Genetic Characterization and Diversity of Rhizobia Isolated from Root Nodules of Green Gram (Vigna radiata L.) found in Central Plateau of India

In agri-ecosystem, microorganisms play an important role, especially the group of rhizobacteria which are associated for promotion of plant growth and development. Rhizobia play a significant role in agricultural ecosystem through symbiotic association with a wide range of leguminous plant (Orrell and Bennet, 2013) and are commercially exploited. Several rhizobial strains have been identified for various objectives viz., to enhance the mineral uptake, production of phytohormones, nitrogen fixation, detoxification of metals and neutralizing biotic and abiotic stress, producing volatile organic compounds (VOCs) and enzymes to prevent diseases Koskey et al., (2018).
 
Legume-rhizobia association is a host specific symbiosis hence the need to identify the strains and the diversity of rhizobia associated with specific type of legume are essential for better bio-prospecting of the associated benefits Batista et al., (2015). A wide range of variation is present among the distribution of rhizobia and greatly affected by geographical locality, edaphic factors and environmental variation with time and space Martiny et al., (2006), Cao et al., (2014). Diverse heterogenous group of rhizobia consist of  alpha and beta groups and have different geographical distributions. Currently, there are 40 recognized rhizobia species, within 7 genera, which belong to the class ‘Alphaproteobacteria’ (AlloRhizobium, AzoRhizobium, BradyRhizobium, MesoRhizobium, Methylobacterium, Rhizobium and SinoRhizobium) and two to the class ‘Betaproteobacteria’ (Burkholderia and Ralstonia) Kosaky et al., (2018).
 
Recently agriculture trend has become the use of sustainable farming practiced that are environmentally safe, eco-friendly, efficient, cost effective and reachable to farmers field. Demand of rhizobia as a bio-fertilizer in tropical and sub-tropical part of India has increased due to beneficial effect associated for crop yield as well as soil fertility Barman et al., (2017). However lack of awareness, unavailability of  appropriate and efficient strains, erroneous government policies, lack of quality assurance, absence of supportive infrastructure and limited research the diverse and elite rhizobia strain associated with the plants is the constraints of the utilization of bio-fertilizers in green gram production Mahdi et al., (2010). Greengram (Vigna radiata L.) is a popular legume crop cultivated in the country and well fit into many cropping systems, including rice and sugarcane under both rainfed and irrigated conditions. In India, very limited work was conducted on rhizobia diversity associated with green gram in central plateau and hills with constricting environmental conditions, hence based on our present need we proposed this study. The results on distribution and molecular variation among the native rhizobia isolates will be used for selection of novel rhizobia strains for green gram crop in the country as well as and development of bio-fertilizer technology.  

Now a days, molecular techniques have been developed to aid the traditional phenotypic and morpho-cultural techniques in distinguishing the different microbialgenera, species and strains (Koskey et al., 2018). In addition to determine the genetic diversity of native rhizobia the 16S rRNA which are highly conserved, have been sequenced to determine the taxonomic position of different rhizobia strains (Rahmani et al., 2011). 16S rRNA primer is used for phylogenetic taxonomy and identification of bacterial species. Therefore, the present study aimed at isolation and morpho-molecular characterization of rhizobia from root nodule of green gram cultivated in central part of India.

Field trapping of rhizobia
 
The present study was performed at Biotechnology Centre, Jawaharlal Nehru Krishi Vishwa Vidhalya, Jabalpur, India. Field trapping of rhizobia was carried out during January to August 2018 from various locations of central India with contrasting agro climatic conditions. The rhizobia trapping were done from central part of India (Table 1). Sampling was done randomly by carefully uprooting five plants from middle of the field with the help of a sterile spade and trowel along with a soil block of  ~20 cm  diameter up to a depth of about 20 cm. The clump of soil and root were uplifted carefully, placed in sterile aluminum foil and keep in refrigerator at 4°C and next day used it the isolation of the Rhizobia. Further nodule were detached from the root and kept in onkrage tube and extraction was done.
 

 
Isolation of rhizobia from root nodules
 
Healthy and pink nodules were selected for the isolation of rhizobia using standard protocol described by Somasegaran and Hoben (1994). A total 40 rhizobia were obtained and maintained on screw-capped YEMA slant bottles and preserved at 4°C.
 
Morphological characterization
 
Rhizobia isolates were examined for morphological characterization such as colony morphology, cell shape, border, muscocity, transparency, colour and gram staining using standard microbiological techniques described by Somasegaran and Hoben (1994).
 
