In Addition to Damaging the Plasma Membrane, Phenolic Monoterpenoid Carvacrol Can Bind to the Minor Groove of DNA of Phytopathogenic Fungi to Potentially Control Tea Leaf Spot Caused by Lasiodiplodia theobromae.

Carvacrol expresses a wide range of biological activities, but the studies of its mechanisms focused on bacteria, mainly involving the destruction of the plasma membrane. In this study, carvacrol exhibited strong activities against several phytopathogenic fungi and demonstrated a novel antifungal mechanism against Lasiodiplodia theobromae. RNA sequencing indicated that many genes of L. theobromae hyphae were predominately induced by carvacrol, particularly those involved in replication and transcription. Hyperchromic, hypsochromic, and bathochromic effects in the UV-visible absorption spectrum were observed following titration of calf thymus DNA (ctDNA) and carvacrol, which indicated the formation of a DNA-carvacrol complex. Circular dichroism (CD) spectroscopy indicated that the response of DNA to carvacrol was similar to that of 4',6-diamidino-2-phenylindole (DAPI) but different from that of ethidium bromide (EB), implying the ionic bonds between carvacrol and ctDNA. Fluorescence spectrum (FS) analysis indicated that carvacrol quenched the fluorescence of double-stranded DNA (dsDNA) more than single-stranded DNA, indicating that carvacrol mainly bound to dsDNA. A displacement assay showed that carvacrol reduced the fluorescence intensity of the DNA-DAPI complex through competition with DAPI, but this did not occur for DNA-EB. The FS assay revealed that carvacrol bound to the AAA sequence on the minor groove of ds-oligonucleotides. The hydroxyl of carvacrol was verified to bind to ctDNA through a comparative test in which structural analogs of carvacrol, including thymol and 4-ethyl-1,2-dimethyl, were analyzed. The current study indicated carvacrol can destruct plasma membranes and bind to the minor groove of DNA, inhibiting fungal proliferation by disturbing the stability of dsDNA.

Integrated Transcriptome and Proteome Analysis Reveals that the Antimicrobial Griseofulvin Targets Didymella segeticola Beta-Tubulin to Control Tea Leaf Spot.

Because effective control measures are lacking, tea leaf spot caused by Didymella segeticola results in huge tea (Camellia sinensis) production losses on tea plantations in Guizhou Province, southwestern China. Screening for natural antimicrobial agents with higher control effects against this pathogen and studying their modes of action may contribute to disease management. Here, Penicillium griseofulvum-derived antimicrobial griseofulvin (GSF) can inhibit the hyphal growth of D. segeticola strain GZSQ-4, with a half-maximal effective concentration of 0.37 µg/ml in vitro and a higher curative efficacy at a lower dose of 25 µg/ml for detached tea twigs. GSF induces deformed and slightly curly hyphae with enlarged ends, with protoplasts agglutinated in the hyphae, and higher numbers of hyphal protuberances. GSF alters hyphal morphology and the subcellular structure's order. The integrated transcriptome and proteome data revealed that the transport of materials in cells, cellular movement, and mitosis were modulated by GSF. Molecular docking indicated that beta-tubulin was the most potent target of GSF, with a binding free energy of -13.59 kcal/mol, and microscale thermophoresis indicated that the dissociation constant (Kd) value of GSF binding to beta-tubulin 1, compared with beta-tubulin 2, was significantly lower. Thus, GSF potentially targets beta-tubulin 1 to disturb the chromosomal separation and fungal mitosis, thereby inhibiting hyphal growth.

Sequencing, Functional Annotation, and Interaction Prediction of mRNAs and Candidate Long Noncoding RNAs Originating from Tea Leaves During Infection by the Fungal Pathogen Causing Tea Leaf Spot, Didymella bellidis.

