Diversity of Indigenous Symbiotic Nitrogen Fixing Bacteria from Blackgram [Vigna mungo (L.) Hepper] Cultivated in Rice Fallows

Legume nodulating bacteria (LNB), also known as rhizobia produce root nodules on leguminous plants where in nitrogen fixation takes place. In agriculture, this symbiotic relationship is significant as it provides nitrogenous fertilizers to the leguminous crops. Traditionally rhizobia were exclusively members of the Rhizobiaceae family in the Alphaproteobacteria class of bacteria, which includes the genera Rhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Allorhizobium and Sinorhizobium (Sprent, 2001; Sawada et al., 2003).  
        
Further reports revealed that large number of diazotrophs have the ability to nodulate and fix atmospheric N₂ in legumes in addition to rhizobia (Willems, 2006). These include Methylobacterium and Devosia belong to Alphaproteobacteria as well as Burkholderia and Ralstonia of the Betaproteobacteria class. Chen et al., (2005) isolated several species of Betaproteobacteria from the root nodules of leguminous plants. Strains of Burkholderia have been isolated from legumes such as Mimosa sp., Macroptilium atropurpureum and Cyclopia sp (Pandey et al., 2005: Elliott et al., 2007a and b). 
        
In addition to Alphaproteobacteria and Betaproteobacteria bacteria belong to class Gammaproteobacteria are also reported to nodulate legumes (Benhizia et al., 2004; Kan et al., 2007: Shiraishi et al., 2010).
        
However, relatively less information is available on the genetic diversity of indigenous rhizobia nodulating blackgram cultivated in rice fallows.  Hence the present study was carried out to know the diversity of symbiotic native nitrogen fixing bacteria isolated from the root nodules of Blackgram grown in rice fallows using partial 16S rRNA sequencing.

Study site
 
This study was conducted at the Acharya Nagarjuna University, Guntur, Andhra Pradesh, India in 2015.
 
Isolation of native rhizobia from blackgram root nodules
 
In the present study, the nodulated roots of mature Vigna mungo plants cultivated in rice fallows were collected. Rhizobium strains were isolated from freshly collected healthy root nodules on yeast extract mannitol agar (YEMA) medium with 0.1% Congo red. The pure cultures of all isolates were maintained on YEMA slants and preserved at 4°C (Vincent, 1970). The identity of the strains was confirmed by tests such as Gram staining, growth on culture media such as YEMA with Congo red (Vincent, 1970, 1982),  Hofer’s alkaline broth and  glucose peptone agar (Vincent, 1970), acid production, ketolactose test (Bernaertz and Deley, 1963) and nodulating ability on homologous hosts (Somasegaran and Hoben, 1985).
 
Morphological observation
 
The bacterial strains inoculated on YEMA plates were incubated for 48 h at room temperature. The individual colonies were characterized based on their shape, colour, texture, elevation, margin and mucilage production by using standard microbiological methods as described by Vincent (1970; 1982) and Somasegaran and Hoben (1985).
 
PCR amplification and partial sequencing of 16S rRNA gene
 
The amplification of PCR and sequencing of 16S rRNA gene of the four isolates VM-2, VM-8, VM-9 and VM-15 was done by using commercial service of Macrogen Inc. Korea.
 
Phylogenetic analysis of bacterial strains
 
The gene sequences of VM-2, VM-8, VM-9 and VM-15 were submitted to BLAST for comparison with Gen Bank sequences (National Center for Biotechnology Information, 2010) employing the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/GenBank/HYPERLINK “http://www.ncbi.nlm.nih.gov/GenBank/).Only”). For the phylogenetic analysis, Gene Sequences greater than 600 bp in length were used.  Sequence alignment was performed with Clustal W and the phylogenetic tree was constructed using the Molecular Evolutionary Genetics Analysis (MEGA) software version 5 (Tamura et al., 2011). A bootstrap confidence analysis was performed with 1,000 repetitions.

Isolation of indigenous Rhizobium strains
 
Indigenous Rhizobium strains were isolated from effective root nodules of blackgram cultivated in rice fallows on YEMA medium with Congo red. All the strains were negative for the production of 3-ketolactose from lactose. The characteristics of rhizobia isolated from blackgram root nodules were in conformity with the earlier reports for Rhizobium (Vincent, 1970; Somasegaran and Hoben, 1985).  The similar type of reports was also made by Kalita et al., (2006) in blackgram, greengram and pigeonpea, Saeki et al., (2005); Sharma Mahaveer et al., (2010) in soybean, Solaiman et al., (2010) in chick pea and in horse gram by Mishra et al., (2017).
        
