Occurrence of Fludioxonil Resistance in Penicillium digitatum from Citrus in California

Penicillium digitatum is the causal agent of green mold, one of the most important postharvest diseases of citrus (Citrus spp.). Fludioxonil can be used alone or in combination with azoxystrobin for the control of green mold and other postharvest diseases on citrus. Baseline sensitivity to fludioxonil in P. digitatum populations from California citrus packinghouses has been previously established (Kanetis et al. 2008). To monitor resistance to fludioxonil in P. digitatum, 20 Penicillium spp. isolates were obtained from decayed oranges by isolation from decayed tissue and 22 isolates were recovered from potato dextrose agar plates amended with 0.5 mg/liter fludioxonil (Kanetis et al. 2006) and exposed to packinghouse air for 5 min each sampling time during March to July 2013 in two California citrus packinghouses. All isolates were single-spore cultured and identified as P. digitatum based on morphological characters (Pitt 1979). The sequence analysis of the internal transcribed spacer (ITS) region, using the primers ITS1/ITS4, was conducted to confirm the species-level identification. A MegaBLAST search showed that the sequences of all isolates had 99% homology (E-value = 0.0) with that of P. digitatum deposited at GenBank (Accession No. AF033471.1). In a mycelial growth assay, 15 of the 42 P. digitatum isolates were able to grow at 0.5 μg/ml, a previously established discriminatory concentration (Kanetic et al. 2006). EC50 values (the effective concentration that inhibits fungal growth by 50% relative to the control) of fludioxonil for the resistant isolates were >100 mg/liter following a method described by Li and Xiao (2008). To assess whether fludioxonil at the label rate was able to control fludioxonilresistant isolates, ‘Eureka’ lemons were wounded with a stainless steel rod (1 × 2 mm) and inoculated with 10 μl of conidial suspensions (6 × 10 conidia/ml) of a representative sensitive or a resistant isolate. After 4 h, inoculated fruit were dipped for 30 s in either a formulated product of fludioxonil, Graduate (50% active ingredient; Syngenta Crop Protection, Research Triangle Park, Raleigh, NC) at 1,198 mg/liter, or water as a control, and then stored at 20°C in air for 7 days. There were four 20-fruit replicates for each treatment and the experiment was performed twice. All inoculated water-dipped fruit were decayed, while fludioxonil completely controlled green mold on fruit inoculated with the fludioxonil-sensitive isolate. However, 100% fruit inoculated with the fludioxonil-resistant isolate and treated with fludioxonil were decayed. Fludioxonil-insensitive isolates have been reported to occur in natural populations of P. digitatum before its commercial use, but at very low frequency (1.4 to 2.5 × 10) (Kanetis et al. 2006). This is the first report of fludioxonil resistance in P. digitatum collected from commercial citrus packinghouses after the introduction of the fungicide on the market. These fludioxonil-resistant isolates were obtained from packinghouses where the fungicide had been used during citrus packing for 2 consecutive years, approximately for a 3-month period in each year, indicating that fludioxonil-resistant individuals emerged quickly in P. digitatum populations. Our results show that fludioxonil failed to control green mold incited by the fludioxonil-resistant isolate, suggesting that appropriate fungicide resistance management practices need to be implemented to ensure adequate control.