茎基腐亚洲镰孢菌侵染抗感小麦品种的组织病理学观察

小麦茎基腐是由多种镰孢菌侵染的世界性土传病害,亚洲镰孢菌(Fusarium asiaticum)是我国冬小麦主产区茎基腐镰孢菌的优势种群,对小麦生产造成巨大损失。本研究利用绿色荧光蛋白报告基因标记亚洲镰孢菌,研究其侵染抗感小麦的病理组织学过程,建立了茎基腐病菌与寄主互作的直观性的研究体系,对病害防治及抗病育种具有重要意义。基于PEG-CaCl_2介导原生质体转化法将gfp导入亚洲镰孢菌株CF0915,对转化子进行荧光表达、PCR验证、遗传稳定性、生长特性及致病力分析,选取与野生型表现相近的转化子进行侵染分析。结果表明,绿色荧光蛋白基因(gfp)与潮霉素基因(hyg)PCR扩增表明gfp已整合入真菌基因组中,转化子菌丝与分生孢子表现强烈绿色荧光信号,gfp能够在转化子中稳定遗传,菌落形态、生长速度及致病力与野生型菌株无显著差异;将gfp标记病菌分生孢子接种感病品种1 d后,大量孢子附着于根毛及根表皮细胞开始萌发,接种2 d后观察到抗性品种分生孢子萌发;感病品种接种3 d后,菌丝直接侵入表皮细胞或沿表皮细胞间层定殖生长,扩展至皮层组织,8 d后菌丝从根部迅速扩展至茎基部,至第10 d大量菌丝充塞根皮层细胞,叶鞘维管束也被菌丝侵染,并产生大量大型分生孢子,植株表现褐色病斑,14 d后根部及茎维管束被大量菌丝体填充,而后产生大量厚垣孢子,至25 d大部分感病品种幼苗萎蔫死亡;与感病品种相比,抗性品种在整个侵染过程中表现时间滞后。本研究对引起茎基腐病的亚洲镰孢菌侵染小麦的组织学过程观察,为病菌致病机理的阐释及抗病资源的利用提供了重要理论依据。

Histological observation of wheat infected by Fusarium asiaticum, the causal agent of wheat crown rot disease

Wheat crown rot disease caused by Fusarium spp. was one of worldwide-spread and devastating disease for wheat production. F. asiaticum was one of the dominant pathogen of wheat crown rot disease in major winter wheat production area in China. It’s beneficial to the pathogenesis analysis and resistance breeding through histological observation of infection process. F. asiaticum labeled by green fluorescent protein (GFP) for studying infection process in wheat was a direct method for in vivo observation of interaction between pathogen and host. Gene gfp was transferred into F. asiaticum strain CF0915 via PEG-CaCl₂ mediated transformation method. Based on analysis of fluorescence expression, PCR verification, genetic stability, growth parameters and pathogenicity analysis, transformants CF0915-GFP resembling the wild-type strain was chosen to investigate infection process on wheat cultivars of different resistance. The results showed that transformants CF0915-GFP showed bright green fluorescence, and gfp gene was integrated into the genome of F. asiaticum by PCR amplification verification and inherited normally and stably, the growth characters and pathogenicity were comparable to wild strain. Numerous conidiospores attached root hairs and root epidermis of wheat and germinated at 1 day after inoculation in susceptible cultivar ‘Yangmai 158’, until 2 dpi germination of conidiospores was observed in moderately-resistant cultivars ‘CI12633’ and ‘Sumai 3’. The hyphae infected root hairs and grooves along junctions of adjacent epidermal cells and then spread into cortex of roots at 3 dpi. The hyphae spread fast onto leaf shealth from roots at 8 dpi, while at 10 dpi massive hyphae invaded into root cortex cells and epidermal cells of leaf shealth and meanwhile abundant macroconidiaspores were observed, brown disease spots appeared on basal stems of susceptible cultivar. Massive hyphae were filled in xylems vessels of roots and leaf shealths at 14 dpi, subsequently numerous chlamydospores were observed in the epidermal cells of leaf shealth. Until 25 dpi most seedlings appeared wilt and dead. Compared to susceptible cultivar, infection process was retarded later in moderately-resistant cultivars. In this study the histopathological observation of infection process of F. asiaticum in wheat provided an important theoretical basis for disease control and resistance resource utilization.