广西红花檵木炭疽病病原鉴定及其生物学特性

Identification and biological characteristics of pathogen causing anthracnose of Loropetalum chinense var.rubrum in Guangxi

【目的】明确广西地区红花檵木(Loropetalum chinense var.rubrum)炭疽病病原菌的分类地位和生物学特性,为该病害的正确诊断及科学防治提供理论依据。【方法】采用组织分离法分离红花檵木病叶的病原菌,通过活体接种法确定其致病性,结合形态学特征和多基因序列[核糖体转录间隔区(ITS)、肌动蛋白(ACT)、β-微管蛋白(TUB2)、甘油醛-3-磷酸脱氢酶(GAPDH)、几丁质合酶(CHS-1)和钙调蛋白(CAL)]分析,确定引起红花檵木炭疽病的病原菌。采用菌落生长速率法测定不同温度、pH、碳源、氮源和杀菌剂对病原菌菌丝生长的影响。【结果】从广西红花檵木炭疽病病叶组织中分离获得6株形态一致并具有致病性的真菌菌株,菌落近圆形,中部灰色至深灰色,边缘灰白色,气生菌丝体疏松、絮状,分生孢子单细胞、长椭圆形,与果生刺盘孢(Colletotrichum fructicola)形态一致。对代表性菌株H6的ITS、ACT、TUB2、GAPDH、CHS-1和CAL基因序列进行PCR扩增,并在NCBI中进行BLAST比对分析,发现这些序列与多个果生刺盘孢菌株的序列同源性达99%~100%。使用这些基因序列构建多基因系统发育进化树,发现菌株H6与果生刺盘孢聚于同一最小分支,因此确定红花檵木炭疽病病原菌为果生刺盘孢。生物学特性测定结果表明,最适宜菌株H6菌丝生长的温度为28.0℃,最适pH为8.0,最适碳源和氮源分别为木糖醇和L-脯氨酸;对菌株H6菌丝生长有较强抑制效果的杀菌剂有吡唑醚菌酯、啶氧菌酯、乙蒜素、多菌灵和咪鲜胺。【结论】结合形态学与分子生物学鉴定方法,将广西地区红花檵木炭疽病的病原菌鉴定为果生刺盘孢(C.fructicola),明确了最适宜该病原菌生长的温度、pH、碳源和氮源等条件及对该病原菌具有较强抑制效果的几种杀菌剂。

【Objective】The study aimed to clarify the classification and biological characteristics of the pathogen causing anthracnose of Loropetalum chinense var.rubrum in Guangxi,so as to provide a theoretical basis for the diagnosis and scientific control of the disease.【Method】The pathogen was isolated by tissue separation method and its pathogenicity was verified by living leaf inoculation tests.Furthermore,the pathogen was identified by its morphological characteristics and phylogenetic analysis of multi-locus[internal transcribed spacer(ITS),actin(ACT),(β-tubulin)TUB2,glyceraldehyde-3-phosphate dehydrogenase(GAPDH),chitin synthase(CHS-1)and calmodulin(CAL)]sequences.The effects of different temperature,pH,carbon source,nitrogen source and fungicide on the mycelial growth of pathogen were measured by colony growth rate method.【Result】Six fungal strains with consistent morphology and pathogenicity were isolated from diseased leaf tissue.The colony was nearly round,gray to dark gray in the middle,and gray white at the edge.The aerial mycelium was loose and flocculent.And the conidia were single celled and long oval.The morphological characteristics were consistent with those of Colletotrichum fructicola.The ITS,ACT,TUB2,GAPDH,CHS-1 and CAL gene sequences of representative strain H6 were amplified by PCR and analyzed by BLAST in NCBI.The results showed that these sequences had 99%-100%homology with several C.fruticola strains.Furthermore,these genes were used to construct phylogenetic trees.Strain H6 was found in the same branch as C.fructicola.Therefore,the pathogen was identified as C.fruticola.The results of biological characteristic tests showed that the optimum temperature,pH,carbon and nitrogen sources for mycelial growth of strain H6 were 28.0°C,8.0,werexylitol and L-proline,respectively.Pyraclostrobin,picoxystrobin,ethylicin,carbendazim and prochloraz had strong inhibitory effect on mycelial growth of strain H6.【Conclusion】The pathogen causing anthracnose of L.chinense var.rubrum in Guangxi was identified as C.fructicola by morphological and molecular biological methods.The conditions of temperature,pH,carbon source and nitrogen source that were most suitable for the growth of the pathogen were verified.At the same time,several fungicides with strong inhibitory effect on the pathogen were also determined.