基于rDNA-ITS和大亚基的甘蔗凤梨病菌分子鉴定及序列分析

【目的】分析甘蔗凤梨病菌Ceratocystis paradoxa（de Seynes）Moreau]核酸r DNA-ITS和大亚基区域序列,为甘蔗凤梨病菌的快速、有效分子检测提供技术支持。【方法】从发生凤梨病的新台糖22号（ROC22）地下种茎分离获得1株菌株FL-1,根据柯赫氏法则测定其致病性;将该病菌在PDA培养基进行培养,观察其菌落形态特征,并在光学显微镜下观察分生孢子梗、厚垣孢子、分生孢子的形态特征。用引物ITS1/4和NL1/4分别对该菌株的核酸rD NA-ITS和大亚基序列进行扩增,以MEGA软件的Neighor-joining方法分别构建系统发育树,并用p-distance进行遗传距离分析。【结果】分离菌株FL-1为甘蔗凤梨病致病菌,具有典型的节孢子和厚垣孢子,形态特征与奇异长喙壳菌（C.paradoxa）一致。从菌株FL-1基因组DNA中分别扩增获得499 bp rD NA-ITS和563 bp大亚基片段,其序列均与C.paradoxa其他菌株的同源性为100.0%。系统聚类分析结果表明,两条序列均与C.paradoxa聚为一个独立簇。遗传矩阵分析结果表明,rD NA-ITS和大亚基均与C.paradoxa聚合在一起,种内遗传距离分别为0-0.017和0-0.008,而Ceratocystis属内种间遗传距离分别为0.015-0.106和0.002-0.059。结合分子检测结果和形态学特征,将FL-1鉴定为C.paradoxa。【结论】来自甘蔗的FL-1菌株是C.paradoxa复合群的成员,并被划分在进化复合群的分枝1上。rD NA-ITS和大亚基适合用于甘蔗凤梨病的PCR检测及分子标记。

Identification and sequences analysis of Ceratocystis paradoxa causing sugarcane pineapple disease based on rDNA-ITS and large subunit

【Objective】The nucleotide sequences of r DNA-ITS and large subunit of Ceratocystis paradoxa（de Seynes） Moreau causing sugarcane pineapple disease, were analyzed in order to provide technological supports for rapid and effective molecular detection of the pathogen in sugarcane production. 【Method 】A representative strain FL-1 was isolated from ROC22 underground seedcane with pineapple disease. The pathogenicity of strain FL-1 to sugarcane plants was determined by artificial inoculation based on Koch ＇s postulates. The morphological characteristics of FL-1colony grown on PDA medium were observed, as well as its conidiophores, aleurioconidia and conidia under light microscope. The nucleotide sequence regions of r DNA-ITS and large subunit were amplified by using primers ITS1/4 and NL1/4. The phylogenetic tree based on ITS and large subunit sequences of strain FL-1, respectively, were constructed by using Neighbor-joining method, and their phylogenetic distance were analyzed using the p-distance program in software MEGA. 【 Result 】 The isolate FL- 1 was determined as the pathogen causing sugarcane pineapple disease ac-cording to the pathogenicity test, and it was charactered by typical arthrospores and aleurioconidia, which was consisted with that of C. paradoxa. The nucleotide sequences of r DNA-ITS and large subunit amplified from genome DNA of isolate FL-1 were 499 and 563 bp in length, respectively. And they shared 100.0% of homology with the fungus of C.paradoxa. The r DNA-ITS sequence was clustered with C. paradoxa in an absolute evolution group in the phylogenetic tree, as well as the large subunit sequence. The genetic distance matrix results showed that r DNA-ITS and large subunit sequences of isolate FL-1 were genetically clustered with C. paradoxa, and the intraspecific genetic distance were0-0.017 and 0-0.008, respectively. While, the interspecific genetic distance of Ceratocystis were 0.015-0.106 and0.002-0.059, respectively. Therefore, FL-1 strain was identified as C. paradoxa based on