贵州省黄果树旅游区柑橘叶斑病病原菌的鉴定与分析

Identification and analysis of the pathogens causing citrus leaf spot in Huangguoshu scenic area

柑橘叶斑病是黄果树旅游区柑橘重要产区和母本园区的重要病害,自2017年起对其进行了柑橘病害调查及病原菌鉴定,并从病害样品中分离、纯化了多个菌株,进行了菌落形态、子实体、产孢细胞、分生孢子和子囊孢子等形态的观察及分子生物学的鉴定。依据柯赫氏法则,在室内或自然条件下,研究了代表性菌株ZHGS-2017-010对柑橘叶片和果实的致病性。依据18S核糖体RNA基因(SSU)、核糖体内转录间隔区(rDNA-ITS1-5.8S-ITS2)、28S核糖体rRNA基因(LSU)、延伸因子-1α(EF-1α)和β-微管蛋白(β-tub),采用PAUP软件及最大简约法,进行了多基因系统发育树分析。结果表明,子实体为黑色,不规则球形。产孢细胞为圆柱形,淡蓝色或透明,大小为(42.5±5.3)μm×(4.4±1.0)μm。分生孢子,无色或淡蓝色、光滑、无隔膜,呈梭形或纺锤形,大小为(26.5±2.4)μm×(6.5±0.8)μm。基于SSU、ITS、LSU、EF1-α、TUB的系统发育树研究表明,代表性菌株ZHGS-2017-010与参考菌株Botryosphaeria dothidea CBS 110302和CMW 800菌株在系统发育树上聚为一支,其自举支持率为91%,表明引起该地区柑橘病害的病原为葡萄座腔菌(Botryosphaeria dothidea)。致病性试验表明,代表性菌株ZHGS-2017-010可导致柑橘产生叶斑和果实腐烂。

Citrus leaf spot is an important disease in Huangguoshu citrus production area.In order to identify the pathogen(s)causing the disease,we isolated several strains from diseased samples,observed the colonies,conidiomata,conidiogenous cells,conidia,and ascospore,then identified these strains using the molecular biology method.According to Koch's rule,the pathogenicity tests of a representative strain ZHGS-2017-010 were conducted on the leaves and fruits of citrus under both laboratory field conditions.Phylogenetic analysis was conducted by PAUP software with the method of Maximum parsimony based on multi-locus sequences of 18S RNA gene(SSU),rDNA-ITS1-5.8S-ITS2(ITS),28S rRNA(LSU),the translation elongation factor 1-α(EF-1α)andβ-tubulin(β-tub)genes.The results indicated that fruiting body was black and irregular spheroidal;conidiogenous cells were hyaline,cylindrical,with the size of(42.5±5.3)μm×(4.4±1.0)μm;conidia was colorless or hyaline,smooth,aseptate,fusoid or ellipsoid,with the size of(26.5±2.4)μm×(6.5±0.8)μm.Phylogenetic analysis based on multi-locus sequences of SSU,ITS,LSU,EF-1α,andβ-tub indicated that the representative strain ZHGS-2017-010 was clustered into the clade of Botryosphaeria dothidea CBS 110302 and CMW 800,with a bootstrap level of 91%.This study indicated the pathogen causing citrus leaf spot is Botryosphaeria dothidea in this region,and the representative strain ZHGS-2017-010 could induce leaf spot and fruit rot of citrus.