Symbiotic effectiveness of different indigenous Bradyrhizobium strains on selected Rj-genes harboring Myanmar soybean cultivars

1 Laboratory of Plant Nutrition, Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. 2 Laboratory of Plant Nutrition, Division of Molecular Biosciences, Department of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. 3 Department of Agronomy, Yezin Agricultural University, Yezin, Naypyitaw, Myanmar.


INTRODUCTION
Soybean (Glycine max L.) has become an important crop in Myanmar, as it plays a significant role in human, animal and plant nutrition.It can be grown in various parts of the country.The majority of soybean production is located in the Shan State, followed by the Mandalay, Sagaing, Ayeyarwady, and Bago regions (CSO, 2006).Soybean acreage has gradually increased since 1995 to 1996, but the productivity of soybean (1.51 tons ha -1 ) is still low compared to the world average yield (2.52 tons ha -1 ) (MOAI, 2013).In Myanmar, the Department of Agricultural Research (DAR) has been producing improved soybean cultivars, such as Yezin-3 and Yezin-6, to replace local varieties and to increase soybean production.Moreover, Bradyrhizobium strains such as TAL 377, TAL 379 and TAL 102, produced by Nitrogen Fixation for Tropical Agricultural Legumes (NifTAL), are being used as inoculants.The Plant Pathology Section of the DAR initiated rhizobial inoculant research and production.However, effective, locally adapted Rhizobium strains are not readily available on the Myanmar market.Therefore, exotic strains must be replaced by those that are indigenous, as they have adapted to local environmental conditions and are readily available.The symbiotic effectiveness of native Bradyrhizobium strains on different Myanmar soybean cultivars has been reported (Aung, 2007;Soe and Yamakawa, 2013a).
The responses of soybean cultivars vary with the Rhizobium strains.While some cultivars are fully compatible with other Rhizobium, others cannot form nodules even though the Rhizobium belongs to certain serogroups of Bradyrhizobium (Van et al., 2007).This might be due to nodulation regulatory genes called Rj genes found in cultivars.The Rj genotypes (non-Rj, rj 1 , Rj 2 , Rj 3 and Rj 4 ) have been found to exist in nature (Devine and Kuykendall, 1996).The nitrogen fixation rate varies among both cultivars and inoculant strains.Yamakawa et al. (1999Yamakawa et al. ( , 2003) ) reported on the higher nodulation ability of Rj 2 Rj 3 Rj 4 .Soe andYamakawa (2013a, 2013c) reported that indigenous strains are effective on Yezin-6 (non-Rj) and Yezin-3 (Rj 4 ).Therefore, it is necessary to select strains that are compatible with different Rj-genotypes to promote soybean production through enhancing symbiotic nitrogen fixation.
Inoculation with symbiotic rhizobia for higher biological nitrogen fixation is a common agronomic practice for agricultural production in other countries.In Myanmar, the majority of farmers rely on nitrogenous fertilizer for crop production, although it is very expensive (Than and Han, 1988).An alternative to nitrogenous fertilizer is the use of effective and efficient strains of N-fixing bacteria.Rhizobium inoculant production is relatively inexpensive and affordable to most farmers (Than et al., 1987).Inoculation of soybean with Rhizobium is essential to increase productivity.
In Myanmar, researchers have been emphasizing the selection of inoculants to promote soybean production.
However, the selection of strains compatible with cultivars grown in Myanmar is still limited.To foster soybean productivity, it is necessary to select strains compatible with cultivars.Therefore, the goal of the present study was to screen strains for nitrogen fixation by using Yezin-6 (non-Rj) and to evaluate the symbiotic effectiveness of selected Bradyrhizobium strains on different Rj-gene harboring Myanmar soybean cultivars.

Origin of Bradyrhizobium strains
The Bradyrhizobium japonicum strain USDA110 was obtained from the Laboratory of Plant Nutrition, Kyushu University.Indigenous Bradyrhizobium strains were obtained from a previous experiment ( Htwe et al., 2015a).The strains were selected from different groups identified in a phylogenetic tree.The origins of the isolates are provided in Table 1.

