Stereo-selective cardiac toxicity induced by metconazole via oxidative stress and the wnt/ß-catenin signaling pathway in zebrafish embryos.

Metconazole (MEZ), a chiral triazole fungicide, produces enantioselective adverse effects in non-target organisms. Among MEZ's isomers, cis-MEZ displays robust antimicrobial properties. Evaluating MEZ and cis-MEZ's toxicity may mitigate fungicide usage and safeguard non-target organisms. Our study evaluated the toxicity of MEZ and its cis-isomers at concentrations of 0.02, 0.2, 2, and 4 mg L-1. We report stereoselectivity and severe cardiovascular defects in zebrafish, including pericardial oedema, decreased heart rate, increased sinus venous and bulbous arteries distances, intersegmental vessel defects, and altered cardiovascular development genes (hand2, gata4, nkx2.5, tbx5, vmhc, amhc, dll4, vegfaa, and vegfc). Further, MEZ significantly increased oxidative stress and apoptosis in zebrafish, primarily in the cardiac region. Isoquercetin, an antioxidant found in plants, partially mitigates MEZ-induced cardiac defects. Furthermore, MEZ upregulated the Wnt/ß-catenin pathway genes (wnt3, ß-catenin, axin2, and gsk-3ß) and ß-catenin protein expression. Inhibitor of Wnt Response-1 (IWR-1) rescued MEZ-induced cardiotoxicity. Our findings highlight oxidative stress, altered cardiovascular development genes, and upregulated Wnt/ß-catenin signaling as contributors to cardiovascular toxicity in response to MEZ and cis-MEZ treatments. Importantly, 1R,5S-MEZ exhibited greater cardiotoxicity than 1S,5R-MEZ. Thus, our study provides a comprehensive understanding of cis-MEZ's cardiovascular toxicity in aquatic life.

Residual behavior of dinotefuran and its metabolites during Huangjiu fermentation and their effects on flavor.

Yellow rice wine (Huangjiu) is a traditional Chinese alcoholic beverage. However, there is a risk of pesticide residues in Huangjiu due to pesticide indiscriminate use. In this study, the residues of dinotefuran and its metabolites during Huangjiu fermentation and their effects on flavor substances were studied. The initial concentrations of dinotefuran ranged from 856.3 to 1874.9 µg/L, and its half-life was no more than 3.65 d. At 24 d of Huangjiu fermentation, the terminal residues of dinotefuran, 1-methyl-3-(tetrahydro-3-furylmethyl)urea (UF) and 1-methyl-3-(tetrahydro-3-furylmethyl)guanidine (DN) were 195.1-535.3 µg/L, 38.33-48.70 µg/L and 37.8-74.1 µg/L, respectively. Twenty potential degradation compounds were identified by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS), and their toxicity was evaluated. Finally, the effect of dinotefuran on physicochemical properties and total phenol content of Huangjiu were analyzed. The risk of rancidity was significantly increased and bitter amino acids were formed. These findings provide a guidance and the safe production of Huangjiu.

Characterization of the Endophytic Bacillus subtilis KRS015 Strain for Its Biocontrol Efficacy Against Verticillium dahliae.

Endophytes play important roles in promoting plant growth and controlling plant diseases. Verticillium wilt is a vascular wilt disease caused by Verticillium dahliae, a widely distributed soilborne pathogen that causes significant economic losses on cotton each year. In this study, an endophyte KRS015, isolated from the seed of the Verticillium wilt-resistant Gossypium hirsutum 'Zhongzhimian No. 2', was identified as Bacillus subtilis by morphological, phylogenetic, physiological, and biochemical analyses. The volatile organic compounds (VOCs) produced by KRS015 or its cell-free fermentation extract had significant antagonistic effects on various pathogenic fungi, including V. dahliae. KRS015 reduced Verticillium wilt index and colonization of V. dahliae in treated cotton seedlings significantly; the disease reduction rate was ∼62%. KRS015 also promoted plant growth, potentially mediated by the growth-related cotton genes GhACL5 and GhCPD-3. The cell-free fermentation extract of KRS015 triggered a hypersensitivity response, including reactive oxygen species (ROS) and expression of resistance-related plant genes. VOCs from KRS015 also inhibited germination of conidia and the mycelial growth of V. dahliae, and were mediated by growth and development-related genes such as VdHapX, VdMcm1, Vdpf, and Vel1. These results suggest that KRS015 is a potential agent for controlling Verticillium wilt and promoting growth of cotton.

