Isolation , identification and molecular characterization of Rhizobium species from Sesbania bispinosa cultivated in Bangladesh

The investigation was carried out to study the characteristics of rhizobial strains isolated from the leguminous plant, Sesbania bispinosa growing in different regions of Bangladesh. Forty-four isolates were studied for biochemical and molecular characteristics. Isolates were able to utilize different carbohydrates. All isolates showed complete resistance to cloxacillin and penicillin G that results in increased survivability of rhizobial populations in antibiotic stressed conditions. Isolates were able to form nodule in the plant infection test. The majority of the strains showed positive results for nodC and nifH gene amplification which are the typical characteristics of Rhizobium species. Genetic relatedness was assessed by comparing the sequences of 16S rRNA. Two distinct clusters were seen in the dendrogram constructed by the Complete Linkage method. The isolates R7, R8, R17, R33 and R3 were distinct from the 20 reference strains. The first cluster was phylogenetically distinct from the reference strains and might have evolved from a distinct lineage. Isolate R4 was placed adjacent to Rhizobium cnuense. So, the findings may represent new species of Rhizobium. This study helps to identify an ideal strain of Rhizobium from S. bispinosa that can be function as biofertilizer when released in the soil and contribute to sustainable agricultural practices by improving yields.


INTRODUCTION
Nitrogen is one of the most abundant nutrient supplement for plant growth and is not directly available to plants because of its strong triple covalent bond.Increasing global population demands large food production which can be achieved by using plant beneficial microorganisms.Rhizobia are plant growth promoting bacteria that plays a key role in enhancing nitrogen fixation ability of leguminous plants by converting nitrogen to ammonia and provide organic nitrogenous compound to the plants.Rhizobia form nodules on root to fix nitrogen where nod factor and flavonoids initiate nodulation (Oldroyd, 2013).Nodulins are nod gene protein that are associated with Nod factor assembly and nod gene is the determinant of host range (Perret et al., 2000).The nifH gene is selected as a nitrogen fixation marker.Rhizobia take up carbon sources derived for plant photosynthesis.Rhizobial isolates harbour plasmid.Mulligan et al. (1985) demonstrated that nodulation genes, the regulatory genes, and the nitrogen fixing genes are located on large symbiotic plasmid.Antibiotics accumulation in soils after excretion from feces and urine was reported by Ji et al. (2012).These antibiotics are lethal to susceptible rhizobial population and decreased rhizobial persistence in the soil and ultimately total nitrogen fixation.Cole andElkan (1973, 1979) demonstrated that antibiotic resistance is a plasmid-born character.Cole et al. (1973) and Frioni et al. (2001) reported that greatest difference of antibiotics tolerance was found between fast and slow growing rhizobia.The antibiotic-resistant Rhizobium occupies large number of nodules in legumes (Belachew, 2010;Gemell et al., 1993).Sesbania is an important green manure that fixes large quantities of N 2 .In the wet season, Sesbania bispinosa grew rapidly, exhibiting high level of nitrogen accumulation in soil Ladha et al. (1992) and Ndoye et al. (1988).S. bispinosa has not been much investigated regarding the rhizobial infection in Bangladesh.Carbohydrate utilization test is used to characterize rhizobia.The utilization of different carbohydrates serve as a diagnostic tool in the differentiation of Rhizobium species (El-Idrissi et al., 1996;Graham et al., 1991).Molecular methods including polymerase chain reaction (PCR) and 16S rRNA sequencing are more reliable for identification of bacterial isolates.
The aim of study was to characterize forty-four Rhizobium species isolated from S. bispinosa and also to identify the antibiotic resistant Rhizobium which has increased survivability and can be used as biofertilizer to increase productivity.

Isolation of Rhizobium strains
Pink and healthy root nodules of S. bispinosa were washed in water.It was then transferred to 3% H2O2 solution and soaked for 2-3 min.To remove the traces of H2O2 solution, nodules were rinsed 5-6 times in sterile distilled water.Nodules were washed in 70% ethanol and then sterile glass rod was used to crush the nodule.Nodule suspension was streaked on yeast-mannitol agar (YMA) plates and incubated at 30°C for 24 h (Agrawal et al., 2012).Well isolated typical single colonies were restreaked on freshly prepared YMA plates in order to obtain pure cultures and colony characteristics were observed.

