Application of PCR technique for detection soft rot Erwinia in Taiwan

• Main Authors: Tsai, Chia-Ling, 蔡佳玲
• Other Authors: Hsu Shih-Tien
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/65854317737212626148

碩士 === 國立中興大學 === 植物病理學系 === 86 === The polymerase chain reaction (PCR) using primer pair 5A / 5B(designed from the sequences of E. chrysanthemi (Ech) pecS gene,controlling the expression of the pectinase, cellulase and pigmentationgenes) was applied to identify Ech: A a specific 500 bp DNA fragment wasamplified using the template DNAs of all 25 Ech isolates tested. Whenusing a primer pair Ec1 / Ec2 (designed from E. carotovora pel gene,encoding pectic lyase) in PCR reaction, a specific 434 bp DNA fragmentwas amplified from the templat e DNAs from all 20 Ecc isolates tested.In the detection of rotten plant tissues artificially inoculated withEch or Ecc by the PCR assay, pretreatedment with 0.5 N NaOH, 0.5 N NaOH+5 % PVP (polyvinypyrrolidone) agents, or by differential speeds ofcentrifugation may increase detection sensitivity depending on kinds ofplant species tested in this study. The presence in Ech or Eccpopulation of non-target bacteria including Pseudomonas putida, P.aeruginosa, and some other bacteria showed no effect on the sensitivityof the PCR assay. But the irrigation water from agricultural fields inChanghua and Taichung areas, or the extracts of soil and peat mosscontained in PCR reaction mixture reduced the sensitivity of thedetection assay. The treatment of extracts from soil and peat moss bypassing through Sephadex G-200 improved the sensitivity of PCR assay.The PCR assay using the primer pairs 5A / 5B or Ec1 / Ec2 with theproper conditions used in this study was successfully applied toinvestigate the etiology of soft rot plant tissues collected either fromthe markets or from crop fields. Among 36 samples collected frommarkets, two samples were diagnosed to be Ech and 34 samples were Eccpathogen, and these results agreed with those by diagnosis with theselective media. Among 22 samples from the crop fields, 3 samples werediagnosed to be Ech, and 9 samples were Ecc pathogen. Out of 10 sampleswhich were not detectable using PCR assay, 6 samples of soft rot gingerswere caused by non erwinia bacteria as diagno sed by the selectivemedia, and the rest 4 samples were Ecc diagnosed by the selective mediaapproach. In conclusion, the PCR technique using primer pairs 5A / 5Band Ec1 / Ec2 has a great potential to be used for rapid and correctdiagnosis of Ech and Ecc causing diseases.