淡紫紫孢菌微菌核发酵滤液生防活性及代谢组学分析

doi:10.16409/j.cnki.2095-039x.2022.04.008

摘要/Abstract

摘要： 淡紫紫孢菌是一种重要的生防真菌,在特定培养条件下可产生抗逆结构-微菌核。本文通过室内生测和温室盆栽试验,研究了淡紫紫孢菌微菌核发酵滤液对植物病原菌和根结线虫的抑制作用。结果表明,微菌核发酵滤液具有较强的杀线抑菌活性,其中对核盘菌生长抑制率达到70.5%,并显著降低菌核数量,滤液稀释10倍后对根结线虫二龄幼虫的致死率达到50%以上。用量为10 mL/株时对根结线虫病防效达到44.9%,黄瓜株高和茎粗分别增加21.6%和38.4%。同时,基于LC-MS构建了淡紫紫孢菌微菌核培养代谢组。分析表明,微菌核形成过程中芥酸、胡椒酚和泛酸等抑菌物质以及丁酸、咪唑乙酸等杀线化合物相对含量显著增加。本研究为揭示淡紫紫孢菌生防机制奠定了基础,为微菌核制剂的研发提供了理论依据。

关键词: 淡紫紫孢菌, 微菌核, 根结线虫, 发酵滤液, 代谢组

Abstract: Purpureocillium lilacinum is an important biocontrol fungus,which can produce a kind of resistant structure -microsclerotia under given culture conditions.In this study,the control efficiencies of microsclerotia fermentation filtrate of P.lilacinum against plant fungal pathogens and root-knot nematode were investigated by antagonism in the lab and pot experiment in greenhouse.The results showed that the filtrate of microsclerotia had strong inhibition to nematodes and pathogenic fungi.The growth inhibition rate to Sclerotinia sclerotiorum reached 70.5%,and the yield of sclerotia significantly decreased.The nematicidal activity of the filtrate to the juveniles of root-knot nematode was over 50% when the filtrate was diluted 10 times.In greenhouse,when 10 mL/seedling was used,the control efficiency against root-knot nematode in cucumber reached 44.9%,and the height and stem diameter of the seedlings increased by 21.6% and 38.4%,respectively.Additionally,the metabolome of P.lilacinum microsclerotia fermentation was constructed basing on LC-MS analysis,and the results showed that the relative contents of anti-microbial compounds,such as erucic acid,piperol and pantothenic acid,and the metabolites with nematicidal activities such as butyric acid and imidazolacetic acid,increased significantly.This study lays a foundation to understand the biocontrol mechanisms of P.lilacinum and facilitates the development of fungal agents composed of microsclerotia.

Key words: Purpureocillium lilacinum, microsclerotia, root-not nematode egg, fermentation filtrate, metabolome

中图分类号:

S476

范乐乐, 郭妍, 赵雪, 孙漫红, 李世东. 淡紫紫孢菌微菌核发酵滤液生防活性及代谢组学分析[J]. 中国生物防治学报, 2022, 38(4): 821-830.

FAN Lele, GUO Yan, ZHAO Xue, SUN Manhong, LI Shidong. Biocontrol Activities and Metabolome Analysis of Microsclerotia Fermentation Filtrate of Purpureocillium lilacinum[J]. Chinese Journal of Biological Control, 2022, 38(4): 821-830.

