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Title: Development of a greenhouse-based inoculation protocol for the fungus Colletotrichum cereale pathogenic to annual bluegrass (Poa annua)

item BEIRN, LISA - Rutgers University
item WANG, RUYING - Rutgers University
item CLARKE, BRUCE - Rutgers University
item Crouch, Jo Anne

Submitted to: PeerJ
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/14/2015
Publication Date: 8/13/2015
Citation: Beirn, L., Wang, R., Clarke, B.B., Crouch, J. 2015. Development of a greenhouse-based inoculation protocol for the fungus Colletotrichum cereale pathogenic to annual bluegrass (Poa annua). PeerJ. 3:e1153 DOI:10.7717/peerj1153

Interpretive Summary: Colletotrichum cereale is a fungus that causes deadly diseases of cool-season grasses, especially those that are used as turfgrasses for golf courses, sports fields, parks and lawns. For almost 20 years, scientists have attempted to learn how this fungus causes disease, but have been unable to develop a reliable way to artificially infect plants except when grown in outside field plots. Performing these studies outdoors rather than in contained indoor environments increases the risk of spreading the disease. In this work, we developed and tested a new and reliable method to infect turfgrass plants with C. cereale that can be performed indoors under controlled environmental conditions. The ability to perform reproducible experimental studies of C. cereale within indoor growth chambers will help scientists to identify the processes that allow the pathogen to infect plants and cause disease, without the risking further spread of the disease by working with it outdoors.

Technical Abstract: The fungus Colletotrichum cereale incites anthracnose disease on Poa annua (annual bluegrass) turfgrass. Anthracnose disease is geographically widespread highly destructive, with infections by C. cereale resulting in extensive turfgrass 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. The lack of a reliable greenhouse-based inoculation protocol performed under controlled environmental condition is a primary obstacle in the study of C. cereale and anthracnose disease. The objective of this study was to develop a consistent and reproducible inoculation protocol for the two major genetic lineages of C. cereale. By adapting successful field-based protocols and combining with components of existing inoculation procedures, the method we developed consistently produced C. cereale infection of two susceptible P. annua biotypes. Approximately 7 to 10 days post-inoculation, inoculated 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 re-isolated from symptomatic tissue. Real-time PCR detection analysis based on the Apn2 marker confirmed the presence of the fungus in host tissue, with both lineages of C. cereale detected from all inoculated plants. When misting was withheld from the protocol, 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, enabling future scientifically reproducible investigations of the biology, infectivity and lifestyle of this important agronomic pathogen.