贝莱斯芽胞杆菌LY7对辣椒炭疽病菌的抑菌作用及其抑菌机制初探

doi:10.16409/j.cnki.2095-039x.2025.02.044

摘要/Abstract

摘要： 辣椒炭疽病是辣椒的三大病害之一，由于辣椒炭疽病是由炭疽菌属多个不同种真菌造成，单一药剂难以达到良好防治效果，本研究探索其生物防治途径，以期为防治该病害提供更多有效措施。前期从辣椒叶片中分离获得1株对辣椒炭疽病菌Colletotrichum scovillei具有良好抑制作用的贝莱斯芽胞杆菌Bacillus velezensis LY7，通过比色法明确其生物学特性，并结合生长速率法优化其最佳发酵条件，通过固相微萃取气质联用（SPME?GC?MS）对菌株LY7的挥发物进行鉴定并进行毒力测定；通过盐酸沉淀法对菌株LY7的抗菌脂肽进行分离纯化，并使用液质联用（LC-MS）进行鉴定，分离鉴定了菌株LY7产生的抑菌活性物质，通过田间防效试验测定其防治效果。研究结果表明，菌株LY7的生长条件可以满足其在田间生产实践中的应用，以葡萄糖为碳源、酵母粉+蛋白胨为氮源的YSP培养基，按1%（v/v）接种，在30℃、pH 5.0、160 r/min发酵72 h为最佳发酵条件，且其代谢物的稳定性较好。菌株LY7可以产生2-十一醇、2-十一酮、2-十二酮等6种挥发性物质，其中2-十一醇对辣椒炭疽病菌的毒力较高，EC₅₀为6.73 μL/mL；菌株LY7可以产生脂肪酰胺类物质（FA） 42种、异戊醇脂质类物质（PR） 8种、鞘氨醇类物质（SP） 3种、甘油磷脂类物质（GP） 2种、季铵碱类物质（QA） 1种，共56种潜在抗菌脂肽。田间防效试验表明，菌株LY7稀释80倍发酵液灌根防治效果最高，达到76.70%，显著高于对照药剂咪鲜胺处理。研究结果为后续菌株的开发应用奠定了基础。

关键词: 辣椒炭疽病菌, 贝莱斯芽胞杆菌, 挥发性物质, 抗菌脂肽, 田间防效

Abstract: Pepper anthracnose is one of the three major diseases of pepper. Due to the genetic diversity of Colletotrichum pathogen complexes, single-site fungicides are difficult to achieve good control effects. This study explored its biological control methods in order to provide more effective measures for the prevention and control of pepper anthracnose. A strain of Bacillus velezensis LY7 with good inhibitory effect on Colletotrichum scovillei was isolated from pepper leaves. Its biological characteristics were determined by colorimetry, and the optimal fermentation conditions were optimized by growth rate method. The volatiles of the strain LY7 were identified by solid phase microextraction gas chromatography-mass spectrometry (SPME?GC?MS) and assessed for virulence. In addition, the antifungal lipopeptide was isolated and purified by hydrochloric acid precipitation method, and identified by liquid chromatography-mass spectrometry (LC-MS). Further, the antifungal active substances produced by strain LY7 were isolated and identified, and the control effect was determined by field experiment. The results showed that the growth conditions of strain LY7 could meet its application in field production practice. The optimal fermentation conditions were as follows: YSP medium with glucose as carbon source, yeast powder and peptone as nitrogen source, 1% (v/v) inoculation, 30 ℃, pH 5.0, 160 r/min for 72 h, and the stability of its metabolites was better. Strain LY7 could produce 6 volatile substances, including 2-undecanol, 2-undecanone and 2-dodecanone, among which 2-undecanol has higher toxicity to C. scovillei with an EC₅₀ of 6.73 μL/mL. In addition, strain LY7 can also produce 56 potential antibacterial lipopeptides that inhibit the occurrence of C. scovillei, including 42 fatty amides (FA), 8 isoamyl alcohol lipids (PR), 3 sphingosines (SP), 2 glycerophospholipids (GP), and 1 quaternary ammonium alkali (QA). Field experiments showed that the 80-fold diluted fermentation broth of strain LY7 had the highest control effect, reaching 76.70%, which was significantly higher than the treatment of prochloraz in the field. The results of this study laid a foundation for the future development and application of the strain.