Biochemical characterization
 
Biochemical test was done for all forty rhizobia isolates with standard techniques and its includes growth on glucose peptone agar, growth on YEMA with bromothymol blue, IAA production test, phosphate solubilisation test, ammonia production test, carbohydrate fermentation test, citrate utilization test, catalase test.
 
Gram staining
 
Gram reaction was carried out for bacterial isolates to classify them in two groups i.e., gram positive and gram negative as protocol given by Vincent, (1970). Young 2-3 days old pure cultures of YEMB were smeared on slides. The wet smear was air dried heat fixed and then gram stained. Prepared slides were observed under oil immersion on a compound microscope (Leica Microsystems).
 
Growth on glucose peptone agar
 
Growth on glucose peptone agar was done according to the protocol given by Kleczkowska et al., (1968). Isolated rhizobia were cultured in peptone glucose agar medium and plates were incubated at 28±2°C for 72-96 hours.
 
Growth on YEMA with bromo thymol blue
 
The production of acid or alkali was determined in YEMA medium containing bromo thymol blue according to the protocol given by Vincent, (1970). One ml of 1 per cent Bromothymol blue (BTB) was prepared and added to 10 ml of YEMA medium and poured in sterile petri plates then allowed to solidify. A loopful of Rhizobia isolates were inoculated on the media plates and incubated at 28±2°C for 7 days in dark. The isolates that changed the green colour of YEMA-BTB medium to blue were considered as alkaline producers and slow growers.
 
IAA production test
 
IAA production by the isolates was studied by the method of Hartmann et al., (1983).
 
Phosphates solubilisation test
 
Phosphate solubilisation activity was done by prepared plated with Pikovskaya’s media and isolates were spot inoculated on the centre of agar plate aseptically. All the plates were incubated at 28 ± 2°C for 5-6 days. A clear zone around a growing colony indicated phosphate solubilisation (Pikovaskya, 1948).
 
Ammonia production test
 
Ammonia production of different isolates was done by methods of Laslo et al., (2012).
 
Carbohydrate fermentation and citrate utilization test
 
Both the tests were performed by methods of manual (Aneja, 2003), (APHA, 1998) and Bergey’s manual (1964). While catalase test was performed to study the presence of catalase enzyme in bacterial colonies. Pure isolates (24 h old) were taken on glass slides and one drop of H₂O₂ (30 per cent) was added. Appearance of gas bubble indicated the presence of catalase enzyme (Rangaswami and Bagyaraj, 1993). The effect of temperature was performed in terms of growth. Rhizobia were grown in YEMA medium on petriplates and incubated at three different temperatures viz., 28°C, 37°C and 48°C for 3 days and the growth was noted.
 
Genotype characterization
 
Molecular study was carried out selected twenty rhizobia isolates. The analysis of genetic relatedness of the native rhizobia isolates from Central Plateau of India was carried out using sequence analysis of amplified 16s rDNA genes
 
Genomic DNA isolation
 
Total genomic DNA extraction of twenty rhizobia isolates was carried with minor modification of a method outlined by Wilson (2001). PCR amplification was carried out in 20 μl reaction with 12.6 μl nuclease free water, 2 μl buffer (Fermentas), 0.4 μl dNTP (10mm), 1 μl of each 27F and 1492F primers, 1.6 μl MgCl₂ (10mm), 0.4 μl Taq DNA Polymerase (Fermentas) and 2 μl of DNA template. 16S rRNA primers 27F-5’-AGAGTTTGATCCTGGCTCAG-3’ and 1492R-5’ TACCTTGTTTTACGACTT-3’ sequenced were used.
 
DNA sequencing using 27F and 1492R forward and reverse primers
 
PCR amplicon of the 20 selected rhizobia isolates were sent for sequencing to Agri genome labs Pvt Ltd. Kochi, Kerala, India. Sequence results obtained from the sequencing lab were used to assess the genetic relatedness. Sequences were blast at National Centre on Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast) for similarity search. Phylogenetic relationship analysis was performed using neighbor joining method with 1000 bootstrap value using DARwin software.
 

