Sensitivity Profile to Pyraclostrobin and Fludioxonil of Alternaria alternata from Citrus in Italy

Alternaria brown spot (ABS), caused by Alternaria alternata, is one of the main citrus diseases that causes heavy production losses and reductions in fruit quality worldwide. The application of chemical fungicides has a key role in the management of ABS. In this study, 48 isolates of A. alternata collected from citrus orchards since 2014 were tested in vitro for their sensitivity to pyraclostrobin and fludioxonil, the latter being temporarily registered in Italy since 2020. Pyraclostrobin sensitivity was determined using spore germination and mycelial growth assays. The effective concentration inhibiting 50% of fungal growth (EC50) was determined for each isolate. The sensitivity assays showed that the majority of A. alternata isolates tested were sensitive to pyraclostrobin. EC50 values of fludioxonil in a mycelial growth assay indicated that 100% of isolates were sensitive to this fungicide. The analysis of the cytochrome b gene showed that none of the 40 isolates with a different sensitivity profile had the G143A mutation, and the subgroup of 8 isolates analyzed by real-time PCR did not carry the G137R and F129L mutations. A subset of four more sensitive and two reduced-sensitive isolates was chosen to assess sensitivity on detached citrus leaves treated with pyraclostrobin at the maximum recommended label rate. Disease incidence and symptom severity were significantly reduced, with a small reduction reported in leaves inoculated with the reduced-sensitive isolates. Furthermore, there was no correlation between sensitivity and fitness parameters evaluated in vitro (mycelium growth and sporulation rate). These findings help the development of monitoring resistance programs and, consequently, set up effective anti-resistance strategies for managing ABS on citrus orchards.