向日葵黄萎病种子带菌研究

Detection of contamination of sunflower seed coats with Verticillium dahliae

为探究向日葵种子是否能够携带黄萎病菌进行远距离传播,以随机挑选的7个向日葵品种为研究材料,以种皮DNA为模板,利用真菌通用引物ITS1/ITS4和ITS区内侧的黄萎菌特异引物进行巢氏聚合酶链式反应(polymerase chain reaction,PCR),将所获得的PCR条带测序后在Gen Bank中进行比对。结果表明,供试的向日葵品种的种皮中所扩增出的PCR条带均来自黄萎病菌(Verticillium dahliae)。为了进一步确定上述检测结果,另挑选9个不同向日葵品种的种子,对其种皮带菌率进行测定。结果表明,供试的9个向日葵品种的种皮带菌率介于10.0%~25.0%之间,其中田间采集的3638C新鲜种子带菌率最低,仅为10.0%,而实验室留存的赤029X115R种子带菌率最高,为25.0%。为了进一步验证PCR实验结果的准确性,利用表达绿色荧光蛋白(green fluorescent protein,GFP)的黄萎病菌株接种向日葵植株根部,在荧光显微镜下对种皮进行观察。结果表明,在接种向日葵花盘上收获的种子的种皮上可以观察到GFP荧光信号。上述研究结果表明,向日葵种子的种皮可以携带黄萎病菌,而巢氏PCR可以作为一种检测向日葵黄萎病种子是否带菌的快速而可行的方法。

Sunflower Verticillium Wilt is a soil-borne disease caused by Verticillium dahliae, which can infect a variety of crops, resulting in a serious decline in both yield and quality. The conidia and microsclerotia in sunflower debris are the principal sources of infection. A nested polymerase chain reaction(PCR) was used to detect the contamination of V. dahliae in sunflower seed coats. Seven sunflower varieties were selected to isolate DNA from their seed coats, and then performed a nested PCR by using specific primers of V. dahliae and the universal primers of fungi(ITS1/ITS4). The PCR products were sequenced then BLAST-searched in Gen Bank.In order to confirm the PCR results, green fluorescent protein(GFP) labeled V. dahliae was used to inoculate sunflower roots. The seed coats were then removed from inoculated sunflower seeds and detected GFP signal under a fluorescence microscope. The results showed that all the amplified PCR bands belonged to V. dahliae isolated from the seed coats of the tested sunflower cultivars. Meanwhile, variable seed contamination ratios were found among different sunflower varieties. The lowest and highest contamination ratios were observed in3638 C(10.0%) and Chi029 X115 R(25.0%) varieties, respectively. Moreover, the GFP signals obtained on the seed coats of inoculated sunflowers further supported the PCR results. Based on the above results, we confirm that the sunflower seed coat is an important tissue for V. dahliae colonization, and it is the main carrier for the long distance transmission of the sunflower Verticillium Wilt. Additionally, we conclude that the nested PCR method is fast and highly reliable to detect V. dahliae contamination in sunflower seed coats.