Isolation and inoculation of diazotrophic bacteria in rice ( Oryza sativa L . ) grown in Vitoria da Conquista-BA

1 State University of Southwest Bahia UESB, Estrada do Bem Querer km 4 – P. O. Box 45, Postal Code 45031-900, Vitoria da Conquista (BA), Brazil. 2 Plant Production Graduate Program, North Fluminense State University (RJ), Brazil. 3 Plant Science, State University of Southwest Bahia – UESB, Brazil. 4 Forest Engineer, Casa Familiar Rural de Presidente Tancredo Neves, Km 315, Presidente Tancredo Neves – Ba – CEP: 45416-000, Brazil. 5 Plant Production Graduate Program, North Fluminense State University (RJ), Brazil. 6 Embrapa Agrobiology, Embrapa Agrobiologia, BR 465, Km 7, Seropédica-RJ – CEP:23891-000, Brazil. 7 Department of Plant and Animal Science (DFZ), State University of Southwest Bahia – UESB, Brazil.


INTRODUCTION
Oryza sativa L. rice is a plant that belongs to the family of Poaceae, being the third-largest cereal crop in the world, only exceeded by corn and wheat.Rice is a cereal of basic human diet, representing approximately 20% of the world energy intake and 15% of protein intake (Kennedy and Burlingame, 2003).Among the various ways of increasing plant production, the supply of fertilizers stands out, while nitrogen being the most important element for obtaining high productivity, because it acts as the synthesis of proteins and enzymes that guarantee the life of the plant.Nitrogen is an element that is easily lost through leaching, volatilization and denitrification in soilplant system (Fageria and Baligar, 2005), irrational use of nitrogenous fertilizer burden the costs of production and can cause negative effects to the environment, such as contamination of water courses and increasing the concentration of greenhouse gases.Alternative sources of nitrogen supply to plants, such as the biological nitrogen fixation (BNF), can supplement or even replace the use of nitrogenous fertilizers in agricultural processes.The BNF is one of the most important processes known in nature, being performed by prokaryotic organisms.Diazotrophic bacteria are able to reduce the N 2 atmosphere making it understandable to the plants (Reis et al., 2006).
The actuation of diazotrophic bacteria regarding the biological fixation of nitrogen was verified initially between the legume plants, a better result is achieved in the soybean crop, where the BNF can provide up to 94% of the N, required by most productive cultivars (Hungria et al., 2006).In non-leguminous plants, the process of BNF is not as efficient as in soybean crop, numerous experiments have demonstrated that plants of the family Poaceae present significant potential, responding with an increase in production when inoculated with diazotrophic bacteria (Baldani et al., 2002;Guimarães et al., 2003;Campos et al., 2003;Guimarães et al., 2010).Bacteria capable of fixing atmospheric nitrogen, such as the genera Herbaspirillum, Burkholderia and Azospirillum have been isolated from various grasses in several experiments (Perin et al., 2006;Rodrigues et al., 2006;Reis Junior et al., 2004).
The inoculation with N-fixing diazotrophic bacteria is a viable alternative for the producer, with significant increases in yield and reduction in the use of nitrogen fertilizers with lower costs.The isolation of diazotrophic bacteria native of Bahia and their inoculation in rice plants may have bigger effects on the BNF, than bacteria from other regions of Brazil, as these may have greater adaptability to the conditions of the state's productive regions.
This study aimed to isolate and inoculate diazotrophic bacteria efficient as the BNF in rice (Oryza sativa L.) cultivar BRS Tropical cultivated in Vitoria da Conquista, and with potential to be use in the Southwest region of Bahia.

