摘要
以棉花黄萎病菌Bp2培养液中毒素蛋白免疫BALB/C小白鼠制备抗血清,建立了可特异性检测棉花黄萎病菌的间接ELISA方法。所制备的毒蛋白抗血清效价为1∶204800,对毒蛋白的最低检测量达7.81μg.L-1。可以与江苏省各地采集到的棉花黄萎病菌的培养液发生特异性反应,而与其它致病菌培养液反应呈阴性。用该方法对81份江苏省抽样的棉子进行检测,12份呈阳性反应。取检测为阳性反应的这些棉子培养液针刺接种棉苗也全都发病。这些结果表明所建立的间接ELISA方法可用于棉子携带黄萎病菌的快速检测。
Verticillium wilt of cotton is a widespread and destructive disease that is caused by the fungus pathogen Verticilliurn dahliae. Except condign climate and lack of resistant varieties, one of the important reasons made the disease more and more serious is seed-borne pathogen. To establish an effective method to detect Verticilliurn dahliae in cottonseed, an indirect ELISA method was developed. The toxin of Verticililliurn dahliae strain Bp2 was used asantigen to prepare polyclonal antiserum. An indirect ELISA method was developed using alkaline phosphatase-conjugated goat anti-mouse antibody as secondary antibody according to standard ELISA procedures. If the value of P/N≥2, the sample was positive. P/N means the rate of OD405 of sample and OD405 of negative control. The titre of the antiserum was 1 ： 204800 and the toxin could be detected as low as 7.81 ng·mL^-1. All of Verticililliurn dahliae strains collected from different cities in Jiangsu Province performed positive reaction, and no cross-reactivity was observed among the isolates representing other species of fungi such as Rhizoctonia solani , Fusariurn rnonili forme , Fusariurn grarninearum , Sclerotinia sclerotiorurn , Magnaporthe grisea. The Verticililliurn dahliae strain Bp2 or T9 was cultured after 1 day, the toxin could be detected in the culture solution. Any of these two strains was cultured after 3 days, the value of P/N was 9.05, 8.82, respectively, and this high value could remain 11 days. So Verticilillium dahliae in cottonseed could be deduced by detecting the toxin with this developed indirect ELISA. 81 cottonseed samples were randomly chosen from different seed companies and were detected if there was Verticilliurn dahliae in them. 100 g cottonseeds from each sample were washed by glide. Coated cottonseeds were washed till the coating chemical was moved clearly. Then the cottonseeds were dried in room tem perature and separated by five shares each. Each share was put in 150 mL Czapek medium and then cultured for three days. The culture medium was centrifuged for 30 min at 5000 r ·min^-1 , and the supernatant was tested using indirect ELISA. 12 out of 81 cottonseed samples performed positive reaction. Cotton seedlings were infected Verticillium wilt by inoculated with the culture solution of the cottonseed samples that performed positive reaction. These results demonstrated that method of indirect ELISA could be used for quickly detection of Verticillium dahliae in cottonseeds.
出处
《棉花学报》
CSCD
北大核心
2006年第6期327-331,共5页
Cotton Science
基金
国家"十五"攻关项目(2001-BA509B05-02)
江苏省农业三项工程项目(SX(2004)098)
关键词
间接ELISA
棉子
黄萎病菌
毒素
indirect ELISA
cottonseed
Verticillium dahliae
toxin
