Pathogen-induced expression of harpinPss increases resistance in tobacco against fusarium oxysporum f. sp. nicotianae

HarpinPss (encoded by the hrpZ gene), a proteinaceous elicitor produced by Pseudomonas syringae pv. syringae, induces cell death in plants through hypersensitive response (HR). With an aim to generate transgenic tobacco resistant to fungal diseases, hrpZ was expressed in a secretable form, tagged with the signal peptide (SP) of PR1a, under the constitutive 35S promoter (P35S) or pathogen-inducible promoters (PIPs) like phenylalanine ammonia lyase (PAL), osmotin (OSM), and hypersensitive-related (HSR) promoters. The constitutive expression of the secretable form of hrpZ did not permit regeneration of transformed cells due to harpinPss-induced cell death. Transformants were recovered at a low frequency (2-6%) from leaf discs infected with Agrobacterium harbouring the SP-hrpZ driven by PIPs due to wound-induced leaky expression of harpinPss. The transgenic lines were confirmed by PCR using transgene-specific primers for SP-hrpZ. The expression of hrpZ under PIPs in transgenic lines was confirmed by Western blotting after challenging the leaves with Fusarium oxysporum f. sp. nicotianae. RT-PCR analysis also confirmed the expression of SP-hrpZ driven by PIPs in transgenic tobacco upon infection with F. oxysporum f. sp. nicotianae. The expression of harpinPss in these transgenic lines was accompanied by expression of defense-response genes such as PR1, PR2, PR3, HSR and HIN1. Transgenic tobacco plants showed enhanced resistance to F. oxysporum f. sp. nicotianae. Our findings suggest the potential use of an elicitor gene (hrpZ), driven by PIPs (PAL, OSM, and HSR) for the development of resistant plants

Understanding plant-beneficial microbe interactions for sustainable agriculture

A better understanding of the specific molecular interactions between plants and microbes is crucial to develop newer strategies for sustainable agriculture. The productivity of wide range of agricultural crops under decreasing land resources and shrinking biological potential of the soil need to be improved. Search for useful microorganisms associated with the plants has been highly productive for sustainable agriculture. We take a close look at the current level of molecular interactions that mostly involve specific molecular patterns of microbes and their cognate receptors in plants and development of efficient biofertilizers for improving crop yields. This article covers the broader aspects of plant-microbe interactions with more focus on plant growth promoting rhizobacteria (PGPR). Further upcoming strategies to understand the plant-PGPR interactions are discussed

Synthesis, x-ray crystal structures and biological evaluation of some mono- and bi-cyclic 1,3-diazetidin-2-ones: non-natural β -lactam analogues

Mono- and bi-cyclic 1,3-diazetidin-2-ones (aza-β -lactams) are synthesised and evaluated as non-natural analogues of β -lactams. The aza-β -lactams are designed on the principle that their reaction with active site serine hydroxy will form a carbamoyl-enzyme intermediate that is sluggish to hydrolysis. The synthesis of racemic mono- and bi-cyclic aza-β -lactams is carried out starting from pyrimidinone 18 which is transformed to the densely functionalised substrate 20. The chemical reactivity of tricarbonyl 20 for selective functional group manipulation was first assessed and then it was transformed to amino alcohol 24. Cyclisation of 24 affords aza-carbapenams and its homologation followed by aldol cyclisation provides access to aza-carbacephams. The X-ray structures of aza-carbapenam 35 and aza-carbacepham 42 suggest that the structural requirements for biological activity in β -lactams are fulfilled. An unexpected ozonolysis product, phenol 52 resolves spontaneously during crystallisation and its crystal structure was also determined. The biological activity of the novel mono- and bi-cyclic aza-β -lactams was evaluated with potent gram-positive bacterial strain, Bacillus subtilis and compared with β -lactam antibiotics, ampicillin and penicillin G. Of the 19 aza-β -lactams tested, eight compounds show inhibition better than the standards while another eight are of comparable activity. This study shows that aza-β -lactams represent a novel and non-natural lead towards serine peptidase inhibitors

Phylloplane bacteria increase seedling emergence, growth and yield of field-grown groundnut (Arachis hypogaea L.)