Inoculation of diazotrophic bacteria experiment
The experiment was conducted in a greenhouse at the Campus of the State University of Southwest Bahia (UESB), in Vitoria da Conquista, located in the southwest region of the state, at 14°51' South latitude, 40°50' west longitude, at height of 928 m.
The first 20 cm from a Yellow Latosol (Oxisol), flat terrain, with medium texture, coming from the experimental field UESB were used, with the following chemical characteristics: pH 5.4, 22-3 g dm - 3 of organic matter, 2.0 mg dm -3 of phosphorus and 0.1 cmolc dm -3 of potassium.Pots were used with 9kg capacity.The correction of soil fertility was performed according to the recommendations of the use of lime and fertilizers in Minas Gerais (Ribeiro et al., 1999) for upland rice, higher than the recommendation of the correction of soil fertility according to the Manual of Fertilization and Liming for the state of Bahia.
The experimental design was entirely randomized and treatments arranged in a factorial design (4 x 2), where the first factor had doses applied as urea (0, 20, 60 and 100 kg of N ha -1 ) and the second factor had the presence or absence of inoculant.Eight treatments were performed with six replicates for Tropical BRS and BRS genotypes, with four plants per pot.
The inoculation was done on the day of sowing.The inoculum was from Embrapa Agrobiology, containing a strain of Herbaspirillum seropedicae ZAE 94 (deposited in a culture collection of diazotrophic bacteria Embrapa Agrobiology with the code BR 11417).The seeds were wrapped in peat and dried in the shade, until the time of planting, and 10 g of peat were used per kg of seed.Seedling emergence occurred 10 days after planting.
The nitrogen fertilization was divided three times for the doses of 60 and 100 kg ha -1 of N, half of the fertilizer was applied at planting, 1/4 10 days after emergence days seedlings and 1/4 20 days after second application.At dose 20 kg, ha -1 of N was divided only twice, because the quantity was small to divide.After the emergence of 60 days, an assessment was made to determine the plant height, number of tillers and the accumulation of fresh and dry weight of plants.
Data were analyzed with SAEG 8.0 (Euclydes, 1983), according to their normality (Lilliefors test) and homogeneity of variance test (Cockran & Bartlet).Given the presuppositions, data were analyzed by ANOVA.The separation of means was done using the statistical test LSD at 5% probability.Test typically used to mean separation, can be applied to the data, the experiment also had a few treatments.The same test is not very restrictive as Tukey.Regression analysis was used to describe the response of plants to different levels of fertilization.For testing, the statistical program Sisvar 5.0 was used (Ferreira, 2003).

Isolation of diazotrophic bacteria
Absolute control of each genotype of rice and brought to Embrapa Agrobiology was collected for the realization of the isolation of diazotrophic bacteria.The samples were separated in aerial part and roots, disinfested superficially in tap water and then distilled water.These were macerated in blender in saline solution, followed by serial dilutions and inserted into vials containing culture media semi-specific nitrogen free, NFb for Azospirillum spp.(Döbereiner et al., 1995), JNFb for Herbaspirillum spp.(Döbereiner et al., 1995), JMV for Burkholderia spp.(Baldani et al., 2000) and LGI for Azospirillum amazonense (Döbereiner et al., 1995).
The flasks were incubated at 30°C for 7 days, being considered positive for those counts that showed typical aerotáxica film, near the surface of the medium.The count of the population of diazotrophic bacteria was held by the most probable number (MPN) using the Mc Crady table for 3 repetitions per dilution (Döbereiner et al., 1995).Vials that showed film formation were used in the isolation and purification of bacteria according to Dobereiner et al. (1995) and Baldani et al. (2000).
Morphology of the colonies was observed through the growth of isolated solid media, semi-speciphic (NFB, JNFB and VMY) and rich media (potato dextrose agar), observed morphological characteristics of the colonies such as, borders, coloring and texture (Döbereiner et al., 1995).
The nitrogenase activity of isolated was assessed by acetylene reduction technique (ARA), described by Boddey et al. (1990).The bacteria were grown in bottles with a capacity of 15 ml, containing 5 ml of semi-solid medium semi-specific without pH indicator.These flasks were incubated at 30°C for 48 h.After the formation of the film, the bottles were sealed with rubber stoppers pierceable subseal type sterile, 1 ml syringe of air was taken from each bottle and 1 ml of acetylene was also injected.The flasks were incubated at 30°C for an hour and, later, 0.5 ml of the gas phase was introduced into the gas chromatograph with flame ionization, Perkin Elmer model F11 was use to determine the concentration of ethylene in the sample.After determining the ARA, the vials were homogenized in a shaker table, until the complete homogenisation of the film was gotten.
The production capacity of the hormone Auxin (IAA) was analyzed by the microplate method (Sarwar and Kremer, 1995). 1 µL of bacterial culture was used, cultivated previously for 24 h in DYGS medium, and inoculated into 20 ml of DYGS medium supplemented with L-tryptophan, the final concentration of 200 µg ml -1 by test tube was gotten.The tubes were kept in the dark, under 150 RPM agitation, with constant temperature of 33°C.Aliquots of 1 ml was withdrawn after 42 h of cultivation, and centrifuged at 10,000 rpm for 15 min.
In U-type plate 96 wells, an aliquot of 150 µl of the supernatant was mixed with 100 µl of 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 under environmental temperature, and read absorbance were made in a microplate reader (Labsystem iems reader MF, Labsystem) at a wavelength of 540 nm.Quantification of indole compounds was evaluated using the calibration curve prepared with serial dilutions of IAA standards (10-80 µg ml -1 ).
For standardization of the samples, the results were expressed in µg ml -1 of IAA per unit of protein.All samples were analyzed in triplicate IAA on boards in U of 96 wells, and the result was due to an average of 3 replicates.For the determination of total protein, the vials were thawed and homogenized again, Elisa plates in U was used at a rate of 20 μl for the sample, 30 μl of sterile distilled water and 50 μl of 1 m NaOH to lyse the cells, then heated for 5 min at 100°C.
To this solution, 900 µl of reagent of Bradford was added, the plates were hustling in vortex and incubated for 30 min at room temperature.After the incubation, the reading of the absorbance at 595 nm was carried out in a spectrophotometer (Bradford et al., 1976).The protein concentration was determined using the standard curve obtained by the absorbance values of known amounts of BSA (bovine serum albumin) at the following concentrations: 0, 2, 4, 8, 12, 16 and 25 mg.ml -1 .All samples were analyzed in triplicate and the results obtained by an average of the three readings.
The data were analyzed with the SAEG 8.0 (Euclydes, 1983) regarding normality (Lilliefors test) and homogeneity of variance test (Cockran and Bartlet).The separation of means was done using the Scott Knott statistic test at 5% probability.For testing, statistics program Sisvar 5.0 was used (Ferreira, 2003).

