棉花黄萎病菌毒素ELISA检测方法的建立及其应用

Development of ELISA for a Verticillium Dahliae Phytotoxin and its Application

以纯化的棉花黄萎病菌毒素(PLPC)免疫新西兰白兔制备了PLPC特异性抗血清,建立了可特异性检测棉花黄萎病菌毒素的A蛋白双抗体夹心ELISA方法。应用建立的ELISA方法测定了病菌培养滤液、人工接种幼苗和大田病株不同组织中的毒素,结果表明:不同产毒能力菌株(VD 8和VD-5)在25℃下振荡培养3d后就能在培养滤液中检测到毒素,这比生物测定要早2～3d。将VD-8菌株的孢子以灌根方式接种泗棉3号幼苗5d后,就能从接种幼苗的茎杆和叶柄中检测到毒素,这比幼苗自然发病显症要早3～5d。在测定的30份大田病株茎杆、叶柄、叶脉样品中,阳性样品率为100%。这些结果表明建立的ELISA方法可用于菌株产毒能力的测定和大田棉花黄萎病的早期诊断。

A specific polyclonal antiserum was prepared against a purified phytotoxin from Verticillium dahliae of cotton, and a double antibody sandwich ELISA procedure was developed for detection of Verticillium dahliae phytotoxin (PLPC) in culture filtrates, different issues of inoculated cotton seedlings and naturally diseased plants. Results showed that the PLPC in culture filtrates was detectable by the developed ELISA when 2 strains (VD8 and VD5) were grown in Czapek's medium under shaking condition at 25℃ only for 3 days, which is 1～2 days earlier than that of the bioassay. The PLPC in stems and petioles of seedlings (cv. Simian 3) could be detected by the ELISA when the seedlings were inoculated with the spores (1×107 spores·ml1) of strain VD8 by watering roots only for 5 days, which are 3～5 days earlier than the time of symptom appearance. The rate of positive samples was 100% by the ELISA among 30 stems, petioles and veins from naturally diseased cottons. These results demonstrate that the developed ELISA can be used for early detection of PLPC in culture filters and early diagnosis of verticillium wilt of cotton. 