Establishment of the basis on molecular taxonomy of phytopathogenic fungi

采用脂肪酸分析和限制性片段长度多态性（RFLP）分析相结合的生化方法，对5种丝核菌（Rhizoctonia spp.）利用气液色谱法对水稻纹枯病菌（R.solani）、水稻纹枯病菌（R. ericidae）、水稻纹枯病菌（R.oryzae-sativae）、烟霉病菌（R.fumigata）和白叶枯病菌（R.candida）等7个种内类群（包括13个种内类群）进行了鉴定。鉴定的主要脂肪酸是棕榈酸、棕榈油酸、油酸和亚油酸。全细胞脂肪酸的百分比组成的基础上的主成分分析显示，一致的低变异性之间的单一物种的菌株和5个丝核菌属之间的显着差异。相似的结果，主成分分析提出了低变异性之间的菌株相同的AG和7个AG之间的差异的R. solani。平均连锁聚类分析也表明， 关于我们 同一种丝核菌的菌株和同一株立枯丝核菌的同一AG菌株在树状图上形成独立的聚类，根据聚类图可以区分不同种丝核菌和不同AG的立枯丝核菌。然而，所有的ISG属于AG-1，AG-2和Ag-4分离物区分的平均连锁聚类分析。根据脂肪酸组成的百分比，AG-1有3个ISG，AG-2有5个ISG，AG-4有2个ISG。对5个丝核菌属和7个代表立枯丝核菌13个ISG的AG-1 IA菌株的28 S rDNA基因进行了RFLP分析。PCR扩增产物为1.4和1.8 kirobase对片段。用限制性内切酶消化28 SrDNA后获得的RFLP图谱因所用的4种酶MspI、HhaI、Sau 3AI和HaeIII而异。MspI和Hhal酶切的RFLP图谱显示5种丝核菌的特异性片段多态性。在PCR扩增的rDNA产物的变化和多态性的4种限制性内切酶消化也观察到7个AGs的R. solani。这些差异主要存在于AG-1和AG-2的ISG之间。从RFLP图谱中得到的树状图显示，来自AG-1和AG-2的ISG可以各自被细分为不同的组，这些组与其他AG的大多数分离物远缘。立枯丝核菌AG-1 IA菌株与其它立枯丝核菌ISG和AG菌株具有特异的RFLP图谱，且与其它ISG和AG菌株存在系统发育差异，脂肪酸分析和RFLP分析结果表明，立枯丝核菌AG-1 IA菌株与其它立枯丝核菌ISG和AG菌株在系统发育上存在显著差异。少

Biochemical methods consisting of fatty acid analysis and restriction fragment length polymorphism (RFLP) analysis were performed for the objective indicator for identification and characterization of Rhizoctonia spp.and R.solani AGs.Percentage composition of whole-cellular fatty acids of 5 Rhizoctonia spp. (R.solani, R.oryzae, R.oryzae-sativae, R.fumigata and R.candida) and 7 AGs including 13 intraspecific groups (ISGs) of R.solani were characterized by gas-liquid chromatography. The major fatty acids identified were palmitic, palmitoleic, oleic and linoleic acids. Principal componet analysis based on the percentage composition of whole-cellular fatty acids revealed consistently low variability among isolates of a single species and significant differences among 5 Rhizoctonia spp.Similar results by principal component analysis were presented low variability among isolates of an identical AG and differences among seven AGs of R.solani. Average linkage cluster analysis also showed that … More the isolates in the same species of Rhizoctonia spp.and an identical AG of R.solani formed individual clusters on the dendrogram, and each species of Rhizoctonia and different AGs R.solani couls be distinguished based on the dendrogram. However, all of the ISGs belonging to the AG-1, AG-2 and Ag-4 isolates were differentiated by the average linkage cluster analysis. Based on percentage composition of fatty acids, 3ISGs in AG-1,5 ISGs in AG-2, and 2 ISGs in AG-4, were presented respectively. Isolates of R.solani AG-1 IA showed specific and significant composition of whole-cellular fatty acids from other AGs and ISGs of R.solani.Isolates of 5 Rhizoctonia spp.and 7 AGs representing 13 ISGs of R.solani were characterized by the RFLPs analysis of a 28S rDNA gene amplified by the polymerase chain reaction (PCR). Amplification products by PCR reaction were 1.4-and 1.8-kirobase pair fragments. RFLP profiles obtained after digestion of 28S rDNA with the restriction enzymes were different depending on the 4enzymes, MspI,HhaI,Sau3AI and HaeIII,used. The RFLP profiles by the digestion with MspI and Hhal showed specific restriction fragments polymorphism among 5 Rhizoctonia spp. Variations in the PCR-amplified rDNA products and the polymorphisms on digestion with 4 restriction enzymes were also observed among 7 AGs of R.solani. These differences were maily conseved among ISGs of AG-1 and AG-2. A dendrogram derived from the RFLP profiles showed that ISGs from AG-1 and AG-2 can each be subdivided into distinct groups, that are distantly related to the majority isolates of the other AGs. Isolates of R.solani AG-1 IA showed specific RFLP profiles and phylogenetic differences from the other ISGs and AGs of R.solani.These two nethods, fatty acid analusis and RFLP analysis, clearly concluded the significant difference among isolates of Rhizoctonia spp. Less