中国农业科学 ›› 2023, Vol. 56 ›› Issue (22): 4453-4466.doi: 10.3864/j.issn.0578-1752.2023.22.008

• 植物保护 • 上一篇    下一篇

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

郭宁(), 孙华, 马红霞, 刘树森, 张海剑, 石洁(), 郑晓娟, 董跃广   

  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
  • 收稿日期:2023-08-02 接受日期:2023-08-27 出版日期:2023-11-16 发布日期:2023-11-17
  • 通信作者: 石洁,E-mail:shij99@163.com
  • 联系方式: 郭宁,E-mail:guoning433@163.com。
  • 基金资助:
    河北省省级科技计划(20326516D); 国家现代农业产业技术体系(CARS-02)

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

GUO Ning(), SUN Hua, MA HongXia, LIU ShuSen, ZHANG HaiJian, SHI Jie(), ZHENG XiaoJuan, DONG YueGuang   

  1. 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
  • Received:2023-08-02 Accepted:2023-08-27 Published:2023-11-16 Online:2023-11-17

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(Trichoderma spp.)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(Pythium inflatum)、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无影响,且当Tr21发酵滤液浓度为20×稀释液时对玉米种子的萌发和生长促进效果最好。盆栽试验结果表明,Tr21发酵滤液浓度为5×稀释液时,对3种腐霉茎腐病的室内防治效果最佳,分别为60.67%、63.15%和59.66%。用Tr21的5×稀释液进行种子处理,当药种质量比例为1﹕100时,对腐霉茎腐病的防治效果最高,达82.25%。【结论】获得一株有效防治玉米腐霉茎腐病的木霉菌株Tr21,经鉴定该菌为非洲哈茨木霉,该菌株发酵滤液可导致腐霉菌菌丝畸形、断裂、细胞膜受损、内含物溢出等,是一株具有开发前景的生防微生物。

关键词: 腐霉菌, 茎腐病, 玉米, 非洲哈茨木霉, 生物防治

Abstract:

【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 stain showed that the cell membrane of three Pythium species was damaged by Tr21 fermentation broth, and the PI dye was more likely to penetrate the damaged cell membrane into the mycelium and stain the mycelia red. The results of nucleic acid and protein leakage showed that the absorbance values of the mycelia treated by the fermentation broth changed greatly. After treatment for 5 h, the OD260 increased by 0.08 and OD280 increased by 0.10, 0.11 and 0.10, respectively, indicating that the membrane of the mycelia was damaged, leading to the overflow of mycelia contents. The different concentrations of Tr21 fermentation broth had no effect on the germination characteristics of maize seeds, and the 20× diluent had the best effect on germination and growth of seeds. The results of pot experiment showed that 5× diluted fermentation broth of Tr21 had the best control efficacy on Pythium stalk rot caused by three Pythium species, which was 60.67%, 63.15% and 59.66%, respectively. The control efficacy on Pythium stalk rot of 5× diluent was the highest, reaching 82.25%, with a mass ratio of 1﹕100 (5× diluent to seed).【Conclusion】An effective T. afroharzianum strain Tr21 was obtained for preventing and controlling of maize Pythium stalk rot. The fermentation broth of Tr21 can lead to mycelia malformation, breakage, cell membrane damage and contents leakage, etc. In conclusion, the T. afroharzianum strain Tr21 is a promising biocontrol microbial.

Key words: Pythium, stalk rot, maize, Trichoderma afroharzianum, biological control