Polyphasic characterization of bacteria obtained from upland rice cultivated in Cerrado soil Part of the Master's Dissertation of the first author approved by the Molecular Science Post-Graduation Program of the Goiás State University.

Lívia Fabiana Braga Fênix Araújo de Oliveira Eva Aparecida Prado do Couto Karina Freire d'Eça Nogueira Santos Enderson Petrônio de Brito Ferreira Claudia Cristina Garcia Martin-Didonet About the authors

ABSTRACT

This work aimed to characterize 20 isolates obtained from upland rice plants, based on phenotypic (morphology, enzymatic activity, inorganic phosphate solubilization, carbon source use, antagonism), genotypic assays (16S rRNA sequencing) and plant growth promotion. Results showed a great morphological, metabolic and genetic variability among bacterial isolates. All isolates showed positive activity for catalase and protease enzymes and, 90% of the isolates showed positive activity for amylase, catalase and, nitrogenase. All isolates were able to metabolize sucrose and malic acid in contrast with mannitol, which was metabolized only by one isolate. For the other carbon sources, we observed a great variability in its use by the isolates. Most isolates showed antibiosis against Rhizoctonia solani (75%) and Sclerotinia sclerotiorum (55%) and, 50% of them showed antibiosis against both pathogens. Six isolates showed simultaneous ability of antibiosis, inorganic phosphate solubilization and protease activity. Based on phylogenetic analysis of the 16S rRNA gene all the isolates belong to Bacillus genus. Under greenhouse conditions, two isolates (S4 and S22) improved to about 24%, 25%, 30% and 31% the Total N, leaf area, shoot dry weight and root dry weight, respectively, of rice plants, indicating that they should be tested for this ability under field conditions.

Keywords:
Oryza sativa; PGPR; Associative bacteria; Bacterial diversity

Introduction

Beneficial endophytic microorganisms can live inside plants without causing damage to the host. Among them, many bacteria colonize the intercellular spaces throughout the whole plant, including seeds.11 Liu Y, Zuo S, Xu L, Zou Y, Song W. Study on diversity of endophytic bacterial communities in seeds of hybrid maize and their parental lines. Arch Microbiol. 2012;194:1001-1012. Intercellular spaces contain sources of carbohydrates and minerals, such as: nitrogen, phosphorus, calcium, potassium, and chlorine, as well as other metabolites as organic acids, facilitating bacterial development and colonizing.22 Gamalero E, Glick BR. Mechanisms used by plant growth-promoting. In: Maheshwari DK, ed. Bacteria in Agrobiology: Plant Nutrient Management. Berlin/Heidelberg: Springer-Verlag; 2011:17-46.

Among several microorganisms colonizing plant tissues, plant growth-promoting rhizobacteria (PGPR) were also found.33 Kiersztyn B, Siuda W, Chróst RJ. Persistence of bacterial proteolytic enzymes in lake ecosystems. FEMS Microbiol Ecol. 2012;80:124-134. These PGPRs provide beneficial effects, such as: mineral nutrition improvement, increase on the tolerance of biotic and abiotic stresses, root development promotion, and suppression of soil borne diseases.22 Gamalero E, Glick BR. Mechanisms used by plant growth-promoting. In: Maheshwari DK, ed. Bacteria in Agrobiology: Plant Nutrient Management. Berlin/Heidelberg: Springer-Verlag; 2011:17-46.

Also, these bacteria can be involved on nitrogen fixation, inorganic phosphate solubilization, iron complexation, phytohormones synthesis and plant pathogen control, involving many enzymes on these processes.22 Gamalero E, Glick BR. Mechanisms used by plant growth-promoting. In: Maheshwari DK, ed. Bacteria in Agrobiology: Plant Nutrient Management. Berlin/Heidelberg: Springer-Verlag; 2011:17-46.,44 Fernandes Junior PI, Lima AA, Passos SR, et al. Phenotypic diversity and amylolytic activity of fast growing rhizobia from pigeonpea [Cajanus cajan (L.) MILLSP.]. Braz J Microbiol. 2012;43:1604-1612. Thus, plants under influence of PGPR can be greater, stronger, more productive and healthier.33 Kiersztyn B, Siuda W, Chróst RJ. Persistence of bacterial proteolytic enzymes in lake ecosystems. FEMS Microbiol Ecol. 2012;80:124-134.,55 Isawa T, Yasuda M, Awasaki H, Minamisawa K, Shinozaki S, Nakashita H. Azospirillum sp. strain B510 enhances rice growth and yield. Microbes Environ. 2010;25:58-61.,66 Ji SH, Gururani MA, Chun S-C. Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars. Microbiol Res. 2014;169:83-98. Besides, the assessment of biochemical characteristics in bacteria allows inferences about their adaptive capacity to the environment and about the plant-bacteria interaction, since it involves several genes and mechanisms, such as chemoattraction and biofilm formation, among other activities.33 Kiersztyn B, Siuda W, Chróst RJ. Persistence of bacterial proteolytic enzymes in lake ecosystems. FEMS Microbiol Ecol. 2012;80:124-134.

