向日葵黑茎病双重PCR检测实用化研究

Leptosphaeria lindquistii is one of the important quarantine fungal diseases of sunflower. So far, it is mainly identified and characterized by its morphological traits at home and abroad. Singlet and multiplex PCR molecular detections of Leptosphaeria lindquistii based on its ITS and Actin gene have also been reported. But most of them were detection methodological studies adopting purified fungal strains. However, the application studies concerning with the detection sensitivity, actual detection of infected sunflower tissues have not yet been done. In this research, the strain BXC1 of Leptosphaeria lindquistii separated from the rural field of Baxiang Village, Xinjiang Province and preliminarily identified by its ITS amplification and sequencing, was used for duplex PCR detection, sensitivity test and mock detection of mixed sunflower DNA and the fungal DNA. Then, the duplex PCR detections on the seedlings of the compatible and incompatible sunflower varieties challenged by BXC1 with different inoculation methods were conducted when its typical symptom appeared. Results showed that both the genomic DNA of BXC1 and its mixture with sunflower genomic DNA could produce the fungal ITS and the Actin gene specific bands, 580 bp and 255 bp respectively by the duplex PCR. In addition, the mixture DNA could also produce the sunflower ITS specific band (ca.740 bp), indicating the excellent specificity of this established duplex PCR detection of Leptosphaeria lindquistii. Using the gradually diluted BXC1 DNA or sunflower DNA or the mixture of both as templates to perform the duplex PCR, a sensitivity of 0.05ng fungal DNA/μL in 20μL PCR reaction was detected; either the compatible or the incompatible sunflower varieties, intact seedling inoculated or needle prick inoculated, with or without typical symptoms, all the inoculated stem portions could produce the fungal ITS and the Actin gene specific bands by the established duplex PCR detection. These results indicate that the duplex PCR detection of Leptosphaeria lindquistii is specific, reliable, convenient and can be used directly for the quick detection of Leptosphaeria lindquistii pathogen in the infected sunflower tissues.