Integrated enhancer regulatory network by enhancer-promoter looping in gastric cancer.

Enhancer cis-regulatory elements play critical roles in gene regulation at many stages of cell growth. Enhancers in cancer cells also regulate the transcription of oncogenes. In this study, we performed a comprehensive analysis of long-range chromatin interactions, histone modifications, chromatin accessibility and expression in two gastric cancer (GC) cell lines compared to normal gastric epithelial cells. We found that GC-specific enhancers marked by histone modifications can activate a population of genes, including some oncogenes, by interacting with their proximal promoters. In addition, motif analysis of enhancer-promoter interacting enhancers showed that GC-specific transcription factors are enriched. Among them, we found that MYB is crucial for GC cell growth and activated by the enhancer with an enhancer-promoter loop and TCF7 upregulation. Clinical GC samples showed epigenetic activation of enhancers at the MYB locus and significant upregulation of TCF7 and MYB, regardless of molecular GC subtype and clinicopathological factors. Single-cell RNA sequencing of gastric mucosa with intestinal metaplasia showed high expression of TCF7 and MYB in intestinal stem cells. When we inactivated the loop-forming enhancer at the MYB locus using CRISPR interference (dCas9-KRAB), GC cell growth was significantly inhibited. In conclusion, we identified MYB as an oncogene activated by a loop-forming enhancer and contributing to GC cell growth.

The Identification and Role of the Key Mycotoxin of Pestalotiopsis kenyana Causing Leaf Spot Disease of Zanthoxylum schinifolium.

Leaf spot is a common disease of Zanthoxylum schinifolium (Z. schinifolium), which can seriously harm the plant's ability to grow, flower, and fruit. Therefore, it is important to identify the mechanism of leaf spot caused by Pestalotiopsis kenyana (P. kenyana) for thorough comprehension and disease control. In this study, to verify whether the mycotoxins produced by P. kenyana cause leaf spot disease, the best medium for P. kenyana, namely PDB, was used. The mycotoxins were determined by ammonium sulfate precipitation as non-protein substances. The crude mycotoxin of P. kenyana was prepared, and the optimal eluent was eluted with petroleum either/ethyle acetate (3:1, v/v) and purified by silica gel column chromatography and preparative high-performance liquid chromatography to obtain the pure mycotoxins PK-1, PK-2, and PK-3. The PK-3 had the highest toxicity to Z. schinifolium, which may be the primary mycotoxin, according to the biological activity test using the spray method. The physiological and biochemical indexes of Z. schinifolium plants treated with PK-3 mycotoxin were determined. Within 35 days after mycotoxin treatment, the results showed that the protein content and malondialdehyde content of leaves increased over time. The soluble sugar and chlorophyll content decreased over time. The superoxide dismutase activity and catalase activity of the leaves increased first and then decreased, and the above changes were the same as those of Z. schinifolium inoculated with the spore suspension of the pathogen. Therefore, it is believed that the mycotoxin pestalopyrone could be a virulence factor that helps P. kenyana induce the infection of Z. schinifolium. In this study, the pathogenic mechanism of Z. schinifolium leaf spot was discussed, offering a theoretical foundation for improved disease prevention and control.

Integrated Transcriptome and Metabolome Analysis Reveals the Molecular Mechanism of Rust Resistance in Resistant (Youkang) and Susceptive (Tengjiao) Zanthoxylum armatum Cultivars.

Chinese pepper rust is a live parasitic fungal disease caused by Coleosporium zanthoxyli, which seriously affects the cultivation and industrial development of Z. armatum. Cultivating and planting resistant cultivars is considered the most economical and environmentally friendly strategy to control this disease. Therefore, the mining of excellent genes for rust resistance and the analysis of the mechanism of rust resistance are the key strategies to achieve the targeted breeding of rust resistance. However, there is no relevant report on pepper rust resistance at present. The aim of the present study was to further explore the resistance mechanism of pepper by screening the rust-resistant germplasm resources in the early stage. Combined with the analysis of plant pathology, transcriptomics, and metabolomics, we found that compared with susceptible cultivar TJ, resistant cultivar YK had 2752 differentially expressed genes (DEGs, 1253 up-, and 1499 downregulated) and 321 differentially accumulated metabolites (DAMs, 133 up- and 188 down-accumulated) after pathogen infection. And the genes and metabolites related to phenylpropanoid metabolism were highly enriched in resistant varieties, which indicated that phenylpropanoid metabolism might mediate the resistance of Z. armatum. This finding was further confirmed by a real-time quantitative polymerase chain reaction analysis, which revealed that the expression levels of core genes involved in phenylpropane metabolism in disease-resistant varieties were high. In addition, the difference in flavonoid and MeJA contents in the leaves between resistant and susceptible varieties further supported the conclusion that the flavonoid pathway and methyl jasmonate may be involved in the formation of Chinese pepper resistance. Our research results not only help to better understand the resistance mechanism of Z. armatum rust but also contribute to the breeding and utilization of resistant varieties.

