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Antagonistic endophytic bacteria associated with nodules of soybean (Glycine max L.) and plant growth-promoting properties

Abstract

A total of 276 endophytic bacteria were isolated from the root nodules of soybean (Glycine max L.) grown in 14 sites in Henan Province, China. The inhibitory activity of these bacteria against pathogenic fungus Phytophthora sojae 01 was screened in vitro. Six strains with more than 63% inhibitory activities were further characterized through optical epifluorescence microscopic observation, sequencing, and phylogenetic analysis of 16S rRNA gene, potential plant growth-promoting properties analysis, and plant inoculation assay. On the basis of the phylogeny of 16S rRNA genes, the six endophytic antagonists were identified as belonging to five genera: Enterobacter, Acinetobacter, Pseudomonas, Ochrobactrum, and Bacillus. The strain Acinetobacter calcoaceticus DD161 had the strongest inhibitory activity (71.14%) against the P. sojae 01, which caused morphological abnormal changes of fungal mycelia; such changes include fracture, lysis, formation of a protoplast ball at the end of hyphae, and split ends. Except for Ochrobactrum haematophilum DD234, other antagonistic strains showed the capacity to produce siderophore, indole acetic acid, and nitrogen fixation activity. Regression analysis suggested a significant positive correlation between siderophore production and inhibition ratio against P. sojae 01. This study demonstrated that nodule endophytic bacteria are important resources for searching for inhibitors specific to the fungi and for promoting effects for soybean seedlings.

Keywords:
Endophytes; Soybean; Antagonisis; Phytophthora sojae; Plant growth-promoting potential

Introduction

The root nodules of legume plants are symbiotic organs induced by soil bacteria known as rhizobia. As part of the root system, root nodules harbor symbiotic bacteria and many endophytes, including Agrobacterium tumefacien,11 De Lajudie P, Willems A, Nick G, et al. Agrobacterium bv. 1 strains isolated from nodules of tropical legumes. Syst Appl Microbiol. 1999;22:119-132.A. rhizogenes,22 Murugesan S, Vijayakumar R, Panneerselvam A. Characterization of Agrobacterium rhizogenes isolated from the nodules of some leguminous plants. World Appl Sci J. 2011;15:32-37.Phyllobacterium, Stenotrophomonas, Enterobacteriaceae,33 Kan FL, Chen ZY, Wang ET, Tian CF, Sui XH, Chen WX. Characterization of symbiotic and endophytic bacteria isolated from root nodules of herbaceous legumes grown in Qinghai-Tibet Plateau and in other zones of China. Arch Microbiol. 2007;188:103-115.Bacillus species,44 Saini R, Dudeja SS, Giri R, Kumar V. Isolation, characterization, and evaluation of bacterial root and nodule endophytes from chickpea cultivated in Northern India. J Basic Microbiol. 2013;53:1-8.Bacillus, Bordetella, Curtobacterium, and Pantoea.55 Sturz AV, Christie BR, Matheson BG, Nowak J. Biodiversity of endophytic bacteria which colonize red clover nodules, roots, stems and foliage and their influence on host growth. Biol Fertil Soils. 1997;25:13-19. Aside from their diversity, which has been studied extensively, the effect of nodule endophytes on host legumes was revealed. The nodule endophytic Agrobacterium strains specifically inhibit the nodulation of Rhizobium gallicum on the common bean (Phaseolus vulgaris L.)66 Mrabet M, Mnasri B, Romdhane SB, Laguerre G, Aouani ME, Mhamdi R. Agrobacterium strains isolated from root nodules of common bean specifically reduce nodulation by Rhizobium gallicum. FEMS Microbiol Ecol. 2006;56:304-309. or facilitate the unspecific nodulation of Sinorhizobium meliloti on woody legumes.77 Liu J, Wang ET, Ren DW, Chen WX. Mixture of endophytic Agrobacterium and Sinorhizobium meliloti strains could induce nonspecific nodulation on some woody legumes. Arch Microbiol. 2010;192:229-234. Some nodule endophytes that belong to Bacillus, Bordetella, Curtobacterium, or A. rhizogenes could promote the growth or nodulation of red clover.55 Sturz AV, Christie BR, Matheson BG, Nowak J. Biodiversity of endophytic bacteria which colonize red clover nodules, roots, stems and foliage and their influence on host growth. Biol Fertil Soils. 1997;25:13-19. These phenomena are similar to that of endophytes isolated from other parts of plants and could benefit host plants by producing phytohormones, 1-aminocyclopropane-1-carboxylase (ACC) deaminase, and antibiotic compounds, as well as by fixing nitrogen, solubilizing phosphate, or suppressing phytopathogens through the competence of invasion sites.88 Khan Z, Kim SG, Jeon YH, Khan HU, Son SH, Kim YH. A plant growth promoting rhizobacterium, Paenibacillus polymyxa strain GBR-1, suppresses root-knot nematode. Bioresour Technol. 2008;99:3016-3023.

