Black gram ( Vigna mungo L. Hepper ) is one of the main leguminous crops that provide chief source of food. Several Bradyrhizobium species are able to induce effective nodules in black gram cultivars. In the present study, we characterized forty isolates of indigenous black gram bradyrhizobia from Myanmar based on the sequence analysis of the bacterial 16S rRNA gene. The sequence analysis confirmed that all isolates were categorized and identified as the genus Bradyrhizobium and they were conspecific with B . elkanii , B . sp. , B . liaoningense, B. japonicum and B . yunamingense . Almost all the collected isolates from major black gram growing regions of Nyaunglebin Bago Regio, Chaungzon Mon State, Sittwe Rakhine State, Danubyu Ayeyarwady Region and Launglon Tanintharyi Region were identified as B . liaoningense . At Danubyu Ayeyarwady Region and Pyinmanar Nay Pyi Taw Region, most of the strains were identified as B . japonicum . On the other hand, more or less all the isolates from Launglon Tanintharyi Region and Hpa-an Kayin State were related to B . elkanii . However, all B . sp. strains were found in Salingyi Sagaing Region black gram growing region. This is the first report describing Bradyrhizobium strains that were isolated from soil samples of major black gram growing areas in Myanmar. Evaluation of the effectiveness of Myanmar Bradyrhizobim strains isolated from soil samples of major black gram growing areas of Myanmar for plant growth and nitrogen fixation w as studied in pot experiments with completely randomized design and three replicates. The nodule dry weight, shoot dry weight and acetylene reduction activity of the plant inoculated with Bradyrhizobium elkanii LauBG38 were significantly higher in ARA per plant, nodule and shoot dry weights than the other tested isolates in both Yezin -4 and Yezin-7 black gram varieties. We expect that Myanmar Bradyrhizobium elkanii LauBG38 will be able to use as Biofertilizer for black gram cultivars.
Grain legumes play an important nutritional role in the diet of millions of people in the developing countries [
Nitrogen (N) fixation through legume-Rhizobium symbiosis is important for enhancing agricultural productivity and is therefore of great economic interest [
Rhizobial inoculant can be used to substitute the nitrogenous fertilizers in food legume crops. Recently, peat-based root nodule bacterial inoculants containing TAL strains are using as biofertilizer in seven legumes distributed by Ministry of Agriculture and Irrigation, Myanmar [
Commercial production of Rhizobium inoculant has been developed for several decades. The main objective of using Rhizobium inoculant is to substitute the nitrogenous fertilizers in food legume production. It is cheaper and lighter in weight than urea and easier to use for the farmers [
Myanmar farmers have used, and continue to use, rhizobial inoculants when sowing legumes, but the practice is currently not extensive. The Department of Agricultural Research, Ministry of Agriculture, Livestock and Irrigation is responsible for producing inoculants in Myanmar for their distribution to farmers. Production by DAR peaked during the 1980s at 600 - 700,000 packets annually. Current production is < 100,000 packets, due to limitations in the whole supply chain from production and quality assurance to distribution to demand. Myanmar farmers use nitrogenous (N) fertilizers sparingly, particularly on legume crops. Thus, low-nodulation induced N deficiencies of the legumes are not remedied by inputs of fertilizer N and the value of lost production could exceed $100 million annually [
Several studies also reported significant increase growth parameters and yield due to the inoculation of rhizobial isolates in chickpea [
For this reason, we aimed to isolate indigenous root nodule bacteria from collected soil samples of major black gram growing areas of Myanmar, to identify the phylogenetic diversity of indigenous black gram-nodulating bradyrhizobia in Myanmar based on sequence analysis of the 16S rRNA region of the isolates, and to evaluate the effectiveness of indigenous Myanmar Bradyrhizobium strains for plant growth and nitrogen fixation of Myanmar black gram varieties are required for investigation.