Association between PGPRs and root of rice plants has been studied and many isolates showing effects on rice growth were found.55 Isawa T, Yasuda M, Awasaki H, Minamisawa K, Shinozaki S, Nakashita H. Azospirillum sp. strain B510 enhances rice growth and yield. Microbes Environ. 2010;25:58-61. The application of this technology on rice chain production can provide benefits for crop growth and reduce production costs, as well as helping on the reduction of environmental risks.55 Isawa T, Yasuda M, Awasaki H, Minamisawa K, Shinozaki S, Nakashita H. Azospirillum sp. strain B510 enhances rice growth and yield. Microbes Environ. 2010;25:58-61.,77 Ikeda AC, Bassani LL, Adamoski D, et al. Morphological and genetic characterization of endophytic bacteria isolated from roots of different maize genotypes. Microb Ecol. 2013;65:154-160.

Furthermore, the identification and characterization of bacteria as PGPR provide insights for understanding the composition of bacterial communities associated with rice plants grown under Cerrado conditions. However, studies with focus on the isolation and characterization of bacteria from Cerrado soils able to promote plant growth of upland rice are still scarce as well as, necessary for this crop. Thus, this study aimed to characterize bacterial isolates obtained from rice roots based on biochemical and genetic characteristics and, to determine their ability to promote plant growth aiming their use as inoculant for upland rice.

Material and methods

Bacterial isolates

The isolates evaluated in this study were obtained from rice roots by Rezende88 Rezende SS. Bactérias diazotróficas: isolamento, diversidade e caracterização em plantas do arroz no cerrado de Goiás. Trabalho de Conclusão de Curso (Graduação em Farmácia) - UEG-UnCET; 2007. and are available at the Collection of Microorganisms and Multifunctional Fungi of Embrapa Rice and Beans.

Morphological characterization and Gram coloring assays

Morphological characterization was performed according to Vermelho et al.99 Vermelho AB, Pereira AF, Coelho RRR, Souto-Padrón T. Práticas de Microbiologia. Rio de Janeiro: Guanabara Koogan; 2006. based on shape, border, surface, consistence and elevation of the colonies. Gram coloring was performed according to Louvet et al.1010 Louvet J-N, Attik G, Dumas D, Potier O, Pons M-N. Simultaneous Gram and viability staining on activated sludge exposed to erythromycin: 3D CLSM time-lapse imaging of bacterial disintegration. Int J Hyg Environ Health. 2011;214:470-477.

Biochemical assays

All biochemical trials were performed on a completely randomized design in triplicate under laboratory conditions. For the evaluation of carbon sources use (maleic acid, malic acid, nicotinic acid, D-arabinose, D-fructose, D-glucose, mannitol, mio-inositol, sucrose and sorbitol), each isolate was inoculated in Petri dishes containing solid King B medium added with each different carbon sources and incubated (28 °C; 2 d). After incubation, we checked out the growth of the isolates according to Hungria et al.1111 Hungria M, Vargas MAT, Suhet AR, Peres JRR. Fixação biológica do nitrogênio na soja. In: Araújo RS, Hungria M, eds. Microorganismos de importância agrícola. Brasília: Embrapa-SPI; 1994:9-89.

