Studies on the identification of Bacillus subtilis Y1336 and Streptomyces candidus Y21007 by molecular technique and its effectiveness on inhibition of plant pathogens

• Main Authors: Chia-Ping Shih, 石佳平
• Other Authors: Hui-Liang Wang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/45188755080533775887

碩士 === 國立高雄師範大學 === 生物科技系 === 96 === Bacillus subtilis are known as a biopesticide which can inhibit plant diseases because they can secrete iturin A. In this study, we design four pairs of primers to amplify partial sequence of ituA, ituB, ituC and ituD genes in iturin A operon. Genomic DNA of ten Bacillus subtilis strains were obtained by these primers for PCR test, partial sequence of ituA, ituB and ituD genes of nine strains are produced, and partial sequence of ituC gene of seven strains were also obtained. In addition, according to 3ʹ conserved sequence of 16S rDNA and 5ʹ conserved sequence of 23S rDNA of genus Bacillus, we design a pair of primers Bacillus 16S F and Bacillus 23S R to proceed PCR test. In electrophorisis analysis six of seven Bacillus subtilis strains were amplified. So the five pair of primers can be used as a molecular tool to identify Bacillus subtilis. In RAPD analysis six pairs of random primers were able to differentiate Bacillus subtilis Y1336 from other nine Bacillus subtilis strains. Many species of genus streptomyces can secrete antibiotic borrelidin to inhibit plant disease. In the study, we design five pair of primers to amplify borrelidin genes of genomic DNA of Streptomyces candidus Y21007, and partial sequence of borA3, borA4 and borA5 genes of borrelidin synthesis were amplified. We also amplified ITS sequences of Streptomyces candidus Y21007 by using Streptomyces 16S F and Streptomyces 23S R. 370 bp cDNA were sequenced and compared with other ITS sequences in NCBI. It has the highest identity to ITS of Streptomyces griseus. In this study, we also test the effect of inhibition of Bacillus subtilis Y1336 and Streptomyces candidus Y21007 to Phytophthora capsici Leonian, Phytophthora palmivora, Botryodiplodia theobromae Pat., Botrytis cinerea, Fusarium oxysporum, Botyodiplodia theobromae and Phygophthora capsici L on PDA plates. They could inhibit plant pathogens well except Phytophthora capsici Leonian. However a mutant strain which were irradiated by UV light and could produce more borrelidin with better effect on inhibiting Phytophthora capsici Leonian.