Evaluation of diazotrophic bacteria from rice varieties ( Oryza sativa L . ) grown in Rio de Janeiro State , Brazil

The indiscriminate application of nitrogen fertilizer in order to increase rice crop productivity contributes to environmental degradation. One of the possible alternatives to ease up this problem is the use of sustainable and cost-effective technologies such as biofertilizers to promote plant growth. This work aimed to isolate, characterize and select diazotrophic bacteria associated with rice cultivars EPAGRI-109 and BRS TROPICAL. About 39 bacteria belonging to the genera Azospirillum, Herbaspirillum, Paenibacillus, Ideonella and Pseudomonas were isolated. Based on the best results of acetylene reduction assay (ARA), indolic compounds production and phosphate solubilization, the isolates were selected as potential plant growth promoters which were tested on rice cultivars IR 42, EPAGRI 109 and BRS TROPICAL under axenic condition and the most efficient isolates promoted up to 31% of dry weight of the evaluated rice cultivars. Two promising isolates were selected in this work (Paenibacillus species strain EP3-16N and Nitrospirillum species strain EP4-1L), however, more studies are necessary to confirm this capacity in field conditions.


INTRODUCTION
Rice (Oryza sativa L.) has been playing an important nutritional role for more than half of the world population, and about 486.67 million ton of rice was consumed worldwide in 2016/2017 (United States Department of Agriculture [USDA], 2018).Brazil is the world's 10 th largest rice producer and it is produced mostly in the wetlands ecosystem of the Southern region under flooded condition (USDA, 2018).
Rice cropping requires considerable amounts of nutrients for development and growth and, nitrogen is particularly important for rice cultivation.Association of this crop with several nitrogen-fixing bacteria might contribute a part of nitrogen required by the plant (Choudhury and Kennedy, 2005).
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License losses associated to rice cropping.The BNF occurs in some prokaryotic organisms, such as, bacteria and cyanobacteria which are capable of converting N 2 into NH 3 (Baca et al., 2000;Baldani and Baldani, 2005;Kraiser et al., 2011).
The enzyme nitrogenase which is present in free-living or symbiotic diazotrophic bacteria is extremely versatile as it transforms N 2 into ammonia and catalyzes the reduction of several other substrates such as acetylene.As a consequence, the reduction of acetylene (C 2 H 2 ) to ethylene (C 2 H 4 ) became the basis of indirect estimation of nitrogenase activity (Boddey et al., 1990;Stoltzfus et al., 1997;Moreira and Siqueira, 2006;Mus et al., 2016).
These microorganisms colonize the interior and exterior of plant organs exerting beneficial functions (in addition to BNF) to diseases either directly or indirectly by antagonizing the pathogenic fungi through siderophores, chitinase and antibiotics production as well as phosphate solubilization, phytohormones production, etc (Downing et al., 2000;Baldani et al., 2002;Lee et al., 2004;Videira et al., 2009;Figueredo et al., 2010).
Indole acetic acid (IAA) is the most abundant phytohormone that regulates various aspects of plant developmental functions (Teale et al., 2006) reflected by the complexity of their biosynthesis, transport and signaling pathways (Santner et al., 2009).Studies have revealed great variability of auxin production among these strains; also, their inoculation promotes plant growth (Asghar et al., 2002;Khalid et al., 2004;Kuss et al., 2007;Richardson et al., 2009).
Solubilization of phosphates is presented by many bacteria and fungi in soil and in association with different plant species altogether.The metabolic processes involved in P solubilization and mineralization include excretion of hydrogen ions, release of organic anions, siderophores and phosphatases (Richardson, 2001).Solubilization of phosphate compounds is very common in vitro, but it has not been reported frequently because of technical difficulties in uncontrolled environment (Khan et al., 2007;Estrada et al., 2013).
Rice cultivars EPAGRI-109 and BRS TROPICAL are recommended for planting in Rio de Janeiro State and their associations with plant growth promoting bacteria have been previously reported (Kuss et al., 2007;Viana et al., 2015); however, no data is available to diazotrophic bacteria associated with these cultivars in Rio de Janeiro State.
According to some authors, native isolates of diazotrophic bacteria may contribute to greater nitrogen gains in Poaceae since it is locally adapted to environmental conditions (Santos et al., 2015;Viana et al., 2015).
The work aimed to isolate, characterize and select diazotrophic bacteria associated with rice cultivars IR-42, EPAGRI-109 and BRS TROPICAL grown in Rio de Janeiro State.

