New Native Rhizobia Strains for Inoculation of Common Bean in the Brazilian Savanna

Maximization of biological nitrogen fixation in the common bean (Phaseolus vulgaris L.) crop depends on the genetic characteristics related to the plant, the symbiotic efficiency of rhizobia, and environmental factors. The objective of this study was to evaluate the performance of rhizobia selected beforehand from Cerrado (Brazilian tropical savanna) soils in Mato Grosso do Sul. The experiments were conducted in 2007 in the municipalities of Aquidauana, Anaurilândia, Campo Grande, and Dourados, all located in the state of Mato Grosso do Sul. All procedures established followed the current recommendations of the Brazilian Ministry of Agriculture (Ministério de Agricultura, Pecuária e Abastecimento – MAPA), in accordance with the “official protocol for assessing the feasibility and agronomic efficiency of strains, and inoculant technologies linked to the process of biological nitrogen fixation in legumes”. The program for selection of rhizobia for inoculation in bean plants resulted in identification of different strains with high symbiotic efficiency, competitiveness, and genetic stability, based on the Embrapa Agropecuária Oeste collection of multifunctional microorganism cultures. In previous studies, 630 isolates of Rhizobium were evaluated. They were obtained from nodules of leucaena (380) or dry beans (250) from 87 locations, including 34 municipalities in Mato Grosso do Sul. Three of them stood out from the others: CPAO 12.5 L2, CPAO 17.5 L2, and CPAO 56.4 L2. Inoculation of these strains in bean plants demonstrated economic viability and high potential for obtaining a more effective inoculant suitable for trading purposes.


