Beirn Lisa A, Wang Ruying, Clarke Bruce B, Crouch Jo Anne
Department of Plant Biology & Pathology, Rutgers University , New Brunswick, NJ , USA.
Systematic Mycology & Microbiology, USDA-ARS , Beltsville, MD , USA.
PeerJ. 2015 Aug 13;3:e1153. doi: 10.7717/peerj.1153. eCollection 2015.
The fungus Colletotrichum cereale incites anthracnose disease on Poa annua (annual bluegrass) turfgrass. Anthracnose disease is geographically widespread throughout the world and highly destructive to cool-season turfgrasses, with infections by C. cereale resulting in extensive turf loss. Comprehensive research aimed at controlling turfgrass anthracnose has been performed in the field, but knowledge of the causal organism and its basic biology is still needed. In particular, the lack of a reliable greenhouse-based inoculation protocol performed under controlled environmental conditions is an obstacle to the study of C. cereale and anthracnose disease. Our objective was to develop a consistent and reproducible inoculation protocol for the two major genetic lineages of C. cereale. By adapting previously successful field-based protocols and combining with components of existing inoculation procedures, the method we developed consistently produced C. cereale infection on two susceptible P. annua biotypes. Approximately 7 to 10 days post-inoculation, plants exhibited chlorosis and thinning consistent with anthracnose disease symptomology. Morphological inspection of inoculated plants revealed visual signs of the fungus (appressoria and acervuli), although acervuli were not always present. After stringent surface sterilization of inoculated host tissue, C. cereale was consistently re-isolated from symptomatic tissue. Real-time PCR detection analysis based on the Apn2 marker confirmed the presence of the pathogen in host tissue, with both lineages of C. cereale detected from all inoculated plants. When a humidifier was not used, no infection developed for any biotypes or fungal isolates tested. The inoculation protocol described here marks significant progress for in planta studies of C. cereale, and will enable scientifically reproducible investigations of the biology, infectivity and lifestyle of this important grass pathogen.
禾生炭疽菌可引发一年生早熟禾草坪草的炭疽病。炭疽病在全球范围内广泛分布，对冷季型草坪草具有高度破坏性，禾生炭疽菌的感染会导致大面积草坪草损失。针对控制草坪草炭疽病已开展了全面的田间研究，但仍需要对病原菌及其基本生物学特性有所了解。特别是，缺乏在可控环境条件下进行的可靠的温室接种方案是研究禾生炭疽菌和炭疽病的一个障碍。我们的目标是为禾生炭疽菌的两个主要遗传谱系开发一种一致且可重复的接种方案。通过调整先前成功的田间方案并结合现有接种程序的组成部分，我们开发的方法在两种易感一年生早熟禾生物型上始终能引发禾生炭疽菌感染。接种后约7至10天，植株出现与炭疽病症状相符的黄化和变稀现象。对接种植株的形态学检查发现了真菌的可见迹象（附着胞和分生孢子盘），不过分生孢子盘并非总是存在。对接种的寄主组织进行严格的表面消毒后，始终能从有症状的组织中重新分离出禾生炭疽菌。基于Apn2标记的实时PCR检测分析证实了寄主组织中存在病原体，所有接种植株均检测到了禾生炭疽菌的两个谱系。当不使用加湿器时，所测试的任何生物型或真菌分离株均未发生感染。本文所述的接种方案标志着禾生炭疽菌的植物体内研究取得了重大进展，并将使对这种重要的禾本科病原菌的生物学、感染性和生活方式进行科学可重复的研究成为可能。
