中国主要茶区茶树炭疽菌系统发育学

doi:10.3864/j.issn.0578-1752.2015.24.007

摘要/Abstract

摘要： 【目的】炭疽菌能够侵染茶树叶片并造成病叶干枯、脱落，论文旨在分离、鉴定中国主要茶区茶树炭疽病的病原菌，为茶树病害防治工作提供科学依据。【方法】利用单孢分离法对采自中国15省（市、自治区）茶区的茶树炭疽病病叶进行炭疽菌分离，并对其ITS、TUB2两个位点进行PCR扩增和测序，测序结果采用MEGA 6.0软件以邻接法（Neighbor-Joining，NJ）构建多基因位点系统发育树。对分离的菌株在马铃薯葡萄糖琼脂培养基（potato dextrose agar，PDA）上的形态特征进行描述。对龙井43和中茶108茶树品种叶片进行有伤和无伤处理后接种代表性菌株，测试其致病性。【结果】以茶树上分离的炭疽菌菌株的2个基因位点序列（ITS、TUB2）构建多基因位点系统发育树，Colletotrichum boninense CBS 123755作为外群，结果表明所有菌株聚为3支，其中45个菌株与C. camelliae聚为一族、28个与C. fructicola聚为一族、5个与C. siamense聚为一族，所有菌株均归属于C. gloeosporioides复合种，并且C. camelliae在中国茶区分布范围最广，为茶树炭疽菌的优势种。形态特征结果表明，C. camelliae在PDA培养基上生长速度平均11.8 mm·d^-1，菌落白色，气生菌丝致密，绒毛状，中央凸起，边缘整齐；菌核、刚毛未见；分生孢子梗、产孢细胞未见；分生孢子透明，光滑，圆柱状，两端钝圆或基部渐尖，（8—15）µm×（3—6）µm；菌丝附着胞褐色，棍棒状，不规则状，有分枝，（8—10.5）µm×（6.5—8）µm；分生孢子附着胞未见。C. fructicola在PDA培养基上生长速率平均6.76 mm·d^-1，菌落正面白色，背面中央褐色，边缘白色，气生菌丝致密，绒毛状；菌核、刚毛未见；分生孢子梗透明，有隔，具分枝，产孢细胞圆柱状，7—18 µm，顶端直径1—2 µm；分生孢子透明，圆柱状，两端钝圆，部分中部溢缩，（10—15）µm×（3—3.5）µm；菌丝附着胞深褐色，圆形，近圆形，不规则形，有分枝，（6.5—8）µm×（3.5—5.5）µm；分生孢子附着胞褐色或深褐色，圆形，（5—7）µm×（5—6.5）µm。C. siamense在PDA培养基上生长速率平均7.6 mm·d^-1，菌落白色，气生菌丝致密，绒毛状；菌核、刚毛未见；分生孢子梗透明，有隔，具分枝；产孢细胞圆柱状，8—16 µm，顶端直径1—2 µm；分生孢子透明，圆柱形，梭形，两端钝圆，部分中部有溢缩，（9.5—13.5）µm×（3—3.5）µm；菌丝附着胞深褐色，球形，棒状，不规则形，（5—8）µm×（3—5.5）µm。致病性测试结果表明，C. camelliae能侵染龙井43品种茶树有伤叶片，但对龙井43无伤叶片和中茶108有伤、无伤叶片无致病性，而C. fructicola和C. siamense 2个种对2个品种茶树有伤、无伤叶片均无致病性。【结论】基于系统发育学和形态学结合的方法对中国15省（市、自治区）部分茶区茶树病叶病原菌鉴定，基本明确C. camelliae为中国茶树炭疽菌优势种。