Soil samples were collected from eight major black gram growing regions of Myanmar (
Region of Hpa-an Kayin State (16˚53'N 97˚38'E) with a pH 5.13 was Latritic Soils [
Soil sampling site | Soil Classification19) | Location | Climate20) (Avg. Temp; RF) |
---|---|---|---|
Nyaunglebin Bago Region | Meadow Soil | 17˚57'N 96˚43'E | 27.3˚C, 3292 mm |
Danubyu Ayeyarwady Region | Alluvial Soil | 17˚22'N 95˚27'E | 26.8˚C, 2250 mm |
Chaungzon Mon State | Meadow Soil | 16˚23'N 97˚32'E | 26.8˚C, 4772 mm |
Launglon Tanintharyi Region | Latritic soils | 14˚05'N 98˚12'E | 26.6˚C, 5594 mm |
Sittwe Rakhine State | Meadow Soil | 20˚30'N 93˚20'E | 25.7˚C, 4664 mm |
Pyinmanar Nay Pyi Taw Region | Meadow Alluvial Soil | 19˚45'N 96˚12'E | 27.0˚C, 1302 mm |
Salingyi Sagaing Region | Meadow and Meadow Alluvial Soil | 21˚58'N 95˚05'E | 27.5˚C, 803 mm |
Hpa-an Kayin State | Latritic soils | 16˚53'N 97˚38'E | 27.5˚C, 4284 mm |
Sources: 19)Shein, H.A. The soil types and characteristics of Myanmar. Department of Agriculture, Ministry of Agriculture, Livestocks and Irrigation: Nay Pyi Taw, Myanmar, 2015. 20)Aung, L.L.; Zin, E.E.; Theingi, P.; Elvera, N.; Aung, P.P.; Han, T.T.; Oo, Y.; Skaland, R.G. Myanmar Climate Report published by Department of Meteorology and Hydrology Myanmar, Ministry of Transport and Communications, Government of the Republic of the Union of Myanmar, 2017.
Soil samples | Physicochemical property | ||||
---|---|---|---|---|---|
Soil pH (Soil: H2O; 1:2.5) | Total N (%) | Mineralizable N (g/kg) | Total P2O5 (%) | Total K2O (%) | |
Nyaunglebin Bago Region | 5.04 | 0.31 | 1.31 | 0.23 | 1.14 |
Danubyu Ayeyarwady Region | 6.70 | 0.12 | 0.91 | 0.06 | 0.73 |
Chaungzon Mon State | 4.69 | 0.23 | 1.57 | 0.10 | 1.74 |
Launglon Tanintharyi Region | 4.79 | 0.73 | 1.62 | 0.08 | 2.98 |
Sittwe Rakhine State | 5.91 | 0.16 | 2.07 | 0.05 | 0.42 |
Pyinmanar Nay Pyi Taw Region | 6.01 | 0.25 | 0.95 | 0.09 | 0.76 |
Salingyi Sagaing Region | 6.97 | 0.05 | 0.42 | 0.03 | 0.16 |
Hpa-an Kayin State | 5.13 | 0.16 | 0.75 | 0.07 | 0.75 |
Soil sample analyses were performed in the plant nutrition laboratory, faculty of agriculture, Kyushu University, Japan.
The collected soil samples were analyzed at Plant Nutrition Laboratory, Faculty of Agriculture, Kyushu University, Fukuoka, Japan. For each collected soil sample, soil pH H2O (1:2.5 soil: H2O) was measured using a pH meter (HM-10P; DKK-TOA Corp., Tokyo, Japan). Soil samples were also digested using the salicylic acid-sulfuric acid-hydrogen peroxide method [
One gram of each composite soil sample was diluted with 99 ml of sterilized one-half strength modified Hoagland nutrient solution (MHN) in a 200 mL conical flask. The flasks were shaken on a rotary shaker at 120 rpm for one hour to prepare a well-mixed soil suspension. The culture pots (1 L volume) were filled with 1 L of vermiculite and 0.6 L of MHN nutrient solution. The pots were covered with aluminum foil and autoclaved at 121˚C for 20 min. For surface sterilization, the seeds were soaked in a 2.5% sodium hypochlorite solution for 5 min, rinsed five times with 10 ml of 99.5% ethanol and washed five times with sterilized MHN nutrient solution to remove traces of sodium hypochlorite and ethanol. Myanmar Yezin-7 black gram variety, common use in Myanmar was used as trap host for all soil samples.
The culture pots using black gram Yezin-7 variety with eight soil suspensions and control pot were prepared. The seeds were surface-sterilized and planted in the sterilized vermiculite pots. 5 ml aliquot of soil suspension was inoculated per seed. The control was planted without inoculation to assess the possibility of contamination. The plants were cultivated in the incubator (25˚C and 16 hours light) for four weeks. Autoclaved deionized water was poured when the original weight of the pots decreased by around 300 g.
