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

在这三年的研究项目中，成功地将一些土壤传播的植物病原体转化为特定的标记基因，并通过检测引入的标记基因的表达来监测它们在土壤（感染前）和植物（感染宿主植物后）中的侵染行为。为了评价该方法的可行性或有效性，作者选取了传统培养方法难以区分的尖孢镰刀菌的不同菌种，对每种菌种进行了实际的追踪和区分。本文研究了尖孢霉(f.o.y。番茄属植物；f. p. fragarea, f. oxy。f. p.甜瓜和f. oxy。分别用抗潮霉素基因、β -葡糖醛酸酶基因、荧光素酶基因和绿色荧光蛋白基因4个不同的标记基因对spinaciae F. sp.进行转化。通过电穿孔法将这些基因导入真菌的微分生孢子中，原位杂交检测基因的表达。在本系统的实际方法中，将寄主植物移栽到已引入基因标记病原体的土壤中，并在种植后以确定的间隔从土壤和植物组织中检测感染的病原体。本方法通过检测荧光标记探针对每个标记基因特异性杂交所发出的不同荧光，使我们能够同时区分镰刀菌的不同种类。因此，笔者在本项目研究中成功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.