Scoping Meta-Review of Methods Used to Assess Artificial Intelligence-Based Medical Devices for Heart Failure.

Artificial intelligence and machine learning (AI/ML) are playing increasingly important roles, permeating the field of medical devices (MDs). This rapid progress has not yet been matched by the Health Technology Assessment (HTA) process, which still needs to define a common methodology for assessing AI/ML-based MDs. To collect existing evidence from the literature about the methods used to assess AI-based MDs, with a specific focus on those used for the management of heart failure (HF), the International Federation of Medical and Biological Engineering (IFMBE) conducted a scoping meta-review. This manuscript presents the results of this search, which covered the period from January 1974 to October 2022. After careful independent screening, 21 reviews, mainly conducted in North America and Europe, were retained and included. Among the findings were that deep learning is the most commonly utilised method and that electronic health records and registries are among the most prevalent sources of data for AI/ML algorithms. Out of the 21 included reviews, 19 focused on risk prediction and/or the early diagnosis of HF. Furthermore, 10 reviews provided evidence of the impact on the incidence/progression of HF, and 13 on the length of stay. From an HTA perspective, the main areas requiring improvement are the quality assessment of studies on AI/ML (included in 11 out of 21 reviews) and their data sources, as well as the definition of the criteria used to assess the selection of the most appropriate AI/ML algorithm.

Identification and Characterization of the BBX Gene Family in Bambusa pervariabilis × Dendrocalamopsis grandis and Their Potential Role under Adverse Environmental Stresses.

Zinc finger protein (ZFP) transcription factors play a pivotal role in regulating plant growth, development, and response to biotic and abiotic stresses. Although extensively characterized in model organisms, these genes have yet to be reported in bamboo plants, and their expression information is lacking. Therefore, we identified 21 B-box (BBX) genes from a transcriptome analysis of Bambusa pervariabilis × Dendrocalamopsis grandis. Consequently, multiple sequence alignments and an analysis of conserved motifs showed that they all had highly similar structures. The BBX genes were divided into four subgroups according to their phylogenetic relationships and conserved domains. A GO analysis predicted multiple functions of the BBX genes in photomorphogenesis, metabolic processes, and biological regulation. We assessed the expression profiles of 21 BBX genes via qRT-PCR under different adversity conditions. Among them, eight genes were significantly up-regulated under water deficit stress (BBX4, BBX10, BBX11, BBX14, BBX15, BBX16, BBX17, and BBX21), nine under salt stress (BBX2, BBX3, BBX7, BBX9, BBX10, BBX12, BBX15, BBX16, and BBX21), twelve under cold stress (BBX1, BBX2, BBX4, BBX7, BBX10, BBX12, BBX14, BBX15, BBX17, BBX18, BBX19, and BBX21), and twelve under pathogen infestation stress (BBX1, BBX2, BBX4, BBX7, BBX10, BBX12, BBX14, BBX15, BBX17, BBX18, BBX19, and BBX21). Three genes (BBX10, BBX15, and BBX21) were significantly up-regulated under both biotic and abiotic stresses. These results suggest that the BBX gene family is integral to plant growth, development, and response to multivariate stresses. In conclusion, we have comprehensively analyzed the BDBBX genes under various adversity stress conditions, thus providing valuable information for further functional studies of this gene family.

Strong Opponent of Walnut Anthracnose-Bacillus velezensis and Its Transcriptome Analysis.

Walnut is a significant economic tree species worldwide. Walnut anthracnose, caused by the pathogen Colletotrichum gloeosporioides, greatly reduces walnut production and economic benefits. Our study showed that Bacillus velezensis effectively halted the growth of C. gloeosporioides, inducing noticeable abnormalities such as hyphal breakage and distortion, thereby curtailing the pathogen's virulence. A 50-100 times dilution of B. velezensis fermentation broth, applied every two to three days, served as an efficient protective layer for walnut leaves and fruits against C. gloeosporioides infection. Transcriptomic analysis of B. velezensis unveiled its dynamic response against C. gloeosporioides. On the second day, B. velezensis upregulated a significant number of differentially expressed genes related to the synthesis of metabolic products, amino acid biosynthesis, and motility. On the fourth day, continuous synthesis of metabolic products and amino acids, along with differential expression of spore-related genes, was observed. By the sixth day, the focus shifted towards environmental adaptation and carbon source utilization. Throughout the process, B. velezensis likely employed strategies such as the release of metabolic products, increased chemotaxis, and nutrient competition to exert its antagonistic effect on C. gloeosporioides. Fluorescence quantitative results showed that 15 primer pairs were up-regulated and 15 were down-regulated, with a 100% similarity rate to transcriptome sequencing results, confirming their authenticity. These findings provided a foundation for the widespread application of B. velezensis as a biocontrol agent in agriculture and forestry.