Sampling
Two rice cultivars (O.sativa L.), EPAGRI-109 and BRS TROPICAL, and their rhizospheric soils were used for enumeration and isolation of diazotrophic bacteria.Samples from cultivar EPAGRI-109 were obtained at field condition while those of cultivar BRS TROPICAL were derived from seeds cultivated at greenhouse condition because during the isolation none was planted in the field.
EPAGRI-109 plants were collected during January 2013 100 days of growth (DAG) and 120 DAG during the vegetative growth stage in Campo dos Goytacazes, RJ, Brazil field.
Seeds of BRS TROPICAL were inoculated with Sp 245 to evaluate if the inoculation would alter bacteria groups associated with rice plants.The seeds of BRS TROPICAL were sown in a randomized experimental design in the greenhouse using 12 pots each containing 16 seeds and 20 kg un-sterile soil of an Ultisol soil collected from Embrapa Agrobiologia experimental field at Seropédica, RJ.The treatments were inoculated and un-inoculated with Azospirillum brasilense strain Sp 245 with 4 replicates.

Isolation of diazotrophic bacteria
EPAGRI-109 and BRS TROPICAL plants were individually wrapped in plastic bags and brought to the Laboratory of Grasses at Embrapa Agrobiologia, RJ for the extraction of soil adhered to roots (rhizospheric soil), shoots and roots.Diazotrophic bacteria were isolated using the serial dilution technique.10 g rhizospheric soil was transferred to flasks containing 90 ml saline solution of K2HPO4 (100 mg L −1 ), MgSO4 (50 mg L −1 ), NaCl, (20 mg L −1 ), CaCl2.2H2O(50 mg L −1 ), and FeEDTA (16.4 mg L −1 ).The roots and shoots were washed in tap water and then surface disinfested in chloramine T (1%), chopped into 10-cm pieces and 10 g were transferred to flasks containing 90 ml saline solution.After homogeneization by blending, each sample was diluted up to 10 -6 level with saline solution.Thereafter, 0.1 ml of each dilution extract was used to inoculate vials containing 5 ml JNFb (Döbereiner et al., 1995), Nfb (Döbereiner et al., 1995) and LGI (Magalhães et al., 1983) semisolid media.The diazotrophs population per gram of fresh tissue was enumerated by the Most Probable Number (MPN) technique according to the McCrady's table (Döbereiner et al., 1995).

Acetylene reduction activity (ARA)
Isolates from EPAGRI-109 were obtained using JNFb, NFb and LGI nitrogen free semi-solid media and those from BRS TROPICAL were obtained using JNFb and NFb semi-solid media.The positive growth was evaluated after the appearance of a typical diazotrophic bacterial pellicle in the subsurface of the medium after incubation for 7 to 10 days at 30°C.The bacteria were purified on potato agar medium (Döbereiner et al., 1995).
ARA was performed to estimate nitrogenase activity following Boddey et al. (1990).Isolates were cultured in NFb, JNFB or LGI semi-solid (without bromothymol blue) and incubated at 30°C, then evaluated after 24, 48, 72 and 96 h in different flasks.Flasks were sealed with rubber caps, injected with 10% acetylene (v/v of gas phase) and incubated for 1 h at 30°C.Aliquots of 0.5 ml of gas samples were analysed through a Perkin Elmer F11 model gas chromatograph with Porapak N column (50 cm, 40°C).Diazotrophic bacteria A. brasilense Sp245, Nitrospirillum amazonense CBAmC and Herbaspirillum seropedicae ZAE94 were used as positive control.Total proteins were determined according to Bradford method (Bradford et al., 1976).Absorbance was recorded through a Labsystems iEMS Microplate Reader MF at 595 nm and protein contents were estimated using bovine serum albumin (BSA) calibration curve (7.5 to 150.0 μg ml -1 ) as external standard.

