First Report of Moldy Core of Sweet Tango Apples from New Zealand Caused by Alternaria arborescens.

Moldy core is a fungal disease of apple fruits that is characterized by mycelial growth in the seed locules and is sometimes accompanied by penetration of the immediate surrounding flesh. The disease can go undetected until the fruit is cut open, as no external symptoms appear on the fruit. Alternaria, Aspergillus, Cladosporium, Coniothyrium, Epicoccum, Phoma and Stemphylium are some of the common pathogens associated with moldy core (Serdani et al. 2002; Gao et al. 2013; McLeod 2014). The disease is more common in apple cultivars with an open calyx, where spores may initiate infections during the growing season or at the post-harvest storage stage (Spotts et al. 1988). In 2018, a shipment of 'Sweet Tango' apples from New Zealand to Scotian Gold Co-operative Ltd., Nova Scotia, Canada, was found to be affected by moldy core. Moderate to severe moldy core symptoms were observed when 10 apples were cut open (Figure S1). In comparison, 'Sweet Tango' apples grown in Nova Scotia showed no moldy core symptoms when 10 random fruits were cut open. Small pieces of the diseased fruit tissue from the core region were surface-disinfected for 1 min in 1% NaOCl, rinsed three times with sterilized water and placed onto potato dextrose agar (PDA) dishes. The PDA dishes were incubated in dark at 22 oC and single spore isolation was carried out to fresh PDA dishes. These isolate produced colonies of regular shape, tan black with prominent white gray margin and gray colour conidia (Figure S2 AB). The colonies turn dark black after 3 weeks of growth on PDA. Mycelia were septate and conidia were oval or obclavate or club-shaped with a tapering end with 4-6 longitudinal and transverse septa (Figure S2 C-D). The size of conidia ranges from 12.5-20 x 8.7-12.5 µM on 20 days old PDA dishes. Based on the size and shape of conidia and other morphological characteristics the isolated fungi were identical to Alternaria spp. (Simmons 2007). To assess the identity of the isolated pathogen species by multi-locus sequence analysis, genomic DNA was extracted from the pure cultures of two isolates (5.8 and 8) using the E.Z.N.A. SP Fungal DNA Kit (Omega Bio-Tek). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH), major allergen (Alt a 1), OPA10-2, the internal transcribed spacer (ITS) region of ribosomal DNA and the translation elongation factor 1-α (TEF1-α) region from two Alternaria spp. isolates (5.8 and 8) were amplified and sequenced using primers gpd1/2 (Berbee et al. 1999), A21F/A21R (Gabriel 2015), OPA10-2/ OPA10-2L (Andrew et al. 2009), ITS1/ITS4 (White et al. 1990) and EF1-up /EF1-low (O'Donnell et al. 1998) respectively. The resulting sequences of both isolates were deposited in the NCBI GenBank (GAPDH; MW411052, MW411053, Alt a 1; MW411050, MW411051, OPA10-2; MW415762, MW415763, ITS; MK140445, MT225559, TEF1-α; MT305773 and MT305774 ). Sequences of GAPDH, Alt a 1, OPA-10-2, ITS and TEF1-α genes of both isolates were identical to each other and showed 100 %, 100 %, 99.21 %, 100% and 100% identity to A. arborescens S. (AY278810.1, AY563303.1, KP124712.1, KY965831.1, KY965831.1) respectively. Identity with reference strain CBS 102605 confirms that both of the isolated strains 5.8 and 8 are A. arborescens. The pathogenicity of the two A. arborescens isolates were confirmed by artificially inoculating healthy 'Sweet Tango' fruit by dispensing the conidial suspension directly on the seed locule. Briefly, surface-disinfected fruits were air-dried for 5 min and then peeled using a sterilized knife and cut transversally. Each half of the fruit was inoculated with 100 µl of conidial suspensions (∼1 × 104 conidia/ml) in potato dextrose broth (PDB) and incubated at 22 °C in a humid chamber for 7-10 days, or until symptoms with visible mycelial growth were observed. The control fruits were treated with 100 µl of sterilized PDB. Both A. arborescens isolates produced visible moldy core symptoms on the inoculated 'Sweet Tango' fruits, whereas no symptoms were observed on the control fruits (Figure S1). The experiment was repeated three times with at least three replicates with similar results. A. arborescens was successfully re-isolated from the artificially-inoculated fruits to complete Koch's postulates. To our knowledge, this is the first report of Alternaria arborescens causing moldy core disease in 'Sweet Tango' apples from New Zealand. Acknowledgments We thank Eric Bevis for his help in sample preparation for DNA sequencing, Willy Renderos for pathogenicity assay. We also thank Joan Hebb (Scotian Gold Cooperative Ltd.,) for providing the apple sample for this study. This research was made possible through financial support from Agriculture and Agri-Food Canada. The authors(s) declare no conflict of interest. Literature Cited Andrew M., Peever T.L., Pryor B.M. An expanded multilocus phylogeny does not resolve species among the small-spored Alternaria species complex. 2009. Mycologia. 101:95-109. Berbee, M. L. et al. 1999. Cochliobolus phylogenetics and the origin of known, highly virulent pathogens, inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase gene sequences Mycologia. 91:964. Gabriel, M.F. I. Postigo, A. Gutiérrez-Rodríguez, E. Suñén, C.T. Tomaz, J. Martínez 2015. Development of a PCR-based tool for detecting immunologically relevant Alt a 1 and Alt a 1 homologue coding sequences. Medical Mycology. 53 (6):636-642. Gao, L. L., Zhang, Q., Sun, X. Y., Jiang, L., Zhang, R., Sun, G. Y., Zha, Y. L., and Biggs, A. R. 2013. Etiology of moldy core, core browning, and core rot of Fuji apple in China. Plant Dis. 97:510-516. Kerry, O'Donnell, H.C. Kistler, E. Cigelnik, R.C. Ploetz. 1998. Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies. PNAS. 95: 2044-2049. McLeod, A. 2014. Moldy core and core rots. Pages 40-41 in: Compendium of Apple and Pear Diseases and Pests, 2nd ed. T. B. Sutton, H. S. Aldwinckle, A. M. Agnello, and J. F. Walgenbach, eds. American Phytopathological Society, St Paul, MN. Serdani, M., Kang, J. C., Peever, T. L., Andersen, B., and Crous, P. W. 2002. Characterization of Alternaria species groups associated with core rot of apples in South Africa. Mycol. Res. 106:561-569. Simmons, E. G. 2007. Alternaria: an identification manual. CBS Biodiversity Series. 6:780 pp. Spotts, R. A., Holmes, R. J., and Washington, W. S. 1988. Factors affecting wet core rot of apples. Australas. Plant Pathol. 17:53-57. White, T. J., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, eds. San Diego, CA: Academic Press. Woudenberg, J. H. C., et al. 2015. Alternaria section Alternaria: Species, formae speciales or pathotypes. Stud. Mycol. 82:1-21.