Studies on the development of differential medium and its differential mechanism for plant pathogenic Xanthomonas spp.

• Main Authors: Sung, Ai-Ning, 宋靄寧
• Other Authors: Lee, Yung-Ann
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/46863053810749855394

碩士 === 輔仁大學 === 生命科學系碩士班 === 96 === Xanthomonas spp. is an important seed-borne bacterial pathogen. Planting seed free of Xanthomonas spp. contamination is an important disease management strategy. The use of healthy seed is critical to control this bacterial disease. We have developed a differential medium of Xanthomonas spp., mTBM-42. On the mTBM-42, Xanthomonas spp. appeared as glistening, raised, and green colonies. Colonies of Xanthomonas spp. were surrounded by a large clear zone and a smaller milky zone. Yellow-pigmented non-xanthomonads isolated from plant leaves and seeds grew on the differential medium as yellow, flat, and without clear and milky zones. The mTBM-42 differential medium was used to isolate plant pathogen Xanthomonas spp. from infected seed. The purpose of this study was to improve the differential medium and study on its differential mechanism for plant pathogenic Xanthomonas spp. First, mTBM-42 were simplified for LA-based LSTB. On the LSTB, colonies of Xanthomonas spp. were green, glistening, raised, and surrounded by zone of smaller milky. According to the results, the hydrolysis of Tween 80 was related to green colonies and milky zone of Xanthomonas spp. on mTBM-42. A Tween 80-hydrolyzing esterase gene (estA) was cloned from Xanthomonas campestris pv. campestris XCC1-1. Sequence analysis of XCC-estA gene revealed high sequence identity to all of Xanthomonas tested., but no similar sequence of yellow-pigmented non-xanthomonads. We disigned a PCR primer pair, base on the XCC-estA gene. The PCR primer pair was specific to the genus Xanthomonas. Therefore, we can use the differential medium with the specific primers for detection of the plant pathogenic Xanthomonas spp. in plant seeds. Constructed E. coli BL21 (DE3) and E.coli Rosetta (DE3) pLysS could express a protein about 66 kDa, named XCC-EstA. Colonies of E. coli containing XCC-estA gene were green and surrounded by zone of smaller milky on mTBM-42. XCC-EstA antibodies can be used to detect the presence of XCC-EstA. The results showed that XCC-EstA was localized in the cell membrane. We produced estA gene-knock out XCC mutants. XCC mutant still have a zone of milky on mTBM-42, but the zone formation rate and colony morphology are different from wild type. According to the above results show that the esterase gene cloned from X. campestris pv. campestris XCC1-1, XCC-estA, was related to milky zone and green colony formation.