Seasonal changes in the abundance Fusarium proliferatium, microbial endophytes and nutrient levels in the roots of hybrid bamboo Bambusa pervariabilis × Dendrocalamopsis grandis.

Plant root pathogens invade the soil around plant roots, disturbing the systemic balance, reducing plant defenses, and causing severe disease. At present, there are few studies on the severity of plant diseases caused by pathogen invasion in different seasons and how pathogens affect root microecology. In this study, we compared the levels of nutrients in the root tissues of the two groups of plants. We used 16S and ITS amplicon sequencing with Illumina NovaSeq 6000 to compare seasonal changes in the composition and structure of microbial communities from healthy roots of bamboo Bambusa pervariabilis × Dendrocalamopsis grandis and roots infected by the soilborne pathogen Fusarium proliferatum. We have found that the invasion of the pathogen led to a substantial decrease in nutrient elements in bamboo roots, except for nitrogen. The pathogen presence correlated with seasonal changes in the bamboo root microbiome and decreased bacterial richness in diseased plants. The root microbial community structure of healthy plants was more stable than that of their diseased counterparts. Furthermore, we identified the lesion area and relative abundance of F. proliferatum were significant predictors of disease progression. The potassium tissue content and the disease lesion area were identified as factors linked with the observed changes in the bamboo root microbiome. This study provides a theoretical foundation for understanding the seasonal dynamics F. proliferatum, an economically important soilborne pathogen of hybrid bamboo grown in Sichuan Province, China.

Procyanidin B2 alleviates uterine toxicity induced by cadmium exposure in rats: The effect of oxidative stress, inflammation, and gut microbiota.

Environmental exposure to hazardous materials causes enormous socioeconomic problems due to its deleterious impacts on human beings, agriculture and animal husbandry. As an important hazardous material, cadmium can promote uterine oxidative stress and inflammation, leading to reproductive toxicity. Antioxidants have been reported to attenuate the reproductive toxicity associated with cadmium exposure. In this study, we investigated the potential protective effect of procyanidin oligosaccharide B2 (PC-B2) and gut microbiota on uterine toxicity induced by cadmium exposure in rats. The results showed that the expression levels of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were reduced in utero. Proinflammatory cytokines (including tumor necrosis factor-α, interleukin-1ß and interleukin-6), the NLRP3 inflammasome, Caspase-1 and pro-IL-1ß were all involved in inflammatory-mediated uterine injury. PC-B2 prevented CdCl2-induced oxidative stress and inflammation in uterine tissue by increasing antioxidant enzymes and reducing proinflammatory cytokines. Additionally, PC-B2 significantly reduced cadmium deposition in the uterus, possibly through its significant increase in MT1, MT2, and MT3 mRNA expression. Interestingly, PC-B2 protected the uterus from CdCl2 damage by increasing the abundance of intestinal microbiota, promoting beneficial microbiota, and inhibiting harmful microbiota. This study provides novel mechanistic insights into the toxicity of environmental cadmium exposure and indicates that PC-B2 could be used in the prevention of cadmium exposure-induced uterine toxicity.

Verification of the Interaction Target Protein of the Effector ApCE22 of Arthrinium phaeospermum in Bambusa pervariabilis × Dendrocalamopsis grandis.

The study of interaction proteins of the pathogen A. phaeospermum effector protein is an important means to analyze the disease-resistance mechanism of Bambusa pervariabilis × Dendrocalamopsis grandis shoot blight. To obtain the proteins interacting with the effector ApCE22 of A. phaeospermum, 27 proteins interacting with the effector ApCE22 were initially identified via a yeast two-hybrid assay, of which four interaction proteins were obtained after one-to-one validation. The B2 protein and the chaperone protein DnaJ chloroplast protein were then verified to interact with the ApCE22 effector protein by bimolecular fluorescence complementation and GST pull-down methods. Advanced structure prediction showed that the B2 protein contained the DCD functional domain related to plant development and cell death, and the DnaJ protein contained the DnaJ domain related to stress resistance. The results showed that both the B2 protein and DnaJ protein in B. pervariabilis × D. grandis were the target interaction proteins of the ApCE22 effector of A. phaeospermum and related to the stress resistance of the host B. pervariabilis × D. grandis. The successful identification of the pathogen effector interaction target protein in B. pervariabilis × D. grandis plays an important role in the mechanism of pathogen-host interaction, thus providing a theoretical basis for the control of B. pervariabilis × D. grandis shoot blight.

Leaf spot of Alnus cremastogyne Caused by Colletotrichum gloeosporioides in Sichuan, China.