参考文献

[1] Jatala P. Biological control of plant-parasitic nematodes[J]. Annual Review of Phytopathology, 1986, 24(1):453-489.
[2] 孙漫红,刘杏忠,晋治波.淡紫拟青霉对大豆胞囊线虫卵及2龄幼虫的影响[J].植物保护学报, 2002, 29(1):57-61.
[3] Walters S A, Barker k R. Efficacy of Paecilomyces lilacinus in suppressing Rotylenchulus reniformis on tomato[J]. Journal of Nematology, 1994, 26(4):600-605.
[4] 卯婷婷,陶刚,赵兴丽,等. 4种微生物菌剂对辣椒主要病害的生物防治作用[J].中国生物防治学报, 2019, 36(2):258-264.
[5] 袁和奇,徐鹏程,肖海艳,等.淡紫紫孢菌防治采后番茄果实灰霉病[J].中国生物防治学报, 2017, 33(4):525-530.
[6] 肖炎农,王明祖,王道本.淡紫拟青霉几丁质酶对南方根结线虫的影响[J].中国生物防治, 1997, 13(1):30-32.
[7] Park J O, Hargreaves J R, Mcconville E J, et al. Production of leucinostatins and nematicidal activity of Australian isolates of Paecilomyces lilacinus(Thom) Samson[J]. Letters in Applied Microbiology, 2004, 38(4):271-276.
[8] Sharma A, Sharma S, Yadav S, et al. Role of Karanja deoiled cake based medium in production of protease and fatty acids by Paecilomyces lilacinus 6029[J]. Journal of Bioscience and Bioengineering, 2014, 118(3):270-271.
[9] Seo Y, Kim T H. Control of Meloidogyne incognita using mixtures of organic acids[J]. The Plant Pathology Journal, 2014, 30(4):450-455.
[10] 王明祖,周华众,付艳平,等. 36-1菌对植物病原真菌的拮抗性[J].中国生物防治, 1996, 12(1):20-23.
[11] Wang, G, Liu Z G, Lin R M, et al. Biosynthesis of antibiotic leucinostatins in bio-control fungus Purpureocillium lilacinum and their inhibition on Phytophthora revealed by genome mining[J]. PLoS Pathogens, 2016, 12(7):e1005685.
[12] Meléndez-González C, Murià-González M J, Anaya A L, et al. Acremoxanthone E, a novel member of heterodimeric polyketides with a bicyclo[3.2.2] nonene ring, produced by Acremonium camptosporum W. GAMS (Clavicipitaceae) endophytic fungus[J]. Chemistry Biodiversity, 2015, 12(1):133-147.
[13] 郝泽婷,郝晓慧,阎婧,等.淡紫拟青霉次级代谢产物的研究进展[J].生物技术通报, 2018, 34(10):11-17.
[14] 刘杏忠,罗科,张青文.淡紫拟青霉发酵液生理活性的初步鉴定[J].北京农业大学学报, 1991(2):48, 68.
[15] Elsherbiny E A, Taher M A, Elsebai M F. Activity of Purpureocillium lilacinum filtrates on biochemical characteristics of Sclerotinia sclerotiorum and induction of defense responses in common bean[J]. European Journal of Plant Pathology, 2019, 155(1):39-52.
[16] Song Z Y, Shen L, Zhong Q, et al. Liquid culture production of microsclerotia of Purpureocillium lilacinum for use as bionematicide[J]. Nematology, 2016, 18(6):719-726.
[17] Biles C L, Holland M, Ulloa-Godinez M, et al. Pyrenochaeta terrestris microsclerotia production and pigmentation on onion roots[J]. Hortscience, 1992, 27(11):1213-1216.
[18] Coleysmith J R, Cooke R C. Survival and germination of fungal sclerotia[J]. Annual Review of Phytopathology, 1971, 9(1):65-92.
[19] Townsend B B, Willetts H J. The development of sclerotia of certain fungi[J]. Transactions British Mycological Society, 1954. 37(3):213-221.
[20] Villamizar L F, Nelson T L, Jones S A et al. Formation of microsclerotia in three species of Beauveria and storage stability of a prototype granular formulation[J]. Biocontrol Science and Technology, 2018, 28(12):1097-1113.
[21] 殷幼平,黄姗,宋章永,等.莱氏野村菌CQNr01微菌核的人工诱导培养[J].中国农业科学, 2012, 45(23):4801-4807.
[22] Jackson M A, Jaronski S T. Production of microsclerotia of the fungal entomopathogen Metarhizium anisopliae and their potential for use as a biocontrol agent for soil-inhabiting insects[J]. Mycological Research, 2009, 113(Pt 8):842-850.