The activity of the enzymes citrate lyase and urease was measured through qualitative assays by inoculating the isolates on solid Simmons citrate and urea medium and observing the blue and red colors of the culture medium, respectively.99 Vermelho AB, Pereira AF, Coelho RRR, Souto-Padrón T. Práticas de Microbiologia. Rio de Janeiro: Guanabara Koogan; 2006. Nitrate reductase and nitrogenase enzymes were qualitatively assayed in semi-solid medium.1212 Döbereiner J, Baldani VLD, Baldani JI. Como isolar e identificar bacterias diazotroficas de plantas. Brasilia, DF: EMBRAPA-SPI; 1995:60 pp. Catalase activity was assayed according to Vermelho et al.99 Vermelho AB, Pereira AF, Coelho RRR, Souto-Padrón T. Práticas de Microbiologia. Rio de Janeiro: Guanabara Koogan; 2006. For amylase and protease activities, we observed the formation of a translucid halo around the colonies, formed by the isolates inoculated on M9 solid medium containing 1% of starch and 2% of milk protein, respectively.1313 Oliveira NA, Oliveira LA, Andrade JS, Chagas Júnior AF. Atividade enzimática de isolados de rizóbia nativos da amazônia central crescendo em diferentes níveis de acidez. Ciênc Tecnol Aliment. 2006;26:204-210. Cellulase activity was determined according to Cattelan et al.1414 Cattelan A, Hartel P, Furhmann F. Screening for plant growth promoting rhizobacteria to promote early soybean growth. Soil Sci Soc Am J. 1999;63:1670-1680.

Solubilization of inorganic phosphate assay was performed using three replicates. Solubilization ability was confirmed based on the observation of a translucid halo around the colonies formed by the isolates inoculated on NBRI-P1515 Nautiyal CS. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett. 1999;170:265-270. and Pikovskaya medium.1616 Pikovskaya RI. Mobilization of phosphorous in soil in connection with vital activity of some microbial species. Mikrobiologya. 1948;17:362-370.

Antagonistic test

The antagonistic ability of the isolates was performed using three replicates, under in vitro conditions against the fungi Sclerotinia sclerotiorum and Rhizoctonia solani on Petri dishes containing BDA medium according to Hammami et al.1717 Hammami I, Hsouna AB, Hamdi N, Gdoura R, Triki MA. Isolation and characterization of rhizosphere bacteria for the biocontrol of the damping-off disease of tomatões in Tunisia. C R Biol. 2013;336:557-564.

Clustering analysis of the isolates based on morpho-physiological data

Morphological, enzymatic activity, carbon source use and antibiosis data were transformed into a binary matrix and submitted to cluster analysis performed by the software NTSYSpc®2323 Rolf J. NTSYS-PC: Numerical Taxonomy and Multivariate Analysis System, Version 1.4. New York: Exeter; 1988. using UPGMA as grouping algorithm and Jaccard as similarity coefficient.

16S rRNA gene sequencing analysis

Based on the clustering analysis of the isolates based on morpho-physiological data 11 isolates (S2, S4, S6A, S17, S22, S26, S29, S37, S41, S63 and S105) were selected for 16S rRNA gene sequencing. The DNA of the isolates was obtained according to Laranjo et al.1818 Laranjo M, Machado J, Young JPW, Oliveira S. High diversity of chickpea Mesorhizobium species isolated in a Portuguese agricultural region. FEMS Microbiol Ecol. 2004;48:101-107. The 16S rRNA gene was amplified by PCR using the primers Y1 and Y3.1818 Laranjo M, Machado J, Young JPW, Oliveira S. High diversity of chickpea Mesorhizobium species isolated in a Portuguese agricultural region. FEMS Microbiol Ecol. 2004;48:101-107.,1919 Young JPW, Downer HL, Eardly BD. Phylogeny of the phototrophic Rhizobium strain BTAi1 by polymerase chain reaction-based sequencing of a 16S rRNA gene segment. J Bacteriol. 1991;173:2271-2277. The amplicons were purified and used on the sequencing reaction according to Laranjo et al.1818 Laranjo M, Machado J, Young JPW, Oliveira S. High diversity of chickpea Mesorhizobium species isolated in a Portuguese agricultural region. FEMS Microbiol Ecol. 2004;48:101-107. Sequences coding for the partial 16S rRNA gene of the isolates S2, S4, S6A, S17, S22, S26, S29, S37, S41, S63 and S105 was obtained and when submitted to the GenBank database (www.ncbi.nlm.nih.gov) received the accession numbers KX855959.1, KX855958.1, KX197211.1, KX197210.1, KX855960.1, KX197209.1, KX855957.1, KX197208.1, KX197214.1, KX197206.1 and KX197207.1, respectively.