Alnus cremastogyne Burk, a broad-leaved tree endemic to south-western China, has both ecological and economic value. The tree is widely used in furniture, timber, windbreaks and sand fixation, and soil and water conservation (Tariq et al. 2018). In December 2020, a new leaf spot disease was discovered on A. cremastogyne in two plant nurseries in Bazhong City (31°15' to 32°45N, 106°21' to 107°45'E), with 77.53% disease incidence. Among the infected trees, 69.54% of the leaves were covered with symptoms of the disease. The typical symptoms initially appeared as irregular brown necrotic lesions, while some lesions were surrounded by a light yellow halo. As the disease progressed, the number of necrotic lesions increased, and lesions gradually expanded and coalesced (Fig. 1). Finally, the disease caused the leaves of A. cremastogyne to wither, curl, die, and fall off. Ten symptomatic leaves were collected from 5 different trees in the two plant nurseries. The leaves with symptoms of leaf spot disease were collected and cut from the junction between the diseased and the healthy tissues. The infected tissues from 10 samples were cut into small 2.5 × 2.5 mm pieces. Infected tissues was sterilized in 3% NaClO solution for 60 s followed by 75% ethanol for 90 s, rinsed three times in sterile water, blot-dried with autoclaved paper towels, and then cultured on potato dextrose agar (PDA) at 25℃ for 4 to 8 days in 12 h/12 h light/dark conditions. After 8 days, the colony diameter reached 71.2 to 79.8 mm. The colonies were initially light pink, and then turned white with pale orange beneath. The conidia were single-celled, aseptate, colorless, cylindrical, straight, bluntly rounded at both ends, and measured 11.6 to 15.9 × 4.3 to 6.1 µm (n = 100). These morphological characteristics were consistent with the description of Colletotrichum gloeosporioides (Pan et al. 2021). For molecular identification, the genomic DNA of a representative isolate, QM202012, was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The internal transcribed spacer (ITS), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified with primers ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone & Kohn, 1999) and GDF/GDR (Templeton et al. 1992), respectively. Sequences were deposited in GenBank (ITS: OL744612, ACT: OL763390, and GAPDH: OL799166). BLAST results indicated that the ITS, ACT, and GAPDH sequences showed >99% identity with C. gloeosporioides sequences in NCBI (GenBank NR160754, MG561657, and KP145407). Identification was confirmed by Bayesian inference using Mr Bayer (Fig 2) A conidial suspension (1 × 106 conidia/ml) was used to test pathogenicity on the leaves of 4-year-old A. cremastogyne plants (10 plants). Fifteen leaves of each plant (10 pots in total) were inoculated with the spore suspension on the leaves. The same number of control leaves was sprayed with sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 25°C under 16 h/8 h photoperiod and 67 to 78% relative humidity. The symptoms observed on the inoculated plants were similar to those of the original diseased plants, with 100% of the inoculated plants being infested with brown leaf spots, but the controls remained symptom-free. C. gloeosporioides was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times, showing similar results each time, confirming Koch's postulates. To our knowledge, this is the first report of leaf spot on A. cremastogyne caused by C. gloeosporioides in China. This finding indicates that C. gloeosporioides may become a serious threat to A. cremastogyne production in Bazhong City and helps to further examine and prevent leaf spot disease in A. cremastogyne growing areas in Bazhong City.

Drought stress-mediated differences in phyllosphere microbiome and associated pathogen resistance between male and female poplars.

Phyllosphere-associated microbes play a crucial role in plant-pathogen interactions while their composition and diversity are strongly influenced by drought stress. As dioecious plant species exhibited secondary dimorphism between the two sexes in response to drought stress, whether such difference will lead to sex-specific differences in phyllosphere microbiome and associated pathogen resistance between male and female conspecifics is still unknown. In this study, we subjected female and male full siblings of a dioecious poplar species to a short period of drought treatment followed by artificial infection of a leaf pathogenic fungus. Our results showed that male plants grew better than females with or without drought stress. Female control plants had more leaf lesion area than males after pathogen infection, whereas drought stress reversed such a difference. Further correlation and in vitro toxicity tests suggested that drought-mediated sexual differences in pathogen resistance between the two plant sexes could be attributed to the shifts in structure and function of phyllosphere-associated microbiome rather than the amount of leaf main defensive chemicals contained in plant leaves. Supportively, the microbiome analysis through high-throughput sequencing indicated that female phyllosphere enriched a higher abundance of ecologically beneficial microbes that serve as biological plant protectants, while males harbored abundant phytopathogens under drought-stressed conditions. The results could provide potential implications for the selection of suitable poplar sex to plants in drought or semi-drought habitats.

Nigrospora musae Causing Taxus chinensis var. mairei stem blight in Sichuan, China.