Aim: To isolate and characterize groundnut-associated bacterial isolates for growth promotion of groundnut in field. Methods and Results: Three hundred and ninety-three groundnut-associated bacteria, representing the geocarposphere, phylloplane and rhizosphere, and endophytes were applied as seed treatment in greenhouse. Maximum increase in plant biomass (up to 26%) was observed following treatment with a rhizosphere isolate identified as Bacillus firmis GRS 123, and two phylloplane isolates Bacillus megaterium GPS 55 and Pseudomonas aeruginosa GPS 21. There was no correlation between the production of L-tryptophan-derived auxins and growth promotion by the test isolates. Actively growing cells and peat formulations of GRS 123 and GPS 55, and actively growing cells of GPS 21, significantly increased the plant growth and pod yield (up to 19%) in field. Rifampicin-resistant mutants of GRS 123 and GPS 21 colonized the ecto- and endorhizospheres of groundnut, respectively, up to 100 days after sowing (DAS), whereas GPS 55 was recovered from both the habitats at 100 DAS. Conclusion: Seed bacterization with phylloplane isolates promoted groundnut growth indicating the possibility of isolating rhizosphere beneficial bacteria from different habitats. Significance and Impact of the Study: Identification of phylloplane bacteria as effective plant growth-promoting rhizobacteria (PGPR) broadens the spectrum of PGPR available for field applicatio

Biological control of late leaf spot of peanut (Arachis hypogaea) with chitinolytic bacteria

Late leaf spot (LLS), caused by Phaeoisariopsis personata, is a foliar disease of groundnut or peanut (Arachis hypogaea) with high economic and global importance. Antifungal and chitinolytic Bacillus circulans GRS 243 and Serratia marcescens GPS 5, selected among a collection of 393 peanut-associated bacteria, were applied as a prophylactic foliar spray and tested for control of LLS. Chitin-supplemented application of B. circulans GRS 243 and S. marcescens GPS 5 resulted in improved biological control of LLS disease. Supplementation of bacterial cells with 1% (wt/vol) colloidal chitin reduced lesion frequency by 60% compared with application of bacterial cells alone, in the greenhouse. Chitin-supplemented application of GRS 243 and GPS 5 also resulted in improved and stable control of LLS in a repeated field experiment and increased the pod yields by 62 and 75%, respectively, compared with the control. Chitin-supplemented application of GPS 5 was tested in six on-farm trials, and the increase in pod yields was up to 48% in kharif (rainy season). A 55-kDa chitinase was purified from the cell-free culture filtrate of GPS 5 by affinity chromatography and gel filtration. Purified chitinase of S. marcescens GPS 5 (specific activity 120 units) inhibited the in vitro germination of P. personata conidia, lysed the conidia, and effectively controlled LLS in greenhouse tests, indicating the importance of chitinolysis in biological control of LLS disease by GPS

Chitin-supplemented foliar application of Serratia marcescens GPS 5 improves control of late leaf spot disease of groundnut by activating defence-related enzymes

Chitinolytic Serratia marcescens GPS 5 and non-chitinolytic Pseudomonas aeruginosa GSE 18, with and without supplementation of chitin, were tested for their ability to activate defence-related enzymes in groundnut leaves. Thirty-day-old groundnut (cv. TMV 2) plants pretreated with GPS 5 and GSE 18 (with and without supplementation of 1% colloidal chitin) were challenge inoculated after 24 h with Phaeoisariopsis personata, the causal agent of late leaf spot (LLS) disease of groundnut. GPS 5 and GSE 18, applied as a prophylactic spray, reduced the lesion frequency by 23% and 67%, respectively, compared with control. Chitin supplementation had no effect on the control of LLS by GSE 18, unlike GPS 5, which upon chitin supplementation reduced the lesion frequency by 64%, compared with chitin alone. In a time course study the activities of chitinase, β-1,3- glucanase, peroxidase and phenylalanine ammonia lyase were determined for the different treatments. There was an enhanced activity of the four defence-related enzymes with all the bacterial treatments when compared with phosphate buffer and colloidal chitin-treated controls. In correlation to disease severity in bacterial treatments, chitin-supplemented GSE 18 was similar to GSE 18, whereas chitin-supplemented GPS 5 was much more effective than GPS 5, in activation of the defence-related enzymes. The high levels of enzyme activities following chitin-supplemented GPS 5 application continued up to the measured 13 days after pathogen inoculatio

Management of late leaf spot of groundnut (Arachis hypogaea) with chlorothalonil-tolerant isolates of Pseudomonas aeruginosa

Fifteen groundnut-associated bacterial isolates that inhibited by >90% the in vitro conidial germination of Phaeoisariopsis personata, causal agent of late leaf spot disease of groundnut, were applied as a prophylactic spray (108 cfu mL-1) and tested for control of the disease in the glasshouse. Two groundnut seed-associated bacterial isolates, GSE 18 and GSE 19, identified as Pseudomonas aeruginosa, reduced the lesion frequency (LF) by up to 70%. A 90-day-old peat-based formulation of P. aeruginosa GSE 18 reduced LF measured 15 days postinoculation by up to 60%. Both P. aeruginosa GSE 18 and GSE 19 were tolerant to chlorothalonil (Kavach®) up to 2000 µg mL-1 in LB broth. In glasshouse trials, GSE 18 and GSE 19 tested in combination with reduced concentrations of chlorothalonil were highly efficient in management of the disease. The disease was completely controlled by chlorothalonil (>250 µg mL-1), and in the presence of GSE 18 or GSE 19, 100 µg mL-1 chlorothalonil was equally effective. Application of rifamycin-resistant mutants of GSE 18 or GSE 19 together with chlorothalonil significantly increased the survival of these isolates in the groundnut phylloplane. In the field, a combination of GSE 18 and 500 µg mL-1 chlorothalonil reduced disease severity comparable to 2000 µg mL-1 chlorothalonil alone. Use of chlorothalonil-tolerant pseudomonads together with a quarter concentration of the recommended field dose of chlorothalonil doubled pod yield compared with the untreated unsprayed contro

