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Universidade Federal do Rio Grande do Sul, Instituto de Biociencias, Departamento de Genetica. Av. Bento Goncalves,
9500 Caixa Postal 15.053, CEP 91501-970, Porto Alegre, RS, Brazil
b
Fundacao Estadual de Pesquisa Agropecuaria (FEPAGRO), Rua Goncalves Dias 570, 90130-060 Porto Alegre, RS, Brazil
article info
abstract
Article history:
Several strains of bacilli, mainly species of the genera Bacillus and Paenibacillus, displaying
important plant growth promoting (PGP) characteristics were isolated from seven distinct
rice production zones of the Rio Grande do Sul State, Brazil. Of 296 isolates, 155 were from
24 January 2008
rhizospheric soil and 141 from bulk soil. In order to evaluate the diversity among the isolates
of each bacterial population the Shannon index was used on a 70% similarity basis. Diversity
indices varying from 2.27 to 5.51 were obtained. Using principal coordinate analysis (PCA) to
correlate bacterial diversity with soil parameters, it was found that soil pH was the
Keywords:
characteristic most closely related to bacilli diversity. The bacilli isolated were also analyzed
Bacillus
for some PGP activities. Of those 296 isolates, 94 and 148 produced between 0.1 and 30 mg of
Paenibacillus
indole-3-acetic acid (IAA) ml1 in vitro after 72 and 144 h of incubation, respectively. Twenty-
two isolates were able to solubilize phosphate and 32 isolates produced siderophores.
rhizobacteria (PGPR)
Paenibacillus and Bacillus genera were the most prominent groups in the rhizosphere and
Nitrogen fixation
soil populations analyzed. Paenibacillus borealis was the most frequent species in both
PCR-RFLP
locations. The isolate SVPR30, identified by 16S rRNA gene sequence analysis as a strain
of Bacillus sp., was chosen for in vivo greenhouse experiments and proved to be very efficient
in promoting a significant increase in the roots and shoot parts of rice plants. The identification and isolation of PGP bacilli from temperate and subtropical soils, which combine the
ability to fix nitrogen with the production of substances capable to promote the plant
growth, could significantly increase productivity of grain crops in Brazil.
# 2008 Elsevier B.V. All rights reserved.
1.
Introduction
* Corresponding author at: Universidade Federal do Rio Grande do Sul, Instituto de Biociencias, Departamento de Genetica. Av. Bento
Goncalves, 9500 Caixa Postal 15.053, Predio 43312, sala 207b, CEP 91501-970, Porto Alegre, RS, Brazil. Tel.: +55 51 3308 9813;
fax: +55 51 3308 7311.
E-mail address: lpassaglia@terra.com.br (L.M.P. Passaglia).
0929-1393/$ see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsoil.2008.01.006
312
2.2.
2.
2.1.
DNA isolation
DNA was directly extracted from bacterial cultures by a directlysis method that consisted of boiling the samples for 5 min at
100 8C in 200 ml 0.1 M NaCl. DNA was electrophoresed on 0.8%
agarose ethidium bromide gel.
2.3.
Guaba
Hulha Negra
Osorio
Santana do Livramento
Santa Vitoria do Palmar
Sao Lourenco do Sul
Uruguaiana
exc: exchangeable.
Clay
(%)
pH H2O
P
(mg kg1)
K
(mg kg1)
Al exc
(mg kg1)
Ca exc
(mg kg1)
Mg exc
(mg kg1)
Fe
(%)
13
18
7
22
13
15
20
6.9
4.3
5.5
5.1
4.8
5.1
4.7
18
6
18
18
14
18
2.6
48
121
67
222
75
115
127
0
107.9
0
9.0
36.0
27.0
36.0
840
940
640
2160
640
540
2180
145.9
206.6
182.3
401.1
182.3
218.8
510.5
0.1
0.6
0.1
0.2
0.2
0.3
1.0
Organic
matter (%)
1.4
0.5
0.8
2.6
2.4
3.8
2.9
2.4.