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13 Lin L, Qiao YS, Ju ZY, et al. Isolation and characterization of endophytic Bacillus subtilis Jaas ed1 antagonist of eggplant Verticillium wilt. Biosci Biotechnol Biochem. 2009;73:1489-1493.
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As a major legume crop, soybean (Glycine max L.) plays an important role in sustainable agriculture and in the economy of many countries. Soybean has a great nitrogen-fixing ability due to its symbiosis with rhizobia in root nodules. The presence of Bradyrhizobium japonicum, B. liaoningense, B. yuanmingense, B. elkanii,1515 Man CX, Wang H, Chen WF, Sui XH, Wang ET, Chen WX. Diverse rhizobia associated with soybean grown in the subtropical and tropical regions of China. Plant Soil. 2008;310:77-87.,1616 Han LL, Wang ET, Han TX, et al. Unique community structure and biogeography of soybean rhizobia in the saline-alkaline soils of Xinjiang, China. Plant Soil. 2009;324:291-305.B. huanghuaihaiense,1717 Zhang YM, Li YJ, Chen WF, et al. Bradyrhizobium huanghuaihaiense sp. nov., an effective symbiotic bacterium isolated from soybean (Glycine max L.) nodules. Int J Syst Evol Microbiol. 2012;62:1951-1957.B. daqingense,1818 Wang JY, Wang R, Zhang YM, et al. Bradyrhizobium daqingense sp. nov. isolated from nodules of soybean grown in Daqing City of China. Int J Syst Evol Microbiol. 2013;63:616-624.B. pachyrhizi, B. iriomotense, B. canariense,1919 Zhang YM, Li YJ, Chen WF, et al. Biodiversity and biogeography of rhizobia associated with soybean plants grown in the North China Plain. Appl Environ Microbiol. 2011;77:6331-6342.Sinorhizobium fredii, and S. sojae2020 Li QQ, Wang ET, Chang YL, et al. Ensifer sojae sp. nov., isolated from root nodules of Glycine max grown in saline-alkaline soils. Int J Syst Evol Microbiol. 2011;61:1981-1988. has been reported in China, which is the center of origin of soybean.2121 Lie TA, Gktan D, Engin M, Pijnenborg J, Anlarsal E. Co-evolution of the legume-rhizobium association. Plant Soil. 1987;100:171-181.,2222 Wu LJ, Wang HQ, Wang ET, Chen WX, Tian CF. Genetic diversity of nodulating and non-nodulating rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in different ecoregions of China. FEMS Microbiol Ecol. 2011;76:439-450. Similar to other plants, endophytic bacteria have been isolated from different parts of soybean,1919 Zhang YM, Li YJ, Chen WF, et al. Biodiversity and biogeography of rhizobia associated with soybean plants grown in the North China Plain. Appl Environ Microbiol. 2011;77:6331-6342.,2323 Ashby SF. Some observations on the assimilation of atmospheric nitrogen by a free living soil organism, Azotobacter chroococcum of Beijerinck. J Agric Sci. 1907;2:35-51.

24 Minamisawa K, Seki T, Onodera S, Kubota M, Asami T. Genetic relatedness of Bradyrhizobium japonicum field isolates as revealed by repeated sequences and various other characteristics. Appl Environ Microbiol. 1992;58:2832-2839.

25 Chen WX, Yan GH, Li JL. Numerical taxonomic study of fast-growing soybean rhizobia and a proposal that Rhizobium fredii be assigned to Sinorhizobium gen. nov.. Int J Syst Bacteriol. 1988;38:392-397.
-2626 Zhao L, Fan MC, Zhang DH, et al. Distribution and diversity of rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in Northwest China. Syst Appl Microbiol. 2014;37:449-456. and some of these parts showed antagonistic and growth-promoting potential.2727 Dalal J, Kulkarni N. Antagonistic and plant growth promoting potentials of indigenous endophytic bacteria of soybean (Glycine max (L) Merril). Curr Res Microbiol Biotechnol. 2013;1:62-69.

28 Kuklinsky-Sobral J, Araújo WL, Mendes R, Pizzirani-Kleiner AA, Azevedo JL. Isolation and characterization of endophytic bacteria from soybean (Glycine max) grown in soil treated with glyphosate herbicide. Plant Soil. 2005;273:91-99.
-2929 Senthilkumar M, Swarnalakshmi K, Govindasamy V, Lee YK, Annapurna K. Biocontrol potential of soybean bacterial endophytes against charcoal rot fungus, Rhizoctonia bataticola. Curr Microbiol. 2009;58:288-293. Diverse endophytic bacteria, including Pantoea, Serratia, Acinetobacter, Bacillus, Agrobacterium, and Burkholderia, have also been isolated from soybean nodules.3030 Li JH, Wang ET, Chen WF, Chen WX. Genetic diversity and potential for promotion of plant growth detected in nodule endophytic bacteria of soybean grown in Heilongjiang province of China. Soil Biol Biochem. 2008;40:238-246. However, antagonistic endophytic bacteria within nodules of soybean for P. sojae in Henan Province have not been sufficiently studied.

On the basis of the above mentioned background knowledge and considering the nodule endophytes as a new bacteria resource with potential in biotechnology, we conducted this study (1) to screen antagonistic endophytic bacteria from soybean nodules against P. sojae; (2) to explore the potential plant-beneficial traits of endophytic bacteria; and (3) to assay the seedling growth response of soybean to the inoculation of endophytic bacteria.

Materials and methods

Collection of root nodules, soil samples, phytopathogenic fungus, and soybean seeds

Nodules from cultivated soybean were collected from July to August 2012, when the plants were blooming. Samples were obtained from fields of 14 sites subordinate to 9 districts of Henan Province, China (map available as Supplementary Fig. 1 Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjm.2017.06.007. ).2121 Lie TA, Gktan D, Engin M, Pijnenborg J, Anlarsal E. Co-evolution of the legume-rhizobium association. Plant Soil. 1987;100:171-181.,2222 Wu LJ, Wang HQ, Wang ET, Chen WX, Tian CF. Genetic diversity of nodulating and non-nodulating rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in different ecoregions of China. FEMS Microbiol Ecol. 2011;76:439-450. Three healthy root nodules with similar sizes were excised from the lateral roots of each plant. Soil debris was brushed away from the nodules, and the nodules were stored in sterile plastic bags at 4 °C until they were processed for isolation within 24 h.

In each site, soil cores were sampled at five locations with a depth of 15-20 cm and 5 cm away from the taproots, which were bulked and thoroughly mixed to form composite samples. Soil samples were stored in loosely tied plastic bags at 4 °C. Soil texture was defined according to the international institution triangle coordinate graph, and soil pH was determined as described in Zhao et al.3131 Zhao LF, Deng ZS, Yang WQ, Cao Y, Wang ET, Wei GH. Diverse rhizobia associated with Sophora alopecuroides grown in different regions of Loess Plateau in China. Syst Appl Microbiol. 2010;33:468-477.

A phytopathogenic fungus, P. sojae 01, was provided by the College of Life Sciences of Northwest A & F University in China and was incubated on potato dextrose agar plate (PDA: extract of 200 g potato, 20 g of glucose, 18 g of agar, 1 L of distilled water) at 30 °C for 3 days and maintained at 4 °C for temporary storage.