Key words: pepper anthracnose, Bacillus velezensis, volatile substances, antibacterial lipopeptide, field control effect

中图分类号:

S476

吕红, 邹晓璐, 徐紫璐, 秦楠, 殷辉, 任璐, 赵晓军. 贝莱斯芽胞杆菌LY7对辣椒炭疽病菌的抑菌作用及其抑菌机制初探[J]. 中国生物防治学报, 2025, 41(5): 1104-1118.

Lü Hong, ZOU Xiaolu, XU Zilu, QIN Nan, YIN Hui, REN Lu, ZHAO Xiaojun. Antifungal Effect and Mechanism of Bacillus velezensis LY7 on Colletotrichum scovillei[J]. Chinese Journal of Biological Control, 2025, 41(5): 1104-1118.

参考文献

[1] 联合国粮食及农业组织. https://www.fao.org/home/zh.
[2] Than P P, Jeewon R, Hyde K D, et al. Characterization and pathogenicity of Colletotrichum species associated with anthracnose on chilli (Capsicum spp.) in Thailand[J]. Plant Pathology, 2008, 57(3): 562-572.
[3] Damm U, Cannon P F, Liu F, et al. The Colletotrichum orbiculare species complex: Important pathogens of field crops and weeds[J]. Fungal Diversity, 2013, 61: 29-59.
[4] Dean R, Van Kan J A L, Pretorius Z A, et al. The top 10 fungal pathogens in molecular plant pathology[J]. Molecular Plant Pathology, 2012, 13(4): 414-430.
[5] Diao Y Z, Zhang C, Liu F, et al. Colletotrichum species causing anthracnose disease of chili in China[J]. Persoonia-Molecular Phylogeny and Evolution of Fungi, 2017, 38(1): 20-37.
[6] Miliute I, Buzaite O, Baniulis D, et al. Bacterial endophytes in agricultural crops and their role in stress tolerance: a review[J]. Zemdirbyste Agriculture, 2015, 102(4): 465-478.
[7] Zheng Y K, Qiao X G, Miao C P, et al. Diversity, distribution and biotechnological potential of endophytic fungi[J]. Annals of Microbiology, 2016, 66: 529-542.
[8] Nguyen A D, Wang S L, Trinh T H T, et al. Plant growth promotion and fungal antagonism of endophytic bacteria for the sustainable production of black pepper (Piper nigrum L.)[J]. Research on Chemical Intermediates, 2019, 45: 5325-5339.
[9] Algam S A, Xie G L, Coosemans J. Delivery methods for introducing endophytic Bacillus into tomato and their effect on growth promotion and suppression of tomato wilt[J]. Plant Pathology Journal, 2005, 4: 69-74.
[10] Zheng X, Zhou M, Yoo H, et al. Spatial and temporal regulation of biosynthesis of the plant immune signal salicylic acid[J]. Proceedings of the National Academy of Sciences, 2015, 112(30): 9166-9173.
[11] Chowdhury S P, Hartmann A, Gao X W, et al. Biocontrol mechanism by root-associated Bacillus amyloliquefaciens FZB42-a review[J]. Frontiers in Microbiology, 2015, 6: 780.
[12] Kai M. Diversity and distribution of volatile secondary metabolites throughout Bacillus subtilis isolates[J]. Frontiers in Microbiology, 2020, 11: 559.
[13] Zhao P, Li P, Wu S, et al. Volatile organic compounds (VOCs) from Bacillus subtilis CF-3 reduce anthracnose and elicit active defense responses in harvested litchi fruits[J]. AMB Express, 2019, 9: 119.
[14] Wu S, Wu S. Processivity and the mechanisms of processive endoglucanases[J]. Applied Biochemistry and Biotechnology, 2020, 190(2): 448-463.
[15] Fukamizo T. Chitinolytic enzymes catalysis, substrate binding, and their application[J]. Current Protein and Peptide Science, 2000, 1(1): 105-124.
[16] Onaga S, Taira T. A new type of plant chitinase containing LysM domains from a fern (Pteris ryukyuensis): roles of LysM domains in chitin binding and antifungal activity[J]. Glycobiology, 2008, 18(5): 414-423.