Cultivation
The 1 L pots were filled with vermiculite and 0.6 L of half-strength modified Hoagland nutrient (MHN) solution (Nakano et al., 1997).The pots were autoclaved at 120°C for 20 min.Control pots were also prepared to check for contamination.For surface sterilization, the seeds were soaked in 2.5% sodium hypochlorite solution for 5 min, rinsed with 10 mL of 99.5% ethanol five times and washed with sterilized MHN solution five times to remove the traces of sodium hypochlorite and ethanol.Five surface sterilized seeds were sown in the pots.
The single pure colony of the B. japonicum strain USDA110, and indigenous strains from an A1E plate, were cultured in A1E liquid media (Kuykendall, 1979) and incubated on a rotary shaker at 30°C for 7 days.One milliliter of liquid culture of each isolate was diluted with 99 mL of sterilized Hoagland solution to prepare abacterial suspension of about 10 7 cells mL -1 .Each seed was inoculated with 5 mL of bacterial suspension.The plants were cultivated in an environmentally controlled room (25°C and 75% relative humidity) for 4 weeks.Watering was done as necessary.Autoclaved deionized water was used in this study.The first screening experiment was performed from February 2015 to March 2015.From first screening experiment, five effective strains showing the higher nitrogenase activity compared to B. japonicum USDA110 and other indigenous strains in Yezin-6 (non-Rj) soybean cultivar were selected for second screening experiment.These selected five strains were evaluated for symbiotic efficacy on different Rj-*Corresponding author.E-mail: aungzawhtwe333@gmail.com.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License gene-harboring soybean cultivars designated as Yezin-6 (non-Rj), Yezin-9 (Rj3), Yezin-10 (Rj2Rj3) and Yezin-11 (Rj4) from June 2015 to July 2015.Htwe et al. (2015b) stated that Yezin-6 (non-Rj), Yezin-9 (Rj3), Yezin-10 (Rj2Rj3) and Yezin-11 (Rj4) showed the higher nitrogen fixation and nodulation due to inoculation with B. japonicum USDA110.Therefore, these four efficient soybean cultivars for nitrogen fixation were used to evaluate the symbiotic effective of selected strains for second screening experiment.From second screening experiment, three different strains showing higher nitrogenase activity in Yezin-6 (non-Rj) and Yezin-11 (Rj4) were selected for further experiment.Symbiotic effectiveness of these three selected strains was tested on Yezin-3 (Rj4) and Yezin-8 (non-Rj) in August 2015.This experiment aimed to check the effectiveness of these selected strains on other Rj4 and non-Rj genotypes soybean cultivars.Nodulation, nitrogenase activity, shoot dry weight and root dry weight were measured from three plants per pot at 28 days after sowing.

Acetylene reduction assay
The soybean plants were cut at the cotyledonary nodes and the roots with intact nodules placed in 100 mL conical flasks.Flasks were sealed with a serum stopper and injected with 12 mL of (C2H2) gas to replace air with acetylene.One-mL subsamples were analyzed for ethylene (C2H4) concentration at 5 and 65 min after injecting with C2H2 gas, using a flame ionization gas chromatograph (GC-14A, Shimadzu, Kyoto, Japan) equipped with a stainless steel column (3 mm diameter, 0.5 m long) as described by Soe and Yamakawa (2013a).After the assay, nodules were counted.Shoots, roots and nodules were collected separately and oven dried at 70°C for 24 h to record dry weights.

Statistical analysis
Data were analyzed using the STATISTIX 8 software package (Analytical Software, Tallahassee, FL, USA) and the means were compared by Tukey's HSD test with a P value < 0.05 taken to indicate statistical significance.

