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ItemConditional expression of harpin < inf > Pss < /inf > causes yeast cell death that shares features of cell death pathway with harpin < inf > Pss < /inf > -mediated plant hypersensitive response (HR)( 2001-01-01)Conditional expression of harpinPss causes yeast cell death that shares features of cell death pathway with harpinPss-mediated plant hypersensitive response (HR). Pseudomonas syringae pv. syringae 61 hrpZ gene encodes harpinPss, a 34.7 kD extracellular protein that elicits a hypersensitive response (HR) in plants. Conditional expression of either full-length or truncated hrpZ sequences under the GAL1 promoter caused cell death in Saccharomyces cerevisiae Y187. Plating of pYEUT-hrpZ transformants on a medium containing galactose resulted in complete inhibition of colony formation, whereas their growth on a glucose-based medium was unaffected. Western blot analysis confirmed the expression of harpinPss in yeast cells transformed with pYEUT-hrpZ and grown in galactose-containing medium. A time-dependent decline in the percentage of trypan blue-excluding cells in cultures of pYEUT-hrpZ transformants was observed when cultured on galactose-containing medium. Similarly, the number of viable cells reduced to about 50% within 6 h. There were similarities in the harpinPss-mediated cell death in plants and yeast cell death (YCD). Galactose-induced cell death in pYEUT-hrpZ transformants of S. cerevisiae Y187 was suppressed by a protein kinase inhibitor K252a (10 μM). The viability of pYEUT-hrpZ transformants was prolonged in the presence of 100 U ml-1 catalase suggesting a role for the oxidative burst in YCD that was further supported by the flow cytometric patterns of propidium iodide uptake by yeast cells. Overall, it appears that yeast provides a useful model system to understand the molecular mechanism of harpinPss-mediated cell death. © 2001 Academic Press.
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ItemChitin-supplemented formulations improve biocontrol and plant growth promoting efficiency of Bacillus subtilis AF 1( 2001-08-27)Formulations of a chitinolytic biocontrol and a plant growth promoting Bacillus subtilis AF 1 were prepared in peat, in peat supplemented with either 0.5% chitin or Aspergillus niger mycelium, or in spent compost obtained from Agaricus bisporus cultivation and were evaluated for biocontrol of two fungal pathogens and plant growth promoting activities on pigeon pea and groundnut. A steady increase in cell numbers of introduced B. subtilis AF 1 was observed in all the formulations at 30°C. The increase in cell numbers was about 5.0 log units. Peat or spent compost inoculated with physiologically active and dormant states of B. subtilis AF 1 showed different time period requirements to attain maximum cell numbers. The presence of chitin or A. niger (in peat) or A. bisporus (in spent compost) as supplement in the carrier material improved the multiplication of B. subtilis AF 1. When used as seed treatments, formulations of AF 1 in peat supplemented with chitin or chitin-containing materials showed better control of A. niger (causing crown rot of groundnut) and Fusarium udum (causing wilt of pigeon pea) than AF 1 culture alone, in both groundnut and pigeon pea. Bacillus subtilis AF 1 formulations promoted seed germination and biomass of both ground-nut and pigeon pea even under pathogen pressure. Survival of AF 1 on fresh culture-treated and formulation product-treated plants was similar in pathogen-infested soil.
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ItemConstitutive expression of hrap gene in transgenic tobacco plant enhances resistance against virulent bacterial pathogens by induction of a hypersensitive response( 2002-01-01)Hypersensitive response-assisting protein (HRAP) has been previously reported as an amphipathic plant protein isolated from sweet pepper that intensifies the harpinPss-mediated hypersensitive response (HR). The hrap gene has no appreciable similarity to any other known sequences, and its activity can be rapidly induced by incompatible pathogen infection. To assess the function of the hrap gene in plant disease resistance, the CaMV 35S promoter was used to express sweet pepper hrap in transgenic tobacco. Compared with wild-type tobacco, transgenic tobacco plants exhibit more sensitivity to harpinPss and show resistance to virulent pathogens (Pseudomonas syringae pv. tabaci and Erwinia carotovora subsp. carotovora). This disease resistance of transgenic tobacco does not originate from a constitutive HR, because endogenous level of salicylic acid and hsr203J mRNA showed similarities in transgenic and wildtype tobacco under noninfected conditions. However, following a virulent pathogen infection in hrap transgenic tobacco, hsr203J was rapidly induced and a micro-HR necrosis was visualized by trypan blue staining in the infiltration area. Consequently, we suggest that the disease resistance of transgenic plants may result from the induction of a HR by a virulent pathogen infection.
