Molecular characterization of boscalid- and penthiopyrad-resistant isolates of Didymella bryoniae and assessment of their sensitivity to fluopyram

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. Copyright © 2011 Society of Chemical Industry