Evaluation of plant growth promotion

Based on genotypic characterization, four isolates were selected to be evaluated under greenhouse conditions as compared to a no-inoculated treatment (NI). Seeds of rice cv. Aimoré were sown in 8 L pots filled with soil and arranged in a random block design with four replicates. At sowing time, seeds were inoculated with peat-based inoculant containing 1 × 108 cell g-1 of each isolate and reference strain. Rice plants were harvested at 40 days after emergence (DAE) to determine leaf area (LA), root volume (RV), root dry weight (RDW), shoot dry weight (SDW) according to Ferreira2020 Ferreira EPB, Barbosa LHA, Knupp AM, et al. Identification of high nodulation efficiency among wild genotypes of common beans. Annu Rep Bean Improv Coop. 2010;53:170-171.,2121 Ferreira EPB, Stone LF, Didonet AD. Green manure species and sowing time effects on the agronomic performance of common bean. Agron J. 2013;105:1721-1727. and, the total N (N-Total) using the Kjedahl method, as described by Silva and Queiroz.2222 Silva DJ, Queiroz AC. Análise de alimentos: métodos químicos e biológicos. 3rd ed. Viçosa, MG: UFV; 2006, 235 pp.

Data analysis

For the 16S rRNA-based phylogeny study, the sequences obtained were submitted to NCBI BLAST against a non-redundant nucleotide database for getting homologous sequences.2424 Altschul SF, Gish W, Miller M, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403-410. Sequences showing degree of similarity were aligned using the CLUSTAL W program.2525 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997;25:4876-4882. The evolutionary history was inferred by using the maximum likelihood method,2626 Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A. 2004;101:11030-11035. with tree consensus being inferred from 500 replicates using bootstrap.2727 Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985;39:783-791. All positions containing gaps and missing data were eliminated. There were a total of 1312 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.2828 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725-2729.

Data obtained from the greenhouse experiment were subjected to analysis of variance. When F was significant, the Scott-Knott test of means was applied with a 5% probability using SISVAR statistical software.

Results

Analysis based on the morphological characteristics of the isolates resulted in four different groups (A, B, C and D). The groups were composed by 5, 9, 2 and 4 isolates, respectively (Table 1). Isolates of groups A and B show the same morphological characteristics, except for Gram coloration, by which the isolates of group A are Gram-, while isolates of group B are Gram+. Similarly, isolates of the group C are Gram-, and isolates of group D are Gram+. Additionally, isolates of groups A and B show different morphological characteristics compared to isolates of groups C and D (Table 1).

Table 1
Morphological characteristics on King B medium of endophytic isolates obtained from rice plants.

The results of carbon source use and enzyme activity showed a great metabolic diversity among the isolates (Table 2).

Table 2
Metabolic characteristics of endophytic isolates obtained from rice plants.

All the isolates were able to use sucrose and malic acid as carbon source, while only the isolate S22 was able to use mannitol. Also, 45% and 35% of the isolates were able to use fructose and mio-inositol, respectively. Besides, the isolate S22 was able to use nine of the ten carbon sources evaluated, while the isolate S97 used just two of these sources (Table 2).

All the isolates showed catalase activity, while 90% of the isolates showed amylase, phosphatase and nitrogenase activity. The isolates S8A, S22, S35 and S105 showed activity of seven enzymes, and isolates S5 and S19 showed activity of four (Table 2).

For the phosphate solubilization assay, only the results obtained from the Pikovskaya medium are shown, since we did not observe growth of the isolates in the NBRI-P medium. About 75% of the isolates were able to solubilize inorganic phosphate (Table 3). These isolates showed a SI of about 111% in average, with the greatest values been observed for isolates S2 (133.86%) and S5 (133.6%). Isolates S8b and S63 did not present solubilization ability.

Table 3
Solubilization index (SI) for inorganic phosphate assayed in Pikovskaya medium, protease enzymatic index (PEI) and antibiosis assays against Sclerotinia sclerotiorum (Ss) and Rhizoctonia solani (Rs) of endophytic isolates obtained from rice plants. Mean values of three replicates.

The general mean observed for PEI was about 1.92. Six isolates (S6, S4, S29, S17, S37 and S2) showed PEI greater than 2, while the isolate S105 showed the smallest value for PEI (Table 3).

