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Detection of powdery mikiew fungus from winter buds by the polymerase chai* reaction using species-specifrc prinerset.

使用物种特异性 prinerset 通过聚合酶 chai* 反应检测冬芽中的粉状 mikiew 真菌。

基本信息

批准号：
09660055
负责人：
SATO Yukio
金额：
$ 2.11万
依托单位：
College of Technology, Toyama Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

Powdery mildew fungi generally overwinter by cleistothecla. On the other hand, the powdery mildew fungus of crape myrtle (Lagerstroemia indica) is known to overwinter in winter buds of the plant. If the fungus latently infecting the winter buds can be detected, we can obtain the information as to when the winter buds are infected with infection frequency of the winter buds. This may clarify the life cycle of this fungus, which leads to the possibility of disease forecasting. We thus tried to develop the detection method of powdery mildew from the winter buds of crape myrtle by the polymerase chain reaction using species-specific primer set. The results are as follows.1)DNA sequences of the ITS regions including 5.8S rDNA were determined for various powdery mildew fungi Based on the sequences, four primers specific to the powdery mildew fungus (Erysiphe australiana) of crape myrtle were constructed 2) Only the DNA of the powdery mildew of crape myrtle was amplified by the specific primer set, which indicates the usefulness of the primer set 3) Some faint bands were detected when fungal isolates from leaves of crape myrtle were subjected to the PCR amplification using the specific primer set. Some of these bands could not be removed even if the annealing temperature was changed 4)PCR-RFLP using the restriction enzyme Xhol I was useful to differentiate foe crape myrtle powdery mildew from the fungal isolates. 5) The minimum limit of detection the primer set SA2/SA3 was 0.0025pg DNA for the crape myrtle fungus, 6) Danas successfully extracted from the leaves of crape myrtle by using Dneasy Plant Mini- Kit combined with cleaning by Hepese.

白粉病真菌一般以闭囊藻越冬。另一方面，众所周知，紫薇（Lagerstroemia indica）的白粉病真菌在植物的冬芽中越冬。如果能够检测到潜伏侵染冬芽的真菌，就可以获得冬芽何时被侵染的信息以及冬芽的侵染频率。这可能会阐明这种真菌的生命周期，从而带来疾病预测的可能性。因此，我们尝试开发使用物种特异性引物组的聚合酶链反应检测紫薇冬芽白粉病的方法。结果如下： 1）确定了多种白粉病真菌ITS区的DNA序列，包括5.8S rDNA 在此基础上构建了4个针对紫薇白粉病真菌（Erysiphe australiana）特异的引物 2）该特异性引物组仅扩增了紫薇白粉病菌的DNA，说明该引物组的有用性 3）一些微弱的引物组当使用特定引物组对紫薇叶中的真菌分离物进行 PCR 扩增时，检测到条带。即使改变退火温度，其中一些条带也无法去除。 4) 使用限制性酶 Xhol I 的 PCR-RFLP 可用于区分紫薇白粉病和真菌分离株。 5)引物组SA2/SA3对紫薇真菌的检测最低限为0.0025pg DNA。6)利用Dneasy Plant Mini-Kit结合Hepese清洗，成功从紫薇叶中提取Danas。