苹果梨(Pyrus bretchneideri Rehd.)黑斑病潜伏侵染及其预先合成抗菌物质的变化和诱导

【作者】 李永才；

【导师】 张福泉；

【作者基本信息】 甘肃农业大学 ， 果树学， 2000， 硕士

【摘要】 黑斑病是造成甘肃省苹果梨（Pyrus bretchneideri Rehd）贮藏期间的主要病害。本研究采用普通病理、组织病理及病理生理等研究方法系统的研究了苹果梨黑斑病的主要致腐病原链格孢（Alternaria alternata）的侵染规律、途径及方式，提取、分离、鉴定了果皮中预先合成的抗菌物质。此外，还初步研究了采后钙、壳聚糖、水杨酸及热水处理对苹果梨黑斑病的影响及诱导果皮中抗菌物质生成的关系。 苹果梨黑斑病为典型的潜伏侵染性病害，采后冷藏60d，表面光滑无任何症状，自采后90d开始，皮孔或萼筒伸出黑灰色菌丝，进而其周围组织坏死形成黑斑。至采后100d，黑斑病的发病率达28.86％。经鉴定其致腐病原为链格孢（Alternaria alternata,(Fr)Keissl）。 链格孢在采前可经两条途径侵入苹果梨果实组织而潜伏：一是在花朵开放时经花柱侵入，在花后50天定殖于花柱的病原菌通过萼室间组织陆续进入心室，至采收时，心室中链格孢出现率可达45％，二是自果实膨大期开始经果皮侵入而潜伏于皮下组织，至采收时萼端、中部、梗端链格孢的带菌率分别为40％、24％和42.8％。 皮孔是链格孢侵入果实组织的主要通道。粘附于皮孔的孢子首先萌发产生菌丝，随后菌丝进一步增殖侵染，使皮孔表层细胞解体，同时侵入点表皮及内侧3-4层细胞迅速栓化，限制了菌丝的扩展，使其失去独立性形成椭圆形、卵形、多角形细胞聚集在一起的“拟薄壁细胞”，并进一步发展成为“微菌核”，在病程中表现出一段时间的休眠。 膨大期、采收期、采后100d的苹果梨果皮的乙醇（甲醇）—二氯甲烷粗提取液经TLC分离，以链格孢为指示菌进行生物活性分析均有不同比移值的抑菌带出现，采用气—质联用法（GC/MS）对其成分进一步分析表明，三个时期抑菌带成分存在差异。其中棕榈酸甲酯、油酸甲酯、亚油酸甲酯、鱼鲨烯（三十碳六烯）在膨大期含量最高，随着成熟和衰老的进行，含量逐渐降低，至采后100d，其含量分别为膨大期的1.8％、7.3％、19％和26.6％。推测它们可能是生长期间苹果梨果皮中存在的主要抗菌物质。邻苯二甲酸酯类是采收期和采后100d果皮中含量相对较高的抗菌物质，其中采后100d检 出了6种邻苯二甲酸酯，相对含量高达60％。它们可能为采后果皮中主要 的抗菌物质。 6oCaCI。、lo壳聚糖、45t 20min和50℃10 min热水处理均能有效 地控制苹果梨的黑斑病。而水杨酸处理对苹果梨黑斑病的控制作用不明显。 通过对上述最佳采后处理的苹果梨果皮提取液做TLC生物活性分析，发现 除6％CaCI。外，l％壳聚糖、45C 20min和 50’C 10 min热水处理均可诱导 产生新的抑菌带。更多还原

【Abstract】 Alternaria rot caused by Aliernaria alternaia is a main disease which occurs during Pingguoli pear storage in Gansu province. The occurrence and infection pathway of the pwhogen were examined with the methods of pathology, etiology and histology, and the preformed antifungal compounds were extracted, isolated and identificated from the peel of the fruit with the method of pathologic physiology. It wa~ also surveyed the effect of postharvest treatment with CaCI2, chitosan, salicylic acid and hot water on incidence of Alternaria rot and relationship betwt:en posthanest treatment and induction producing antifungal compounds of frui:s. Alternaria rot of Pingguoli pear belonged to the typical latent infection. At cold condition(Ot,RH 85-90%), fruit surface showed sound after 60 days storage; however, black-gray hyphae grew from lenticels or calyx tube after 90 days storage, The tissue collapsed and resulted in visible black spot as the hyphae spreaded. The incidence of Alternaria rot reached 28.86% till 100 days, The fungi isolated from the~.e black spot were identified as Alternaria alternata (Fr) Keissl A. allernata vias able to infect initially through two pathways during growing season and remain latent or quiescent states. The fungi colonized firstly on styles at blossoming. The colonized fungi grew into carpel cavities progressive after 50 days of petal fall. The clonization of the pathogen could be 45% in carpel cavity till the harvest time. The fUngi also attacked fruit surface and remained latent in underlying epidermal cells during fruit development. The clonization of the pathogen at calyx, side and stem of fruit is 40%, 24% and 42.8% respectively at harvest time. 66 A. alternata infected the fruit through lenticels. The conidia attached on lenticel surface geiminated and formed hyphae, which collapsed superficial cell of lenticel. The development of the hyphae was limited due to the lignification of epiderm and its below -4 cells of layers soon, which forced the hyphae to a structure as parenchyma cells which might develope a structure of aggregated cells resembling microsclerotia later and remained in dormancy. Ethanol (methyl alcohol) ichloromethane crude extract from the peel at enlarging stage, harvest and after 100 days cold storage was separated by thin? layer cbromatographic(TLC) plates. The isolated antifungal compounds were detected with conidia of A. ulternata and the inhibition zones at three stages showed difference. By using gas-chromatography and mass spectroscopy (GC/MS) the coripounds in the inhibition zones were seperated and identified. Palmitate methyl, oleic acid methyl, linolenic acid methyl and squalene appeared at the three stages, all their concentration were the highest at the fruit enlarging stage and decreased rapidly at fruit maturation and aging. These compounds might be suggested as a main antifungal compounds in the growing fruits. The phthalate alkyl esters were i elative higher concentration at harvest and after 100 days cold storage, There were 6 phthalat e alkyl esters identified from the peel of after 100 days cold storage, and the relative concentration were up to 60%. These esters might be suggested as the antifungal compounds of postharvest pear. Alternaria ro; of Pingguoli pear could be controled with 6% CaCl2~ 1% chitosan 45 20mm and 50 10mm hot water; however,the fruit treated with salicylic acid had no effect on the rot. By TLC bioassay of the crude extracts from t更多还原