‘芽黄’红瑞木炭疽病病原菌的分子鉴定

‘芽黄’红瑞木炭疽病在上海普遍发生。本研究从上海迪士尼园区采集疑似炭疽病的红瑞木病叶,进行了分离培养,获得2种培养形态的炭疽菌,致病性测定证明其为致病菌。联合核糖体转录间隔区、3-磷酸甘油醛脱氢酶、钙调蛋白和β-微管蛋白2多基因序列,使用最大似然法和贝叶斯分析法分别构建了多基因系统进化树。结果显示,分离获得的2株炭疽菌菌株分别与暹罗炭疽菌Colletotrichum siamense和胶孢炭疽菌C.gloeosporioides聚集在一个分支,辅以形态学特征,鉴定引起‘芽黄’红瑞木炭疽病的病原菌为暹罗炭疽菌和胶孢炭疽菌,这是国内关于红瑞木炭疽病的首次报道。     

海南4个地区暹罗炭疽菌群体遗传结构分析

对分离自海南4个地区13种寄主植物上暹罗炭疽菌的ITS-CAL-GAPDH 3基因序列进行群体遗传结构分析。95条暹罗炭疽菌多基因序列可定义为16个单倍型,其中,单倍型H5为主要单倍型,分布于所有寄主植物。AMOVA分析显示,遗传变异主要发生在种群内;病菌未经历过大规模的种群扩张过程。研究结果表明,暹罗炭疽菌种群具有丰富的遗传多样性。     

一种新油茶炭疽病原多基因序列鉴定

从湖南、广西油茶叶上分离获得5株能侵染油茶树的病原菌。通过柯赫氏法则证明该病原菌为油茶炭疽病的致病菌。病菌菌落圆形,初期为白色,逐渐变为浅灰色;气生菌丝由白色和灰白色渐变为深灰色绒状;菌落背面中心颜色深,外部颜色浅;菌丝划伤后在PDA培养基上能产生橘黄色的分生孢子堆,分生孢子为单胞、直、圆柱状、无色、光滑、两头钝圆或一端稍尖,分生孢子大小(11~15)μm×(4~5)μm;菌丝体附着胞不规则状,浅褐色,(8~12)μm×(5.5~7)μm。采用形态学结合多基因(核糖体内转录间隔区(ITS)、钙调蛋白-CAL、3-磷酸甘油醛脱氢酶-GAPDH)系统学的方法对它们进行鉴定,结果发现,5个菌株均为暹罗刺盘孢(Colletotrichum siamense)。这是国内油茶上C.siamense的首次报道。     

暹罗炭疽菌中脂滴包被蛋白Cap20与乙酸激酶CsAck的互作研究

暹罗炭疽菌（Colletotrichum siamense）是一种重要的病原真菌,是我国橡胶树炭疽病（Colletotrichum leaf disease, CLD）的田间优势病原种,也是热带、亚热带地区许多其他农林作物炭疽病的主要病原种。脂滴是细胞的中性脂,存在于绝大多数真核细胞中,是细胞内甘油三酯的主要贮存形式。脂滴在细胞内可以参与脂质代谢、膜转运、蛋白降解和信号传导等。包被蛋白（perilipin）是脂滴表面最丰富的脂质相关蛋白,主要存在于动植物体内,对细胞内脂滴代谢起着重要的调控作用。Cap20是暹罗炭疽菌中包被蛋白的同源蛋白,前期证实Cap20是一个致病相关蛋白,它参与了炭疽菌附着胞膨压形成、脂滴数量和致病性。了解其互作蛋白和互作的生物学意义可能为深入了解脂滴包被蛋白参与的致病分子机制具有重要意义。课题组前期通过酵母双杂交技术在暹罗炭疽菌cDNA文库中筛选到一个与Cap20互作的候选蛋白乙酸激酶,本研究克隆了该乙酸激酶的编码基因,并进行了蛋白结构和同源蛋白聚类分析。结果表明,乙酸激酶编码基因的DNA大小为1312 bp,包括一个内含子,cDNA为1248 bp,编码415个氨基酸,含有一个乙酸激酶结构域,命名为CsAck。然后构建含6×his标签的原核表达载体PET32a-CsAck,利用其和含GST标签的pGEX6p-1-Cap20（先前构建）进行蛋白表达和纯化,获得含有6×His标签的融合蛋白CsAck和含有GST标签的Cap20。通过Pull down验证了Cap20和CsAck蛋白之间的体外互作。最后,构建含有潮霉素转移酶基因HPH的表达载体PXY203-CsAck-S和含有氯嘧磺隆抗性基因ILV1的表达载体pCB1532-GFP-Cap20,将它们共同转化暹罗炭疽菌野生型菌株获得转化子。Co-IP（Co-immunoprecipitation）的结果证实Cap20和CsAck在暹罗炭疽菌中可以相互作用。该结果证实了致病性相关蛋白Cap20和乙酸激酶CsAck在体外和体内均可发生蛋白互作,可为进一步研究Cap20在暹罗炭疽菌中的致病功能和调控机制奠定基础。     