Taxus chinensis var. mairei is the endemic, endangered, and first-class protected tree species in China. This species is considered as an important resource plant because it can produce Taxol which is an effective medicinal compound against various cancers (Zhang et al., 2010). Stem blight was observed in two plant nurseries in Ya'an (102°44'E,30°42'N), Sichuan province in April 2021. The symptoms first appeared as round brown spots on the stem. As the disease progressed, the damaged area gradually expanded into an oval or irregular shape, which was dark brown. About 800 square meters of planting area were investigated and the disease incidence was up to approximately 64.8%. Twenty obviously symptomatic stems which exhibited the same symptoms as above were collected from 5 different trees in the nursery. To isolate the pathogen, the symptom margin was cut into small blocks (5 x 5 mm), and the blocks were surface sterilized in 75% ethanol for 90 s and 3% NaClO solution for 60 s . Finally incubated on Potato Dextrose Agar (PDA) at 28℃ for 5 days. Ten pure cultures were isolated by transferring hyphal and the three strains (HDS06, HDS07 and HDS08) were selected as representative isolates for further study. Initially, colonies on the PDA of three isolates were white and cotton-like, and then gradually turned gray-black from the center. After 21 days, conidia were produced and were smooth-walled, single-celled, black, oblate, or spherical, measuring 9.3 to 13.6 × 10.1 to 14.5 µm in size (n = 50). Conidia were present at the tip of conidiophores on hyaline vesicles. These morphological features were generally consistent with those of N. musae (Wang et al., 2017). To validate the identification, DNA were extracted from the three isolates, followed by the amplification of transcribed spacer region of rDNA (ITS), the translation elongation factor EF-1 (TEF-1), and the Beta-tubulin (TUB2) sequences with the respective primer pairs ITS1/ITS4 (White et al., 1990), EF-728F/EF-986R (Vieira et al., 2014) and Bt2a/Bt2b (O'Donnell et al., 1997) .The sequences were deposited in GenBank with the accession numbers ON965533, OP028064, OP028068, OP060349, OP060353, OP060354, OP060350, OP060351 and OP060352, respectively. Phylogenetic analysis of combined ITS, TUB2, and TEF genes using the Mrbayes inference method showed that the three isolates clustered with Nigrospora musae as a distinct clade (Fig. 2). Combine with morphological characteristics and phylogenetic analysis, three isolates were identified as N. musae. 30 2-year-old healthy potted plants of T. chinensis were used for pathogenicity test. 25 of these plants were inoculated by injecting 10 µL of the conidia suspension (1 × 106 conidia/mL) into stems and then wrap around the seal to moisturize. The remaining 5 plants were injected with the same amount of sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 25°C and 80% relative humidity. After 2 weeks, the inoculated stems developed lesions similar to those observed in the field, whereas controls were asymptomatic. N. musae was re-isolated from the infected stem and identified by both morphological characteristics and DNA sequence analysis. The experiments repeated three times showed similar results. As far as we know, this is the first report of N. musae causing T. chinensis stem blight in the world. The identification of N. musae could provide a certain theoretical basis for field management and further research of T. chinensis.

Brown leaf spot of Cunninghamia lanceolata Caused by Colletotrichum kahawae in Sichuan province, China.

Chinese fir (Cunninghamia lanceolata) is an important timber species that has been widely cultivated in southern China. It is extensively applied in medicine, environmental monitoring, furniture, urban (e.g., street trees) and rural landscaping, windbreak forest, soil and water conservation. In January 2022, distinct leaf spot symptoms were observed in Chinese fir in Hongya Forestry (29°45'N, 103°11'E) Meishan City, Sichuan Province, China. Field surveys showed that the disease was widespread, with around 70% disease incidence. The typical symptoms initially appeared as yellowish-brown necrotic lesions on the margin of the leaves. Subsequently, lesions gradually expanded and developed into larger necrotic areas with red-brown irregular shape. The lesions later expanded throughout the leaf. Infected leaves turned dark brown and wilted, leading to seeding's death. Diseased leaves with typical symptoms were collected for pathogen isolation and identification. Infected tissues from ten samples were cut into small pieces of 2 × 2 mm. Infected tissues were surface disinfected with 3% sodium hypochlorite and 75% ethanol for 30s and 60s, respectively, and rinsed with sterile water 3 times. They were blotted dry with autoclaved paper towels and incubated on potato dextrose agar (PDA) with streptomycin sulfate (50 µg/mL) for 5 ~ 8 days at 25°C. and 12 h light/dark period. The diameter of the colonies reached 65.7 to 75.9 mm, with a gray to black center, and white edges while the reverse sides were gray to orange. Conidia were single-celled, colorless, straight, cylindrical, bluntly rounded at both ends, Conidia dimensions varied from, 7.3 µm to 15.7 µm in length and 3.3 µm to 6.1 µm in width (n = 100). For molecular identification, the genomic DNA of isolate SM2290708, SM229070801 and SM229070802 were extracted using a fungal genomic DNA extraction kit (Beijing Solarbio Science & Technology Co., Ltd., City, China). The internal transcribed spacers of the ribosomal RNA (ITS) [ITS1/ITS4 (White et al., 1990), calmodulin (CAL) (Weir et al., 2012), ß-tubulin (TUB2) (O'Donnell et al., 1997), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Templeton et al. 1992) were amplified. Sequences were deposited in GenBank (ITS: ON564877, OQ535027 and OQ535028; CAL: ON583827, OQ538101 and OQ538102; TUB2: ON583830, OQ538104 and OQ538105; and GAPDH: ON583831, OQ538108 and OQ538109). BLAST results showed that our ITS, CAL, TUB2 and GAPDH sequences were >99% identical to the corresponding sequences of Colletotrichum kahawae deposited at NCBI (GenBank JX010231, JX009642, JX010444, and JX010012). Identification was confirmed by Bayesian inference using MrBayes (Fig 2). The conidial suspension (1 × 106 conidia/ml) was used for inoculation by spraying leaves of ten 3-year-old Chinese fir plants for pathogenicity test. Fifteen leaves of each plant were inoculated. An equal number of control leaves was sprayed with sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 28°C under a 16 h/8 h photoperiod and in 73% to 79% relative humidity. After fifteen days, the symptoms observed on the inoculated plants were similar to those of the original diseased plants, but the controls remained asymptomatic. Colletotrichum kahawae was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of brown leaf spot on C. lanceolata caused by C. kahawae in China. The results of this study provide basic information for diagnosis of the pathogen and developing prevention strategies to manage C. lanceolata leaf spot disease.

