Original£ºCotton Science£¬2006£¬18(6)£º327-331
Indirect ELISA Method for Detection of Verticillium Abstract£ºApart from condign climate and lack of resistant varieties£¬one of the important reasons causing Verticillium wilt more and more serious is seed-orne pathogen. To establish an effective method to detect Verticillium dahliae in cottonseeds, we have developed an indirect ELISA method£®The toxin of Verticillium dahliae strain Bp2 was used as antigen to prepare polyclonal antiserum£®The indirect ELISA method was developed by using alkaline phosphatase-onjugated goat antimouse antibody as secondary antibody according to standard ELISA procedures£®If the value of P/N (ratio of OD405 of a sample to that of negative control) ¡Ý 2£¬the sample was positive£®The titer of the antiserum was 1©s204800 and the toxin could be detected as 1ow as 7.81 ¦Ìg¡¤L-1£®A11 of Verticillium dahliae strains, collected from different areas in Jiangsu Province, performed positive reaction. 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 . After the Verticillium dahliae strain Bp2 or T9 was cultured for one day£¬or the toxin could be detected in the culture solution£®Two of strains were cultured after three days£¬the value of P/N was 9.05 and 8.82, respectively£¬and these high values could remain for 11 days£®So Verticillium dahliae in cottonseeds could be deduced by detecting the toxin with this developed indirect ELISA£®81 cottonseed samples, randomly chosen from different seed companies, were detected if there was Verticillium dahliae with them. Firstly, 100 g cottonseeds from each sample were washed by glide£®Coated cotton-seeds were washed till the chemical coating was moved clearly£®Then, the cottonseeds were dried at room temperature and separated to five shares each£®Each share was put in 150 mL Czapek£§s 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 by using indirect ELISA. 12 out of 81 cottonseed samples performed positive reaction£®Cotton seedlings were infected by Verticillium wilt inoculated with the culture solution of the cottonseed samples that performed positive reaction£®These results demonstrated that the method of indirect ELISA could be used for quickly detection of Verticillium dahliae in cottonseeds£®
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