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.

Gray blight disease is a serious disease of tea (Camellia sinensis (L.) Kuntz), for which there is currently no effective control or preventative measure apart from fungicides. Screening for effectiveness of a natural anti-microbial against this pathogen and identifying its mode of action could contribute to the management of this disease. Antifungal activity of the anti-microbial Ningnanmycin (NNM) from Streptomyces noursei var. xichangensis against the pathogen causing gray blight disease, Pseudopestalotiopsis camelliae-sinensis (F. Liu & L. Cai) strain GZHS-2017-010 was confirmed in vitro by the mycelial growth rate method. Optical microscopy, scanning electron microscopy and transmission electron microscopy were used to observe morphological change of hyphae of Ps. camelliae-sinensis treated with NNM. RNA sequencing, bioinformatics and quantitative real-time PCR were used to identify genes in the hyphae which were differentially expressed in response to treatment with NNM. Thirty-eight genes from sixteen pathways, known as targets of antifungal agents, were used to investigate gene expression in hyphae at the half-maximal effective concentration (EC50) dosage, EC30, and EC70 for 1, 7, or 14 h. The results indicated that NNM can inhibit the growth of hyphae in vitro, with an EC50 of 75.92 U/mL, inducing morphological changes in organelles, septa, and extracellular polysaccharides, targeting ribosomes to disturb translation in protein synthesis, and influencing some biosynthetic functions of the hyphae.

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D. segeticola is a significant plant pathogen that causes leaf spot on tea (Camellia sinensis) in Guizhou Province, China, leading to significant losses in production (Zhao et al. 2018). Although genome sequences of D. segeticola have been published (Ren et al. 2019), no data of transcriptome and microRNA of the pathogen and host during infection are available. Here, we report the high-quality transcriptome and microRNA of D. segeticola infection using the platform of Illumina Hiseq 4000 or Illumina Hiseq 2500. The number of annotated unigenes is 10894 and 33932 at several databases for mycelia and tea. PC-3p-9306445_2, nta-MIR6149a-p3_2ss13GA18CG, mdm-MIR535a-p3_2ss13GA18CG, cst-MIR11334-p3_2ss13GA18CG, et al can target more gene, with the number of the target genes being 31, 21, 16 and 11. The sequence of transcriptome and microRNA of D. segeticola will provide an important resource for researchers studying pathogenic mechanism and disease resistance.

Gray blight disease is one of the most destructive diseases of tea plants and occurs widely in the tea-growing areas of the world. It is caused by several fungal phytopathogens, of which Pseudopestalotiopsis camelliae-sinensis is the main pathogen in China. The environmentally friendly antimicrobial, phenazine-1-carboxylic acid (PCA), a metabolite of the natural soil-borne bacteria Pseudomonas spp., can inhibit a range of fungal crop diseases. In this study, we determined that PCA was active against Ps. camelliae-sinensis in vitro. We studied the mode of action of PCA on hyphae using a microscopic investigation, transcriptomics, biochemical methods, and molecular docking. The results of scanning and transmission electron microscopy indicated that PCA caused developmental deformity of mycelia and organelle damage, and it significantly decreased the accumulation of exopolysaccharides on the hyphal surface. The transcriptome revealed that 1705 and 1683 differentially expressed genes of Ps. camelliae-sinensis treated with PCA were up-regulated or down-regulated, respectively, with genes associated with ribosome biogenesis, oxidative phosphorylation, and encoding various proteins of N-glycan biosynthesis being significantly up-regulated. Up-regulation of nine genes related to N-glycan biosynthesis of Ps. camelliae-sinensis in response to PCA treatment was confirmed by reverse transcription qPCR. The enzymatic activity of catalase and superoxide dismutase of hyphae was significantly decreased by PCA treatment. Our results indicated that exposure to PCA resulted in expression changes in oxidoreductase genes, accumulation of reactive oxygen species, and decreased activity of catalase, with concomitant damage to the fungal cell membrane and cell wall.