After carefully uprooting, the five nodules (≥2 mm in diameter) were collected per pot. For surface sterilization, the nodules were soaked in 70% ethanol for 3 min, 2.5% sodium hypochlorite (NaClO) solution for 15 min and washed five times with 0.9% autoclaved sodium chloride (NaCl) solution. The surface sterilized nodules were transferred separately into the autoclaved small test tubes and crushed. For every sample, a loopful of the suspension was streaked on Yeast Extract Mannitol Agar (YMA) plates containing 25-µg Congo red [
For DNA extraction, the collected isolated were streaked onto A1E agar plates and incubated at 30˚C for 7 days. A single pure colony of each isolate from A1E plates was cultured in AIE liquid medium at 30˚C for 5 days to obtain the required optimum density (0.4 < OD600 nm < 0.6). Total DNA was extracted using ISOPLANT (Nippon gene, Tokyo, Japan), following instructions from the manufacturer. The DNA concentrations were calculated using NIH Image 1.62 (National Institutes of Health, Bethesda, MD, USA) after agarose gel electrophoresis (0.3% agarose gel in 1 TAE buffer), staining with ethidium bromide (Toyobo, Tokyo, Japan), and destaining in 1 TAE buffer.
The primers 16S-F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 16S-R2 (5’-CGGCTACCTTGTTACGACTT-3’) were used to amplify the 16S rRNA region of mesorhizobia. The PCR reaction consisted of a pre-run at 94˚C for 5 min, denaturation at 94˚C for 30 s, annealing at 60˚C for 30 s, and extension at 72˚C for 1 min [
For homology searches, sequences were compared with the DNA Data Bank of Japan (DDBJ) using the Basic Local Alignment Search Tool (BLAST) program [
The nucleotide sequences of 16S rRNA genes of 40 Bradyrhizobium strains were deposited in the DDBJ under the set of accession numbers LC515811 to LC515850.
Myanmar black gram (Vigna mungo L.) cultivars, Yezin-4 and Yezin-7 were collected from Food Legumes Section, Department of Agricultural Research, Yezin, Myanmar. The black gram Yezin-7 variety was the most widely grown cultivar in Myanmar and used for screening of the 40 indigenous Bradyrhizobium strains. For effectiveness of selected Bradyrhizboium strains, Black gram Yezin-4 and Yezin-7 cultivars from Myanmar were investigated. The purified forty Bradyrhizobium strains were cultured in A1E liquid media [
The purified forty indigenous bradyrhizobial strains were screened for nitrogen fixing effectiveness on Myanmar black gram Yezin-7 in pots with vermiculite and MHN solution were covered with aluminum foil and autoclaved at 121˚C for 20 min. Black gram Yezin-7 seeds were surface sterilized [
The five Bradyrhizobium strains of B. elkanii HpaBG5, B. liaoningense HpaBG6, B. liaoningense HpaBG7, B. liaoningense HpaBG12, B. elkanii LauBG38 (
Isolates | Genus and species | Dendrogram cluster | Shape | Size (mm) |
---|---|---|---|---|
HpaBG1 | Bradyrhizobium elkanii | Be1 | UF | 1.5 |
