Development of specific PCR primers for the detection of Rhizoctonia solani AG 2-2 LP from the leaf sheaths exhibiting large-patch symptom on zoysia grass.
Development of specific PCR primers for the detection of Rhizoctonia solani AG 2-2 LP from the leaf sheaths exhibiting large-patch symptom on zoysia grass.
复制标题
开发特异性 PCR 引物，用于检测结缕草表现出大斑块症状的叶鞘中的立枯丝核菌 AG 2-2 LP。
DOI：
10.1016/s0378-1097(04)00016-3
复制
发表时间：
2004
影响因子：
2.1
通讯作者：
M. Hyakumachi
中科院分区：
文献类型：
--
作者：
T. Toda;T. Mushika;M. Hyakumachi
采用聚合酶链反应（PCR）技术对结缕草上引起大面积病害的立枯丝核菌AG 2-2 LP菌株进行了检测。根据随机扩增多态性DNA（RAPD）-PCR扩增的区域设计特异性引物。15个引物和3个培养类型的R.使用solaniAG 2-2（IIIB、IV和LP型）进行RAPD-PCR。RAPD-PCR的带型分析表明，3种培养类型的菌株有明显的区别。RAPD-PCR结果构建的树状图表明，AG 2-2的3个培养类型分别聚为一类。利用引物A09对LP分离物进行PCR扩增，选择其中一个仅在LP分离物中出现的片段设计特异性引物。引物对A091-F/R从LP分离株的纯真菌DNA中得到单一产物，但从其他两种类型（IIIB和IV）R. solaniAG 1、2-1、2-3、2-tulip、3-10和BI分离物以及其它草坪草真菌病原体。引物对A091-F/R也从病叶鞘中扩增出单一产物，该产物与LP分离物的纯DNA产物一致。引物对A091-F/R没有从健康叶鞘中产生PCR产物。用引物A091-F/R对结缕草叶鞘进行PCR扩增，检出率高于常规分离技术。结果表明，利用特异引物A091-F/R建立的PCR技术可用于大斑病结缕草叶鞘LP分离物的快速检测。
Detection ofRhizoctonia solaniAG 2-2 LP isolates causing large-patch disease on zoysia grass was done using polymerase chain reaction (PCR). Specific primers were designed based on an amplified region using random amplified polymorphic DNA (RAPD)-PCR. Fifteen primers and three cultural types ofR. solaniAG 2-2 (types IIIB, IV and LP) were used for RAPD-PCR. The banding patterns by RAPD-PCR showed that the three cultural types were clearly distinguishable. A dendrogram constructed from the results of RAPD-PCR showed that the three cultural types of AG 2-2 clustered separately. The sequence of one PCR-amplified region which appeared only in LP isolates using primer A09 was selected for designing specific primers. Primer pair A091-F/R gave a single product from pure fungal DNA of LP isolates but not from those of the other two types (IIIB and IV),R. solaniAG 1, 2-1, 2-3, 2-tulip, 3–10 and BI isolates and other turfgrass fungal pathogens. Primer pair A091-F/R also gave a single product from diseased leaf sheaths and this product was in accordance with those of pure fungal DNA of LP isolates. Primer pair A091-F/R did not yield PCR product from healthy leaf sheaths. The frequencies of detection of LP isolates from leaf sheaths of zoysia grass using PCR with primer pair A091-F/R were higher than those of the conventional isolation technique. These results showed that the PCR-based technique using specific primers A091-F/R is useful for the rapid detection of LP isolates from leaf sheaths of zoysia grass exhibiting large-patch symptoms.