Evaluation of the efficiency of the isolates according to FBN on agronomic characteristics
For the evaluation experiment of efficiency isolates about the agronomic characteristics, FBN used completely randomized design, factorial scheme 4 x 5 willing treatments, where the first factor had the doses applied (0, 20, 60 and 100 kg N ha -1 ) and the second factor had the inoculation of Herbaspirillum seropedicae strain with ZAE 94 (deposited in the collection of cultures of diazotrophic bacteria by Embrapa Agrobiologia with code BR11417), the three strains isolated from the first experiment showed greater efficiency in the production of Auxin and acetylene reduction for each medium and the absence of inoculation.The nitrogen fertilizer in the form of urea, was divided twice, half in planting and the other half 60 days after the emergence of plants.20 treatments with 4 repetitions for the cultivar BRS Tropical were performed.The experiment was conducted in pots containing 9 kg of soil, conducted with 4 plants in each.Viana et al. 2849 The best strains of each semi-specific medium were selected to prepare the inoculum and subsequently, the BRS Tropical inoculation was carried out.Strains were grown in 50 ml medium DYGS stirring at 150 rpm, with temperature of 30°C for 24 h.The peat was already prepared and sterile, making the mixture of 15 ml of the bacterial suspension to 35 g of peat.Before planting, the seeds were wrapped in peat and dried in the shade at the rate of 10 g per kg of peat seed.
Assessments were made 60 days after seedling emergence, in which plant height, number of tillers and grain during harvest was measured.The data were analyzed with the SAEG 8.0 (Euclydes, 1983) regarding their normality (Lilliefors test) and homogeneity of variance test (Cockran and Bartlet).The separation of means was done using the statistical LSD test at 5% probability, and the use of regression analysis describe the response of plants to different levels of fertilization.For testing, the statistical program Sisvar 5.0 was used (Ferreira, 2003).

