一种自溶性产酶溶杆菌新菌株LE16对温室番茄灰霉病的抑制作用

Suppression of tomato gray mold by a new strain of autolytic Lysobacter enzymogenes (LE16) in greenhouse

摘要:

【目的】我国人多地少，设施农业日益普及，温室栽培保障了番茄的有效供给，在温室中种植的番茄容易发生灰霉病，造成减产。生物防治产品对人畜安全，环境友好，但亟待增加生防微生物种类，提高防效。【方法】以自主分离的自溶性产酶溶杆菌(Lysobacter enzymogenes) LE16为对象，利用纯培养试验明确其发酵液对灰霉病菌(Botrytis cinerea)的拮抗作用；生物化学、液相色谱-质谱联用和转录组技术从生防菌合成分泌的胞外水解酶、抗菌物质和病菌基因差异表达等方面，揭示生防菌拮抗病原真菌的机制；温室盆栽试验评估菌株LE16发酵液对番茄灰霉病的防治潜力。【结果】LE16能合成分泌铁载体、多种与抗病性相关的酶类(包括磷酸酶、蛋白酶、纤维素酶、β-1,3-葡聚糖酶和溶菌酶)以及9种抗真菌物质(毒菌素D、N-十一烷基苯磺酸、利福布汀、纳奈霉素、替加环素、米诺环素、间甲酚、肉桂酸和环戊酮)和激活植物系统获得性抗性的P-水杨酸。LE16发酵液粗提物显著抑制灰霉病菌生长繁殖，抑制率为34.99%-100%，显著高于产酶溶杆菌HYP18和撕裂蜡孔菌HG2011。此外，LE16发酵液显著诱导灰霉病菌基因的差异表达，涉及蛋白质合成、DNA复制与修复、信号转导等多个生物过程、细胞成分和分子功能，以及甘氨酸、丝氨酸、苏氨酸和赖氨酸等代谢通路。在温室试验中，LE16发酵液显著增加了番茄叶片的抗氧化酶(超氧化物歧化酶和过氧化物酶)活性，降低了病菌对细胞膜的伤害作用，对灰霉病的抑制效果为72.54%-74.42%，略低于化学农药嘧霉胺。【结论】LE16能通过合成分泌胞外水解酶、铁载体、抗真菌活性物质，诱导植物产生抗病性等多种机制，防治温室番茄灰霉病，具有潜在的应用前景。

Abstract:

[Objective] Facility agriculture has experienced a surge in populairty in China which features a large population and little arable land.Greenhouse cultivation guarantees tomato security in China.However,greenhouse tomato are prone to gray mold (induced by Botrytis cinerea),leading to huge yield loss.Biocontrol is safe for human being and animal and environmentally friendly.It is urgent to seek more biocontrol microorganisms to improve the control effect.[Methods] In the current series of experiments,the biocontrol potentitial of a self-isolated new strain (LE16) of autolytic Lysobacter enzymogenes was explored.To be specific,pure culture was carried out to test the antagonism of LE16 fermentation broth against B.cinerea.Biochemical analysis,liquid chromatography-mass spectrometry,and transcriptome techniques were used to clarify the synthesis and secretion of extracellular hydrolases and antifungal compounds of LE16,and differentially expressed genes in B.cinerea,with a view to elucidating the antifungal mechanism.The inhibitory effect of LE16 fermentation broth on tomato gray mold was evaluated with the pot experiment in a greenhouse.[Results] LE16 synthesized and released siderophore,a variety of extracellular enzymes related to disease resistance (including phosphatase,protease,cellulase,β-1,3-glucanase and lysozyme),9 antifungal compounds (ustiloxin D,N-undecylbenzenesulfonic acid,rifabutin,nanaomycin,tigecycline,minocycline,m-cresol,cinnamic acid and cyclopentanone),and P-salicylic acid that is able to stimulate systemic acquired resistance in plants.The crude extract of LE16 fermentation broth significantly inhibited the growth and reproduction of B.cinerea,and the inhibition rate was 34.99%-100%,much higher than those of L.lysobacterium HYP18 and Ceriporia lacerata HG2011 on solid culture.Additionally,LE16 fermentation broth significantly induced the differential expression of genes in B.cinerea.The differentially expressed genes were involved in biological processes,cellular components,molecular functions (e.g.,protein synthesis,DNA replication and repair,and signal transduction),and metabolic pathways related to proteins,glycine,serine and threonine.In the greenhouse experiment,LE16 fermentation broth significantly enhanced the activity of antioxidant enzymes,including superoxide dismutase and peroxidase,in tomato leaves,and alleviated the damage to cell membranes caused by the pathogen.The inhibitory effect against gray mold varied from 72.54% to 74.42%,slightly lower than that of the chemical pesticide pyrimethanil.[Conclusion] LE16 suppresses the incidence of tomato gray mold in the greenhouse by multiple mechanisms,such as the synthesis and release of extracellular hydrolases,siderophore and antifungal compounds,and the induction of resistance to disease in the plants.Thus,LE16 shows a promising potential in controlling tomato gray mold in the greenhouse.