Among the four rhizobial strains isolated, three strains (VM-2, VM-8 and VM-9) were identified as slow growers and alkali producers and VM-15 as fast grower and acid producer.  Fast-growing rhizobial strains turned the medium (YEM-BTB medium) into yellow (acid) and slow-growing rhizobial strains turned the medium into blue (alkali). Similar type of results were observed in indigenous Rhizobium isolated from faba bean and chick pea (Alemayehu, 2009; and Solaiman et al., 2010). Nodulation tests confirmed that all the isolates produced effective nodules on blackgram roots in 15-20 days after inoculation. 
 
Morphological observation
 
Colony characters of the isolates were studied by growing them on standard YEMA (Fig 1). The colony characters were recorded after 48 h of incubation. The Rhizobium colonies on Congo red medium appeared as white creamy white with low to medium mucilage, round, transparent, smooth in texture and convex with entire margin (Table 1). These characters are in agreement with colony characters of Rhizobium species reported by Kreig and Holt (1984) and Holt et al., (1994).
 

 

          
Phylogenetic analysis of the four representative isolates
 
The phylogenetic analysis of the four gene sequences of 16S r RNA of VM-2, VM-8, VM-9 and VM-15 was blasted against nucleotide database of the NCBI and the sequences were aligned with the set of published sequences on the basis of the conserved primary sequence and also by nucleotide BLAST similarity search analysis. Based on the 16S rRNA gene sequences, the strain VM-2 showed a close relation with Rhiobium sp., strain, VM-8 and VM-9 with Bradyrhizobium sp. and VM-15 with Achromobacter sp. (Fig 2-5). The 16S rRNA sequences were deposited in NCBI with the accession numbers KJ 704783 (VM-2), KJ 704784 (VM-8), KJ 704785 (VM-9) and KJ 501696 (VM-15).
 

 

 

 

        
The above results clearly indicate that the strains belong to Rhizobiaceae (VM-2), Bradyrhizobiaceae (VM-8, VM-9) and Alcaligenaceae (VM-15) families which are phylogenetically distinct.
        
The phylogenetic tree was constructed using the maximum parsimony method.
        
Less information is available on the genetic diversity of native rhizobia nodulating green gram and blackgram. Bradyrhizobia nodulating green gram in the subtropical region of china are genetically diverse from bradyrhizobia of cowpea (Zhang et al., 2007).
        
In addition to Bradyrhizobium, unidentified slow growers were also isolated from blackgram cultivated in south India (Saleena et al., 2001), while few strains isolated from mungbean and blackgram in Thailand were closely related to Bradyrhizobium japonicum (Yokoyama et al., 2006).
        
The studies of Chinnaswamy et al., (2009) revealed that Blackgram, Greengram and Cowpea cultivated in various agro-climatic regions of India are nodulated by Bradyrhizobium yuanmingense.
        
From the studies of Ademir Sergio et al., (2015) it was reported that legume Phaseolus lunatus (Lima bean) in Brazil are nodulated by Rhizobium etli/Rhizobium phaseoli.

In recent years, Brevundimonas, Devosia, Methylobacterium, Ochrobactrum and phyllobacterium  which do not belong to Rhizobiaceae have been reported to produce nodules and fix atmospheric nitrogen (Chen et al., 2005; Willems, 2006: Sprent, 2008).
        
Burkholderia, Achromobacter, Cupriavidus and Ralstonia which belong to Betaproteobacteria are able to form nodules and fix atmospheric nitrogen (Willems, 2006). The ability of these Betaproteobacteria to form nodules and fix atmospheric N₂ in host plant is further confirmed by the existence of nod genes and nif genes similar to rhizobia belongs to alphaproteobacteria located on a symbiotic plasmid (Chen et al., 2003b, 2005).  
        
Nodulation by Achromobacter in different legumes such as Prosopis juliflora (Benata et al., 2008), Astragalus villossimus (Shakirov and Khakimov, 2010), Mucuna bracteata (Salwani et al., 2012), cowpea (Guimaraes et al., 2012) and in soybean (Wedhastri et al., 2013) was reported earlier. In the present investigation Achromobacter sp. (VM-15) was reported for the first time to nodulate blackgram.

From the partial 16s rRNA sequence analysis, the diazotrophic microsymbionts isolated from the root nodules of blackgram exhibited high genetic diversity. This suggests that the diversity of bacteria that can nodulate legume blackgram is high and includes Rhizobium, Bradyrhizobium and Achromobacter.

The authors are grateful for the financial support granted by the University Grants Commission (File No 39-398/2010 SR). The authors also thank Dr. E. Prabhavathi for technical support.