First Report of Epicoccum sorghinum Causing Leaf Spot on Paeonia suffruticosa in China.

Tree peony ( Andr.; Paeoniaceae) is highly valued in Chinese culture for their ornamental and medicinal benefits in anti-inflammation, anti-arrhythmia, activating the immune system and protecting the cardiovascular system (Zhai et al. 2020). In May from 2019 to 2021, leaf spot symptoms were observed on tree peony in an area of 0.8 hm2 in Bozhou, Anhui Province, China; with a disease incidence of 35%. Initially, black-brown spots (0.5-2.4 mm) appeared on the leaves, as the disease developed, the spots enlarged to form round, black lesions. Small pieces of tissue taken from the margins of lesions were surface-disinfected by 75% ethanol for 20 s followed by 0.1% mercuric chloride for 3 min, rinsed with distilled sterile water for three times, placed on potato dextrose agar (PDA) at 25 ºC for 7 days in the dark.Fifteen single spore isolates were obtained as described (Dong 2009). When cultured on PDA for 10 days, colonies were white, villose, then became gray-brown, with the reverse side becoming reddish-brown; Chlamydospores globose or irregular shaped, dark brown, unicellular or multicellular (4.3 to 12.2 × 13.1 to 33.7 µm) (n = 200). Pycnidia were black-brown, mostly spheroid, and 86 to 128 × 110 to 197 µm (n = 50); Conidia were hyaline, ellipsoidal, unicellular, aseptate, and 4.1 to 5.2 × 1.8 to 2.4 µm (n = 200) . The DNA of a representative isolate AF21-1-2 was extracted using the CTAB protocol (Doyle and Doyle 1987). The rDNA internal transcribed spacer (ITS), β-tubulin (TUB) and 28s nrDNA (LSU) genes were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass et al. 1995), and LR5/LR0R (Vilgalys and Hester, 1990), respectively (OK485136, OK631973, and ON358331). BLASTn analysis showed that the ITS, TUB, and LSU sequences had 99% (497/501 bp), 98% (530/539 bp), and 99% (878/882 bp) identity with the isolates ESCZO20 (MN944541), CZ01 (MT254851) and Dl16-338 (LN907481), respectively. Maximum likelihood phylogenetic tree revealed that AF21-1-2 clustered with isolates. For pathogenicity test, leaves of 3-year-old healthy tree peony plant in the field were inoculated with 10 conidia/ml spore suspension (prepared with 30-day-old cultures on PDA), added with 0.1% (v/v) Tween-80. Ten surface-sterilized leaves per plant were sprayed with 30 ml suspension until runoff, a total of 3 plants were inoculated. Leaves sprayed with sterilized water serve as a negative control. Each plant was covered with a plastic bag to maintain high relative humidity at 15 to 23°C and was monitored daily for symptom development. Typical symptoms appeared on more than 80% of the leaves after 7 days of inoculation, while control leaves remained symptomless. Cultures isolated from inoculated leaves had the same morphological and molecular traits as those that were previously used to test Koch's postulates. Based on the morphological and molecular characteristics, the causal agent on tree peony was identified as (Aveskamp et al. 2010). To our knowledge, this is the first report of causing leaf spot on tree peony in China. The disease will cause serious economic loss for tree peony if it is not managed properly. This report may provide the basis for diagnosis and control of the disease.