First report of Penicillium crustosum causing blue mold on stored apple fruit in Serbia

Authors: VICO, IVANA - University Of Belgrade; Gaskins, Verneta; DUDUK, NATASA - University Of Belgrade; VASIC, MILJAN - University Of Belgrade; Yu, Jiujiang; PETER, KARI - Pennsylvania State University; Jurick, Wayne

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2014
Publication Date: 5/7/2014
Publication URL: http://handle.nal.usda.gov/10113/59691
Citation: Vico, I., Gaskins, V.L., Duduk, N., Vasic, M., Yu, J., Peter, K.A., Jurick II, W.M. 2014. First report of Penicillium crustosum causing blue mold on stored apple fruit in Serbia. Plant Disease. DOI: 10.1094/PDIS-02-14-0179-PDN.

Interpretive Summary: Blue mold, which is caused by a number of related fungi that grow on apple and pear fruit during long term storage, results in significant economic losses. It is a consistent problem for the apple growing, packing and processing industry because it makes fruit unmarketable for consumption and processing in the US and worldwide. Several isolates of fungi causing blue mold were collected in 2013 from a commercial packing and storage facility in Serbia. These isolates were characterized and identified using nucleic acid-based methods. This study determined several distinguishing factors that can be used by growers to visually identify the fungus. In addition, the strains that were isolated can be utilized by other scientists to design rapid nucleic-acid based detection methods, which will help reduce losses due to storage decay.

Technical Abstract: Penicillium crustosum Thom causes blue mold on pome fruits and is also regularly found on cheese, nuts and soil. The fungus produces an array of mycotoxins that impact human health, including penitrem A, roquefortine C, terrestric acid, and cyclopenol. In January and February 2013, decayed apples, ‘Golden Delicious’ and ‘Jonagold’ (Malus x domestica Borkh.) with blue mold symptoms were collected from cold storages in Svilajnac and Bela Crkva, Serbia. Decayed areas were light to medium brown and decayed tissue was soft and watery with a sharp margin between the diseased and healthy areas. One isolate from each cultivar was designated JP2 and JBC7 and further characterized. Conidiophores of both isolates were terverticillate, stipes were septate with rough walls, and phialides were ampulliform. Conidia were smooth, born in columns, and were spherical to subglobose. Conidial dimensions for JP2 were 3.2-(3.73)-4.56 x2.64-(3.32)-4.3µm and for JBC7 were 3.1-(3.65)-4.46 x 2.81-(3.31)-4.27µm (n=50). The isolates were cultured on Czapek yeast autolysate agar (CYA), Malt extract agar (MEA), and Yeast extract sucrose agar (YES) media and incubated at 25°C for 7 days. No growth was observed when these isolates were incubated at 37°C. Mycelia were white with heavy sporulation yielding grayish green-colored colonies on all media. Colonies were radially sulcate and velutinous, with clear exudate, and produced a yellow to orange reverse on CYA and YES. On MEA, colonies were plane, low, and mycelia subsurface with conidia having a dry powdery appearance. Crusts of conidial masses formed after 10 or more days. Both isolates were identified as P. crustosum Thom using morphological characters according to Pitt and Hocking, and Frisvad and Samson. Species level identification was completed by isolating genomic DNA followed by amplification of the ß-tubulin locus using gene specific primers via conventional PCR. MegaBLAST analysis of the 2X consensus nucleotide sequences revealed that JP2 and JBC7 were 99% identical to P. crustosum culture collection isolate IBT 21518 (Genbank Accession JN112030.1). Koch’s postulates were examined using two apple cultivars ‘Idared’ and ‘Kolacara’. Ten fruit per cultivar, per isolate were inoculated on two sides of each fruit; 20 fruit were used as water-only inoculated controls. Fruit were washed with soap and water, surface sanitized with 70% ethanol, and placed into polyethylene boxes. Using a finishing nail, 4-mm wounds were created and inoculated with 50 µl of a 3,000,000/ml conidial suspension or Tween-treated sterile distilled water. Boxes with inoculated and control fruit were stored at 25°C for 10 days. The inoculated fruit developed soft watery lesions, with defined edges that were identical to the original symptoms, while the control fruit were symptomless. The fungus was reisolated from infected tissue and showed the same morphological characteristics as the original isolates thus completing Koch’s postulates. This is the first report of P. crustosum causing postharvest blue mold decay on apple fruit obtained from storage in Serbia and indicates that P. crustosum is an emerging pathogen for the Serbian pome fruit growing and packing industry.