On the antibiosis assays against S. sclerotiorum, 11 isolates were able to inhibit the growth of the fungi, while against R. solani 14 isolates inhibited the fungi growth. Besides, the isolates S6A, S25, S26, S29, S32, S35, S37, S41, S97 and S105 showed antibiosis against both fungi (Table 3).

The similarity analysis based on morphological characteristics, enzymatic activity, carbon source use and antibiosis assay revealed six clusters formation at 60% of similarity, indicating great diversity among the 20 isolates regarding these traits (Fig. 1).

Fig. 1
Consensus dendrogram obtained by combining the morphological, enzymatic activity, carbon source use and antibiosis data among 20 endophytic isolates obtained from upland rice plants. Dendrogram was generated by the algorithm UPGMA and similarity matrix obtained from the use of Jaccard coefficient.

The isolate S63, on group G6, showed a 40% similarity to the other isolates, followed by the isolates of the group G1. The greatest similarity was about 90%, observed between the isolates S5 and S37. These isolates grouped with other eight isolates on group G2 (Fig. 1).

The 16S rRNA gene sequencing analysis revealed that all the evaluated isolates showed high similarity with the genus Bacillus as compared with other plant growth-promoting genera (Fig. 2).

Fig. 2
Maximum likelihood phylogeny of the 16S rRNA gene showing the relationships among endophytic isolates obtained from rice plants (in bold) with different genera of plant growth-promoting rhizobacteria. GenBank accession numbers are shown in parentheses. Bar, 20 nt substitutions per 1000 nt.

The phylogeny analysis considering only the genus Bacillus showed that the isolates S6A, S17, S26, S37 and S22 are closely related with the strains EGE-B-Li-6 and St05 of B. subtilis and B. methylotrophicus, respectively, while the isolates S2 and S41 are closely related with different strains of B. methylotrophicus and B. valezensis. On the other hand, the isolates S63 and S105 clustered with the strain FBZ42 of B. valezensis. Also, the isolate S29 showed high similarity with B. vallismortis and B. amyloliquefaciens. This analysis also revealed that the isolate S4 is an out group, showing low similarity with the different strains of the genus Bacillus. Even belonging to Bacillus genus (Fig. 2) and showing 98% of identity on the BLAST analysis with the strain SL 8 of B. amyloliquefaciens, the specie of this isolate is still uncertain needing further studies to its determination (Fig. 3).

Fig. 3
Maximum likelihood phylogeny of the 16S rRNA gene showing the relationships among endophytic isolates obtained from rice plants (in bold) with Bacillus sp. reference strains. GenBank accession numbers are shown in parentheses. Bar, 2 nt substitutions per 1000 nt.

Based on the results of the phylogenetic analysis, the isolates S4, S22, S29 and S105 were chosen to be evaluated as plant growth promoters under greenhouse conditions. Results showed a clear effect of the isolates on the root development of upland rice plants cv. Aimoré (Fig. 4).

Fig. 4
Root development of upland rice cv. Aimoré as affected by endophytic isolates obtained from rice plants.

The plant growth promotion ability of the isolates was also observed over root dry weight (RDW), leaf area (LA), shoot dry weight (SDW) and total N (N-Total) accumulated on the shoots of rice plants as observed in Table 4. The isolates S4 and S22 promoted higher values of RDW, LA, SDW and N-Total as compared to NI treatment (Table 4).

Table 4
Effect of different isolates on the leaf area (LA), root dry weight (RDW), shoot dry weight (SDW) and total N (N-Total) of upland rice cv. Aimoré as compared to a no-inoculated treatment (NI). Mean values of four replicates.

Discussion

The morphological analysis revealed four different morphological groups with the prevalence of isolates showing irregular shape, wrinkled border and Gram+ bacteria as well (Table 1). The genus Bacillus sp. comprises Gram+ bacteria and several species of this genus show colony morphology quite similar to those found in this study.2929 Realpe ME, Hernández CA, Agudelo CI. Especies del género Bacillus: morfología macroscópica y microscópica. Biomédica. 2002;22:106-109.

