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灰葡萄孢寄生菌Clpse01的杀线活性研究OA北大核心CSTPCD

The Nematicidal Activity of Clonostachys pseudochoroleuca Clpse01 on Botrytis cinerea

中文摘要英文摘要

在用灰葡萄孢菌Botrytis cinerea培养松材线虫的过程中,发掘了一株具有杀线活性的灰葡萄孢寄生菌Clpse01.本文在光学显微镜下对菌株Clpse01的形态和其对灰葡萄孢菌的生长影响进行了观察;采用ITS4/ITS5、LR5/LR0R、Bt2a/Bt2b和EF1-688F/EF1-1251R引物进行了分子生物学鉴定;同时测试了菌株Clpse01对松材线虫的杀线活性及对松材线虫卵孵化能力和繁殖能力的影响;最后测试了菌株Clpse01的胞外蛋白浓度,胞外β-葡萄糖苷酶活性和胞外几丁质酶活性.根据形态学和分子生物学结果,将菌株Clpse01鉴定为假卡鲁卡枝惠霉Clonostachys pseudochroleuca.菌株Clpse01可以缠绕、侵入灰葡萄孢菌的菌丝以获得营养来源,使灰葡萄孢菌的菌丝生长受到抑制.将2000条松材线虫接种到长满菌株Clpse01的培养基上,8 d后仅分离出6条/皿活线虫,而对照灰葡萄孢菌培养基上分离的线虫数为33066条/皿活线虫;菌株Clpse01发酵液处理松材线虫48 h后的校正死亡率为88%,浓度为1×108孢子/mL的孢子悬液处理松材线虫24h后的校正死亡率为88.33%;菌株Clpse01 2X稀释发酵液24 h对卵的孵化抑制率为100%.发酵培养6h后,菌株Clpse01胞外蛋白浓度为1782.917μg/mL,胞外β-葡萄糖苷酶为9.99U/mL,胞外几丁质酶含量为0.8792 U/mL,并随发酵时间推移而升高.菌株Clpse01通过寄生松材线虫的食物来源灰葡萄孢菌和影响松材线虫的繁殖、存活和卵的孵化起作用,胞外β-葡萄糖苷酶、几丁质酶是毒杀松材线虫的两个重要生理因子.

A parasitic fungus with nematocidal activity,Clpse01,was found while pine wood nematodes(Bursaphelenchus xylophilus)were feeding on Botrytis cinerea media.Clpse01 morphologies and parasite evidences were seen under the microscope.Multiple genes,including ITS,large subunit ribosomal DNA,β-tublin and elongation factor,were used for further limiting by the phylogenic tree.Clpse01's nematocidal effectiveness against pine wood nematodes,as well as its effects on egg hatching and reproduction,were investigated.The Clpse01 fermentation fluid was analyzed for extracellular protein concentrations,β-glucosidase activity,and chitinase activity.Clpse01 was identified as Clonostachys pseudochloleuca using a combination of morphological and molecular phylogenetic approaches.C.pseudochroleuca Clpse01 was discovered entangling and infiltrating B.cinerea hyphae,acquiring nutrients from them while limiting B.cinerea normal growth.Inoculating 2000 pine wood nematodes into media with full growth of isolate Clpse01 resulted in only 6 alive ones after 8 days,while nematodes isolated from the control medium of B.cinerea reached 33066.The adjusted mortality rate of pine wood nematode treated with C.pseudochroleuca Clpse01 fermentation broth for 48 h was 88.0%,while that treated with 1×108 conidiospore suspensions per milliliter for 24 h was 88.33%.In double diluent fermentation broth for 24 hours,egg hatching was completely inhibited.After 6 hours of fermentation,the Clpse01 liquid contained 1782.917μg/mL of total extracellular protein,9.99 U/mL of β-glucosidase,and 0.8792 U/mL of chitinase.Protein concentrations increased with time.In general,C.pseudochroleuca Clpse01 functions by parasitizing B.cinerea,the feeding fungus,and influencing B.xylophilus reproduction,survival,and egg hatching.

徐勇;彭子嘉;徐正梅;邵雅坤;蓝燕;徐清华;余仲东

西北农林科技大学林学院,杨凌 712100||修水县农业局,九江 332400西北农林科技大学林学院,杨凌 712100先正达(中国)投资有限公司,上海 200120

植物保护学

松材线虫;灰葡萄孢菌;假卡鲁卡枝惠霉;寄生真菌;生防真菌

pine wood nematode;Botrytis cinerea;Clonostachys pseudochroleuca;parasitic fungus;biocontrol fungus

《中国生物防治学报》 2024 (001)

186-195 / 10

国家重点研发项目(2023YFC2604803)

10.16409/j.cnki.2095-039x.2023.02.066

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