CD4 + T cells ferroptosis is associated with the development of sepsis in severe polytrauma patients.

BACKGROUND: Immunological disorder remains a great challenge in severe poly-trauma, in which lymphopenia is an important contributor. The purpose of present study is to explore whether ferroptosis, a new manner of programmed cell death (PCD), is involved in the lymphocyte depletion and predictive to the adverse prognosis of severe injuries. PATIENTS AND METHODS: Severe polytrauma patients admitted from January 2022 to December 2022 in our trauma center were prospectively investigated. Peripheral blood samples were collected at admission (day 1), day 3 and day 7 from them. Included patients were classified based on whether they developed sepsis or not. Clinical outcomes, systematic inflammatory response, lymphocyte subpopulation, CD4 + T cell ferroptosis were collected, detected and analyzed. RESULTS: Notable lymphopenia was observed on the first day after severe trauma and failed to normalize on the 7th day if patients were complicated with sepsis, in which CD4 + T cell was the subset of lymphocyte that depleted most pronouncedly. Lymphocyte loss was significantly correlated with the acute and biphasic systemic inflammatory response. Ferroptosis participated in the death of CD4 + T cells, potentially mediated by the downregulation of xCT-GSH-GPX4 pathway. CD4 + T cells ferroptosis had a conducive predicting value for the development of sepsis following severe trauma. CONCLUSIONS: CD4 + T cells ferroptosis occurs early in the acute stage of severe polytrauma, which may become a promising biomarker and therapeutic target for post-traumatic sepsis.

Stereoselective cardiotoxic effects of metconazole on zebrafish (Danio rerio) based on AGE-RAGE signalling pathway.

Metconazole (MEZ) is a novel chiral triazole fungicide that is widely used to prevent and control soil-borne fungal pathogens and other fungal diseases. However, it has a long half-life in aquatic environments and thus poses potential environmental risks. This study evaluates the acute and stereoselective cardiotoxicity of MEZ in zebrafish (Danio rerio) embryos. In addition, transcriptomics, real-time quantitative PCR, enzyme activity determination, and molecular docking are performed to evaluate the molecular mechanisms underlying the cardiotoxicity of MEZ in zebrafish. MEZ decreases the heart rate while increasing the pericardial oedema rate; additionally, it induces stereoselective cardiotoxicity. 1S,5S-MEZ exhibits stronger cardiotoxicity than 1R,5R-MEZ. Furthermore, MEZ increases the expression of Ahr-associated genes and the transcription factors il6st, il1b, and AP-1. Heart development-related genes, including fbn2b, rbm24b, and tbx20 are differentially expressed. MEZ administration alters the activities of catalase, peroxidase, and glutathione-S-transferase in zebrafish larvae. Molecular docking indicates that 1R,5R-MEZ binds more strongly to the inhibitor-binding sites of p38 in the AGE-RAGE signalling pathway than to other MEZ enantiomers. Studies conducted in vivo and in silico have established the enantioselective cardiotoxicity of MEZ and its underlying mechanisms, highlighting the need to evaluate the environmental risk of chiral MEZ in aquatic organisms at the enantiomeric level.

Two zinc finger proteins, VdZFP1 and VdZFP2, interact with VdCmr1 to promote melanized microsclerotia development and stress tolerance in Verticillium dahliae.

BACKGROUND: Melanin plays important roles in morphological development, survival, host-pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae, increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear. RESULTS: We identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9, a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae, as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1. Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae. CONCLUSIONS: Our results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae.

