贝莱斯芽胞杆菌5YN8生物被膜在防治番茄灰霉病过程中的功能研究

doi:10.16409/j.cnki.2095-039x.2022.06.010

摘要/Abstract

摘要： 番茄灰霉病是由灰葡萄孢菌Botrytis cinerea引起的番茄上重要的土传病害之一，危害番茄果实、叶、茎等部位，严重制约番茄的安全生产，微生物菌剂的施用被认为是防治该病害的有效措施。前期研究发现贝莱斯芽胞杆菌5YN8能够显著防治番茄灰霉病，本研究通过构建相关基因突变体，探究生物被膜在5YN8防病过程中的作用，结果显示生物被膜增强突变体具有更致密的褶皱，而减弱突变体生物被膜光滑。进一步研究发现，菌株5YN8及其突变体处理14 d后，生物被膜减弱突变显著减少细菌在叶面的定殖，而增强突变则提升其定殖。另外，生物被膜减弱突变体防病效果显著降低，∆spo0A对灰霉病防效仅为16.02%，而生物被膜增强突变体∆abrB、∆sinR防病效果显著增强，分别达80.77%、78.94%。相关性分析表明，5YN8的抑菌率、叶面定殖与其防效显著正相关。综合结果表明，5YN8生物被膜的形成依赖保守基因的调控，通过改变生防菌定殖及抑菌能力影响对番茄灰霉病防效。上述研究将为生防菌5YN8的功能修饰增强及在番茄灰霉病的应用提供理论基础。

关键词: 番茄灰霉病, 贝莱斯芽胞杆菌, 生物防治, 生物被膜, 定殖

Abstract: Gray mold disease is one of the important soil borne diseases of tomato,which is caused by Botrytis cinerea.It endangers the fruit,leaf,stem and other parts of tomato and seriously restricts the safe production of tomato.In the present,the application of microbial agents is considered to be an effective measure to control this disease.Previously,it was found that Bacillus velezensis 5YN8 could significantly control gray mold disease in tomato.In this study,we constructed mutants of biofilm-related genes to explore the role of biofilm in the disease control.The results showed that the biofilm-induced mutants formed more robust biofilms with significant more wrinkles,while the biofilm-impaired mutants formed weaker biofilms with smooth surfaces and less wrinkles.Further results demonstrated that the colonization of the biofilm-impaired mutants on the leaf was significantly reduced 14 days after bacterial treatment,meanwhile,the leaf colonization of the biofilm-induced mutants was significantly increased.The disease control effect of biofilm-impaired mutant∆spo0A significantly reduced,with the efficacy of only 16.02%.On the contrary,the control effect of∆abrB and∆sinR significantly increased,with the efficacies of 80.77% and 78.94%,respectively.Correlation analysis indicated that there was a significant positive correlation between the inhibition zone,the bacterial colonization and the control efficacy of 5YN8.To sum up,it was found that the formation of 5YN8 biofilm depended on the regulation of conserved genes,and the control effect of B.cinerea in tomato was affected by colonization and antagonism of 5YN8.The above results will provide a theoretical basis for the functional modification and enhancement of the biocontrol agent 5YN8 and its application in B.cinerea.

Key words: gray mold disease, Bacillus velezensis, biological control, biofilm, colonization

中图分类号:

S476

司方洁, 任金瑶, 黄涛祥, 俞仪阳, 郭坚华, 蒋春号. 贝莱斯芽胞杆菌5YN8生物被膜在防治番茄灰霉病过程中的功能研究[J]. 中国生物防治学报, 2022, 38(5): 1223-1230.

SI Fangjie, REN Jinyao, HUANG Taoxiang, YU Yiyang, GUO Jianhua, JIANG Chunhao. The Function of Bacillus velezensis 5YN8 Biofilm in Controlling of Botrytis cinerea in Tomato[J]. Chinese Journal of Biological Control, 2022, 38(5): 1223-1230.

