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Genetic and symbiotic diversity of nitrogen-fixing bacteria isolated from soils under 1
agriculture use in the Western Amazon using cowpea as the trap plant 2
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Running title: N2 fixing bacteria in agriculture soils of Amazonia 4
Amanda Azarias Guimarãesa, Paula Marcela Duque Jaramillob, Rafaela Simão Abrahão 5
Nóbregaa, Ligiane Aparecida Florentinob, Karina Barroso Silvaa and Fatima Maria de 6
Souza Moreiraa, b, # 7
aSetor de Biologia, Microbiologia e Processos Biológicos do Solo, Soil Science graduate 8
programme, Departamento de Ciência do Solo, Universidade Federal de Lavras, Campus 9
UFLA, 37200-000 Lavras, Minas Gerais, Brazil; bMicrobiologia Agrícola graduate 10
programme, Departamento de Biologia, Universidade Federal de Lavras, Campus UFLA, 11
37200-000 Lavras, Minas Gerais, Brazil 12
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#Corresponding author: [email protected]
Present address: Paula Marcela Duque Jaramillo, Departamento de Biologia Celular,
Universidade de Brasília, 70910-900 Brasília, Distrito Federal, Brazil; Rafaela Simão
Abrahão Nóbrega, Universidade Federal do Piauí, Campus Professora Cinobelina Elvas,
64.900-000 Bom Jesus, Piauí, Brazil.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01303-12 AEM Accepts, published online ahead of print on 13 July 2012
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Abstract: Cowpea is a legume of great agronomic importance that establishes symbiotic 17
relationships with nitrogen-fixing bacteria. However, little is known about the genetic and 18
symbiotic diversity of these bacteria in distinct ecosystems. Our study evaluated the genetic 19
diversity and symbiotic efficiency of 119 bacterial strains isolated from agriculture soils in 20
the Western Amazon using cowpea as a trap plant. These strains were clustered into 11 21
cultural groups according to growth rate and pH. The 57 non-nodulating strains were 22
predominantly fast growing and acidifying, indicating a high incidence of endophytic 23
strains in the nodules. The other 62 strains, authenticated as nodulating bacteria, exhibited 24
variable symbiotic efficiency, with 68% of strains promoting a significant increase in shoot 25
dry matter of cowpea when compared with the control with no inoculation and low levels of 26
mineral nitrogen. Fifty genotypes with 70% similarity and 21 genotypes with 30% 27
similarity were obtained through BOX-PCR clustering. The 16S rRNA gene sequencing of 28
strains representative of BOX-PCR clusters showed a predominance of bacteria from the 29
genus Bradyrhizobium, however with high species diversity. Rhizobium, Burkholderia, and 30
Achromobacter species were also identified. These results support observations of cowpea 31
promiscuity and demonstrate the high symbiotic and genetic diversity of rhizobia species in 32
areas under cultivation in the Western Amazon. 33
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Keywords: nodulation, Vigna unguiculata, legume-nodulating bacteria, biodiversity, 35
biological nitrogen fixation, symbiotic promiscuity 36
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INTRODUCTION 40
The Brazilian Amazon Forest covers the states of Acre, Amapá, Amazonas, 41
Maranhão, Mato Grosso, Pará, Rondônia, Roraima, and Tocantins, corresponding to 60% 42
of the national territory and an area of approximately 5,000,000 km2. Although the diversity 43
of the fauna and flora of this extensive region is well studied, little is known about its soil 44
microbiota. The few existing studies on the subject suggest a high level of diversity among 45
the nitrogen-fixing bacteria that nodulate different species of legumes found in this region 46
(8;13; 14; 18). Several studies further indicate the potential of this area to harbor currently 47
undescribed cultivable and non-cultivable prokaryotes (7; 10; 19). 48
Several studies that have examined the diversity of the nitrogen-fixing Leguminosae 49
nodulating bacteria have used cowpea (Vigna unguiculata (L.) Walp) as the trap plant 50
species. Cowpea is an important agronomic plant; it is also considered promiscuous, 51
capable of establishing symbiotic relationships with a variety of nodulating bacteria (20) at 52
varying degrees of efficiency (14). Because of symbiotic promiscuity, it has long been 53
assumed that cowpea did not respond well to inoculation with field-selected strains. 54
However, experiments using Amazonian strains of Bradyrhizobium have shown significant 55
