大粒咖啡炭疽病病原菌形态和分子鉴定及杀菌剂筛选  

Morphological and Molecular Identification of Colletotrichum kahawae Causing Anthracnose of Coffea liberica and Fungicide Selection for the Disease Management

作　　者：李学俊 任圆 陈红梅 曲鹏 杜华波 何鹏搏 谭万忠 LI Xuejun;REN Yuan;CHEN Hongmei;QU Peng;DU Huabo;HE Pengbo;TAN Wanzhong(College of Tropic Crop,Yunnan Agricultural University,Pu’er,Yunnan 655099,China;College of Plant Protection,Yunnan Agricultural University,Kunming,Yunnan 650100,China;Yunnan Yanshan Craneberry Agriculture Co.,Ltd.,Wenshan,Yunan 663100,China;West Yunnan University,Lincang,Yunnan 677000,China)

机构地区：[1]云南农业大学热带作物学院,云南普洱655099 [2]云南农业大学植物保护学院,云南昆明650100 [3]云南砚山曼悦莓农业有限公司,云南文山663100 [4]滇西科技师范学院,云南临沧677000

出　　处：《热带作物学报》2024年第6期1273-1281,共9页Chinese Journal of Tropical Crops

基　　金：云南省科技厅重点研发计划项目——云南省咖啡产业绿色发展国际联合研发中心(No.202303AP140010);云南省教育厅基础研究项目(No.2023Y0911)。

摘　　要：大粒咖啡是重要的遗传种质资源和风景园林植物,在我国部分地区有少量种植。咖啡炭疽病是影响咖啡生长发育和产量的重要真菌病害。本研究对感染炭疽病的大粒种咖啡叶片采用组织分离法进行分离,纯化后进行组织培养,利用柯赫氏法则验证其致病性,结合菌株的形态学特征和多基因序列系统发育分析对病原菌进行鉴定;用菌落生长速率法对10种药剂进行抑菌率测定。分离纯化获得4个真菌纯培养菌株,通过科赫氏法则实验结果确认,菌株KFTJ01是导致大粒种咖啡炭疽病的病原菌。采用PCR扩增成功获得菌株KFTJ01的rDNA-ITS、CAL和Tub2基因序列,其长度分别为554、489、706 bp。分别上传到NCBI GenBank进行blastn比对。结果表明,菌株KFTJ01与卡哈瓦刺盘孢(Colletotrichum kahawae)菌株WZ-135(MN856281.1)、CREADC-ER2212(MT409131.1)和YMTJ4(MK569149.1)的相似率分别达99.62%、100%、100%。用KFTJ01菌株和下载的刺盘孢属同源种菌株的3个DNA序列构建系统演化树,菌株KFTJ01与卡哈瓦刺盘孢(C.kahawae)聚集于同一分枝上(可信度为98%),因此将菌株KFTJ01鉴定为卡哈瓦刺盘孢。这是我国的一个检疫对象物种,在咖啡类作物上尚未报道,应引起高度重视。杀菌剂筛选试验结果显示,70%甲基硫菌灵WP和25%甲硫·乙唑醇SA可完全抑制KFTJ01的生长(抑菌率均为100%),抑菌中浓度(EC_(50))分别为0.0607、0.0809 mg/mL,表明这2种药剂对抑制病菌非常经济有效。由此可见,引起大粒种咖啡炭疽病的病原菌为卡哈瓦刺盘孢,这是在我国报道的一种新记录咖啡炭疽病病原菌;甲硫·乙唑醇和甲基硫菌灵对病原菌具有很好的抑菌效果。本研究结果可为大粒咖啡炭疽病的准确诊断和防控提供重要参考依据。The present work was conducted to identify the pathogen causing anthracnose of Liberica coffee(Coffea liberica)plants and to screen fungicides against the disease pathogen.The leaves of C.liberica with anthracnose symptoms were sampled from fields for fungal isolation and purification.Four pure fungal isolates were obtained and only KFTJ01 was verified pathogenic to the coffee leaves by tests following Koch’s Postulates.The rDNA-ITS sequence(554 bp),beta-tubulin gene(tub2)and calmodulin gene(CAL)were successfully amplified via PCR from genomic DNA of KFTJ01 and sequenced.Blastn analysis of the the three DNA sequences showed that KFTJ01 was identical to the strain WZ-135(99.62%,MN856281),CREADC-ER2212(100%,MT409131)and YMTJ4(100%,MK569149)of Colletotrichum kahawae.KFTJ01 was also clustered with C.kahawae WZ-135 strain on the same end-branch of the phylogenic tree(bootstrap confidence is 98%).Therefore,the pathogen of Liberica coffee was identified as C.kahawae which is a quarantine species and has never been recorded in China.Fungicide selection experiments showed that among the 10 chemicals,methionine-acetazolyl and methyl thiobacillam almost completely(100%)suppressed the colony growth of KFTJ01.The median effective concentration calculated from the functions was 0.0607 mg/mL and 0.0809 mg/mL,respectively,indicating that both the chemicals were very effective against the anthracnose pathogen.The results are important references for the field diagnosis of the coffee plant anthracnose and for controlling the disease epidemics.

关 键 词：大粒咖啡 炭疽病 卡哈瓦刺盘孢 多基因序列分析 杀菌剂 

分 类 号：S435.712[农业科学—农业昆虫与害虫防治]