HpaBG2 | Bradyrhizobium elkanii | Be1 | UF | 1.5 |
HpaBG3 | Bradyrhizobium elkanii | Be1 | UF | 1.5 |
HpaBG4 | Bradyrhizobium sp. | Bs1 | UF | 1.5 |
HpaBG5 | Bradyrhizobium elkanii | Be1 | UF | 1.5 |
ChaBG6 | Bradyrhizobium liaoningense | Bl1 | EP | 1.5 |
ChaBG7 | Bradyrhizobium liaoningense | Bl2 | EP | 1.5 |
ChaBG8 | Bradyrhizobium liaoningense | Bl1 | EP | 1.5 |
ChaBG9 | Bradyrhizobium japonicum | Bo1 | EP | 1.5 |
ChaBG10 | Bradyrhizobium liaoningense | Bl1 | EP | 1.5 |
NyaBG11 | Bradyrhizobium liaoningense | Bl1 | EP | 1.5 |
NyaBG12 | Bradyrhizobium liaoningense | Bl1 | EP | 1.5 |
NyaBG13 | Bradyrhizobium liaoningense | Bl1 | EP | 1.5 |
NyaBG14 | Bradyrhizobium liaoningense | Bl1 | EP | 1.5 |
NyaBG15 | Bradyrhizobium liaoningense | Bl1 | EP | 1.5 |
DanBG16 | Bradyrhizobium liaoningense | Bl2 | EP | 2.0 |
DanBG17 | Bradyrhizobium japonicum | Bo1 | EP | 2.0 |
DanBG18 | Bradyrhizobium japonicum | Bo1 | EP | 2.0 |
DanBG19 | Bradyrhizobium japonicum | Bo1 | EP | 2.0 |
DanBG20 | Bradyrhizobium japonicum | Bo1 | EP | 2.0 |
SalBG21 | Bradyrhizobium sp. | Bs2 | UF | 1.5 |
SalBG22 | Bradyrhizobium yunamingense | By1 | UF | 1.5 |
SalBG23 | Bradyrhizobium sp. | Bs2 | UF | 1.5 |
SalBG24 | Bradyrhizobium sp. | Bs2 | UF | 1.5 |
SalBG25 | Bradyrhizobium sp. | Bs2 | UF | 1.5 |
SitBG26 | Bradyrhizobium liaoningense | Bl2 | EP | 1.5 |
SitBG27 | Bradyrhizobium liaoningense | Bl2 | EP | 1.5 |
SitBG28 | Bradyrhizobium liaoningense | Bl1 | EP | 1.5 |
SitBG29 | Bradyrhizobium japonicum | Bo1 | EP | 1.5 |
SitBG30 | Bradyrhizobium liaoningense | Bl2 | EP | 1.5 |
PyiBG31 | Bradyrhizobium japonicum | Bo1 | EP | 2.0 |
PyiBG32 | Bradyrhizobium japonicum | Bo1 | EP | 2.0 |
PyiBG33 | Bradyrhizobium japonicum | Bo1 | EP | 2.0 |
PyiBG34 | Bradyrhizobium sp. | Bs2 | EP | 2.0 |
PyiBG35 | Bradyrhizobium japonicum | Bo1 | EP | 2.0 |
LauBG36 | Bradyrhizobium elkanii | Be1 | UF | 1.5 |
LauBG37 | Bradyrhizobium elkanii | Be1 | UF | 1.5 |
LauBG38 | Bradyrhizobium elkanii | Be1 | UF | 1.5 |
LauBG39 | Bradyrhizobium liaoningense | Bl2 | UF | 1.5 |
LauBG40 | Bradyrhizobium elkanii | Be1 | UF | 1.5 |
Bl1 = Bradyrhizobium liaoningense cluster 1; Bl2 = Bradyrhizobium liaoningense cluster 2; Bo1 = Bradyrhizobium ottawaense cluster 1; Bs1 = Bradyrhizobium sp. cluster 1; Bs2 = Bradyrhizobium sp. cluster 2; By1 = Bradyrhizobium yunamingense cluster 1; Be1 = Bradyrhizobium elkanii cluster 1; UP = Undulated-pulvinate, EP = Entirely-pulvinate.
potential efficiency on ARA per plant. The experiment was performed in completely randomized design with three replicates. The inoculation and growing condition of pot experiment were also conducted as the above experiment. ARA per plant, nodule, root and shoot dry weight were determined after four weeks. Data were analyzed using the STATISTIX 8 software (Analytical Software, Tallahassee, FL, USA), and treatment means were compared by Tukey’s HSD test (P < 0.05) for the collected parameters.
