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Potentiality of Bacillus subtilis as biocontrol agent for management of anthracnose disease of chilli caused by Colletotrichum gloeosporioides OGC1.

ABSTRACT: A soil bacterium, Bacillus subtilis, isolated from the rhizosphere of Chilli, showed high antagonistic activity against Colletotrichum gloeosporioides OGC1. A clear inhibition zone of 0.5-1 cm was observed in dual plate assay. Microscopic observations showed a clear hyphal lysis and degradation of fungal cell wall. In dual liquid cultures, the B. subtilis strain inhibited the C. gloeosporioides up to 100 % in terms of dry weight. This strain also produced a clear halo region on chitin agar medium plates containing 0.5 % colloidal chitin, indicating that it excretes chitinase. The strain also produced other mycolytic enzymes-glucanase and cellulase, demonstrated by a clear zone of hydrolysis of yeast cell wall glucan (YCW 0.1 % v/v) and carboxymethylcellulose (CMC 0.1 % v/v). In liquid cultures, the strain showed appreciable levels of chitinase, glucanase and cellulase activities and hydrolytic activity with C. gloeosporioides OGC1 mycelia as the substrate. The role of the B. subtilis strain in suppressing the fungal growth in vitro was studied in comparison with a UV mutant of that strain, which lacked both antagonistic and hydrolytic activity. The mycolytic enzyme mediated antagonism of B. subtilis was further demonstrated by heat inactivation (70-100 °C), treatment with trypsin and TCA of the crude enzyme extract which lacked antifungal property also. Treatment of the chilli seeds with Bacillus sp. culture showed 100 % germination index similar to the untreated seeds. The treatment of the seed with co-inoculation of the pathogen with Bacillus sp. culture showed 65 % reduction in disease incidence by the treatment as compared to the seed treated with pathogen alone (77.5 %).

SUBMITTER: Ashwini N

PROVIDER: S-EPMC3964249 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAs one of the main pathogens causing tea anthracnose disease, Colletotrichum gloeosporioides has brought immeasurable impact on the sustainable development of agriculture. Given the adverse effects of chemical pesticides to the environment and human health, biological control has been a focus of the research on this pathogen. Bacillus altitudinis GS-16, which was isolated from healthy tea leaves, had exhibited strong antagonistic activity against tea anthracnose disease.MethodsThe antifungal mechanism of the endophytic bacterium GS-16 against C. gloeosporioides 1-F was determined by dual-culture assays, pot experiments, cell membrane permeability, cellular contents, cell metabolism, and the activities of the key defense enzymes.ResultsWe investigated the possible mechanism of strain GS-16 inhibiting 1-F. In vitro, the dual-culture assays revealed that strain GS-16 had significant antagonistic activity (92.03%) against 1-F and broad-spectrum antifungal activity in all tested plant pathogens. In pot experiments, the disease index decreased to 6.12 after treatment with GS-16, indicating that strain GS-16 had a good biocontrol effect against tea anthracnose disease (89.06%). When the PE extract of GS-16 treated mycelial of 1-F, the mycelial appeared deformities, distortions, and swelling by SEM observations. Besides that, compared with the negative control, the contents of nucleic acids, protein, and total soluble sugar of GS-16 group were increased significantly, indicating that the PE extract of GS-16 could cause damage to integrity of 1-F. We also found that GS-16 obviously destroyed cellular metabolism and the normal synthesis of cellular contents. Additionally, treatment with GS-16 induced plant resistance by increasing the activities of the key defense enzymes PPO, SOD, CAT, PAL, and POD.ConclusionsWe concluded that GS-16 could damage cell permeability and integrity, destroy the normal synthesis of cellular contents, and induce plant resistance, which contributed to its antagonistic activity. These findings indicated that strain GS-16 could be used as an efficient microorganism for tea anthracnose disease caused by C. gloeosporioides.