The seeds of soybean (G. max L.) cultivar Zhonghuang 13, which is the principle cultivar used in the sampling region, were bred by the Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences.

Isolation and purification of soybean nodule endophytes

Bacteria were isolated from root nodules according to a standard method as described by Ma et al.3232 Ma Y, Mani R, Yong ML, Helena F. Inoculation of endophytic bacteria on host and non-host plants. Effects on plant growth and Ni uptake. J Hazard Mater. 2011;195:30-237. and Miller et al.3333 Miller KI, Chen Q, Man-Yuen SD, Roufogalis BD, Neilan BA. Culturable endophytes of medicinal plants and the genetic basis for their bioactivity. Microb Ecol. 2012;64:431-449. A single colony of the isolate was repeatedly streaked on the same medium and examined with a microscope. Pure cultures were preserved on plates at 4 °C for temporary storage or in sterile vials with 30% (v/v) glycerol for long-term storage at -80 °C. To confirm if the surface sterilization process was successful, several surface-sterilized nodules were rolled over nutrient agar (NA) plates and aliquots of water from final rinse solutions and then plated onto NA plates.3434 Deng ZS, Zhao LF, Kong ZY, et al. Diversity of endophytic bacteria within nodules of the Sphaerophysa salsula in different regions of Loess Plateau in China. FEMS Microbiol Ecol. 2011;76:463-475. Plates without any contaminants were considered effectively surface-sterilized, and the corresponding plates were used for the isolation of endophytes.

Screening of antagonistic endophytic bacteria

The antifungal activity of endophytes against pathogenic fungus P. sojae 01 was detected by using the point inoculation method.3535 Zhao LF, Xu YJ, Sun R, Deng ZD, Yang WQ, Wei GH. Identification and characterization of the endophytic plant growth prompter Bacillus cereus strain MQ23 isolated from Sophora alopecuroides root nodules. Braz J Microbiol. 2011;42:567-575. Spores of fungal cultures were inoculated on PDA plates, and a small block of agar with fungal mycelia cut with a sterile puncher (Ø = 4 mm) was placed in the center of a fresh plate. Tested strains were spot inoculated on the edge of PDA plates approximately 25 mm from the center. After incubation at 28 °C for 7 days, the inhibition zone was measured. Fungal mycelia that were cultivated without inoculation were included as control.3636 Geetha R, Falguni S, Anjana JD, Archana G. Enhanced growth and nodulation of pigeon pea by co-inoculation of Bacillus strains with Rhizobium spp.. Bioresour Technol. 2008;99:4544-4550. Experiments were performed in triplicate for each bacterial isolate.

Secondary screening of antifungal activity was performed similar to the primary screening method, but bacteria were spot inoculated as bacterial suspension (OD600 ≈ 1). Antagonistic activities were evaluated by measuring inhibition zones between pathogens and tested bacteria.

Microscopic observation of phytopathogenic fungi mycelia

To determine the effect of endophytic bacteria on pathogenic fungus, treated and untreated pathogenic fungi were cultured for 2 days on PDA medium. The morphological changes of pathogenic fungus caused by endophytes were examined under an optical epifluorescence microscope (BX50 Olympus) at 200-fold magnification and compared with the structures of the control groups. The mycelium of each pathogenic fungus on the growth PDA medium was directly examined and photographed from the plates by using a digital camera (Olympus).

Sequencing and phylogenetic analysis

The total genomic DNA was extracted from the culture of nodule isolates by using the previous method.3737 Moulin L, Béna G, Boivin-Masson C, St Pkowski T. Phylogenetic analyses of symbiotic nodulation genes support vertical and lateral gene co-transfer within the Bradyrhizobium genus. Mol Phylogenet Evol. 2004;30:720-732. The 16S rRNA gene was amplified from the genomic DNA by PCR with the universal forward primer P1 (5′-CGGGAT CCA GAG TTT GAT CCT GGC TCA GAA CGA ACG CT-3′) and reverse primer P6 (5′-CGGGAT CCT ACGGCT ACC TTG TTA CGA CTT CAC CCC-3′), respectively, which corresponded to the positions of 8-37 bp and 1479-1506 bp in Escherichia coli 16S rRNA gene.3838 Van Berkum P, Beyene D, Eardly BD. Phylogenetic relationships among Rhizobium species nodulating the common bean (Phaseolus vulgaris L.). Int J Syst Evol Microbiol. 1996;46:240-244. An aliquot of PCR product of isolates was directly sequenced by Sangon Biotech (Shanghai) Co., Ltd. using the same primers mentioned above. Acquired and related sequences were matched with ClustalX1.81 software, imported into Bioedit 4.8.4, and manually corrected. A phylogenetic tree was constructed using the Jukes-Cantor model and the neighbor-joining method3939 Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406-425. in TREECON package (version 1.3b).4040 Van de Peer Y, De Wachter R. Construction of evolutionary distance trees with TREECON for Windows: accounting for variation in nucleotide substitution rate among sites. Comput Appl Biosci. 1997;132:227-230. The similarity of each tested strain was computed by using the DNAMAN application (version 6.0.3.40, Lynnon Corporation). The acquired 16S rRNA gene sequences were submitted to NCBI GenBank (http://www.ncbi.nlm.nih.gov/). The GenBank accession numbers of the sequences obtained in this study are KF843714-KF843719.

Siderophore production

Bacteria were cultured in Luria-Bertani (LB) broth at 30 °C with shaking at 130 rpm until the exponential growth phase (OD600 ≈ 1) was achieved. The production of siderophores by the bacteria was determined according to the chrome azurol-S (CAS) analytical method.4141 Schwyn B, Neilands JB. Universal chemical assay for the detection and determination of siderophore. Anal Biochem. 1987;160:47-56. The supernatant was obtained by centrifugation at 9000 × g for 10 min and then mixed with 1 mL of CAS assay solution.4242 Manjanatha MG, Loynachan TE, Atherly AG. Tn5 mutagenesis of Chinese Rhizobium fredii for siderophore overproduction. Soil Boil Biochem. 1992;24:151-155. A medium mixed with the CAS assay solution at a 1:1 ratio was included as blank control, and the difference of OD630 between the treatment and blank was estimated as values of siderophore production.4343 Sun LN, Zhang YF, He LY, et al. Genetic diversity and characterization of heavy metal-resistant-endophytic bacteria from two copper-tolerant plant species on copper mine wasteland. Bioresour Technol. 2010;101:501-509. Experiments were performed in triplicate.

