应用real-time PCR定量检测小麦条锈菌潜伏侵染量方法的建立

 小麦条锈病是我国小麦主要病害之一。快速、及时地诊断与定量监测处于潜育状态下的病叶,对准确估计越冬、越夏后的病情,制定正确的防治方案具有重要的意义。根据小麦条锈菌Puccinia striiformis的β-tubilin基因序列设计对该病原菌的种具有特异性的引物betaf/betar,并分别在普通PCR和real-time PCR扩增时对该引物的特异性和灵敏性进行了测定。结果表明该引物对小麦条锈菌特异性高,可稳定扩增出243 bp的目标条带。Real-time PCR的灵敏度为普通PCR的100倍。应用此特异性引物,建立了real-time PCR测定系统,定量测定了条锈菌在小麦叶片接种后组织内的DNA随时间的变化。结果表明,在接种后12 h,可在小麦叶片内检测到条锈菌,且条锈菌在小麦叶片内潜育期间随时间呈指数增长。接种第6 d后叶片内的菌量有明显的增加。建立的小麦条锈菌的real-time PCR早期定量测定方法,为及时、快速监测小麦条锈病在潜育期间的发病规律以及为该病的预测、防治提供依据。

Quantification of latent infections of wheat stripe rust by using real-time PCR

Wheat stripe rust caused by Puccinia striiformis is one of the most important wheat diseases in China. Early detection and monitoring of the development of latent infection are important in disease prediction and management. In this study, P. striiformis-specific primer pair, betaf/betar, was designed according to the sequence of β-tubilin gene to detect the pathogen existing in wheat leave as latent infection. Specificity of the designed primer pair was confirmed by testing the genome DNA of various species of wheat pathogens. A 243 bp fragment of genome DNA was stably amplified from P. striiformis, but not from the other wheat pathogens. The real-time PCR assay was generated to quantify the target DNA. Sensitivities of regular PCR and real-time PCR assay were compared by using serial dilutions of template DNA. The sensitivity of real-time PCR was about 100-times higher than that of regular PCR. The dynamics of P. striiformis in wheat seedlings were monitored as latent infection by using the real-time PCR assay. The result showed that P. striiformis could be detected as early as 12 h after inoculation and developed as exponential growth before the symptom appeared. This study demonstrates a potential application of real-time PCR in prediction and management of wheat stripe rust epidemics.