results in soils from Minas Gerais (24). These strains are currently approved for cowpea 56
inoculation by the Ministry of Agriculture, Livestock and Supply (Ministério da 57
Agricultura, Pecuária e Abastecimento, or MAPA) and have been successfully tested in 58
other parts of the country (2). Thus, evaluation of the symbiotic diversity and efficiency of 59
native strains represents an important step towards obtaining novel inoculant strains. 60
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Cultural characteristics have been used successfully for the initial characterization 61
and screening of nodulating bacteria; however, molecular techniques, such as BOX-PCR 62
and 16S rRNA gene sequencing, are strongly recommended because their results are more 63
precise in terms of identification and the evaluation of diversity. 64
The purpose of our study was to evaluate the cultural, genetic and symbiotic 65
diversity of nitrogen-fixing bacteria isolated from cowpea nodules (Vigna unguiculata (L.) 66
Walp) taken from soils under agricultural use in the region of Upper Solimões river, 67
Western Amazon. 68
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MATERIALS E METHODS 70
Strains origin. Strains were obtained from the area between coordinates 4°21’-71
4°26’ S and 69°36’-70°1’ W in the municipality of Benjamin Constant, Amazonas State, 72
which encompasses the town of Benjamin Constant and the localities of Guanabara II and 73
Nova Aliança. This area, known as upper Solimões, is located in the triple frontier of 74
Brazil, Colombia, and Peru. 75
The sampling area includes six windows: windows 1 and 2 in Guanabara II, 76
windows 3, 4, and 5 in Nova Aliança, and window 6 in Benjamin Constant, where several 77
studies on biodiversity and soils have been conducted (http://www.biosbrasil.ufla.br/). 78
These windows were chosen to include the different land use systems in the region: primary 79
forest, secondary forest (late regeneration state), secondary forest (early regeneration state), 80
agroforestry systems, agriculture, and pasture. In each window, sampling points were 81
placed 100 m apart and, in some cases, 50 m apart, totaling 98 sampling points. Soil 82
samples were collected in March 2004, and each composite sample consisted of 12 simple 83
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samples: four sampled in a 3-m radius and eight in a 6-m radius from the sampling point, at 84
a depth of 0-20 cm. 85
A total of 119 strains previously isolated in 2004 from nodules were used in this 86
study. Nodules were surface disinfected by a brief immersion in 95% alcohol, followed by 87
a longer immersion (3 minutes) in H2O2 and washing in several rinses of sterile water (27). 88
These nodules were obtained after the inoculation of soil samples collected from agriculture 89
sampling points, in cowpea cultivar BR14 Mulato serving as the trap plant species. Soil 90
samples were collected at a depth of 0.0-0.20 m from the following sampling points under 91
agriculture systems: 18, 19, 21, 26, 27, 28 and 32 at window 2 (Guanabara II); 49 at 92
window 4 (Nova Aliança) and 72 at window 5 (http://www.biosbrasil.ufla.br/). The ranges 93
of chemical characteristics of the soil samples at depth 0-20 cm collected in this land use 94
system were as follows: pH in water: 4,7 - 6,0; K+ : 42 - 136 mg dm-3; P: 2,3 - 9,3 mg dm-3; 95
S: 2,1 - 10,3 mg dm-3; Al 3+: 0 - 5,4 cmolc dm-3; Ca2+ : 5,6 - 17,5 mg dm-3; Mg2+ : 1,6 - 3,7 96
mg dm-3. Micronutrient levels were as follows: Fe2+: 10,2 - 162,0 mg dm-3; Zn2+ : 1,9 - 11,5 97
mg dm-3; Mn2+ : 20,9 - 116,4 mg dm-3; B: 0,3 - 0,6 mg dm-3 and Cu2+ :0,7 a 1,8 mg dm-3. 98
The organic matter contents varied from 1.4 to 2.2 dag·kg-1, H+Al from 2,6 to 21,4; SB 99
from 8,3 to 21,3 cmolc dm-3; V from 32,4 to 85,5%. Further details on the fertility of these 100
soils compared with other local land use systems are available in Moreira et al. (17). 101
Amendments, fertilizers or pesticides have not been applied to any of the LUS and there is 102
no record of using commercial bacterial inoculants for legumes. 103
Legume-nodulating species of the following genera were found at the soil sampling 104
sites 18, 19, 21, 26, 27, 28 e 32 (Guanabara II): Acacia, Entada, Inga, Mimosa, Swartzia 105
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and Tachigali, being those of the tree species Inga edulis the most abundant. In sampling 106
points 49 and 72 (Nova Aliança) only Piptadenia sp. occurs. 107
The following cultural characteristics of each strain were evaluated in petri dishes 108
