First Report of Epicoccum nigrum Causing Brown Leaf Spot in Tea in Guizhou Province, China

Abstract

Brown leaf spots were observed on tea [Camellia sinensis (L.) Kuntze] in Sinan County (27.74 °N, 108.35 °E) and Kaiyang County (27.96 °N, 107.34 °E), Guizhou Province, China, from 2018 to 2020. For the leaf spots with the typical symptoms, the disease incidence was estimated to range between 56% and 61%, respectively. The disease severity was estimated to range from 39 to 43 across 12 tea plantations, respectively. The disease initially occurred at the margins of leaf tips, and the lesions expanded gradually, being dark brown and irregularly shaped and became necrotic. To identify the causal organism, two leaves from each of 15 tea twigs, one or two per plantation, were detached from 8- or 10-year-old tea plants on each of 12 plantations. Samples taken from the lesion margins were sterilized with 75% ethanol followed by 0.5% NaOCl, placed on potato dextrose agar (PDA), and then incubated at 25oC in darkness for 5 days (Wang et al. 2020). For each sample, hyphal tips from the margin of a growing colony were successively transferred to fresh PDA, and pure cultures were obtained. Three representative strains were grown on PDA, malt extract agar (MEA), and oatmeal agar (OA) plates. The colonies had smooth margins and abundant mycelia on all three media, with the colony colors being from gray to light purple on PDA, white on MEA, and purplish-red on OA at 5 days post-inoculation. At 20 days post-inoculation on MEA, stromata began to gradually form, which were droplet-like, 100 to 2,000 μm in diameter, and semi-immersed on the medium’s surface. Black sporodochia were produced on the surfaces of stromata. Conidiophores were aggregated in sporodochia, densely compacted, and dark brown. Conidia were globose or pyriform, dark, multicellular, and measured 22.95 ± 3.59 × 19.82 ± 3.13 μm (n = 50) in diameter. The morphological characteristics of the mycelia and reproductive structures of the strains were identical to those of Epicoccum nigrum. The internal transcribed spacer (ITS) region of rDNA, and the partial 28S large subunit rDNA (LSU), RNA polymerase II second largest subunit (RPB2), and beta-tubulin (TUB) genes of these strains were amplified using the primers V9G/ITS4 (De Hoog and Gerrits van den Ende 1998; White et al. 1990), LR0R/LR5 (Rehner and Samuels 1994), RPB2-5F2/fRPB2-7cR (Sung et al. 2007), and TUB2Fd/TUB4Rd (Woudenberg et al. 2009), respectively, and deposited in GenBank (accession no. MW646378, MW291537, MW602293, and MW602295 for ITS, LSU, RBP2, and TUB, respectively). A maximum parsimony phylogenetic analysis indicated that the representative strains clustered with E. nigrum CBS 173.73 (Chen et al. 2017). Pathogenicity tests were performed on 5-year-old potted tea and on 10-year-old C. sinensis cv. Fuding-dabaicha in the field. Mycelial plugs (6-mm diam.) and a conidial suspension (106 conidial/mL) were applied on punctured leaves using a sterile needle and non-punctured leaves. Inoculation with only a PDA plug or sterile water served as controls. Brown spots appeared on the wounded sites of tea leaves at 2 days post-inoculation. No symptoms were observed on the non-wounded leaves or wounded leaves inoculated with PDA plugs lacking mycelia. The re-isolated pathogen from diseased plants was identical to the purified strain ACCC39731 used for inoculation, with re-isolation frequency being 85.0%. To our knowledge, this is the first report of E. nigrum causing leaf spot on tea plants in China, and our findings will be useful for its management and further research.

Isolation and identification of Epicoccum nigrum as the causal agent of brown spot disease in Solanum tuberosum in China

“…The disease symptoms caused by representative strain DXPH showed necrotic lesions at the wound site on wounded leaves and dark brown lesions at the leaf margins of unwounded leaves, which may result from the germinating conidia penetrating host plants through wounds and stomata, and eventually facilitating the development of disease (Allen et al, 1983). However, a previous report indicated that E. nigrum isolated from tea leaves could only cause brown spots at the wounded sites of tea leaves (Yin et al, 2022), which may indicate differences in susceptibility of different cultivars or the pathogenic diversity of E. nigrum isolated from different host plants. The obvious brown spots on potato leaves in this pathogenicity test occurred at 4 days postinoculation (dpi), which was earlier than those on loquat leaves (10 dpi; Wu et al, 2017).…”

“…The disease symptoms, on the other hand, were similar to those caused by Epicoccum sp. (Yin et al, 2022). Subsequently, based on the standards of conidial germination described by Pascual et al (2003), the collected black dots generated abundant conidia, which germinated and produced germ tubes in PDB medium after 7 h. In general, the germination of spores indicated that the organism had the capacity for transmission and re‐infection (Palma‐Guerrero et al, 2008).…”

“…Multigene phylogenetic analysis is often conducted to identify a species within a genus (Suleimanova et al, 2021). In the case of E. nigrum , the ITS, Tub2 , LSU and RPB2 genes have been commonly used for identification (Yin et al, 2022). In this study, the ITS, RPB2 and Tub2 genes were used to identify the pathogenic isolate DXPH, and BLAST results showed that DXPH sequences were 100%, 100% and 99% identical to the sequences of E. nigrum (CBS 140523), and a multilocus phylogenetic tree further confirmed this conclusion.…”

“…It can also inhibit the development of the sugarcane pathogens F. verticilioides , Xanthomomas albilineans , Ceratocystis paradoxa and Colletotrichum falcatum in vitro by increasing the root system biomass and antifungal compound production of host plants (Favaro et al, 2012). In addition, it can cause leaf spots on cowpea (Deepika et al, 2020), loquat (Wu et al, 2017) and tea (Yin et al, 2022), fruit rot on deciduous holly (Lin et al, 2018) and three cultivated dragon fruits (Taguiam et al, 2020) and seed disease of pea (Wilman et al, 2014) as a pathogen. E. nigrum may therefore have opposite effects in agricultural production, as an aggressive disease or a biological control agent.…”

“…Generally, the disease symptoms caused by E. nigrum on plant leaves are dark brown lesions, which expand from the margin of leaf tips and eventually become necrotic (Yin et al, 2022). Recently, a new brown spot disease on the margins of potato leaves was discovered in Dingxi, Gansu Province, China, and some black dots appeared on both upper and lower surfaces of diseased leaves, similar to those described above for E. nigrum .…”