参考文献

[1] 韩君,范怀峰,王海娜,等.防治灰霉病药剂的开发进展[J].农药研究与应用, 2011(3):5-10.
[2] 董金荣,王玉楼,吉同銮,等.保护地番茄主要病害综合防治技术初探[J].安徽农学通报, 2011, 17(16):104, 158.
[3] 李宝聚,朱国仁,关天舒,等.节能日光温室中番茄灰霉病发生规律的研究[J].植物保护, 2003, 29(2):26-29.
[4] Barret M, Morrissey J P, O'Gara F. Functional genomics analysis of plant growth-promoting rhizobacterial traits involved in rhizosphere competence[J]. Biology and Fertility of Soils, 2011, 47(7):729-743.
[5] Bais H P, Fall R, Vivanco J M. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[J]. Plant Physiology, 2004, 134(1):307-319.
[6] Chu F, Kearns D B, Branda S S, et al. Targets of the master regulator of biofilm formation in Bacillus subtilis[J]. Molecular Microbiology, 2006, 59(4):1216-1228.
[7] Bai U, Mandic-Mulec I, Smith I. SinI modulates the activity of SinR, a developmental switch protein of Bacillus subtilis, by protein-protein interaction[J]. Genes& Development, 1993, 7(1):139-148.
[8] Chu F, Kearns D B, Mcloon A, et al. A novel regulatory protein governing biofilm formation in Bacillus subtilis[J]. Molecular Microbiology, 2008, 68(5):1117-1127.
[9] Kearns D B, Chu F, Branda S S, et al. A master regulator for biofilm formation by Bacillus subtilis[J]. Molecular Microbiology, 2005, 55(3):739-749.
[10] Jiang C H, Liao M J, Wang H K, et al. Bacillus velezensis, a potential and efficient biocontrol agent in control of pepper gray mold caused by Botrytis cinerea[J]. Biological Control, 2018, 126:S466746282.
[11] Romero D, de Vicente A, Rakotoaly R H, et al. The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca[J]. Molecular Plant-Microbe Interactions, 2007, 20(4):430-440.
[12] Davey M E, O'Toole G A. Microbial biofilms:from ecology to molecular genetics[J]. Microbiology and Molecular Biology Reviews, 2000, 64(4):847.
[13] Gryczan T J, Contente S, Dubnau D. Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis[J]. Journal of Bacteriology, 1978, 134(1):318-329.
[14] Kearns D B, Chu F, Rudner R, et al. Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility[J]. Molecular Microbiology, 2004, 52(2):357-369.
[15] Chu F, Kearns D B, Mcloon A, et al. A novel regulatory protein governing biofilm formation in Bacillus subtilis[J]. Molecular Microbiology, 2008, 68(5):1117-1127.
[16] Branda S S, Gonzalez-Pastor J E, Ben-Yehuda S, et al. Fruiting body formation by Bacillus subtilis[J]. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(20):11621-11626.
[17] Chen Y, Cao S, Chai Y, et al. A Bacillus subtilis sensor kinase involved in triggering biofilm formation on the roots of tomato plants[J]. Molecular Microbiology, 2012, 85(3):418-430.
[18] Chen Y, Yan F, Chai Y, et al. Biocontrol of tomato wilt disease by Bacillus subtilis isolates from natural environments depends on conserved genes mediating biofilm formation[J]. Environmental Microbiology, 2013, 15(3):848-864.
[19] Gueroutfleury A M, Frandsen N, Stragier P. Plasmids for ectopic integration in Bacillus subtilis[J]. Gene, 1996, 180(1-2):57-61.
[20] Morikawa M. Beneficial biofilm formation by industrial bacteria Bacillus subtilis and related species[J]. Journal of Bioscience& Bioengineering, 2006, 101(1):1-8.
[21] Chen Y, Cao S, Chai Y, et al. A Bacillus subtilis sensor kinase involved in triggering biofilm formation on the roots of tomato plants[J]. Molecular Microbiology, 2012, 85(3):418-430.
[22] Bais H P, Fall R, Vivanco J M. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[J]. Plant Physiology, 2004, 134(1):307-319.
[23] 张虎,肖静,原梨萍,等. spoIIE基因缺失对克劳氏芽孢杆菌淀粉酶酶活的影响[J].食品工业科技, 2019, 40(1):131-135.