First Report of Alternaria alternata Causing Postharvest Decay on Apple Fruit During Cold Storage in Pennsylvania.

Alternaria rot, caused by Alternaria alternata (Fr.) Keissl., occurs on apple fruit (Malus × domestica Borkh) worldwide and is not controlled with postharvest fungicides currently registered for apple in the United States (1). Initial infections can occur in the orchard prior to harvest, or during cold storage, and appear as small red dots located around lenticels (1). The symptoms appear on fruits within a 2 month period after placement into cold storage (3). In February 2013, 'Nittany' apple fruit with round, dark, dry, spongy lesions were collected from bins at commercial storage facility located in Pennsylvania. Symptomatic apples (n = 2 fruits) were placed on paper trays in an 80 count apple box and immediately transported to the laboratory. Fruit were rinsed with sterile water, and the lesions were superficially disinfected with 70% ethanol. The skin was removed with a sterile scalpel, and tissues underneath the lesion were cultured on potato dextrose agar (PDA) and incubated at 25°C with constant light. Two single-spore isolates were propagated on PDA, and permanent cultures were maintained on PDA slants and stored at 4°C in darkness. Colonies varied from light gray to olive green in color, produced abundant aerial hyphae, and had fluffy mycelial growth on PDA after 14 days. Both isolates were tentatively identified as Alternaria based on multicelled conidial morphology resembling "fragmentation grenades" that were medium brown in color, and obclavate to obpyriform catentulate with longitudinal and transverse septa attached in chains on simple conidiophores (2). Conidia ranged from 15 to 60 μm (mean 25.5 μm) long and 10 to 25 μm (mean 13.6 μm) wide (n = 50) with 1 to 6 transverse and 0 to 1 longitudinal septa per spore. To identify both isolates to the species level, genomic DNA was extracted from mycelial plugs and gene specific primers (ALT-HIS3F/R) were used via conventional PCR to amplify a portion of the histone gene (357 bp) (Jurick II, unpublished). Amplicons were sequenced using the Sanger method, and the forward and reverse sequences of each amplicon were assembled into a consensus representing 2× coverage. A megaBLAST analysis revealed that the isolates were 99% identical to Alternaria alternata sequences in GenBank (Accession No. AF404617), which was previously identified to cause decay on stored apple fruit in South Africa. To prove pathogenicity, Koch's postulates were conducted using organic 'Gala' apples. The fruit were surface disinfested with soap and water and sprayed with 70% ethanol to runoff. Wounds, 3 mm deep, were done using a sterile nail and 50 μl of a conidial suspension (1 × 10 conidia/ml) was introduced into each wound per fruit. Fruit were then stored at 25°C in 80 count boxes on paper trays for 21 days. Water only was used as a control. Ten fruit were inoculated with each isolate or water only (control) and the experiment was repeated once. Symptoms of decay observed on inoculated were 'Gala' apple fruit were identical to the symptoms initially observed on 'Nittany' apples obtained from cold storage after 21 days. No symptoms developed on fruit in the controls. A. alternata was re-isolated 100% from apple inoculated with the fungus, completing Koch's postulates. A. alternata has been documented as a pre- and postharvest pathogen on Malus spp. (3). To our knowledge, this is the first report of postharvest decay caused by A. alternata on apple fruit during cold storage in Pennsylvania. References: (1) A. L. Biggs et al. Plant Dis. 77:976, 1993. (2) E. G. Simmons. Alternaria: An Identification Manual. CBS Fungal Biodiversity Center, Utrecht, the Netherlands, 2007. (3) R. S. Spotts. Pages 56-57 in: Compendium of Apple and Pear Diseases, A. L. Jones and H. S. Aldwinkle, eds. American Phytopathological Society, St. Paul, MN, 1990.