毛豆露菌病之發生及分子偵測

The occurrence of downy mildew of vegetable soybean and its detection by PCR

"自2000年起，台灣高雄五號毛豆果莢發生不知名病害，經鑑定為露菌病。2001年於雲林縣元長鄉發現R6階段果莢外表並不呈現病斑，與健康果莢比較，僅在種子突出部位稍微變黃，但剝開果莢後可觀察到其內部組織變紅褐色，佈滿菌絲與卵孢子，種子表面亦覆蓋有菌絲與卵孢子，鏡檢證實是由Peronospora manshurica (Naoum.) Syd. ex Gaum.所引起。田間採回R6階段高雄五號毛豆果莢內之P. manshurica卵孢子，以無菌水調成2,000 oospores/ml，將卵孢子懸浮液接種至高雄五號、十號品種毛豆之2葉齡幼苗，經14日後，未能觀察到幼葉出現病斑及被覆孢子囊梗及孢子囊。卵孢子在水瓊脂（water agar）平板上經14日培養，仍未見發芽。但卵孢子以2,3,5-triphenyltetrazolium chloride染色，約34%仍具有活性。田間採集之R6階段罹病莢果，在實驗室內經風乾30日後，取出種子播種，經14日後，亦未能觀察到幼葉出現病斑或被覆孢子囊梗及孢子囊。P. manshurica以引子對ITS1/ITS4，經由PCR之後，增幅出888 bp片段，由此序列設計引子對PM3/PM4，經由PCR用於偵測P. manshurica時，可在感染露菌病之豆莢得到一283 bp產物，其解序結果確含括在r DNA之5.8S至ITS2。利用此專一性引子對進行PCR增幅時，其靈敏度可達1ng之基因體核酸的濃度。"

"Downy mildew, a consistently occurring foliar disease of soybean, caused some defoliation, reduces seed quality and size, and may lower yield. Kaohsiung No.5, a new cultivar of vegetable soybean, developed in 1996, was found that downy mildew infected not only leaves but also pods and seeds in spring season from 2000 to 2001. This study confirmed that the stained pods and seeds of vegetable soybean that was caused by P. manshurica. The sequence data showed only 1 % nucleotide difference in the ITS1, 5.8S, and ITS2 sequences of Peronospora manshurica in this study and P. manshurica from NCBI accession no. AB021711 posted by Saito, M. in 1999. A specific detection method based on polymerase chain reaction（PCR）was developed for identification of P. manshurica. Samples were subjected to 30 cycles of amplification. Each cycle consisted of denaturation for 1 min at 92℃, annealing for 45s at 55℃, and primer extension for 30s at 72℃. Amplified product of DNA was examined in an agarose gel stained with ethidium bromide. With the primer pair PM3/PM4, an expected 283-bp fragment was amplified only from vegetable soybean pods infected with P. manshurica, but not from healthy seeds of vegetable soybean or other tested fungi. The minimal amount of template DNA could be amplified was about 1 ng."