methods for soil microorganisms. In: A.L.
Page, R.H. Miller, and D.R. Keeney, editors, Methods of soil analysis:
Chemical and microbial properties. 2nd ed. ASA and SSSA Pub. Madison,
WI. p. 718-802.
Yandigeri, M.S., K.K. Meena D. Singh, N. Malviya, D.P. Singh, M.K. Solanki,
A.K. Yadav, and D.K. Arora. 2012. Drought-tolerant endophytic
actinobacteria promote growth of wheat (Triticum aestivum) under water
stress conditions. Plant Growth Regul. 68: 411420. doi:10.1007/s10725-
012-9730-2
Yanni, Y.G., R.Y. Rizk, F.K. Abd-El-Fattah, A. Squartini, V. Corich, A.
Giacomini, F. de Bruijn, J. Rademaker, J. Maya-Flores, P. Ostrom,
M.Vega-Hernandez, R.I. Hollingsworth, E. Martinez-Molina, P.
Mateos, E. Velazquez, J. Wopereis, E. Triplett, M. Umali-Garcia, J.A.
Anarna, B.G. Rolfe, J.K. Ladha, J. Hill, R. Mujoo, P.K. Ng, and F.B.
Dazzo. 2001. The beneficial plant growth-promoting association of
Rhizobium leguminosarum bv. trifolii with rice roots. Aust. J. Plant
Physiol. 28: 845-870.
Yanni, Y.G., R.Y. Rizk, V. Corich, A.Squartini, K.Ninke, S. Philip-Hollingsworth,
G. Orgambide, F. deBruijn, R. Stoltzfus, D. Buckley, T. Schmidt, P.F.
Mateos, J.K. Ladha, and F.B.Dazzo. 1997. Natural endophytic association
between Rhizobium leguminosarum bv. trifolii and rice roots and
assessment of its potential to promote rice growth. Plant Soil 194: 99-114.
doi:10.1023/A:1004269902246
Zahir, Z.A., A. Munir, H.N. Asghar, B. Shahroona, and M. Arshad. 2008.
Efectiveness of rhizobacteria containing ACC-deaminase for growth
promotion of peas (Pisum sativum) under drought conditions. J. Microbiol.
Biotechnol. 18: 958-963.
Zaidi, A., M.S. Khan, M. Ahemad, and M. Oves. 2009. Plant growth promotion
by phosphate solubilizing bacteria. Acta Microbiol. Immunol. Hung. 56:
283-284. doi:10.1556/AMicr.56.2009.3.6

You might also like