Although Tongguan capsule (TGC) is used in the treatment of coronary atherosclerotic disease, the exact mechanism remains unclear.Network pharmacology and experimental validation were applied to examine the mechanism of TGC for treating myocardial ischemia-reperfusion injury (MIRI).-/- mice were divided into three groups (n = 6): Sham group, I/R group, and TGC group (1 g/kg/d). To further verification, HCAEC cells were subjected to hypoxia-reoxygenation (H/R) to establish in vitro model.The components and candidate targets were searched based on various databases such as TCMSP, TCMID, BATMAN-TCM. The binding ability was determined by molecular docking. The ischemia-reperfusion (I/R) model was constructed by ligating the left anterior descending (LAD) coronary artery. APOEIn vivo, TGC improved LVEF and FS, reducing infarct size. In vitro, Hoechst 33258 staining result showed TGC inhibited apoptosis. Compare with the H/R model, TGC treatment increased the levels of GSK-3β, LC3, and Beclin1, while decreasing the expression of mTOR and p62 (p < 0.05).The compounds, such as quercetin, luteolin, tanshinone IIA, kaempferol and bifendate, were obtained after screening. The affinity values of the components with GSK-3β, mTOR, Beclin-1, and LC3 were all <-5 kcal/mol. The findings revealed that TGC exerted a cardioprotective effect by up regulating autophagy-related proteins through the mTOR pathway, which may be a therapeutic option for MIRI. However, there are still some limitations in this research. It is necessary to search more databases to obtain information and further demonstrated through randomized controlled trials for generalization. Reperfusion therapy is a milestone achievement in the management of acute myocardial infarction (AMI) , Salvia miltiorrhiza Bunge (Lamiaceae), Borneol, and Leech, play the role on promoting blood circulation, invigorating qi, replenishing energy and improving metabolism in TCM  may have its own unique role in the treatment of MIRI , TCMID (http://119.3.41.228:8000/tcmid/) and BATMAN-TCM (http://bionet.ncpsb.org.cn/batman-tcm/). The targets of the compounds were obtained through TCMSP, Swiss TargetPrediction (http://swisstargetprediction.ch/), and STITCH (http://stitch.embl.de/). Simultaneously, the UniProt database (https://www.uniprot.org/) was applied for gene name standardization.The TGC active ingredients, with drug-likeness (DL) ≥0.18 and oral bioavailability (OB) ≥30%, were screened from TCMSP (https://www.genecards.org), Online Mendelian Inheritance in Man , and TTD (http://db.idrblab.net/ttd/). The overlapping genes with MIRI and active components were identified as candidate targets.The targets related to ‘myocardial ischemia-reperfusion injury’ were retrieved through the GeneCards (https://string-db.org/), and the condition was set as Homo sapiens with a confidence of 0.95 to obtain the relevant information on protein interactions. PPI network was constructed through Cytoscape (V3.7.1) and CytoNCA plug-in was devoted to calculate the topology index, including betweenness centrality (BC), closeness centrality (CC), degree centrality (DC), eigenvector centrality (EC), local average connectivity-based method (LAC), and network centrality (NC). The genes greater than the median of each topological index were regarded as core targets.The candidate targets were imported into String  and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the core targets.With https://pubchem.ncbi.nlm.nih.gov/), and 3D chemical structures were created by ChemBioDraw software. Protein structures were searched from PDB (https://www.rcsb.org/) according to UniProt ID. Water molecules and ligands of protein were removed by Discovery Studio. AutoDock Vina software was used for molecular docking with the binding site of the protein’s own ligand as the active pocket.The 2D structures of important compounds were obtained from PubChem  were performed in compliance with the regulations of the Second Affiliated Hospital of Guangzhou University of Chinese Medicine institutional animal care  and conducted according to the AAALAC and IACUC guidelines : Sham group, I/R group, and TGC group (1 g/kg/d). During I/R surgery, the left anterior descending (LAD) coronary artery was ligated with 8–0 nylon suture. The ST segment elevation on the electrocardiogram and the color change of myocardial tissue indicated that myocardial ischemia was successfully established. Sutures were released after 45 min to induce 24 h of reperfusion. Mice in sham group underwent similar surgery without LAD coronary artery occlusion. All mice were given intragastric administration 5 days before surgery. The TGC group was administered TGC , while the sham group and the I/R group were