Diversity index
2.5.
2.6.
313
Siderophore production
2.7.
Phosphate solubilization
2.8.
314
2.9.
2.10.
In vivo plant growth promotion experiment with a
native PGPR isolate
A plant growth experiment was carried out with Oriza sativa
plants, variety BR-IRGA 409, according to Mariano and Silveira
(2005) and Dobereiner et al. (1995). Pure bacterial cultures were
grown in King B medium at 28 8C and diluted to a final
concentration of 109 cfu ml1 in sterile distilled water. Rice
seeds were surface-sterilized in 70% ethanol for 2 min and
1.2% sodium hypochlorite for 10 min, and rinsed 10 times in
sterile tap water. Pots (15 cm 20 cm) were sterilized with
0.7% sodium hypochlorite solution, filled with sterile vermiculite and seeded. Plants were inoculated with 1 ml aliquots of
the bacterial cultures by directly irrigating the substrate. The
following treatments were investigated: (1) negative control:
plants were irrigated only with distilled water; (2) positive
control: plants were irrigated with a 50 ml mixture (v/v) of two
mineral fertilizer solutions [Solution 1 was prepared with
4.2 g l1 MgSO4, 1.4 g l1 K2HPO, and 5.8 g l1 KNO3; Solution 2
was prepared with 8.5 g l1 Ca (NO3)2]; (3) type strain: plants
were inoculated with P. polymyxa ATCC 10343 strain; (4)
isolated strain: plants were inoculated with the SVPR30 strain.
Treatments (3) and (4) were irrigated only with distilled water.
Three seeds were placed at the same depth (2.5 cm below the
soil surface) in all pots. Fifteen and thirty days after sprouting,
three plants of each treatment were dried at 65 8C to constant
weight. Next, dry weight, root and plant sizes were determined. Data obtained from the different treatments were
statistically analyzed using the Tukey test at P = 0.05. The
experiment was conducted three times using a completely
randomized design in a greenhouse (12-h photoperiod) with
nine plants per treatment. The results obtained in these
experiments were very similar; therefore, data from only one
experiment are presented.
3.
Results
3.1.
the nifH gene. Of those 296 isolates, 155 were from rhizospheric
soil and 141 from bulk soil, representing, respectively, 52.4%
and 47.6% of the bacilli isolated.
Strains of nitrogen-fixing bacilli belonging to different
species were discriminated by the RFLP-PCR of the nifH gene
approach using the two selected restriction enzymes, HaeIII
and TaqI. The RFLP patterns obtained allowed the construction of a dendrogram for each site sampled. The analysis of
each dendrogram revealed that certain genotypes were typical
of the rhizospheric soil or of the bulk soil (data not shown).
High genetic diversity was found within the different
populations of bacilli: Sao Lourenco do Sul (H0 = 5.51), Santa
Vitorio do Palmar (H0 = 5.23), Guaba (H0 = 4.56), Santana do
Livramento (H0 = 3.39), Uruguaiana (H0 = 2.82), and Hulha
Negra (H0 = 2.27). It was not possible to calculate the diversity
index for the Osorio site, since all isolates presented identical
band patterns in nifH-PCR-RFLP analysis, indicating that the
bacilli population in this locality was very uniform and
composed of strains belonging to the same genus or even to
the same species.
Principal coordinate analysis was used to investigate
relationships between bacterial diversity (H0 ) and abiotic soil
parameters. The first two dimensions of PCA (PCA1 and PCA2)
explained 77.63% of the total variation, with component 1
accounting for 51.56% and component 2 for 26.07% of the
variance (Fig. 1). This analysis showed that pH, clay, and
organic matter contents were the major soil factors affecting
diversity of the six different populations of bacilli (Fig. 1).
