蒙氏肠球菌ZC-05对猕猴桃细菌性溃疡病防效评价及生防机制初探

doi:10.16409/j.cnki.2095-039x.2026.02.014

摘要/Abstract

摘要： 猕猴桃细菌性溃疡病被喻为猕猴桃的“癌症”，迄今缺乏有效防治方法。研究前期获得一株分离自猕猴桃枝条的猕猴桃细菌性溃疡病生防细菌——蒙氏肠球菌Enterococcus mundtii ZC-05，本文用绿色荧光蛋白（GFP）标记该菌株，研究其定殖特性；通过林间试验验证该菌株的实际防病效应；并基于全基因组测序分析探究其生防机制。结果表明，采用原生质体融合技术，可有效转化菌株ZC-05并成功获得带有GFP标记的转化子ZC-05-GFP。共聚焦显微镜观察发现，菌株ZC-05-GFP可在注干2周后在猕猴桃茎中定殖，并在叶表观察到。林间试验证实，菌株ZC-05菌悬液注干猕猴桃后可显著抑制猕猴桃细菌性溃疡病的发生，防效为50.96%，显著高于化学杀菌剂噻菌铜（13.46%）(P<0.05)。对菌株ZC-05的全基因组测序分析发现，该菌株含有7个T6SS效应蛋白基因，荧光定量PCR分析结果表明，编码抑菌毒性蛋白的基因Gm-1574在与猕猴桃细菌性溃疡病病原菌Psa-Y共培养后显著上调表达，可能对菌株ZC-05发挥生防效果起重要作用。本研究明确了菌株ZC-05的定殖特性，证实了菌株对猕猴桃细菌性溃疡病的林间防病效果，初步探究了菌株的生防机制，为生防注干菌剂的制备和实际开发利用提供了依据。

关键词: 猕猴桃细菌性溃疡病, 蒙氏肠球菌, 荧光标记, 生防机制

Abstract: Kiwifruit bacterial canker, often termed the “cancer” of kiwifruit, currently lacks effective control methods. In this study, a potential biocontrol strain Enterococcus mundtii ZC-05, previously isolated from stem of Kiwifruit, was labeled with green fluorescent protein(GFP) to investigate its colonization characteristics. Results indicated that protoplast fusion was an effective method for transforming strain ZC-05, successfully yielding the GFP-labeled transformant ZC-05-GFP. Confocal microscopy observation revealed that ZC-05-GFP colonized kiwifruit stems two weeks after trunk injection and reach the leaf via the sap flow. Field trials demonstrated that injection of ZC-05 suspension significantly reduced the incidence of kiwifruit bacterial canker, achieving a control efficacy of 50.96%, which was substantially higher than that of the copper-based bactericide Thiodiazole-Copper(13.46%)(P<0.05). Whole-genome sequencing of ZC-05 identified seven genes encoding type VI secretion system(T6 SS) effector proteins. Furthermore, quantitative real-time PCR analysis indicated that the gene Gm-1574, encoding an antibacterial toxic protein, was significantly up-regulated after co-culture with Pseudomonas syringae pv. actinidiae(Psa-Y), the pathogen of Kiwifruit bacterial canker, suggesting its potential important role in the biocontrol activity of ZC-05. This study clarifies the colonization characteristics of ZC-05, confirms its field efficacy against kiwifruit bacterial canker, and preliminarily elucidates its biocontrol mechanisms. These findings provide a basis for the development and practical application of trunk-injected biocontrol agents in the future.

Key words: kiwifruit bacterial canker, Enterococcus mundtii, GFP labeling, biocontrol mechanism

中图分类号:

S476

顾佳颖, 尹志诚, 李南羿, 蔡小剑, 张昕. 蒙氏肠球菌ZC-05对猕猴桃细菌性溃疡病防效评价及生防机制初探[J]. 中国生物防治学报, 2026, 42(3): 611-620.