Magnetic functionalized graphene oxide combined with ultra-high performance liquid chromatography for trace detection of succinate dehydrogenase inhibitor fungicides in food.

In this study, an efficient, sensitive, and convenient magnetic solid-phase extraction method combined with ultra-high performance liquid chromatography-tandem mass spectrometry (MSPE-UHPLC-MS/MS) was developed for the simultaneous determination of 19 succinate dehydrogenase inhibitor fungicide residues in six different food matrices The synthesized tetraethylenepentamine magnetic graphene oxide nanocomposite showed the advantages of good dispersibility, large specific surface area (113.93 m2 /g) and large pore volume (0.25 cm3 /g), making it an ideal succinate dehydrogenase inhibitor pretreatment adsorbent. The MSPE-UHPLC-MS/MS method showed linearity in the range of 5.0-800.0 µg/kg, with a correlation coefficient (R2 ) > 0.99, and a limit of quantification of 5 µg/kg. The recovery of succinate dehydrogenase inhibitor fungicides was in the range of 71.2%-119.4%. The MSPE method is simple, rapid, and efficient, making it an ideal alternative to sample pretreatment in the determination of trace succinate dehydrogenase inhibitor fungicides in complex matrices.

Potential Risks of Tebuconazole during Wine Fermentation at the Enantiomer Level Based on Multiomics Analysis.

The enantioselectivity and potential risks of tebuconazole enantiomers (R-tebuconazole and S-tebuconazole) in wine fermentation were investigated in this study using Cabernet Sauvignon grapes. Tebuconazole was mainly degraded during the alcoholic fermentation stage, and no obvious transformation between R-tebuconazole and S-tebuconazole was observed. Selective degradation between these two enantiomers occurred, with R-tebuconazole degrading faster than S-tebuconazole. The residual tebuconazole inhibits glucose metabolism and the unsaturated fatty acid formation in the wine fermentation system and inhibits gene expression in the late phase of Saccharomycetales, affecting its cell wall formation. Overall, the findings highlight that R-tebuconazole exhibited a higher risk than S-tebuconazole in these processes. These insights are potentially exploitable to understand chiral pesticides at the enantiomer level using multiomics technology in food-processing systems.

Functional Characterization of Verticillium dahliae Race 3-Specific Gene VdR3e in Virulence and Elicitation of Plant Immune Responses.

Verticillium dahliae is a soilborne fungal pathogen that causes disease on many economically important crops. Based on the resistance or susceptibility of differential cultivars in tomato, isolates of V. dahliae are divided into three races. Avirulence (avr) genes within the genomes of the three races have also been identified. However, the functional role of the avr gene in race 3 isolates of V. dahliae has not been characterized. In this study, bioinformatics analysis showed that VdR3e, a cysteine-rich secreted protein encoded by the gene characterizing race 3 in V. dahliae, was likely obtained by horizontal gene transfer from the fungal genus Bipolaris. We demonstrate that VdR3e causes cell death by triggering multiple defense responses. In addition, VdR3e localized at the periphery of the plant cell and triggered immunity depending on its subcellular localization and the cell membrane receptor BAK1. Furthermore, VdR3e is a virulence factor and shows differential pathogenicity in race 3-resistant and -susceptible hosts. These results suggest that VdR3e is a virulence factor that can also interact with BAK1 as a pathogen-associated molecular pattern (PAMP) to trigger immune responses. IMPORTANCE Based on the gene-for-gene model, research on the function of avirulence genes and resistance genes has had an unparalleled impact on breeding for resistance in most crops against individual pathogens. The soilborne fungal pathogen, Verticillium dahliae, is a major pathogen on many economically important crops. Currently, avr genes of the three races in V. dahliae have been identified, but the function of avr gene representing race 3 has not been described. We investigated the characteristics of VdR3e-mediated immunity and demonstrated that VdR3e acts as a PAMP to activate a variety of plant defense responses and induce plant cell death. We also demonstrated that the role of VdR3e in pathogenicity was host dependent. This is the first study to describe the immune and virulence functions of the avr gene from race 3 in V. dahliae, and we provide support for the identification of genes mediating resistance against race 3.