All isolates were able to use sucrose and malic acid as a carbon source (Table 2). The ability to metabolize sucrose indicates that these isolates have enzyme invertase, which catalyses the hydrolysis of sucrose, allowing those isolates to use this compound as an energy source.3030 Reid SJ, Abratt VR. Sucrose utilisation in bacteria: genetic organisation and regulation. Appl Microbiol Biotechnol. 2005;67:312-321. Malate, the ionized form of malic acid, is a second preferred carbon source for B. subtilis.3131 Kleijn RJ, Buescher JM, Chat L, Jules M, Aymerich S, Sauer U. Metabolic fluxes during strong carbon catabolite repression by malate in Bacillus subtilis. J Biol Chem. 2010;285:1587-1596.

We also found the enzyme amylase in most of the isolates (Table 2). Amylases are widely distributed in bacteria and fungi and are classified by the action of exoenzymes, endoenzymes and enzyme debranching.44 Fernandes Junior PI, Lima AA, Passos SR, et al. Phenotypic diversity and amylolytic activity of fast growing rhizobia from pigeonpea [Cajanus cajan (L.) MILLSP.]. Braz J Microbiol. 2012;43:1604-1612.

Bacteria utilize citrate through the enzyme citrate lyase. In addition, those bacteria can produce other organic acids derivatives, which can act chelating aluminum and phosphorus molecules.3232 Mailloux RJ, Lemire J, Kalyuzhnyi S, Appanna V. A novel metabolic network leads to enhanced citrate biogenesis in Pseudomonas fluorescens exposed to aluminum toxicity. Extremophiles. 2008;12:451-459. In our study, among the positive bacteria for citrate lyase only one did not show the capacity to solubilize inorganic phosphate, indicating that for the other bacteria a synergy between these activities may occur.

Among the isolates, ten of them showed activity for nitrate reductase and nitrogenase, and three for urease activity (Table 2). These enzymes are determining factors related to biological nitrogen fixation and nitrogen metabolism.22 Gamalero E, Glick BR. Mechanisms used by plant growth-promoting. In: Maheshwari DK, ed. Bacteria in Agrobiology: Plant Nutrient Management. Berlin/Heidelberg: Springer-Verlag; 2011:17-46. The presence of these enzymes indicates that these isolates show metabolic pathways involved in N assimilation and, thus they can contribute to plant growth.77 Ikeda AC, Bassani LL, Adamoski D, et al. Morphological and genetic characterization of endophytic bacteria isolated from roots of different maize genotypes. Microb Ecol. 2013;65:154-160.

On the other hand, only two isolates did not show inorganic phosphate solubilization capacity (Table 3). Besides, among the bacteria studied in this study, the highest inorganic phosphate solubilization index (SI) was about 134%. Several soil bacteria can solubilize inorganic phosphate and provide P for plant growth, increasing P utilization efficiency.3333 Bhattacharyya PN, Jha DK. Plant growth-promoting rhizobacteria (PGPR): emergence in agriculture. World J Microbiol Biotechnol. 2012;28:1327-1350. Bacteria like Pseudomonas and Acetobacter can show SI of 260% and 483%, respectively.3434 Joseph S, Jisha MS. Buffering reduces phosphate solubilizing ability of selected strains of bacteria. World J Agric Sci. 2009;5:135-137.

On studies with bacteria isolated from Brazilian soils, the average SI was 170.5% and the bacteria LMG1222, which belongs to the genus Burkholderia was the most efficient, with a SI of 251%.3535 Marra LM, Oliveira SM, Soares CRFS, Moreira FMS. Solubilization of inorganic phosphates by inoculant strains from tropical legumes. Sci Agric. 2011;68:603-609. The mean SI observed for the tested isolates in this study was 122.9%, with the highest values around 133% for isolates S2 and S5 (Table 3).

Another important tool for plant growth-promoting bacteria is their ability to produce proteases. The production of protease by bacteria is often studied and has great importance for industrial use and on the understanding of the activities occurring in the soil environment.33 Kiersztyn B, Siuda W, Chróst RJ. Persistence of bacterial proteolytic enzymes in lake ecosystems. FEMS Microbiol Ecol. 2012;80:124-134. According to Oliveira et al.,1313 Oliveira NA, Oliveira LA, Andrade JS, Chagas Júnior AF. Atividade enzimática de isolados de rizóbia nativos da amazônia central crescendo em diferentes níveis de acidez. Ciênc Tecnol Aliment. 2006;26:204-210. bacteria showing protease enzymatic index (PEI) ≥2 are considered good protease producers.

