玉米腐霉茎腐病生防木霉菌株的筛选、鉴定及防治效果分析  

Screening,Identification and Control Efficacy Analysis of Trichoderma Strains Against Maize Pythium Stalk Rot

作　　者：郭宁[1] 孙华[1] 马红霞[1] 刘树森 张海剑[1] 石洁[1] 郑晓娟 董跃广 GUO Ning;SUN Hua;MA HongXia;LIU ShuSen;ZHANG HaiJian;SHI Jie;ZHENG XiaoJuan;DONG YueGuang(Plant Protection Institute,Hebei Academy of Agriculture and Forestry Sciences/Key Laboratory of Integrated Pest Management on Crops in Northern Region of North China,Ministry of Agriculture and Rural Affairs/IPM Innovation Center of Hebei Province/International Science and Technology Joint Research Center on IPM of Hebei Province,Baoding 071000,Hebei)

机构地区：[1]河北省农林科学院植物保护研究所/农业农村部华北北部作物有害生物综合治理重点实验室/河北省农业有害生物综合防治技术创新中心/河北省作物有害生物综合防治国际科技联合研究中心,河北保定071000

出　　处：《中国农业科学》2023年第22期4453-4466,共14页Scientia Agricultura Sinica

基　　金：河北省省级科技计划(20326516D);国家现代农业产业技术体系(CARS-02)。

摘　　要：【目的】筛选对玉米腐霉茎腐病病原菌具有抑制作用的木霉(Trichodermaspp.)菌株,明确其分类地位以及对腐霉茎腐病的防治效果和抑菌机理,为腐霉茎腐病生防制剂的研发提供菌种资源。【方法】采用菌丝生长速率法测试候选木霉菌株对肿囊腐霉(Pythiuminflatum)、强雄腐霉(P.arrhenomanes)和芒孢腐霉(P.aristosporum)的抑制作用,筛选拮抗菌株;通过形态学和分子生物学特性确定Tr21菌株的分类地位;采用常规抑菌方法观察Tr21对腐霉菌丝形态的影响;采用溴化丙啶(PI)染液检测法及对不同处理时间菌丝上清液中蛋白和核酸吸光值的检测,分析Tr21发酵液对腐霉菌细胞膜通透性的影响;通过不同浓度Tr21发酵滤液浸种试验,检测Tr21发酵滤液对玉米种子发芽性状的影响;通过温室盆栽试验和田间人工接种试验,明确Tr21对腐霉茎腐病的防治效果。【结果】从实验室保存的109株木霉菌中,筛选到7株木霉菌对肿囊腐霉、强雄腐霉和芒孢腐霉具有拮抗活性,抑制率均>60%,其中Tr21菌株对3种腐霉菌的抑制率达到100%,其5×、10×和20×稀释液对3种腐霉菌的抑制率均达到100%。50×稀释液对3种腐霉菌的最低抑制率也达到55.56%。经形态学和分子生物学鉴定,Tr21为非洲哈茨木霉(T.afroharzianum)。显微镜观察显示Tr21发酵滤液能够造成腐霉菌菌丝变粗、菌丝分枝增多、节点缩短、断裂、内含物溢出等畸形现象。PI荧光染色试验显示Tr21发酵滤液导致3种腐霉菌的细胞膜受损,PI染液更易穿透受损的细胞膜进入到菌丝体内,使菌丝染成红色。核酸、蛋白泄露试验发现发酵滤液处理过的菌丝吸光值变化较大,处理5 h后,肿囊腐霉、芒孢腐霉和强雄腐霉菌丝的OD260均增加了0.08,OD280分别增加了0.10、0.11和0.10,表明腐霉菌菌丝细胞膜受损或其完整性被破坏,导致菌丝内含物外溢。不同浓度Tr21发酵滤液对玉米种子的发芽性状�【Objective】The objective of this study is to screen Trichoderma strains which have inhibitory effect on the Pythium spp.causing maize stalk rot,and to clarify their taxonomic status,control efficacy and antifungal mechanism.This study will provide important resources for the research and development of biocontrol agent against Pythium stalk rot.【Method】For the antagonistic strains screening,the inhibitory effect of Tichoderma strains on P.inflatum,P.arrhenomanes and P.aristosporum was tested by measuring the mycelia growth.The taxonomic status of Tr21 was determined by morphological and molecular characteristics.The effect of Tr21 on the mycelia morphology of Pythium spp.was observed in the laboratory.In order to analyze the effect of Tr21 fermentation broth on the membrane permeability of Pythium spp.,propyridine bromide(PI)dye solution was used to stain,and the absorbance values of protein and nucleic acid in mycelia supernatant at different treatment times were detected.The effect of Tr21 fermentation broth on germination characteristics of maize seeds was tested by seed soaking with different concentrations of fermentation broth.The control efficacy of Tr21 on stalk rot was confirmed through greenhouse pot and field inoculation experiments.【Result】From the 109 strains of Trichoderma spp.,seven strains were screened with antagonistic activity against P.inflatum,P.arrhenomanes and P.aristosporum,and the inhibition rate was above 60%.The inhibition rate of Tr21 to three Pythium species reached 100%,the inhibition rate of 5×,10×and 20×diluent to three Pythium species reached 100%,and the inhibition rate of 50×diluent to three Pythium species was also more than 55.56%.Tr21 strain was identified by morphological and molecular biology as T.afroharzianum.The results of microscopic observation showed that the fermentation broth of Tr21 could cause mycelial malformations,such as rough mycelia,increased mycelial branching,shortened nodes,and overflow of mycelia contents.The result of PI fluorescence stai

关 键 词：腐霉菌 茎腐病 玉米 非洲哈茨木霉 生物防治 

分 类 号：S435.13[农业科学—农业昆虫与害虫防治] S476[农业科学—植物保护]