Tea leaf spot caused by Didymella bellidis can seriously reduce the productivity and quality of tea (Camellia sinensis var. sinensis) leaves in Guizhou Province, southwest China. Analysis of the relationship between messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) of tea could provide insights into the plant-pathogen interaction. In this study, high-throughput sequencing of mRNAs and lncRNAs from tea leaves during infection by D. bellidis was conducted using the Illumina Novaseq 6000 platform. Infection by D. bellidis hyphae resulted in up- or downregulation of 553 and 191 of the differentially expressed mRNAs (DEmRNAs), respectively. As the S gene number (total number of genes with significantly differential expression annotated in the specified Gene Ontology [GO] database), three were enriched with respect to the defense response to the fungus at the biological process level. Expression of the DEmRNAs peroxidase 21 (TEA000222.1) and mcht-2 (TEA013240.1) originating from tea leaves were upregulated during challenge by D. bellidis hyphae, whereas expression of the LRR receptor-like serine/threonine-protein kinase ERECTA (TEA016781.1) gene was downregulated. The infection of D. bellidis hyphae resulted in up- or downregulation of 227 and 958 of the differentially expressed lncRNAs (DElncRNAs). The DEmRNAs associated with uncharacterized LOC101499401 (TEA015626.1), uncharacterized protein (TEA014125.1), structural maintenance of chromosomes protein 1 (TEA001660.1), and uncharacterized protein (TEA017727.1) occurred as a result of cis regulation by DElncRNAs MSTRG.20036, MSTRG.3843, MSTRG.26132, and MSTRG.56701, respectively. The expression profiling and lncRNA/mRNA association prediction in the tea leaves infected by D. bellidis will provide a valuable resource for further research into disease resistance.

Metabolome and transcriptome profiling unveil the mechanisms of light-induced anthocyanin synthesis in rabbiteye blueberry (vaccinium ashei: Reade).

BACKGROUND: Blueberry is one of the most important fruit crops worldwide. Anthocyanin is an important secondary metabolites that affects the appearance and nutritive quality of blueberries. However, few studies have focused on the molecular mechanism underlying anthocyanin accumulation induced by light intensity in blueberries. RESULTS: The metabolic analysis revealed that there were 134 significantly changed metabolites in the natural light compared to the control, and flavone, flavonol, and anthocyanins were the most significantly increased. Transcriptome analysis found 6 candidate genes for the anthocyanin synthesis pathway. Quantitative reverse transcription PCR (qRT-PCR) results confirmed changes in the expression levels of genes encoding metabolites involved in the flavonoid synthesis pathways. The flavonoid metabolic flux in the light intensity-treatment increased the accumulation of delphinidin-3-O-arabinoside compared to under the shading-treatment. Furthermore, we performed qRT-PCR analysis of anthocyanin biosynthesis genes and predicted that the gene of VcF3'5'H4 may be a candidate gene for anthocyanin accumulation and is highly expressed in light intensity-treated fruit. Through the co-expression analysis of transcription factors and anthocyanin synthesis pathway genes, we found that the VcbHLH004 gene may regulate VcF3'5'H4, and then we transformed VcbHLH004 heterologously into tomato to verify its function. CONCLUSION: These results provide novel insights into light intensity regulation of blueberry anthocyanin accumulation and represent a valuable data set to guide future functional studies and blueberry breeding.

Multi-Omics Analysis Reveals that the Antimicrobial Kasugamycin Potential Targets Nitrate Reductase in Didymella segeticola to Achieve Control of Tea Leaf Spot.

Because of the lack of effective disease management measures, tea leaf spot-caused by the fungal phytopathogen Didymella segeticola (syn. Phoma segeticola)-is an important foliar disease. The important and widely used agricultural antimicrobial kasugamycin (Ksg), produced by the Gram-positive bacterium Streptomyces kasugaensis, effects high levels of control against crop diseases. The results of this study indicated that Ksg could inhibit the growth of D. segeticola hyphae in vitro with a half-maximal effective concentration (EC50) of 141.18 µg ml-1. Meanwhile, the curative effect in vivo on the pathogen in detached tea leaves also demonstrated that Ksg induced some morphological changes in organelles, septa, and cell walls as observed by optical microscopy and by scanning and transmission electron microscopy. This may indicate that Ksg disturbs biosynthesis of key metabolites, inhibiting hyphal growth. Integrated transcriptomic, proteomic, and bioinformatic analyses revealed that differentially expressed genes or differentially expressed proteins in D. segeticola hyphae in response to Ksg exposure were involved with metabolic processes and biosynthesis of secondary metabolites. Molecular docking studies indicated that Ksg may target nitrate reductase (NR), and microscale thermophoresis assay showed greater affinity with NR, potentially disturbing nitrogen assimilation and subsequent metabolism. The results indicated that Ksg inhibits the pathogen of tea leaf spot, D. segeticola, possibly by binding to NR, disturbing fungal metabolism, and inducing subsequent changes in hyphal growth and development.