Indolic compounds production
Total indolic compounds produced by isolates were quantified in microplates as described by Sarwar and Kremer (1995).For this test, 1 ml of bacterial culture previously cultured for 24 h in DYGS medium (Rodrigues Neto et al., 1986) was inoculated into 5 ml of DYGS medium supplemented with L-tryptophan at the final concentration of 100 μg ml -1 .The tubes remained in the dark under agitation of 150 rpm at a temperature of 30°C for 48 h. 1 ml aliquots were removed and centrifuged at 5000 ° g for 15 min.
In microplate 96-well, 150 μl aliquot of the supernatant was mixed with 100 μl of the Salkowski reagent (1 ml of 0.5 M FeCl3 in 49 ml of 35% perchloric acid) previously prepared.The samples remained in the dark for 30 min at room temperature and the absorbance reading was done on a microplate reader (Labsystem IEMS Reader MF, Labsystem) to a wavelength of 540 nm.The quantification of indole compounds was evaluated using a calibration curve prepared with serial dilutions of synthetic IAA standards (5 to 100 μg tryptophan ml -1 ).Cultures of Gluconacetobacter diazotroficus (PAL5), A. brasilense (Sp245), H. seropedicae (ZAE94) and N. amazonense (CBAmc) were used as positive controls.Concentration was estimated with a standard calibration curve of indole acetic acid (10 to 80 mg ml -1 ).Values are presented as mg of indolic compounds per mg of proteins, determined by the Bradford method (Bradford et al., 1976).After the determination of ARA, 20 μl of pellicle and culture medium mix was added into 20 μl 1 M NaOH and 60 μl of sterile distilled water and placed in a water bath at 90°C for 5 min to promote cell disruption.Subsequently, 900 μl of the Bradford reagent was added, the tubes were mixed by vortex and incubated for 3 min at room temperature.

Molecular characterization
Total DNA extraction was performed using cetyl trimethylammonium bromide (CTAB) protocol as described in Sambrook et al. (1989).The isolates were inoculated in liquid DYGS medium at 30°C for 24 h under constant agitation and the bacterial culture was transfered to 2 ml microtube centrifuged at 4,000 x g for 8 min.The pellet was suspended in 567 μl T₁₀E₁ (10 mM Tris pH 8.0 and 1 mM NA₂EDTA) and homogenized vigorously; further, 30 μl of 10% SDS was added and homogenized by inversion with 3 μl of Proteinase K (20 mg ml -1 ) and then incubated at 65°C for 20 min.
da Silva et al. 2353 After 100 μl of 5 M sodium chloride was added and homogenized by inversion, an additional 80 μl of CTAB / NaCl (10% CTAB in 0.7 M NaCl) was added and the incubation was carried out at 65°C for 20 min.700 μl phenol-chloroform-alcohol isoamyl alcohol (25:24:1) was added and left overnight.The material was centrifuged for 10 min at 4°C at 13,000 × g.The supernatant was transferred to a new microtube and 700 μl chloroform-isoamyl alcohol (24:1) was added and centrifuged for 10 min at 4°C at 13,000 × g.The supernatant was thereafter recovered and transferred to a new 1.5 ml microtube to which 0.6 volume of ice-cold isopropanol was added.The material was incubated at 20°C for 30 min.Afterwards, the material was again centrifuged for 10 min at 4°C at 13,000 × g and the supernatant was discarded.The material was further subjected to centrifugation for 10 min at 4°C to 13,000 x g, with 70% ice-cold ethanol.The material was dried at room temperature and then suspended in 100 μl T₁₀E₁ (10 mM Tris pH 8.0 and 1 mM NA₂EDTA).
The DNA quantification and qualification was assessed by electrophoresis on 0.7% agarose gel.5 μl of sample was used together with 2 μl of sample buffer (0.25% bromophenol blue and 40% sucrose), along with 3 μl of 1 kb Plus DNA ladder standard (invitrogen® Cat.No. 10787-018).The samples were migrated in 80 v for 90 min in 1X TAE buffer (0.04 M Tris acetate and 1 mM EDTA).The gel was stained with an ethidium bromide solution (0.5 μg ml -1 ) and visualized with ultraviolet light on KODAK® Gel Logic 100 Photodocumentator (KODAK Scientific Imaging Systems, Cat.No. 172.8468) and one computer.Gels were analyzed using the KODAK® 1D Image Analysis (KODAK Molecular Imaging Systems, Cat.Nr. 811.2344).
For identification and phylogenetic analysis, PCR products of each isolate were purified using Wizard® genomic DNA purification kit (cat.A1120, PROMEGA Corporation, USA) and then sequenced at the Genomic Laboratory of Embrapa Agrobiologia.Sequences were deposited in the GenBank at accession numbers KT619165 -KT619177.

Statistical analysis
The normality (Lilliefors test) and homogeneity of variance (Bartlet's test) of the data were processed with SAEG 8.0 program (Euclydes, 1983).Analysis of variance was performed using the SISVAR 5.0 program (Ferreira, 2003) and a comparison of means by the Scott-Knott test at a 5% probability (Scott and Knott, 1974).