Nitrogen fixation and nifH gene amplification

The fixation of atmospheric nitrogen by the bacterium was tested qualitatively using Ashby's N-free medium (NFM: 10 g of mannitol, 0.2 g of KH2PO4, 0.2 g of MgSO4·7H2O, 0.2 g of NaCl, 0.1 g of CaSO4·2H2O, 5 g of CaCO3, pH 7.0-7.5, 1.8 g agar in l L of distilled water).2323 Ashby SF. Some observations on the assimilation of atmospheric nitrogen by a free living soil organism, Azotobacter chroococcum of Beijerinck. J Agric Sci. 1907;2:35-51. Plates were incubated at 28 °C for 3 days, and strains that grew normally in NFM media were defined as candidates of nitrogen fixers.3030 Li JH, Wang ET, Chen WF, Chen WX. Genetic diversity and potential for promotion of plant growth detected in nodule endophytic bacteria of soybean grown in Heilongjiang province of China. Soil Biol Biochem. 2008;40:238-246.,4444 Teng SS, Liu Y, Zhao L. Isolation, identification and characterization of ACC deaminase-containing endophytic bacteria from halophyte Suaeda salsa. Acta Microbiol Sin. 2010;50:1503-1509. Experiments were performed in triplicate. The forward primer nifH40F (5′-GGN ATC GGC AAG TCS ACS AC-3′), reverse primernifH817R (5′-TCR AMC AGC ATG TCC TCS AGC TC-3′),and the procedure described by Vinuesa et al.4545 Vinuesa P, Leon-Barrios M, Silva C, et al. Bradyrhizobium canariense sp. nov., an acid-tolerant endosymbiont that nodulates endemic genistoid legumes (Papilionoideae: Genisteae) from the Canary Islands, along with Bradyrhizobium japonicum bv. genistearum, Bradyrhizobium genospecies alpha and Bradyrhizobium genospecies beta. Int J Syst Evol Microbiol. 2005;55:569-575. were used for nifH gene specific amplification by PCR. PCR products were separated by horizontal electrophoresis in 1% (w/v) agarose gels, and patterns were visualized.

Indole acetic acid (IAA) production

IAA production was estimated by inoculating a bacterial suspension (1 × 108 cfu mL-1) in 10 mL LB broth that contained l-tryptophan (100 µg mL-1) and shaken at 30 °C for 72 h in the dark. Five milliliters of each culture were centrifuged (20 min, 6000 × g), and IAA production was measured as indolic compounds in 2 mL of supernatant mixed with 2 mL of Salkowski reagent, and the absorbance was read at 535 nm after 30 min incubation in the dark.4646 Glickman E, Dessaux Y. A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Appl Environ Microbiol. 1995;61:793-796. A standard curve was used for calibration to quantify. Three replicates were performed for each IAA synthesis measurement.

Plant inoculation studies with endophytic bacteria

Antifungal endophytic bacteria were cultured in TY agar medium at 30 °C to the mid-log phase.4747 Hao X, Xie P, Johnstone L, Miller SJ, Rensing C, Wei G. Genome sequence and mutational analysis of plant-growth-promoting bacterium Agrobacterium tumefaciens CCNWGS0286 isolated from a zinc-lead mine tailing. Appl Environ Microbiol. 2012;78:5384-5394. Cells were pelleted by centrifugation (3440 × g, 10 min at 4 °C), washed twice with a sterile saline solution, and prepared for bacterial suspensions (approximately 108 cfu mL-1). The treatment of soybean seeds was the same as the surface sterilization of nodules.3131 Zhao LF, Deng ZS, Yang WQ, Cao Y, Wang ET, Wei GH. Diverse rhizobia associated with Sophora alopecuroides grown in different regions of Loess Plateau in China. Syst Appl Microbiol. 2010;33:468-477. In each sterile Petri dish, 30 surface-sterilized seeds were placed separately on moist filter paper for germination at 28 °C. Germinated seeds were immersed in bacterial suspension for 8 h. Experiments were conducted in triplicate. The control was immersed with sterile water.

Inoculated seedlings were sown in pots filled with 190 g sterilized vermiculite and then incubated in the greenhouse with a photoperiod of 16 h daylight at 22 °C, a night temperature of 20 °C, and 65% relative humidity. After the first main leaf appeared, each seedling was inoculated with 108 cfu of the tested strain every week, and sterilized water was poured every 3 days to maintain relative humidity.4747 Hao X, Xie P, Johnstone L, Miller SJ, Rensing C, Wei G. Genome sequence and mutational analysis of plant-growth-promoting bacterium Agrobacterium tumefaciens CCNWGS0286 isolated from a zinc-lead mine tailing. Appl Environ Microbiol. 2012;78:5384-5394. Seedlings without inoculation were included as blank control. Plants were harvested after 6 weeks, and root length, fresh weight, and chlorophyll content were determined.

Statistical analysis

Data collected from growth promotion and endophytic inoculation experiments were examined with ANOVA using the IBM SPSS 17.0 package (by the Data Theory Scaling System Group, Faculty of Social and Behavioral Sciences, Leiden University, The Netherlands). The effects of six endophytic bacteria on shoot length, root length, fresh weight, and chlorophyll content of soybean seedlings were analyzed with GraphPad Prism 5.01 software.

Results

Isolation and screening of antagonistic bacteria

A total of 276 bacterial isolates were obtained, of which 31 showed significant inhibition (inhibition ratio >42%) against P. sojae 01 in the initial and secondary screenings (Supplementary Table S1 Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjm.2017.06.007. ). Six isolates that showed more than 63% inhibition for mycelial growth of P. sojae 01 on PDA plate were selected for further study. These isolates were DD161 (71.14% inhibition), DD176 (70.30%), DD198 (68.43%), DD222 (64.32%), DD201 (63.40%), and DD234 (63.16%).

