双孢蘑菇疣孢霉病的发病过程及病原菌的核相研究

The development of Agaricus bisporus wet bubble disease and the nuclear phase of pathogen

摘要:

[目的]确定有害疣孢霉的传播途径，明确双孢蘑菇受有害疣孢霉侵染后发病症状和微观形态变化，以及有害疣孢霉的核相。[方法]将有害疣孢霉喷施于培养料及覆土材料的不同深度，观察记录双孢蘑菇的发病情况；将有害疣孢霉接种于不同生长阶段的双孢蘑菇子实体，观察记录其发病情况；使用光学显微镜及扫描电镜观察双孢蘑菇子实体受有害疣孢霉侵染前后的形态变化；通过DAPI（4'，6-二脒基-2-苯基吲哚）染色的方法对有害疣孢霉核相进行观察。[结果]将有害疣孢霉接种于培养料及覆土层的不同深度得到双孢蘑菇发病率如下：覆土层表面 > 覆土层中间 > 覆土与培养料交界处 > 培养料中间层；有害疣孢霉可以侵染双孢蘑菇的任意阶段，将其接种于原基直径小于3 mm子实体表面时，得到不能正常分化的“马勃状”组织；对有害疣孢霉的侵染过程进行观察得到：其孢子可粘附于双孢蘑菇表面，并萌发长出芽管，接种处双孢蘑菇表面产生褐色病斑，双孢蘑菇菌丝体发生质壁分离，最后菌丝体膨大，细胞壁变薄甚至溢裂，菌丝体内部中空；有害疣孢霉产生两种类型的分生孢子，Ⅰ类无隔膜含1个细胞核；Ⅱ类具1隔膜含2个细胞核，2个细胞核被隔膜分开；细胞核的第1次有丝分裂发生于分生孢子母细胞中；厚垣孢子由上下2个细胞构成，上胞中含有2个细胞核。下胞含1-2个细胞核。有害疣孢霉的厚垣孢子萌发可产生1-2个芽管，芽管中细胞核的数目不断变化，一般0-2个细胞核。[结论]双孢蘑菇受其侵染后发生显著的细胞学变化；我们对有害疣孢霉做遗传分析时，进行单孢分离需挑取无隔膜的分生孢子为实验材料进行遗传分析。

Abstract:

[Objective] We studied the dynamic nuclear behavior of Hypomyces perniciosus on axenic culture and its disease progression after infection on different growth stages of Agaricus bisporus. [Methods] Infection process was initiated by inoculating different stages of A. bisporus fruit body, and different depths of compost and casing soil with H. perniciosus. Disease progression was studied by observing symptoms on the fruit body using light microscopy and scanning electron microscopy. The nuclear behavior of H. perniciosus was determined by observation using fluorescence light microscopy after binding of DNA specific fluorochrome dye (DAPI:4, 6-Diamidino-2-phenylindole dihydrochloride) to the nuclei. [Results] Inoculating H. perniciosus on different depths of compost and casing soil resulted in different disease rate as follows:on the surface of casing soil>in the center of casing soil>between the casing soil and the compost>in the center of compost. H. perniciosus can infect any stage of fruit body development, when young primordial (up to 3 mm) was infected, large, irregular and tumorous fungal masses were formed. H. perniciosus directly penetrated A. bisporus without the formation of appressorium-like structures. The germination of the conidia led to a necrotic brown lesion symptom on A. bisporus at the beginning stages of disease development. The mycelium of A. bisporus plasmolysed, hydropically degenerated, cytoplasmolysed, emptied of mycelium cytosol and eventual death as the disease advanced. H. perniciosus produced two types of conidia. Group I conidia had no septa, colorless and smooth containing one nucleus. Group II didymoconidium had septa, containing two nuclei, separated by septa. The first round of mitosis occurred in conidia with no nucleus in the germinal tube. Another kind of asexual spore for thicker cell wall wart convex chlamydospore, chlamydospore had two cells. The upper cell had two nuclear while the basal cell had one or two nuclear, when germinated, it produced one or two germinal tubes. The number of nuclear in the germinal tube was irregular, usually contained 0 to 2 nuclear. [Conclusion] H. perniciosus can infect any part of the A. bisporus fruit body and can cause tremendous cytology changed. If we perform single spore isolation to do genetic analysis, one must isolate conidia with no septa.