| Summary: | 碩士 === 國立中興大學 === 植物病理學系 === 87 === Sixty different random primers were used to find specific DNA fragments of Acidovorax avenae subsp. citrulli by using random amplified polymorphic DNA (RAPD). A specific DNA fragment (about 300bp in size) of A. avenae subsp. citrulli amplified by the primer OPD-11 was cloned into the pCRII-TOPO vector, and sequenced to design primer pairs SL1/SR1. The primer pairs could amplify a distinct band of 194 bp that was specific to A. avenae subsp. citrulli isolated from watermelon, muskmelon and bitter gourd by polymerase chain reaction (PCR). And there was no any DNA fragment amplified with primer pairs SL1/SR1 from any other tested bacteria belonging to 16 species and 7 genus. The minimum amount of DNA from A. avenae subsp. citrulli that could be amplified by PCR was 100pg. Sensitivity of PCR for detection of cells of A. avenae subsp. citrulli with primer pairs SL1/SR1 was 1.2’102 cfu, and could be raised to 1.2 cfu by re-amplification of diluted standard PCR products (Second PCR). Nontarget bacteria WS, MS1 and MS2 did not affect the efficiency of specific amplification of A. avenae subsp. citrulli in PCR assay with primer pairs SL1/SR1. PCR technique with primer pairs SL1/SR1 identifies cultures of A. avenae subsp. citrulli within 4hours and detected the bacterium in diseased tissues and infested seeds of watermelon and muskmelon. The results indicated that the primer pairs SL1/SR1 could be a useful tool for rapid identification and detection of A. avenae subsp. citrulli in diseased plant tissues infected with A. avenae subsp. citrulli, and could also be potentially used for detection of contaminated seeds.
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