GU Jiaying, YIN Zhicheng, LI Nanyi, CAI Xiaojian, ZHANG Xin. Evaluation of Control Efficacy of Enterococcus mundtii ZC-05 against Bacterial Canker of Kiwifruit and Preliminary Study on Its Biocontrol Mechanism[J]. Chinese Journal of Biological Control, 2026, 42(3): 611-620.

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参考文献

[1] Asif M, Liang S, Renjian H, et al. A stitch in time:Sustainable and eco-friendly solutions for kiwifruit bacterial canker[J]. Physiological and Molecular Plant Pathology, 2024, 136(1):102506.
[2] Gan K, Sawa T, Ayumi I, et al. Evaluation of various cultivars of Actinidia species and breeding source Actinidia rufa for resistance to Pseudomonas syringae pv. actinidiae biovar 3[J]. Journal of General Plant Pathology, 2018, 84(6):399-406.
[3] 钟曼茜,翟舒嘉,刘伟,等.我国即食猕猴桃产业发展现状、问题与对策[J].中国果树, 2023, 65(2):122-127.
[4] Pereira C, Costa P, Pinheiro L, et al. Kiwifruit bacterial canker:an integrative view focused on biocontrol strategies[J]. Planta, 2021, 253(2):49.
[5] Zhang Y, Chen Y, Ma J, et al. Controlling citrus Huanglongbing based on soil remediation and biocontrol[J]. European Journal of Plant Pathology, 2024,169(2):379-393.
[6] 田涛,亓雪晨,王琦,等.芽孢杆菌绿色荧光蛋白标记及其在小麦体表定殖的初探[J].植物病理学报, 2004, 34(4):346-351.
[7] Stuurman N, Bras C P, Schlaman H R M, et al. Use of green fluorescent protein color variants expressed on stable broad host range vectors to visualize rhizobia interacting with plants[J]. The American Phytopathological Society, 2000, 13(11):1163-1169.
[8] Prieto P, Mercado-blanco J. Endophytic colonization of olive roots by the biocontrol strain Pseudomonas fluorescens PICF7[J]. FEMS Microbiology Ecology, 2008, 64(2):297-306.
[9] 杨潇湘,黄小琴,张蕾,等.绿色荧光蛋白标记解淀粉芽孢杆菌Bam22在油菜体内的定殖[J].中国农学通报, 2022, 38(1):125-130.
[10] Tian R, Tian Y, Mi Q, et al. Histocytological analysis reveals the biocontrol activity of a rhizospheric bacterium Pseudomonas rhizophila Z98 against kiwifruit bacterial canker[J]. Pesticide Biochemistry and Physiology, 2025, 208(1):106251.
[11] Wang H., Wang N., Tan Y, et al. Paenibacillus polymyxa YLC1:a promising antagonistic strain for biocontrol of Pseudomonas syringae pv. actinidiae,causing kiwifruit bacterial canker[J]. Pest Management Science, 2023, 79(11):4357-4366.
[12] Li W, Lee S Y, Cho Y J, et al. Mediation of induced systemic resistance by the plant growth-promoting rhizobacteria Bacillus pumilus S2-3-2[J].Molecular Biology Reports, 2020, 47(11):8429-8438.
[13] Shao X, Wu Q, Li L, et al. Adapting the inoculation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from branch microbiome[J]. Molecular Plant Pathology, 2024, 25(1):e13399.
[14] 尹志诚,顾佳颖,李南羿,等.猕猴桃溃疡病生防菌群的构建及其生防效应[J].林业科学, 2024, 60(12):101-110.
[15] Peter A M, Lara B, Ben C, et al. Re-programming of Pseudomonas syringae pv. actinidiae gene expression during early stages of infection of kiwifruit[J].BioMed Central Genomics, 2018, 19(1):822.
