First Report on Resistance to Pyraclostrobin, Thiophanate-methyl, Fenhexamid and Boscalid in Botrytis cinerea from Eucalyptus Seedlings in Florida Greenhouses.

Botryotinia fuckeliana de Bary (anamorph Botrytis cinerea Pers.) is an ubiquitous plant pathogen causing gray mold disease on more than 200 crops grown in the field or in greenhouses. Eucalyptus seedlings originating from three different greenhouses showing stem lesions were submitted to the Gulf Coast Research and Education Center Disease Clinic in June 2012. Ten single spore isolates of B. cinerea were obtained and tested for sensitivity using spore germination and germ tube elongation assays described previously (4). Fungicides tested were pyraclostrobin at 100 μg/ml (Cabrio, BASF, Research Triangle Park, NC), thiophanate-methyl at 100 μg/ml (Topsin-M, UPI, King of Prussia, PA), fenhexamid at 1 and 50 μg/ml (Elevate, Arysta Life Sciences, Cary, NC), fludioxonil at 0.1 and 10 μg/ml (Medallion, Syngenta Crop Protection, Research Triangle Park, NC), and iprodione at 5 and 50 μg/ml (Rovral, Bayer CropScience, Greensboro, NC) on 1% malt extract agar (MEA, 10 g malt extract and 15 g agar), and to cyprodinil at 1 and 25 μg/ml (Vanguard, Syngenta Crop Protection) on 0.5% sucrose agar (4). Sensitivity to the succinate dehydrogenase inhibitors (SDHIs) boscalid at 5 μg/ml (Endura, BASF), penthiopyrad at 1 and 3 μg/ml (Fontelis, DuPont Crop Protection, Willington, DE), and fluopyram at 3 μg/ml (Luna Privilege, Bayer CropScience) was evaluated on yeast bacto acetate agar (YBA) (3). The discriminatory dose for boscalid was adapted from (2) whereas those used for penthiopyrad and fluopyram were developed in this study. Isolates were grown on malt yeast extract agar for 7 to 10 days and spore suspensions were prepared in sterile distilled water and diluted to 10⁶ conidia/ml. Respective media in 9-cm petri dishes were seeded with 7-μl droplets from each isolate allowing testing for all isolates on one plate. Two plates were used for each fungicide and sensitivity tests were repeated twice. Germination and germ tube growth were assessed microscopically after 16 to 24 h incubation at 22°C. The frequency of isolates resistant to two, three, and four fungicides was 90, 60, and 10%, respectively. Nine isolates (90%) were resistant to thiophanate-methyl and pyraclostrobin, simultaneously, whereas six (60%) and two isolates (20%) were resistant to boscalid and fenhexamid, respectively. All boscalid-resistant isolates were also resistant to pyraclostrobin and thiophanate-methyl, but one fenhexamid-resistant isolate was sensitive to the other three fungicides. Eight isolates that germinated at 5 μg/ml iprodione but not at 50 μg/ml were considered sensitive. All isolates were sensitive to the SDHIs penthiopyrad and fluopyram as well as to cyprodinil and fludioxonil. To our knowledge, this is the first report of resistance to pyraclostrobin, thiophanate-methyl, fenhexamid, and boscalid in B. cinerea from eucalyptus seedlings in Florida. The absence of resistance to fludioxonil and iprodione is likely because these fungicides are not registered in nurseries as well as fluopyram and penthiopyrad which were developed only recently. Management practices should be developed to limit the selection and spread of additional resistant populations in eucalyptus nurseries as has occurred in Florida strawberries where multi-fungicide resistance is widespread (1). References: (1) A. Amiri et al. Plant Dis. 97:393, 2013. (2) M. Leroch et al. Appl. Environ. Microbiol. 79:159, 2013. (3) G. Stammler and J. Speakman. J. Phytopathol. 154:508, 2006. (4) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.