Functional Annotation of circRNAs in Tea Leaves After Infection by the Tea Leaf Spot Pathogen, Lasiodiplodia theobromae.

Tea leaf spot, caused by Lasiodiplodia theobromae, is an important disease that can seriously decrease the production and quality of tea (Camellia sinensis (L.) O. Kuntze) leaves. The analysis of circular RNA (circRNA) in tea leaves after infection by the pathogen could improve understanding about the mechanism of host-pathogen interactions. In this study, high-performance sequencing of circRNA from C. sinensis Fuding-dabaicha leaves that had been infected with L. theobromae was conducted using the Illumina HiSeq 4000 platform. In total, 192 and 153 differentially expressed circRNAs from tea leaves were significantly up- and downregulated, respectively, after infection with L. theobromae. A gene ontology analysis indicated that the differentially expressed circRNA-hosting genes for DNA binding were significantly enriched. The genes with significantly differential expressions that were annotated in the specified database (S genes) were σ factor E isoform 1, triacylglycerol lipase SDP1, DNA-directed RNA polymerase III subunit 2, WRKY transcription factor WRKY24, and regulator of nonsense transcripts 1 homolog. A Kyoto Encyclopedia of Genes and Genomes analysis indicated that the significantly enriched circRNA-hosting genes involved in the plant-pathogen interaction pathway were Calmodulin-domain protein kinase 5 isoform 1, probable WRKY transcription factor 33, U-box domain-containing protein 35, probable inactive receptor-like protein kinase At3g56050, WRKY transcription factor WRKY24, mitogen-activated protein kinase kinase kinase YODA, SGT1, and protein DGS1. Functional annotation of circRNAs in tea leaves infected by L. theobromae will provide a valuable resource for future research on host-pathogen interactions.

Two effective clinical prediction models to screen for obstructive sleep apnoea based on body mass index and other parameters.

BACKGROUND AND OBJECTIVE: The diagnosis of obstructive sleep apnea (OSA) relies on polysomnography which is time-consuming and expensive. We therefore aimed to develop two simple, non-invasive models to screen adults for OSA. METHODS: The effectiveness of using body mass index (BMI) and a new visual prediction model to screen for OSA was evaluated using a development set (1769 participants) and confirmed using an independent validation set (642 participants). RESULTS: Based on the development set, the best BMI cut-off value for diagnosing OSA was 26.45 kg/m2, with an area under the curve (AUC) of 0.7213 (95% confidence interval (CI), 0.6861-0.7566), a sensitivity of 57% and a specificity of 78%. Through forward conditional logistic regression analysis using a stepwise selection model developed from observed data, seven clinical variables were evaluated as independent predictors of OSA: age, BMI, sex, Epworth Sleepiness Scale score, witnessed apnoeas, dry mouth and arrhythmias. With this new model, the AUC was 0.7991 (95% CI, 0.7668-0.8314) for diagnosing OSA (sensitivity, 75%; specificity, 71%). The results were confirmed using the validation set. A nomogram for predicting OSA was generated based on this new model using statistical software. CONCLUSIONS: BMI can be used as an indicator to screen for OSA in the community. We created an internally validated, highly distinguishable, visual and parsimonious prediction model comprising BMI and other parameters that can be used to identify patients with OSA among outpatients. Use of this prediction model may help to improve clinical decision-making.

First Report of Stagonosporopsis caricae Causing Chayote Leaf Spot in Guizhou Province, China.