INTRODUCTION
Common bean (Phaseolus vulgaris L.), hereinafter "bean", is a crop of great social and economic importance in Brazil because, in addition to being one of the main sources of protein for the population, it is a source of income for thousands of rural producers, especially family farmers.Considering all three systems of bean production, at a low, medium, and high technological level, corresponding to the first, second, and third crop seasons, the average yield obtained in 2013 was 789, 1061, and 2466 kg ha -1 , respectively (IBGE, 2015).Among the different aspects related to the cropping systems and bean production, N management stands out due to high fertilizer costs (Cantarella, 2007).Moreover, the possibility of environmental contamination through adoption of unsuitable techniques and inadequate management must be considered, as shown by several authors, such as Straliotto et al. (2002) and Pelegrin et al. (2009).In this context, management of N fertilization is one of the main problems in the bean crop, since insufficient application rates may limit its productive potential and excessive N rates are not only costly, but can also promote negative environmental impact (Santos et al., 2003).
Biological nitrogen fixation (BNF) process can, at least partially, supply the requirements of this crop and ensure greater yield levels, up to 2500 kg ha -1 (Hungria et al., 2000;Pelegrin et al., 2009).Nevertheless, factors such as soil acidity, high concentrations of toxic Al and the competitive ability of rhizobia native to Brazilian soils can affect the symbiosis between rhizobia strains and bean crop, causing low efficiency in the inoculation process (Mercante et al., 1999;Vargas et al., 2000;Ferreira et al., 2013).Issues related to genetic instability of many rhizobia strains may cause a loss of efficiency or nodulation capacity under stress conditions, which may explain, in part, the considerable diversity in results obtained from inoculation of bean under field conditions (Peres et al., 1994;Hungria and Vargas, 2000;Hungria et al., 2003;Pelegrin et al., 2009).
Several species of Rhizobium have been described as capable of creating nodules and, in most cases, fixing N 2 in symbiosis with bean: R. leguminosarum bv.phaseoli (Jordan, 1984), R. tropici (Martínez-Romero et al., 1991), R. etli bv.phaseoli (Segovia et al., 1993), R. gallicum (bv.gallicum and bv.phaseoli), R. giardinii (bv.giardinii and bv.phaseoli) (Amarger et al., 1997), R. leucaenae (Ribeiro et al., 2012), R. freirei (Dall 'Agnol et al., 2013), and R. paranaense (Dall 'Agnol et al., 2014).An important feature of the bean microsymbiont rhizobia genome is the presence of DNA repeated sequences found in the symbiotic plasmid regions, pSym containing the structural nitrogenase gene, nif, or the nodulation gene, nod (Girard et al., 1991;Segovia et al., 1993).These copies are usually needed for expression of the BNF process and effectiveness, but they also represent recombination sites where genomic rearrangements occur, which can lead to loss of properties in these strains (Girard et al., 1991), providing the explanation for the frequent occurrence of loss of ability to create nodules and to fix N 2 in various strains.
Studies carried out in large group strains of R. tropici, including R. leucaenae, R. freirei, and R. paranaense showed that this specie had greater genetic stability than the other bean microsymbiont, probably due to a single nifH gene copy (Piha and Munns, 1987;Martínez-Romero et al., 1991).Estimates of rearrangement frequency show that R. tropici can be 100 times more stable than R. etli and, when considering plasmid loss, the stability of R. tropici can be a hundred times higher than R. etli (Ferreira et al., 2013).Recent studies involving analysis of symbiotic and non-symbiotic genes of 15 rhizobia strains selected in programs of four distinct Brazilian geographical regions showed that all these strains clustered with R. tropici type A or type B and suggested that the strain selection in this species could reflect evolutionary strategies to maximize nodulation and BNF in bean (Pinto et al., 2007).
In this context, programs for continuous selection of genetically more stable native strains, adapted to local climatic conditions, with greater competitiveness in Brazilian soils and tolerant to environmental stresses, can significantly contribute to achieve more Rev Bras Cienc Solo 2017;41:e0150120 efficient fixation systems and, consequently, increases in bean yield and even contribute to reduction in N fertilizer application that increases production costs and negatively affects the environment.
In previous works several native rhizobia strains capable of fixing nitrogen in bean plants have already been selected by the Embrapa in Cerrado soils in the state of Mato Grosso do Sul.This study was carried out with the objective to evaluate the strains agronomic efficiency in order to indicate commercial inoculants compositions to the bean culture.
The tillage procedures for setting up the experiments in the Aquidauana, Anaurilândia, and Campo Grande areas combined one plowing with two harrowings; in the Dourados area, the no tillage system was used.The cultivar Pérola of the Carioca commercial group was used in the four experiment locations.Seeds were sown manually by distributing 15 seeds per meter, aiming to obtain approximately 10 plants m -1 .Seeds were sown on April 4, April 17, April 19, and May 5, 2007 in Dourados, Anaurilândia, Campo Grande, and Aquidauana, respectively.Fertilization in the municipalities of Anaurilândia, Dourados, and Campo Grande consisted of the application of 300 kg ha -1 of the formulation 00-20-20 (N-P-K), whereas in Campo Grande, the fertilization was 270 kg ha -1 of the same formulation.For the control with N fertilization, 40 kg ha -1 N (urea) was applied at sowing and the same amount manually in topdressing at 30 days after emergence.
Rev Bras Cienc Solo 2017;41:e0150120 The trials were carried out following the technical control recommendations for pests and diseases, without irrigation.The following characteristics were analyzed: number of nodules (NN), following the methodology proposed by Cardoso et al. (2009); dry matter of nodules (DMN) and dry matter of shoots (DMS) of plants in full flowering, after drying in an oven at 60 °C until achieving constant weight; N content in the shoots determined by the Kjeldahl method (semi micro), according to Malavolta et al. (1997); 100 grain weight; and crop yield.Estimates were made on the basis of experimental plots using the two central rows, eliminating the first two plants from the ends of each row.Yield was calculated after correcting for moisture of 13 %.

Rhizobia strains used as reference for comparison
Rhizobia strains commercially recommended in Brazil and used in all the experiments belong to the group of Rhizobium tropici, CIAT 899 Inoculation with bacteria was performed using a product prepared at a density of 10 9 cells g -1 , which was applied to the seeds at the rate of 500 g per 50 kg of seeds.Before inoculation, 300 mL of sugar solution at 10 % (m/v) were added to improve inoculant adhesion on the seed surface.

