First report of Leaf Blight of Liquidambar formosana Hance caused by Alternaria tenuissima in China.

Abstract

Liquidambar formosana Hance is widely planted in urban landscapes in China owing to its ornamental red leaves. In June 2020, a distinctive leaf spot disease was observed on L. formosana in Nanjing Forestry University, Jiangsu Province of China (32°4'49"N, 118°48'56"E). Approximately 61% (14 out of 23) of the trees displayed leaf spots. The diseased symptoms included irregularly distributed spots that showed black or dark brown, and occasionally with pale green halo. Two representative trees were selected for sampling and five leaves with typical symptoms were selected randomly for isolation. The tissues from the margin of the lesions (0.2 cm × 0.2 cm) were cut and disinfected in 1% sodium hypochlorite for 90 s, rinsed in sterile water twice for 30 s, and dried with sterile paper. Then, 20 tissues were incubated on 2% potato dextrose agar (PDA) supplemented with 100 mg/L Ampicillin Sodium and incubated in the dark at 25℃ for 4 days. Seventeen single-spore fungi were isolated from lesion tissues as described by Woudenberg et al. (2013). The colony morphology of 17 isolates was extremely similar, so 3 isolates (NFUA01, NFUA02, and NFUA03) were selected randomly for further study. Colonies on PDA were circular, gray, and slightly raised loose cotton mycelium, while the reverse side was olive green in the center with white margins. Conidiophores were brown, simple or branched, and produced numerous conidia in short chains. Conidia were obclavate or ellipsoid, brown, with 1-5 transverse septa and 0-3 longitudinal septa, and measured 7.1 to 32.5 × 3.3 to 13.3 μm (n=50). The morphological observations were consistent with the description of the genus Alternaria sp. (Woudenberg et al. 2013). Six gene fragments, including SSU, LSU, ITS, GAPDH, RPB2 and EF-1 region, were amplified and sequenced. The primers of six nuclear loci were used by NS1 / NS4((White et al. 1990), LSU1Fd (Crous et al. 2009)/ LR5 (Vilgalys & Hester 1990), V9G (De Hoog & Gerrits van den Ende 1998)/ ITS4 (White et al. 1990), gpd1 / gpd2 (Berbee et al. 1999), RPB2-5F2 / fRPB2-7cR (Liu et al. 1999), and EF1-728F / EF1-986R (Carbone & Kohn 1999). The sequences were submitted in GenBank (SSU, ON237470 to ON237472; LSU, ON237464 to ON237466; ITS, ON197354 to ON197356; GAPDH, ON237476 to ON237478; RPB2, ON237467 to ON237469; EF-1, ON237473 to ON237475). BLAST result showed that SSU, LSU, ITS, GAPDH, RPB2, and EF-1 sequences of NFUA01, NFUA02, and NFUA03 were identical to A. tenuissima at a high level (>99%, Table 1). A maximum likelihood and Bayesian posterior probability analysis were performed by IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (Guindon et al. 2010; Ronquist et al. 2012). The representative strains which selected for Phylogenetic analyses were chosen from the strains which mentioned by Woudenberg et al (2013) and obtained the sequences from NCBI. The concatenated sequences placed NFUA01, NFUA02 and NFUA03 in the clade of Alternaria tenuissima with a high confidence level (ML/BI= 100/1). A pathogenicity assay was done using isolate NFUA01 on 3-year-old L. formosana seedlings. L. formosana leaves were wounded by a sterilized needle (0.5-mm-diam), and inoculated with spore suspension (106 conidia/mL), and L. formosana leaves inoculated with sterile water were used as the control. Each treatment had 5 leaves, and incubated at 25℃ under high moisture conditions. The experiments were conducted three times. Seven days after inoculation, leaves inoculated with spore suspension showed brown leaf blights resembling the original disease symptoms, whereas the control remained healthy. The fungus was reisolated from the lesions and was confirmed as A. tenuissima based on morphologically characteristics and ITS sequence analysis. To our knowledge, this is the first report of A. tenuissima associated with leaf blight on L. formosana. The finding provides clear pathogen information for further evaluation of the disease control strategies.