Isolation and characterization of symbiotically defective pyrimidine and amino acid auxotroph of Mesorhizobium ciceri in chickpea.

Transposon Tn5 induced, four Mesorhizobium ciceri auxotroph were isolated and characterized. Unlike its wild type parent (TL 620), all four mutants were found defective for amino acid and pyramiding biosynthesis. The auxotroph mutants were characterized and found TL130 as cytosine and uracil, TL 196 for guanine, cytocine, uracil and riboflavin, TL 141 as serine and TL 38 as argentine defective. Symbiotic characterization of these mutants revealed phenotypic deformities and deficiencies in biological nitrogen fixation. All the four auxotrophic mutants were characterized as nod+/fix+ nature with reduced nitrogenase activity of 42.2, 26.3 and 17.13% respectively as compared to the wild type which is further supported by sub cellular examination of the nodules section by TEM study.

Isolation and screening of phlD (+) plant growth promoting rhizobacteria antagonistic to Ralstonia solanacearum.

Tomato (Lycopersicon esculentum) is important widely grown vegetable in India and its productivity is affected by bacterial wilt disease infection caused by Ralstonia solanacearum. To prevent this disease infection a study was conducted to isolate and screen effective plant growth promoting rhizobacteria (PGPR) antagonistic to R. solanacearum. A total 297 antagonistic bacteria were isolated through dual culture inoculation technique, out of which forty-two antagonistic bacteria were found positive for phlD gene by PCR amplification using two primer sets Phl2a:Phl2b and B2BF:BPR4. The genetic diversity of phlD (+) bacteria was studied by amplified 16S rDNA restriction analysis and demonstrated eleven groups at 65% similarity level. Out of these 42 phlD (+) antagonistic isolates, twenty exhibited significantly fair plant growth promoting activities like phosphate solubilization (0.92-5.33%), 25 produced indole acetic acid (1.63-7.78 µg ml(-1)) and few strains show production of antifungal metabolites (HCN and siderophore). The screening of PGPR (phlD (+)) for suppression of bacterial wilt disease in glass house conditions was showed ten isolated phlD (+) bacteria were able to suppress infection of bacterial wilt disease in tomato plant (var. Arka vikas) in the presence R. solanacearum. The PGPR (phlD (+)) isolates s188, s215 and s288 was observed to be effective plant growth promoter as it shows highest dry weight per plant (3.86, 3.85 and 3.69 g plant(-1) respectively). The complete absence of wilt disease symptoms in tomato crop plants was observed by these treatments compared to negative control. Therefore inoculation of tomato plant with phlD (+) isolate s188 and other similar biocontrol agents may prove to be a positive strategy for checking wilt disease and thus improving plant vigor.

Phenotypic and molecular characterization of indigenous rhizobia nodulating chickpea in India.

In a combined approach of phenotypic and genotypic characterization, 28 indigenous rhizobial isolates obtained from different chickpea growing regions in peninsular and northern India were analyzed for diversity. The field isolates were compared to two reference strains TAL620 and UPM-Ca142 representing M. ciceri and M. mediterraneum respectively. Phenotypic markers such as resistance to antibiotics, tolerance to salinity, temperature, pH, phosphate solubilization ability, growth rate and also symbiotic efficiency showed considerable diversity among rhizobial isolates. Their phenotypic patterns showed adaptations of rhizobial isolates to abiotic stresses such as heat and salinity. Two salt tolerant strains (1.5% NaCl by T1 and T4) with relatively high symbiotic efficiency and two P-solubilising strains (66.7 and 71 microg/ml by T2 and T5) were identified as potential bioinoculants. Molecular profiling by 16S ribosomal DNA Restriction Fragment Length Polymorphism (RFLP) revealed three clusters at 67% similarity level. Further, the isolates were differentiated at intraspecific level by 16S rRNA gene phylogeny. Results assigned all the chickpea rhizobial field isolates to belong to three different species of Mesorhizobium genus. 46% of the isolates grouped with Mesorhizobium loti and the rest were identified as M. ciceri and M. mediterraneum, the two species which have been formerly described as specific chickpea symbionts. This is the first report on characterization of chickpea nodulating rhizobia covering soils of both northern and peninsular India. The collection of isolates, diverse in terms of species and symbiotic effectiveness holds a vast pool of genetic material which can be effectively used to yield superior inoculant strains.

Mutation in the lysA gene impairs the symbiotic properties of Mesorhizobium ciceri.

A Tn5-induced mutant of Mesorhizobium ciceri, TL28, requiring the amino acid lysine for growth on minimal medium was isolated and characterized. The Tn5 insertion in the mutant strain TL28 was located on a 6.8-kb EcoRI fragment of the chromosomal DNA. Complementation analysis with cloned DNA indicated that 1.269 kb of DNA of the 6.8-kb EcoRI fragment restored the wild-type phenotype of the lysine-requiring mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to lysA gene of different bacteria. The lys (-) mutant TL28 was unable to elicit development of effective nodules on the roots of Cicer arietinum L. There was no detectable level of lysine in the root exudates of chickpea. However, addition of lysine to the plant growth medium restored the ability of the mutant to produce effective nodules with nitrogen fixation ability on the roots of C. arietinum.