[17] Bonmatin J M, Laprévote O, Peypoux F. Diversity among microbial cyclic lipopeptides: iturins and surfactins. Activity-structure relationships to design new bioactive agents[J]. Combinatorial Chemistry & High Throughput Screening, 2003, 6(6): 541-556.
[18] Buchoux S, Lai-Kee-Him J, Garnier M, et al. Surfactin-triggered small vesicle formation of negatively charged membranes: a novel membrane-lysis mechanism[J]. Biophysical Journal, 2008, 95(8): 3840-3849.
[19] Maget-Dana R, Peypoux F. Iturins, a special class of pore-forming lipopeptides: biological and physicochemical properties[J]. Toxicology, 1994, 87(1-3): 151-174.
[20] Deleu M, Paquot M, Nylander T. Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes[J]. Biophysical Journal, 2008, 94(7): 2667-2679.
[21] Yamamoto S, Shiraishi S, Suzuki S. Are cyclic lipopeptides produced by Bacillus amyloliquefaciens S13-3 responsible for the plant defence response in strawberry against Colletotrichum gloeosporioides?[J]. Letters in Applied Microbiology, 2015, 60(4): 379-386.
[22] Cao S, Zheng Y, Yang Z, et al. Effect of methyl jasmonate on the antifungal activity of Colletotrichum acutatum infection in loquat fruit and the possible mechanisms[J]. Postharvest Biology and Technology, 2008, 49(2): 301-307.
[23] Ghorbanpour M, Omidvari M, Abbaszadeh-Dahaji P, et al. Mechanisms underlying the protective effects of beneficial fungi against plant diseases[J]. Biological Control, 2018, 117: 147-157.
[24] Xu S, Liu Y X, Cernava T, et al. Fusarium fruiting body microbiome member Pantoea agglomerans inhibits fungal pathogenesis by targeting lipid rafts[J]. Nature Microbiology, 2022, 7(6): 831-843.
[25] Ren L, Zhou J, Yin H, et al. Antifungal activity and control efficiency of endophytic Bacillus velezensis ZJ1 strain and its volatile compounds against Alternaria solani and Botrytis cinerea[J]. Journal of Plant Pathology, 2022, 104(2): 575-589.
[26] Gao Z F, Zhang B J, Liu H P, et al. Identification of endophytic Bacillus velezensis ZSY-1 strain and antifungal activity of its volatile compounds against Alternaria solani and Botrytis cinerea[J]. Biological Control, 2017, 105: 27-39.
[27] 马东丽, 刘晓峰, 任璐, 等. 醉鱼草内生细菌ZJ1生防机制初探[J]. 山西农业科学, 2021, 49(5): 634-638.
[28] 高振峰. 内生细菌ZSY-1对番茄灰霉病和早疫病的防治及促生效果研究[D]. 太原: 山西农业大学, 2018.
[29] 国家质量技术监督局. GB/T 17980.33-2000. 农药田间药效试验准则(一)杀菌剂防治辣椒炭疽病[S]. 北京: 中国标准出版社, 2000.
[30] Gauvry E, Mathot A G, Couvert O, et al. Effects of temperature, pH and water activity on the growth and the sporulation abilities of Bacillus subtilis BSB1[J]. International Journal of Food Microbiology, 2021, 337: 108915.
[31] Xing Y X, Wei C Y, Mo Y, et al. Nitrogen-fixing and plant growth-promoting ability of two endophytic bacterial strains isolated from sugarcane stalks[J]. Sugar Technology, 2016, 18: 373-379.
[32] Tilocca B, Cao A, Migheli Q. Scent of a killer: microbial volatilome and its role in the biological control of plant pathogens[J]. Frontiers in Microbiology, 2020, 11: 509409.
[33] Audrain B, Farag M A, Ryu C M, et al. Role of bacterial volatile compounds in bacterial biology[J]. FEMS Microbiology Reviews, 2015, 39(2): 222-233.
[34] Schmidt R, Cordovez V, De Boer W, et al. Volatile affairs in microbial interactions[J]. The ISME Journal, 2015, 9(11): 2329-2335.