with a culture medium (three petri dishes with culture medium by each strain) containing 109
mannitol, yeast extract, mineral salts, and bromothymol blue at pH 6.8 (medium 79) (4) 110
similar to the well known YMA (27): growth rate measured by time to appearance of 111
isolated colonies [fast: 2-3 days (d); intermediate: 4-5 d; slow: 6-10 d; or very slow: more 112
than 10 d]; alteration of culture medium pH (acidification, alkalinization, and 113
neutralization) according to Moreira et al. (18); exopolysaccharide production (minimal, 114
light, moderate, heavy); and colony color according to Jesus et al. (8). Only pH and growth 115
rate were used to determine groups of phenotypic similarity. The distribution of strains in 116
different cultural groups and relative efficiency classes was analyzed graphically using 117
descriptive statistics. 118
Strain authentication and symbiotic efficiency. To examine nodulation capacity 119
(authentication), i.e., the ability to establish symbiosis with its original host, and symbiotic 120
efficiency of the 119 nitrogen-fixing bacteria strains isolated from cowpea nodules (trap 121
species), one experiment was performed in a greenhouse at the Laboratory of Soil 122
Microbiology, Department of Soil Science, Federal University of Lavras. The experiment 123
was conducted over a period of 35 days (November, 3rd to December, 8th, 2008). During 124
this period maximum daily temperature registered varied from 20 to 34ºC and the relative 125
air humidity varied from 70 to 80%. 126
Cowpea (BR17 Gurgueia cultivar) was grown in 500-mL recyclable amber glass 127
bottles wrapped in aluminum foil with a four-fold dilution of modified Hoagland nutrient 128
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solution (5). The inoculated plants and the uninoculated control plants had a low nitrogen 129
concentration (5.25 mg·L-1) in the nutrient solution, which is considered a starting dose for, 130
and not an inhibitor of the process of biological nitrogen fixation. The following quantities 131
of stock solutions were added to 4 L of water: 0.4 mL of 236.16 g·L-1 CaN2O6·4H2O; 0.1 132
mL of 115.03 g·L-1 NH4H2PO4; 0.6 mL of 101.11 g·L-1 KNO3; 2.0 mL of 246.9 g·L-1 133
MgSO4·7H2O; 3.0 mL of 87.13 g·L-1 K2SO4; 10 mL of 12.6 g·L-1 CaH4P2O8·H2O; 200 mL 134
of 1.72 g·L-1 CaSO4·2H2O; 1 mL of 10 g·L-1 FeCl3; and 1 mL of micronutrients (2.86 mg·L-135
1 H3BO3; 2.03 mg·L-1 MnSO4·4H2O; 0.22 mg·L-1 ZnSO4·7H2O; 0.08 mg·L-1 CuSO4·5H2O; 136
and 0.09 mg·L-1 Na2MoO4·H2O). 137
Controls without inoculation and with nitrogen supplementation were also included. 138
In the control with nitrogen supplementation complete Hoagland solution was used, with 139
52.5 mg·L-1 nitrogen. 140
Two strips of filter paper 2 cm wide and of a length corresponding to the height of 141
the bottle were placed inside each bottle to promote contact between the nutrient solution 142
and the cowpea seeds, in addition to a small amount of cotton in the mouth of the bottle to 143
support the seed. Subsequently, all bottles were autoclaved for 40 min at 1.5 kg/cm2 and 144
127°C. 145
Cowpea seeds were surface sterilized with 98% alcohol for 30 s and with 2% 146
sodium hypochlorite for 2 min. Seeds were subsequently washed six times with sterile 147
distilled water, immersed in water for 1 hr, and then placed in petri dishes with moistened 148
sterile cotton in a growth chamber at 28°C for 24 hr, or until radicle emission, at which 149
point they were transferred to bottles containing the nutrient solution. 150
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To generate the treatments, liquid medium 79 (4) was inoculated with bacterial cells 151
previously grown on solid medium 79 using a platinum needle and was incubated at 28°C 152
with constant agitation for 3 days for fast growing strains, 5 days for intermediate growing 153
strains and 8 days for slow growing strains. At planting, each seed was inoculated with 1 154
mL of culture containing about 109 cells. 155
The study was completely randomized and performed in triplicate. Three positive 156
controls inoculated with the reference strains UFLA 03–84, INPA 03–11B (24), and BR 157
3267 (15), which had been approved as cowpea inoculants by the Ministry of Agriculture 158
(http://www.in.gov.br/visualiza/index.jsp?data=10/08/2004&jornal=1&pagina=17&totalAr159
quivos=72), and two uninoculated negative controls with low and high nitrogen content (as 160
described previously) were used in each experiment. 161
To evaluate the symbiotic efficiency of nitrogen-fixing bacteria, plants were 162
harvested 35 days after the commencement of experiments to determine the dry matter of 163
