First report of Alternaria alternata causing leaf spot on Toona ciliate in China.

Toona ciliate is an excellent timber and ornamental tree cultivated in China (Li et al. 2018). In May 2018, a leaf spot disease was observed on the foliage of T. ciliate in Nanchang city, Jiangxi province. Disease incidence averaged approximately 40%. Initial symptoms were small, brown spots with yellow halos, then the spots gradually enlarged and coalesced to form large lesions. To identify the pathogen, thirty pieces (5 × 5 mm) from the lesion margins were surface sterilized in 70% ethanol (30 s), then in 3% NaOCl (1 min), and finally rinsed three times with sterile water. The pieces were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation. Fourteen strains with similar morphological characters were isolated, and three representative isolates (MT-2, MT-5, MT-8) were used for morphological and molecular characterization. The colonies on PDA were gray to brown after 7 days. Ovoid or elliptical conidia were brown to light-brown in color with a short beak, 1-5 diaphragms, and 0-3 mediastinum. The diameter of these conidia were thick (18.2-47.4×7.9-15.1 µm, n= 100). The morphological characteristics of three isolates matched those of Alternaria sp. with straight or curved primary conidiophores with obclavate, long ellipsoid conidia (Woudenberg et al. 2013). The internal transcribed spacer (ITS) regions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), small subunit (SSU), large subunit (LSU), RNA polymerase second largest subunit (RPB2), translation elongation factor 1-alpha (TEF1) (Woudenberg et al. 2013) and Alternaria major allergen gene (Alt a 1) (Woudenberg et al. 2014) were amplified by using the following primer pairs ITS1/ITS4, GPD-1/GPD-2, NS1/NS4, LR0R/LR05, RPB2-5F2/fRPB2-7cR, EF1-728F/EF1-986R and Alt-f/Alt-r, respectively. The sequences were deposited in GenBank (ITS: ON459540, ON459541, ON459542; GAPDH: ON427936, ON427937, ON427938; SSU: ON422107, ON422108, ON422109; LSU: ON422110, ON422111, ON422112; RPB2: ON427939, ON427940, ON427941; TEF1: ON427933, ON427934, ON427935; Alt a 1: ON427942, ON427943, ON427944). A maximum likelihood and Bayesian posterior probability-based analyses using IQ-tree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, GAPDH, SSU, LSU, RPB2, TEF1, Alt a 1) placed three isolates in the clade of Alternaria alternata (Fr.) Keissl. The three isolates were identified as A. alternata based on morphological and molecular characteristics. For pathogenicity tests, 10 T. ciliate plants (two leaves each, n=20) grown outdoors were pin-pricked with a sterile needle and inoculated with a drop of spore suspension (106 conidia per mL) in July. Another 20 healthy leaves were inoculated with sterile water as the control. All the inoculated leaves were wrapped with plastic bags to keep them moist for 2 days. The pathogenicity tests were repeated twice. The resulting symptoms were similar to those on the original infected plants, whereas the control leaves remained asymptomatic for 10 days after inoculation. The same fungus was re-isolated from the lesions, confirming Koch's postulates. The pathogen was previously reported to cause leaf spots on Aquilegia flabellata (Garibaldi et al. 2022), Chrysanthemum morifolium (Luo et al. 2022), Liriodendron chinense × tulipifera (Jin et al. 2021) and so on. To our knowledge, this is the first report of A. alternata associated with leaf spot disease on T. ciliate in China. This disease may potentially decrease the value of ornamental T. ciliate plants under favorable conditions and proper management strategies should be applied.