百合枯萎病病原菌的分离鉴定及其接种浓度对发病程度的影响  

Isolation and identification of lily Fusarium wilt pathogen and the effect of spore suspension concentration on the extent of disease

作　　者：任胜林 梅慧玲 李映萱 杜彭涛 文涛 乔亦铸 张赫 麦远愉 董银霜 李静 黄启为 REN Shenglin;MEI Huiling;LI Yingxuan;DU Pengtao;WEN Tao;QIAO Yizhu;ZHANG He;MAI Yuanyu;DONG Yinshuang;LI Jing;HUANG Qiwei(College of Resources and Environmental Sciences/Jiangsu Provincial Key Laboratory for Organic Solid Waste Utilization/Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization/National Engineering ResearchCenter for Organic-Based Fertilizers,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学资源与环境科学学院/江苏省固体有机废弃物资源化高技术研究重点实验室/江苏省有机固体废弃物资源化协同创新中心/国家有机类肥料工程技术研究中心,江苏南京210095

出　　处：《南京农业大学学报》2020年第6期1097-1106,共10页Journal of Nanjing Agricultural University

基　　金：国家公益性行业(农业)科研专项(201503110);国家自然科学基金项目(31972506);江苏省重点研发计划(现代农业)项目(BE2018338);江苏省农业科技自主创新资金[CX(18)3095]。

摘　　要：[目的]江苏宜兴是我国百合生产的传统优势产区,百合枯萎病是严重阻碍百合产业化健康发展的一个关键因子。通过分离鉴定百合枯萎病的致病菌株,接种不同浓度的病原菌孢子悬液,研究百合的发病程度,为江苏宜兴百合枯萎病的生物防治提供科学基础。[方法]采集宜兴百合种植区典型枯萎病发病严重的植株及鳞茎分离致病菌株,通过柯赫氏法则回接试验初步确定致病性,对致病性强的菌株进行DNA提取、PCR扩增和测序,基于病原菌rDNA-ITS基因序列和GenBank中已有相关尖孢镰刀菌序列,应用MEGA 7.0软件构建系统发育树;采用刺伤百合鳞片接种不同浓度的病原菌孢子悬液与在土壤中接种不同浓度病原菌孢子悬液的方法,评估不同浓度孢子悬液对百合枯萎病发病程度的影响。[结果]从典型枯萎病严重发病的植株上共分离得到4株病原菌株:P-OF、P-PF、P-RF和P-BF,分离率分别为25%、20%、10%和5%。柯赫氏法则回接试验表明:接种菌株P-OF、P-PF百合发病率为100%,鳞片病情指数分别为92.50、48.25、15.00、30.00;盆栽试验表明:接种菌株P-OF的百合植株最先发病,初步确定菌株P-OF的致病性最强;通过形态学和分子生物学对菌株P-OF的鉴定结果表明,从典型枯萎病严重发病百合植株中所分离的病原菌为尖孢镰刀菌(Fusarium oxysporum)。百合枯萎病的病情指数与接种病原菌孢子悬液浓度相关,百合鳞片接种试验和盆栽试验结果表明,土壤病原菌浓度为103 CFU·g^-1,是导致百合植株发病的临界浓度,当土壤病原菌浓度低于105 CFU·g^-1时,病情指数随接种浓度增加而增加,当土壤病原菌浓度高于105 CFU·g^-1时,病情指数不再显著增加。[结论]从江苏宜兴百合枯萎病的典型发病植株中分离到4株病原菌株,其中菌株P-OF的致病性最强,经鉴定为尖孢镰刀菌百合专化型(Fusarium oxysporum sp.f.lilii),导致百合植株发病的临界土壤[Objectives]Lily(Lilium spp.)is one of the dominant traditional production areas in Yixing City,Jiangsu Province,China.Fusarium wilt is a key stunting factor for the healthy development of lily production.The isolation and identification of the pathogenic strains of lily Fusarium wilt in Yixing City,inoculation of spore suspensions of pathogens of different concentrations and the relationship between occurrence degree and the concentration of spore suspensions of pathogens were carried out for providing scientific basis in biological control technology of lily Fusarium wilt in Yixing.[Methods]The pathogenic strains were isolated from the plants with severe blight disease and bulbs in the lily field of Yixing.The pathogenicity was preliminarily determined by the Koch’s rule backhaul experiment.The highly pathogenic strains were subjected to DNA extraction,PCR amplification and sequencing.The phylogenetic dendrogram was constructed based on the rDNA-ITS gene sequence of the pathogens and the related Fusarium oxysporum sequence in GenBank by using MEGA 7.0 software.The incidence of lily Fusarium wilt was evaluated by inoculating experiments both in inoculating pathogen spore suspension to the soil and in wounded inoculating pathogen spore suspension on the plants.[Results]A total of 4 pathogenic strains of P-OF,P-PF,P-RF and P-BF were isolated from severely blighted plants,with isolation rates of 25%,20%,10%and 5%,respectively.The back-to-back experiments of the law showed that the incidence of P-OF and P-PF was 100%,and the scale index was 92.50,48.25,15.00 and 30.00,respectively.The results of pot experiment showed that the treatment of lily plants inoculated with P-OF was the first to develop the disease.This hinted that the pathogenicity of P-OF might be the strongest.The identification of P-OF by morphology and molecular biology showed that the pathogen isolated from lily plants with severe wilt was Fusarium oxysporum.The results of lily scale inoculation and pot experiment showed that the concentration of so

关 键 词：百合 枯萎病 病原菌分离 孢子悬液浓度 病情指数 发病率 

分 类 号：S432.4[农业科学—植物病理学]