shoots (DMS), number of nodules (NN), and dry matter of nodules (DMN). After the 164
determination of NN, the shoots and nodules were placed in paper bags and dried in a 165
forced air oven (65-70°C) to a constant weight for the determination of dry matter content. 166
The relative efficiency of each treatment was calculated using the following formula: 167
RE = (inoculated DMS/DMS with N) x 100 168
where RE was the relative efficiency, inoculated DMS was the dry matter of shoots after 169
inoculation with respective strain, and DMS with nitrogen was the dry matter of shoots in 170
the treatment that received high amount of mineral nitrogen. 171
All data were tested for normality. The results were analyzed by analysis of variance 172
(ANOVA) with the number of nodules (NN) transformed to the square root of (x + 1) as 173
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recommended by SAS Learning Edition 2.0. Mean values were grouped by the Scott-Knott 174
test (23) at 5% significance using SISVAR. 175
Characterization of genetic diversity by BOX-PCR. The genetic diversity of the 176
62 authenticated strains was evaluated by BOX-PCR. The following type and reference 177
strains were included: Cupriavidus taiwanensis (LMG19424T), Burkholderia sabiae 178
(BR3405), Azorhizobium doebereinerae (BR5401T), Bradyrhizobium sp. (UFLA03-84), 179
B.elkanii (INPA 3-11B), Mesorhizobium plurifarium (BR3804), and Azorhizobium 180
caulinodans (ORS571T). 181
To prepare the samples, isolated colonies from strains grown in medium 79 were 182
placed in 2-mL microtubes containing 1 mL of ultrapure sterile water, heated to 100°C for 183
10 min, and then cooled on ice. A 25-μL amplification reaction was performed with the 184
following components: 9.45 μL of ultrapure sterile water; 1.25 μL of 100 mM dNTPs; 5.0 185
μL of Gitschier 5X buffer (21); 0.4 μL of 20 mg·mL-1 BSA; 2.5 μL of 100% DMSO; 1.0 μL 186
of 0.3 µg·μL-1 BOX primer (5’-CTACGGCAAGGCGACGCTGACG-3’) (26); 0.4 μL of 187
5U·μL-1 Taq DNA polymerase (Fermentas); and 5.0 μL of DNA and the cycling programs 188
were as previously described (21). The amplified fragments were separated by 189
electrophoresis at 45V on a 20 x 20-cm 1.5% agarose gel in 0.5X TAE buffer for 15 hr at 190
room temperature. The 1 kb Plus DNA Ladder (InvitrogenTM) was used as a molecular 191
weight marker. The gel was stained with ethidium bromide and photographed. 192
The genetic diversity of the strains was analyzed by the presence or absence of 193
polymorphic bands in the gel. The data were grouped by the UPGMA (Unweighted Pair 194
Group Mean Arithmetic Method) algorithm and Jaccard coefficient using BioNumerics 6.5 195
(Applied Maths, Sint-Martens-Latem, Belgium). 196
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Characterization of genetic diversity by sequencing of the 16S rRNA gene. A 197
total of 23 authenticated strains, including at least one from each of the 8 cultural groups 198
and representatives of the nine genotypes determined by BOX-PCR at 30% similarity were 199
randomly selected for sequencing of the 16S rRNA gene. Bacteria were grown in medium 200
79 at 28°C for the predetermined growth interval of each strain until logarithm phase. 201
Genomic DNA was extracted from cell cultures according protocol of the extraction kit ZR 202
Fungal/Bacterial DNA (Zymo Research Corp). 203
A 5-μL aliquot of extracted DNA was added to a 50-μL PCR reaction containing 0.2 204
mM dNTP, 2.5 mM MgCl2, 0.2 µM 27F primer (5’-AGAGTTTGATCCTGGCTCAG-3’) 205
(11), 0.2 µM 1492R primer (5’-GGTTACCTTGTTACGACTT-3’) (11), 1 U Taq DNA 206
polymerase (Fermentas), 1X PCR buffer, and ultrapure sterile water. Amplification was 207
performed in an Eppendorf thermal cycler under the following conditions: one initial 208
denaturation step at 94°C for 5 min; 40 cycles of denaturation at 94°C for 40 s, annealing at 209
55°C for 40 s, and extension at 72°C for 1.5 min; and a final extension at 72°C for 7 min. 210
The amplified products were separated on a 1% agarose gel, stained with ethidium bromide, 211
and visualized on a transilluminator. Purification of the products was carried out with 212
Microcon™ (Millipore) filters. Sequencing was performed with primer 27F in a 3730xl 213
sequencer. 214
The quality of sequences was verified using Phred and submitted to BLAST for 215
comparison with GenBank sequences (National Center for Biotechnology Information, 216
2010) using the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov 217
/Genbank/). Only sequences greater than 600 bp in length were used in the phylogenetic 218