Biochemical testing for identification
All isolates were processed for oxidase test as described by Kovaks (1956); catalase and nitrate reduction test as determined by Graham and Parker (1964).Urea agar slants were used to determine the urease activity that was incubated for 7 days at 30°C (Christensen, 1946).Isolates were also tested for indole production as described by Lowe (1962), gelatin liquefication and motility test as mentioned by Arora (2003), and ONPG (O-nitro phenyl---Dgalactoside) test as described by Cappuccino (2007).For carbohydrate utilization tests, the basal medium used was that of Bishop et al. (1976) and for carbohydrate utilization test, different carbohydrates were substituted for mannitol, and KN03 (0.6 g/Iiter) was used as the nitrogen source.

Determination of the antibiotic susceptibility
Bacterial suspension was prepared on nutrient broth and lawn on Muller-Hinton agar plates by using sterile cotton swab.Antibiotic discs were placed aseptically and plates were then incubated at 30°C for 24 h.The diameter was measured in millimeters after incubation.Name of the antibiotic and their concentration are given in Table 1.

Plant infection test
Plant infection test was performed to confirm whether the isolates were able to form nodule and fix atmospheric nitrogen in modified Jensen's Agar medium.Seed was surface sterilized by covering with 3% hydrogen peroxide solution and left for 2 min.After washing three times in sterile water, surface sterilized seeds of S. bispinosa were spread onto surface of water agar plates.Plates were inverted and left in dark until germination occurred for 2 days to 1 week.Germinated seeds were aseptically transferred to big size test tubes containing modified Jensen's agar medium and roots were pointing downward with the contact of the agar.When leaves appeared, 1 ml of rhizobial suspension was added at their base.Plants were grown in light for approximately 8 weeks.

Detection of nifH and nodC genes
Amplification was performed in a 12.5µL reaction volume for each specimen containing 2.5 µL (100 ng/µL) of template DNA.The reaction volume was prepared by mixing the following reagents: 7 µL of DEPC treated water, 1 µL of 20 mM MgCl2, 0.2 µL of 10 mM dNTP, 0.625 µL of 10 mM forward and 0.625 µL of 10 mM reverse primer, 0.05 µL of Taq polymerase and 2.5 µL of template DNA.Mastermix was transferred to each PCR tube and then the corresponding DNA samples (2.5 µL) were added to each tube.For the amplification of the nifH gene, the PCR contents were subjected to initial denaturation at 95°C for 3 min followed by 35 cycles of denaturation at 95°C for 45 s.Annealing, elongation and final extension phase were maintained at 63°C for 45 s, 72°C for 45 s and 72°C for 7 min, respectively.An identical regime was  (Perret and Broughtn, 1998) and nodC (forward 5′-GCCATAGTGGCAACCGTCGT-3′ and reverse 5′-CTCGCCGCTGCAAGT-3′) (Jacob et al., 1985).