Poor competitiveness of Bradyrhizobium in pigeon pea root colonisation in Indian soils

Background Pigeon pea, a legume crop native to India, is the primary source of protein for more than a billion people in developing countries. The plant can form symbioses with N2-fixing bacteria, however reports of poor crop nodulation in agricultural soils abound. We report here study of the microbiota associated with pigeon pea, with a special focus on the symbiont population in different soils and vegetative and non-vegetative plant growth. Results Location with respect to the plant roots was determined to be the main factor controlling the microbiota followed by developmental stage and soil type. Plant genotype plays only a minor role. Pigeon pea roots have a reduced microbial diversity compared to the surrounding soil and select for Proteobacteria and especially for Rhizobium spp. during vegetative growth. While Bradyrhizobium, a native symbiont of pigeon pea, can be found associating with roots, its presence is dependent on plant variety and soil conditions. A combination of metagenomic survey, strain isolation and co-inoculation with nodule forming Bradyrhizobium spp. and non-N2 fixing Rhizobium spp. demonstrated that the latter is a much more successful coloniser of pigeon pea roots. Conclusions Poor nodulation of pigeon pea in Indian soils may be caused by a poor Bradyrhizobium competitiveness against non-nodulating root colonisers such as Rhizobium. Hence, inoculant strain selection of symbionts for pigeon pea should not only be based on their nitrogen fixation potential but more importantly on their competitiveness in agricultural soils

Biological control of crown rot of groundnut by Trichoderma harzianum and T. viride

Crown rot of groundnut (Arachis hypogaea) caused by Aspergillus niger is prevalent in warm and dry climatic zones and its incidence ranges from 2% to 14% (Pande and Narayana Rao 2000). The pathogen attacks groundnut plants at all the growth stages and causes pre-emergence rotting in seeds, soft rot in emerging seedlings, and crown rot in mature plants. Thus, management ofcrown rot by fungicides is difficult and expensive. Biological control of plant diseases is cost effective and environmentally safe compared to fungicides. Also, the biocontrol agent once established persists in the soil for longer periods and offers disease protection even in the consecutive crop seasons (Mew and Rosales 1986). Trichoderma spp are antagonistic to a wide range of phytopathogenic fungi and are able to control economically important diseases in several crop plants (Papavizas 1985). Trichoderma harzianum and Bacillus subtilis AF 1 were tested to control the incidence ofcrown rot in groundnut and varying levels of disease control were obtained with these biocontrol agents (Lashin et al. 1989, Podile 2000). Bacillus subtilis AF 1 induced production of lipoxygenase and altered the phytoalexin metabolism in groundnut seedlings (Podile 2000). We report the results of the in vitro antagonistic potential of 16 Trichoderma isolates against A. niger and the efficacy of the selected isolates to control A. niger infection under greenhouse conditions in comparison with a fungicide

Pseudomonas aeruginosa inhibits the plant cell wall degrading enzymes of Sclerotium rolfsii and reduces the severity of groundnut stem rot

Three hundred and ninety-three groundnut-associated bacterial strains, applied both as seed treatment and soil amendment, were evaluated for control of stem rot disease (caused by Sclerotium rolfsii) of groundnut in a controlled environment. Twelve strains significantly (P=0.01) reduced the incidence of stem, rot of which groundnut seed endophytes Pseudomonas aeruginosa GSE 18 and GSE 19 reduced the seedling mortality by 54% and 58%, compared to the control. In dual cultures, the 12 biocontrol strains reduced the mycelial growth of S. rolfsii by 32%-74% as compared to the control. Cell-free culture filtrates of P. aeruginosa GSE 18 and GSE 19 inhibited the activity in vitro of the cell wall-degrading enzymes (CWDE) polygalacturonase and cellulase by S. rolfsii up to a maximum of 55% and 50%, respectively, when measured 6 days after inoculation. Pseudomonas aeruginosa GSE 18 and GSE 19 with a known tolerance to thiram, a commonly used seed dressing fungicide, suppressed the growth of S. rolfsii, inhibited the activity of CWDE, and reduced the incidence of stem rot, suggesting the usefulness of these biocontrol strains as components in the integrated management of groundnut stem rot