Root rot Caused by Setophoma terrestris on Illicium verum in Yunnan Province, China.

Star anise (Illicium verum Hook. f.), a genus of star anise in the family Magnoliaceae, is an important cash crop of "medicinal and food" origin, mainly from China. In August 2021, root rot of I. verum was first observed on more than 80% of the plants grown within a 500 hectares area in Wenshan city, Yunnan Province. At the early stage of the disease, the phloem of the root was dark yellow-brown, and the leaves turn yellow. With further disease development, the whole root became black (Fig. 1a, 1b), and the leaves gradually fall off, affecting the growth, yield and eventually caused death of the whole plant. A total of 20 root samples were collected from typical symptomatic plant roots with 20 years old in Wenshan City (23°18'12″N, 103°56'98″E) and were cut into 2 × 2 mm pieces at the junction of infected and healthy tissue. Each sample was surface-sterilized with 3% NaClO and 75% alcohol for 60 s before rinsing three times with distilled water. The sterile filter paper (5×5 cm) was used to dry the tissue, and samples were cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml). Plates were incubated at 25°C in the dark in the incubator. From 9 isolates obtained in culture, 7 exhibited the morphology described by Boerema et al. (Boerema et al. 2004) for Setophoma sp. The hyphae were hyaline and septate (Fig.1c). After 14 days of culture on V8 juice agar, white round colonies are formed, but there is no groove in the middle of the colonies (Fig.1d), and transparent, oval, or cylindrical conidia were produced, 6.0-8.0 x 2.5 to 4.0 um (Fig.1e). DNA was extracted from a representative isolate BJGF-04 for molecular identification using a fungal genomic DNA extraction kit (Solarbio, Beijing, China). Polymerase chain reactions (PCRs) were performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990) and primers T1/ß-Sandy-R for the ß-tubulin gene (TUB) region (Yang et al. 2017) and primers NL3/ LR5 for 28S large subunit rDNA (LSU) region (Hu et al. 2021) and NS1/ NS4 for 5.8S large subunit rDNA (SSU) region (Mahesha et al. 2021). Newly generated representative sequences were deposited in GenBank: ITS sequence (ON645256), TUB sequence (ON854484), and LSU sequence (ON644445), SSU sequence (ON644451). were sequenced and blasted, showing 99 to 100% sequence homology with known S. terrestris. Pathogenicity was performed using one-year asymptomatic plants of I. verum. A conidial suspension (1 x 106 conidia/ml) collected from V8 juice cultures with 0.05% Tween buffer was poured at a volume of 10 ml/plant. Three individual seedlings were used as replicates for each treatment, and sterile water was used as the negative control. All plants were placed in an artificial climate incubator at 25°C under 90% relative humidity. After 20 days, all inoculated plants showed symptoms identical to those described above, whereas controls remained healthy. Setophoma terrestris was reisolated from the infected roots, which was confirmed by morphological and molecular identification, which completed Koch's postulates. To our knowledge, this is the first report of S. terrestris as a causal agent of root rot on I. verum in China.

Changes in the Histology of Walnut (Juglans regia L.) Infected with Phomopsis capsici and Transcriptome and Metabolome Analysis.