Experiment of inoculation of diazotrophic bacteria
The results showed significant differences for inoculation and nitrogen levels on all variables for BRS Tropical (Figure 1a, b, c, d).In all variables, the trend of behavior occurred in a linear fashion with increasing doses of N.
Inoculation with H. seropedicae increased by 3.5, 6.0, 6.8 and 2.6% compared to control treatment at doses 0, 20, 60 and 100 kg ha -1 of N, respectively for the variable height of rice plants of cultivar BRS Tropical (Figure 1a).For variable number of tillers, the increase was 22.7, 27.8, 31.5 and 12.5% compared to treatment without inoculation with doses of 0, 20, 60 and 100 kg (Figure 1b).The inoculation with diazotrophic bacteria promoted an increase in the number of tillers, probably leading to gains in dry matter production and grain for BRS Tropical.
The accumulation of weight was 32, 36.2, 70.5 and 55.7%, and the dry mass was 27.4, 28.2, 50.8 and 44.2% higher in regarding the treatment without inoculation doses for 0, 20, 60 and 100 kg ha -1 N, respectively (Figure 1c, d).The results for BRS Tropical also showed an increase in dry matter accumulation, with gains of 28%.Cultivating BRS, introduced different behavior of Wildcard MG cultivate BRS inoculated treatment Tropical, which is less than the treatment without inoculation and tendency of quadratic behavior, suggesting that there is a maximum dose of N which ensures the quantitative increase of the studied variables.Treatment without inoculation to cultivate, followed the same tendency of cultivating BRS Tropical, where the largest dose of nitrogen applied was responsible for increase of the variables examined (Figure 1 e, f, g, h).Inoculation with strain ZAE 94 provided height of 4.5% less plants to plants that did not receive inoculation (Figure 1 e).
The highest dose of nitrogen applied without inoculation provided increased tillering in 124% compared to treatment without inoculation and no added nitrogen.In inoculated treatment, the maximum number of tillers was observed with the addition of 60 kg.ha -1 of N, with an   1f).The accumulation of weight was 9.7%, 17.6%, 7.1 and 38.6%, and the dry mass was 18.5%, 11.5%, 60.7% and 20.1 below in relation to treatment without inoculation to the doses 0, 20, 60 and 100 kg ha-1 of N, respectively (Figure 1 g, h).Cultivating BRS MG wildcard, inoculated with the strain ZAE 94, plus 20 kg ha -1 of N, showed gains in dry matter accumulation around 31%, compared to N-free treatment, but 12% lower than the treatment without inoculation, with the same amount of N applied (Figure 1f).The control treatment showed height of plants 3.4% greater than the treatment medium inoculated with A. brasilense, and 2% greater than that with A. lipoferum.It was observed that in this experiment, cultivate BRS MG Curing did not answer inoculation with strain of diazotrophic bacteria ZAE 94.

Isolation of Diazotrophic bacteria
In the experiment of isolation of diazotrophic bacteria, populations of diazotrophic bacteria culturing media MV, NFB and JNFB were found.In bacterial populations, LGI was not found.Through the technique of MPN (most probable number), the number of bacteria found in the roots of the plants were higher than the values found in the aerial part of plants (Table 1).
The isolates found in cultivating tropical BRS (Table 1) were from the three means of cultivation used in the experiment, where about 55% of the isolates were from non-disinfested plant roots.Of the total of 31 isolates, 9 were grown in medium JMV from the roots of the plants, and then cultured in medium 8 NFB, and the rest of the isolates were grown in middle JNFB, where 13 were isolated from aerial parts of plants and only 1 was isolated from roots.
The isolation of diazotrophic bacteria coming from the cultivars BRS Topical and BRS MG wildcard, grown in Vitória da Conquista-BA, has obtained 50 isolates, of which 18% were classified as similar to those in the genus Burkholderia, 16% similar to the genus Azospirillum and 66% similar to those of Herbaspirillum.The results found in the three formats evaluated for the production of indole-3-acetic acid (IAA) presented values between 0.261 and 4.347 g.mg µ -1 protein.The largest concentrations of indole compounds produced were found in isolates similar to Herbaspirillum spp., followed by Burkholderia spp.and Azospirillum spp.
The ability to make the BNF, via the nitrogenase activity as measured by acetylene reduction technique (ARA), showed that all the selected isolates were able to reduce acetylene to ethylene, proving the efficiency of these bacteria as the potential to fix atmospheric nitrogen to a greater or lesser intensity.The variability among the isolates occurred in the range of the 199.717 and 16.559 mmol/ml protein for an hour before the incubation.

