建立的筛选体系尽管解决了致病性相关突变体快速筛选的问题，但由于接种苗数较少，可能出现部分假阳性，这是由于在接种均一的前提下，棉花幼苗个体间对病原菌敏感性不一致和少量突变体悬浮液孢子浓度偏低（产孢能力受限的突变体）造成的。解决方法是将筛选出靶标突变体再次鉴定，以本研究鉴定的致病性相关突变体的比例（1344个突变体中69个致病力显著下降）测算，16000个突变体约有800多个致病力相关突变体，仅需不到1个循环即可获得重复验证结果，提高筛选的准确性；或者采用定量菌液法验证致病下相关突变体的可靠性，如蛭石沙土无底纸钵定量蘸菌液法[24]。同时，应用本筛选体系还需注意两个问题：一是筛选致病力下降不明显的突变体准确性偏差，建议对获得的靶标突变体进行二次筛选验证；二是对于致病力增强的突变体需要通过观察发病进程的来判断，且最好选用耐或者抗病的品种作为鉴定材料。

　　4结论

　　明确了引起棉花黄萎病的大丽轮枝菌孢子悬浮液浓度为大于5.0×105孢子/mL，建立了大丽轮枝菌的标准化培养和孢子悬液制备方法，甘油菌种活化培养5d，标准培养瓶扩大培养9d，确定了25mL灭菌水为适宜的孢子洗脱体积，定量制备的孢子悬浮液适合于接种棉花幼苗并引起黄萎病。在此基础上，建立了大丽轮枝菌致病相关突变体的快速筛选体系。应用该体系测试表明，1人1个循环7个流程可完成1344个突变体的快速筛选体系，鉴定周期仅54d，工作量仅为21人日，极大地缩短了筛选周期。同时，测试获得69个致病力显著下降的突变体，对筛选到的致病性相关突变体进行定量菌液接种验证，结果一致。研究建立的快速筛选体系适用于T-DNA随机插入突变体库的筛选鉴定，这将为后续大丽轮枝菌致病性相关突变体的批量筛选提供保障。

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