First report of pyrimethanil resistance in Botrytis cinerea from stored apples in Pennsylvania

Authors: YAN, HAIJUAN - Jiangsu Academy Agricultural Sciences; Gaskins, Verneta; Luo, Yaguang - Sunny; KIM, YONG-KI - Pace International, Llc - Usa; Jurick, Wayne

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/20/2013
Publication Date: 1/15/2014
Publication URL: http://handle.nal.usda.gov/10113/58991
Citation: Yan, H., Gaskins, V.L., Luo, Y., Kim, Y., Jurick II, W.M. 2014. First report of pyrimethanil resistance in Botrytis cinerea from stored apples in Pennsylvania. Plant Disease. DOI:10.1094/PDIS-11-13-1120-PDN.

Interpretive Summary: Gray mold is caused by a fungus that occurs on apple fruit in the field and during long term cold storage. It is a main problem for the apple growing industry and is controlled by fungicides. However, the number of chemicals used to control this fungus are limited and it is imperative that the most effective materials are used to combat decay and minimize losses. Resistance to postharvest fungicides in the gray mold fungus was discovered for the first time in Pennsylvania at a commercial pome fruit packinghouse. The results from this research are valuable to stakeholders (i.e. apple growers, packers, and processors) so that they can use the most effective chemicals to limit decay, maintain fruit quality, and reduce losses. The fungal strains that were characterized in this study can also be used by industry or university scientists to design cutting edge nucleic-acid based tests to detect Penbotec-resistant gray mold strains rapidly and accurately to help aid in stakeholder fungicide management decisions.

Technical Abstract: Botrytis cinerea Pers.: Fr. (teleomorph Botryotinia fuckeliana [de Bary] Whetzel) causes gray mold on apple fruit which is an economically significant disease. A survey of commercial packinghouses in Washington State revealed that gray mold accounted for 28% of the decayed apples in storage. Fungicides are the primary method of control as host resistance in commercial apple cultivars is lacking. In February 2013, gray mold was observed at ~5% incidence for commercially packed ‘Gala’ apple fruit that had been treated with Penbotec® (active ingredient: pyrimethanil) prior to controlled atmosphere storage in Pennsylvania. Eight infected apple fruit were collected, placed in 80 count boxes on cardboard trays, and stored at 4ºC. Isolates were obtained from decayed apple tissue, placed on potato dextrose agar (PDA) Petri plates, and incubated at 20ºC with natural light. Eight single spore isolates were identified as Botrytis cinerea based on cultural characteristics. Species level identification was executed by obtaining mycelial genomic DNA, amplifying the Internal Transcribed Spacer (ITS) rDNA with ITS4/ITS5 primers, and sequencing the amplicon directly. MegaBLAST analysis of the 2X consensus for the 8 isolates revealed 100% identity to B. cinerea ITS sequences in Genbank (Accession # KF156296.1 and JX867227.1) with E values of 0.0, thus confirming the morphological identification. Minimum inhibitory concentration (MIC) was determined using conidial suspensions (10,000 spores/ml) and a range (0 – 500 µg/ml) of technical grade pyrimethanil on three replicated 96-well microtiter plates for each experiment. Conidial germination was inhibited at 250 µg/ml for all eight isolates and the experiment was conducted four times. Mycelial growth analysis, using a plug of actively growing mycelium, was conducted three times using technical grade pyrimethanil with three plates per experiment. Five isolates grew at 250 µg/ml (highly resistant), while three did not (moderately resistant). To assess resistance in vivo, organic ‘Gala’ apples were rinsed with soap and water, sprayed with 70% ethanol, placed on trays and allowed to air dry. Apples were wounded with a sterile finishing nail, inoculated with 20 µl of a conidial suspension (10,000 spores/ml) of either a moderately or a highly resistant isolate and dipped in Penbotec® at 500 µg/ml or sterile water for 30 seconds. Fruit were stored in 100 count boxes at 22ºC for 5 days and decay incidence and severity were recorded. Ten fruit composed a replicate per treatment and the experiment was repeated. Water only controls were symptomless and water-dipped inoculated fruit had 100% decay. Penbotec®-treated fruit had 100% decay incidence and mean lesion diameters of 37.6 (+/- 13.1 mm) for the highly, and 35.7 (+/- 9.0 mm) for the moderately resistant isolate. This is the first report of pyrimethanil resistance in B. cinerea from decayed apples collected from a commercial packinghouse in Pennsylvania. The results indicate that pyrimethanil resistance has developed in B. cinerea which can result in control failures on Penbotec®-treated fruit during storage. Furthermore, it emphasizes the need for additional tools to manage gray mold on apple fruit.