The forty root nodule bacteria were isolated from eight different soil samples from major black gram growing areas in Myanmar (
Neighbor-joining trees for each gene had similar overall tree topologies. Groups were selected on the basis of the minimum standard changes between named species in the 16S rRNA phylogram (
The seven clusters were identified in the phylogenetic tree including four clusters of B. liaoningense (Bl1 and Bl2), two clusters of B. sp. (Bs1 and Bs2), one cluster of each B. japonicum (Bj1), B. yunamingense (By1) and B. elkanii (Be1) (
Almost all the collected isolates from Nyaunglebin Bago Region, Chaungzon Mon State and Sittwe Rakhine State black gram growing regions were identified as B. liaoningense. At Danubyu Ayeyarwady Region and Pyinmanar Nay Pyi Taw Region, most of the strains were identified as B. japonicum. On the other hand, more or less all the isolates from Launglon Tanintharyi Region and Hpa-an Kayin State were related to B. elkanii. However, all B. sp. strains were found in Salingyi Sagaing Region black gram growing region (
In this study, cluster Bl1 and Bl2 were observed in major black gram growing areas Nyaunglebin Bago Regio, Chaungzon Mon State, Sittwe Rakhine State, Danubyu Ayeyarwady Region and Launglon Tanintharyi Region. In Hpa-an Kayin State, Salingyi Sagaing Region and Pyinmanar Nay Pyi Taw Region, cluster Bs1 and Bs1 were found but in Chaungzon Mon State, Danubyu Ayeyarwady Region, Sittwe Rakhine State and Pyinmanar Nay Pyi Taw Region, cluster Bj1 was distributed. However, By1 cluster was only distributed in Salingyi Sagaing Region and Be1 cluster was scattered in both Hpa-an Kayin State and Launglon Tanintharyi Region of black growing areas of Myanmar (
In the screening experiment, the effective strains were determined their potential ability in the N fixation analyzed by means of ARA per plant. Each bacterial strain that responded on black gram Yezin-7 was expressed in
On the other hand, the B. elkanii LauBG38 showed the highest nodule and shoot dry weights of 9.17 mg plant−1, 0.17 g plant−1 in Yezin-4 and 10.97 mg plant−1, 0.18 g plant−1 in Yezin-7 black gram variety, respectively. In Yezin-4 variety, inoculation with B. elkanii LauBG38 gave significant higher in nodule dry weight than B. liaoningense ChaBG6, B. liaoningense ChaBG7, and B. liaoningense NyaBG12. In Yezin-7 variety, B. elkanii LauBG38 gave the highest nodule dry weight among the tested strains and this strain was significant difference from those given by B. liaoningense ChaBG6 and B. liaoningense NyaBG12 (
The symbiotic association between legumes and rhizobia is one of the most important contributors to the world's supply of biologically fixed nitrogen to agriculture. Effective symbiosis can only be achieved when the nodules are formed
Treatment | Yezin-4 | Yezin-7 | ||||
---|---|---|---|---|---|---|
NDW (mg∙plant−1) | SDW (g∙plant−1) | ARA (µmole C2H4 h−1∙plant−1) | NDW (mg∙plant−1) | SDW (g∙plant−1) | ARA (µmole C2H4 h−1∙plant−1) | |
HpaBG5 | 6.83 ab | 0.15 ab | 0.19 ab | 7.83 ab | 0.17 ab | 0.23 ab |
ChaBG6 | 5.50 b | 0.14 ab | 0.17 ab | 5.93 b | 0.15 ab | 0.15 b |
ChaBG7 | 5.77 b | 0.14 ab | 0.18 ab | 7.30 ab | 0.16 ab | 0.22 ab |
NyaBg12 | 4.63 b | 0.11 b | 0.10 b | 4.80 b | 0.12 b | 0.12 b |
LauBG38 | 9.17 a | 0.17a | 0.36 a | 10.97 a | 0.18 a | 0.41 a |
Means in each column followed by different letters differed significantly at P < 0.05 (Tukey’s test), NDW, SDW means nodule and shoot dry weight per plant and ARA means C2H4 produced per hour per plant, NDW and ARA of non-inoculated treatment is zero for both black gram varieties, SDW of non-inoculated treatment is 0.05 g plant−1 for both back gram varieties.
by effective rhizobia. The symbiotic relationship between rhizobia and black gram has not been extensively analyzed. Therefore, this is the first report investigation of addressing the genetic diversity and evaluation the effectiveness of indigenous Bradyrhizobium strains for Myanmar black gram cultivars.
