中国滇南地区莲雾炭疽病病原菌的形态学和分子生物学鉴定

Morphological and Molecular Identification of the Pathogen Causing Wax Apple Anthracnose in Southern Yunnan,China

莲雾炭疽病是国内外莲雾种植区的常年流行病及引起严重经济损失的一种重要真菌病害,但不同地区的病原菌种可能不同.该研究主要描述云南省南部发生的莲雾炭疽病症状,分离纯化病原菌并鉴定其种类.经观察,此莲雾炭疽病的典型病斑为椭圆或不规则形,中央区灰白色,周围褐色,最外围有一黄色晕圈;后期病斑扩展形成不规则大斑或枯斑;病害侵染果实引起腐烂变质.通过分离培养和纯化获得7个真菌菌株,柯赫氏致病性试验表明从西双版纳和普洱两地样品中获得的LWTJ2和LB4为致病菌株.两菌株形态特征相同,在马铃薯葡萄糖琼脂培养基(PDA)上的菌落为圆形,白色或淡黄白色,边缘光滑齐整,表面呈绒毛状,气生菌丝发达;后期菌落颜色稍加深,中央区灰黑色并产生大量蛋橙色分生孢子团;在显微镜下观察,病菌菌丝透明,分隔,呈锐角或近直角分枝;分生孢子无色,长椭圆形,两端盾圆,大小为9.8~18.0(平均13.9±2.1)μm×4.5~6.0(平均5.5±1.0)μm;未观察到病菌的有性型.采用ITS1/ITS4通用引物通过PCR扩增获得两菌株的rDNA-ITS序列均为545 bp(GenBank登录号为OL963924和OL413460).Blast-n比对和系统演化树分析表明,两个菌株Blast-n相似度及在演化树上聚集于同一末端分枝的置信限均为100%;两者与暹罗刺盘孢Collectotrichum siamense WZ-365菌株(Acc.No.MN856443)对比的相似度为99.08%,在系统演化树中与暹罗刺盘孢聚于同一末端分枝的置信限为96%.该研究较详细地观察记载了滇南地区莲雾炭疽病的症状及病原菌的形态特征,根据形态学与rDNA-ITS序列的Blast-n对比和演化树分析鉴定获得的LWTJ2和LB4菌株均属于暹罗刺盘孢C.siamense.这是莲雾炭疽病的一种新病原真菌,其进一步证明了莲雾炭疽病病原菌的多样性,也为该病害的诊断监测和有效控制提供了科学依据.

Wax apple(Syzygium samarangense)is an important and high-economic value fruit tree planted broadly in the tropic and subtropic countries.The anthracnose is a fungal disease that is widespread in the wax-apple growing areas and usually causes serious yield reductions.In 2021,wax apple anthracnose was observed during the surveys of diseases of tropical fruit trees in Sipsongpanna and Pu er,Yunnan,China and found that the disease was widespread and serious.Therefore,the present study was conducted to investigate the symptom of disease,isolate and identify the pathogen of disease.Typical spot of the anthracnose on wax apple leaves observed in this study was oval or irregular,with a gray-white center surrounded by a brown band and yellow halo.Later,the central necrotic tissue was dried and fallen,leaving holes on the leaf.The pathogen of the disease also infected the fruits and caused fruit rot during storage.Seven fungal isolates were obtained from diseased leaf tissues.Laboratory tests with Koch s Postulates verified that LWTJ2 from Pu er and LB4 from Sipsongpanna was the pathogen causing wax apple anthracnose.The pathogenic isolates were morphologically indistinguishable.After cultured on potato glucose agar(PDA)medium plates at 25℃for 6 d,the colonies were round with complete edges and prosperous aerial hyphae,surface smooth and villous,white or light-yellow colored.At 12 d after cultivation,the colony became pale-white with gray-black center,producing plenty of orange-colored conidia.Under microscope,the hyphae were hyaline,septate,and branched at near right-angles.Conidia were colorless,long-oval or cylinder-shaped with round ends,and sized 9.8-8.0(mean 13.9±2.1)μm×4.5-6.0(mean 5.5±1.0)μm,with obvious oily droplet inside the spore cell.Teleomorph of the fungus was not detected throughout the study.Two rDNA-ITS sequences(both 545 bp)were amplified respectively from LWTJ2 and LB4 by PCR with ITS1/ITS4 universal primers and registered in NCBI GenBank with assigned accession numbers of OL963924 and OL413460.Blast-n analysis of rDNA-ITS sequences and phylogenetic tree analysis showed that LWTJ2 and LB4 isolates were the same fungal species(max.ident.and confidence=100%)and both isolates were identical to the isolate WZ-365(Acc.No.MN856443)of Colletotrichum siamense(max.ident.=99.08%and confidence=96%).To conclude,the present study recorded and described detailly the anthracnose symptoms and morphological characteristics of its pathogen.C.siamense was identified as the pathogen causing wax apple anthracnose.Results of the present work further demonstrated the species diversity of wax apple anthracnose pathogens and provided important reference and scientific foundation for diagnosis,monitoring and management of this disease.