Phylogenetic Diversity and Evaluation the Effectiveness of Indigenous Bradyrhizobium Strains for Myanmar Black Gram ( Vigna mungo L. Hepper) Cultivars

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 areas of Myanmar for plant growth and nitrogen fixation was 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.


Introduction
Grain legumes play an important nutritional role in the diet of millions of people in the developing countries [1]. Black gram (Vigna mungo L. Hepper) is a short duration crop that belongs to grain legumes family and rich in protein [2]. It is an important legume crop in Asia [3]. In Myanmar, it is one of the major exportable crops and it is the second largest cultivated Legume crop as well [4].
During 2017-2018, black gram growing areas in Myanmar was about 9.77 million hectares with a production of 1.37 metric tons, and the average yield was 1.41 metric ton•ha −1 [5].
Nitrogen (N) fixation through legume-Rhizobium symbiosis is important for enhancing agricultural productivity and is therefore of great economic interest [6]. Leguminous crops have a reputation for maintaining soil fertility since it can assimilate nitrogen from the atmosphere through symbiotic biological N 2 fixation (BNF) with Rhizobia [7]. Biological nitrogen fixation (BNF) is an important component of sustainable agriculture [8], and rhizobial inoculants have been applied frequently as biofertilizers. 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 [9]. However, the effectiveness of symbiotic N 2 fixation may be an important factor to take into consideration through successful management of symbiosis between black gram cultivars and effective bradyrhizobial strains.
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 [10]. To improve high quality inoculant production, the evaluation of highly effective rhizobial strains for specific legume is one of the principle obligations [11] [12]. Indigenous rhizobial strains also play an important role since they have adapted to local environmen-tal conditions. Therefore, the investigation of effective indigenous rhizobial strains should be considered for current inoculant production in Myanmar.
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 [13].
Several studies also reported significant increase growth parameters and yield due to the inoculation of rhizobial isolates in chickpea [14] in mung bean and in

Analysis of Collected Soil Samples
The collected soil samples were analyzed at Plant Nutrition Laboratory, Faculty of Agriculture, Kyushu University, Fukuoka, Japan. For each collected soil sample, soil pH H 2 O (1:2.5 soil: H 2 O) 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 [21]; then, total N was examined using the indophenol method [22], and total P was tested using the ascorbic acid method [23]. Total K was analyzed using an atomic absorption spectrophotometer (Z-5300; Hitachi, Tokyo, Japan) afterward samples were digested.
To analyze mineralizable N, we firstly used the soil incubation method [24] followed by the indophenol method [22].

DNA Extraction, PCR Analysis and Phylogenetic Analysis
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 < OD 600 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 [26]. The cycle was repeated for 33 cycles, followed by a final extension at 72˚C for 10 min. PCR products were purified using the Wizard Gel and PCR Clean-up System (Promega, Madison, WI, USA). Purified PCR products (≥50 ng•µL −1 ) were subjected to direct sequencing by Macrogen (Tokyo, Japan), using the primer set described above. Raw sequence results were edited using MEGA version 6-software [27] to create 16S sequence fragments.
For homology searches, sequences were compared with the DNA Data Bank of Japan (DDBJ) using the Basic Local Alignment Search Tool (BLAST) program [28]. To construct the phylogenetic tree, sequences of type strains and closely related strains of Bradyrhizobium genospecies were retrieved from the BLAST database. All selected sequences including type strains and closet strains were aligned using the CLUSTALW function of the MEGA version 6-software [27]. After alignment, a phylogenetic tree was constructed according to the neighbor-joining method [29]. The phylogenetic tree was bootstrapped with 1000 replications of each sequence to evaluate the tree topology for reliability. Genetic distances were calculated using the Kimura two-parameter model [30].

Nucleotide Sequence Accession Numbers
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.

