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The fungal pathogen Colletotrichum truncatum is a potential bioherbicide agent for management of the noxious weed scentless chamomile (Metricaria perforate) in western Canada. Isolates of the potential biocontrol agent from scentless chamomile cannot be separated morphologically from the pathogen that causes anthracnose on lentil and field pea, raising questions about crop safety for its use as a bioherbicide agent in the region. Internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) of C. truncatum from scentless chamomile and several crop species were examined for genetic differences. ITS-rDNA fragments of 63 isolates were amplified using the universal primers Its4 and Its5 and sequenced. The rDNA fragment was 571 bp in size for isolates from scentless chamomile, 566 bp from lentil, 558 or 559 bp from field pea isolates and 555 bp from soybean. Phylogenetic analysis using parsimony and neighbour-joining methods separated these putative C. truncatum isolates into four groups that coincided with host origin. All of the isolates of scentless chamomile fell within a single cluster regardless of their geographic origins, and were most closely related to C. truncatum isolates from lentil. Morphologically, the isolates from scentless chamomile, lentil and field pea were similar in conidial shape and dimension, whereas soybean isolates had substantially longer and more falcate conidia and slightly bigger appressoria. Based on the sequence information, strain-specific PCR primers were designed for isolates of C. truncatum from each host and used to amplify specific rDNA fragments (markers). This molecular technique was sensitive and strain-specific for detection and differentiation of Colletotrichum isolates from scentless chamomile, lentil, field pea and soybean, and may be used as a tracking and monitoring tool in environmental risk assessment for the bioherbicide agent. [ABSTRACT FROM AUTHOR]