棉花落叶型黄萎病菌的检测技术

DETECTION OF DEFOLIATED STRAIN OF VERTICILIUM DAHLIAE

采用Nit硝酸还原酶缺陷型突变体技术,共得到棉花黄萎病菌Nit突变体301个,其中A型155个,B型97个,C型29个。共检测到落叶型棉花黄萎病菌11株,除以前报道的6株外,发现了5个新菌株。针刺接种后,在棉株上均引起典型的落叶型症状。通过聚丙烯酰胺凝胶电泳比较落叶型和非落叶型的8个代表菌株的蛋白质电泳图谱的差异,发现在浓度为7.5％的凝胶中,落叶型菌株有两条特异性的蛋白,其迁移率分别为Rf_5=0.135,Rf_9=0.405。用非落叶型菌株DF_4、落叶型菌株SY_(12)菌体的可溶性蛋白制备获得两个抗血清。用间接ELISA法检测,即能区分棉花黄萎病菌和立枯、枯萎、炭疽病菌,落叶型和非落叶型黄萎病菌均呈阳性反应,但落叶型黄萎病菌的OD值高于非落叶型黄萎病菌。

301 non-nitrate utilizing (Nit) mutants were obtained by nitrate reductase deficiency. The Nit mutants could be divided into four phenotypes by their growth on different suppliment minimal agar media. 155 mutants of Nit A (Nit 1) and 97 mutants of Nit B (Nit M) were majority of the Nit mutants. The number of Nit C mutants was 29. 11 strains of defoliated Verticillium dahliae were detected. Among them, five were new records. Typical defoliating symptom was obtained after pinned infection, which were corresponded to Puhalla's VE Group 1. All other strains were classified into non-foliating VC Group. The pathogenicity of the five strains was evaluated under controlled conditions. They caused typical defoliating symptom on cotton 20 days after inoculation. The PAGE result of six defoliation strains was compared with two non-defoliating strains. Two specific protein bands were found in 7.5% gel with Rf5 = 0.135, Rf9 = 0.405. Two antisera were obtained by im-mulizing four rabbits with the soluble protein of Verticillium dahliae strains DF4 (non-defoliating strain) and SY12 ( defoliating strain), respectively. The antisera were purified by saturated (NH4)2SO4. When the antisera were diluted to 1:800, the strains of V. dahliae could be distinguished from the strains of Rhissoctonia solani, Colletotrichum gossypii and Fusarium oxysporum f. sp. vasinfectum by both antisera. The OD value of defoliating strains was higher than the non-defoliation strains.