Phomopsis capsici (P. capsici) causes branch blight of walnuts, which leads to significant economic loss. The molecular mechanism behind the response of walnuts remains unknown. Paraffin sectioning and transcriptome and metabolome analyses were performed to explore the changes in tissue structure, gene expression, and metabolic processes in walnut after infection with P. capsici. We found that P. capsici caused serious damage to xylem vessels during the infestation of walnut branches, destroying the structure and function of the vessels and creating obstacles to the transport of nutrients and water to the branches. The transcriptome results showed that differentially expressed genes (DEGs) were mainly annotated in carbon metabolism and ribosomes. Further metabolome analyses verified the specific induction of carbohydrate and amino acid biosynthesis by P. capsici. Finally, association analysis was performed for DEGs and differentially expressed metabolites (DEMs), which focused on the synthesis and metabolic pathways of amino acids, carbon metabolism, and secondary metabolites and cofactors. Three significant metabolites were identified: succinic semialdehyde acid, fumaric acid, and phosphoenolpyruvic acid. In conclusion, this study provides data reference on the pathogenesis of walnut branch blight and direction for breeding walnut to enhance its disease resistance.

Soil cadmium stress affects the phyllosphere microbiome and associated pathogen resistance differently in male and female poplars.

Microorganisms associated with the phyllosphere play a crucial role in protecting plants from diseases, and their composition and diversity are strongly influenced by heavy metal contaminants. Dioecious plants are known to exhibit sexual dimorphism in metal accumulation and tolerance between male and female individuals. Hence, in this study we used male and female full-siblings of Populus deltoides to investigate whether the two sexes present differences in their phyllosphere microbiome structures and in their associated resistance to the leaf pathogenic fungus Pestalotiopsis microspora after exposure to excess soil cadmium (Cd). We found that Cd-treated male plants grew better and accumulated more leaf Cd than females. Cd stress reduced the lesion areas on leaves of both sexes after pathogen infection, but male plants exhibited better resistance than females. More importantly, Cd exposure differentially altered the structure and function of the phyllosphere microbiomes between the male and female plants, with more abundant ecologically beneficial microbes and decreased pathogenic fungal taxa harbored by male plants. In vitro toxicity tests suggested that the sexual difference in pathogen resistance could be attribute to both direct Cd toxicity and indirect shifts in the phyllosphere microbiome. This study provides new information relevant for understanding the underlying mechanisms of the effects of heavy metals involved in plant-pathogen interactions.

Colletotrichum fructicola Causal agent of Shot-Hole Symptoms on Leaves of Prunus sibirica in China.

Prunus sibirica L. (Siberian apricot) is a member of the Rosaceae family and an ecologically important tree species in China (Buer et al., 2022). Shot hole symptoms on the leaves were observed in five Siberian apricot groves in Chengdu (103.81 E, 30.97 N), Sichuan province in July 2020. The symptoms first appeared as small purplish-brown spots with yellow rings around them. As the disease progressed, the damaged area (diameter 1.5-3.0 cm) became necrotic and fell off. The disease incidence was about 60% and the disease index was 28.6 of leaves in the grove. in most severe cases. Fifteen symptomatic leaves were collected from 5 different trees in an orchard. Pathogen isolation was performed from symptomatic leaf tissue (5 × 5 mm) though surface disinfection (in 70% ethanol and 2% NaClO) and incubation on Potato Dextrose Agar (PDA) at 28℃ for 3 days. Overall 10 isolates with similar colony morphology were obtained from the 15 infected tissue pieces, and three representative isolates (XCK 2-4) were selected for further study. Colonies of the isolates on PDA were initially cottony, pale white to grayish-green with abundant aerial hyphae and produced conidial masses after 7 days. Conidiogenous cells were clavate and aggregated in acervuli. Conidia were smooth-walled, single-celled, straight, and slightly obtusely rounded at both ends, 12.8 to 18.7 × 4.3 to 5.7 µm in size (Fig. 1). The morphological characteristics of the three isolates were consistent with the description of species in the Colletotrichum gloeosporioides complex. DNA was amplified using the following primers pairs for the internal transcribed spacer (ITS) region of rDNA and partial sequences of beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and translation elongation factor (TEF-1), respectively: ITS1/ITS4, T1/Bt2b, GDF/GDR, CHS-F/CHS-R, and EF-F/EF-R (Vieira et al., 2014). Accession numbers (MW228049, MW284974, MW284976, MW284975 and MW284977, respectively) were obtained afterepositing all the resulting sequences in GenBank. Nucleotide blast showed 99 to 100% identities with Colletotrichum fructicola (GenBank accessions nos. MZ961683, MW284974, MN525881, MN525860, MF627961). Phylogenetic analysis of combined ITS-TUB-GAPDH genes using the Mrbayes inference method showed that the three isolates clustered with three reference isolates of C. fructicola as a distinct clade (Fig. 2). To verify Koch's postulates, ten 3-year-old healthy potted plants of P. sibirica were inoculated by spraying a conidial suspension (6 × 105 conidia/mL) of isolate XCK2 on both sides of leaves, and the control leaves were sprayed with sterile water. Then, all treatments were placed in a moist environment (25±2°C, 80% relative humidity, natural light). The inoculated plants showed typical symptoms of plants with natural infections, while the controls remained asymptomatic after 14 days. The pathogen C. fructicola was re-isolated from all inoculated plants, and the culture and fungus characteristics were the same as those of the original isolate. Colletotrichum fructicola was not isolated from the control plants. The results indicated that C. fructicola is the causal agent of the disease. Colletotrichum fructicola was reported as a leaf pathogen on Camellia chrysantha in China (Zhao et al., 2021). This is the first report of C. fructicola causing P. sibirica leaf shot-hole in the world. The identification of C. fructicola could provide relevant information for applying management strategies and research on the Siberian apricot disease.

