Isolation of rhizobium from moth bean root nodules
The research was carried out at Oilseeds Research Station, Tindivanam, Villupuram district, Tamil Nadu, India during 2018 to 2020. Moth bean root samples with nodules were collected during
rabi 2017 from five villages
viz., Eraiyanur (12.2087°N, 79.6708°E), Konkarampattu (12.2210°N, 79.5287°E), Mannur (13.0217°N, 79.9574°E), Modaiyur (12.2344°N, 79.4915°E) and Rettanai (12.1958°N, 79.5519°E) of Villupuram district in Tamil Nadu. Unlike other legumes moth bean nodules are easily detachable and the plants were carefully uprooted by scooping it along with the soil in appropriate moisture. Fresh, healthy and larger sized nodules were carefully detached from each plant for study. The selected nodules were in ivory colour. The colour and freshness of the nodules indicated that the rhizobia are in active stage. The nodules were first washed thoroughly with sterile distilled water and were surface sterilized with 70% ethanol and 0.1% mercuric chloride and again washed thrice with sterile distilled water. The collected nodules were used for isolation and further morphological and physiological characterization.
Serial dilutions and plating technique
The root nodules were crushed in a test tube having sterile distilled water using glass rod. A turbid extraction was obtained at the end. Serial dilution was prepared from extraction of bacteroid solution. The extract was serially diluted up to 10-6 dilution and 10-4 to 10-6 dilutions were selected for plating. From the suspension 0.1 mL was spread onto the YEMA plates and incubated at room temperature. The same procedure was followed for the nodule samples collected from different locations and the isolates were named as MB (Table 1).
Morphological characterization
All the selected isolates were examined for the morphological, physiological and biochemical characterization as per the standard procedures given by
Barthalomew and Mittewer (1950). Strains of rhizobia were identified by observing their growth on different solid and liquid media for their confirmation. Morphological and microscopic characteristics of all the isolates were investigated after an incubation period of 7 days at 28°C on yeast extract mannitol agar medium. Individual colonies were characterized for shape, size, colour, opacity, elevation, edge and microscopic features of the isolates were studied by Gram’s staining technique.
Biochemical characterization
The most important rhizobial presumptive test
viz., growth on YEMA and congo red medium, growth on glucose peptone agar, catalase activity test, citrate utilization test, Keto-lactose test and growth on YEMA containing BTB medium were performed
(Singha et al., 2015) as presented in Table 1.
Growth on YEMA with congo red
In general, rhizobia produced white colonies, whereas contaminants like
Agrobacterium absorbs the dye strongly. Bacteria isolated from moth bean nodules were streaked on plates and incubated at 28°C for 48 hours.
Growth on glucose peptone agar (GPA) medium
Glucose peptone agar medium with Bromo-thymol blue (indicator) is widely used for isolating pure rhizobial colonies. The bacterial isolates were streaked on glucose peptone agar plates and incubated at 28°C for 48 hours. Rhizobia showed no growth or very poor growth on glucose peptone agar medium and caused very little change in pH Profuse growth in glucose peptone agar medium indicated contamination.
Keto-lactose test
Keto-lactose agar medium was prepared and bacterial isolates were streaked on plates. Inoculated plates were then incubated at 28°C for 48 hours. Plates were then flooded with Benedict’s reagent, incubated at 25°C and results were observed after 1 hour.
Catalase activity test
The presence of the enzyme catalase in the rhizobial isolates was examined by suspending one loopful of the organism in a drop of 3 per cent H
2O
2 on a glass slide. Production of bubbles indicated a positive result or vice-versa.
Citrate utilization test
The ability of the isolates to utilize citrate was determined by the growth in Simmon’s Citrate Agar (SCA). A distinct colour from green to blue referred to a positive utilization of citrate by the isolates.
Production of exopolysaccharide
The production of exopolysaccharide by each isolate was noted. The appearance of the colonies (
i.e., gummy, watery, translucent to a thick dense consistency, milky creamy appearance, opacity, presence of dark centres
etc.) was observed (
Grönemeyer et al., 2014;
Paudyal and Gupta, 2017).
Bromothymol blue test
Bromo-thymol blue test was done by inoculating the bacteria on YEM agar medium containing 0.025% bromothymol blue and the plates were incubated for 3 days (Be for acid or alkaline reaction. After incubation, positive sample showed yellow colour due to acid production.
16S rRNA sequencing
Genomic DNA from the efficient bacterium was isolated by using the HipurA® Bacterial Genomic DNA Purification Kit (M/s. Hi Media Laboratories, Mumbai, India). Bacterial genomic DNA was then amplified with 16S rRNA gene target universal primers 27F (5’AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) by a thermocycler (Mastercycler® nexus, Eppendorf India Private Limited). The PCR reaction mixture (25 µL) contained 20 ng of template DNA, 0.3 µL of 1 U
TaqDNA polymerase (Sigma, India), 2.5 µL of 1x Taq DNA polymerase buffer (Sigma, India), 2 mM magnesium chloride (Sigma, India), 400 µM dNTPs (Sigma, India), 10 pmol of forward and reverse primer and the final volume was made up with sterilized deionized water. The thermoprofile condition of PCR consisted of initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 50°C for 30 sec, extension at 72°C for 1 min and a final extension at 72°C for 10 min and hold at 4°C. Genomic DNA was viewed and documented using a gel documentation and analysis system (Vilber E-box, Germany).
The fluorescent dye terminator method was used for obtaining the nucleotide gene sequences of 16S rRNA, which was then purified by the Millipore-Montage dye removal kit. Sanger sequencing was performed at the M/s. Barcode Biosciences, Bengaluru, India. The 16S rRNA sequences of the isolates obtained from the automatic sequencer were then aligned and identified using the e-server, EzTaxon (
http://eztaxon-e.ezbiocloud.net/)
(Yoon et al., 2017) to determine their closest relative. The neighbour joining phylogenetic tree was constructed with the bootstrap values of 1000 replicates by using the MEGA version.
Nodulation study
Small sized pots were filled with 3 kg of sterile soil. Uninoculated plants served as negative controls and MB-1, MB-2 and MB-7 was the positive control. The seeds were surface sterilized with 1 per cent mercuric chloride. Seeds were inoculated with 1 ml of YEM broth containing approximately 109 cfu mL
-1 and sterile water source was used. Twenty-five days after inoculation, plants were removed from the pots and the presence or absence of nodules assessed.
Pot culture studies
Pot culture experiment was conducted with six treatments and four replications and arranged in a completely randomized design. Twenty-four pots were filled with sterile soil, each having 7 kg of sterile soil. Surface sterilized seeds were used. Rhizobial isolates MB-1 and MB-2 was used for this study and these were compared with the standard rhizobial isolates
viz., COG15 used for groundnut and BMBS14 culture used for green gram and black gram. Seeds were treated with the respective rhizobial isolate (2 x 109 cfu ml
-1) and shade dried seed were sown (5 seeds pot-1) in the pots. The pots were labelled and watered regularly. Biometric observation
viz., plant height, root length, number of nodules, nodules dry weight, pod yield per plant, haulms yield was recorded and statistically analysed (Fig 1).