Economic analysis
In economic analysis, only the costs of the inoculant, the fertilizer (urea), and application operations (in the planting furrow and in topdressing) were considered.Other costs, such as the operation of soil tillage, planting, fertilization with P and K, and crop treatments were not considered because they were common to all treatments.The price of urea in Mato Grosso do Sul in December 2014, was US$ 518.08 Mg -1 or US$ 1.15 kg -1 of N (Conab, 2015).The application cost was estimated considering 0.1 Mh ha -1 (machine hour per hectare) or US$ 1.15 ha -1 and the inoculant cost was US$ 1.89 ha -1 .The bean price considered the average price of different regions of the state of Paraná because Paraná is a producing region and a traditional provider of product price.The average price on February 6, 2015 was US$ 54.59 per 60 kg bag of Carioca beans or US$ 0.91 per kg (Agrolink, 2015).
Using these data as a reference, comparisons were made (considering increase in yield, cost of production, increase in gross returns, and increase in net returns) between the treatments and the absolute control (without N fertilization and without inoculation with rhizobia).The increase in yield corresponded to the difference in grain production achieved in the treatments considered and in the absolute control.The increase in gross returns was determined by the increase in yield multiplied by the value paid for the commercial product.The increase in net returns was the difference between the growth of gross return minus the costs of inoculant and nitrogen fertilizer and their application.
The results were subjected to simple analysis of variance by location and to joint analysis in situations in which the relation of residues of each variable mean squares was less than or equal to 7.0, as recommended in Banzato and Kronka (1992).When necessary, the averages were compared by the Tukey test and the Scott-Knott test at 5 % probability.

RESULTS AND DISCUSSION
The environmental variability among the experimental sites was evident (Table 2).This characteristic, as determined by the Brazilian Ministry of Agriculture (Ministério de Agricultura, Pecuária e Abastecimento -MAPA) by Normative Instruction DAS 13 of March 25, 2011, is one of the fundamental criteria used to assess the feasibility and agronomic efficiency of inoculants and technologies associated with the biological nitrogen fixation (BNF) process in legumes for the purpose of selection and recommendation of bean crop inoculants in Brazil.
The NN were lower in areas with N fertilization (Table 3), indicating a negative effect of N fertilization on nodulation.The BNF would be an alternative, however due to high energy cost, it is not the preferable path to N assimilation.The availability of mineral N can affect the rhizobium-legume interaction, producing no nodules or even stopping BNF in plants with nodules (Schultze et al., 1994;Mercante et al., 1999).
The ability to increase formation of nodules is an important factor in the selection process aiming to increase BNF, although it is not considered as a measure of the nodule efficiency.In this regard, CPAO 17.5 L2, CPAO 12.5 L2, and CIAT 899 stood out in Aquidauana, H 12 in Anaurilândia, CPAO 56.4 L2 in Campo Grande, and CPAO 12.5 L2, CIAT 899, CPAO 17.5 L2, and H12 in Dourados.In general analysis, the strains H12, CPAO 17.5 L2, CPAO 12.5 L2, CPAO 2.11 L, CIAT 899, and CPAO 56.4 L2 were the ones that stood out best (Table 3).It should be emphasized that the potential of the native strains evaluated were similar to the commercial strains H12 and CIAT 899, and superior to PRF 81 (Table 3).
All inoculated treatments promoted greater nodulation than the control and the treatments that used mineral fertilization in Aquidauana, although the latter two did not differ between themselves (Table 4).In Anaurilândia, the strains H 12 and CPAO 2.11 L stood out in plant nodulation.In Campo Grande, no differences were observed between the treatments studied, whereas in Dourados, the strains CPAO 12.5 L2, CIAT 899, CIAT 17.5 L2, and CPAO 56.4 L2 stood out.When analyzed together (Table 3), all the strains and CIAT 899 and H 12 had higher nodule weight, and the lowest values were observed in the treatment of inoculation with the strain PRF 81, the control, and the treatment that contained mineral N. Silva et al. (2009) also observed a linear decrease in DMN as the mineral N supply increased.According to these authors, there is a range of nitrate leaf concentrations where large reductions in nodule size are observed.The presence of nodules in the control treatment indicates the existence of native strains of Rhizobium spp. in all places; however, both the number of nodules and dry weight of nodules were lower than in plants inoculated with the strains tested, indicating that selection of efficient strains is required to achieve maximization of BNF in the bean crop. (1)Data processed by (x+1) 1/2 for statistical analysis.Means followed by the same letter in the line do not differ by the Tukey test at 5 % probability.
The production of DMS was equal among sites and among treatments (Table 4).In the treatments inoculated with different strains, the production of shoot dry matter was similar to the treatment with N fertilization (80 kg ha -1 of N), although they did not differ from the control.These results showed the efficiency of the native rhizobia population for nodule creation and N fixation, and the unfeasibility of N fertilization, which increases the cost of production and leads to possible environmental problems.
Regardless of the location, the treatments did not show differences in the N contents of shoot dry matter (Tables 2 and 3).In this regard, Lemos et al. (2003) also found no differences in N concentrations in the dry matter between genotypes of bean which received or did not receive inoculation with rhizobia.The lack of difference between the treatments and the control with N fertilization (80 kg ha -1 of N) differs from the results of Binotti et al. (2010), who observed an increase in N concentrations in dry matter.However, this result indicates that there was restriction in the use of the N applied, possibly from losses due to other processes, such as leaching and volatilization, among others, or the efficiency of native strains in biological N fixation and also in supplying plant needs.
With the exception of Dourados, the 100 grain weight of the control treatment was lower compared to the other treatments (Table 4).These results are similar to multiple studies that have demonstrated the positive effect of N on 100 grain weight (Rapassi et al., 2003;Silva et al., 2004;Alvarez et al., 2005;Teixeira et al., 2005;Binotti et al., 2009).However, Bassan et al. (2001) observed higher values in the inoculated treatments in relation to the non-inoculated ones and attributed the result to the non-existence of efficient native strains.Thus, it can be inferred that the strains evaluated in this study were also competitive and efficient in supplying N, enabling achievement of 100 grain weight equivalent to that obtained in the treatment with N fertilization at 80 kg ha -1 .4) obtained in the different treatments at each experimental location can be regarded as satisfactory because sowing was carried out at the end of the period (1) Data processed by (x+1) 1/2 for statistical analysis.