Isolation of diazotrophic bacteria
The MPN counts of cultivar EPAGRI-109 varied from 10 4 cells g root frw -1 and 10 6 cells g frw -1 in rhizospheric soil while the shoot values were 10 3 to 10 4 cells g root frw -1 .Interestingly, root associated bacteria of un-inoculated plants of BRS TROPICAL of the greenhouse were similar to EPAGRI-109 grown under field condition.In addition, number of bacteria in shoots and rhizospheric soil were higher in plants inoculated with A. brasilense Sp245 (Table 1).
The microbial population intimately associated with the rice plants was of 10 6 cells g frw -1 in cultivar EPAGRI 109 and in un-inoculated BRS TROPICAL plants.When BRS TROPICAL plants were inoculated with A. brasilense Sp245 the MPN values for shoots grossly doubled over in un-inoculated plants.Pedraza et al. (2009) also observed bacterial MPN increased on phyllosphere of rice cultivars in response to inoculation of Azospirillum strains.The inoculation treatment affected the bacterial counts in shoots and rhizosphere because A. brasilense Sp245 effectively colonized and established at these sites and altered the population of diazotrophs.
We obtained isolates in shoots (10), roots (8) and rhizospheric soil (9) from BRS TROPICAL and only isolates in shoots (9) and roots (3) from cultivar EPAGRI-109 (Table 2).Hardoim et al. (2011) observed that some rice cultivars have a strong influence on the population composition of bacterial communities in their rhizosphere, selecting similar bacterial communities while other genotypes select divergent bacterial communities.Both bacterial adaptation and plant genotype contribute to the formation of bacterial communities associated with rice roots.This behavior could also be explained by the development stage which influences different exsudates production in roots (Chaparro et al., 2014).
Although, N-free semi solid medium was used for diazotyroph isolation, all isolates from EPAGRI-109 were not able to grow and/or reduce acetylene to ethylene under the experimental conditions.Some isolates from EPAGRI-109 showed higher ARA than A. brasilense Sp245 (Figure 1A).In contrast, isolates from BRS TROPICAL showed lower ARA than A. brasilense Sp245, although 16S rDNA homology showed that TR-I1 is closely related to this species (Figure 1A, Table 4).Variations of ARA values among the isolates of the same species have been reported previously by several authors (Staal et al., 2001;Videira et al., 2012;Estrada et al., 2013).Although an indirect technique, ARA was used for indirect nitrogen fixation ability measurement because it is inexpensive and practical but since several factors (such as pH, carborn source, temperature and dissolved oxygen) can interfere with the performance of the isolates it have been used mostly to qualify than quantify their BNF potential.

Phosphate solubilization
41% of isolates was able to solubilize inorganic phosphates in vitro; the solubilization halo of isolates  varied from 0.33 to 2.45 mm during the four evaluations (Figure 3).These values are in the same range of those observed for A. brasilense Sp245 and N. amazonense CBAmC used as positive control.Phosphates solubilization was higher for G. diazotrophicus PAL5, Burkhoderia tropica Ppe8 and H. seropedicae ZAE94.However, no trend of P solubilization by the isolates could be established until 16 th day of evaluation.Some isolates produced greater solubilization halo on first day and others produced bigger halos of solubilization on the last day of analysis.
Phosphorus (P) is important in several physiological processes of plants, especially in photosynthesis, carbon metabolism and membrane formation, being a structural component of many coenzymes, phospho-proteins, phospholipids, DNA of all living organisms and responsible for the transfer and storage of energy which is used for growth and reproduction (Anand et al., 2016).
Despite this, about 95 to 99% of the phosphorus present in the soils is unavailable to the plants due to the fixation of P that is adsorbed on the mineral particles of the soil or precipitated by the ions Al 3+ and Fe 3+ in solution (Wu, 2005;Sharma et al., 2013;Anand et al., 2016).
Soil microorganisms play a key role in the soil P dynamics and subsequent soil P availability.Phosphates solubilizers, especially bacteria, increase the solubilization of insoluble phosphorus compounds through the release of phosphatic and phytase enzymes and enzymes that are present in various soil micro-organisms (Vassileva et al., 2000;Anand et al., 2016).