Screening of effective bacterial strains by Yezin-6 (non-Rj) for N fixation
The cultivar Yezin-6 (non-Rj) was inoculated with 25 Bradyrhizobium strains isolated in Myanmar and the strain USDA110, and compared for nodulation and nitrogenase activity.They were not significantly different in terms of shoot or root dry weight (Table 2).The highest nodule counts were found in the plants inoculated with B. japonicum USDA110.The highest nodule dry weights were obtained from the plants inoculated with Bradyrhizobium spp.SHY6-1 and B. elkanii SAY3-4, but they were only significantly different from the strain Bradyrhizobium spp.AHY3-9.
In this screening experiment, the strains were compared for their ability to fix N, in terms of acetylene reduction activity (ARA) per plant.The nitrogenase activity of each bacterial strain on the cultivar Yezin-6 (non-Rj) is described in   3).These results confirmed that B. liaoningense SMY3-1 and B. japonicum SAY3-7 were superior in terms of the ability to fixN, and B. elkanii AHY3-1 was superior for nodulation.
In cultivar Yezin-9 (Rj 3 ), nodule dry weight differed significantly among the inoculated Bradyrhizobium strains (Table 4), although nodule number, shoot dry weight and root dry weight N activity were not significantly different.Among the tested strains, B. japonicum SAY3-7 produced a significantly greater nodule mass.Bradyrhizobium japonicum SAY3-7 had the highest N fixing ability among inoculated strains, although it was not superior for nitrogenase activity.These results indicated that the B. Japonicum strain SAY3-7 was more effective in terms of nodulation, N fixation and plant growth for the cultivar Yezin-9 (Rj 3 ).
There was significant difference in nodule number and nodule dry weight for the cultivar Yezin-10 (Rj 2 Rj 3 ), inoculated with Bradyrhizobium strains.Bradyrhizobium elkanii AHY3-1 produced a sgnificantly greater number of nodules and higher nodule dry weight, although there were no significant differences with B. liaoningense SMY3-1.Nitrogenase activity and the shoot and root dry weights were not significantly different among the inoculated strains (Table 5).These results confirmed that B. elkanii AHY3-1 was more effective in nodule formation on the roots of Yezin-10 (Rj 2 Rj 3 ).
For Yezin-11 (Rj 4 ), the nitrogenase activity, nodule number and nodule dry weight were significantly different among the Bradyrhizobium strains (Table 6).A higher level of N fixation, in term of nitrogenase activity, was observed for Yezin-11 (Rj 4 ) inoculated with B. japonicum SAY3-7and B. elkanii AHY3-1, although they were not significantly different from Yezin-11 (Rj 4 ) inoculated with B. liaoningense SMY3-1, B. yuanmingense SMY6-10 or B. japonicum USDA110.B. elkanii AHY3-1 produced a significantly greater number of nodules compared to the other strains, but it was not significantly different from B. japonicum USDA110.Moreover, B. elkanii AHY3-1 produced a significantly higher nodule dry weight compared with the other strains.Shoot and root dry weights were not significantly different among the Bradyrhizobium strains (Table 6).These results demonstrated that the strains B. elkanii AHY3-1 and B. japonicum SAY3-7 were more effective in terms of nitrogen fixation on Yezin-11 (Rj 4 ).Moreover, it was found that B. elkanii AHY3-1 has very efficientnodulation, as indicated by superior nodule counts and higher nodule dry weight.
For Yezin-8 (non-Rj), nodule dry weight, shoot dry weight and nitrogenase activity were different among the Bradyrhizobium strains (Table 7).Inoculation with B. japonicum SAY3-7 resulted in significantly higher nitrogenase activity among the tested strains.Significantly greater nodule dry weight and shoot weight were obtained from the plants inoculated with B. japonicum SAY3-7.Nodule number and root dry weight were not significantly different among the Bradyrhizobium strains (Table 8).These results highlighted B. japonicum SAY3-7 as having greater N fixing ability.