analysis. Sequence alignment was performed with ClustalW, and the phylogenetic tree was 219
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constructed using the neighbor-joining method in the Kimura 2 model (9) using the 220
parameters in MEGA version 4 (25). A bootstrap confidence analysis was performed with 221
1000 repetitions. 222
RESULTS 223
The 119 strains examined here were phenotypically clustered according to growth 224
rate (time for appearance of visible isolated colonies) and the ability to change the pH of 225
the culture medium. A total of 11 distinct phenotypes were observed: (FA) fast-growth, 226
total medium acidification; (FN) fast-growth, no alteration of medium pH; (FAL) fast-227
growth, medium alkalinization; (FAN) fast-growth, acidification localized, i.e. around the 228
colonies; (IA) intermediate growth, total medium acidification; (IN) intermediate growth, 229
no alteration of medium pH; (IAL) intermediate growth, total medium alkalinization; (IAN) 230
intermediate growth, acidification localized pH, i.e. around the colonies; (SA) slow growth 231
and medium acidification; (SN) slow growth, no alteration of medium pH; (SAL) slow 232
growth, medium alkalinization (Figure 1). 233
In greenhouse experiment, nodulation was not observed for the control treatments 234
(without inoculation and 52.5 mg L-1 or 5.25 mg L-1 of mineral nitrogen), indicating the 235
absence of contamination. This result allowed for the authentication of symbiosis and the 236
evaluation of the symbiotic efficiency of the selected strains. Our results showed that 237
cowpea established symbiosis with 62 of the 119 strains tested (51%). Strains UFLA 03-238
214, UFLA 03-142, UFLA 03-200, UFLA 03-183, and UFLA 03-195, along with the 239
reference strains UFLA 03-11b and BR 3267, demonstrated the highest means for nodule 240
numbers. 241
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All of the inoculation treatments resulted in a shoot dry mass statistically different 242
to that of the nitrogen control. This included those inoculated with the reference strains 243
UFLA 03-84, UFLA 03-11B, and BR 3267. The mean shoot dry mass of the control with 244
no inoculation and a low level of mineral nitrogen (5.25 mg L-1) was 0.28 g, while that of 245
the control with the optimal dosage of mineral nitrogen for plant development (52.5 mg L-1) 246
was 0.95 g. 247
Figure 2 shows the relative efficiency (RE %) of the strains clustered into groups 248
according to the Scott-Knott test with 5% similarity: efficient (group “b”), intermediate 249
efficiency (groups “c” and “d”), low efficiency (group “e”), and inefficient (groups “f”, “g”, 250
and “h”). The last cluster represents 30% of the isolates studied; these strains did not differ 251
significantly from each other or exhibit lower values than the control without inoculation 252
and a low level of nitrogen. The remaining isolates were clustered with the reference strains 253
UFLA 03-84 (group e), INPA 03-11B (group d), and BR 3267 (group b). 254
The analysis of genetic diversity using BOX-PCR revealed high diversity in 52 of 255
the 62 strains that established symbiosis with cowpea. The DNA from UFLA 03-178, 256
UFLA 03-181, UFLA 03-185, UFLA 03-186, UFLA 03-188, UFLA 03-189, UFLA 03-257
190, UFLA 03-200, UFLA 03-219, and UFLA 03-222 was not sufficiently amplified by 258
BOX-PCR, and these strains were not included in the clustering (Figure 3). Fifty genotypes 259
were grouped with 70% similarity (Figure 3), though the majority consisted of a single 260
strain. Similarity to the reference and the type strains BR 5401 T (Azorhizobium 261
doebereinerae), ORS 571T (Azorhizobium caulinodans), LMG 19424T (Cupriavidus 262
taiwanensis), BR 3405 (Burkholderia sabiae), BR 3804 (Mesorhizobium plurifarium), 263
UFLA 03-84 (Bradyrhizobium sp.), and INPA 03-11B (Bradyrhizobium elkanii) was lower 264
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than 50%. Only two groups with 100% similarity were formed: UFLA 03-148/UFLA 03-265
176 and UFLA 03-173/UFLA 03-150. 266
Figure 4 shows the comparison of 16S rRNA gene sequences from 23 strains that 267
nodulated cowpea, representing eight cultural groups (RA, RAL, IA, IN, IAL, SA, SN, and 268
SAL) and nine genotypes. Groups were formed according to BOX-PCR profiles with 30% 269
similarity to the sequences of known species of the α- and β-Proteobacteria deposited in 270
GenBank; similarities between the studied strains and the GenBank strains ranged from 97 271
to 100% (Table 1). In addition to various species of Bradyrhizobium, GenBank sequence 272
