Phenotypic and molecular characterization of resistance to the SDHI fungicide fluopyram in populations of Alternaria alternata from pistachio orchards in California

Abstract

In this study, the sensitivity of 902 isolates of A. alternata collected before and after the commercial use of fluopyram were assessed using mycelial growth tests. Distribution of 50% effective concentration (EC50) values for 116 A. alternata isolates (population A), collected in 2011 from an experimental orchard that had been treated with fluopyram products or boscalid showed that 86% of the isolates displayed different levels of resistance to boscalid while 87 (76%) and 28 (24%) showed sensitivity and different levels of resistance to fluopyram, respectively. Among the last group of isolates, only 5 and 23 showed moderate (MR) and low (LR) resistance to fluopyram, respectively. The sensitivity to fluopyram was further evaluated in agar medium at the discriminatory dose of 5 μg/ml fluopyram for a total of 786 A. alternata isolates collected in 2012 from commercial pistachio orchards with (173 isolates, population B; 373 isolates, population C) or without a history of use of fluopyram products (240 isolates, population D). Among the 240 isolates from 31 boscalid-treated orchards, 93 and 7% showed sensitivity (S) and low resistance (LR) to fluopyram, respectively, while 65 and 33 were resistant (R) and LR to boscalid, respectively. Analyses of the sensitivities to boscalid and fluopyram of the 546 isolates from populations B and C collected from orchards exposed to fluopyram or other SDHIs showed fluopyram-resistant isolates did not occur at the same frequency as boscalid resistance. In both populations, the frequencies of boscalid resistance isolates in all plots, exposed or not to fluopyram products, were significantly higher than those observed for fluopyram. Sequence analyses of the A. alternata SDHB, C, and D proteins showed the presence of several amino acid substitutions (SDHB: H277Y/R/L, P230 A/R/I/F,D, N235D/T/E/G; SDHC: H134R, S135R; SDHD: D123E, H133P) in boscalid- and fluopyram-resistant phenotypes. Current data provided further evidences of the good performance of fluopyram and the various and interactive impacts of SDH mutations on the levels of resistance to SDHIs. Although their frequency is still very low, fluopyram-resistant phenotypes could quickly build up in Alternaria populations under continual selection pressure by SDHI-containing products and ultimately affect fluopyram efficacy. As fluopyram products become extensively used in pistachio orchards, continual monitoring of Alternaria sensitivities to fluopyram and other active ingredients in these mixtures (tebuconazole in particular) will be required to detect any shifts in sensitivity that could result in disease control failures.