Population structure,pathogenicity, and fungicide sensitivity of Colletotrichum siamense from different hosts in Hainan,China

Areca palm, rubber tree, and coffee are always planted adjacent to each other or intercropped with each other, and Colletotrichum siamense was found to be the dominant species of Colletotrichum from these crops in Hainan, China. To study the population structure, pathogenicity, and fungicide sensitivity of C. siamense from these three crops in Hainan, isolates were first identified by their morphological characteristics and multilocus phylogeny. Then both permutational multivariate analysis of variance (PERMANOVA) and pairwise F_ST analysis showed that the original host and geographical origin (counties) had significant effects on genetic variation in the C. siamense population, explaining 5.6% and 16.3% of genetic variation, respectively. There were significant genetic differentiations between coffee and rubber tree populations but the genetic differentiation was small (F_ST = 0.04), while significant differentiations were observed among all populations from different counties except those between Haikou and Chengmai. Pathogenicity analysis using artificial inoculation showed that isolates had significantly higher disease incidence and diseased lesion diameter on wounded leaves than on nonwounded leaves. However, the original host of an isolate and geographical origin did not significantly affect the pathogenicity of the C. siamense populations from these three hosts. In vitro tests showed that there were no significant differences in the sensitivity of C. siamense populations to carbendazim, prochloraz, difenoconazole, and propiconazole from different original hosts and geographical origins. The present study suggests that host specialization of C. siamense has not occurred yet in coffee, rubber tree, and areca palm.     

油茶暹罗刺盘孢菌群体遗传结构分析

暹罗刺盘孢菌是油茶炭疽病病原之一,在我国多个油茶产区均有分布。研究油茶暹罗刺盘孢菌群体遗传结构可为全面、有效防治油茶炭疽病害提供理论依据。本研究对分离自海南、江西、湖南、广西4省(自治区)6个地区暹罗刺盘孢菌菌株的ITS、CAL和GAPDH 3个基因的序列进行群体遗传结构分析。根据拼接的上述3个基因的序列,57个暹罗刺盘孢菌菌株可定义为13个单倍型,其中单倍型H7为主要单倍型,分布于本研究所涉及的所有地区。病菌不同地理种群间的遗传分化较大,AMOVA分析显示,遗传变异主要发生在种群内,病菌未经历过大规模的种群扩张。研究结果表明油茶炭疽病原暹罗刺盘孢菌种群具有丰富的遗传多样性。     

Identification of Colletotrichum siamense causing litchi pepper spot disease in mainland China

Litchi (Litchi chinensis) pepper spot disease results in black spotting symptoms on litchi fruits. This disease was first observed on litchi cultivar Guiwei, in Guangzhou, China, in 2009, and then found widespread in other litchi-growing regions of China. Colletotrichum isolates were consistently recovered from typical black spot lesions of diseased fruits with frequency ranging from 83% to 100%. Three representative Colletotrichum isolates from Maoming, Guangzhou and Shenzhen were selected for identification and pathogenicity testing in the field. Based on morphology and phylogenetic analysis using the ribosomal internal transcribed spacer region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), actin (ACT), β-tubulin (TUB2) and glutamine synthetase (GS) gene sequences, the three isolates were identified as C. siamense. In the pathogenicity experiments, typical symptoms appeared on the inoculated litchi fruits, including black spots and green patches around these black spots. These symptoms were consistent with the symptoms originally observed in the field. Colletotrichum siamense was successfully reisolated from the typical black spot lesions of the inoculated litchi fruits. To the authors' knowledge, this is the first report on characterization of C. siamense as the causal agent of litchi pepper spot disease in mainland China by successful inoculation on fruits under field conditions.     