comparisons revealed strains with high similarity to Rhizobium (UFLA 2-186 and UFLA 2-273
188) of the α-Proteobacteria, and to Burkholderia (UFLA 2-216) and Achromobacter 274
(UFLA 03-205, UFLA 03-183, UFLA 03-206, and UFLA 03-202) of the β-Proteobacteria. 275
DISCUSSION 276
Cowpea is a relevant food crop and it is extremely useful for diversity studies 277
because of its promiscuity. Our results from the current study support the observed 278
promiscuity of this plant species through the demonstration of high symbiotic and genetic 279
diversity among the bacterial strains studied. 280
The non-authenticated strains isolated from nodules (i.e., those strains that did not 281
nodulate) were predominantly fast-growing and acidifying strains, indicating the presence 282
of endophytic bacteria that grew faster than the rhizobia during the isolation process (12). 283
The nodules were not senescent because they were stiff with no observed decomposition 284
and were harvested from actively growing plants. 285
Our results for the symbiotic efficiency of the inoculant strains treatments, UFLA 286
03-84 (low efficiency), INPA 3-11b (intermediate efficiency) and BR 3267 (efficient), 287
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which should be similar to the treatment with high amount of mineral N, may be related to 288
their different tolerances to the high temperatures. During the period in which the 289
experiments were performed, the temperature outside the greenhouse was approximately 290
35ºC (http://www.inmet.gov.br), indicating that the indoor temperature was even higher. 291
Temperatures above 34ºC are one factor that may affect the infection process of nodulating 292
bacteria. Plants fertilized with mineral nitrogen show higher tolerance to abiotic stress than 293
plants that must acquire this nutrient through biological nitrogen fixation (28) which can 294
also explain the highest mean of the control treatment with high mineral N supply regarding 295
the treatments that received the efficient inoculant strains mentioned above. 296
The BOX-PCR results suggest high genetic diversity among the nodulating strains 297
isolated from soil under agricultural use and corroborate the results from previous studies in 298
which higher diversity was observed in cultivated lands than in primary forests (8, 13). This 299
finding may be explained by the greater demand for nitrogen that arises in cultivated lands; 300
demand stimulates nodulation and, consequently, the proliferation of rhizobia (16). 301
The high genetic diversity (Figures 3 and 4) of the strains observed in the present 302
study was similar to that reported previously (13) for the diversity of bacteria trapped by the 303
siratro (Macroptilium atropurpureum) trap plant in the same sampling points. However, 304
these authors also reported a higher level of diversity among the nitrogen-fixing bacteria of 305
legumes (Bradyrhizobium, Azorhizobium, Mesorhizobium, Sinorhizobium, Rhizobium, and 306
Burkholderia). In contrast, our study found higher diversity within the Bradyrhizobium and 307
Achromobacter species. The higher prevalence of bacteria exhibiting slow growth and the 308
ability to turn the pH of the culture medium alkaline or neutral is characteristic of 309
Bradyrhizobium species that nodulate cowpea and has been observed previously (3; 29). 310
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Window 2 (Guanabara II) contained most of the sampling points responsible for 311
providing the largest number of strains capable of establishing symbiosis with cowpea. This 312
area also accounts for the largest number of bacterial species identified, which in turn may 313
be related to the high diversity of host legume plant species present in the area. 314
Two strains each of Bradyrhizobium (UFLA 03-145 and UFLA 03-150) and 315
Achromobacter (UFLA 03-205 and UFLA 03-202) (Table 1) were among the strains 316
considered to be efficient. Bradyrhizobium is also the genus to which the strains UFLA 3-317
84, INPA 3-11B and Br 3267 approved as inoculants by MAPA, belong. 318
Strains identified as belonging to Bradyrhizobium showed 100% similarity to as 319
many as four different species (Table 1). However, 16S rRNA gene sequencing does not 320
offer good species-level resolution among members of the Bradyrhizobium, thus requiring 321
further testing to identify species belonging to this genus (30). For example, during the 322
identification of Bradyrhizobium pachyrhizi and B. jicamae by 16S rRNA sequence 323
