Screening And Identification Of Bacterial Antagonists For Inhibition Of Appressorium Formation Of Colletotrichum Capsici And Antifungal Protein Isolation Of Strain C-D6

Anthracnose caused by Colletotrichum capsici is a major disease of tropical vegetables such as Capsicum annuum and Brassica parachinensis and caused heavy agriculture losses in south China. In order to explore new biocontrol strategy of anthracnose, potential bacterial biocontrol agents which can inhabit the formation of appressorium, was isolated and identified, and isolation of antifungal protein and detection of antibiotic gene were conducted. The results are as follows:1. Five strains, showed obvious antagonism to appressorium formation, were obtained from soils collected from Guangdong and Sichuan Province. These bacteria cells are rod-shaped, Gram-positive, and formed elliptic endospores. The 16SrDNA of 5 isolates were amplified using universal primers 8F/1492R and sequenced. These sequence were submitted for a BLAST search in the NCBI database, strain CF3, CG4, CH9 and EF4 showed 99% homology with the sequences of two reference isolates of Bacillus subtilis (Accession Nos. GQ861468 and FJ263018). The four strains and Bacillus subtilis formed a group in phylogenetic tree of the 16SrDNA sequences. Strain BB2 showed 98% similarity with the sequences of B. thuringiensis and B.cereus (Accession Nos. HQ917121, FJ483513) by BLAST search. Using specific primers YyaO-F/TetB-R of B. subtilis, a 600bp DNA fragment were amplified from strain CF3, CG4, CH9 and EF4. Base on morphological characteristics, analysis of 16SrDNA sequence and analysis of specific genes, strain CF3, CG4, CH9 and EF4 were identified as B. subtilis. Strain BB2 could not be defined any species by analysis of 16SrDNA sequence.2. A antifungal protein which can inhibit the appressorium formation was isolated from B. subtilis strain C-D6. The crude active protein was isolated from C-D6 culture filtrates by precipitated with 20% ammonium sulfate saturation, dialysis (MWCO 7000d) and Sephadex G-75 chromatography. The crude extract protein showed two peaks (280nm UV detection) in G-75 chromatography and only peak I has antifungal activity. The peak I was further purified by Q-Fastlow anion-exchange chromatography, three major peaks were yielded in chromatographic profile and only peakâ…¡has the biological activity. By HPLC detection, the peakâ…¡was a high purity protein and its molecular weight was about 32kDa.3. The ability of 31 bicontrol strains of Bacillus spp. to inhibit growth of C. capsici were tested and their inhibition rate ranged from 42.17% to 61.68%. These strains were iditified using specific primer PCR technique. The results indicated that 14 strains (P-H10, C-D6, C-G13, C-E1, A-H11, D-A4, H-E4, K-F7, B-G4,1B6, CF3, CG4, CH9 and EF4) and 5 strains (P-F7, D-E9, C-A1,1D11 and 4A6) could be amplified by specific primer YyaO-F/TetB-R of B. subtilis and YyaR-F/TetB-R of B. amyloliquefaciens, respectively. Detection of antibiotic gene of 31 strains were conducted using 10 specific primers. The results indicated that the numbers of strain contained Iturins, Bacilysin, Fengycin and TasA gene were 9,4,2 and3, respectively.