DISCUSSION
Nitrogen is an essential nutrient for plant growth.However, its availability is one of the major limiting factors in plant growth and development (Newbould, 1989).Therefore, current agricultural practices primarily rely on chemical fertilizers to increase productivity (Peoples et al., 2009;Jensen et al., 2012).The heavy use of chemical fertilizers in agriculture is of global concern: alternatives to chemical fertilizers are urgently needed (Sharma and Kumawat, 2011).Biological nitrogen fixation technology is now one such alternative, as it reduces the need for chemical nitrogenous fertilizer (Stacey et al., 2006).Therefore, rhizobia have been used extensively in agriculture to enhance nitrogen fixation (Elkan, 1992).
Soybean, in symbiosis with Bradyrhizobium, has the ability to fix nitrogen at a rate of up to 300 kg N ha -1 under favorable conditions (Smith and Hume, 1987).Optimizing this symbiosis can both increase yields and improve soil fertility, thus reducing the costs and environmental impacts caused by nitrogen fertilizer application (Peoples et al., 2009;Canfield et al., 2010).Therefore, increasing the use of legume crops and identifying important factors related to nodulation are needed to reduce the use of nitrogenous fertilizers and improve the sustainability of agriculture (Peoples et al., 2009;Canfield et al., 2010;Jensen et al., 2012).Selection of an efficient strain is considered to be one of the major factors affecting symbiotic N fixation.
Selection of Rhizobium strains suitable for each legume cultivar is essential in inoculant production.To produce inoculants for each recommended cultivar of soybean, several strains and isolates of rhizobia were initially screened in the greenhouse for their symbiotic effectiveness and nitrogen fixation ability.Then, selected strains and isolates were produced as inoculants and tested for nodulation, nitrogen fixation ability and yield response (Boonkerd and Singleton, 2002).Screening experiments were performed in a greenhouse, under controlled conditions, to select the most effective strains for each cultivar.
The symbiotic effectiveness of 25 Bradyrhizobium strains on Yezin-6 (non-Rj) was determined, as cultivars harboring non-Rj genes are compatible with all types of Bradyrhizobial strains (Ishizuka et al., 1991).The top four strains showing higher nitrogenase activity were B. japonicum SAY3-7, B. elkanii AHY3-1, B. liaoningense SMY3-1 and Bradyrhizobium spp.AHY3-6.Moreover, B. yuanmingense SMY6-10 belonged to different species from top four strains was also selected.Since the effectiveness of Rhizobium strains varies, the first step for inoculant production is to obtain the most effective strain for N fixation for the legumes to be inoculated (Kucey et al., 1988).Therefore, these five strains were selected for the next experiment.
Five strains were evaluated for their symbiotic effectiveness on four soybean cultivars: Yezin-6 (non-Rj), Yezin-9 (Rj 3 ), Yezin-10 (Rj 2 Rj 3 ) and Yezin-11 (Rj 4 ).When they were compared fornitrogenase activity, it was found that all tested Myanmar Bradyrhizobial strains were more effective on Yezin-6 (non-Rj)and Yezin-11 (Rj 4 ), but not on Yezin-9 (Rj 3 ) and Yezin-10 (Rj 2 Rj 3 ).This might be due to regulatory genes, as Rj genes have the ability to control the compatibility of hosts and rhizobia (Ishizuka et al., 1991;Saeki et al., 2000).In this study, Bradyrhizobium spp.AHY3-6 and B. yuanmingense SMY6-10 strains showed lower N fixation efficiency, in terms of ARA, on all cultivars when compared with other strains.It was clear that the N fixation rates depended on not only cultivars and inoculated bacterial strains, but also on the interaction between cultivar and strain.Graham (2000) showed that nitrogen fixation levels vary among legume cultivars; therefore, the higher efficiency with respect to nitrogen fixation is also dependent on particular combinations of strains and cultivarsin some crops.It has also been reported that the effectiveness of Bradyrhizobium strains varies with soybean genotype (Okereke et al., 2001;Tien et al., 2002).
The top three strains, B. japonicum SAY3-7, B. liaoningense SMY3-1 and B. elkanii AHY3-1,which were shown to be more effective on Yezin-6 (non-Rj) and Yezin-11 (Rj 4 ) cultivars, were tested on other Rj 4 and non-Rj cultivars, identified as Yezin-3 and Yezin-8, respectively, to determine their compatibility and effectiveness.These results indicated that B. japonicum SAY3-7 had the highest nitrogen fixing ability on Yezin-8 (non-Rj), but the nitrogenase activity of that strain was not significantly different from those of B. liaoningense SMY3-1 and B. elkanii AHY3-1 strains on Yezin-3 (Rj 4 ).In Yezin-3 (Rj 4 ) soybean cultivar, B. elkanii AHY3-1 produced a greater number of nodules and nodule mass although nitrogenase activity did not differ among inoculated strains.Adhikari et al. (2013) also stated that strains producing greater nodule mass do not increase N fixation, in terms of ARA.
B. japonicum SAY3-7 had consistently higher nitrogenase activity, especially on non-Rj-and Rj 4 -gene harboring soybean cultivars was selected for Rhizobium inoculant production.Israel et al. (1986) reported that selection of host cultivar-compatible inoculantsis important for increasing nitrogen fixation in soybean.The extent of nitrogen fixation by soybean cultivars may vary depending on the symbiotic effectiveness of Rhizobium strains and their compatibility.Our results supported the findings of other studies which is clearly stated that increases in biological nitrogen fixation in soybean production can be obtained by selecting effective strains and efficient soybean cultivars as cultivar-strain pairs (Duong et al., 1984;Kucey et al., 1988;Thi, 2007;Soe and Yamakawa, 2013a).