Screening of Effective Bradyrhizobial Strains by Myanmar Black Gram Yezin-4 for Nitrogen Fixation
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 [33] and germinated on sterile petri dishes with filter paper. The germinated seeds were sown into the sterilized pot with vermiculite. In the non-inoculated treatment, a control treatment was also provided. The weights of original pots were measured.
The plants were cultivated in incubator (25˚C and 70% relative humidity). During the growing period, sterilized water was irrigated. The plants in each pot were uprooted and carefully washed with water so as not to detach the nodules.
The acetylene reduction assay (ARA) was performed according to Haider et al., 1991 [34] to measure nitrogenase activity. The black gram plants were cut at the cotyledonary nodes. Then, the black gram roots with intact nodules were placed in a 100 mL conical flask and sealed with a serum stopper. A 12 mL aliquot of acetylene (C 2 H 2 ) gas was injected into the flask to replace the air with acetylene.
The flasks containing roots with intact nodules were incubated at room temperature and 1.0 mL subsamples were analyzed at 5 and 65 min, respectively. The ARA value, in terms of ethylene (C 2 H 4 ) production per plant, was measured using a flame ionization gas chromatograph (GC-14A, Shimadzu, Kyoto, Japan) equipped with a stainless steel column (3 mm diameter, 0.5 m length). The column was filled with Porapak R 60 -80 mesh (Nicalai Tesque, Inc., Kyoto Japan).
Column, injection and detection temperatures were 35˚C, 45˚C and 170˚C, respectively. N 2 gas was used as the carrier gas at a flow rate of 45 mL•min −1 . The number of nodules was counted after the assay. Shoots, roots and nodules were collected separately and oven dried for at 70˚C for 48 hours to record the dry weight determination.

Evaluation the Effectiveness of Selected Bradyrhizboium Strains on Two Myanmar Black Gram Cultivars: Yezin-4 and Yezin-7
The five Bradyrhizobium strains of B. elkanii HpaBG5, B. liaoningense HpaBG6, B. liaoningense HpaBG7, B. liaoningense HpaBG12, B. elkanii LauBG38 (Table   3) were selected based on the results of the above screening experiment in their K. M. Soe et al. 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.

Diversity of Indigenous Bradyrhizobium Strains for Myanmar Black Gram (Vigna mungo L.) Cultivars
The forty root nodule bacteria were isolated from eight different soil samples from major black gram growing areas in Myanmar ( Table 1, Table 2 and Figure   1). According to Somasegaran and Hoben, 1994 [25], these strains were proved as pure Bradyrhizobium strains. In YMA plates, the bradyrhizobial colonies reached 1 -3 mm diameter with undulated pulvinate and entirely pulvinate shapes after 5 -7 days incubation (Table 3).
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 (Figure 2), and all groups were well supported in neighbor-joining analyses which had less than 50% bootstrap support in the neighbor-joining tree. The results of the phylogenetic analysis based on the 16S rRNA sequence, indicated that all the 40 isolates belonged to the genus Bradyrhizobium (Table 3 and Figure 2).    Tanintharyi Region of black growing areas of Myanmar (Figure 2).

Screening of Effective Bacterial Strains by Yezin-7 for Nitrogen Fixation
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 Figure 3.    (Table 4). However, shoot dry weight of tested B. elkanii LauBG38 was only significant difference than B. liaoningense NyaBG12 in both Myanmar black gram varieties (Table 4).

Discussion
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  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 [35] [36] [37] [38]. The phylogenetic investigates based on 16S rRNA gene sequences showed that the rhizobial strains nodulating black gram in India were dispersed under the genus Bradyrhizobium [39]. These results were dependable with former reports that showed black gram rhizobia were more closely associated with Bradyrhizobium species [39] [40] [41]. The present study was focused on investigation of 16S rRNA region of 40 Bradyrhizobial strains that were successfully isolated from the different soil samples of major black gram growing areas in Myanmar and proved as pure Bradyrhizobium strains [25].
The genus Bradyrhizobium was proposed by Jordan (1982) [42] for the slowgrowing root nodule bacteria on yeast extract-mannitol agar (YMA) medium.
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 [44] and in Myanmar [53]. Symbiotic N 2 fixation can recompense for absent soil nitrogen (N) and thus potentially save costly mineral N fertilizer [65]. Rhizobial inocula for inoculating legumes increasingly account for differences in symbiotic specificity and effectivity, two parameters that are often correlated [66]. Several studies also reported significant increase growth parameters and yield due to the inoculation of rhizobial isolates in chickpea in Myanmar [14], green gram and black gram in Thailand [67]. In addition, indigenous rhizobial strains also play important role since they have adapted to local environmental conditions.
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 [68]. environment. In grain legume species, genotypic variability affected nodule number or mass or nitrogenase activity [70] pointed out that, through the use of plant genotypes in symbiotic ability, it is possible to identify genes responsible for a particular part of the process, depending on a particular rhizobial strain used. The selection of effective rhizobial strains for cultivated legumes is a critical step in the production of high quality legume inoculant.
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.

Conclusion
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 effec- 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.
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.