Isolation and morpho-cultural characteristics of isolates
 
A total of 40 bacterial isolates were isolated from various agro-climatic zones of Madhya Pradesh. The colony colour varied with creamy, white, milky, yellow, watery, mucoid with round shaped, colonies being observed with firm gummy or smooth muscoid texture to the margin of the colonies (Plate 1). These morphological characteristics approaches were closer to the genus Rhizobium as described by Jordan and Allen (1974). Thirty six isolates were found as rod shaped bacteria and considered as gram negative bacteria whereas four isolates viz., JRB 118 JRB 120, JRB 130 and JRB 144 were gram positive bacteria (Table 2). Similarly, all the isolates were grown on YEMA with congored and incubated in dark for 48-72 hours and observed that 35 isolates did not absorb red colour and considered as gram negative Rhizobium while JRB 111, JRB 118, JRB 130, JRB 134 and JRB 144 absorb red colour on YEMA plate and considered as gram positive  (Plate 1) (Table 2). Previously, Shetta et al., (2011), Gauri et al., (2012), Kang et al., (2013) and Hamza and Albejo, (2017) identified Rhizobium isolates on YEMA media with congored dye. Notably 32 isolates showed very weak growth on glucose peptone agar media and whereas, eight isolates, JRB 111, JRB118, JRB 120, JRB126, JRB 130, JRB 142, JRB 144 and JRB 146 showned strong growth and concluded as they did not belong to Rhizobium species. Previously, Tyagi et al., (2017) and Timmusk et al., (2017) also worked on green gram rhizobia and reported that all the rhizobial isolates showed positive results.
 

 

 
All the isolates were turned BTB indicator from deep green to yellow when grown on YEMA-BTB. Twenty five isolates were shown yellow colour and indicate acidic in nature and the 15 isolates were showed green color which resulted alkaline in nature. Based on the results obtained in regard to extent and growth period on YEMA with Bromothymol blue the bacterial isolates were placed into two broad groups.
 
a) Isolates which showned growth within 3-5 days and the color change on YEMA-BTB from blue to yellow suggested that all isolates were fast growers and fall under the genus Rhizobium species Mamun et al., (2013).

b) Isolates which were showed slow growth after one week and the color change on YEMA-BTB from blue to green were regarded as slow growers i.e., BradyRhizobium species. Previously Sharma et al., (2011) and Hamza and Alebejo, (2017) also defined the nature of bacterial species from different sources.
 
Bio-chemical characterization
 
Fourty isolates were tested for IAA assay in the presence of L- tryptophan in the medium and 38 isolates were shown pink colour in the medium and identifies IAA producer strains, while JRB 111 and JRB 118 had shown negative results in the medium (Table 3). Our results are supported by the work of Elazanty et al., (2015) in Vicia faba and Vigna mungo. For phosphate solubilization all the 40 strains were grown on pikovskaya’s medium and shown an evident of different size of halo zone (Table 2). Our results are in close agreement with the findings of Sridevi et al., (2007) in crotalaria and Mondal and Gena (2020) in cluster bean. All the rhizobia isolates were tested and were found as ammonia excretors. Thirty seven isolates were moderate ammonia excretors as they showed deep yellow colour to brownish colour  whereas JRB 111 and JRB 118 isolates does not produced any colour and concluded as non ammonia excretors (Table 3). Similar to our work, Manasa et al., (2017) revealed in his studies 15 rhizobial isolates for positive for ammonia production.
 

 
Various carbon sources like, phenol red glucose, phenol red lactose and phenol red sucrose broth etc. were used by the bacteria. In our study out 40 Rhizobia, 34 isolates were shown carbohydrate degradation activity by changing the broth colour from red to yellow colour and gas formation in durum tubes while six isolates JRB 111, JRB 118, JRB 120, JRB 130, JRB 144 and JRB 146 showed negative results (Table 3). Earlier, Kucuk et al., (2006) and Wadhwa et al., (2017) were also found positive for utilization of glucose as carbon source among studied Rhizobium strains. In citrate utilization test, only 10 Rhizobia isolates viz., JRB 115, JRB 117, JRB 119, JRB 120, JRB 123, JRB 125, JRB 126, JRB 144, JRB 147 and JRB 148 were shown citrate utilizing activity in the medium by changed colour from green to blue while remaining 30 Rhizobia isolates remained as green in colour (Table 3). Earlier, Hamza and Alebejo, (2017); Deshwal et al., (2014) and Girija et al., (2020) were isolated rhizobia and categorized Rhizobium isolates for citrate utilization activity. Catalase test was performed in 40 strains and all strains formed a bubble on glass slide by adding 2-3 drops of hydrogen peroxide on freshly grown cultures (Table 3). Our results are in accordance with earlier findings of Gachande and Khansole, (2011) and Naz et al., (2009) found positive rhizobia isolates for catalase test. Further, all the isolates were evaluated for  tolerance to three temperature regimes viz., 28°C, 37°C and 48°C. At 28°C all isolates shows excellent growth where as 37°C moderate growth was observed.  At 48°C, thirty seven isolates were shown poor growth and five isolates viz., JRB112, JRB121, JRB131, JRB143 and JRB149 were shown good growth and this resulted that the isolates can survive at extreme environmental conditions. Results of Naz et al., (2009) and Ruiz-Diez et al., (2012) were supported our findings.

