First report of Penicillium expansum isolates resistant to pyrimethanil from stored apple fruit in Pennsylvania

Authors: YAN, HAIJUAN - Jiangsu Academy Agricultural Sciences; Gaskins, Verneta; VICO, IVANA - University Of Belgrade; Luo, Yaguang - Sunny; Jurick, Wayne

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/12/2014
Publication Date: 1/22/2014
Publication URL: http://handle.nal.usda.gov/10113/58989
Citation: Yan, H., Gaskins, V.L., Vico, I., Luo, Y., Jurick II, W.M. 2014. First report of Penicillium expansum isolates resistant to pyrimethanil from stored apple fruit in Pennsylvania. Plant Disease. DOI: 10.1094/PDIS-12-13-1214-PDN.

Interpretive Summary: Blue mold is a plant disease caused by a filamentous fungus that results in decay of apples and pears during cold storage. Blue mold decay is a major problem for the apple growing and packing industry in the US and worldwide, and is mainly controlled by application of fungicides. The number of chemicals approved for use to control this disease is limited (3 total), and those few are rapidly losing their efficacy. In this study, resistance to the postharvest fungicide, Penbotec, in the blue mold fungus was discovered for the first time in Pennsylvania at a commercial pome fruit packinghouse. The results from this research are directly applicable and extremely important to stakeholders (i.e. apple growers, packers, and processors), who must use the most effective chemicals to limit fruit decay during storage, and thereby maintain high quality, marketable fruit that ensure profitability. In addition, the blue mold strains that were characterized in this study can be used by other scientists in the design of nucleic-acid based tests to detect Penbotec-resistant blue mold strains rapidly and accurately.

Technical Abstract: Apples in the United States are stored in low temperature controlled atmosphere for 9–12 months and are susceptible to decay by blue mold. Penicillium spp. cause significant economic losses worldwide and produce mycotoxins that contaminate processed apple products. Blue mold is managed by a combination of cultural practices and the application of pre- and postharvest fungicides. In 2004, a new postharvest fungicide, Penbotec® (active ingredient, pyrimethanil), was registered for use in the United States to control blue mold on pome fruits (1). In this study, ten ‘Red Delicious’ apples with blue mold symptoms were collected in May 2011 from wooden bins at a commercial facility located in Pennsylvania. These fruit had been treated with Penbotec® prior to controlled atmosphere storage to control postharvest decay. Ten single spore Penicillium spp. isolates were analyzed for growth using 96-well microtiter plates containing a minimal medium amended with a range of technical grade pyrimethanil from 0 to 500 µg/ml. Conidial suspensions adjusted to 100,000 conidia per ml were added to three 96 well plates for each experiment; all experiments were repeated three times. Nine resistant isolates had prolific mycelial growth at 500 µg/ml, which is 1000 times the discriminatory dose that inhibited baseline sensitive P. expansum isolates from Washington State (1). However, one isolate (R13) had limited conidial growth, but no mycelial proliferation, at 0.5 µg/ml, and was categorized as mildly resistant. One resistant (R22) and the mildly resistant (R13) isolate, selected on the basis of their different sensitivities to pyrimethanil, were further analyzed in vitro and in vivo. Both isolates were identified as P. expansum via conventional PCR using ß-tubulin gene specific primers according to Sholberg et al. (2). Analysis of the 2X consensus amplicon sequences from R13 and R22 matched perfectly (100% identity and 0.0 E value) with other P. expansum accessions in Genbank including #JN872743.1, which was isolated from decayed apple fruit from Washington State. To determine if pyrimethanil applied at the labeled rate of 500 µg/ml would control R13 or R22 in vivo, organic ‘Gala’ apple fruit were wounded, inoculated with 50 µl of a conidial suspension (10,000 conidia per ml) of either isolate, dipped in Penbotec® fungicide or sterile water, and stored at 25C for 7 days. Twenty fruit composed a replicate within a treatment and the experiment was performed twice. Non-inoculated water only controls were symptomless, while water-dipped inoculated fruit had 100% decay with mean lesion diameters of 36.8 (+/- 2.68 mm) for R22 and 38.5 (+/- 2.61 mm) for R13. The R22 isolate caused 30% decay with 21.6 mm (+/- 5.44 mm) lesions when inoculated onto Penbotec®-treated apples, while the R13 isolate had 7.5% decay incidence with mean lesion diameters of 23.1 (+/- 3.41 mm). The results from this study demonstrate that P. expansum pyrimethanil-resistant strains are virulent on Penbotec®-treated apple fruit and have the potential to manifest in decay during storage. To the best of our knowledge, this is the first report of pyrimethanil resistance in P. expansum from Pennsylvania, which is a major apple growing region for the US. Moreover, these results illuminate the need to develop additional chemical, cultural, and biological methods to control this fungus.