嗜果刀孢菌寄主范围的测定及PCR快速检测

Host Determination of The Wilsonomyces Carpophilus of Wild Apricot Forest of Tianshan and Establishment of a Rapid Detection System

[目的]探讨嗜果刀孢菌对新疆其他主要树种的潜在危害,提高该病害的诊断和检测效率。[方法]本文将嗜果刀孢菌通过离体叶片接种至6科19种植物的健康叶片上,初步测定其致病性和寄主范围;参考tef1序列设计了嗜果刀孢菌的特异性引物;通过常规PCR和实时荧光PCR对76株嗜果刀孢菌和22株其它真菌进行特异性和灵敏度检测,并优化实时荧光PCR的反应体系;另外,应用所设计的特异性引物对田间自然发病样本和人工接种7、10、13、16、19、21、24、48 h后的野杏叶片样品进行常规PCR检测。[结果]15种植物叶片在接种嗜果刀孢菌5 d后出现明显病斑,不同寄主的病斑大小存在极显著性差异(P<0.01),其中新疆小叶白蜡的病斑面积最大,达28.99 mm^(2),而紫叶矮樱、黄果山楂、新疆杨、鞑靼桑等4种植物叶片未发病。本研究设计的特异性引物W0404-14-F/W0404-14-R扩增出的特异性条带为113 bp,对照及其它真菌无扩增条带。常规PCR检测的灵敏度为5.99×10^(-1)ng·μL^(-1)。在野杏叶片接种10 h后通过常规PCR可在叶片内检测到嗜果刀孢菌。[结论]嗜果刀孢菌可为害15种其他树种,具有潜在危害性;PCR快速检测方法检测嗜果刀孢菌速度快、特异性强,本研究首次探索了嗜果刀孢菌潜在寄主范围及PCR快速检测方法,为由嗜果刀孢菌引起的穿孔病的诊断及检测提供了理论依据。

[Objective]To explore the potential hazard of W.carpophilus on other major tree species in Xinjiang,and to improve the diagnosis and detection efficiency of this disease,[Method]in this paper,W.carpophilus was inoculated into healthy leaves of 19 plants in six families by ex vivo leaf inoculation,and its pathogenicity and host range were preliminarily determined;Specific primers for W.carpophilus were designed with reference to the tef1 sequence;76 strains of W.carpophilus and 22 other fungi were spe-cifically and sensitively detected by conventional PCR and real-time PCR,and the reaction system of real-time PCR was optimized;In addition,the designed specific primers were used to perform conventional PCR on natural onset samples in the field.Samples and wild apricot leaf samples 7,10,13,16,19,21,24 and 48 h after artificial inoculation were used for routine PCR detection.[Result]The leaves of 15 plants inoculated showed obvious lesions after 5 d of inoculation,with extremely significant differences(P<0.01)in lesion size between different hosts,with the largest lesion size of up to 28.99 mm^(2) in Xinjiang leaflet white wax,and only four plants,namely,Prunus.cistena,Crataegus chlorocarpa,Populus alba var.pyr-midalis,Morus alba var.tatarica,were not involved.The specific primers W0404-14-F/W0404-14-R de-signed in this study were able to amplify a specific band at 113 bp for W.carpophilus but not for other fungi.The sensitivity of the conventional PCR assay was 5.99××10^(-1)ng·μL^(-1).W.carpophilus could be de-tected within the leaves 10 h after inoculation of Wild Apricot Leaves by conventional PCR.[Conclusion]W.carpophilus can harm 15 other tree species and is potentially harmful;PCR assay is rapid and specific for the detection of W.carpophilus.This study is the first time to explore the potential host range of W.car-pophilus and a rapid PCR assay for the diagnosis and detection of porokeratosis caused by W.carpo-philus.