Efficiency of the diazotrophic bacteria as the biological nitrogen fixation (BNF) on the agronomic characteristics
Results showed that 17 B, 37 C and ZAE 94 strains had tendency of quadratic behavior where the dose of 60 kg nitrogen, provided the bigger increase in the variables analyzed, and economy in the amount of N applied.Strain 1A, had the tendency of linear behavior in a negative way, where the increase in the dose of nitrogen provided reduction in variables analyzed, and the control treatment had the tendency of linear behavior, suggesting that increasing the dose of nitrogen ensures the increase of variables.All of these observations were valid for the variables analyzed, except for the number of tillers, where in all treatments the tendency was linear (Figures 2a, b, c, d, e, f).
In a general way, inoculation with diazotrophic bacteria was positive for cultivating Tropical BRS, in relation to control treatment with gains of 9.4 and 3.3% in plant height of 60 DAE, 14.4% in number of tillers, 98% in the dry plants and 3.2% in the grain dry mass weight.The largest dose of N applied in the treatments inoculated, provided smaller gain, in relation to the use of lower doses of nitrogen.The BNF occurs in response to the needs of the plant, and with high level of N-mineral which cause reduction in population of diazotrophic bacteria, and shows no incentive to BNF.
Inoculation showed positive effects and significant superior compared to control treatment, in relation to the dry mass weights.Strain ZAE94, although lower than the inoculations, 37 C and 17 B was the most responsive to increasing doses of N, with an increase of 30% in relation to the treatment without added N. Furthermore, inoculation with strains 17 B and 37 C, presented showed an increase of 11.3 and 9.5%, (Figure 2d).
Results for the production of grain showed that inoculation with different strains of diazotrophic bacteria was positive and statistically superior to control treatment.At 60 kg ha -1 of N, the gains were 7.6 and 28.7% for the 17 strains B and ZAE 94, respectively, in relation to control treatment with the same amount of N applied.Inoculation with strain 37 C was statistically equal to control treatment.
Inoculation with strains 17 B, 37 C and ZAE 94 plus 60 kg ha -1 of N, resulted in an increased production of 174.4,159.4 and 208.5%, respectively.Maximum response on grain production was observed with inoculation with strain ZAE 94 plus 60 kg ha -1 of nitrogen, which was exceeding 200% increase.Treatment without inoculation with diazotrophic bacteria showed higher production to treatments inoculated with strains isolated in the doses 0 and 100 kg.ha -1 of nitrogen, with gains of up to 17.2 and 52.9%, respectively.

Inoculation of diazotrophic bacteria
The results of this study showed that inoculation was beneficial to rice cultivation in the tested conditions according to the results found by Kuss et al. (2008), using the IRGA-420 cultivar at 40 DAS (days after seeding), combination between nitrogen as urea (0, 60 and 120 kg ha -1 ), inoculation with Azospirillum brasilense, isolated UFSM-BD-31-06 and the control, showed no significant differences between treatments for height plants, but inoculation promoted an increase in the variable analyzed.Ramos et al. (2010), also observed an increase in plant height of 21.4% when plants were inoculated with Azospirillum lipoferum (BR strain 11084) and fertilized with 30 kg ha -1 N, and 17.6%, when there was only inoculation of maize plants at 30 days after seeding.
The results are in agreement with Oliveira et al. (2007), where only inoculation with diazotrophic bacteria was able to produce more forage Brachiaria brizantha cv.Marandu due to the increase in the number of tillers per plant.Guimarães et al. (2003), evaluated the effect of inoculation with diazotrophic bacteria in upland rice, and Sala et al. (2005) in wheat plants also observed an increase in the dry weight of shoots in the presence of inoculation, but found no significant differences between treatments as studied in this work.