Short-term exposure of Cannabidiol on Zebrafish (Danio Rerio): Reproductive Toxicity.

Cannabidiol (CBD), a medically active component of hemp, is a popular ingredient in healthcare and personal-care products. The increasing demand for CBD and the legalization of hemp growth may promote chronic exposure of non-target organisms to CBD. In this study, the reproductive toxicity of CBD was investigated on adult zebrafish. With CBD treatment, female zebrafish spawned less with higher natural mortality and malformation rates. Both female and male zebrafish showed a decreased gonadosomatic index with an increased percentage of pre-mature oocytes and sperm and had an increased hepatosomatic index with decreased content of vitellogenin. The value of estrogen/testosterone (E2/T) decreased in female zebrafish and increased in male zebrafish. Sex hormone synthesis genes were downregulated in ovaries and upregulated in testicles, except for cyp11a, in contrast to the other genes. Apoptosis-related genes were upregulated in the zebrafish brain, gonad, and liver. These results show that CBD might damage the reproductive function by inducing an apoptotic response, further inhibiting zebrafish reproductive ability.

Self-adaptive nanoassembly enabling turn-on hypoxia illumination and periphery/center closed-loop tumor eradication.

Solid tumors are regarded as complex evolving systems rather than simple diseases. Self-adaptive synthetic therapeutics are required to cope with the challenges of entire tumors; however, limitations in accurate positioning and destruction of hypoxic niches seriously hinder complete tumor eradication. In this study, we engineer a molecular nanoassembly of sorafenib and a hypoxia-sensitive cyanine probe (CNO) to facilitate periphery/center synergistic cancer therapies. The self-adaptive nanoassembly with cascade drug release features not only effectively kills the peripheral tumor cells in normoxic rims but precisely illuminates hypoxic niches following the reduction of CNO by nitroreductase. More important, CNO is found to synergistically induce tumor ferroptosis with sorafenib via nicotinamide adenine dinucleotide phosphate (NADPH) depletion in hypoxic niches. As expected, the engineered nanoassembly demonstrates self-adaptive hypoxic illumination and periphery/center synergetic tumor eradication in colon and breast cancer BALB/c mouse xenograft models. This study advances turn-on hypoxia illumination and chemo-ferroptosis toward clinical applicability.

Insights into the Biocontrol Function of a Burkholderia gladioli Strain against Botrytis cinerea.

Pathogenic fungi are the main cause of yield loss and postharvest loss of crops. In recent years, some antifungal microorganisms have been exploited and applied to prevent and control pathogenic fungi. In this study, an antagonistic bacteria KRS027 isolated from the soil rhizosphere of a healthy cotton plant from an infected field was identified as Burkholderia gladioli by morphological identification, multilocus sequence analysis, and typing (MLSA-MLST) and physiobiochemical examinations. KRS027 showed broad spectrum antifungal activity against various phytopathogenic fungi by secreting soluble and volatile compounds. KRS027 also has the characteristics of plant growth promotion (PGP) including nitrogen fixation, phosphate, and potassium solubilization, production of siderophores, and various enzymes. KRS027 is not only proven safe by inoculation of tobacco leaves and hemolysis test but also could effectively protect tobacco and table grapes against gray mold disease caused by Botrytis cinerea. Furthermore, KRS027 can trigger plant immunity by inducing systemic resistance (ISR) activated by salicylic acid- (SA), jasmonic acid- (JA), and ethylene (ET)-dependent signaling pathways. The extracellular metabolites and volatile organic compounds (VOCs) of KRS027 affected the colony extension and hyphal development by downregulation of melanin biosynthesis and upregulation of vesicle transport, G protein subunit 1, mitochondrial oxidative phosphorylation, disturbance of autophagy process, and degrading the cell wall of B. cinerea. These results demonstrated that B. gladioli KRS027 would likely become a promising biocontrol and biofertilizer agent against fungal diseases, including B. cinerea, and would promote plant growth. IMPORTANCE Searching the economical, eco-friendly and efficient biological control measures is the key to protecting crops from pathogenic fungi. The species of Burkholderia genus are widespread in the natural environment, of which nonpathogenic members have been reported to have great potential for biological control agents and biofertilizers for agricultural application. Burkholderia gladioli strains, however, need more study and application in the control of pathogenic fungi, plant growth promotion, and induced systemic resistance (ISR). In this study, we found that a B. gladioli strain KRS027 has broad spectrum antifungal activity, especially in suppressing the incidence of gray mold disease caused by Botrytis cinerea, and can stimulate plant immunity response via ISR activated by salicylic acid- (SA), jasmonic acid- (JA), and ethylene (ET)-dependent signaling pathways. These results indicate that B. gladioli KRS027 may be a promising biocontrol and biofertilizer microorganism resource in agricultural applications.