At present, chayote (Sechium edule (Jacq.) Swartz) have been widely planted in Guizhou Province, southwestern China, and the cultivation area in Huishui county ranks first among all the counties or cities in Guizhou Province. Chayote leaf spot was firstly observed in Huishui County (25.99°N, 106.64°E) from April to June in 2019. The disease incidence ranged from 52% to 58%, and the severity of leaf symptoms ranged from 34 to 41% across nine chayote plantations. Such levels disease development lead to considerable enocomic losses. Leaf lesions initially occurred at the leaf margins, and the lesions expanded gradually, becoming dark brown and irregularly shaped. To identify the leaf spot-associated pathogen, the samples were cut from lesion margins, sterilized with 75% ethanol followed by 0.5% sodium hypochlorite for 30 s, rinsed with sterile water three times, and transferred onto potato dextrose agar (PDA). They were then incubated at 25°C in darkness for 5 days. The hyphal tips from the margins of growing colonies were successively transferred to fresh PDA plates for obtaining isolates. All strains grew with a similar morphology on PDA, malt extract agar (MEA), and oatmeal agar (OA) plates, and the colonies presented smooth margins and abundant mycelia on all three media. The colonies were gray to light green on PDA and gray on MEA and OA at 5 days post-inoculation. At 11 days post-inoculation on 10% V8 medium at 25oC with a cycle of 14 h/ultraviolet light and 10 h/night, sexual morph was observed, ascomata pseudothecioid, subglobose, 121 × 142 µm, ostiolate, walls of brown textura angularis, and smooth. Asci were bitunicate, cylindrical to clavate, 7 × 90 µm, 8-spored, ascospores elliptical, straight to slightly curved, 5 × 17 µm, 1-septate, constricted at the septum, sub-hyaline, and smooth. Conidiomata were pycnidial, subglobose, 166 × 258 µm, ostiolate, wall of dark brown to black textura angularis, smooth. Conidia were short, cylindrical or slightly reniform, 6.18 ± 0.67 × 3.51 ± 0.33 µm (n = 50), 0-1 septate, hyaline, smooth. Chlamydospores were subhyaline to dark brown, verruculose or incidentally tuberculate, and solitary or in chains, and 14.16 ± 1.23 × 5.92 ± 0.49 µm (n = 50). The morphological characteristics of the strains were identical to those of Stagonosporopsis caricae (Aveskamp et al. 2010; Sivanesan 1990). The genes or DNA sequences of the partial 28S large subunit rDNA, the internal transcribed spacer, RNA polymerase II second largest subunit, and beta-tubulin were amplified (Liu et al. 1999; Rehner and Samuels 1994; Sung et al. 2007; Vilgalys and Hester, 1990; White et al. 1990; Woudenberg et al. 2009). The sequences were further deposited in GenBank (ITS: MZ619042-MZ619044, LSU: MZ620651-MZ620653, RPB2: MZ673652-MZ673654, and TUB: MZ673649-MZ673651). A phylogenetic analysis confirmed these strains to be identical to S. caricae reference strains CBS 248.90, CBS 282.76, and PD 06/03082531. Pathogenicity tests were performed on potted chayote and five-year-old chayote in the field. Mycelial plugs (6 mm diameter) were applied on wounded chayote leaves. Brown spots appeared on the wounded sites of chayote leaves after inoculation with mycelial plugs. No symptoms were observed on the leaves inoculated with PDA plugs lacking mycelia. The re-isolated pathogen from diseased plants was identical to the representative strains used for inoculation. To our knowledge, this is the first report of S. caricae causing leaf spot on chayote in China, and our findings will be useful for its management and further research.

The Sequence and Integrated Analysis of Competing Endogenous RNAs Originating from Tea Leaves Infected by the Pathogen of Tea Leaf Spot, Didymella segeticola.