HSP70 and TNF Loci Polymorphism Associated with the Posner-Schlossman Syndrome in a Southern Chinese Population.

Previous studies have shown that HLA gene polymorphisms are associated with the pathogenesis of the Posner-Schlossman syndrome (PSS). This study was aimed at evaluating the associations between HLA-III gene polymorphisms and PSS in a southern Chinese Han population. A total of 150 PSS patients and 183 healthy controls were included in this study. Twenty-one single nucleotide polymorphisms (SNPs) of HLA-III genes (including HSP70-1, HSP70-2, HSP70-hom, TNF-α, TNF-ß, C2, and CFB) were genotyped using the SNaPshot technique. Our study showed that the frequencies of G allele at rs909253, A allele at rs1041981, and G allele at rs2844484 of TNF-ß in the patient group were significantly higher than those in healthy controls (Corrected P (P c ) = 0.040, OR = 1.45; P c = 0.033, OR = 1.45; P c = 0.045, OR = 1.58, respectively). The frequency of T allele at rs12190359 of HSP70-1 was significantly lower in PSS patients than those in healthy controls (P c = 0.018 and OR = 0.10). The frequencies of the CCT haplotype of HSP70-1 gene (rs1008438-rs562047-rs12190359) and the ACCCTTT haplotype of HSP70 gene (rs2227956-rs1043618-rs1008438-rs562047-rs12190359-rs2763979-rs6457452) were significantly lower in PSS patients than those in healthy controls (P c = 0.024, OR = 0.10; P c = 0.048, OR = 0.10, respectively). In conclusion, the G allele at rs909253, A allele at rs1041981, and G allele at rs2844484 of TNF-ß gene might be risk factors for PSS, while the T allele at rs12190359 of HSP70-1 gene and specific haplotypes of the HSP70-1 and HSP70 genes might be protective factors for PSS.

Polysaccharide derived from Inonotus obliquus inhibits lipopolysaccharide-induced acute endometritis in mice.

OBJECTIVE: Endometritis bacterial pathogenic condition that affects both humans and animals develops in the inner lining of the uterus. Inonotus obliquus polysaccharide (IOP), an active cocktail of Inonotus obliquus, has been shown to have a relatively wide range of biological activities and can play a role in various diseases. However, from the currently reported article, there is no information about the anti-inflammatory effect of IPO in the symptoms of lipopolysaccharide (LPS)-induced endometritis. Therefore, this study carefully observed the phenomenon of IOP on the symptoms of endometritis induced by LPS in mice, elucidated the protective mechanism of IOP on the body, and clarified the potential mechanism of IOP. METHODS: A total of 72 BALB/c female experimental mice were divided into several groups for comparison. They were the blank control group, the LPS group, the LPS+ IOP group (the effect of IOP dose on mice was also explored, divided into low, medium, and high) and LPS+ amoxicillin group. All groups except control group were infused with LPS into the uterus. The mice of LPS+ IOP groups and LPS+ amoxicillin group were orally administered with IOP or amoxicillin after LPS challenge for 3 hours. Histopathology and myeloperoxidase (MPO) activity were used to detect uterine tissue injury, and cytokine levels were used to measure uterine inflammation. The expression of toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB)-related proteins in the inflammatory signaling pathway was observed. RESULTS: Pathological and MPO activity analyses revealed that IOP relieved LPS-induced uterine tissue injury. Quantitative reverse transcription-polymerase chain reaction was used to detect and quantitatively study the RNA information of mouse cells, which had high accuracy and sensitivity. From the test results, IOP does effectively control the release of pro-inflammatory cytokines such as interleukin-6 (IL-6), IL-1ß, IL-8 and tumor necrosis factor-α (TNF-α), avoiding the body's immune response. Analysis of uterine tissue cell components also confirmed that the expression level of inflammatory mediator-induced nitric oxide synthase (iNOS) was also greatly reduced. Analysis of western blotting results of cell synthesis showed that IOP mainly inhibited the protein expression of TLR4 and myeloid differentiation factor 88 in the body. CONCLUSION: This study proved that the mechanism of action of IOP is to inhibit the TLR4/NF-κB signaling pathway to reduce the release of pro-inflammatory cytokines from body cells, thereby alleviating the symptoms of endometritis induced by LPS. Thus, IOP may act as an effective drug in preventing and curing LPS-induced endometritis.