Sequence analysis of 16S ribosomal RNA (rRNA) has been developed used as one of the most important methods in taxonomy and phylogenic analysis of bacteria [
The genus Bradyrhizobium was proposed by Jordan (1982) [
Previously, Soe et al., 2013 [
The agro-ecological origin of rhizobial inoculants and thus most possibly edaphic and climatic variation are often not considered sufficiently to make inoculation successful. A variety of biotic and abiotic factors, such as host plant, cultivation history, drought, soil pH, salinity, mineral nutrient availability, soil organic carbon content and texture, are known to affect rhizobial diversity and distribution [
A phylogenetic analysis of present study showed that indigenous B. liaoningense as a dominant strain was distributed throughout the five major black gram growing areas of Myanmar with a pH range of 4.69 - 6.70. B. liaoningense strain was isolated from soybean growing areas in China [
Symbiotic N2 fixation can recompense for absent soil nitrogen (N) and thus potentially save costly mineral N fertilizer [
An effective Rhizobium-legume symbiosis largely depends on the presence of a specific and compatible strain in the soil for a particular legume. Several studies have reported a significant increase in green gram and black gram growth parameters and yield due to the inoculation of bradyrhizobial isolates [
In this study, the evaluation the effectiveness of selected Bradyrhizboium strains on two Myanmar black gram varieties, Yezin-4 and Yezin-7 were investigated. It was observed that the inoculation of Bradyrhizobium elkanii LauBG38 gave significantly higher in ARA per plant for nitrogen fixation and nodule dry weight in both black gram varieties. ARA per plant for nitrogen fixation and nodule dry weight of Yezin-7 variety was higher than those of Yeizn-4 black gram. Therefore based on the results of nodulation efficiency of nodule dry weight of the plants Yezin-7 could be used in future experiment as superior host genotypes for high nitrogen fixation. Symbiotic nitrogen fixation depends on interactions among the genotype of the host plant, rhizobial strain genotype and environment. In grain legume species, genotypic variability affected nodule number or mass or nitrogenase activity [
The effectiveness of indigenous Bradyrhizobium strains was observed in Myanmar back gram cultivars using the correct varieties, and proper nodulated bacteria. This is the first report of phylogenetic diversity and evaluation the effectiveness of indigenous Bradyrhizobium strains for Myanmar black gram cultivars. The selected Bradyrhizobium elkanii LauBG38 strain might be considered for rhizobial inoculatns to use as Biofertilizer in Myanmar near future.
This is the first report on describing Bradyrhizobium strains that were isolated from soil samples of major black gram growing areas of Myanmar and the effectiveness of those strains for plant growth and nitrogen fixation of Myanmar Black gram varieties. In total, 40 indigenous bradyrhizobia were successfully isolated and their geographic distribution was determined based on the analysis of the 16S rRNA region. Our results indicated that B. liaoningense strain was widely distributed in the major black gram growing regions of Myanmar whereas B. japonicum was found more abundant in Danubyu Ayeyarwady Region and Pyinmanar Nay Pyi Taw Region. However, B. elkanii weas found more abundant in Launglon Tanintharyi Region and Hpa-an Kayin State. The effectiveness of those strains for plant growth and nitrogen fixation of Myanmar black gram varieties was investigated in the present study. The 40 Bradyrhizobium strains were screened for their effectiveness on Yezin-7 black gram variety and five Bradyrhizobium strains were selected. These selected strains were tested for their effectiveness on Yezin-4 and Yezin-7 black gram varieties. The Bradyrhizobium elkanii LauBG38 was significantly superior in both black gram varieties. All of these experiments were conducted under the control conditions by growing the plants in the sterilized vermiculite with MHN solution. So, the bradyrhizobial strains selected in the control room trials then must be evaluated in the field. Although it is a preliminary study, it can help for the future study in the inoculants production. The further investigation of symbiotic effectiveness of selected indigenous bradyrhizobia strains on Myanmar black gram cultivars will be examined in the field condition. We do hope that Myanmar Bradyrhizobium strains will be able to use as Biofertilizer for black gram cultivars that enhance crop production through nitrogen fixation and yield.
The authors are thankful to Japan Society for the Promotion of Science (JP19F18079) for their financial support of the present study. We are also very grateful to the members of Land Use Division, Department of Agriculture and members of Department of Agricultural Research, Ministry of Agriculture, Livestock and Irrigation, Myanmar who helped for collecting soil samples and black gram seeds for these experiments.
The authors declare no conflicts of interest regarding the publication of this paper.
Soe, K.M., Htwe, A.Z., Moe, K., Abiko, T. and Yamakawa, T. (2020) Phylogenetic Diversity and Evaluation the Effectiveness of Indigenous Bradyrhizobium Strains for Myanmar Black Gram (Vigna mungo L. Hepper) Cultivars. American Journal of Plant Sciences, 11, 285-306. https://doi.org/10.4236/ajps.2020.112022