棉 花 学 报    Cotton Science   2006，18(6)：327-331

Indirect ELISA Method for Detection of Verticillium Dahliae in Cottonseed
LIN Ling¹，CHEN Zhi-shi¹，GONG Wei-rong²，ZHANG Ai-xiang¹，GU Gan-yu¹，GU Ben-kang¹
（1.Institute of Plant Protection，Jiangsu Academy of Agricultural Sciences，Nanjing 210014，China；2. Jiangsu Plant Protection Unit，Nanjing 210013，China）

Abstract：Verticillium wilt of cotton is a widespread and destructive disease that is caused by the fungus pathogen Verticillium 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 Verticillium dahliae  in cottonseed，an indirect ELISA method was developed. The toxin of Verticilillium 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 Verticilillium 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，Fusarium moniliforme，Fusarium graminearum ，Sclerotinia sclerotiorum，Magnaporthe grisea. The Verticilillium 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 Verticillium dahliae in them. 100g 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 temperature 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.
Key words：indirect ELISA；cottonseed；Verticillium dahliae；toxin    [Full Text, 2186KB]