First report of Alternaria tenuissima causing leaf spot disease on Paeonia lactiflora in Korea

Paeonia lactiflora is an herbaceous perennial native to central and eastern Asia. Given its notably showy flowers, P. lactiflora is grown extensively as a garden plant. In July 2023, numerous Alternaria leaf spots were observed affecting approximately 70% of the peony field (46,200m 2 ) at Haman-gun’s Peony Festival (35°20′16.8″N, 128°23′53.3″E) and similar symptoms were also found at a field (20,000m 2 ; 35°34′45.7″N, 128°10′41.1″E), Hapcheon-gun, Gyeongsangnam-do, Korea. The causal pathogens were isolated and have been deposited Korean Agricultural Culture Collection (KACC 411028 and KACC 411029). Small pieces of tissue taken from the margins between healthy and diseased tissues were surface-disinfected in 0.1% NaOCl for 1 min, rinsed with distilled water, placed on water agar (WA) medium, and incubated in the dark at 25°C. Uncrowded chains of Alternaria spores observed to have developed on WA, and single spores were transferred onto potato dextrose agar (PDA), WA, potato carrot agar (PCA) and V8 media and cultured in the dark at 25°C for 6 days. The isolates were characterized by unbranched conidial chains of up to 13 conidia in length, with the occasional formation of one or two lateral branches. Conidia were typically elongated ovoid to obclavate, measuring (11-) 30-43 (-61) × 11-17 μm. They usually had 3-8 transverse septa, and occasionally 0-3 longitudinal septa, some of which are oblique. The genomic DNA was extracted using the PINucle FAST Genomic DNA Extraction Kit (200). Nucleotide sequences were obtained from regions of the internal transcribed spacer (ITS), RNA polymerase II gene (RPB2), translation elongation factor 1-alpha gene (EF-1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Alternaria major allergen gene (Alt α 1) of the isolates, using the primer sets of ITS5/ITS4, gpd1/gpd2, fRPB2-5F/fRPB2-414R, EF1-728F/EF1-986R, and Alt-for/Alt-rev as described by Woudenberg et al. (2015). The sequences have been submitted to the GenBank database. They showed similarity over 98.79% (ITS 100%, RPB2 98.79%, EF-1α 100%, GAPDH 100%, and Alt α 1 98.79%) with those of Alternaria tenuissima JY30 in the GenBank and ML phylogenetic trees established based on their combined sequences showed that the strains clustered into a branch of A. tenuissima (e-Xtra 2). The pathogenicity of the fungus was confirmed by spraying a spore suspension (2 × 10 5 conidia/ml), prepared in sterile distilled water using a hemocytometer, on the nine healthy young leaflets of one compound leaf on each of five two-year-old P. lactiflora seedlings without mechanical damage, whereas five control plants were sprayed with distilled water. All plants were kept at 25°C and 80% relative humidity. Initial symptoms were observed as dark brown spots 7 days after inoculation, with irregularly shaped, light brown spots appearing afterwards. The pathogen was consistently re-isolated from the leaf spots of all treated plants and confirmed based on its conidia and nucleotide sequences. No symptom was observed on the controls. On the basis of its morphological and molecular characteristics, and fulfilling Koch’s postulates, the isolated pathogenic fungus was identified as A. tenuissima. This species was reported as the causal agent of leaf spot in P. lactiflora in China (Sun and Huang 2017). To the best of our knowledge, this is the first confirmed report of P. lactiflora leaf spot by A. tenuissima in Korea, thereby highlighting the need for further research on measures for the control of this pathogen.