First Report of Leaf Spots and Necrosis Caused by Paraphaeosphaeria recurvifoliae on Yucca gloriosa in China

Yucca gloriosa L. has been introduced to China as a garden plant because of its tolerance to low temperatures and attractive tubular flowers. In June 2018 and 2019, leaf spot and tissue necrosis appeared on Y. gloriosa plants at Chengyang campus and Laoshan Yangkou scenic area, both in Qingdao, Shandong Province, China. The initial symptoms were observed on the middle and lower older leaves as small, round or oval, dark brown leaf spots. Then, the leaf spot gradually expanded and became oval or irregularly round, with a clear dark brown border (leaf spot size: about 0.5 to 5 cm long and 0.5 to 1.5 cm wide). Dark fruiting bodies were observed in the center of the leaf spot. The disease symptoms subsequently spread to upper leaves of the plant, inducing necrosis and wilting of whole leaves. To determine an agent of this disease, 10 symptomatic leaves were collected from Chengyang campus and Laoshan Yangkou scenic area. Small pieces of tissue from each infected leaf were excised, surface disinfested in 75% ethanol solution for 30 s, and placed on potato dextrose agar (PDA) at 25°C in the dark for 5 days. Fungal cultures were then transferred to new PDA. For morphological description, the cultures were incubated at 27 ± 2°C for 10 days. Colonies on PDA (200 g/liter extract of boiled potatoes, 20 g/liter glucose, and 15 to 20 g/liter agar) at first were whitish to cream colored or greyish, and they reached an average diameter of 60 mm. Small black pycnidia were buried in mycelia at the late growth stage. Pycnidia were brown, ovoid, superficial to partly immersed, and 85 to 155 μm in diameter. Conidia produced were unicellular and were spherical or elliptic (5.5 × 4.6 μm in average size, n = 80), usually from hyaline to dark brown. The observed morphological features matched those described for Paraphaeosphaeria recurvifoliae (Lee et al. 2005; Pieczul and Perek 2016). For further identification, the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of nuclear rDNA was amplified from the extracted genomic DNA with primers ITS1 and ITS4 (Berbee et al. 1999), followed by the sequencing of the PCR product (569 bp). All rDNA sequences of tested isolates were identical. One was deposited in GenBank (accession no. MN121340). BLAST analysis revealed more than 99% identity to the sequence of P. recurvifoliae (AY957476 and KT350983). To confirm Koch’s postulates, 12 Y. gloriosa plants (about 2 years old) were randomly collected from Chengyang campus. P. recurvifoliae spores were placed on the surface of 24 leaves (about 5 months old) of six Y. gloriosa plants, with 10 μl of P. recurvifoliae conidial suspension (2.6 × 10⁵ conidia/ml) for each leaf. Distilled water was individually placed on the surface of 24 control leaves. The experiments were performed in a greenhouse (27 ± 2°C, 75 ± 5% relative humidity, and a photoperiod of 14 h light/10 h dark). After 10 to 15 days, the inoculated plants developed symptoms similar to those observed in naturally infected plants, whereas control plants were symptomless. The percent incidence and disease severity index (Islam and Borthakur 2012; Zhao et al. 2014) of leaves inoculated with strain MN121340 were 87.5% and 43.75, respectively. The fungal pathogen was reisolated, and the resulting ITS sequence was identical to the inoculated P. recurvifoliae. Based on these results, the disease on Y. gloriosa was caused by P. recurvifoliae. To our knowledge, this is the first report of P. recurvifoliae occurrence on Y. gloriosa in China. This finding raises awareness and concern about this emerging disease, and control strategies should be followed.