Establishment of Monitoring System for Soil-borne Plant Pathogens by Gene Marking

在这三年的研究中，一些土传植物病原菌成功地转化了特异的标记基因，并通过检测引入的标记基因的表达来监测它们在土壤中(感染前)和植物中(感染后)的侵染行为。为了评价该方法的可行性和有效性，作者采用了常规培养方法无法区分的尖孢镰刀菌不同菌种，并实际尝试对每一种真菌进行追踪和鉴别。在本研究中，尖孢镰刀菌(F.oxosum(F.oxy.)F.sp.番茄属(Lycopsici，F.oxy)F.sp.碎片区，F.oxy。F.sp.甜瓜和F.oxy。F.sp.分别用潮霉素抗性基因、β-葡萄糖苷酸酶基因、荧光素酶基因和绿色荧光蛋白基因转化菠菜)。通过电穿孔将这些基因导入这些真菌的微孢子中，并用原位杂交检测基因的表达。在本系统的实际方法中，将寄主植物移植到已导入基因标记病原菌的土壤中，并在种植后一定时间间隔从土壤和植物组织中检测受感染的病原菌。本方法使我们能够通过检测用于每个标记基因的特异杂交的荧光标记的探针发出的不同荧光来同时区分镰刀菌的不同形态。因此，在本课题研究中，作者成功地建立了丝状土传植物病原真菌的基因标记监测方法。

In this reseasrch project for these three years, some soil-borne plant pathogens were successfully transformed with specific marker genes and their infection behavior in soil (before infection) and in planta (after infection into host plants) was monitored by detecting expression of the introduced marker genes. In order to evaluate feasibility or effectiveness of the present method, the author adopted different formae speciales of Fusarium oxysporum whic were indistinguishable one another by the conventional cultural method, and actually attempted to trace and distinguish each fungus. In this study, four formae speciales of F.oxysporum (F.oxy. f. sp. lycopersici, F.oxy. f. sp. fragarea, F.oxy. f. sp. melonis and F.oxy. f. sp. spinaciae) were separately transformed with four different marker genes, hygromycin-resistant gene, beta-glucuronidase gene, luciferase gene and green fluorescent protein gene. These genes were introduced into microconidia of these fungi by electroporation and the gene expression detected using an in situ hybridization. In the actual approach of the present system, host plants were transplanted in soil into which the gene marked pathogens had been introduced and the infected pathogens were detected from both soil and plant tissues as definate intervals after planting. The present method enabled us to simultaneously distinguish different formae speciales of the Fusarium pathogen by detecting different fluorescences emitted from the fluorochrome-labelled probes used for specific hybridization of each marker gene. Thus, the author succeeded in establishing the gene-marking method for monitoring filamentous soil-borne phytopathogenic fungi in the present project research.