Microscopic observation of phytopathogenic fungus

The colonies of pathogenic fungus were more inhibited after 4 days of culturing with endophytic bacteria on PDA medium compared with the control. Microscopic observation showed that the fungal mycelia presented morphological changes in the treatment of endophytic bacteria. The treated fungus became fractured (Fig. 1B) orlysed (Fig. 1C) and were wrapped with biofilm formed by the bacteria (Fig. 1B and C) unlike the control (Fig. 1A). In addition, for the mycelia treated by endophytic bacteria, the hyphae ends became protoplast balls (Fig. 1D and G) or split (Fig. 1G) even though they were not wrapped by biofilm. Some aerial hyphae showed sarciniform wrapped around each other (Fig. 1E) and twisted (Fig. 1E), as well as a fractured and spherical protoplastend. Furthermore, some aerial hyphae became thin, transparent, and bent, and formed transparent liquid droplets (Fig. 1F) under the action of endophytic bacteria.

Fig. 1
Morphological changes of the mycelia of plant pathogenic fungi upon interaction with endophytes isolated from soybean nodules. (A) Normal mycelia of Phytophthora sojae 01(CK); (B) mycelia became wrapped with biofilm formed by endophytic bacteria DD222 (1); (C) mycelia became fractured (2), lysis (3) under effect by endophytic bacteria DD161; (D) hyphae end became protoplast concentration and formed a ball (4, 5, 6, and 7) for mycelia unwrapped by biofilm under the action of DD201; (E) some aerial hyphae showed sarciniform (8) wrapped around each other (8) and twisted (9, 10) under the action of DD198. (F) Aerial hyphae became thin, transparent, and bent, and formed transparent liquid droplets (11, 12, 13, and 14) under the action of endophytic bacteria DD234; (G) hyphae end became split ends (15, 17) and protoplast concentration appeared spherical (16) under the action of endophytic bacteria DD176.

Phylogenetic analysis of antagonistic endophytic bacteria

The phylogeny of 16S rRNA genes indicated that six endophytic antagonists belonged to five genera, as shown in Fig. 2 and Table 1. DD198 showed 99.9% similarity with Enterobacter cloacae XJU-PA-7 (EU733519). DD161 had 100% sequence similarities with Acinetobacter calcoaceticus. DD201 was 100% similar to Pseudomonas putida, DD234 was 100% similar to Ochrobactrum haematophilum, and DD222 and DD176 presented 100% similarities with Bacillus amyloliquefaciens and Bacillus cereus, respectively.

Fig. 2
Neighbor joining tree based on alignment of nucleotide sequences of the 16S rRNA gene from tested strains (shown in bold) and reference strains. GenBank accession numbers were placed in parentheses. Bootstrap values greater than 50% were indicated. Scale bar represents the number of substitutions per site.

Table 1
Phylogenetic similarity and plant-growth promoting properties of endophytic bacteria.

Characterization of potential plant-beneficial traits of endophytic bacteria

Table 1 summarizes the results of PGP trait evaluation in vitro. Except for DD234, the other five strains showed the capacity to produce siderophore and IAA, as well as the capacity to fix nitrogen. Different biosyntheses of siderophores were found among the strains. DD161, DD176, and DD198 produced 54.3, 48.3, and 20.7 µg mL-1 of siderophores, respectively, while DD222, DD201, and DD234 produced less than 5 µg mL-1 of siderophores in the same experimental conditions. Regression analysis showed a significant positive correlation between siderophore production and inhibition ratioagainst P. sojae 01 (R = 0.9643, p = 0.0019 < 0.05) (details available in Supplementary Fig. S2 Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjm.2017.06.007. ). IAA production of DD201 was significantly higher (16.5 µg mL-1) than that of the other five (<3 µg mL-1).

Seedling growth response to the inoculation of endophytic bacteria

The results in Fig. 3 showed that inoculations with DD176 significantly increased (19.2%, p < 0.05) the shoot length of soybean seedlings. Inoculations with DD176, DD161, and DD198 resulted in a significant increase in root length (38.32%, 36.23%, and 29.82%, respectively) (p < 0.05), fresh weight of plants (36.45%, 20.47%, and 17.00%, respectively) (p < 0.05), and chlorophyll content (36.73%, 17.09%, and 13.75%, respectively). Overall, these results showed that inoculation of the tested endophytic bacteria significantly improved the growth of soybean seedlings.

Fig. 3
Effect of six endophytic bacteria on shoot length (A), root length (B), fresh weight (C), and chlorophyll content (D) of soybean seedlings. Each value is the mean of 10 replicates. Bars represent the standard deviations of mean. Statistical significance was determined at p < 0.05 according to Tukey's test. Asterisk represents significant difference.

Discussion

Currently, endophytic microorganisms are believed to be an important bioresource for modern agriculture4848 Peixoto-Neto PAS, Azevedo JL, Araújo WL. Microorganismos endofíticos: interações com plantas e potential biotecnológico. Biotecnol Cienc Desenv. 2002;29:62-76. because of the beneficial effects of endophytes on plant growth promotion, biocontrol, and disease resistance. As a part of the root system, root nodules are also a habitat for endophytes. However, this habitat is different from other parts of plants because the endophytes in this habitat have to compete and co-exist with symbiotic bacteria and help the plants through certain mechanisms. In this study, a significant inhibitory activity against pathogenic fungus P. sojae 01 was found among 11.2% (31/276) of the nodule endophytic bacteria (Table 1 and Supplementary Table S1 Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjm.2017.06.007. ). The high proportion of fungal antagonistic bacteria in the root nodules revealed in this study and in previous studies4949 Lin TX, Zhao LF, Yang Y, Guan Q, Gong MF. Potential of endophytic bacteria isolated from Sophora alopecuroides nodule in biological control against Verticillium wilt disease. Aust J Crop Sci. 2013;7:139-146. demonstrated that antagonistic activity might be a universal mechanism through which nodule endophytic bacteria can help host plants.