广州市沙田柠檬炭疽病病原菌种类的分子鉴定

【目的】明确沙田柠檬炭疽病的病原菌种类,为沙田柠檬炭疽病的田间防治提供指导。【方法】对采集的炭疽病样本进行分离纯化培养,获得 ST2-1、ST2-2、ST2-3、ST3-1、ST3-2、ST3-3、ST4-1、ST4-2和 ST4-3 等 9 株沙田柠檬炭疽病病原菌。分别采用针刺和喷洒接种的方法测定上述菌株的致病性,并分别克隆其核糖体内转录间隔区（ITS）、β 微管蛋白（TUB2）、钙调蛋白（CAL）、肌动蛋白（ACT）、核糖体大亚基（LSU）、甘油醛 -3- 磷酸脱氢酶（GAPDH）和几丁质合成酶 1（CHS1）基因部分片段进行多基因联合鉴定。【结果】ST2-1、ST2-2、ST2-3、ST3-1、ST3-2、ST3-3、ST4-1、ST4-2 和 ST4-3 均 具 有 较 强 致 病 性, 其 中 ST2-1、ST2-2 和 ST2-3 的生物学特征和进化树表明它们属于暹罗刺盘孢菌（Colletotrichum siamense）,ST3-1、ST3-2和 ST3-3 属于兰花刺盘孢菌（Colletotrichum cliviicola）,ST4-1、ST4-2 和 ST4-3 属于果生刺盘孢菌（Colletotrichum fructicola）。【结论】综合形态学特征和分子生物学鉴定结果,引起沙田柠檬炭疽病的病原菌有暹罗刺盘孢菌、兰花刺盘孢菌和果生刺盘孢菌,其中暹罗刺盘孢菌仅分离自沙田柠檬发病的果实,兰花刺盘孢菌和果生刺盘孢菌分离自沙田柠檬发病的叶片,其中兰花刺盘孢菌能在柑橘类植物上引起炭疽病为国际上首次报道。     

广东咖啡炭疽病病原菌初步鉴定及防治药剂筛选

【目的】明确引起广东咖啡炭疽病的病原菌种类并筛选防治药剂。【方法】采用组织分离法对采集到的疑似炭疽病咖啡叶片进行分离,单孢纯化后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(ITS、TUB2、GAPDH、CHS、ACT和GS)的系统发育分析对病原菌进行鉴定;利用菌丝生长速率法测定4种常用杀菌剂对病原菌的抑制效果。【结果】分离得到的菌株为果生炭疽菌Colletotrichum fructicola、暹罗炭疽菌C. siamense和芭蕉生炭疽菌C. musicola,致病性测定结果显示其均能侵染叶片。咪鲜胺、吡唑醚菌酯和甲基硫菌灵3种杀菌剂对两株强致病性炭疽病菌(果生炭疽菌C. fructicola CA-13和暹罗炭疽菌C. siamense CA-16)的抑制效果最强,其EC_(50)值均小于0.1 mg/L。【结论】广东咖啡炭疽病的优势病原菌为果生炭疽菌C. fructicola和暹罗炭疽菌C. siamense,咪鲜胺、吡唑醚菌酯和甲基硫菌灵可作为防治咖啡炭疽病的首选药剂。     

海南橡胶树暹罗刺盘孢菌群体遗传结构分析

暹罗刺盘孢菌Colletotrichum siamense是引起海南橡胶树炭疽病的重要病原之一,为了明确海南橡胶树暹罗刺盘孢菌群体的遗传结构,本研究对海南5个市(县)的橡胶树暹罗刺盘孢菌的ITS序列进行分析。结果表明,58个病菌样品可推导出11种单倍型,其中单倍型H11包含样品数最多且在5个市(县)均有分布,其次为单倍型H4,在4个市(县)有分布。遗传分化指数(Fst)表明保亭种群与其他种群的遗传分化较大。AMOVA分析显示,遗传变异主要发生在种群内,占总变异的93.9%。对所有地理种群病原菌ITS序列的核苷酸不配对进行分析,表明病菌未经历过大规模的种群扩张。系统发育分析表明,来自相同地区的病菌单倍型并没有聚在一起。研究结果表明海南橡胶树暹罗刺盘孢菌种群具有丰富的遗传多样性。