Occurrence and Extent of Boscalid Resistance in Populations of Alternaria alternata from California Pistachio Orchards.

Alternaria late blight (ALB) caused by Alternaria spp. is an annual disease problem in California pistachio and requires repeated applications of fungicides to prevent significant losses of pistachio foliage and nut quality. From 2003 onward, the succinate dehydrogenase inhibiting fungicide boscalid has played a key role in ALB management. The development of boscalid resistance in A. alternata populations was monitored from 2005 to 2012 in pistachio producing areas in California. A total of 1,765 single-spore isolates, collected from commercial and experimental pistachio orchards with or without a history of boscalid exposure, were tested in a radial growth assay in agar media amended with the discriminatory dose of 10 µg/ml of boscalid. The frequency of boscalid-resistant isolates in 2005 was 12% but increased significantly and remained stable toward the end of the survey period. Most of the resistant isolates exhibited a high level of resistance (R) to boscalid with percent of mycelial growth inhibition (PGI) values between 0 and 50%, whereas significantly fewer isolates had an intermediate level (IR) of resistance (50 < PGI < 75%). The frequency of sensitive (S) isolates (75 < PGI < 100%) was generally the highest in orchards with no history of boscalid usage, whereas mean incidences of boscalid-resistant populations of Alternaria were 81, 92.4, 80.2, and 98%, in 2006, 2007, 2011, and 2012, respectively, in orchards that received a high number (at least three per season) of boscalid spray applications. In comparison, none to relatively low frequencies (0 to 12%) of resistance were observed in populations with no or limited exposure to Pristine, suggesting an air-movement of resistant spores through wind from treated to nontreated areas. In 2012, boscalid-resistant isolates were found practically in every sampled location in all counties, with the orchards in Fresno, Madera, Tulare, and King Counties being the locations with the highest frequencies of resistance (100%). Monitoring of A. alternata AaSDHB, AaSDHC, and AaSDHD mutations in 286 boscalid-resistant phenotypes identified 11 mutations, leading to amino acid substitutions in AaSDHB (seven mutations: H277Y/R/L, P230A/R, N235D/T), AaSDHC (one mutation: H134R), and AaSDHD (three mutations: D123E, H133R/P), with AaSDHB mutations being the most prevalent (80%) ones throughout the survey period. The majority of isolates carrying these mutations exhibited the R phenotype toward boscalid. The increased prevalence of boscalid resistance in populations of A. alternata is a likely contributing factor to the inability of pistachio farmers to successfully control ALB with Pristine. Other factors implicated in the rapid and widespread occurrence of A. alternata boscalid-resistant populations in California pistachios are further discussed.