315
Table 2 IAA and siderophore production, and phosphate solubilization abilities of bacilli isolates
Sampled site
Number
of isolates
analyzed
Siderophore
production
Phosphate
solubilization
1130,
72 h
>30,
72 h
0.110,
144 h
1130,
144 h
>30,
144 h
1. Guaba
Rhizosphere
Soil
20
20
0
0
0
0
4
0
1
0
0
0
16
13
2
0
0
0
2. Hulha Negra
Rhizosphere
Soil
22
20
10
0
0
0
17
7
0
0
0
0
17
7
5
0
0
0
3. Osorio
Rhizosphere
Soil
19
21
1
0
2
0
1
0
0
1
0
0
5
2
1
1
0
0
4. Santana do
Livramento
Rhizosphere
20
11
Soil
21
Rhizosphere
22
13
Soil
20
Rhizosphere
23
Soil
20
Rhizosphere
Soil
28
20
1
0
0
1
10
8
3
4
0
0
14
4
5
12
0
0
296
32
22
78
16
01
114
34
02
5. Santa Vitoria
do Palmar
6. Sao Lourenco
do Sul
7. Uruguaiana
Total
3.2.
Table 3 Number of isolates that displayed more than one plant growth promoting trait at the same time per samples site
Sampled site
Siderophore
production and
phosphate
solubilization
Phosphate
solubilization
and IAA
production
IAA, siderophore
production and
phosphate
solubilization
1. Guaba
Rhizosphere
Soil
0
0
0
0
0
0
0
0
2. Hulha Negra
Rhizosphere
Soil
0
0
0
0
10
0
0
0
3. Osorio
Rhizosphere
Soil
2
0
0
0
1
0
0
0
4. Santana do Livramento
Rhizosphere
Soil
7
0
0
0
4
2
0
0
Rhizosphere
Soil
0
1
0
0
3
4
1
0
Rhizosphere
Soil
5
1
1
0
1
1
1
0
7. Uruguaiana
Rhizosphere
Soil
0
1
0
0
1
0
0
0
17
27
Total
316
Fig. 2 Dendrogram based on UPGMA cluster analysis with the NTSYS-PC program using the nifH-PCR-RFLP data obtained
from 34 representative isolates. The profiles obtained with restriction endonucleases HaeIII and TaqI are shown. Each
isolate is identified by the sampled site (Gu: Guaba, HN: Hulha Negra, Li: Santana do Livramento, SVP: Santa Vitoria do
Palmar, SLS: Sao Lourenco do Sul, and U: Uruguaiana) and the place of sampling (S: soil; R: rhizosphere). All these isolates
had their 16S rRNA gene partially sequenced and were grouped according to its genus or species designation.
3.3.
Paenibacillus sp.
Bacillus sp.a
Paenibacillus borealis
Paenibacillus rhizosphaerae
Bacillus pocheonensis
Paenibacillus pabuli
Paenibacillus graminis
Bacillus sp.b
Paenibacillus thiaminolyticus
Paenibacillus dendritiformis
31.6
32.9
14.8
7.8
5.8
3.2
3.2
0.7
ND
ND
ND = Not detected.
Bacillus strains with different nifH-PCR-RFLP profiles.
b
Bacillus strains with different nifH-PCR-RFLP profiles.
a
Soil
48.2
35.5
9.2
ND
ND
ND
ND
ND
5.0
2.1
317
Root
(mm)
Shoot
(mm)
Dry matter
(mg)
93 b
190 a
193 a
135 b
180 ab
203 a
30 a
38 a
47 a
235 a
220 a
47 a
151 d
216 c
250 b
168 d
230 c
260 b
59 a
64 a
72 a
298 a
336 a
82 a
3.4.
4.
Discussion
4.1.
318
4.2.
4.3.
4.4.
5.
Conclusion
Acknowledgments
The authors are grateful to Dr. Fernanda Bered for its
statistical support. This work was financed by a grant and
fellowships from Conselho Nacional de Desenvolvimento
Cientfico e Tecnologico (CNPq/Brazil).
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