| Summary: | 碩士 === 輔仁大學 === 生命科學系碩士班 === 97 === The most common bacterial diseases in Taiwan Phalaenopsis are soft rot and brown spot, caused by Acidovorax avenae subsp. cattleyae and Erwinia chrysanthemi. Recently, we found another bacterial disease. It has similar symptom to soft rot or brown spot. It will confuse us in disease diagnosis. The purpose of this study was to identify the pathogenic bacterium and to develope deferential medium or detective paper of A. avenae subsp. cattleyae, E. chrysanthemi and the bacterium. Furthermore we discussed the symptom of pathogenic bacterium and it’s yellow toxin affect the host. We used cloning, sequencing and sequence alignment of the 16S rRNA gene and found the unknown bacterium has 99-100% idendity of Burkholderia gladioli. But the 16S rRNA gene sequences of B. gladioli, B. plantarii and B. glumae are similar to each other. The identity of the isolated bacterial strains was confirmed by species-specific polymerase chain reaction using the primer pair B. plantarii (Bpl-PL), B. glumae (Bglu-GL), B. gladioli (Bgla-LP, Bgla-GLA), revealed only Bgla-LP and Bgla-GLA amplified correct fragment. We identified the isolated bacterial strains using physiological and biochemical tests, showed it is also similar to B. gladioli, but nitrate reduction test had difference. We inoculated B. gladioli strains in Taiwan Phalaenopsis, found that all isolates have pathogenicity. We have developed a differential medium of E. chrysanthemi, NGM. A. avenae subsp. cattleyae, E. chrysanthemi and B. gladioli grew on the differential medium as white, blue green and yellow. We have developed a selective paper to detect E. chrysanthemi in plant soft rot tissues. We found that Phalaenopsis tissue infected with B. gladioli or E. chrysanthemi present yellow or blue halo in the paper. There were two symptoms when we inoculated B. gladioli strains in Taiwan Phalaenopsis. One of them was smaller, extending slower was similar with A. avenae subsp. cattleyae. Another one was larger, extending faster was similar with E. chrysanthemi. B. gladioli strains have different morphology in Luria agar, Nutrient agar, NG, and NGM medium. By using pulsed-field gel electrophoresis for molecular typing of XbaI enzyme digestion of six isolated bacterial strains, it was found that the isolates from Taiwan Phalaenopsis strains contain at least 4 molecular types. We knew that B. gladioli can produce a yellow toxin called toxoflavin. Discussing the relationship between the toxin and Phalaenopsis symptom infected by B. gladioli. B. gladioli were grown in medium, centrifuged and filtered to get the supernatant. We confirm it was toxoflavin by thin layer chromatography (TLC) using retention factor (Rf). We designed two specific primer pairs from B. glumae toxoflavin, ToxA and ToxB. After amplification, all of B. gladioli strains could amplify correct fragment of ToxB, but only a few strains could amplify correct fragment of ToxA. We inoculated the supernatant in Phalaenopsis found that will produce similar symptom with bacterium inoculation.
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