Our results revealed five good protease producers bacteria (Table 3). Isolate S6 showed PEI about 3, which is very close to the PEI values showed by Bacillus megaterium and Corynebacterium renale (3.1 and 4.3, respectively) isolated from Jacaranda decurrens plants, isolated from soils of the state of Goiás, Brazil.3636 Carrim AJI, Barbosa EC, Vieira JDG. Enzymatic activity of endophytic bacterial isolates of Jacaranda decurrens Cham. (Carobinha-do-campo). Braz Arch Biol Technol. 2006;49:353-359.

The enzymatic diversity displayed by endophytic bacteria isolated from upland rice grown in Cerrado indicates that it is possible to develop a strategy to benefit the selection of microorganisms as biofertilizers, improving their potential as plant growth promoters for rice crop. Thus, the greater the extent of enzymatic pathways presented by the microorganisms, the greater will be the generated benefit. Also, the success of the plant-bacterium interaction and competition with other microorganisms favors the establishment of those microorganisms in such environments.33 Kiersztyn B, Siuda W, Chróst RJ. Persistence of bacterial proteolytic enzymes in lake ecosystems. FEMS Microbiol Ecol. 2012;80:124-134.

The ability of endophytic bacteria strains to grow using carbon source present in soil and plants as substrate indicates their ability to survive and compete in the environment. Organic acids such as maleic and nicotinic acid can also be exuded by the roots of plants, playing an important role in the selection of the associated microbiota. Nicotic and maleic acids showed suppressive effect over most of the studied isolates (Table 2), which corroborates the findings of Fernandes Junior et al.44 Fernandes Junior PI, Lima AA, Passos SR, et al. Phenotypic diversity and amylolytic activity of fast growing rhizobia from pigeonpea [Cajanus cajan (L.) MILLSP.]. Braz J Microbiol. 2012;43:1604-1612.

Among the evaluated bacteria, 55% and 70% showed antibiosis against S. sclerotiorum and R. solani, respectively (Table 3). Antibiosis may be triggered by many processes, including competition and production of metabolites.3737 Cavaglieri L, Orlando J, Rodriguez MI. Biocontrol of Bacillus subtilis against Fusarium verticillioides in vitro and at the maize root level. Res Microbiol. 2005;156:748-754. Isolates S4, S6, S22, S25, S29 and S105 showed antibiosis, solubilization of inorganic phosphate and protease activities (Table 3). According to Park et al.,3838 Park K-H, Lee C-Y, Son H-J. Mechanism of insoluble phosphate solubilization by Pseudomonas fluorescens RAF15 isolated from ginseng rhizosphere and its plant growth-promoting activities. Lett Appl Microbiol. 2009;49:222-228. the solubilization of inorganic phosphate and protease activity may be involved in the antibiosis activity of bacteria against fungi. These bacteria showed a very close similarity based on their morphological and enzymatic activity, carbon source use and antibiosis data. Isolates S4, S6, S25 and S29 shared about 62% of similarity on group G2; isolates S22 and S105 about 65% of similarity on group G4. The similarity between groups G2 and G4 was about 52% (Fig. 1).

Most of the isolates evaluated in this study, especially S4 and S22, show different mechanisms involved in the plant growth promotion, besides the presence of enzymes activity related to adaptation to different environmental conditions, as those of Cerrado. In this way, the results of this study represent a great scientific contribution since many metabolic pathway and action mechanisms can be studied from a few number of isolates. Besides, the contribution from the agricultural point of view is highly promising for the Brazilian Cerrado because there are none biological products registered for the upland rice crop.

