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Pseudostellaria heterophylla (family Caryophyllaceae) is a perennial herbaceous plant. Its tuberous roots are highly valued in traditional Chinese medicine. It is mainly cultivated in a geo-authentic production zone located in the Guizhou, Anhui, Shandong, and Fujian provinces of China (Zhao et al. 2016). The herb is widely used for treating lung diseases and as a spleen tonic (Pang et al. 2011). A severe leaf black spot disease was observed on P. heterophylla in China, from 2018 to 2020. Plants displayed water-soaking symptoms in the early stage of infection, then the watery areas turned brown-red and a black mold appeared on the lesions. At a later stage, the leaf spots showed concentric rings surrounded by a yellow halo, and the initial infection site became dry and necrotic (Supplementary Figure S1). Nine infected plants were collected from three cultivation fields in Shibing County (N 27°4'21", E 108°8'0"), Guizhou province, on April 13th, 2019. The fungus was consistently isolated from symptomatic leaves on potato dextrose agar (PDA) medium according to the method described by Larran et al (2002). A total of 22 isolates were obtained, including 7 isolates of Arcopilus and 15 isolates of Trichoderma. The growth rates of isolate MJ2-2b on PDA and oatmeal agar (OA) medium were 3 to 5 mm/day at 25 °C (Supplementary Figure S2A and S2B). Mycelium of isolate MJ2-2b was dense, yellowish-brown on PDA, while it was sparse, bright-red on OA. Also, the mycelium secreted brownish-red pigment on both PDA and OA. Ascomata when mature were water drop and limoniform. Lateral hairs were brown, erect or flexuous, tapering towards the tips. Ascospores when mature were greyish-white to grey, limoniform, or fusiform to pyriform (Supplementary Figure S2C and S2D). Further, the beta-tubulin gene (Tub2) of the fungus was amplified by using primer pairs T1 and TUB4Rd as described by Wang et al (2016) and subjected to sequencing. NCBI nucleotide BLAST results showed that sequences from seven isolates had a 99.86% identity with A. aureus (strain ChL-C, GenBank accession No. MG889987.1) (Supplementary Figure S2F). Molecular phylogenetic analysis by maximum likelihood method using MEGA 7 confirmed that the fungal isolate clustered with A. aureus. Hence, the causal agent was identified as A. aureus based on morphological and molecular characteristics. The sequence was deposited in GenBank (accession No. MW531453). Pathogenicity tests were conducted on 15-day old tissue-cultured seedlings according to Ghanbary et al (2018) (Supplementary Figure S3). Leaves of 16 seedlings were inoculated with 1×1 mm 5-day-old PDA-grown mycelial plugsof the fungal isolate. The experiment was repeated 3 times. After 10 days, the inoculated leaves showed the same symptoms observed on plants in the field. The associated fungal pathogen was consistently re-isolated from the inoculated seedlings and identified by Tub2 gene sequencing. At present, there are no reports of A. aureus causing disease of plants. To the best of our knowledge, this is the first report of leaf black spot disease on P. heterophylla caused by A. aureus in China.

In recent years, the tea plant (Camellia sinensis [L.] Kuntze) has been widely planted in Guizhou Province, China, with a cultivated area of 350,000 ha in 2017. In November 2018, leaf spots were observed on tea plants in Yuqing County (27.22°N, 107.88°E). Disease incidence was estimated at 74 to 82%, and disease severity was estimated at 42 to 48% in 12 tea plantations. Early symptoms on infected leaves were light brown lesions, which gradually enlarged to brown, scattered, round, elliptical or irregular lesions. The central area of each lesion later became grayish and surrounded by an inconspicuous yellow halo. Neighboring lesions coalesced to form larger lesions at the margin of the leaves. In order to identify the pathogen(s) causing this disease, lesion margins of the sampled leaves (n = 10) were surface sterilized with 75% ethanol for 30 s, soaked in 0.5% sodium hypochlorite for 5 min, rinsed three times in sterilized water, plated on potato dextrose agar (PDA), and incubated for 3 to 5 days in darkness at 25°C. The hyphal tips from the edge of growing colonies were picked and transferred to fresh PDA plates. Colonies were initially pale yellow and gradually became grayish-white, producing masses of flocculent aerial hyphae. At 22 days postinoculation, black, spherical, scattered or clustered pycnidia were evident, 50 to 290 μm in diameter. Their shape was globose or irregular, with one to two ostioles. Pycnidia were nonpapillate or slightly papillate, with the pycnidial wall consisting of two to three layers of cells. Unicellular and hyaline conidia (n = 50) were ellipsoidal or obovate in shape, with mean ± SD dimensions of 6.1 ± 1.1 (4.1 to 9.0) × 2.9 ± 0.6 (1.8 to 4.1) μm, and were usually rounded at each end. On the basis of these morphological characteristics, the isolates were initially identified as Didymella bellidis (Xu et al. 2016; Zou et al. 2020). To confirm the identity, the internal transcribed spacer (ITS), the partial 28S large subunit rDNA (LSU), and the beta-tubulin (TUB) gene of three representative isolates were amplified and sequenced (Chen et al. 2017), respectively. The sequences were deposited in GenBank with accession numbers MN274963, MN274964, MN274965 (ITS); MN274966, MN274967, MN274968 (LSU); and MN274969, MN274970, MN274971 (TUB). Phylogenetic analysis confirmed these isolates to be identical to D. bellidis reference strain D. bellidis CBS 714.85. To fulfill Koch’s postulates, healthy leaves on tea plant twigs (C. sinensis ‘Fuding-Dabai Cha’) were separately inoculated with mycelial plugs from PDA cultures and conidial suspension (1 × 10⁶ conidia/ml, 20 to 30 μl) for three isolates. PDA plugs without mycelia and buffer with no conidia were used as a control. Each treatment contained five replicates. The pathogenicity test was performed three times independently. Brown leaf spots gradually turned into black irregular lesions at 1 day postinoculation; the center of the lesion became darker and necrotic at 5 days postinoculation. The control groups showed no symptoms. The pathogen was reisolated from inoculated samples and identified by morphology and molecular biology. To the best of our knowledge, this is the first report of this fungus causing a tea leaf spot in China and seriously reducing both the productivity and the quality of tea leaves. Our findings will be useful for its management and further research.