拮抗内生球形赖氨酸芽孢杆菌诱导采后苹果抗病机理及关键信息码的研究

Pathogen infection is the main reason of postharvest diseases of apple. Antagonistic endophytic bacteria, as a kind of important biological control microbes, can inhibit pathogen, induce fruit tissue producing defense reaction and improve disease resistance. It is the focus in the field of biological control due to its safety and environment-friendly. Endophytic Lysinibacillus sphaericus (Genebank accession No. KY565423) obtained from the preliminary studies showed antagonistic effects on main pathogenic molds of apple. The project will study the mechanism of induced resistance in postharvest apple by endophytic bacterium. The antagonistic metabolites are screened, physiological indexes of the growth and cell membrane permeability of pathogenic molds treated with the active metabolites are determined and metabolic enzyme systems including protection enzymes of pathogenic molds, regulatory enzymes in the Embden-Meyerhof-Parnas pathway and oxidoreductases in the tricarboxylic acid cycle are tested. Furthermore, influence of antagonistic bacterium elicitors on cell tissue resistance, secondary metabolites, enzyme activities and pathogenesis related proteins are analyzed to make sure of phenotypic characteristics and pathways, and then the mechanism of improving immunity will be verified. The key information codes including elicitors and receptors are clear. The research achievements will enrich the basic theory of biological control by antagonistic microbes and provide scientific basis for the physiological and biochemical actions of postharvest apples and the effective control of apple fungal diseases.

病原菌侵染是造成采后苹果致病的主要原因。拮抗内生菌作为一类重要的生防微生物，能抑制病原菌，诱导苹果组织产生防御反应，提高果实抗病性，安全、环保，已成为采后果实生物防治领域的研究热点。前期研究中获得的内生球形赖氨酸芽孢杆菌（Genebank登录号KY565423），对苹果病害主要病原菌具有拮抗作用。课题基于探究该内生菌诱导采后苹果抗病机理为导向，筛选该拮抗菌代谢产物；分析活性产物对病原菌生长和细胞膜透性等生理指标的影响、对病原菌保护酶、糖酵解调解酶和三羧酸氧化还原酶等酶系统的拮抗作用；研究拮抗菌诱导采后果实细胞结构抗性、次生代谢途径、酶活性和病程相关蛋白的变化，确定诱导苹果组织获得抗性的表型特征和途径，探明免疫力提高的机理；查明防御响应过程的激发子与受体等关键信息码。研究成果丰富了拮抗菌生物防治病害的基础理论，为揭示受诱导后苹果生理生化活动、有效防治苹果真菌病害提供科学依据。

病原菌侵染是采后苹果等果实致病的主要原因。项目以拮抗内生菌为生防菌，通过抑制病原菌、诱导寄主产生防御反应及产生拮抗物质提高抗病能力，阐明内生菌对病原菌的抑制机理，探明拮抗内生菌诱导采后果实抗病机制。. 重要结果如下：①筛选拮抗菌与活性物质，包括球形赖氨酸芽孢杆菌 Ya6及胞外多糖和银杏多糖、假单胞杆菌R6及菌体蛋白和白果蛋白、高地芽孢杆菌 Q7及发酵脂肽、贪噬菌R1、红球菌 FY-5及发酵黄酮和银杏叶黄酮、银杏酸等，优化制备条件，并进行结构表征。②研究抑菌活性，通过对扩展青霉等真菌和金黄色葡萄球菌等细菌的抑菌性研究，获得抑菌谱、抑制浓度等参数。③ 拮抗菌和活性物质对病原菌的抑制机理研究，结果表明拮抗菌破坏病原菌细胞膜通透性，加速膜脂氧化，抑制真菌产孢子能力，菌体蛋白和核酸外泄，菌体破裂。从代谢酶分析，影响病原菌的POD等代谢酶，阻碍营养物质利用，表明是通过作用于病原菌酶系统或是抑制代谢酶合成进行抑制。④对采后苹果贮藏的影响研究，结果为内生菌及代谢产物对果实重量、硬度、褐变、VC等品质指标有积极作用，通过抑制病原菌、增强抗性酶活性来减轻疾病指数、减少营养流失，延缓衰老、延长货架期。⑤拮抗菌诱导采后苹果抗病性机制研究，结果表明拮抗菌对果实PPO等酶、MDA、β-1,3-葡聚糖酶、总酚、木质素、总RNA等关键激发因子均有诱导作用。通过诱导抗性酶来增强机体抗性，促进生长，减轻病原菌损害。拮抗菌干预采后果实糖代谢及呼吸途径，维持较高水平的蔗糖、果糖、葡萄糖、总糖，抑制蔗糖转化酶NI、AI和合成酶SS活性，增强蔗糖磷酸合成酶SPS活性；增强糖酵解和磷酸戊糖途径，提高抗性；抑制三羧酸途径，进而抑制总呼吸途径，延缓呼吸峰值。. 项目从代谢酶、糖代谢、呼吸代谢等系统阐述拮抗菌诱导采后苹果抗性的特征和途径，成果丰富了病害防治、采后果实获得抗性的理论，为拮抗内生菌生物防治采后病害提供科学依据。