咖啡褐斑病菌的分离鉴定及其培养特性测定

Isolate,Identification and Cultivation Characteristics of the Pathogen of Coffee Leaf Brown Eye Spot

为了明确调查中发现在咖啡苗圃以及咖啡幼树中出现的一种疑似咖啡褐斑病的病原菌以及发生条件,本研究通过采集咖啡病叶,按照柯赫氏法则进行分离,同时利用引物ITS1/ITS4及H3F/R对病原菌进行分子鉴定,最后利用平板培养法测定不同温度、光照和pH对菌丝体生长的影响。最终共分离获得5个分离物,其菌落特征、分生孢子梗及分生孢子形态无明显差异,形态特征与尾孢相一致。5个菌株均能对中粒种咖啡致病,再分离获得的菌株其菌落形态、分生孢子梗及分生孢子特征均符合尾孢特征。ITS及组蛋白H3基因序列分子鉴定结果表明,ITS树不能鉴别5个菌株的分类地位,尤其CF13的分类地位。而基于H3基因序列构建的系统发育树中,5个待鉴定菌株与11株咖啡生尾孢菌C.coffeicola聚为一支。结合分离物形态特征,将其鉴定为咖啡生尾孢菌(C.coffeicola)。培养特性测定结果表明:该菌生长的温度范围为13℃~35℃,最适温度为25℃~28℃;菌丝体生长的pH值为3~11,最适pH值为6~7;光照条件对菌丝体生长的影响不明显。

A suspected brown eye spot disease was founded in coffee nurseries and young coffee trees during the investigation.In order to identify the pathogen and their occurrence conditions,this study was conducted by collecting typical diseased coffee leaves,isolating and observing according to Koch's Rule,and using primers ITS1/ITS4 and H3F/R to identify the pathogen.On the other hand,the influence of different temperatures,light intensities and pH levels on mycelium growth was measured on PDA plate.Finally,a total of 5 isolates were obtained,and there was no significant difference in the conidiophores and conidia morphology of the isolates.All the five strains were pathogenic to Robusta coffee,and the morphology characteristics of the re-isolated strains were consistent with those that were earlier observed.The molecular identification results of ITS and histone H3 gene sequences showed that ITS tree could not identify the taxonomic status of five strains,especially CF13.In the phylogenetic tree constructed based on the H3 gene sequence,the sequence of the five strains was closely related to all the 11 sequences of Cercospora coffeicola,and clustered into one clade.Finally,combined with the results of morphological observation,the target strain was identified as C.coffeicola.The results showed that the growth characteristics analysis revealed that the optimal conditions for laboratory culturing of C.coffeicola isolates comprise temperature ranging from 13℃to 35℃,and the optimum temperature was 25℃to 28℃.The pH of mycelium growth was 3 to 11,and the optimum pH was 6 to 7.The effect of light on mycelium growth was not obvious.