Development of Molecular and Greenhouse-Based Resources to Facilitate Controlled in Planta Studies of Colletotrichum Cereale.

Anthracnose, caused by the fungus Colletotrichum cereale, is a destructive disease of Poa annua putting green turf.  Considerable progress has been made developing an integrated program of best management practices to control anthracnose in the field; however, very little is known about the biology of this fungus.  Advancements in this area are hindered by the lack of available tools to conduct in planta studies.   Our objective was to develop key tools required for experimental studies of this pathogen: (a) a reproducible greenhouse inoculation protocol; and (b) a real-time PCR protocol to detect C. cereale directly from infected tissue.  P. annua biotypes 98226 and 99112 were inoculated with a conidial suspension (10^-6) of C. cereale and placed in a mist chamber inside a growth chamber for 24 h. Two different fungal isolates were tested.  Misting occurred for a duration of 1 h every 2 h.  Following misting, plants remained in the growth chamber under conditions of 12 h daylight (500 μE m^-2s^-1), 80% RH, 30°C day, and 26°C night.  Inoculations were repeated twice and included negative controls.  Plants exhibited chlorosis and thinning 10-14 d after inoculation.  Morphological inspection revealed signs of the fungus (appressoria and occasionally acervuli).  After stringent surface sterilization with 10% NaClO and 70% EtOH, C. cereale was re-isolated from symptomatic tissue.  To further confirm infection, we developed a real-time PCR assay based on the Apn2 molecular marker.  This assay was 100% effective at detecting the pathogen from 722 samples including DNA extracted from cultured isolates, symptomatic plant tissue, and herbarium specimens.  Using this protocol, C. cereale infection was verified in all inoculated plants (average cycle threshold value = 32.47).  These tools mark significant progress for in planta studies of C. cereale and will serve as a foundation for investigating the biology, infectivity, and lifestyle of this important pathogen.