祼脚菇属菌抑制柑橘采后病原的活性物质及其作用机理研究

The citrus fruits is largest fruit and third largest trade agricultural product in the world. Citrus output of China ranks first. Jiangxi province is the main citrus producing areas. Postharvest decay rate of citrus fruits reaches up to 20%~40%. Chemical fungicides are used to manage the decay disease. This leads to some problems such as environmental pollution, pathogenic resistance and fruit edible safety. Alternative biological fungicides are urgently needed...We have collected hundreds of wild larger fungi, obtained 59 of their pure cultures and screened a edible larger fungus with strong resistance to main citrus postharvest pathogens blue and green molds. EC50 of ethyl acetate extracts of fermentation broth against two pathogens is 69.25 µg/mL and 56.70 µg/mL respectively. The larger fungus is identified as Gymnopus sp...The objectives of the project are to ① optimize further culture conditions of the larger fungus, which adjust to produce active substance against blue and green molds; ② isolate its monomers and identify their chemical structure; ③ evaluate the effect of in vivo and in vitro active substance on antifungi and disease-control; ④ elucidate the action mechanism against citrus postharvest pathogens blue and green molds by observation effect of active substance on microstructure and ultra-structure of pathogen cells (hyphae, spores) and an important organelle mitochondria, investigation the change of cell membrane permeability, integrity and energy metabolism resulted from active substance, inducing resistance of citrus fruit to pathogens blue and green disease. The finding will be hopeful to provide lead compounds with novel structure and unique mode of action for the development of biological fungicides to manage citrus postharvest pathogens blue and green molds.

柑橘是世界第一大水果、第三大贸易农产品，中国柑橘产量居世界首位，江西是柑橘的生产大省。柑橘采后腐烂率高达20%～40%，现主要靠化学杀菌剂防治，由此引起环境污染、病原抗药性、果品食用安全等问题，迫切需求可替代的生物源杀菌剂。.我们从南昌梅岭采集到百余种野生大型真菌，获得了其中59种纯培养物，筛选出具有强抗柑橘采后主要病原青、绿霉的一株可食用大型真菌，其发酵液乙酸乙酯粗提物EC50分别为69.25µg/mL和56.70µg/mL,鉴定为祼脚菇属一菌株。.本项目拟对该菌株进一步优化培养条件，调控生产抗青、绿霉的活性物质；分离其单体，解析其化学结构；评价活性物质体内、外抗菌控病效果，观测其对病原菌丝、孢子、线粒体的显微和超微结构的影响，导致细胞膜通透性、完整性和能量代谢的变化，诱导柑橘果对病原的抗性，阐明其抑制青、绿霉的作用机理；为开发柑橘采后用生物杀菌剂提供结构新颖、作用方式独特的先导物。

柑橘采后腐烂率在20%～40%，其90%腐烂是由青、绿霉病害引起，造成柑橘产业经济损失。现主要靠化学杀菌剂防治，由此引起环境污染、病原抗药性、果品安全等问题，寻求可替代的生物源杀菌剂显得迫切需要。本项目从筛选的具有防效的祼脚菇0612-9出发，鉴定菌株；优化其高产活性物质的培养基和条件；确定提取分离工艺并鉴定其结构；评价其体内、外抗菌控病效果；观测其对病原显微和超微结构的影响，以及胞膜通透性和完整性变化；监控其对病原抗氧化和三羧酸循环相关酶活以及相关物质含量的动态；识别其靶位；诱导柑橘果对病原菌抗性；阐明了其抑制青、绿霉菌的作用机理。. 该菌株为白绒柄裸脚伞。培养基组成（g/L）为玉米粉30、麦芽糖10、蛋白胨15、磷酸二氢钾2、七水硫酸镁1，条件：pH为5.0、接种3×7mm菌块、装液100ml/250ml、28℃、160r/min，周期从11d缩短到9d,菌球数和菌丝干重分别为优化前的21.4倍和2.43倍；后续发酵接种种龄9d、接种量7.5%、转速120 r/min、周期12d缩短至5d,产量比优化前提高8.28%。发酵液依次过四层纱布、50和100目筛，4℃静置，L型”管虹吸下层清液；D-101大孔树脂动态吸附，水洗，乙酸乙酯解吸；再经正、反相硅胶柱和半制备液相色谱分离，创制的浇注法薄层生物自显影法作为引导。鉴定出构型新化合物胡萝卜烷型倍半萜5-表黄褐铁菌素和它的异构体黄褐铁菌素,结构新化合物命名为白绒柄裸伞酸甲酯，以及2-乙基-2，3-二氢苯甲酸酯[1，4]二恶英。. 活性物质主要是通过作用于青、绿霉病原胞膜上麦角固醇靶位，影响其合成和功能发挥，导致病原细胞膜失去通透性，内含物外泄，膜脂过氧化增强，三羧酸循环受阻、能量代谢紊乱。病原菌丝产生扭曲、螺旋状、表面塌陷皱缩、畸形干瘪，细胞壁增厚、质壁分离，胞膜内折形成层状结构、皱缩，细胞器紊乱、模糊，胞质不均、碎渣样，至空腔化凋亡。另外还诱导柑橘增强相关抗性酶活性，提高抗氧化物质含量，减缓宿主膜脂过氧化进程等。