645 Research Article Received: 28 February 2011 Revised: 30 July 2011 Accepted article published: 31 August 2011 Published online in Wiley Online Library: 10 November 2011 (wileyonlinelibrary.com) DOI 10.1002/ps.2311 Molecular characterization of boscalid- and penthiopyrad-resistant isolates of Didymella bryoniae and assessment of their sensitivity to fluopyram Herv ´ e F Avenot, * Anna Thomas, Ronald D Gitaitis, David B Langston Jr and Katherine L Stevenson Abstract BACKGROUND: Didymella bryoniae has a history of developing resistance to single-site fungicides. A recent example is with the succinate-dehydrogenase-inhibiting fungicide (SDHI) boscalid. In laboratory assays, out of 103 isolates of this fungus, 82 and seven were found to be very highly resistant (B VHR ) and highly resistant (B HR ) to boscalid respectively. Cross-resistance studies with the new SDHI penthiopyrad showed that the B VHR isolates were only highly resistant to penthiopyrad (B VHR -P HR ), while the B HR isolates appeared sensitive to penthiopyrad (B HR -P S ). In this study, the molecular mechanism of resistance in these two phenotypes (B VHR -P HR and B HR -P S ) was elucidated, and their sensitivity to the new SDHI fluopyram was assessed. RESULTS: A 456 bp cDNA amplified fragment of the succinate dehydrogenase iron sulfur gene (DbSDHB) was initially cloned and sequenced from two sensitive (B S -P S ), two B VHR -P HR and one B HR -P S isolate of D. bryoniae. Comparative analysis of the DbSDHB protein revealed that a highly conserved histidine residue involved in the binding of SDHIs and present in wild-type isolates was replaced by tyrosine (H277Y) or arginine (H277R) in the B VHR -P HR and B HR -P S variants respectively. Further examination of the role and extent of these alterations showed that the H/Y and H/R substitutions were present in the remaining B VHR -P HR and B HR -P S variants respectively. Analysis of the sensitivity to fluopyram of representative isolates showed that both SDHB mutants were sensitive to this fungicide as the wild-type isolates. CONCLUSION: The genotype-specific cross-resistance relationships between the SDHIs boscalid and penthiopyrad and the lack of cross-resistance between these fungicides and fluopyram should be taken into account when selecting SDHIs for gummy stem blight management. c  2011 Society of Chemical Industry Keywords: gummy stem blight; carboxamide; iron sulfur subunit; amino acid substitutions; fungicide site of action 1 INTRODUCTION Gummy stem blight (GSB) of cucurbits is caused by the fungus Didymella bryoniae (Auersw.) Rehm [syn.: Mycosphaerella citrullina (C.O. Sm.) Gross. and M. melonis (Pass.) Chiu & J.C. Walker] and its anamorph Phoma cucurbitacearum (Fr.:Fr.) Sacc. 1,2 The disease is most common in tropical and subtropical areas of the world and is also a serious disease of cucurbits in the United States. 1,3,4 It can affect all parts of watermelon seedlings and mature plants. On watermelon seedlings, initial symptoms consist of water-soaked lesions that first appear on the cotyledons. 2,5,6 These are then followed by the colonization of the hypocotyl and crown by the fungus. On young seedlings, crown lesions can partially or totally girdle plant stems. On field-grown watermelons, symptoms of GSB include crown blight, stem cankers and extensive defoliation. In addition to the foliar phase (GSB), the fungus can also cause a fruit rot (black rot). 2,5,6 In the southeastern United States, GSB is the most widespread and destructive disease of watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai]. 3–5 Management options for this disease include using disease-free seed, rotating crops, deep turning of diseased tissue, timing irrigation events to reduce periods of leaf wetness and applying protectant and systemic fungicides. 1,3,7 Of these management options, application of preventive fungicides is the most effective. However, effective chemical management of gummy stem blight has been hindered by the development of fungicide-resistant isolates, particularly with fungicides with site- specific modes of action. In fact, resistant isolates of D. bryoniae to benzimidazoles and the quinone-outside-inhibiting (QoI) fungicides were detected at high frequencies in watermelon fields. This has resulted in the decline in efficacy of these fungicides to the extent that they are no longer recommended for gummy stem blight control. 4,8 – 11 Because GSB-resistant cultivars of watermelon ∗ Correspondence to: Herv´ e F Avenot, Department of Plant Pathology, Uni- versity of Georgia, Tifton Campus, PO Box 748, Tifton, GA 31793, USA. E-mail: havenot@uga.edu Department of Plant Pathology, University of Georgia, Tifton, GA, USA Pest Manag Sci 2012; 68: 645–651 www.soci.org c  2011 Society of Chemical Industry