Tea leaf spot, caused by Didymella segeticola, is an important disease which negatively affects the productivity and the quality of tea leaves. During infection by the pathogen, competing endogenous RNAs (ceRNAs) from tea leaves could contribute to achieving pathogenicity. In this study, circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs), constituting ceRNAs, which share binding sites on microRNAs (miRNAs), and messenger RNAs (mRNAs) from infected and uninfected leaves of tea (Camellia sinensis 'Fuding-dabaicha') were sequenced and analyzed, and the identity and expression levels of the target genes of miRNA-mRNA and miRNA-lncRNA/circRNA were predicted. Analysis indicated that 10 mRNAs were bound by 20 miRNAs, 66 lncRNAs were bound by 40 miRNAs, and 17 circRNAs were bound by 29 miRNAs, respectively. For the regulation modes of ceRNAs, five ceRNA pairs were identified by the correlation analysis of lncRNA-miRNA-mRNA. For instance, expression of the xyloglucan endotransglycosylase gene in infected leaves was downregulated at the level of mRNA through miRNA PC-5p-3511474_3 binding with lncRNA TEA024202.1:MSTRG.37074.1. Gene annotation indicated that expression of this gene was significantly enriched in cell wall biogenesis and in the pathway of plant hormone signal transduction. The functional analysis of ceRNAs isolated from infected tea leaves will provide a valuable resource for future research on D. segeticola pathogenicity.

Integrated mRNA and Small RNA Sequencing for Analyzing Leaf Spot Pathogen Didymella segeticola and Its Host, Tea (Camellia sinensis), During Infection.

Leaf spot on tea plants (Camellia sinensis [L.] Kuntze), caused by the fungus Didymella segeticola (Q. Chen) Q. Chen, Crous & L. Cai (syn. Phoma segeticola), negatively affects the productivity and quality of tea leaves in Guizhou Province, China. Although the genome sequence of D. segeticola has been published, no data on the transcriptome or microRNAs (miRNAs) of the pathogen or host during infection are available. Here, we report on the high-quality transcriptome and miRNA sequences of both D. segeticola and tea during infection, using the Illumina HiSeq 4000 or HiSeq 2500 platforms. Comprehensive expression profiling of the fungal pathogen and its host will provide a resource for future research into trait-specific genes of the pathogen and the host as well as on host-pathogen interactions and on disease resistance mechanisms.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Novel Sulfone Derivative Controls Lasiodiplodia theobromae in Tea Leaf Spot by Reducing the Ergosterol Content.

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.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Integrated mRNA and Small RNA Sequencing for Analyzing Tea Leaf Spot Pathogen Lasiodiplodia theobromae, Under In Vitro Conditions and the Course of Infection.

Lasiodiplodia theobromae is a phytopathogenic fungus, which can cause many different diseases on different crops. The pathogen can cause leaf spot on tea plants (Camellia sinensis), which negatively affects the productivity and quality of tea leaves in tea plantations in Guizhou Province, China. Although the genome sequence of L. theobromae has been published, no data on the transcriptome or small RNA sequences of L. theobromae under in vitro conditions and the course of infection of tea leaf are available. Here, we report the high-quality transcriptome and small RNA sequences of L. theobromae in vitro conditions and the course of infection of tea leaf using the platform of Illumina HiSeq. This comprehensive expression profiling of the fungal pathogen will provide a valuable resource for future research on trait-specific genes of the pathogen, host-pathogen interactions, and disease resistance in the host.

Integration of Transcriptomic and Proteomic Data Reveals the Possible Action Mechanism of the Antimicrobial Zhongshengmycin Against Didymella segeticola, the Causal Agent of Tea Leaf Spot.

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.

Antifungal Activity and Possible Mode of Action of Ningnanmycin Against Tea Gray Blight Disease Pathogen Pseudopestalotiopsis camelliae-sinensis.

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.

Whole Genome Sequence and Gene Annotation Resource for Didymella bellidis Associated with Tea Leaf Spot.

Didymella bellidis is a phytopathogenic fungus that causes leaf spot on tea plants (Camellia sinensis), which negatively affects the productivity and quality of tea leaves in Guizhou Province, China. D. bellidis isolate GZYQYQX2B was sequenced using Pacific Biosciences and Illumina technologies, and assembled into a whole genome of 35.5 Mbp. Transcripts of D. bellidis isolate GZYQYQX2B were predicted from the assembled genome and were further validated by RNA sequence data. In total, 10,731 genes were predicted by integrating three approaches, namely ab initio and homology-based gene prediction, as well as transcriptomics data. The whole-genome sequence of D. bellidis will provide a resource for future research on trait-specific genes of the pathogen and host-pathogen interactions.