Molecular characterization of soil bacteria antagonistic to Rhizoctonia solani, sheath blight of rice.

Bacillus pumillus MTCC7615 has been identified as a potent isolate against Rhizocotonia solani, the fungal pathogen causing sheath blight in rice. The study aimed at probing the role of a 23kb size plasmid pJCP07 of Bacillus pumillus MTCC7615 in its fungal antagonism towards Rhizocotonia solani. Plasmid pJCP07 was found to be involved in production of a fungal antagonistic compound as demonstrated by plasmid curing and conjugational transfer experiments. Tn5 insertional studies further confirmed that the plasmid pJCP07 of Bacillus pumillus MTCC7615 carries some of the gene(s) involved in production of compound antagonistic to Rhizocotonia solani. The plasmid pJCP07 is thus a mobilizable medium-sized plasmid carrying genes responsible for antagonism of Bacillus pumillus MTCC7615 towards Rhizocotonia solani.

Use of nitrogen-fixing bacteria as biofertiliser for non-legumes: prospects and challenges.

The potential of nitrogen-fixing (NF) bacteria to form a symbiotic relationship with leguminous plants and fix atmospheric nitrogen has been exploited in the field to meet the nitrogen requirement of the latter. This phenomenon provides an alternative to the use of the nitrogenous fertiliser whose excessive and imbalanced use over the decades has contributed to green house emission (N2O) and underground water leaching. Recently, it was observed that non-leguminous plants like rice, sugarcane, wheat and maize form an extended niche for various species of NF bacteria. These bacteria thrive within the plant, successfully colonizing roots, stems and leaves. During the association, the invading bacteria benefit the acquired host with a marked increase in plant growth, vigor and yield. With increasing population, the demand of non-leguminous plant products is growing. In this regard, the richness of NF flora within non-leguminous plants and extent of their interaction with the host definitely shows a ray of hope in developing an ecofriendly alternative to the nitrogenous fertilisers. In this review, we have discussed the association of NF bacteria with various non-leguminous plants emphasizing on their potential to promote host plant growth and yield. In addition, plant growth-promoting traits observed in these NF bacteria and their mode of interaction with the host plant have been described briefly.

Characterization of a symbiotically defective serine auxotroph of Mesorhizobium ciceri.

A Tn5-induced mutant strain (TL68) of Mesorhizobium ciceri unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Unlike its wild-type parent (strain TAL620), the mutant had an absolute dependence on serine to grow. Cloning of the DNA region containing Tn5 and sequence analysis showed that Tn5 was inserted into the gene coding for 3-phosphoglycerate dehydrogenase, which catalyses the first step in the serine biosynthetic pathway. The role of serine biosynthesis of M. ciceri in the establishment of nitrogen-fixing symbiosis with chickpea (Cicer arietinum L) was investigated using the mutant TL68. The serA(-) mutant (TL68) was unable to elicit the development of efficient nodules on the roots of Cicer arietinum L. The addition of serine to the plant-growth medium restored the ability of the mutant to nodulate Cicer arietinum, and the nodules were able to fix nitrogen.

Characterization of intrinsic variability of Mesorhizobium ciceri isolates of cultivated fields.

A large number of putative rhizobial isolates were obtained from the root nodules of various chickpea cultivars growing in agricultural research fields. Of these, thirty were selected and characterized for traits, such as, generation time, intrinsic azide resistance and several symbiotic characters.

Biodegradation of beta-cyfluthrin by Pseudomonas stutzeri strain S1.

beta-Cyfluthrin [alpha-cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate] pesticide has been in agricultural use in the recent years for controlling Lepidopteran pests affecting solanaceous crops. The extensive use of synthetic pyrethroids like beta-cyfluthrin has resulted in wide spread environmental contamination. The purpose of this study was to isolate bacteria from soil and to determine their ability to degrade beta-cyfluthrin and identify the intermediates in culture broth using spectroscopy. An aerobic bacterium capable of degrading beta-cyfluthrin was isolated by enrichment culture. The 16S ribosomal DNA sequence of the isolate (strain S1) had 100% identity to the sequence from Pseudomonas stutzeri. Finally products formed during degradation of beta-cyfluthrin have been identified as alpha-cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate (M.W. 341); 4-fluoro-3-phenoxy-alpha-cyanobenzyl alcohol (M.W. 243) and 3(2,2-dichlorovinyl)-2,2-dimethyl cyclopropanecarboxylic acid (M.W. 208).