[16] 李春玲,宋赵依,宗兆锋.非致病性尖镰孢FO47的原生质体融合改良研究[J].西北农林科技大学学报(自然科学版), 2010, 38(10):126-130.
[17] Teh B S, Apel J, Shao Y, et al. Colonization of the intestinal tract of the polyphagous pest Spodoptera littoralis with the GFP-tagged indigenous gut bacterium Enterococcus mundtii[J]. Frontiers in Microbiology, 2016(1), 7:928.
[18] 匡美美,高建有,罗庆,等.湘西州猕猴桃溃疡病发病情况及其影响因素分析[J].湖南农业科学, 2022, 52(1):56-59.
[19] 杨玉文,刘德华,王成梁,等.瓜类细菌性果斑病拮抗菌ZY1的鉴定及其生物学特性[J].中国生物防治学报, 2024, 40(6):1430-1438.
[20] Rio D C, Ares M J R, Hannon G J, et al. Purification of RNA using TRIzol(TRI reagent)[J]. Cold Spring Harbor Protocols, 2010, 2010(6):5439.
[21] Han C, Gu H, Wang J, et al. Regulation of L-Threonine dehydrogenase in somatic cell reprogramming[J]. Stem Cells, 2013, 31(5):953-65.
[22] Chen Q, Yu J J, He J, et al. Isobenzofuranones and isocoumarins from kiwi endophytic fungus Paraphaeosphaeria sporulosa and their antibacterial activity against Pseudomonas syringae pv. actinidiae[J]. Phytochemistry, 2022, 195(1):113050.
[23] De Jong H, Reglinski T, Elmer P A G, et al. Integrated use of Aureobasidium pullulans strain CG163 and acibenzolar-S-methyl for management of bacterial canker in kiwifruit[J]. Plants, 2019, 8(8):287.
[24] Rheinländer P A, Sutherland P W, Elmer P A G. Visualisation of the mode of action of a biological control agent, Aureobasidium pullulans(strain YBCA5)against Pseudomonas syringae pv. actinidiae biovar 3 on the kiwifruit phylloplane[J]. Australasian Plant Pathology, 2021, 50(4):379-388.
[25] Bai J, Liu Y, Liu M, et al. Application of phage therapy against red-fleshed kiwifruit canker[J]. Biological Control, 2022, 169:104893.
[26] 朱海云,马瑜,柯杨,等.猕猴桃细菌性溃疡病生防菌的筛选、鉴定及其防效初探[J].微生物学杂志, 2016, 36(5):90-96.
[27] Biondi E, Schiavi D, Perez F S, et al. Bacillus amyloliquefaciens subsp. plantarum strain D747 to control the kiwifruit bacterial canker disease[J].Proceedings of the X International Symposium on Kiwifruit, 2022, 1332(13):95-102.
[28] Ali M A, Luo J, Ahmed T, et al. Pseudomonas bijieensis strain XL17 within the P. corrugata subgroup producing 2, 4-diacetylphloroglucinol and lipopeptides controls bacterial canker and gray mold pathogens of kiwifruit[J]. Microorganisms, 2022, 10(2):425.
[29] 杜贞娜,晏子英,候忠余,等.猕猴桃溃疡病病原菌的鉴定及生防菌的筛选[J].西南农业学报, 2021, 34(4):755-761.
[30] Daranas N, RosellóG, Cabrefiga J, et al. Biological control of bacterial plant diseases with Lactobacillus plantarum strains selected for their broad-spectrum activity[J].The Annals of applied biology, 2019, 174(1), 92-105.
[31] 殷幼平,袁训娥,李强,等.生防菌枯草芽孢杆菌CQBS03的绿色荧光蛋自基因标记及其在柑橘叶片上的定殖[J].中国农业科学, 2010, 43(17):3555-3563.
[32] Pukatzki S, Ma A T, Sturtevant D, et al. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system[J]. Proceedings of The National Academy of Sciences, 2006, 103(5):1528-1533.
[33] Bernal P, Allsopp L P, Filloux A, et al. The Pseudomonas putida T6SS is a plant warden against phytopathogens[J]. The International Society for Microbial Ecology Journal, 2017, 11(4):972-987