[23] 杨欣,张大敏,李增智.布氏白僵菌微菌核的诱导培养及条件优化[J].菌物学报, 2014, 33(4):827-837.
[24] Kobori N N, Mascarin G M, Jackson M A, et al. Liquid culture production of microsclerotia and submerged conidia by Trichoderma harzianum active against damping-off disease caused by Rhizoctonia solani[J]. Fungal Biology, 2015, 119(4):179-190.
[25] 张家家,李世东,郭荣君,等.淡紫紫孢菌YES和粉红螺旋聚孢霉67-1组合对黄瓜根结线虫病的防效评价[J].中国生物防治学报, 2014, 30(6):787-794.
[26] 聂海珍,孙漫红,李世东,等.棉隆与淡紫拟青霉联合防治番茄根结线虫病的效果评价[J].植物保护学报, 2016, 43(4):689-696.
[27] Dunn W B, Broadhurst D, Begley P, et al. Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry[J]. Nature Protocols, 2011, 6(7):1060-1083.
[28] Zelena E, Dunn W B, Broadhurst D, et al. Development of a robust and repeatable UPLC-MS method for the long-term metabolomic study of human serum[J]. Analytical Chemistry, 2009, 81(4):1357-1364.
[29] Want E J, Masson P, Michopoulos F, et al. Global metabolic profiling of animal and human tissues via UPLC-MS[J]. Nature Protocols, 2013, 8(1):17-32.
[30] Navarro-Reig M, Jaumot J, García-Reiriz A, et al. Evaluation of changes induced in rice metabolome by Cd and Cu exposure using LC-MS with XCMS and MCR-ALS data analysis strategies[J]. Analytical and Bioanalytical Chemistry, 2015, 407(29):8835-8847.
[31] Horai H, Arita M, Kanaya S, et al. MassBank:a public repository for sharing mass spectral data for life sciences[J]. Journal of Mass Spectrometry, 2010, 45(7):703-714.
[32] Thévenot E A, Roux A, Xu Y, et al. Analysis of the human adult urinary metabolome variations with age, body mass index, and gender by implementing a comprehensive workflow for univariate and OPLS statistical Analyses[J]. Journal of Proteome Research, 2015, 14(8):3322-3335.
[33] Chen S Y, Dickson D W, Mitchell D J. Viability of Heterodera glycines exposed to fungal filtrates[J]. Journal of Nematology, 2000, 32(2):190-197.
[34] Verdejo-Lucas S, Viera A, Stchigel A M, et al. Screening culture filtrates of fungi for activity against Tylenchulus semipenetrans[J]. Spanish Journal of Agricultural Research, 2009, 7(4):896-904.
[35] Zaki M J. Effect of fungal culture filtrates on mortality and hatching of Meloidogyne javanica[J]. Pakistan Journal of Biological Sciences, 1999, 2(1):161-163.
[36] 董锦艳,李国红,张克勤.真菌杀线虫代谢物的研究进展[J].菌物系统, 2001, 20(2):286-296.
[37] Sharma A, Sharma S, Mittal A, et al. Evidence for the involvement of nematocidal toxins of Purpureocillium lilacinum 6029 cultured on Karanja deoiled cake liquid medium[J]. World Journal of Microbiology and Biotechnology, 2016, 32(5):1-5.
[38] 林茂松,沈纪冬,文玲,等.淡紫拟青霉代谢产物生理活性物质分析和生物测定[J].云南农业大学学报, 1999, 14(S1):70-74.
[39] 杨媛.淡紫拟青霉代谢产物成分比较及其对番茄生长的影响[D].哈尔滨:东北农业大学, 2019.
[40] Villamizar L F, Barrera G, Marshall S D G, et al. Three-dimensional cellular aggregates formed by Beauveria pseudobassiana in liquid culture with potential for use as a biocontrol agent of the African black beetle (Heteronychus arator)[J]. Mycology, 2020, 12(2):105-118.
[41] Koppenhӧfer A M, Wu S, Kostromytska O S. Microsclerotial granular formulation of the entomopathogenic fungus Metarhizium brunneum and its combinations with hydrogel and imidacloprid against the annual bluegrass weevil (Coleoptera:Curculionidae)[J]. Journal of Economic Entomology, 2020, 113(3):1118-1128.