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ItemWhole cells of Bacillus subtilis AF 1 proved more effective than cell-free and chitinase-based formulations in biological control of citrus fruit rot and groundnut rust( 2004-09-01)In foliar and postharvest biocontrol systems, the use of active metabolites produced by antagonistic microorganisms is advantageous compared with the use of living microorganisms. Chitinases, a major group of hydrolytic enzymes produced by biocontrol agents, are involved in the lysis of cell walls of pathogenic fungi. In the present study, an attempt was made to test the partially purified β-1,4-N-acetylglucosaminidase (NAGase) of a biocontrol strain Bacillus subtilis AF 1 for control of rust in groundnut (caused by Puccinia arachidis) and soft rot in lemons (caused by Aspergillus niger). Four proteins of molecular mass 67, 40, 37, and 32 kDa were isolated from the culture filtrates of AF 1 by affinity chromatography, of which the 67-kDa protein has detectable chitinolytic ability. This protein (NAGase) effectively inhibited the in vitro growth of A. niger in microtitre plates. In the presence of NAGase, germination of urediniospores of P. arachidis was reduced by 96% compared with the control. In a detached leaf bioassay, NAGase reduced the rust lesion frequency by > 60%. NAGase significantly reduced the incidence of soft rot in harvested lemon fruits. However, fresh cells and (or) alginate formulation of AF 1 were more effective than NAGase in control of both of the test plant - pathogen systems.
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ItemBiological control of collar rot disease with broad-spectrum antifungal bacteria associated with groundnut( 2005-02-01)Bacteria associated with 6 habitats of groundnut were evaluated for their broad-spectrum antifungal activity and suppression of collar rot (Aspergillus niger) of groundnut. Three hundred and ninety-three strains were tested against 8 fungal pathogens of groundnut including 5 necrotrophic fungi, Aspergillus flavus, A. niger, Rhizoctonia bataticola, Rhizoctonia solani, and Sclerotium rolfsii, and 3 biotrophic fungi, Cercospora arachidicola, Phaeoisariopsis personata, and Puccinia arachidis. Pseudomonas sp. GRS 175, Pseudomonas aeruginosa GPS 21, GSE 18, GSE 19, and GSE 30, and their cell-free culture filtrates were highly antagonistic to all the test fungi. The cell-free culture filtrates of these bacteria were fungicidal and induced mycelial deformations including hyphal bulging and vacuolization in necrotrophic fungi. The cell-free culture filtrates at 10% (v/v) concentration significantly inhibited the spore germination of biotrophic fungi. In the greenhouse, P. aeruginosa GSE 18 emerged as an effective biocontrol agent of collar rot closely followed by P. aeruginosa GSE 19. The bacterium applied as a seed treatment reduced the pre-emergence rotting and postemergence wilting by > 60%. Pseudomonas aeruginosa GSE 18 effectively colonized the groundnut rhizosphere, both in native and in A. niger infested potting mixtures. Ninety-day-old peat formulation of P. aeruginosa GSE 18 had biocontrol ability comparable with the midlog-phase cells. Pseudomonas aeruginosa GSE 18, tolerant to thiram, in combination with the fungicide had an improved collar rot control. The present study was a successful attempt in selection of broad-spectrum and fungicide tolerant biocontrol agents that can be a useful component of integrated management of collar rot. © 2005 NRC Canada.