First report of Alternaria longipes causing leaf spot on tea in China.

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.

Emergency decision-making model of environmental emergencies based on case-based reasoning method.

Environmental emergencies are characterized by high uncertainty, complex evolution, and potential for serious damage, thus posing enormous pressure and difficulties to the emergency responses of enterprises and governments. Improving the efficiency and quality of emergency decision-making constitutes the primary focus of today's research in this field. This study systematically analyzes the scenario evolution mechanism of environmental emergencies with a multi-dimensional scenario space method, and key scenario factors are identified from disaster-inducing factors, disaster-bearing factors, disaster-pregnant environments, and emergency actions. Based on these, an emergency decision-making model for environmental emergencies (EEEDM) is constructed based on case-based reasoning (CBR). First, different matching algorithms are designed for accurate numerical data, fuzzy semantic data, and symbolic data. The similarity between the target scenario and the historical scenario is calculated, and the historical scenario similarity set is built according to the given threshold value. Finally, the emergency action plan of the scenario is modified with its utility value evaluated. A solution that applies to the target scenario is then obtained. Additionally, the decision-making model proposed in this paper is validated by an example of environmental emergencies. The results show that this model is scientific and reasonable, and it can better realize the multi-dimensional expression and fast matching of the scenarios and meet the decision requirements of "scenario-response". In practice, the model is capable of providing support for relevant departments' emergency decision-making.

Whole Genome Sequences of the Tea Leaf Spot Pathogen Didymella segeticola.

The fungal pathogen Didymella segeticola (basionym Phoma segeticola) causes leaf spot on tea (Camellia sinensis), which leads to a loss in tea leaf production in Guizhou Province, China. D. segeticola isolate GZSQ-4 was sequenced using Illumina HiSeq and Pacific Biosciences (PacBio) RS technologies, and then assembled to approximately 33.4 Mbp with a scaffold N50 value of approximately 2.3 Mbp. In total, 10,893 genes were predicted using the Nonredundant, Gene Ontology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes, and SWISS-PROT databases. The whole-genome sequence of D. segeticola will provide a resource for future research on host-pathogen interactions, determination of trait-specific genes, pathogen evolution, and plant-host adaptation mechanisms.

Correlation of sleep microstructure with daytime sleepiness and cognitive function in young and middle-aged adults with obstructive sleep apnea syndrome.

PURPOSE: To compare microstructural features of sleep in young and middle-aged adults with differing severities of obstructive sleep apnea syndrome (OSAS), and to investigate the relationship between sleep microstructural fragmentation and cognitive impairment, as well as daytime sleepiness, in these patients. METHODS: A total of 134 adults with snoring (mean age, 37.54 ± 7.66 years) were classified into four groups based on apnea-hypopnea index: primary snoring, mild OSAS, moderate OSAS, and severe OSAS. Overnight polysomnography was performed to assess respiratory, sleep macrostructure (N1, N2, N3, and R), and sleep microstructure (arousal, cyclic alternating pattern [CAP]) parameters. Cognitive function and daytime sleepiness were assessed using Montreal Cognitive Assessment (MoCA) and Epworth Sleepiness Scale (ESS). RESULTS: As OSAS severity increased, MoCA gradually decreased and ESS gradually increased. N1%, N2%, and N3% sleep were significantly different between the severe OSAS group and the primary snoring, mild OSAS, and moderate OSAS groups (all P < 0.05). Overall arousal index, respiratory-related arousal index, CAP time, CAP rate, phase A index, number of CAP cycles, and phase A average time differed significantly in the moderate and severe OSAS groups compared with the mild OSAS and primary snoring groups (all P < 0.05). The strongest correlations identified by stepwise multiple regression analysis were between phase A3 index and the MoCA and ESS scores. CONCLUSIONS: Sleep microstructure exhibited significant fragmentation in patients with moderate and severe OSAS, which was associated with decreased MoCA and increased ESS scores. This suggests that phase A3 index is a sensitive indicator of sleep fragmentation in OSAS.