Enantioselective activity and toxicity of chiral acaricide cyflumetofen toward target and non-target organisms.

Cyflumetofen (CYF), a novel chiral acaricide, exert enantiomer-specific effects on target organisms by binding to glutathione S-transferase. However, there is limited knowledge regarding the response of non-target organisms to CYF, including enantioselective toxicity. In this study, we investigated the effects of racemic CYF (rac-CYF) and its two enantiomers (+)-CYF and (-)-CYF on MCF-7 cells and non-target (honeybees) and target (bee mites and red spider mites) organisms. The results showed that similar to estradiol, 1 µM (+)-CYF promoted the proliferation and disturbed the redox homeostasis of MCF-7 cells, whereas at high concentrations (≥100 µM) it exerted a negative effect on cell viability that was substantially stronger than that of (-)-CYF or rac-CYF. (-)-CYF and rac-CYF at 1 µM concentration did not significantly affect cell proliferation, but caused cell damage at high concentrations (≥100 µM). Analysis of acute CYF toxicity against non-target and target organisms revealed that for honeybees, all CYF samples had high lethal dose (LD50) values, indicating low toxicity. In contrast, for bee mites and red spider mites, LD50 values were low, whereas those of (+)-CYF were the lowest, suggesting higher toxicity of (+)-CYF than that of the other CYF samples. Proteomics profiling revealed potential CYF-targeted proteins in honeybees related to energy metabolism, stress responses, and protein synthesis. Upregulation of estrogen-induced FAM102A protein analog indicated that CYF might exert estrogenic effects by dysregulating estradiol production and altering estrogen-dependent protein expression in bees. Our findings suggest that CYF functions as an endocrine disruptor in non-target organisms in an enantiomer-specific manner, indicating the necessity for general ecological risk assessment for chiral pesticides.

Bioaccessibility and Intestinal Transport of Tebuconazole in Table Grape by Using In Vitro Digestion Models.

In this study, the effects of various digestive models, influencing factors and dietary supplements on the bioaccessibility of tebuconazole in table grapes were compared. The Caco-2 cell model was employed to reveal the transfer behavior of tebuconazole. The results indicated that digestion time is the main factor affecting bioaccessibility. With an increase in time, the tebuconazole in grapes was almost completely dissolved, with bioaccessibility reaching 98.5%, whereas dietary fiber reduced bioaccessibility. Tebuconazole undergoes carrier-free passive transport in permeable cells in the Caco-2 cell model. These findings have practical application value for correctly evaluating the harmful level of pollutants in the matrix to human body.

Molecularly engineering a dual-drug nanoassembly for self-sensitized photodynamic therapy via thioredoxin impairment and glutathione depletion.

