First Report of Benzimidazole-Resistant Isolates of Cylindrocladium scoparium in Europe.

Cylindrocladium scoparium Morg. (teleomorph Calonectria morganii Crous, Alfenas & M.J. Wingf.) was detected for the first time in Sicilian ornamental nurseries in 2005 and was responsible for damping-off and leaf spot of mastic tree seedlings (4). In Sicily, C. scoparium has caused extensive losses, and chemical control measures for the disease were necessary, especially in young plants. The benzimidazoles, including the thiophanates, which are transformed to benzimidazoles, are effective at relatively low doses for the inhibition of different species of Cylindrocladium (2). However, in a fungicide trial conducted in a Sicilian nursery on Callistemon cuttings infected by C. scoparium, thiophanate methyl was ineffective. Fourteen isolates of Cylindrocladium sp. were recovered on potato dextrose agar (PDA) from rotted crown and roots of Pistacia lentiscus L. and Callistemon cvs. Laevis, Hanna Ray, Masotti Mini Red, and Rose Opal and cultured on carnation leaf agar. Isolates were identified as C. scoparium based on pyriform-to-broadly ellipsoidal terminal vesicles, conidiophore branching pattern, morphology of conidia and perithecia, as well as their ability to mate with opposite mating types of South African C. scoparium and Italian C. pauciramosum isolates. Perithecia containing viable ascospores developed after approximately 2 months. Three representative isolates were deposited at Fungal Biodiversity Centre, Centraalbureau voor Schimmelcultures (CBS120892, CBS119669, and CBS119670). Sensitivity to benzimidazole was determined by plating mycelial plugs on PDA amended with benomyl or carbendazim at 1, 10, 100, and 500 μg a.i./ml. Four plates were used for each fungicide concentration. Sensitivity was expressed as the minimum inhibitory concentration (MIC) (the lowest fungicide concentration that completely prevented fungal growth). Isolates that did not grow on benzimidazole-amended PDA were classed as sensitive. The experiment was repeated two times. Isolates were considered resistant to benzimidazole if MIC values were greater than 1 μg a.i./ml for benomyl and carbendazim. Of the 14 isolates tested, 13 were resistant to benzimidazole. The benomyl-resistant isolates were also resistant to carbendazim. Among the resistant isolates, five grew in the presence of 10 μg a.i./ml and one isolate grew at 100 μg a.i./ml. Widespread incidence of benzimidazole-resistant isolates in a population of C. pauciramosum was detected in Italy (3). Benzimidazole-resistant isolates of C. scoparium were previously detected in Brazil (1) where the constant use of benomyl in nurseries led to the selection of strains resistant to fungicide at nearly 1,000 μg a.i./ml. To our knowledge, this is the first report of benzimidazole-resistant isolates of C. scoparium in Europe. Because of the high incidence of benzimidazole-resistant isolates detected in our study, good fungicide resistance management practices should be followed. Appropriate cultural, biological, and chemical disease control methods should be combined and implemented to delay further changes in sensitivity of the pathogen. The exclusive use of benzimidazole fungicides in nursery for the control of Cylindrocladium spp. should be avoided. References: (1) A. Alfenas et al. ISPP. Chem. Contr. Newsl. 10:23, 1988. (2) P. W. Crous. Taxonomy and Pathology of Cylindrocladium (Calonectria) and Allied Genera. The American Phytopathological Society, St. Paul MN, 2002. (3) G. Polizzi and A. Vitale. Plant Dis. 85:1210, 2001. (4) G. Polizzi et al. Plant Dis. 90:1110, 2006.