In addition to the antagonism against pathogenic fungi, all six strains produced siderophores and IAA, while five strains were capable of fixing nitrogen. These results demonstrated that nodule endophytic bacteria have diverse functions in the inhibition of phytopathogens and in promoting growth.

Currently, several possible mechanisms are suggested for the inhibition of phytopathogens by endophytic bacteria: (1) competition with pathogens for the ecological niche/substrate (siderophores) in the rhizosphere; (2) production of antibiotics (cyclic lipopeptides, iturin, fengycin)5050 Pertot I, Puopolo G, Hosni T, Pedrotti L, Jourdan E, Ongena M. Limited impact of abiotic stress on surfactin production in planta and on disease resistance induced by Bacillus amyloliquefaciens S499 in tomato and bean. FEMS Microbiol Ecol. 2013;86:505-519. and antifungal substances (2,4-diacetylphloroglucinol); (3) production of extracellular chitinase and laminarinase to lyse fungal cells5151 Mauch F, Mauch-Mani B, Boller T. Antifungal hydrolases in pea tissue. II. Inhibition of fungal growth by combinations of chitinase and β-1,3-glucanase. Plant Physiol. 1988;88:936-942. and degrade fusaric acid produced by fungal pathogens5252 Saharan BS, Nehra V. Plant growth promoting rhizobacteria: a critical review. Life Sci Med Res. 2011;LSMR-21:.; and (4) production of volatile organic compounds(such as 2,3-butanediol and acetoin, which act as signaling molecules to mediate plant-microbe interactions),5353 Ryu CM, Farag MA, Hu CH, et al. Bacterial volatiles promote growth in Arabidopsis. Proc Natl Acad Sci USA. 2003;100:4927-4932. which could strongly inhibit pathogen growth on different hosts5454 Bulgari D, Casati P, Crepaldi P, et al. Restructuring of endophytic bacterial communities in grapevine yellows-diseased and recovered Vitis vinifera L. plants. Appl Environ Microbiol. 2011;77:5018-5022. and elicit plant growth by induced systemic resistance (ISR).5050 Pertot I, Puopolo G, Hosni T, Pedrotti L, Jourdan E, Ongena M. Limited impact of abiotic stress on surfactin production in planta and on disease resistance induced by Bacillus amyloliquefaciens S499 in tomato and bean. FEMS Microbiol Ecol. 2013;86:505-519.,5555 Lugtenberg BJ, Kamilova F. Plant-growth-promoting rhizobacteria. Annu Rev Microbiol. 2009;63:541-556. The results obtained in the current study might indicate evidence of the first and the third mechanisms.

The high correlation (R 2 = 0.93) of siderophores and the fungal inhibition of nodule endophytic bacteria in this study (Supplementary Fig. S2 Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjm.2017.06.007. ) supported the idea that the strong ferrous absorption by endophytic bacteria might be a mechanism for the inhibition of fungal growth. Meanwhile, the formation of biofilm on the hyphae and the morphological changes of the mycelia of the target fungus (Fig. 1) showed that antifungal substances and fungal cell-lysing enzymes might be produced by the endophytic bacteria, as reported by Mauch et al.5151 Mauch F, Mauch-Mani B, Boller T. Antifungal hydrolases in pea tissue. II. Inhibition of fungal growth by combinations of chitinase and β-1,3-glucanase. Plant Physiol. 1988;88:936-942. These results demonstrated that nodule endophytic bacteria are important resources for searching for inhibitors specific to the fungi but without negative effects on symbiotic bacteria.

In accordance with the phylogeny of 16S rRNA genes (Fig. 2), the six most efficient (>63% inhibition) antagonistic strains, which were preliminarily identified as belonging to five genera (Table 1), demonstrated again that the root nodules could be occupied by diverse bacteria. These findings supported the results of other studies.33 Kan FL, Chen ZY, Wang ET, Tian CF, Sui XH, Chen WX. Characterization of symbiotic and endophytic bacteria isolated from root nodules of herbaceous legumes grown in Qinghai-Tibet Plateau and in other zones of China. Arch Microbiol. 2007;188:103-115.,3030 Li JH, Wang ET, Chen WF, Chen WX. Genetic diversity and potential for promotion of plant growth detected in nodule endophytic bacteria of soybean grown in Heilongjiang province of China. Soil Biol Biochem. 2008;40:238-246.,5656 Kumar V, Pathak DV, Dudeja SS, Saini R, Narula S, Anand RC. Legume nodule endophytes more diverse than endophytes from roots of legumes or non legumes in soils of Haryana. India J Microbiol Biotechnol Res. 2013;3:83-92.,5757 Stajković O, De Meyer S, Miličić B, Willems A, Delić D. Isolation and characterization of endophytic non-rhizobial bacteria from root nodules of alfalfa (Medicago sativa L.). Bot Serb. 2009;33:107-114. All these five genera, namely, Acinetobacter, Bacillus, Enterobacter, Ochrobactrum, and Pseudomonas, have been reported previously as nodule endophytes of different legumes, including soybean.33 Kan FL, Chen ZY, Wang ET, Tian CF, Sui XH, Chen WX. Characterization of symbiotic and endophytic bacteria isolated from root nodules of herbaceous legumes grown in Qinghai-Tibet Plateau and in other zones of China. Arch Microbiol. 2007;188:103-115.,5858 Li BJ, Luo M, Zhou J, Kong DJ, Zhang TM. Isolation of endophytic diazotrophic bacteria from several gramineae grasses and determination of their nitrogenase activity. Acta Pratacult Sin. 2008;5:37-42.