Grain yield (Table
(2) Without nitrogen fertilization and without inoculation.
(3) 80 kg ha -1 of N (40 kg at planting and 40 kg in topdressing).Means followed by the same letters in the columns do not differ by the Scott-Knott test at 5 % probability.CV: coefficient of variation.NPP: number per plant.recommended for bean cultivation in accordance with edaphoclimatic zoning.Joint analysis of data (Table 3) showed that inoculation with the strains CPAO 56.4 L2, CPAO 12.5 L2, and N fertilization (80 kg ha -1 of N) were the treatments that showed the highest yields.
There is yet another group formed by the commercial strains (CIAT 899,PRF 81,and H 12) and the native strains (CPAO 17.5 L2 and CPAO 2.11 L), as well as CIAT 899 + 20 kg of N, that differed from the control.
An increase in bean yield was determined by inoculation with strains collected from Central Brazil in relation to the average of the commercial strains CIAT 899 (SEMIA 4077), PRF 81 (SEMIA 4080),and H 12 (SEMIA 4088).All new strains evaluated were superior to those used in the commercial inoculants.The most outstanding were CPAO 56.4 L2 and CPAO 12.5 L2, which exhibited a 25 and 19 % increase bean yield, respectively.There were significant results shown by CPAO 17.5 L2 and CPAO 2.11 L, with increases of 9 and 8 %, respectively.In accordance with the protocol established by the MAPA for analysis of results for indication of strains and their recommendation in Brazil, the native strains CPAO 56.4 L2, CPAO 12.5 L2, CPAO 17.5 L2, and CPAO 2.11 L can be indicated for production of a new bean plant inoculant in Brazil since they resulted in yields higher than those obtained with the inoculants currently marketed.
Due to large variations in the price of N fertilizers and the different forms of supplying N, the use of financial analysis in bean research studies is becoming increasingly common (Binotti et al., 2009;Pelegrin et al., 2009;Binotti et al., 2010).However, an increase in net returns, which should be the ultimate goal of rural producers, is not always linked to technology or a process that enables an increase in yield, as noted by Binotti et al. (2009).These authors found that application rates of ammonium sulfate that led to the highest bean yields did not result in higher values in net return, due to the high costs of N fertilizers.Pelegrin et al. (2009) observed that an increase of 795 kg ha -1 in bean yield with 160 kg ha -1 of N fertilization was financially equivalent to an increase of only 373 kg ha -1 provided by inoculation (Rhizobium tropici CIAT 899-SEMIA4077 sign) associated with only 20 kg ha -1 of N from N fertilization applied at sowing. (1)Data processed by (x+1) 1/2 for statistical analysis.
(3) 80 kg ha -1 of N (40 kg at planting and 40 kg in topdressing).Means followed by the same letters in the columns do not differ by the Scott-Knott test at 5 % probability.CV: coefficient of variation.NPP: number per plant.
Rev Bras Cienc Solo 2017;41:e0150120 CPAO 2.11 L, which led to increases in net return per hectare of US$ 683.21,602.97,478.50 and 471.41,respectively.In this situation, the increases in net return were higher than those obtained from the application of 80 kg ha -1 of N, which was only US$ 450.89 ha -1 .It is interesting to note that the strains used in commercial inoculants (CIAT 899,PRF 81 and H 12) showed increases in net return lower than those obtained from nitrogen fertilization.These results indicate that the new strains evaluated in this study (CPAO 56.4 L2,CPAO 12.5 L2,CPAO 17.5 L2,and CPAO 2.11 L) may be used in the preparation of commercial inoculants for the bean crop.The indication for use of these new strains for all bean producing regions in Brazil also depends on completion of validation tests in all regions.

