Aqbal Singh

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.

Biodegradation of beta-cyfluthrin by Pseudomonas stutzeri strain S1.

Abstractβ-Cyfluthrin [α-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 β-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 β-cyfluthrin and identify the intermediates in culture broth using spectroscopy. An aerobic bacterium capable of degrading β-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 β-cyfluthrin have been identified as α-cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate (M.W. 341); 4-fluoro-3-phenoxy-α-cyanobenzyl alcohol (M.W. 243) and 3(2,2-dichlorovinyl)-2,2-dimethyl cyclopropanecarboxylic acid (M.W. 208).n

Indole acetic acid and ACC deaminase-producing Rhizobium leguminosarum bv. trifolii SN10 promote rice growth, and in the process undergo colonization and chemotaxis

This study focuses on the chemotaxis, colonization and rice growth promoting ability of indole acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing rhizobacteria Rhizobium leguminosarum bv. trifolii SN10, previously isolated from root nodules of Trifolium alexandrium L. We show here that R. leguminosarum bv. trifolii SN10 promote the growth of four different rice varieties grown in India in terms of biomass, root branching and N content. In addition, using scanning electron microscopy and viable cell counts, we provide evidence that the bacteria successfully colonize the root surface of the rice variety which showed maximum growth promotion upon inoculation. Not only this, R. leguminosarum bv. trifolii SN10 exhibit a strong chemotaxis response towards the rice seed and root exudates despite the presence of a bacteriostatic phenolic compound, 7-hydroxycoumarin (umbelliferone). Further, R. leguminosarum bv. trifolii SN10 secretion of phytohormones such as IAA and ACC deaminase suggest the potential of the plant growth promoting rhizobacteria to be used as biofertilizer to enhance rice crop production in the subcontinent.

Natural occurrence of Bacillus thuringiensis in leguminous phylloplanes in the New Delhi region of India

Bacillus thuringiensis (Bt) isolates were present on the phylloplanes of chickpea (Cicer arietinum), pigeon pea (Cajanus cajan), pea (Pisum sativum) and mung bean (Vigna radiata). Bt index (ratio of the number of Bt colonies to the total number of spore-forming colonies per g of leaves) differed significantly among these plants, with the highest (0.20) in the chickpea phylloplane, followed by pigeon pea (0.17). Bt population of the chickpea phylloplane varied with plant age, being maximal in 45-day-old plants. Diversity was observed among Bt isolates for growth (up to 10-fold difference), antibiotic resistance, PCR product profile and toxicity to Helicoverpa armigera. Two isolates with high activity towards H. armigera were found.

Fine mapping of grain length QTLs on chromosomes 1 and 7 in Basmati rice (Oryza sativa L.)

Grain dimensions (length, breadth and length/breadth ratio) are important quality attributes of Basmati rice for its high consumer acceptance. Earlier we identified two significant quantitative trait loci (QTL) intervals on chromosomes 1 and 7 for grain dimensions in Basmati rice using a population of recombinant inbred lines (RILs) from cross between Basmati variety Pusa 1121 and a short grain non-aromatic variety Pusa 1342. For fine mapping of these QTLs, 184 F6 RILs were grown and phenotyped in the normal rice growing season at two different locations. Forty-nine new SSR markers targeting these QTL intervals were tested and nine were found polymorphic between the parents. Using revised genetic maps adding new markers, the grain length QTL qGRL1.1 on chromosome 1 was narrowed down to 108xa0kbp from the earlier reported 6,133xa0kbp. There were total 13 predicted gene models in this interval which includes the probable candidate gene for the exceptionally high grain length of Basmati variety Pusa 1121. Similarly, two tandem QTL intervals qGRL7.1 and qGRL7.2 on chromosome 7 were merged into a single one narrowed down to 2,390xa0kbp from the earlier reported length of 5,269xa0kbp. This region of chromosome 7 also has co-localized QTLs for grain breadth and length to breadth ratio. SSR markers tightly linked to the QTL at a map distance of ≤0.2xa0cM were developed for the qGRL1.1 and qGRL7.1 loci that could be used for marker-assisted breeding. Validation of earlier published markers for the grain length gene GS3 on chromosome 3 showed no difference between Pusa 1121 and Pusa 1342, highlighting the significance of qGRL1.1 and qGRL7.1 for the extra grain length of Basmati variety Pusa 1121.

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.

Fine Mapping of Aroma QTLs in Basmati Rice (Oryza sativa L) on Chromosomes 3, 4 and 8

Aroma is one of the most important quality traits of basmati rice (Oryza sativa L) that leads to high consumer acceptance. Earlier three significant QTLs for aroma, namely aro3-1, aro4-1 and aro8-1, have been mapped on rice chromosomes 3, 4 and 8, respectively, using a population of recombinant inbred lines (RILs) derived from a cross between Pusa 1121 (a basmati quality variety) and Pusa 1342 (a non-aromatic variety). For fine mapping of these QTLs, 184 F6 RILs were grown in the Kharif season of 2005 at New Delhi and Karnal, India. A total of 115 new SSR markers covering the three QTL intervals were designed and screened for parental polymorphism. Of these, 26 markers were polymorphic between parents, eight for the interval aro3-1, eight for the interval aro4-1 and ten for the interval aro8-1, thus enriching the density of SSR markers in these QTL intervals. Revised genetic maps were constructed by adding 23 of these new markers to the earlier map, by giving physical order of the markers in the pseudomolecules a preference. In the revised maps, the interval for QTL aro4-1 could not be improved further but QTL aro3-1 was narrowed down to an interval of 390 kbp from the earlier reported interval of 8.6 Mbp and similarly the QTL aro8-1 was narrowed down to a physical interval of 430 kbp. The numbers of candidate genes in the aro3-1 and aro8-1 intervals have now been reduced to 51 and 66, respectively. The badh2 gene on chromosome 8 was not associated with the aroma QTL on this chromosome.

Rhizobium meliloti purine auxotrophs arenod + but defective in nitrogen fixation

Several purine auxotrophs were isolated inRhizobium meliloti and characterized for their nutritional requirements. They were found to produce small, irregular nodules lacking any detectable nitrogenase activity onMedicago sativa. The symbiotic aberration manifests itself only in the late developmental stage, for, (i) these purine auxotrophs infect theMedicago sativa root hairs by forming normal infection threads, and (ii) the mutants are recovered from the root nodules induced by them. External supplementation of the plant growth substrate with purines or their biosynthetic intermediates fails to restore symbiosis. This, and the failure of complementation of these auxotrophs with the known symbiotic genes, demonstrates that these mutants perhaps define a new set of genes influencing the symbiotic process inRhizobium meliloti.

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 (1.63–7.78xa0μgxa0ml−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 3.69xa0g 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.

Pleiotropic effects of purine auxotrophy inRhizobium meliloti on cell surface molecules

Rhizobial purine auxotrophs have earlier been shown to be defective in symbiosis, though the exact reason for this failure is not clear. Using various dyes that specifically bind different cell surface molecules, we show that there are multiple changes in the cell surface molecules associated with different purine auxotrophs. Affected molecules in different purine auxotrophs that were tested include (i) acidic exopolysaccharides, (ii) cellulose fibrils, and (iii) beta (1–3) glucans. Our results show that the symbiotic deficiency of purine auxotrophs is likely to be a result of these associated changes on the cell surface