山东苹果主产区红富士果面酵母菌多样性及对苹果炭疽病的生防潜能

Colletotrichum gloesporiodes is a major infectious disease of apple in the growing and storage period. And this disease is one of the main causes of yield and quality losses of apple. The traditional chemical control exists fruit pesticide residues, environmental pollution and pathogens resistant to fungicides, etc., Antagonistic yeasts are widely used in biological control of plant diseases due to they has many advantages: genetically stable, broad spectrum antimicrobial, low nutritional requirements, rapid growth and reproduction, and does not produce harmful metabolites, with strong tolerance to a variety of stress and adversity, insensitive to most chemical germicide, and can be used in conjunction with a variety of chemicals and physical methods, etc., Antagonistic yeasts are isolated from apple fruit surface which contains a large number of unknown yeast resources. yeasts are identified based on phenotypic and physiological characteristics and sequence analyses of the 16S rRNA gene, internal transcribed spacer (ITS) regions and 26S rRNA gene D1/D2 domain. Combined with the polymerase chain reaction - single strand conformation polymorphism biotechnology research species and its physiological characteristics of yeasts on apple fruit surface. Screening of a broad spectrum of antagonistic yeast strains using in vitro and in vivo methods. To explore antagonistic mechanism of biocontrol yeast and optimize conditions for improving the effectiveness of biological control.

苹果炭疽病（Colletotrichum gloesporiodes）是苹果生长期和贮藏期的主要侵染性病害，是造成产量和品质损失的主要原因之一。传统化学防治存在农药残留、环境污染以及病原菌抗药性等弊端。拮抗酵母菌因具有遗传稳定，抑菌谱广，营养要求低，生长繁殖快速，不产生对人和寄主有害的代谢产物，对多种胁迫条件和逆境具有较强的耐受力，并且对多数化学杀菌剂不敏感，能与多种化学物质及物理方法结合使用等优点广泛用于植物病害生物防治。本项目基于拮抗菌在生境位的适应能力和有效定殖，从苹果上蕴含的大量未知酵母资源中分离酵母菌株；依据酵母菌形态、生理生化特征以及ITS和26S rRNA基因D1/D2区序列进行鉴定，并结合PCR-SSCP等现代分子生物技术等研究苹果果面酵母菌多样性；采用离体与活体结合筛选出广谱拮抗菌株并探索生防机理、优化生防效力，以期获得新型高效的拮抗酵母菌，为苹果病害的生物防治奠定基础。

在山东主产区采集成熟健康的红富士苹果，采用稀释分离法从苹果样品果面分离纯化得到酵母菌株，依据核糖体26S D1/D2区核苷酸序列的比较，并结合形态学特征，鉴定出酵母菌的分类地位，并对红富士果实表面酵母菌的多样性进行系统进化分析；采用离体与活体结合筛选出拮抗苹果炭疽菌的酵母菌株，探索生防机理并优化生防效力。结果表明，红富士果实表面蕴含丰富的酵母菌资源，共鉴定为13个属20个种；子囊菌酵母占优势，从属于6个属10个种，占酵母总数的58.3%；担子菌酵母从属于6个属9个种，占酵母总数的32.5%；其余为普鲁兰短梗霉等类酵母，占酵母总数的9.2%。红富士果实表面酵母菌的优势属为毕赤酵母属Pichia（3个种）、假丝酵母属Candida（3个种）、隐球酵母属Cryptococcus（3个种），共占酵母总种数的45%。离体防效试验表明，酵母菌株YG3（Cryptococcus flavescens GenBank NO. MF045447）、YG12（Metschnikowia pulcherrima GenBank NO. MF045456）、YG16（Rhodotorula glutinis GenBank NO. MF045460），明显抑制病原菌菌丝的生长，抑制率分别为87.2%、89.6%和91.1%；活体防效试验表明，酵母菌株YG3、YG12、YG16均对苹果炭疽病具有明显的抑制效果，防治效果分别为72.2、77.2、80.4%。拮抗酵母菌对苹果炭疽菌有一定抑制作用，其抑菌效果是多种拮抗机理相互作用的结果，拮抗酵母菌能够在苹果伤口快速生长并维持较高的拮抗活性，与病原菌竞争的营养与空间，分泌胞外裂解酶，诱导提高果实抗性相关的酶活性，来减缓贮藏过程中果实的衰老及病原菌的侵入，并且拮抗酵母菌与肉桂酸、茉莉酸甲酯、壳聚糖等外源性物质结合使用，可以提高生物防治的效果。本研究为苹果病害的生物防治奠定基础，对于苹果产业绿色生产具有重要意义。