Molecular characterization
Amplification of 16S rDNA from all isolates generated fragments in the range of 1500 base pairs.The sequences varied in the range of 1208 to 1479 bp and Blast analysis revealed their phylogenetic relationship, based on similarity to sequences deposited at GenBank (GenBank accession numbers KT619165 -KT619177).The diazotrophic isolates belong to Alfaproteobacteria (Azospirillum and Nitrospirillum species), Betaproteobacteria (Herbaspirillum and Ideonella species) and Gammaproteobacteria (Pseudomonas species) within Proteobacteria.In addition, Firmicutes (Paenibacillus species) within Bacilli was also identified (Table 3).Some authors found these same genera associated with rice plants and closely related to diazothrofic bacteria (Baldani and Baldani, 2005;Breidenbach et al., 2016).

Inoculation experiment
The results of diazotrophic bacteria inoculation under axenic conditions showed significant improvement in dry weight of the inoculated rice cultivars.The shoots of EPAGRI-109 were more responsive to inoculation than BRS TROPICAL and IR42 which did not significantly respond to any treatment (Table 4).The EP3-16N significantly improved shoot wt., that is, about 30% higher than the uninoculated plants and was similar to that of da Silva et al. 2359 nitrogen supplemented cultivar EPAGRI-109.Root dry weight of the cultivar IR42 increased more with inoculation of the isolate EP4-1L and commercial inoculant AZOTOTAL recording 27 and 31% improvement, respectively.However, growth of bacterized BRS TROPICAL and EPAGRI-109 were not statistically different from the uninoculated plants, rather cultivars were negatively affected for inoculation of some bacteria (Table 4).The results support that in addition to the intrinsic characteristics of the bacteria, interactions between genotype and genotype-environment would directly interfere in the growth promotion efficiency of host plants also (Oliveira, 1994;Reis et al., 2000;Kennedy, 2004;Hungria et al., 2016).
However, the results conform to several studies which have shown that in axenic conditions diazotrophic bacteria was promising for field conditions.The in vitro evaluation allows selection of a large number of strains with potential plant growth promoting functions (Baldani et al., 2000;Araújo et al., 2013).
Evaluation under axenic conditions enabled Araújo et al. (2013) to select plant growth promoting isolates and host cultivars for testing in greenhouse, and found dstinct increases in biomass, that is, by 48 and 21% of Cana Forte and Cana Roxa varieties due to inoculation of N. amazonense strains AR3122, respectively.In addition, some varieties respond more to inoculation than others.The bacteria that promote yield with lower nitrogen fertilizer are greatly relevant for sustainable agriculture.
Application of inoculant Azototal containing A. brasilense strains Abv5 and Abv6 has been approved by the Ministry of Agriculture Livestock and Food Supply (MAPA) in Brazil for maize, wheat and rice to reduce nitrogen fertilizers by up to 50% which implies in savings of R$ 1.5 billion per year to farmers.

Conclusion
Thirty-nine isolates of diazotrophic bacteria were isolated from rice cultivars EPAGRI-109 and BRS TROPICAL, about 49% of which occurred from shoots, 28% from roots and 23% in rhizospheric soil.Among them, eight isolates belonged to the genus Azospirillum spp.and two isolates were identified as Herbaspirillum spp.
Paenibacillus spp.strain EP3-16N increased in biomass of plants mainly the cultivar EPAGRI-109 and Nitrospirillum spp.strain EP4-1L in roots of cultivar IR-42.However, more studies are necessary to confirm this capacity in field conditions.
The use of molecular tools must be considered in future studies to elucidate the microorganism diversity and interaction in rice plants.

Figure 2 .
Figure 2. Indolic compounds production of isolates from irrigated rice cultivars at 24, 48 and 72 h of growth in Dygs medium suplemented with L-tryptofhan.

Figure 3 .
Figure 3. Solubilization activity of isolates of irrigated Rice cultivars at 4, 8, 12 and 16 days of growth in NBRIP agar medium.

Table 1 .
MPN of bacteria from shoots, roots and rhizospheric soil of rice cultivar BRS TROPICAL grown at greenhouse condition and MPN of bacteria from shoots, roots and rhizospheric soil of rice cultivar EPAGRI-109 from production area at Campo dos Goytacazes, RJ.Mean values of 3 replicates.

Table 2 .
Isolates from rice cultivars EPAGRI-109 and BRS TROPICAL according to isolation source and semisolid medium.

Table 3 .
Selected isolates and their main characteristics for potential use as plant growth promoter.

Table 4 .
Dry weight of shoots and roots (g) of irrigated Rice cultivars at 30 days after inoculation with diazotrophic bacteria in Hoagland's solution.