油菜黑胫病菌和茎基溃疡病菌的LAMP检测方法的建立

 油菜黑胫病和油菜茎基溃疡病分别由子囊菌Leptosphaeria biglobosa和L. maculans引起。我国油菜产区仅发现L. biglobosa,未发现L. maculans。因而, L. maculans是我国的对外检疫性对象。这两种真菌形态相似,引起的病害症状相似,给田间快速准确鉴定带来难度。本研究基于环介导等温扩增技术(Loop-Mediated Isothermal Amplification, LAMP),建立了快速检测L. biglobosa和L. maculans的方法。根据ITS-rDNA序列设计了5条检测L. biglobosa的LAMP引物和6条检测L. maculans的LAMP引物,检测L. biglobosa和L. maculans的最适温度为65℃,反应时间分别为40 min和50 min。两组引物检测特异性高,检测的模板DNA极限达到fg级,与常规PCR检测相比,LAMP检测L. biglobosa和L. maculans灵敏度分别提高了100倍和1000倍。在病害样品实际检测中,采用两种LAMP体系,分别检测了9株罹病油菜茎秆中的病原菌。结果表明:所有茎秆中的病原菌均为L. biglobosa,没有检测到L. maculans,与特异性PCR检测结果一致。本研究建立的LAMP检测方法为快速高通量检测L. biglobosa和L. maculans奠定了基础。

Development of a LAMP method for detection of Leptosphaeria biglobosa and L. maculans in oilseed rape

Blackleg and stem canker of oilseed rape are two diseases caused by two closely-related ascomycetous fungi, namely Leptosphaeria biglobosa (blackleg) and L. maculans (stem canker). In China, only L. biglobosa has been found in oilseed rape-producing areas, whereas L. maculans has not been reported in this crop. Therefore, L. maculans is officially treated as a quarantine plant pathogenic fungus in China. Both fungi formed pycnidia and conidia with similar morphology, and produced similar symptoms, which can be observed until maturity. It is difficult to identify both diseases quickly and accurately in the field. Therefore, it is imperative to develop a rapid, sensitive and high throughput method for detection and identification of the two fungi. This study was done to establish an easy-to-use detection system based on the loop-mediated isothermal amplification (LAMP) principle. Five specific primers for L. biglobosa and six specific primers for L. maculans were designed based on the ITS-rDNA sequences of the two fungi. Results showed that the established LAMP methods for detecting L. biglobosa and L. maculans are optimally performed at 65°C for 40 min and 50 min, respectively, with high detection specificity. Results of the sensitivity test indicated that threshold of the template DNA for both fungi reached down to the fg level, which was 100 and 1 000 times lower than that in the conventional PCR detection of L. biglobosa and L. maculans, respectively. Results also indicated that the LAMP for L. biglobosa had a positive detection with DNA extracted from diseased stems of oilseed rape showing typical blackleg symptoms, whereas the LAMP for L. maculans had a negative detection from these diseased stem samples. This result suggests that the blackleg disease on oilseed rape was caused by L. biglobosa, but not by L. maculans, and identification of the pathogen (L. biglobosa) in the diseased stems was confirmed by the convenient PCR with L. biglobosa- and L. maculans-specific primers. Therefore, the LAMP techniques developed in this study have a promising potential for detection of both pathogens in diseased tissues of oilseed rape.