The studies on hrpF(+) and hrpF(–) strains of Xanthomonas spp. from cruciferous plants

• Main Authors: Shau-Chang Huang, 黃紹昌
• Other Authors: Yung-An Lee
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/61829468696493827778

碩士 === 輔仁大學 === 生命科學系碩士班 === 101 === The Xan-D medium was used for the isolation of xanthomonads from cruciferous seeds. The wet-shining, convex and yellow-green bacterial colonies on the Xan-D media were supposed to be xanthomonads and chosen for further experiments. The cabbage leaves were inoculated with the isolated xanthomonad strains. Some strains that could cause black rot symptoms were X. campestris pv. campestris (XCC), and some strains causing leaf spot symptoms were X. campestris pv. raphani (XCR). Some other strains that did not produce any disease were non-pathogenic xanthomonads. The isolated XCC and XCR strains could amplify a specific DNA fragment in a PCR using hrpF primers (called XCC or XCR-hrpF(+) strains), but nonpathogenic xanthomonads did not produce any fragments (called Xan-hrpF(–) strains). The Xan-hrpF(–) strains were indeed identified as Xanthomonas spp. by 16S rRNA gene sequence analysis, Biolog and PCR tests. In addition to seeds, XCR-hrpF(+) and Xan-hrpF(–) strains were isolated from radish tissues with bacterial leaf spot symptoms. The molecular typing patterns of isolated XCR-hrpF(+) and Xan-hrpF(–) strains were determined by pulsed-field gel electrophoresis (PFGE), and the results showed that XCR-hrpF(+) and Xan-hrpF(–) strains are heterogeneous. To understand the effect of hrpF on the pathogenicity of XCC-hrpF(+) and XCR-hrpF(+) strains, XCC and XCR hrpF-knockout mutant strains were constructed by EZ-Tn5 transposon mutagenesis and marker exchange methods. The XCC hrpF-knockout strain only reduced the virulence on the inoculated cabbage leaves, but the XCR hrpF-knockout strain did not cause any symptoms and totally lost the pathogenicity. Therefore, the lack of hrpF might not cause pathogenic xanthomonads to lose their pathogenicity. The Xan-hrpF(–) strains were further found that the strains not only lost hrpF but also lost the entire pathogenicity island and some effector protein genes determined by PCR, Southern hybridization and whole genome sequence draft analyses. Xan-hrpF(–) strains did not induce the hypersensitive response (HR) on the tobacco leaves (Nicotiana benthamiana). Although Xan-hrpF(–) strains did not cause diseases, they could survive at least seven days inside the cabbage leaves. Xan-hrpF(–) and XCR hrpF-knockout strains could reduce the symptom development of XCC-hrpF(+) when co-inoculated on cabbage leaves with XCC-hrpF(+), indicating that both Xan-hrpF(–) and XCR hrpF knockout strains could suppress the virulence of XCC-hrpF(+).