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Title:   Author  item
                                            KIM, K - Pace International, Llc - Usaitem
                                            SAITO, SEIYA - University Of Californiaitem
                                            Xiao, Chang-Lin
  Submitted to: Plant Disease
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 4/29/2015
                                    Publication  Date: 10/1/2015
                                    Citation: Kim, Y.K., Saito, S., Xiao, C.L. 2015. Occurrence of fludioxonil resistance in penicillium digitatum from citrus in California. Plant Disease. 99:1447.Interpretive Summary: Penicillium digitatum is the causal agent of green mold, the most important postharvest disease of citrus. Fludioxonil is marketed as either a solo product or in combination with azoxystrobin for control of green mold and other postharvest diseases. The purpose of this study was to monitor resistance to fludioxonil in P. digitatum populations. We collected 42 Penicillium spp. during March to July 2013 in two commercial citrus packinghouses in California. All isolates were identified as P. digitatum and tested for resistance to fludioxonil. Of the 42 P. digitatum isolates, 15 were resistant. Fludioxonil completely controlled green mold on fruit inoculated with the fludioxonil-sensitive isolate, but failed to control the disease incited by the fludioxonil-resistant isolate. This is the first report of fludioxonil resistance in P. digitatum collected from commercial citrus packinghouses after the introduction of the fungicide in the market. These fludioxonil-resistant isolates were obtained from packinghouses where the fungicide had been used during packing of citrus fruit for 2 consecutive years, approximately for a 3-month period in each year, suggesting that fludioxonil-resistant individuals emerged quickly in P. digitatum populations. Our results also indicated that fludioxonil failed to control green mold incited by the fludioxonil-resistant isolate, suggesting that appropriate fungicide resistance management practices need to be implemented.
                                Technical Abstract: Penicillium digitatum is the causal agent of green mold, the most important postharvest disease of citrus (Citrus spp.). Fludioxonil is marketed as either a solo product or in combination with azoxystrobin for control of green mold and other postharvest diseases. Baseline sensitivity to fludioxonil in P. digitatum populations from California citrus packinghouses has been established. To monitor resistance to fludioxonil in P. digitatum populations, 42 Penicillium spp. were obtained from two different sampling methods: 20 were obtained from decayed orange fruit exhibiting green mold like sporulation, and 22 were recovered from potato dextrose agar plates amended with 0.5 μg/ml fludioxonil that were exposed to air during March to July 2013 in two commercial citrus packinghouses in California. All isolates were single-spored and identified as P. digitatum based on the morphological characters and the sequence analysis of the internal transcribed spacer (ITS) region using the primers ITS1/ITS4. A BLAST search showed that the sequences had 99% homology with that of P. digitatum deposited at GenBank (Accession No. AF033471.1). Of the 42 P. digitatum isolates, 15 isolates were able to grow at the discriminatory concentration of 0.5 μg/ml based on a mycelial growth assay.  EC50 values (the effective concentration that inhibits fungal growth by 50% relative to the control) of fludioxonil for the resistant isolates were >100 μg/ml following a method described previously. To assess whether fludioxonil at label rate is still able to control fludioxonil-resistant isolates, ‘Eureka’ lemons were wounded with a stainless steel rod (1 × 2 mm) and inoculated by placing 10 μl of conidial suspensions (6 × 104 conidia/ml) of either a fludioxonil-sensitive or a fludioxonil-resistant isolate at the wound of each fruit.  After 4 h, inoculated fruit were dipped for 30 sec in either Graduate® (Syngenta Crop Protection, Research Triangle Park, Raleigh, NC) at 16 fl oz/100G or water as a control and stored at 20°C for 7 days. There were four 20-fruit replicates for each treatment. The experiment was performed twice. All inoculated fruit treated with water were decayed. Fludioxonil completely controlled green mold on fruit inoculated with the fludioxonil-sensitive isolate, but failed to control the disease incited by the fludioxonil-resistant isolate. 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 - 2.5 × 10-9). This is the first report of fludioxonil resistance in P. digitatum from air-borne spores and decayed citrus fruit collected from commercial citrus packinghouses after the introduction of the fungicide in the market. These fludioxonil-resistant isolates were obtained from packinghouses where the fungicide had been used during packing of citrus fruit for 2 consecutive years, approximately for a 3-month period in each year, suggesting that fludioxonil-resistant individuals emerged quickly in P. digitatum populations. Our results also indicated that fludioxonil failed to control green mold incited by the fludioxonil-resistant isolate, suggesting that appropriate fungicide resistance management practices need to be implemented.


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