Application of modern biotechnology on the detection and diagnosis of plant pathogenic fungi

Abstract

植物檢疫與植物防疫是未來植物保護的工作重點之一。而植物病原真菌之偵測鑑定與診斷技術將是植物檢疫與植物防疫的工作一環“免疫技術(immunologica1 techniques)及核酸技術(nuc1eic acid techniques)進行植物病原真菌偵測、診斷與鑑定為一較新技術“血清技術常利用的方法計以多株抗體、單株抗體為反應基質進行偵測(polyclonal and monoclonal antibodies as reagents)、酵素連結免疫吸附測定分析法(enzyme-linked immunosorbent assay)、免疫螢光及免疫金蛋白標識測定分析（imrnunof-lorescence and immunogold labeling)、核酸技術包括核酸探針(nucleic acid probes)製作，及以偵測診斷為主之核酸探針雜合反應(nucleic acid hybridization based detection techniques)、點雜配分析(dot-blot hybridization assay)、限制酵素輿圖多型性分析(restriction fragment length polymorphisms analysis)、DNA聚合酵素連鎖反應增幅法( DNA polymerase chain reaction ）、任意引子DNA 聚合酵素連鎖反應增幅法(random amplified polymorphic DNA -RAPD)、去氧核醣核酸指紋分析法(DNA fingerprinting)等技術。近年來，由於為使形態相近的不同菌株（種），或同種不同亞種及生理小種的菌株能夠提供便利的鑑別技術，於是利用粒線體核酸之多複本(high copy number)特性與不同菌株問皆會有特殊的差異加以應用核酸限制酵素切割片段多型性(RFLP)分析種問及種內差異性及菌株之問粒線體核酸多型性及變異性，更進一步由粒線體核酸選殖技術篩選出與田間菌株皆可產生交互作用的專一性核酸探針，以期能應用在田間病害之偵測“目前在菌株（種）快速鑑別與類緣關係的研究探討上，以任意引子DNA 聚合酵素連鎖反應技術(RAPD)及DNA 聚合酵素連鎖反應(polymerase chain reaction, PCR)為快速靈敏且可大量應用於實際偵測診斷應用上。目前為止，新技術之應用使植物病原真菌之偵測、診斷與鑑定進入了新里程碑，其實際應用層面及輔助分類鑑定診斷與偵測將是另一值得開發的新科技。 Plant quarantine Plays an important role in the development of plant protection. Detection and diagnosis of plant pathogenic fungi is one of the major work of plant quarantine. Immunological techniques and nucleic acid techniques have been remarkable progress in the development of new techniques for detection and diagnosis of plant pathogenic fungi. Immunological techniques include polyclonal and monoclonal antibodies as reagents, enzyme-linked immunosorbent assay, immunoflorescence and immunogold labeling, and nucleic acid techniques include nucleic acid hybridization based detection techniques, dot-blot hybridization assay, restriction fragment length polymorphisms analysis, DNA polymerase chain reaction, random amplified, DNA-RAPP, DNA fingerprinting. The last 5-10 years have been remarkable progress in the development of new techniques for convenient identification of different isolates of similar morphology and physiological races, by using polymorphisms and variation of mitochondrial DNA, and further cloning of specific DNA probes for the detection and diagnosis of plant pathogenic fungi. At present, random amplified polymorphic DNA and polymerase chain reaction are the most speedy and sensitive methods for practical application on the detection and diagnosis of plantpathogenic fungi. Since now, new biotechnology have been progress for the detection and diagnosis, and further development of new technology for the identification, diagnosis and detection of plant pathogenic fungi