Tea leaf spot, caused by the fungal phytopathogen Didymella segeticola, is an important foliar disease that can cause huge losses in the production and quality of tea, and there are no effective management measures to control the disease. This study screened a natural antimicrobial chemical for its activity against D. segeticola and studied its mode of action. Antifungal activity of the Streptomyces-derived antimicrobial zhongshengmycin (ZSM) against D. segeticola strain GZSQ-4 was assayed in vitro via the mycelial growth rate method. Optical microscopy and scanning and transmission electron microscopy were used to observe the morphological effects on hyphae treated with ZSM, with these studies complemented by transcriptomic, proteomic, and bioinformatic studies to identify the differentially expressed genes or differentially expressed proteins in hyphae treated with ZSM. Correlation analysis of transcriptomic and proteomic data were used to reveal the mode of action. The results indicated that ZSM could inhibit the growth of hyphae in vitro with a half-maximal effective concentration of 5.9 μg/ml, inducing some morphological changes in organelles, septa, and extracellular polysaccharides, targeting ribosomes to disturb translation, affecting the biosynthesis of some hyphal proteins at the messenger RNA and protein levels, and revealing correlations between findings from transcriptomes and proteomes.

Gray blight is a serious disease of tea (Camellia sinensis) for which there is currently no effective control or preventive measure apart from fungicides. Screening for effectiveness of a natural antimicrobial against this pathogen and identifying its mode of action could contribute to the management of this disease. Antifungal activity of the antimicrobial ningnanmycin (NNM) from Streptomyces noursei var. xichangensis against the pathogen causing gray blight disease, Pseudopestalotiopsis camelliae-sinensis 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 changes in hyphae of P. camelliae-sinensis treated with NNM. RNA sequencing, bioinformatics, and quantitative real-time PCR were used to identify genes in the hyphae that were differentially expressed in response to treatment with NNM. Thirty-eight genes from 16 pathways, known as targets of antifungal agents, were used to investigate gene expression in hyphae at the half-maximal effective concentration (EC₅₀), 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.

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.

Diseases caused by fungi can affect the quality and yield of the leaves of tea [Camellia sinensis (L.) Kuntze]. At present, the availability of highly effective and safe fungicides for controlling tea plants remains limited. The objectives of this study were to identify novel compounds with antifungal activities and to determine their molecular mechanisms. A series of sulfone compounds containing 1,3,4-oxadiazole were evaluated in China for their antifungal activities against several pathogens causing foliar diseases and high production losses. Transcriptomics and bioinformatics were used to analyze the differentially expressed genes of Lasiodiplodia theobromae treated with a representative compound, jiahuangxianjunzuo (JHXJZ). Moreover, the effects of JHXJZ on ergosterol content, membrane permeability, cell structure, and seven key genes involved in the ergosterol biosynthetic pathway were investigated. JHXJZ had a strong antifungal activity against L. theobromae in vitro, with an effective concentration giving 50% inhibition of 3.54 ± 0.55 μg/ml, and its curative efficacies on detached tea leaves reached 41.78% at 100 μg/ml. JHXJZ upregulated 899 genes (P < 0.05) and downregulated 1,185 genes (P < 0.05) in L. theobromae. These genes were found to be associated with carbohydrate metabolic processes, which are closely related to steroid biosynthesis in the Kyoto Encyclopedia of Genes and Genomes pathways. Because JHXJZ regulates the key genes of sterol biosynthesis, it decreased the ergosterol content, increased cell-membrane permeability, changed the cellular structure, enhanced the roughness of the surface of the hyphae, and resulted in degradation of the hyphal nuclei and necrosis of the hyphal cytoplasm. Our study demonstrates that JHXJZ is a fungicide with a novel mechanism of action that differs from that of triazole fungicides. JHXJZ has potential for applications in controlling tea plant diseases.[ ] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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 g...

Tea [Camellia sinensis (L.) Kuntze)] have been widely planted in Guizhou Province in recent years, and the cultivation area in the region ranks first among all the provinces or cities in China. Leaf spot disease was an important disease of tea in Kaiyang county, Guizhou Province, which mainly damaged young leaves and shoot of tea and led to a huge loss of the production of tea. The spots initially represented brown and round, and then the diameter of the spot was 4-6 mm during later period, with the color of the center in the spot changing white. Tea leaf spot disease always occurs in early spring and the region with 1300 m altitude. From 2016 to 2019, disease incidence of leaves was estimated at 84% to 92%, and the disease severity on a plant basis was determined to be 64% to 76%, depending on the field. To identify the causal agent of the foliar disease, pieces of the lesion margins were surface sterilized with 75% ethanol for 30 s, followed by 0.5% sodium hypochlorite for 5 min, rinsed with sterile water three times, plated on potato dextrose agar (PDA) and incubated in the dark at 25C for 3 to 5 d. The hyphal tips from the margins of the growing colonies were successively picked and transferred to fresh PDA plates to purify the isolates. The result indicated that the isolates on PDA represented initially round form, and white mycelium. The reverse sides of the isolates firstly displayed light yellow on PDA. Conidiophores represent dark brown, geniculate. Brown conidia, narrow ovoid, length: 22.9 ± 4.5 μm, width: 11.1 ± 1.7 μm, with 4 to 8 transverse septa and with conspicuously ornamented walls. The gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Berbee et al. 1999) and the Alternaria allergen 1 (Alt a1) (Hong et al. 2005) of three strains were amplified, sequenced and deposited in Genbank. Maximum parsimony phylogenetic analysis based on concatenated sequences of combined GAPDH (1-583) and Alt a 1 (588-1065) indicated that the strain AXLKY_2019_010 was identical to reference strain Alternaria longipes strain EGS 30-033, and the clade was supported by 96% bootstrap values. According to the Koch's postulate, the tea leaves were inoculated with PDA plugs with actively growing mycelia using the methods of the puncture, cut and unwound under the laboratory conditions and the natural conditions. Slight yellow spots were gradually formed after 2 d post-inoculation on the inoculated leaves, and the color of the center of the spot changed to be white. With the prolonging of inoculation time, the size of lesion represented to be slightly enlarged. PDA plugs without mycelia were used as a control, and the control group showed no symptoms. The same isolates were consistently reisolated from inoculated leaves. A. longipes can cause leaf blight of carrots in Israel (Vintal et al. 2002), leaf spot of potato in Pakistan (Shoaib et al. 2014) and leaf spot of Atractylodes macrocephala in China (Tan et al. 2012). To our knowledge, this is the first report of A. longipes causing leaf spot on tea in China and our findings will be useful for its management and for further research.