analysis, similarities of 99.1 and 99.4% to Bradyrhizobium elkanii were observed, 324
respectively. Species differentiation was only possible through the phylogenetic analysis of 325
the intergenic spacer (ITS) 16S-23S and the housekeeping genes glnII and atpD, with 326
subsequent confirmation through homology testing (22). 327
Achromobacter strains UFLA 03-202 and UFLA 03-205 were distinctive in terms of 328
their symbiotic efficiency and clustered with the reference strain BR 3267. Benata et al. (1) 329
were the first to report nodulation of Prosopis juliflora by a species of Achromobacter; but 330
species of the genus were reported as human pathogen (6). Here, we report the occurrence 331
of Achromobacter as a cowpea symbiont for the first time. Further studies should be 332
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conducted to evaluate the efficacy of this symbiosis in field conditions as well as to verify 333
the reliable identification to species level. 334
In conclusion, the strains isolated from agricultural soils in the Upper Solimões river 335
region of the Western Amazon showed high genetic and symbiotic diversity. Strains were 336
found with an efficiency similar to those of reference strains approved for cowpea 337
inoculation, demonstrating their potential as inoculants. BOX-PCR was found to be useful 338
for discriminating strains and revealed high diversity among them, especially among 339
species of Bradyrhizobium. Achromobacter species are also able to nodulate cowpea and 340
are efficient in biological nitrogen fixation. 341
ACKNOWLEDGEMENTS 342
We thank CAPES and CNPq for student fellowships, CNPq for a research 343
fellowship and grant, and project GEF/UNEP-GF2715-02 (CSM-BGBD) for financial 344
support. This work presents part of the findings of the international project “Conservation 345
and Management of Below-Ground Biodiversity” implemented in seven tropical 346
countries—Brazil, Cote d’Ivoire, India, Indonesia, Kenya, Mexico, and Uganda. This 347
project is coordinated by the Tropical Soil Biology and Fertility Institute of CIAT (TSBF-348
CIAT with co-financing from the Global Environmental Facility (GEF), and 349
implementation support from the United Nations Environment Program (UNEP). Brazilian 350
Co-executing Institution was Universidade Federal de Lavras in Brazil project CSM-BGBD 351
was named BiosBrasil. Views expressed in this publication are those of their authors and do 352
not necessary reflect those of the authors’ organization, the United Nations Environment 353
Programme and the Global Environmental Facility. 354
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FIGURE 1 Distribution of the 119 strains isolated from soil under agriculture use into 1
culture groups according to time (days) for appearance of isolated colonies and pH 2
change of the medium: (FA) fast-growth, medium acidification; (FN) fast-growth, no 3
alteration of medium pH; (FAL) fast-growth, medium alkalinization; (FAN) fast-growth, 4
acidification localized pH, i.e. around the colonies; (IA) intermediate growth, medium 5
acidification; (IN) intermediate growth, no alteration of medium pH; (IAL) intermediate 6
growth, medium alkalinization; (IAN) intermediate growth, acidification localized pH, 7
i.e. around the colonies; (SA) slow growth and medium acidification; (SN) slow growth, 8
no alteration of medium pH; (SAL) slow growth, medium alkalinization. 9
FIGURE 2 Distribution of 62 nodulating strains into groups by relative efficiency (RE 10
%) according to Scott-Knott test at 5% similarity. RE = (inoculated DMS/DMS with N) 11
x 100, where inoculated DMS is the dry matter of shoots in the treatment with 12
inoculation of respective strain and DMS with N is the dry matter of shoots in the 13
control treatment with mineral nitrogen. The RE of each strain was the mean of three 14
replicates and each replicate had one plant. 15
FIGURE 3 Dendrogram showing the genetic similarity (based on BOX-PCR profiles) 16
of bacterial strains that nodulated cowpea and of type and reference strains of known 17
rhizobia species. These bacteria were isolated from soils under agricultural use in the 18
Western Amazon. Groups were obtained at 70% similarity. *Indicates isolates that have 19
the 16S rRNA gene sequenced (see Table 1). 20
FIGURE 4 Phylogenetic relationships based on 16S rRNA sequences among strains 21
isolated from cowpea nodules and strains representatives of α- and β-Proteobacteria. 22
Phylogeny was determined by the neighbor-joining method. Bootstrap values were 23
based on 1000 trials. 24