59 Tariq M, Hameed S, Yasmeen T, Zahid M, Zafar M. Molecular characterization and identification of plant growth promoting endophytic bacteria isolated from the root nodules of pea (Pisum sativum L.). World J Microbiol Biotechnol. 2014;30:719-725.
-6060 Zakhia F, Jeder H, Willems A, Gillis M, Dreyfus B, de Lajudie P. Diverse bacteria associated with root nodules of spontaneous legumes in Tunisia and first report for nifH-like gene within the genera Microbacterium and Starkeya. Microb Ecol. 2006;51:375-393. However, these endophytes were isolated from soybean nodules collected from different regions of Henan Province, thereby suggesting the antagonistic effect of soybean endophytes on pathogenic fungus of diverse geographical sources and species. This diversity may be attributed to multiple symbiotic relationships in the particular region (the original area of soybean).2121 Lie TA, Gktan D, Engin M, Pijnenborg J, Anlarsal E. Co-evolution of the legume-rhizobium association. Plant Soil. 1987;100:171-181.,2222 Wu LJ, Wang HQ, Wang ET, Chen WX, Tian CF. Genetic diversity of nodulating and non-nodulating rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in different ecoregions of China. FEMS Microbiol Ecol. 2011;76:439-450. During a long evolution period, soybean, rhizobia, and endophytes formed a multiple symbiotic relationship, and soybean provides nutrients and a suitable environment for symbiotic and endophytic bacteria. Rhizobia provided nitrogen nutrition for plants and endophytes, while endophytes strengthened the resistance of plants and symbiotic bacteria against pathogens and bad environmental factors.

In this study, two of the antifungal endophytic bacteria (DD176 and DD222) were identified as Bacillus sp. Bacillus is one of the most abundant rhizosphere bacteria and nodule endophytes.3434 Deng ZS, Zhao LF, Kong ZY, et al. Diversity of endophytic bacteria within nodules of the Sphaerophysa salsula in different regions of Loess Plateau in China. FEMS Microbiol Ecol. 2011;76:463-475.,5858 Li BJ, Luo M, Zhou J, Kong DJ, Zhang TM. Isolation of endophytic diazotrophic bacteria from several gramineae grasses and determination of their nitrogenase activity. Acta Pratacult Sin. 2008;5:37-42.,6060 Zakhia F, Jeder H, Willems A, Gillis M, Dreyfus B, de Lajudie P. Diverse bacteria associated with root nodules of spontaneous legumes in Tunisia and first report for nifH-like gene within the genera Microbacterium and Starkeya. Microb Ecol. 2006;51:375-393. These bacteria could improve the yields of various crops6161 Khan AA, Jilani G, Akhtar MS, Navqi SMS, Rasheed M. Phosphorus solubilizing bacteria: occurrence, mechanisms and their role in crop production. J Agric Biol Sci. 2009;1:48-58.

62 Wang H, Wen K, Zhao X, Wang X, Li A, Hong H. The inhibitory activity of endophytic Bacillus sp. strain CHM1 against plant pathogenic fungi and its plant growth-promoting effect. Crop Prot. 2009;28:634-639.
-6363 Leite HA, Silva AB, Gomes FP, Faria JC, de Souza JT, Loguercio LL. Bacillus subtilis and Enterobacter cloacae endophytes from healthy Theobroma cacao L. trees can systemically colonize seedlings and promote growth. Appl Microbiol Biotechnol. 2013;97:2639-2651. by stimulating plant growth (with hormones) and improving nutrient supply (with phosphate-solubilizing siderophores)or by antagonism against phytopathogens through protease or cellulose production.6464 Kilian M, Steiner U, Krebs B, Junge H, Schmiedeknecht G, Hain R. FZB24 Bacillus subtilis-mode of action of a microbial agent enhancing plant vitality. Pflanzenschutz Nachr Bayer. 2000;1:72-93.

65 Lodewyckx C, Vangronsveld J, Porteous F, et al. Endophytic bacteria and their potential applications. Crit Rev Plant Sci. 2002;21:583-606.
-6666 Li L, Sinkko H, Montonen L, Wei GH, Lindström K, Räsänen LA. Biogeography of symbiotic and other endophytic bacteria isolated from medicinal Glycyrrhiza species in China. FEMS Microbiol Ecol. 2012;79:46-68. Our results showed that B. sp.DD176 and B. sp.DD222 produced siderophore and hormones (IAA) in addition to effectively inhibiting the pathogenic fungus (Table 1). The formation of biofilm and the accompanying morphological changes in the mycelia (Fig. 1) supported the idea that Bacillus spp. could produce several types of enzymes to degrade fungal cell walls, which resulted in a protoplast ball or split ends of the mycelia. Furthermore, B. amyloliquefaciens strains are characterized by high rhizosphere competence and a significant genetic apparatus devoted to the biosynthesis of a wide range of substances with antibiotic activity.5555 Lugtenberg BJ, Kamilova F. Plant-growth-promoting rhizobacteria. Annu Rev Microbiol. 2009;63:541-556.,6767 Stein T. Bacillus subtilis antibiotics: structures, synthesis and specifics functions. Mol Microbiol. 2005;56:845-847.

68 Chen XH, Koumoutsi A, Scholz R, et al. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. J Biotechnol. 2009;140:27-37.

69 Ongena M, Jacques P. Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol. 2008;16:115-125.

70 Ruckert C, Blom J, Chen X, Reva O, Borriss R. Genome sequence of B. amyloliquefaciens type strain DSM7 (T) reveals differences to plant-associated B. amyloliquefaciens FZB42. J Biotechnol. 2011;155:78-85.
-7171 Raaijmakers JM, de Bruijn I, Nybroe O, Ongena M. Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol Rev. 2010;34:1037-1062. All previous studies and our study demonstrated that nodule endophytic Bacillus strains are valuable candidates for exploring biofertilizers.