Photodynamic therapy (PDT) has been extensively investigated as a spatiotemporally noninvasive and controllable modality for cancer treatment. However, the intracellular antioxidant systems mainly consisting of thioredoxin (Trx) and glutathione (GSH) significantly counteract and prevent reactive oxygen species (ROS) accumulation, resulting in a serious loss of PDT efficiency. To address this challenge, we propose that PDT can be improved by precisely blocking antioxidant systems. After molecular engineering and synergistic cytotoxic optimization, a DSPE-PEG2K-modified dual-drug nanoassembly (PPa@GA/DSPE-PEG2K NPs) of pyropheophorbide a (PPa) and gambogic acid (GA) is successfully constructed. Interestingly, GA can effectively destroy intracellular antioxidant systems by simultaneously inhibiting Trx and GSH. Under laser irradiation, the cell-killing effects of PPa is significantly enhanced by GA-induced inhibition of the antioxidant systems. As expected, PPa@GA/DSPE-PEG2K nanoparticles demonstrate potent antitumor activity in a 4T1 breast tumor-bearing BALB/c mouse xenograft model. Such a carrier-free self-sensitized nanotherapeutic offers a novel co-delivery strategy for effective PDT.

Effect of Pathogenic Fungal Infestation on the Berry Quality and Volatile Organic Compounds of Cabernet Sauvignon and Petit Manseng Grapes.

The effect of pathogenic fungal infestation on berry quality and volatile organic compounds (VOCs) of Cabernet Sauvignon (CS) and Petit Manseng (PM) were investigated by using biochemical assays and gas chromatography-ion mobility spectrometry. No significant difference in diseases-affected grapes for 100-berry weight. The content of tannins and vitamin C decreased significantly in disease-affected grapes, mostly in white rot-affected PM, which decreased by 71.67% and 66.29%. The reduced total flavonoid content in diseases-affected grape, among which the least and most were anthracnose-affected PM (1.61%) and white rot-affected CS (44.74%). All diseases-affected CS had much higher titratable acid, a maximum (18.86 g/100 ml) was observed in the gray mold-affected grapes, while only anthracnose-affected grapes with a higher titratable acid level (21.8 g/100 mL) were observed in PM. A total of 61 VOCs were identified, including 14 alcohols, 13 esters, 12 aldehydes, 4 acids, 4 ketones, 1 ether, and 13 unknown compounds, which were discussed from different functional groups, such as C6-VOCs, alcohols, ester acetates, aldehydes, and acids. The VOCs of CS changed more than that of Petit Manseng's after infection, while gray mold-affected Cabernet Sauvignon had the most change. C6-VOCs, including hexanal and (E)-2-hexenal were decreased in all affected grapes. Some unique VOCs may serve as hypothetical biomarkers to help us identify specific varieties of pathogenic fungal infestation.

Simultaneous determination of succinate-dehydrogenase-inhibitor fungicide traces in cereals by QuEChERS preparation and UPLC-MS/MS analysis.

A method for the simultaneous determination of 19 succinate dehydrogenase inhibitor (SDHI) fungicide residues in 8 kinds of cereals was established by combining UHPLC-MS/MS with the improved QuEChERS method. MgSO4 and octadecylsilane (C18) were used as the dispersive-solid phase extraction sorbent. The proposed method had good linearity in the range of 10-100 µg/L with correlation coefficients (R2 > 0.99). The limit of quantification of 19 fungicides was 10 µg/L, which is the minimum addition level of the method. The fortified recoveries of 19 SDHI fungicides at three levels were ranged from 79.57 % to 126.25 %. The developed method was utilized for the analysis of 45 real cereal samples, only 5 samples were detected with SDHI fungicides. The contents of the fungicides detected in the real samples are far lower than the MRL. The results indicated that the proposed method is reliable for detecting SDHI fungicides in cereals.

A polyketide synthase from Verticillium dahliae modulates melanin biosynthesis and hyphal growth to promote virulence.