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Table 1. Origin (sampling point), cultural characteristics, relative efficiency and identification (based on 16S rRNA sequences extant in 1
GenBank) of strains that nodulate and fix nitrogen in symbiosis with cowpea. 2
Strains Sampling point1 Cutural
characteristics2
Relative efficency (RE%)3
Base pairs of 16SrRNA sequence
Most similar sequence found in GenBank
Species Accession number % Similarity
UFLA 03-205 27 FAL 46b 850 Achromobacter xylosoxidans HQ676601 100 850 Achromobacter sp HM151970 100
UFLA 03-183 18 FAL 41c 855 Achromobacter xylosoxidans HQ676601 100 855 Achromobacter sp HM151970 100
UFLA 03-206 27 IA 26f 784 Achromobacter xylosoxidans HQ676601 99 784 Achromobacter sp HM151970 99
UFLA 03-202 26 IN 45b 829 Achromobacter xylosoxidans HQ676601 100 829 Achromobacter sp HM151970 100
UFLA 03-216 32 SN 26f 796 Burkholderia sp. AY914317 97 UFLA 03-173 18 IAL 29f 737 Bradyrhizobium liaoningense EU145999 100
737 Bradyrhizobium yuanmingense AB601663 100 737 Bradyrhizobium japonicum GU552901 100 737 Bradyrhizobium sp HQ233244 100
UFLA 03-144 21 SAL 34e 813 Bradyrhizobium elkanii GU552899 100 813 Bradyrhizobium sp AB531432 100
UFLA 03-139 19A SN 19g 780 Bradyrhizobium elkanii GU433457 100 780 Bradyrhizobium sp AB513461 100 780 Bradyrhizobium pachyrhizi PAC48T AY624135 100
UFLA 03-174 18 SN 40c 869 Bradyrhizobium elkanii GU433457 100 869 Bradyrhizobium sp AB513461 100 869 Bradyrhizobium pachyrhizi PAC48T AY624135 100
3
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1
Strains
Sampling point1 Cutural
characteristics2
Relative efficency (RE%)3
Base pairs of 16SrRNA sequence
Most similar sequence found in GenBank
Species Accession number % Similarity
UFLA 03-143 32 SN 41c 814 Bradyrhizobium elkanii GU552899 100 814 Bradyrhizobium sp HQ233232 100
UFLA 03-149 18 SAL 13h 797 Bradyrhizobium elkanii GU433465 100 797 Bradyrhizobium sp AB513461 100 797 Bradyrhizobium pachyrhizi PAC48T AY624135 100
UFLA 03-214 32 SN 35d
779 Bradyrhizobium elkanii GU433457 100 779 Bradyrhizobium sp AB513461 100 779 Bradyrhizobium pachyrhizi PAC48T AY624135 100
UFLA 03-140 32 SN 40c 765 Bradyrhizobium elkanii GU433457 100 UFLA 03-142 32 SN 37d 741 Bradyrhizobium elkanii GU433457 100 UFLA 03-192 19 SAL 41c 725 Bradyrhizobium elkanii GU433457 100 UFLA 03-182 18 SN 38d 636 Bradyrhizobium elkanii GU433457 100
636 Bradyrhizobium sp GU433446 100 UFLA 03-150 49 IAL 43b 722 Bradyrhizobium japonicum HQ231282 100
722 Bradyrhizobium yuanmingense AB601663 100 722 Bradyrhizobium liaoningense HM446270 100 722 Bradyrhizobium canariense AB195986 100 722 Bradyrhizobium sp DQ113663 100
UFLA 03-147 27 SN 33e 759 Bradyrhizobium sp EU364699 100 759 Bradyrhizobium japonicum FJ025100 100 759 Bradyrhizobium iriomotense EK05T AB300992 100 759 Bradyrhizobium liaoningense FJ418695 100
UFLA 03-197 21 IAL 34e 804 Bradyrhizobium japonicum FJ025100 100 804 Bradyrhizobium liaoningense FJ418695 100 804 Bradyrhizobium sp FJ390936 100
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1
2
Strains
Sampling point1 Cutural
characteristics2
Relative efficency (RE%)3
Base pairs of 16SrRNA sequence
Most similar sequence found in GenBank
Species Accession number % Similarity
UFLA 03-148 26 IN 18g 745 Bradyrhizobium japonicum GU552901 100 745 Bradyrhizobium liaoningense GU433468 100 745 Bradyrhizobium yuanmingense HM446269 100 745 Bradyrhizobium canariense AB195986 100 745 Bradyrhizobium sp EU364719 100
UFLA 03-145 28 SN 44b 803 Bradyrhizobium japonicum GU552901 100 803 Bradyrhizobium liaoningense GU433468 100 803 Bradyrhizobium yuanmingense AB601663 100 803 Bradyrhizobium canariense AB195986 100 803 Bradyrhizobium sp AF514794 100
UFLA 03-186 19A FA 32e 827 Rhizobium sp. HM151908 99 UFLA 03-188 19A FA 37d 725 Rhizobium sp. JF740052 99
1 GPS (www.biosbrasil.ufla.br). 2 Cultural characteristics in medium 79: (FA) fast-growth, medium acidification; (FAL) fast-growth, medium alkalinization; (IA) intermediate growth, medium 3 acidification; (IN) intermediate growth, no alteration of medium pH; (IAL) intermediate growth, medium alkalinization; (SN) slow growth, no alteration of medium pH; (SAL) slow growth, 4 medium alkalinization. 3 Means of relative efficiency based on shoot dry matter (SDM) of inoculated treatment compared with SDM of control with Mineral N by the formula: RE = (SDM 5 inoculated/SDM control mineral N) x 100. The same letters in the same column belong to the same group at a 5% significance level (Scott-Knott test). The RE of each strain was the mean of 6 three replicates and each replicate had one plant. 7 8
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