The isolate DD198 was identified as Enterobacter cloacae, which is a rhizophere bacterium3434 Deng ZS, Zhao LF, Kong ZY, et al. Diversity of endophytic bacteria within nodules of the Sphaerophysa salsula in different regions of Loess Plateau in China. FEMS Microbiol Ecol. 2011;76:463-475.,5858 Li BJ, Luo M, Zhou J, Kong DJ, Zhang TM. Isolation of endophytic diazotrophic bacteria from several gramineae grasses and determination of their nitrogenase activity. Acta Pratacult Sin. 2008;5:37-42.,6060 Zakhia F, Jeder H, Willems A, Gillis M, Dreyfus B, de Lajudie P. Diverse bacteria associated with root nodules of spontaneous legumes in Tunisia and first report for nifH-like gene within the genera Microbacterium and Starkeya. Microb Ecol. 2006;51:375-393.,7272 Ladha JK, Reddy RM. Steps toward nitrogen fixation in rice. In: Ladha LK, Reddy PM, eds. The Quest for Nitrogen Fixation in Rice. Manila: International Rice Research Institute; 1983:33–46. that produces phytohormones, such as ethylene, auxins, cytokinins,6565 Lodewyckx C, Vangronsveld J, Porteous F, et al. Endophytic bacteria and their potential applications. Crit Rev Plant Sci. 2002;21:583-606. siderophores,6363 Leite HA, Silva AB, Gomes FP, Faria JC, de Souza JT, Loguercio LL. Bacillus subtilis and Enterobacter cloacae endophytes from healthy Theobroma cacao L. trees can systemically colonize seedlings and promote growth. Appl Microbiol Biotechnol. 2013;97:2639-2651.,7373 Giongo A, Beneduzi A, Ambrosini A, et al. Isolation and characterization of two plant growth promoting bacteria from the rhizoplane of a legume (Lupinus albescens) in sandy soil. Rev Bras Ciênc Solo. 2010;34:361-369. and fixes nitrogen.7272 Ladha JK, Reddy RM. Steps toward nitrogen fixation in rice. In: Ladha LK, Reddy PM, eds. The Quest for Nitrogen Fixation in Rice. Manila: International Rice Research Institute; 1983:33–46. In the present study, strain E. cloacae DD198 showed significant inhibitory activity against P. sojae 01 in vitro and promoted effects for wheat seedlings with inoculation treatment.

In our study, A. calcoaceticus DD161 possesses the strongest ability to produce siderophores and inhibit the growth of pathogenic fungus, as well as synthesize IAA. The comprehensive effect indicated that the growth of soybean seedling inoculated with DD161 was significantly improved (Fig. 3). Interestingly, only a few reports showed that A. calcoaceticus strains indicated both nitrogen fixation activity and inhibition effect aside from their PGPR activity. Previous reports showed that A. calcoaceticus isolated from rhizosphere of wheat7474 Sturz AV, Matheson BG, Arsenault W, Kimpinski J, Christie BR. Weeds as a source of plant growth promoting rhizobacteria in agricultural soils. Can J Microbiol. 2001;47:1013-1024.,7575 Huddedar SB, Shete AM, Tilekar JN, Gore SD, Dhavale DD, Chopade BA. Isolation, characterization and plasmid pUPI126 mediated indole-3-acetic acid (IAA) productions in Acinetobacter strains from rhizosphere of wheat. Appl Biochem Biotechnol. 2002;102-103:21-39. could synthesize IAA from tryptophan and produce siderophores and phosphate-solubilizing organic acids. Therefore, the A. calcoaceticus strain may improve crop growth and yield on the basis of its biocontrol activity, siderophore production, and nitrogen fixation.7676 Sarode PD, Rane MR, Chaudhari BL, Chincholkar SB. Siderophore genic Acinetobacter calcoaceticus isolated from wheat rhizosphere with strong PGPR activity. Malays J Microbiol. 2009;5:6-12.

Isolate DD201 was identified as Pseudomonas sp., which is an opportunistic bacterium found in terrestrial and aquatic environments, and indicated biotechnological behaviors.7777 Nelson KE, Weinel C, Paulsen IT, et al. Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol. 2002;4:799-808; In this context, P. sp. DD201 showed the highest IAA production (16.5 µmol mL-1) but did not significantly improve the growth of soybean seedlings in inoculation tests (Fig. 3). Previous studies confirmed that IAA could promote plant grow that low concentration and inhibit root growth at high concentration.7878 López-Bucio J, Campos-Cuevas JC, Hernández-Calderón E, et al. Bacillus megaterium rhizobacteria promote growth and alter root-system architecture through an auxin and ethylene-independent signaling mechanism in Arabidopsis thaliana. Mol Plant Microbe Interact. 2007;20:207-217.

79 Persello-Cartieaux F, David P, Sarrobert C, et al. Utilization of mutants to analyze the interaction between Arabidopsis thaliana and its naturally root-associated Pseudomonas. Planta. 2001;212:190-198.
-8080 Keyeo F, Noor O, Shah A, Amir HG. The effects of nitrogen fixation activity and phytohormone production of diazotroph in promoting growth of rice seedlings. Biotechnology. 2011;10:267-273. Presumably, PGP bacteria use IAA as a part of their colonization strategy and as a signal molecule in bacteria-host communication.6666 Li L, Sinkko H, Montonen L, Wei GH, Lindström K, Räsänen LA. Biogeography of symbiotic and other endophytic bacteria isolated from medicinal Glycyrrhiza species in China. FEMS Microbiol Ecol. 2012;79:46-68. These functions might explain the reason for the production of IAA that was common among our six endophytic strains (Table 1).

This study proved that fungal antagonistic strain DD234 was O. haematophilum (Table 1). Ochrobactrum isolates could assist plant nutrient uptake from the soil and prevent plant diseases.8181 Kundu MC, Mandal B. Nitrate enrichment in groundwater from long-term intensive agriculture: its mechanistic pathways and prediction through modeling. Environ Sci Technol. 2009;43:5837-5843. The plant growth-promoting characteristics of siderophore production, IAA production, and phosphate solubilization were found in some strains of this genus8282 Príncipe A, Alvarez F, Castro MG, et al. Biocontrol and PGPR features in native strains isolated from saline soils of Argentina. Curr Microbiol. 2007;5:314-322. and might be the mechanism for increasing host plant growth.8383 Zhao L, Teng SS, Liu YP. Characterization of a versatile rhizospheric organism from cucumber identified as Ochrobactrum haematophilum. J Basic Microb. 2012;52:232-244.

Acknowledgments

This work was supported by projects from the National Science Foundation of China (U1204301), the Foundation for University Key Teacher by the Ministry of Education of Henan Province (2012GGJS166) and the University Key Scientific Research Project of Henan Province (17A180011).

Appendix A Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjm.2017.06.007.

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Publication Dates

  • Publication in this collection
    Apr-Jun 2018

History

  • Received
    5 Jan 2017
  • Accepted
    19 June 2017
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
E-mail: bjm@sbmicrobiologia.org.br