BACKGROUND: During the disease cycle, plant pathogenic fungi exhibit a morphological transition between hyphal growth (the phase of active infection) and the production of long-term survival structures that remain dormant during "overwintering." Verticillium dahliae is a major plant pathogen that produces heavily melanized microsclerotia (MS) that survive in the soil for 14 or more years. These MS are multicellular structures produced during the necrotrophic phase of the disease cycle. Polyketide synthases (PKSs) are responsible for catalyzing production of many secondary metabolites including melanin. While MS contribute to long-term survival, hyphal growth is key for infection and virulence, but the signaling mechanisms by which the pathogen maintains hyphal growth are unclear. RESULTS: We analyzed the VdPKSs that contain at least one conserved domain potentially involved in secondary metabolism (SM), and screened the effect of VdPKS deletions in the virulent strain AT13. Among the five VdPKSs whose deletion affected virulence on cotton, we found that VdPKS9 acted epistatically to the VdPKS1-associated melanin pathway to promote hyphal growth. The decreased hyphal growth in VdPKS9 mutants was accompanied by the up-regulation of melanin biosynthesis and MS formation. Overexpression of VdPKS9 transformed melanized hyphal-type (MH-type) into the albinistic hyaline hyphal-type (AH-type), and VdPKS9 was upregulated in the AH-type population, which also exhibited higher virulence than the MH-type. CONCLUSIONS: We show that VdPKS9 is a powerful negative regulator of both melanin biosynthesis and MS formation in V. dahliae. These findings provide insight into the mechanism of how plant pathogens promote their virulence by the maintenance of vegetative hyphal growth during infection and colonization of plant hosts, and may provide novel targets for the control of melanin-producing filamentous fungi.

A secreted ribonuclease effector from Verticillium dahliae localizes in the plant nucleus to modulate host immunity.

The arms race between fungal pathogens and plant hosts involves recognition of fungal effectors to induce host immunity. Although various fungal effectors have been identified, the effector functions of ribonucleases are largely unknown. Herein, we identified a ribonuclease secreted by Verticillium dahliae (VdRTX1) that translocates into the plant nucleus to modulate immunity. The activity of VdRTX1 causes hypersensitive response (HR)-related cell death in Nicotiana benthamiana and cotton. VdRTX1 possesses a signal peptide but is unlikely to be an apoplastic effector because its nuclear localization in the plant is necessary for cell death induction. Knockout of VdRTX1 significantly enhanced V. dahliae virulence on tobacco while V. dahliae employs the known suppressor VdCBM1 to escape the immunity induced by VdRTX1. VdRTX1 homologs are widely distributed in fungi but transient expression of 24 homologs from other fungi did not yield cell death induction, suggesting that this function is specific to the VdRTX1 in V. dahliae. Expression of site-directed mutants of VdRTX1 in N. benthamiana leaves revealed conserved ligand-binding sites that are important for VdRTX1 function in inducing cell death. Thus, VdRTX1 functions as a unique HR-inducing effector in V. dahliae that contributes to the activation of plant immunity.

The dissipation, processing factors, metabolites, and risk assessment of pesticides in honeysuckle from field to table.

Globally, honeysuckle is consumed as a food and administered as a medicinal agent. However, pesticide residues in honeysuckle limit its application and development of the honeysuckle industry, affecting food safety and endangering human health. Here, the degradation kinetics of 11 typical pesticides, including insecticides, fungicides, and an acaricide, in honeysuckle were investigated. The half-lives of pesticides in Henan and Liaoning fields were 1.90-4.33 and 2.05-4.62 d, respectively. The processing factors (PFs) of these pesticides after oven, sun, and shade drying ranged from 3.52 to 11.2. After decocting, the PFs of the pesticides were <1. Twenty degradation products were identified using ultra high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, and pathways were proposed based on drying and decoction. The ecotoxicities of the degradation products were evaluated using the Toxicity Estimation Software Tool. Finally, the acute hazard indices of these pesticides, as determined via dietary exposure assessment combined with the PFs, were 0.227 and 0.911 for adults and children, respectively. Thus, special populations, such as children, require particularly careful risk control in terms of dietary exposure.