Anthracnose caused by Colletotrichum gloeosporioides is one of most serious fungal diseases on Chinese fir (Cunninghamia lanceolata). Eight fungal endophytes were isolated from a young heathy branch of Chinese fir and screened against the pathogen in vitro. One isolate, designated as SMEL1 and subsequently identified as Epicoccum dendrobii based on morphological and phylogenetic analyses, suppressed mycelial growth of C. gloeosporioides on dual culture plates. Additionally, the metabolites of E. dendrobii significantly decreased the biomass of C. gloeosporioides. E. dendrobii was able to enter the internal tissues of the host plant via stomatal cells. The metabolites of E. dendrobii significantly inhibited conidial germination and appressorium formation, which at least partly explained why the endophyte significantly inhibited lesion development caused by C. gloeosporioides on various host plants. We further confirmed that some components with antifungal activity could be extracted from E. dendrobii using ethyl acetate as an organic solvent. To our knowledge, this is the first report of E. dendrobii as a potential biocontrol agent against a fungal phytopathogen.

Curvularia includes plant pathogenic species with a worldwide distribution and a wide host range, particularly cereals and grass (Poaceae). Chinese fir (Cunninghamia lanceolata) is an important afforestation tree species in southern China, with a high economic value. Leaf blight disease on Chinese fir was discovered in Hunan and Zhejiang, China, and two fungal species were found to be associated with the symptoms. Using morphological and phylogenetic approaches, they were identified as Curvularia spicifera and Curvularia muehlenbeckiae. They were determined to be the pathogens by fulfilling Koch's postulates. Both showed a pathogenicity on Citrus reticulata and Citrus tangerina, which have a similar geographic distribution to Chinese fir. The similar geographic distribution of these hosts may increase the risk of disease. Based on a literature review, leaf blight caused by C. spicifera and C. muehlenbeckiae is recorded here on a gymnosperm for the first time.

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Phyllostachys viridis (Poaceae) is a bamboo species broadly distributed in Asia. It is not only used as raw material in papermaking and food processing, but also used as an evergreen ornamental in the parks and landscapes of China. In February 2019, a leaf spot disease was found on P. viridis in Jurong, Zhenjiang, Jiangsu of China. The incidence rate of this disease was ca. 53%. The disease primarily appeared as orange chlorotic spots on the leaves. The spots enlarged and coalesced to form irregular brown necrotic lesions (Fig. S1A). Symptomatic leaves were collected, and twelve cuttings were made from the margin of the lesions of ca. 3×3 mm, surface-sterilized using 75% ethanol for 1 min, rinsed three times in sterile distilled water, and placed on 2% of potato dextrose agar (PDA) in Petri plates for 5 days at 25°C. Twenty-two fungal isolates were single-spored and stored in the Forest Pathology Laboratory at Nanjing Forestry University. Pathogenicity of all isolates was tested on leaves of P. viridis using mycelial discs. Mycelial plugs (5-mm-diameter discs) of each isolate were taken from the margins of colonies grown on PDA for 5 days and inoculated onto healthy leaves which were surface-sterilized using 75% ethanol and wounded with a sterilized needle. PDA discs without fungi were used to treat the control leaves. The inoculated whole plants were kept in a growth chamber at ca. 25°C at 85% relative humidity. The experiment was repeated twice and there were at least five replicates for each treatment. After 5 days post inoculation, the leaves inoculated with isolate PV2-9 showed necrotic lesions. No lesions were found on the plants inoculated by other fungal isolates and the control PDA plugs (Fig. S1B). The pathogen was re-isolated from the infected leaves, which shared similar morphological characteristics of colonies, aerial mycelia, and conidia with the original inoculated isolate PV2-9. Identification of the fungus was based on both morphological characteristics and molecular methods. Colonies of the isolate PV2-9 on PDA were white, floccose, round, pale grey with pale-reddish pigmentation near the center (Fig. S1C). The colonies attained 65±0.3 mm diam after 7 days at 25 oC. Abundant conidiomata were observed over the medium after 4 weeks (Fig. S1D). Conidiomata were pycnidial, solitary or gregarious, scattered, semi-immersed to erumpent, or superficial uniloculate to multi-loculate, globose to subglobose, ostiolate, 105.2-162.1× 90.3-116.2 μm (x=122.6±14.4 × 97.9±6.8 μm, n=30). Pycnidial wall was comprising 3-7 layers of dark brown to lightly pigmented, 13.4–19.7 μm thick (x =16.4 ± 1.6 μm, n=30) (Fig. S1E, F). Conidiogenous cells were phialidic, hyaline, smooth, flask-shaped to doliiform, lining the conidiomatal cavity (Fig. S1G). Conidia were ellipsoidal to cylindrical, smooth, hyaline, aseptate, 4.0–6.2 × 2.4–3.3 μm (x =4.8 ± 0.5 ×2.5 ± 0.3, n=30), with 0–3 guttules (Fig. S1H). These morphological characteristics matched the Epicoccum poaceicola described by Thambugala et al. (2017). The internal transcribed spacer (ITS) region of rDNA, 28S large subunit ribosomal RNA gene (LSR), RNA polymerase II second largest subunit gene (RPB2), and beta-tubulin gene (TUB) were amplified using the primers published in Raza et al. (2019). The ITS (MT110918), LSU (MT110921), TUB (MT119149), and RPB2 (MT119150) showed 99.3%, 99.8%, 98.1%, and 99.8% identity to the corresponding sequences of E. poaceicola ex-type MFLUCC 15-0448, respectively. Phylogenetic analysis using concatenated sequences of ITS, LSU, TUB and RPB2 also showed that PV2-9 clustered monophyletically with E. poaceicola MFLUCC 15-0448 and supported with a high bootstrap (98%) value (Fig. S2). Based on morphological and phylogenetic data, PV2-9 was identified as E. poaceicola. E. poaceicola has been collected from dead leaves of Poaceae sp. and described as a new species by Hyde et al. (2018). To our knowledge, this is the first report of E. poaceicola causing leaf spot on P. viridis in China. Further studies are needed to assess its impact on P. viridis, as well as monitor and control this disease.

Osmanthus fragrans is a popular ornamental tree species known for its fragrant flowers and widely cultivated in Asia, Europe, and North America. Anthracnose is a disastrous threat to the growth and development of O. fragrans and has caused significant economic losses. To reveal the potential pathogen diversity of anthracnose, 127 isolates of Colletotrichum were isolated from the symptomatic leaves. Morphological studies and multi-locus phylogenetic analyses with the concatenated sequences of internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), actin (ACT), beta-tubulin (TUB2), calmodulin (CAL), and the intergenic region between Apn2 and Mat1-2-1 (ApMat) as well as a pairwise homoplasy index test placed the causal fungi as two new species, Colletotrichum anhuiense (2 isolates) and C. osmanthicola (12 isolates), and three known taxa: C. fructicola (18 isolates), C. gloeosporioides (62 isolates), and C. karstii (33 isolates). Among them, C. gloeosporioides was the most dominant, and C. anhuiense was occasionally discovered from the host tissues. Pathogenicity test in vivo on O. fragrans leaves revealed a significant difference in virulence among these species. Of them, C. gloeosporioides, C. osmanthicola, and C. anhuiense were significantly more virulent than C. fructicola and C. karstii, while C. karstii was the least virulent. To our knowledge, this study was the first to report the pathogen diversity of anthracnose on O. fragrans.