ORIGINAL ARTICLE ISSR fingerprinting for the assessment of the bindweed biocontrol agent Stagonospora convolvuli LA39 after field release De ´ sire ´ e Boss, Monika Maurhofer, Marcello Zala, Genevie `ve De ´ fago, and Patrick C. Brunner Plant Pathology, Institute of Integrative Biology, ETH Zurich, 8092 Zurich, Switzerland Introduction Field bindweed (Convolvulus arvensis [L.] R. Br.) and hedge bindweed (Calystegia sepium L.) are economically import- ant and difficult to control weeds throughout the world (Holm et al. 1977; Schro ¨der et al. 1993). Biological control methods as an alternative to chemical pesticides are of increasing public interest. Fungi belonging to the genus Stagonospora and particularly Stagonospora convolvuli strain LA39, isolated in Long Ashton, UK, emerged as very prom- ising candidates to efficiently control both bindweed spe- cies when asexual spores (conidia) are applied (Pfirter et al. 1997; Guntli et al. 1998; Pfirter et al. 1999b; Guntli et al. 1999; De ´fago et al. 2001). LA39 is a leaf pathogen specific to Convolvulaceae (Pfirter and De ´fago 1998). The fungus is applied with the mycoherbicide approach (Templeton and Smith 1977) using the inundative strat- egy by applying the biocontrol agent in much higher con- centrations than it naturally occurs. Since the potential environmental risks associated with the use of biocontrol agents at high concentrations are of increasing public concern (TeBeest et al. 1992; Teng and Yang 1993; Cook et al. 1996), it is important to have baseline information on the dispersal and survival of the biocontrol agent in the environment. Furthermore, EU risk assessors ask for information about the environmental fate of the biocon- trol organism. It is known that LA39 produces conidia in pycnidia (Pfirter et al. 1999a) and is assumed to spread only over a short distance from the inoculation by Keywords bindweed, biocontrol, Calystegia sepium, Convolvulus arvensis, environmental risk assessment, ISSR fingerprinting, monitoring, Stagonospora convolvuli LA39. Correspondence Patrick C. Brunner, Plant Pathology, Institute of Integrative Biology, ETH Zurich, 8092 Zurich, Switzerland. E-mail: patrick.brunner@agrl.ethz.ch 2007 ⁄ 0238: received 15 February 2007, revised and accepted 12 April 2007 doi:10.1111/j.1472-765X.2007.02177.x Abstract Aims: To develop a molecular identification method based on ISSR fingerprints to monitor the fungal leaf pathogen Stagonospora convolvuli LA39 used to bio- logically control bindweeds after a field release. Methods and results: The developed method proved to be suitable to clearly distinguish LA39 from resident Stagonospora spp. and was applied in two field experiments. First, the environmental persistence of LA39 was assessed in an overwintering experiment. LA39 could be re-isolated from infected bindweed 1 year after field application, but with very low frequency of occurrence. Sec- ondly, LA39 was applied in an area with natural bindweed infestation and re- isolated from infected bindweed. The dispersal of LA39 during one season was poor (4–5 m). Conclusions: ISSR fingerprinting has been shown to be a valuable tool to mon- itor the environmental fate of S. convolvuli in the field. It is concluded that an LA39-based mycoherbicide will have minimal environmental impact caused by the restricted mobility, poor proliferation and poor persistence over seasons of LA39. Significance and impact of the study: Studies about the dispersal and survival of biocontrol agents after field release as well as the development of methods needed for this purpose are indispensable for a comprehensive risk assessment for biocontrol agents. Letters in Applied Microbiology ISSN 0266-8254 244 Journal compilation ª 2007 The Society for Applied Microbiology, Letters in Applied Microbiology 45 (2007) 244–251 ª 2007 The Authors