CONCLUSIONS
Bean inoculation with the native strains CPAO 56.4 L2 and CPAO 12.5 L2 resulted in grain production similar to that obtained with fertilization of 80 kg ha -1 of N.
The strains CPAO 56.4 L2, CPAO 12.5 L2, CPAO 17.5 L2, and CPAO 2.11 L increased bean plant net returns in relation to the absolute control treatment (without N fertilization and without inoculation), to the N control (80 kg ha -1 of N and without inoculation), and to the commercial strains.
The strains CPAO 12.5 L2, CPAO 17.5 L2, and CPAO 56.4 L2 have high potential for obtaining more effective and economical inoculants for use in the bean crop. (1)Yield increase in relation to the control.
Fazenda São José, the Experimental Station of the Universidade para o Desenvolvimento do Estado e da Região do Pantanal (UNIDERP), and the Experimental Station of Embrapa Agropecuária Oeste, respectively.The soils at these different sites were classified as Argissolo Vermelho-Amarelo Distrófico
Native Cerrado rhizobia selected for bean plant inoculationRhizobia selection, based on the Embrapa Agropecuária Oeste collection of cultures of multifunctional microorganisms for bean plant inoculation, resulted in the identification of several isolates with high symbiotic efficiency when inoculated in bean.From assessment of 630 Rhizobium isolates obtained from nodules of leucaena (380) or dry beans (250) from 87 sites including 34 municipalities of Mato Grosso do Sul, four of them stood out from the others based on previous studies: CPAO 2.11 L, CPAO 12.5 L2, CPAO 17.5 L2, and CPAO 56.4 L2.

Table 2 .
Agronomic variables and common bean grain yield (cv. Pérola) in four locations in the state of Mato Grosso do Sul, in 2007 crop season

Table 3 .
Average of number of nodules per plant, dry matter (DMN) of nodules per plant, dry matter of shoots (DMS) per plant, shoot nitrogen (N), 100 grain weight, and common bean (cv.Pérola) grain yield in relation to the different treatments, in four locations in the state of Mato Grosso do Sul (Aquidauana, Anaurilândia, Campo Grande, and Dourados), in 2007 crop season

Table 4 .
Number of nodules per plant, dry matter of nodules (DMN) per plant, dry matter of shoots (DMS) per plant, shoot nitrogen (N), 100 grain weight, and common bean (cv.Pérola) grain yield as a function of the supply of mineral nitrogen and/or inoculation of seeds with different strains of commercial rhizobia and strains selected from soils of Central Brazil (Mato Grosso do Sul), in 2007 crop season

Table 5 .
Variation in financial income in relation to the application of mineral nitrogen and inoculation of bean seeds with different strains of rhizobia in relation to the treatment without inoculation and without fertilization (control), average of sites: Aquidauana-MS, Anaurilândia-MS, Campo Grande-MS, and Dourados-MS, Brazil, in 2007 crop season