RESULTS AND DISCUSSION
Isolates were initially studied for morphological and cultural characteristics.The colonies were circular, convex, gummy with entire margin and no pigmentation on YMA agar plates after 24 h incubation.The isolates were positive for catalase, oxidase, motility, ONPG (Onitro phenyl---D-galactoside) tests and negative for indole production and gelatin liquefication tests (Table 2).These findings were in close agreement with Naz et al. (2009).Isolates also showed positive result for nitrate reductase and urease test.Same test results were found by Sadowsky et al. (1983).They were helpful in presumptive identification of the genus of the isolates.It was also found that isolates were able to grow before 24 h incubation.So, it is concluded that our representative isolates were fast grower.
Carbohydrate utilization capacity deduced that isolates were able to utilize different carbohydrates.Isolates were unable to utilize sucrose, succinate and lactose (Table 3).Same tests results were also observed by Sadowsky et al. (1983).Glenn and Dilworth (1981) reported that  (Fred et al., 1932;Graham and Parker, 1964).These results also confirmed that majority of our isolates were fast grower.Antimicrobial resistance has emerged over last few decades due to abuse of these drugs.Rhizobium was examined for their genetic basis for resistance to antibiotics (Figure 1).All isolates were resistant to penicillin G and cloxacillin and 84.1% isolates were resistant to cephalexin.The resistant isolates might have beta-lactamase enzyme that conferred the resistance property to these isolates.Rhizobium laguerreae resistance to ampicillin, penicillin antibiotics was reported by Saidi et al. (2014).Twenty-five (56.8%) isolates were resistant to kanamycin, 36.4% isolates were resistant to gentamicin and 47.72% isolates were resistant to streptomycin.Streptomycin, kanamycin and gentamicin are aminoglycoside antibiotics and conferred resistance by interrupting protein synthesis.Nineteen (43.2%) isolates were resistant to erythromycin.Since erythromycin molecules are large, they are unable to pass through the outer cell membrane.Prasuna et al. (2014) found a strain that was resistant to many antibiotics (chloramphenicol, erythromycin, kanamycin, neomycin and penicillin G) and these results were in close agreement with our findings.About 38.6, 25 and 4.5% of isolates were resistant to rifampicin, nalidixic acid and polymixin B, respectively (Figure 2).Mihaylova et al. (2014) reported that Rhizobium strains were sensitive to gentamicin and polymyxin B. So, these isolates showed higher antibiotic resistance towards beta-lactamase antibiotics.Antibiotic resistant Rhizobium increases rhizobial survivability in the soil (Naamala et al., 2016).So that, these antibiotics resistant Rhizobium can survive antibiotic stressed conditions and help to increase soil productivity.Presence of nod gene was confirmed by plant infection test.Twenty strains were studied for plant infection test and positive results were obtained for  isolates R3, R5, R7, R8, R10, R12, R17, R18, R24, R32, R35 and R41.
Nitrogenase enzyme, encoded by nif gene, reduced nitrogen to ammona and occuerd in only nitrogen fixing organisms (Singh et al., 2013).The majority of the strains gave PCR amplified product with an approximate length of 781 bp for the nifH gene.The nodC gene with the help of Nod factors initiated the root nodule development in leguminous plants (Barny et al., 1996).When DNA was amplified with nodC specific primer, an amplicon of 500 bp was observed.Not all the strains harboured both nifH and nodC genes.The absence of the amplicon product was probably due to some nucleotide mismatches in nifH and nodC gene which agreed with the result of Laguerre et al. (2001).Some strains (R1, R14, R15, R16, R30, R31, R37 and R39) showed positive result in nodC gene amplification but failed to form nodule in the plant infection test.We concluded that pot experiment might have failed to form nodule for certain limitations such as temperature, humidity etc. which were controlled in the laboratory experiment.
The 16S rRNA sequence of nine isolates were compared with 20 species from the NCBI data base.The sequences were aligned and dendrogram was constructed (Figure 3).In the 16S rRNA sequencing, two distinct clusters were seen.In the first cluster, the isolates R7, R8, R17, R33, R3 were grouped together.On the other hand, the isolates R10, R19, R35 and R4 were clustered together with the 20 reference strains.So, first cluster was phylogenetically distinct from the reference strains and might have evolved from a distinct lineage.Based on the 16S rRNA sequences, the isolates R7, R8 and R17 were indistinguishable from each other.A similar conclusion was applicable for the isolates R10, R19, R35.It could also be inferred that the isolates R10, R19 and R35 exhibited a convincing degree of dissimilarity as compared to the reference strains and hence branched off to form a different cladogroup.The isolate R4 was placed adjacent to Rhizobium cnuense.It is concluded that the findings may represent new species of Rhizobium.Menna et al. (2006) also observed new rhizobial isolates based on the sequencing of 16S rRNA.

Conclusion
Most of the isolates found in this study were fast grower and were able to utilize different carbohydrates.Antibiotic susceptibility test showed that isolates were resistant to antibiotics antibiotics that might help them to survive in antibiotic stress environment.Isolates harbouring nifH and nodC genes might be useful to increase soil fertility.The sequencing results revealed the genetic variability of the isolates.The goal of the present study was to raise the worldwide knowledge of the biodiversity of soil rhizosphere and identified the usefulness of rhizobial population so that isolates can be used as biofertilizers.To identify the genetic diversity of Rhizobium populations, molecular characterization such as fingerprinting techniques are needed.Different environmental stress conditions such as temperature, pH, heavy metals and pesticidal effects needs to be checked prior to the release of Rhizobium among the field populations so that it can survive under the adverse environmental condition and improve soil productivity.

Figure 1 .
Figure 1.Antibiotic susceptibility test of Rhizobium species by disc diffusion test.

Figure 3 .
Figure 3. Dendrogram constructed by the complete linkage method base on 16S rRNA sequences of Rhizobium species.

Table 1 .
Antibiotic used in the susceptibility testing of Rhizobium isolates.

Table 2 .
Biochemical characteristics of isolates.

Table 3 .
Sugar fermentation tests of the isolates.