关键词: 炭疽病, 茶树, 分子系统发育学, 中茶108, 龙井43, 致病性

Abstract: 【Objective】 Anthracnose pathogens can infect tea leaves, causing extensive necrosis of leaves and defoliation. The objective of this study is to identify the pathogens causing anthracnose on tea plants from the major tea areas in China, and to provide an important scientific basis for tea plant disease management in the future. 【Method】 Colletotrichum species associated with anthracnose diseases of tea plants were isolated using single spore isolation and confirmed by DNA sequencing of the partial sequences of β-tubulin and rDNA-ITS region. Neighbor-Joining analysis was performed to establish the multilocus phylogeny using MEGA 6.0. The morphology of isolated strains growing on fresh PDA plates was also described. Pathogenicity of typical isolates was further verified by inoculation assay on the tea varieties of ‘Longjing 43’ and ‘Zhongcha 108’ with and without wound treatments. 【Result】 A total of 78 Colletotrichum strains were isolated from tea plants in 15 provinces (municipality, autonomous region) of China. The 2-locus (ITS, TUB2) dataset for phylogenetic analysis results showed that isolates clustered in three clades, 45 isolates clustered with the ex-type isolate of C. camelliae, 28 isolates clustered with C. fructicola, 5 isolates clustered with C. siamense and all isolates belonged to the C. gloeosporioides species complex. Moreover, the results showed that C. camelliae was the most widely distributed species in the tea regions of China, reflecting that it was the dominant Colletotrichum species on Camellia sinensis. Meanwhile, morphological analysis showed that C. camelliae colonies on PDA growth rate 11.8 mm·d^-1, aerial mycelium white, dense, cottony; setae and sclerotium were not observed; conidiophores and conidiogenous cells were not observed; conidia hyaline, smooth-walled, cylindrical with obtuse ends, sometimes narrowed at the centre or towards the base, (8-15) µm×(3-6) µm; mycelial appressoria brown, clavate, irregularly shaped, branched, (8-10.5) µm×(6.5-8) µm; conidial appressoria were not observed. C. fructicola colonies on PDA growth rate 6.76 mm·d^-1, aerial mycelium white, reverse brown in the centre, white at the margin, dense, cottony; setae and sclerotium were not observed. Conidiophores hyaline, septate, branched; conidiogenous cells hyaline, cylindrical, (7-18) μm, apex (1-2) μm diam. Conidia hyaline, cylindrical, both ends rounded, sometimes narrowed at the centre, (10-15) µm× (3-3.5) µm; mycelial appressoria dark brown, cylindrical, (6.5-8) µm×(3.5-5.5) µm; conidial appressoria dark brown or brown, rounded, (5-7) µm×(5-6.5) µm. C. siamense colonies on PDA growth rate 7.6 mm·d^-1, white, dense, cottony; setae and sclerotium were not observed; Conidiophores hyaline, septate, branched. Conidiogenous cells hyaline, cylindrical, 8-16 μm, apex 1-2 μm diam. Conidia hyaline, cylindrical, fusiformis, sometimes narrowed at the centre, (9.5-13.5) µm×(3-3.5) µm; mycelial appressoria dark brown, rounded, clavate, irregularly shaped, (5-8) µm×(3-5.5) µm. Interestingly, pathogenicity test showed that the C. camelliae could cause anthracnose lesions on the wounded leaves of ‘Longjing 43’ but not on the intact leaves; on the contrary, there were no visible disease symptoms on the ‘Zhongcha 108’ leaves. Moreover, both C. fructicola and C. siamense did not show pathogenicity on these two tea varieties. 【Conclusion】 Based on the phylogenetic analyses and the morphological observation of Colletotrichum strains isolated from tea plants from 15 provinces (municipality, autonomous region) of China, it is concluded that C. camelliae is the main causal agent of Ca. sinensis anthracnose in the major tea areas of China.

Key words: anthracnose, tea plant, molecular phylogeny, Zhongcha 108, Longjing 43, pathogenicity

王玉春，郝心愿，黄玉婷，岳川，王博，曹红利，王璐，王新超，杨亚军，肖斌. 中国主要茶区茶树炭疽菌系统发育学[J]. 中国农业科学, 2015, 48(24): 4924-4935.

WANG Yu-chun, HAO Xin-yuan, HUANG Yu-ting, YUE Chuan, WANG Bo, CAO Hong-li, WANG Lu, WANG Xin-chao, YANG Ya-jun, XIAO Bin. Phylogenetic Study of Colletotrichum Species Associated with Camellia sinensis from the Major Tea Areas in China[J]. Scientia Agricultura Sinica, 2015, 48(24): 4924-4935.

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