Project description:In the present study, Paenibacillus dendritiformis, Trichoderma harzianum, and Trichoderma asperellum were appraised as potential biocontrol agents that induce resistance in chilli (Capsicum annuum) against the devastating pathogen Colletotrichum truncatum, which causes anthracnose. Bright-field and scanning electron micrographs showed the hyphal degradation, lysis, and abnormal swelling in C. truncatum against P. dendritiformis in a dual plate assay. Under greenhouse conditions, chilli seeds pretreated with P. dendritiformis, T. asperellum, T. harzianum, and T. asperellum + T. harzianum by soil soak method inflicted an induced systemic resistance (ISR) in chilli against a C. truncatum-challenged condition. In chilli, the disease index percentage was significantly reduced in the T. asperellum + T. harzianum-treated seeds, followed by the T. harzianum-, T. asperellum-, and P. dendritiformis-treated seeds as compared to the untreated and challenged, respectively. Chilli seeds were primed with T. asperellum + T. harzianum (78.67%), which revealed maximum disease protection under the challenged condition, followed by T. harzianum (70%), T. asperellum (64%), and P. dendritiformis (56%) as compared to untreated and C. truncatum-challenged (6%) condition served as control. The seeds that were pretreated with biocontrol agents (BCAs) inflicted ISR against C. truncatum by enhancing the activity of defence-related enzymes (superoxide dismutase (SOD), peroxidase (POX), polyphenol oxidase (PPO), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and phenylalanine ammonia-lyase (PAL)), accumulating phenolic compounds, and increasing the relative chlorophyll content in chilli. Nitroblue tetrazolium (NBT) and 3,3'-Diaminobenzidine (DAB) stains were used to detect the accumulation of superoxide anion and hydrogen peroxide that appeared nearby the fungal infection sites. The accumulation of reactive oxygen species (O2- and H2O2) in the pathogen-inoculated leaves was a maximum of 48 hpi, followed by P. dendritiformis, T. asperellum, T. harzianum, and T. asperellum + T. harzianum treated tissue upon C. truncatum-challenged condition as compared to the control. Overall, our results showed the potential of T. harzianum, T. asperellum, and P. dendritiformis as biocontrol agents that prevent infection by C. truncatum and inflict an induced systemic resistance in chilli by enhancing the biosynthesis of phenolic compounds, defence and antioxidative enzymes, and reducing the lesion development and reactive oxygen species accumulation. This is the first report of induced systemic resistance against anthracnose in chilli obtained by application of T. harzianum, T. asperellum and P. dendritiformis, through seed priming.

Project description:The ascomycete fungus Colletotrichum truncatum is a major phytopathogen with a broad host range which causes anthracnose disease of chilli. The genome sequencing of this fungus led to the discovery of functional categories of genes that may play important roles in fungal pathogenicity. However, the presence of gaps in C. truncatum draft assembly prevented the accurate prediction of repetitive elements, which are the key players to determine the genome architecture and drive evolution and host adaptation. We re-sequenced its genome using single-molecule real-time (SMRT) sequencing technology to obtain a refined assembly with lesser and smaller gaps and ambiguities. This enabled us to study its genome architecture by characterising the repetitive sequences like transposable elements (TEs) and simple sequence repeats (SSRs), which constituted 4.9 and 0.38% of the assembled genome, respectively. The comparative analysis among different Colletotrichum species revealed the extensive repeat rich regions, dominated by Gypsy superfamily of long terminal repeats (LTRs), and the differential composition of SSRs in their genomes. Our study revealed a recent burst of LTR amplification in C. truncatum, C. higginsianum, and C. scovillei. TEs in C. truncatum were significantly associated with secretome, effectors and genes in secondary metabolism clusters. Some of the TE families in C. truncatum showed cytosine to thymine transitions indicative of repeat-induced point mutation (RIP). C. orbiculare and C. graminicola showed strong signatures of RIP across their genomes and "two-speed" genomes with extensive AT-rich and gene-sparse regions. Comparative genomic analyses of Colletotrichum species provided an insight into the species-specific SSR profiles. The SSRs in the coding and non-coding regions of the genome revealed the composition of trinucleotide repeat motifs in exons with potential to alter the translated protein structure through amino acid repeats. This is the first genome-wide study of TEs and SSRs in C. truncatum and their comparative analysis with six other Colletotrichum species, which would serve as a useful resource for future research to get insights into the potential role of TEs in genome expansion and evolution of Colletotrichum fungi and for development of SSR-based molecular markers for population genomic studies.

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Potentiality of Bacillus subtilis as biocontrol agent for management of anthracnose disease of chilli caused by Colletotrichum gloeosporioides OGC1.

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