Conclusion
In this study, 25 Bradyrhizobium strains were selected and studied for their symbiotic effectiveness on the cultivar Yezin-6 (non-Rj).The five Bradyrhizobium strains, B. japonicum SAY3-7, B. elkanii AHY3-1, B. liaoningense SMY3-1, Bradyrhizobium spp.AHY3-6 and B. yuanmingense SMY6-10, were selected for their greater nitrogen fixing capacity.They were tested on different Rjgene-harboring Myanmar soybean cultivars.B. japonicum SAY3-7, B. liaoningense SMY3-1, and B. elkanii AHY3-1 were more effective on Yezin-6 (non-Rj) and Yezin-11 (Rj 4 ), whereas they were not effective on Yezin-9 (Rj 3 ) or Yezin-10 (Rj 2 Rj 3 ).These three strains were superior in terms of nitrogen fixing ability compared with other indigenous strains and the most widely used exotic strain, B. japonicum USDA110.They were tested on Yezin-8 (non-Rj) and Yezin-3 (Rj 4 ) to evaluate their effectiveness and compatibility.In this experiment, B. japonicum SAY3-7 was found to be the most effective strain for N fixationon .Over all of the experiments, B. japonicum SAY3-7 was the most effective strain for N fixation, as it showed superior nitrogenase activity on Yezin-6 (non-Rj) in the first screening experiment, on Yezin-6 (non-Rj), Yezin-9 (Rj 3 ) and Yezin-11 (Rj 4 ) cultivars in the second experiment, and on Yezin-8 (non-Rj) in the third experiment.According to the results of this study, the most effective strain, B. japonicum SAY3-7, was selected to be used as an inoculant for specific cultivars to increase soybean production in Myanmar.This study was conducted in pots under controlled conditions.B. japonicum SAY3-7 should also be evaluated on Yezin-6 (non-Rj), Yezin-8 (non-Rj) and Yezin-11 (Rj 4 ) and Yezin-3 (Rj 4 ) for symbiotic effectiveness under field conditions.
each column followed by the same letters are not significantly different at P < 0.05 (Tukey's test).NN, nodule number; NDW, nodule dry weight; SDW, shoot dry weight; RDW, root dry weight; ARA, acetylene reduction activity.

Table 1 .
The origins of Bradyrhizobium strains isolated from Myanmar.

Table 2 .
Effect of Myanmar Bradyrhizobial strains on the acetylene-reducing activity, nodulation and plant growth of Yezin-6 soybean cultivar at 28 days after sowing.
abMean values in each column followed by the same letters are not significantly different at P<0.05 (Tukey's test).NN, nodule number; NDW, nodule dry weight; SDW, shoot dry weight; RDW, root dry weight; ARA, acetylene reduction activity.The selection of strains was based on nitrogenase activity.Isolates in bold were selected for second screening experiment.

Table 2 .
The highest ARA per plant was observed for the B. japonicum strain SAY3-7.

Evaluation of the effectiveness of selected Bradyrhizobium strains on different Rj-gene soybean cultivars
were compared for symbiotic effectiveness on four soybean cultivars.For the cultivar Yezin-6 (non-Rj) inoculated with different Bradyrhizobium strains, the nitrogenase activity and nodule number per plant differed significantly among the strains (Table3).Similarly, inoculation with B. liaoningense SMY3-1 showed

Table 3 .
Effect of six different bradyrhizobial strains on acetylene reduction activity, nodulation and plant growth of Yezin-6 soybean cultivar at 28 DAS.Mean values in each column followed by the same letters are not significantly different at P < 0.05 (Tukey's test).NN, nodule number; NDW, nodule dry weight; SDW, shoot dry weight; RDW, root dry weight; ARA, acetylene reduction activity. b

Table 4 .
Effect of six different bradyrhizobial strains on acetylene reduction activity, nodulation and plant growth of Yezin-9 soybean cultivar at 28 DAS.
a Mean values in each column followed by the same letters are not significantly different at P < 0.05 (Tukey's test).NN, nodule number; NDW, nodule dry weight; SDW, shoot dry weight; RDW, root dry weight; ARA, acetylene reduction activity.

Table 5 .
Effect of six different bradyrhizobial strains on acetylene reduction activity, nodulation and plant growth of Yezin-10 soybean cultivar at 28 DAS.Mean values in each column followed by the same letters are not significantly different at P < 0.05 (Tukey's test).NN, nodule number; NDW, nodule dry weight; SDW, shoot dry weight; RDW, root dry weight; ARA, acetylene reduction activity. a

Table 6 .
Effect of six different bradyrhizobial strains on acetylene reduction activity, nodulation and plant growth of Yezin-11 soybean cultivar at 28 DAS.
abMean values in each column followed by the same letters are not significantly different at P < 0.05 (Tukey's test).NN, nodule number; NDW, nodule dry weight; SDW, shoot dry weight; RDW, root dry weight; ARA, acetylene reduction activity.

Table 7 .
Effect of three different bradyrhizobial strains on acetylene reduction activity, nodulation and plant growth of Yezin-3 soybean cultivar at 28 DAS.
a Mean values in each column followed by the same letters are not significantly different at P < 0.05 (Tukey's test).NN, nodule number; NDW, nodule dry weight; SDW, shoot dry weight; RDW, root dry weight; ARA, acetylene reduction activity.

Table 8 .
Effect of three different bradyrhizobial strains on acetylene reduction activity, nodulation and plant growth of Yezin-8 soybean cultivar at 28 DAS.