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【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。
【Objective】 The objective of this study was to analyze the r DNA-ITS and the large subunit region sequences of the nucleic acid of Ceratocystis paradoxa (de Seynes) Moreau and provide technical support for the rapid and effective molecular detection of the pathogen. 【Method】 One strain, FL-1, was isolated from underground stalks of ROC22 in pineapple disease and its pathogenicity was determined according to Koch’s rule. The bacteria were cultured in PDA medium and observed The morphological characteristics of the colonies were observed. Morphological characteristics of conidiophores, chlamydospores and conidia were observed under a light microscope. The primers NA1 / 4 and NL1 / 4 were used to amplify the rD NA-ITS and large subunit sequences respectively. The phylogenetic tree was constructed by using Neighor-joining method of MEGA software and the genetic distances were analyzed by p-distance . 【Result】 The isolate FL-1 was a pathogen of sugarcane pineapple, with typical spores and chlamydospores. Its morphological characteristics were consistent with that of C. pararadoxa. The 499 bp rD NA-ITS and 563 bp large subunit fragments were amplified from the genomic DNA of FL-1 strain respectively. The sequence of the amplified fragment was 100.0% identical to other strains of C.paradoxa. The results of phylogenetic tree analysis showed that both sequences clustered with C. pararadoxa as an independent cluster. Genetic matrix analysis showed that both rD NA-ITS and the large subunit were polymerized with C. pararadoxa, the intraspecies genetic distances were 0 to 0.017 and 0 to 0.008 respectively, while the interspecific genetic distances within the genus Ceratocystis were 0.015-0.106 And 0.002 ~ 0.059. According to the results of molecular detection and morphological features, FL-1 was identified as C.paradoxa. [Conclusion] The FL-1 strain from sugarcane is a member of the C. paradoxa complex and is divided on branch 1 of the evolutionary complex. rD NA-ITS and Large Subunit Suitable for PCR Detection and Molecular Markers of Sugarcane Pineapple Disease.