The phylogenetic analysis of the 16S rRNA gene sequencing, performed for 11 isolates revealed that all of them belong to the genus Bacillus (Fig. 2). The isolate S4 showed low similarity with the different Bacillus spp., remaining as an undefined specie (Fig. 3). The presence of the genus Bacillus associated to rice plants in Brazil was also reported,3939 Beneduzi A, Peres D, Vargas LK, Bodanese-Zanettini MH, Passaglia LMP. Evaluation of genetic diversity and plant growth promoting activities of nitrogen-fixing bacilli isolated from rice fields in South Brazil. Appl Soil Ecol. 2008;39:311-320. but never with the presence of the B. amyloliquefaciens species, as observed in this study. In a study that investigated the associated microbiota with tomato, the presence of B. amyloliquefaciens was related with antibiosis against pathogens.4040 Almoneafy AA, Xie GL, Tian WX, Xu LH, Zhang GQ, Ibrahim M. Characterization and evaluation of Bacillus isolates for their potential plant growth and biocontrol activities against tomato bacterial wilt. Afr J Biotechnol. 2012;11:7193-7201. However, many other factors, such as soil type and plant genotype may influence this plant-microorganism association.3939 Beneduzi A, Peres D, Vargas LK, Bodanese-Zanettini MH, Passaglia LMP. Evaluation of genetic diversity and plant growth promoting activities of nitrogen-fixing bacilli isolated from rice fields in South Brazil. Appl Soil Ecol. 2008;39:311-320.,4141 Ferreira EPB, Dusi AN, Xavier GR, Runjanek NG. Rhizosphere bacterial communities of potato cultivars evaluated through PCR-DGGE profiles. Pesq Agropec Bras. 2008;43:605-612.

In our study, the best results for plant growth promotion under greenhouse conditions were observed for the isolates S4 and S22 (Table 4). As reported before, these isolates show different traits related to plant growth promotion, such as antibiosis, solubilization of inorganic phosphate and protease activity. The isolate S22 increased root dry weight, leaf area, shoot dry weight and N-Total accumulated on rice shoots in about 23%, 21%, 25% and 24%, respectively, as compared to noninoculated treatment. Considering these same parameters, the isolate S4, an unknown specie, promoted an increase of about 40%, 30%, 35% and 24%, respectively (Table 4).

The Brazilian Cerrado is a very challenging environment for cultivated plants, like rice, due to its soil and climate characteristics. Cerrado soils are generally acid and poor in nutrients,4242 Amorim PH, Batalha M. Soil characteristics of a hyperseasonal cerrado compared to a seasonal cerrado and a floodplain grassland: implications for plant community structure. Braz J Biol. 2006;66:661-670. while the rainfall regime is well defined, with a rainy season from October to April and a dry season from May to September with an average annual rainfall of 1460 mm.4343 Silva SC, Heinemann AB, Paz RLF, Amorim AO. Meteorological Information for Research and Agricultural Planning Regarding the Year 2009 and the Municipality of Santo Antônio de Goiás, GO. Santo Antônio de Goiás, Brazil: Embrapa Rice and Beans; 2010. Then, the use of microorganisms as inoculant showing a great variability of traits related with the growth promotion of plants is a very important approach to improve the establishment of rice crops in the Brazilian Cerrado, since plants under influence of PGPR can be greater, stronger, more productive and healthier.33 Kiersztyn B, Siuda W, Chróst RJ. Persistence of bacterial proteolytic enzymes in lake ecosystems. FEMS Microbiol Ecol. 2012;80:124-134.,55 Isawa T, Yasuda M, Awasaki H, Minamisawa K, Shinozaki S, Nakashita H. Azospirillum sp. strain B510 enhances rice growth and yield. Microbes Environ. 2010;25:58-61.,66 Ji SH, Gururani MA, Chun S-C. Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars. Microbiol Res. 2014;169:83-98.

These results point out for further studies on the specie identification of the isolate S4. Also, the isolates S4 and S22 must be tested for their agronomic efficiency under field conditions, aiming to state their potential of use as inoculant for rice.

Conclusions

This study reveals great morphological, metabolic and genetic variability among bacterial isolates obtained from upland rice cultivated in the Cerrado region of the state of Goiás. Those bacteria show many enzymes activity related to adaptation to different environmental conditions, as those of Cerrado. All the isolates belong to the genus Bacillus and exhibit at least one mechanism associated to plant growth promotion. Under greenhouse conditions the isolates S4 and S22 showed high plant growth promotion, indicating that they should be tested for this ability under field conditions.

  • Part of the Master's Dissertation of the first author approved by the Molecular Science Post-Graduation Program of the Goiás State University.

Acknowledgments

The authors thank CAPES, Embrapa Rice and Beans and INCT on Biological Nitrogen Fixation for the financial support. The Department of Biochemistry and Molecular Biology at the Federal University of Paraná and Goiás State University for researcher exchange (BIP).

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Publication Dates

  • Publication in this collection
    Jan-Mar 2018

History

  • Received
    29 Nov 2016
  • Accepted
    27 Apr 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
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