PCR amplification of 16S rRNA region
 
PCR amplification was performed for 20 diverse selected rhizobia isolates by using 27F and 1492R primers (Biorad Thermal Cycler). A single sharp band of ~1500 bp was amplified and visualized in 1.2 per cent agarose gel (Plate 2). As 16S rRNA region is much conserved and no diversity was seen and amplicon size corresponds to the expected size earlier reported by Naz et al., (2009),  Rajendran and Gunasekaran, (2011) and Tyagi et al., (2017).
 

 
Sequencing of 16S rRNA region using 27F forward 1492R reverse primer
 
PCR amplified product 20 µl with 16S rRNA primer was send for sequencing to Agri genome Pvt, Ltd, Kochi, Kerala, India. The identity of 16S rRNA sequences was performed a similarity search in the gene bank database of NCBI (http://www.ncbi.nih.gov.BLAST). The sequencing results were matched as positive that all the strains were shown similarity to Rhizobia sp and identified as Rhizobium and BradyRhizobium spp. Similar results were observed by Kang et al., (2013) and Gilbert et al., (2018).
 
Phylogenetic analysis
 
Phylogenetic relationship analysis was performed using neighbor joining method with 1000 bootstrap value by DARwin method phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Phylogenetic analysis revealed that rhizobia under study were divided into two groups (Fig 1). Major group ‘A’ and minor group ‘B’. Major group ‘A’ is further divided into two subgroups ‘a1’ and ‘a2’. Subgroup ‘a1’ have five species of bacteria viz., BradyRhizobium japonicum, BradyRhizobium japonicum, BradyRhizobium japonicum, BradyRhizobium sp, BradyRhizobium sp. Subgroup ‘a2’ have seven bacteria spp viz., BradyRhizobium sp, BradyRhizobium liaoningense, BradyRhizobium liaoningense, BradyRhizobium elkanii, BradyRhizobium elkanii, BradyRhizobium yuanmingense, and BradyRhizobium yuanmingense.  Minor group ‘B’ divided into two subgroups ‘b1’ and ‘b2’. Subgroup ‘b1’ contained five bacteria spp viz., Rhizobium etli, Rhizobium etli, Rhizobium indigoferae, Rhizobium yanglingense, Rhizobium yanglingense and subgroup ‘b’2 contained three bacteria sp viz., Rhizobium gallicum, Rhizobium gallicum and Rhizobium leguminosarum (Fig 1).  The maximum distance was observed between JRB-119 and JRB-129 and it is due to huge variation among the sampling site viz., Bharghat, Seoni and Ratlam district and they fall in different agroclimatic zones of the state. Similarly, the variation in other strains is due to broad area of soil sampling with huge variation of agro-ecological practices. Previously, Nahar et al., (2012) revealed that the rhizobia isolates of genetic relatedness was assessed by comparing the sequences of 16S rRNA and two distinct clusters were seen in the dendrogram constructed by the complete linkage method.
 

In the present investigation 40 bacterial isolated were isolated from root nodule of green gram growing region of Madhya Pradesh. Isolated species are fast and slow growers and belong to genus Rhizobium and BradyRhizobium. Morphological characterization results were shown resemblance to Rhizobial strains due to failure in absorbance of Congored on YEMA medium, weak growth on glucose peptone agar media. Biochemical results were shown positive results in IAA production, phosphate solubilization, ammonia production, catalase test, citrate utilization test, carbohydrate degradation test and five isolates were shown good survival at extreme temperature conditions i.e., 48°C.  Further 20 diverse Rhizobial strains were selected for PCR amplification using 27F forward and 1492R reverse primers and an amplicon of ~1500bp were observed and sequenced. DNA sequencing results revealed that all the 20 isolates were belonging to genera Rhizobium and BradyRhizobium spp. 

RSS and MS conceived and designed the research and sample collection and participated in drafting the manuscript. SM, RSR and AKS participated in data analyses and wrote the manuscript. All authors read and approved the final manuscript.