【背景】苜蓿假盘菌(Pseudopeziza medicaginis)侵染苜蓿叶片引起的褐斑病是苜蓿的重要病害之一。研究表明，该菌以子囊盘越冬，翌年释放子囊孢子完成初侵染，因此观察该菌子实体的超微结构特征，有利于进一步揭示子囊菌的侵染机制。【目的】明确P. medicaginis子囊孢子萌发、Ca2+信号途径是否参与调控附着胞形成；观察子囊孢子和子实体的超微结构特征。【方法】采用光学显微镜技术，研究不同碳、氮源和Ca2+/CaM信号途径对P. medicaginis子囊孢子萌发、附着胞形成的影响，并采用电镜技术观察子实体和子囊孢子的超微结构。【结果】碳、氮源有利于P. medicaginis子囊孢子的萌发，葡萄糖、麦芽糖、蔗糖、氨基乙酸、酵母粉、尿素、蛋白胨、硝酸铵、硝酸钠等碳、氮源诱导形成附着胞，以尿素诱导效果最明显；Ca2+不仅促进子囊孢子的萌发，显著诱导附着胞的形成，而且高于碳、氮源；10 mmol/L的Ca2+诱导效果最明显，随其浓度的增加，子囊孢子的萌发率和附着胞的形成率下降；低浓度Ca2+螯合剂EGTA诱导形成附着胞，≥1 mmol/L抑制子囊孢子的萌发，外源Ca2+或Ca2+载体A23187能够部分解除EGTA的抑制作用；磷酸酯酶抑制剂Neomycin、Ca2+通道阻断剂Nicardipine、CaM拮抗剂Chloropromazine完全抑制子囊孢子的萌发。成熟的P. medicaginis子实体突破寄主表皮，中间开裂露出子囊，子囊孔口无囊盖，呈横向裂口状。子囊孢子表面具有环形纹饰，细胞核色深，线粒体等细胞器数量多，脂类物质数量多且发达，胞内充满电子致密度高的物质。【结论】碳、氮源和Ca2+可诱导P. medicaginis子囊孢子形成附着胞，钙信号途径参与P. medicaginis子囊孢子的萌发和附着胞形成过程的调控。子实体突破寄主表皮外露呈开裂式，子囊顶端无囊盖，线粒体发达。该研究结果为P. medicaginis的深入研究提供重要细胞学事件。
[Background] Medicago sativa common leaves spot caused by Pseudopeziza medicaginis is a main fungal disease in M. sativa. This strain can overwinter by the formation of apothecia and then release ascospore to complete primary infection. Thus we observed the fruit body under an electron microscope for exploring the colonization mechanism of P. medicaginis. [Objective] To clarify the conditions of ascospore germination, whether the Ca2+ signal pathway is involved in the form of appressoria and observe the ultrastructure of the ascospore and fruit body of P. medicaginis. [Methods] Effect of different types of carbon and nitrogen sources and the different doses of Ca2+ on ascospore germination and appressoria form were studied by optical microscope. The ultrastructure of the fruit body and ascospore were observed by a transmission electron microscope. [Results] Carbon and nitrogen sources benefited the ascospore germination. Glucose, maltose, sucrose, amino acetic acid, yeast powder, urea, peptone, ammonium nitrate, sodium nitrate and so on could induce the formation of appressorium. Urea had the strongest effect. The Ca2+ not only could promoted the germination of ascospores but also could induce the formation of appressorium, the effect was better than adding carbon and nitrogen sources. With Ca2+ concentration increased, the rate of ascospore germination and the rate of appressoria formation increased, the best dose of Ca2+ was 10 mmol/L, once exceed this dose, the rate of ascospore germination and the rate of appressoria formation decreased. With exogenous Ca2+ or A23187 which is Ca2+ carrier addition, the inhibition role of EGTA could partially relieved. Neomycin, an inhibitor of phospholipase, nicardipine which could block Ca2+ channels, chlorpromazine, CAM antagonist could inhibit the germination of ascospores of this strain. The mature P. medicaginis fruit body broke through the epidermis of the host, and the middle part of the fruit body cracked to expose the ascus. The surface of ascospore had a ring-shaped pattern, the nucleus was dark, the number of organelles such as mitochondria is large, the number of lipids was large and developed, and the cells were filled with high electron density materials. [Conclusion] Ca2+ signal pathway was involved in the process of ascospore germination and charge of the appressoria formation of P. medicaginis.
马新,史娟,李杨,韩宇. 苜蓿假盘菌子囊孢子萌发特性及子实体超微结构观察[J]. 微生物学通报, 2020, 47(11): 3564-3576复制