Antagonistic Mechanism Of Bacillus Sp. On Plant Pathogeny And Cloning, Expression Of Antibiotic Protein Gene TasA

Twenty-four strians of Bacillus sp. were isolated from the rhizosphere of tomato plants, and these strains were tested for their antagonistic activities against plant pathogens. Twelve of them (AB, V3, SAI, 3-1, N8-b, V5, 25-1, 25-2, 25-5, 25-9, 25-10 and 25-17) showed strong inhibition activities to several plant fungal pathogens and bacteria. Two strians of B. sp. 25-1, 25-17 with strong antagonistic activities were selected for further tests on their suppression and inhibition mechanism on Colletotrichum musae, Rhizoctonia solani and Phytophthora capsici, especially on C. musae. The result showed that both strains had a strong antagonistic activity against C. musae on agar plates with an apparent inhibition zone. The germination rate of conidium was reduced by 53-69% when the culture filtrates from these two isolates were mixed with condia solution in glass slide. The conidia formation was inhibited on the PDA agar plate with the culture filtrates though the spores germinated and formed their hypha in certain degree. Growth suppression, increasing branches of their offset and swelling in the middle and/or the top of the hyphae were observed with microscope and scanning election microscope. The longer time the treatment, the more severe the hyphae aberration was, some of them were altered as beadlike, some offsets formed cluster-like structure. The hyphal wall became necrosis with the cytoplasm leakage, and some of them finally became global vacuole.TasA protein of Bacillus subtitlis was reported to have a broadspectrum of antibacterial activities. The TasA open reading frame (ORF) was PCR-amplified from 25-17, and cloned into pMD18-T. Sequence analysis of the clone indicated that the TasA ORF consists of 786 nucleotides, and shared 99.24% homology at nucleotide sequence, and 100% identitiy at amino acid sequence between it and the published gene TasA(corresponded with 143163~143948)from Bacillus subtilis(Accession~No:Z99116). The nucleotide sequence variations were base transitions (A/G or C/T substitution), and occurred on their third base of the codons in the ORF. The second structure and the amino acid sequence of TasA protein were predicted by using the softwarerhttp://npsa-pbil.ibcp.fr/cgi-bin/secpred mlr.pl).In order to understand the molecular function of TasA protein, the TasA gene was cloned into expression vector pET-TasA, and expressed in E. coli BL21. A unique 33kD fusion protein product was detected by SDS-PAGE, which indicated the TasA protein was expressed correctly.