[35] Li X, Wang X, Shi X, et al. Antifungal effect of volatile organic compounds from Bacillus velezensis CT32 against Verticillium dahliae and Fusarium oxysporum[J]. Processes, 2020, 8(12): 1674.
[36] Choub V, Won S J, Ajuna H B, et al. Antifungal activity of volatile organic compounds from Bacillus velezensis CE 100 against Colletotrichum gloeosporioides[J]. Horticulturae, 2022, 8(6): 557.
[37] Liu Y, Liu J, Liu M, et al. Comparative non-targeted metabolomic analysis reveals insights into the mechanism of rice yellowing[J]. Food chemistry, 2020, 308: 125621.
[38] Kalinger R S, Pulsifer I P, Hepworth S R, et al. Fatty acyl synthetases and thioesterases in plant lipid metabolism: diverse functions and biotechnological applications[J]. Lipids, 2020, 55(5): 435-455.
[39] Bita C, Gerats T. Plant tolerance to high temperature in a changing environment: scientific fundamentals and production of heat stress-tolerant crops[J]. Frontiers in Plant Science, 2013, 4: 273.
[40] Chowdhury S K, Dutta T, Chattopadhyay A P, et al. Isolation of antimicrobial Tridecanoic acid from Bacillus sp. LBF-01 and its potentialization through silver nanoparticles synthesis: a combined experimental and theoretical studies[J]. Journal of Nanostructure in Chemistry, 2021, 11: 573-587.
[41] Wadsworth J M, Clarke D J, McMahon S A, et al. The chemical basis of serine palmitoyltransferase antifungal activity by myriocin[J]. Journal of the American Chemical Society, 2013, 135(38): 14276-14285.
[42] Yamaji-Hasegawa A, Takahashi A, Tetsuka Y, et al. Fungal metabolite sulfamisterin suppresses sphingolipid synthesis through antifungal activity of serine palmitoyltransferase[J]. Biochemistry, 2005, 44(1): 268-277.
[43] Shao J, Pei Z, Jing H, et al. Antifungal activity of myriocin against Fusarium graminearum and its antifungal activity on deoxynivalenol production in wheat grains[J]. Physiological and Molecular Plant Pathology, 2021, 114: 101635.
[44] Baczewska A H, Dmuchowski W, Jozwiak A, et al. Effect of salt stress on prenol lipids in the leaves of Tilia 'Euchlora' [J]. Dendrobiology, 2014, 72: 177-186.
[45] Bohlmann J, Keeling C I. Terpenoid biomaterials[J]. The Plant Journal, 2008, 54(4): 656-669.
[46] Duraipandiyan V, Indwar F, Ignacimuthu S. Antimicrobial activity of confertifolin from Polygonum hydropiper[J]. Pharmaceutical Biology, 2010, 48(2): 187-190.
[47] Hofius D, Sonnewald U. Vitamin E biosynthesis: biochemistry meets cell biology[J]. Trends in Plant Science, 2003, 8(1): 6-8.
[48] Ashraf M, Foolad M R. Roles of glycine betaine and proline in improving plant abiotic stress resistance[J]. Environmental and Experimental Botany, 2007, 59(2): 206-216.
[49] Feng B, Chen D, Jin R, et al. Bioactivities evaluation of an endophytic bacterial strain Bacillus velezensis JRX-YG39 inhabiting wild grape[J]. BMC Microbiology, 2022, 22(1): 170.
[50] Yan Y, Xu W, Hu Y, et al. Bacillus velezensis YYC promotes tomato growth and induces resistance against bacterial wilt[J]. Biological Control, 2022, 172: 104977.
[51] Nie L J, Ye W Q, Xie W Y, et al. Biofilm: New insights in the biological control of fruits with Bacillus amyloliquefaciens B4[J]. Microbiological Research, 2022, 265: 127196.