Establishment of immunocapture PCR method to detect soil-borne plant pathogen Pythium myriotylum and P. violae

• Main Authors: Chen-Yen Chung, 鍾成晏
• Other Authors: Pi-Han Wang
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/87941952552252686197

碩士 === 東吳大學 === 微生物學系 === 88 === An immunocapture-polymerase chain reaction (IC-PCR) based method was developed to detect Pythium myriotylum Drechsler and P. violae Chester & Hickman which caused ginger rot and carrot cavity spot, respectively. Polyclonal antibody against P. myriotylum was purified from rabbit immune serum with a titer of 1:4000. The cross-reactivity was widespread among 28 Pythium species. and fungal isolates of other 14 genera. There was 0.25%, 0.43%, and 2.5% oospores of P. myriotylum recovered by immunocapture (IC) from 10 g natural soil with 105, 104 or 103 oospores, respectively. Immunocapture was an efficient method to capture a wide range fungal species from soil samples, and then, target fungus could be detected by specific PCR. Twenty oospores or 12.5 fg genomic DNA of P. myriotylum, and 15 fg P. violae genomic DNA could be detected specifically by booster PCR with primer pairs MY5/ITSII and PV1/ITSII. P. myriotylum was able to be detected as few as 102 oospores g-1 from 10 g natural soil sample by IC-PCR. The fungus was detected from five of 19 field soil samples by IC-PCR that could not be isolated by baiting. The IC-PCR technique was a rapid and sensitive method for detecting plant pathogens from soil within 12 hours.