蓝莓炭疽病病原菌鉴定及致病性测定

Identification and Pathogenicity of Pathogen Casuing Anthracnose on Vaccinium

【目的】鉴定辽宁省部分地区疑似炭疽病的蓝莓病株的病原,为蓝莓病害的防控及抗病育种提供依据。【方法】对采自辽宁省兴城市和庄河市的疑似炭疽病的蓝莓发病枝条及叶片进行组织分离、培养,从所分离获得的两类菌株中选择形态学不同的代表菌株LNSW1和B-Cg1进行后续试验和分析。观察两个代表分离菌株在马铃薯葡萄糖琼脂培养基(potato dextrose agar,PDA)上的菌落及分生孢子的形态特征。对代表菌株LNSW1和B-Cg1的真菌核糖体基因进行PCR扩增和测序,采用Mega5.1软件中邻位加入法(neighbor-joining,NJ)进行基于病原菌rDNA-ITS基因序列和GenBank中已有相关炭疽菌序列的系统发育树构建。利用尖孢炭疽菌(Colletotrichum acutatum)和胶孢炭疽菌(C.gloeosporioides)的特异性引物对CgInt2/ITS4和CgInt/ITS4对代表菌株进行特异性扩增,采用菌丝块叶片接种法对8个蓝莓品种进行代表分离菌株的致病性测定。【结果】从辽宁省部分蓝莓产区罹病植株上共分离纯化得到36个菌株,根据各菌株的形态学差异将其分成两个类别,两类别中代表菌株LNSW1和B-Cg1在菌落颜色及分生孢子形态上具有较大差异,且与已报道的炭疽菌属当中的尖孢炭疽菌和胶孢炭疽菌形态特征相似;两个代表分离株的rDNA-ITS序列实际长度为557和547 bp,通过基于真菌核糖体基因的系统发育分析,表明菌株LNSW1与GenBank中尖孢炭疽菌菌株AB729126、KF698729、EU886755聚在同一分支,B-Cg1与胶孢炭疽菌菌株JF923828、KF516931、GU174547聚在同一分支,相似度分别为99%—100%;两对炭疽菌特异性引物扩增结果再次证明所获代表菌株分别为尖孢炭疽菌和胶孢炭疽菌;致病性测定结果表明,尖孢炭疽菌和胶孢炭疽菌菌丝块接种8个蓝莓品种的叶片及枝条均可致病,两类病菌回接症状与田间自然发病症状较为相似,尖孢炭疽菌对蓝莓的致病稍强于胶孢炭疽菌。【结论】通过对辽宁省蓝莓主产区的蓝莓发病枝条及叶片的病原菌进行形态学和分子学鉴定,基本明确辽宁省部分地区的蓝莓炭疽病是由尖孢炭疽菌和胶孢炭疽菌侵染所致。

[ Objective ] The objective of this study is to identify the pathogen causing stem and leaf anthracnose on Vaccinium in Liaoning, and to provide an IPM strategy for disease control and resistance breeding. [Method] Diseased samples were collected from Xingcheng and Zhuanghe County, Liaoning Province and disinfected stem and leaf pieces were placed on acidified potato dextrose agar （PDA） plate. Two represent isolates LNSW1 and B-Cgl were chosen for morphological and molecular study. Isolates were characterized by colony morphology, conidial characteristics under 28~C in dark. The internal transcribed spacer （ITS） region of the ribosomal DNA （ITSI-5,8S-ITS2） of isolates LNSW1 and B-Cgl were amplified and sequenced. The consensus ITS sequences of isolates LNSW1 and B-Cgl were compared with Colletotrichum spp, associated sequences in GenBank. Mega5.1 was used to draw the phylogenetic tree of isolates LNSW1, B-Cgl and other related sequences. Another molecular identification of the two isolates were carried out with amplification of species-specific primer pair Calnt（Calnt2）/ITS4. Pathogenicity of both species was verified by mycelium plug inoculation on eight attached Vaccinium cultivars. [Result] Thirty-six Colletotrichum isolates were collected from Liaoning Province, two different groups were divided by morphology. Represent isolates LNSW1 and B-Cgl were different in morphology of colony and conidia, and were skeptically identified as C. acutatum and C. gloeosporioides. The rDNA-ITS sequences length of isolates LNSW1 and B-Cgl was 557 and 547 bp. The phylogenetic analyses showed that isolate LNSWl and C. acutatum isolates AB729126, KF698729, and EU886755 were divided into one group, and isolate B-Cgl and C. gloeosporioides isolates JF923828, KF516931, and GU 174547 were divided into another group, the homology of each isolate in two groups was between 99%-100%. Additionally, the species-specific amplification results showed that both primer pairs could clearly distinguish the two different isolates. Pathogenicity tests results showed that the two Colletotrichum spp. could both cause anthracnose lesions on leaves and stems of Vaccinium. The pathogenicity of C. acutatum was a little higher than C. gloeosporioides on Northern Highbush Vaccinium. [Conclusion] Identification of the pathogen was based on morphology of colony and conidial, pathogenicity tests, nucleotide sequence of rDNA-ITS and species-specific primer pair Calnt（Calnt2）/ITS4. The typical anthracnose symptoms of Vaccinium in Liaoning Province were described in this study, disease diagnosis and pathogen identification was carried out based on the morphological characteristics, molecular technique and pathogenicity tests. Combined these results, it was confirmed that the causal agent of Vaccinium anthracnose in Liaoning were C. acutatum and C. gloeosporioides.