Isolation of diazotrophic bacteria
The highest numbers of isolates were obtained from the aerial parts of the plants.Rodrigues et al. (2006), observed a greater population of diazotrophic bacteria in roots of rice, compared to the population found in stems and leaves, which was also observed for the bacteria Burkholderia and Azospirillum, however divergent results were found for the genus Herbaspirillum.Brazil et al. (2005), found a higher number of isolates of Azospirillum which was isolated from the roots of irrigated rice plants compared to those isolated from the leaves and stems.Radwan et al. (2004), found that strains of Azospirillum spp.produce three to seven times more indole compounds than Herbaspirillum strains, which differs from the result of this study , where the isolates grown in the middle JNFb supposedly classified as belonging to the gender Herbaspirillum spp.produced on average 3.36 times more than indole compound isolates of Azospirillum.This high variability in the ability to realize the BNF by the technique of acetylene reduction was also observed with bacteria isolated from rice by Rodrigues et al. (2006) and Kuss et al. (2007).To achieve the second experiment, bacteria were selected from each culture medium processed through the combination of the best production results of indole compounds and acetylene reduction capacity of each culture medium.The selected isolates were: 8 A, 17 B and 37 C respectively.

Evaluation of the efficiency of the isolates according to FBN on agronomic characteristics
The application of low recommended doses to the culture, resulted in higher earnings generating economy, since the reduced amount of N was applied, it was reported by Alves et al. (2003), that the use of small doses of N can benefit BNF.
The results found, corroborate the results of Ramos et al. (2010), with significant increases at corn plants height, when they were inoculated with Azospirillum, and higher results even when the inoculation was supplemented with N.
Positive effects of inoculation with diazotrophic bacteria were also found by Hall et al. (2008) they evaluated a similar interaction in wheat, observed greater contribution of inoculation on vegetative period with increase in the number of tillers and consequently higher number of panicles per plant.As well, Guimaraes et al. (2010Guimaraes et al. ( , 2011) ) found that rice plants inoculated and supplemented with N had higher accumulation of dry matter.
Similar results of this study was published by Ferreira et al. (2003), that reported the inoculation of strain ZAE 94, under field conditions, induced increases in grain yields of 38 and 18%, varieties IR42 and IAC4440, respectively, were compared to the control treatment.Divergent results were found by Dotto et al. (2010), who studied the productivity of maize in response to inoculation with H. seropedicae and different levels of N, and observed no significant interactions between inoculation of H. seropedicae and levels of N.
The positive effect of inoculation was also observed by Guimarães et al. (2007), who found increases of up to 30% compared to control treatment without inoculation, depending on the cultivar.Ferreira et al. (2010), also observed that inoculation resulted in higher production of 13% compared to control treatment with IAC in 4440.The results found by Jha et al. (2009) corroborate these results, they show that inoculation with diazotrophic bacteria in rice promotes the growth of plants showing the potential of these bacteria as a promising practice in agriculture can reduce the use of nitrogen fertilizers.

CONCLUSION
The isolation afforded 50 isolates, 19 of BRS and 31 of BRS Tropical.The 1 A, 17 B and 37 C showed a better result in the production of auxin, and the ability to reduce acetylene, depending on the medium used.Inoculation with strains of diazotrophic bacteria guaranteed increase in agronomic variables studied.With a greater verified contribution of inoculation when associated with mineral N fertilization.The estimated grain production has shown that inoculation with diazotrophic bacteria provided, there is an increase of up to 208.5%.

Figure 1 .
Figure1.Regression analysis of variables plant height, number of tillers, accumulation for fresh and dry matter plants, depending on the levels of nitrogen (0, 20, 60 and 100 kg ha -1 ) in BRS Tropical (a, b, c, d) and BRS MG Curinga (e, f, g, h) inoculated or not with strain ZAE 94, under greenhouse conditions (average of 4 plantas.vaso - and 6 repetitions).Vitoria da Conquista, 2012.

Figure 2 .
Figure 2. Regression analysis of variable plant height at 60 DAE, final plant height, number of tillers, dry matter accumulation of plants and grains and grain production, depending on the levels of nitrogen (0, 20, 60 and 100 kg.ha -1 ), BRS Tropical inoculated with strains 1 A, 17 B, 37 C, 94 ZAE and no inoculation under greenhouse (average of 4 plantas.vaso - and 4 repetitions).Vitoria da Conquista, 2012.

Table 1 .
Population of diazotrophic bacteria present in plants by the most probable number technique (MPN).