administered the same volume of normal saline.The operations were completed in the Laboratory Animal Center of the Second Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine. Mice were randomly divided into 3 groups , and heart function was assessed by transthoracic echocardiography . After measurement, left ventricular ejection fraction (LVEF) and fractional shortening (FS) were recorded.After echocardiography was completed, the infarct size of the heart was determined by Evans blue-TTC staining. Briefly, the LAD coronary artery was sutured, and Evans blue solution  was injected into heart vessels through the aorta. The heart was quickly removed and snap-frozen at −20 °C for 30 min, and then was cut into 1 mm thick slices and placed into TTC solution  at 37 °C for 15 min. The sections were fixed with a 4% paraformaldehyde solution. Image-Pro Plus software was devoted to assess infarct size (IA), area at risk (AAR), and total left ventricular area (LV), from which IA/AAR and IA/LV were calculated.2/95% air at 37 °C. Before the experiment, the cells were regularly subcultured until they reached 80% confluence.HCAECs from BeNa Culture Collection, China, were cultured in DMEM  supplemented with 10% FBS  and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO2, 5% CO2, 1% O2) for 3 h and then transferred to 10% fetal bovine serum  medium in a normoxic incubator for 1h. Cell in TGC group was treated with 100 μg/mL TGC solution for 7 h before oxygen-glucose deprivation (OGD). Cells in the control group were cultured under normal conditions.Cells were then divided into a control group, H/R group, and TGC group. Cells in H/R group were cultured in glucose-free medium with hypoxia  2 times and rapidly immobilized with 4% paraformaldehyde. The Hoechst 33258 solution  was then used to stain the cells for 15 min at room temperature. The images were observed under a fluorescence microscope .When performing immunofluorescence, the cell washing and immobilization steps were similar to those in Hoechst 33258 staining. Then, the cells were permeabilized in 0.5% Triton X-100 for 5 min and sealed in 5% BSA for 2 h. After being incubated with LC3 primary antibody  at 4 °C overnight, the cells were treated with fluorescently labeled secondary antibody  and DAPI  at room temperature. The images were observed under a fluorescence microscope, and the expression level was analyzed based on the fluorescence intensity.After H/R, the cells were dissolved in RIPA buffer containing protease and phosphoprotein inhibitors. A BCA kit  was used to detect the protein content. The protein samples were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% bovine serum albumin, the PVDF membrane was incubated with primary antibody at 4 °C overnight and with secondary antibody  at room temperature for 1 h. The primary antibodies were GAPDH , GSK-3beta , P-GSK-3β , mTOR , P-mTOR , Beclin1 , and p62 . The band development was performed with an ECL kit  and the results were analyzed by Image J software.p < 0.05.All data were expressed as the mean ± SD and statistically analyzed by GraphPad Prism 8 software. Statistical significance analysis was performed using one-way ANOVA. The difference was considered statistically significant at Based on TCMSP, TCMID, and BATMAN, 91 active compounds met the screening conditions. A total of 1143 targets of the active compounds were searched from TCMSP, Swiss TargetPrediction, and STITCH. 1302 MIRI-related targets were identified according to GeneCards, OMIM, and TTD. A total of 393 overlapping genes both in MIRI and TGC were recognized as candidate targets. By calculating the median of topological indexes, 95 core targets were obtained, including mTOR, GSK-3β, AKT1, PIK3CA, and PIK3R1 .quercetin, luteolin, tanshinone IIA, kaempferol, and bifendate.In the network , 90 compGO enrichment analysis  indicateKEGG analysis revealed that TGC might improve MIRI by regulating autophagy-related proteins in the mTOR pathway. To further verify the interaction of TGC with targets in this pathway , we perfp < 0.05). After TGC treatment, these two parameters were significantly increased (p < 0.05), suggesting that TGC was beneficial to improve cardiac function after MIRI , indicating that TGC could reduce the proportion of myocardial infarction area . It was further suggested that TGC inhibited apoptosis of coronary endothelial cells by regulating autophagy-related proteins in the mTOR pathway.As shown in western blot , after tNetwork pharmacology analysis suggested that the important active ingredients in TGC, including quercetin, luteolin, tanshinone IIA, kaempferol and bifendate improved MIRI through mTOR pathway and autophagy. Experiment has further determined that TGC reduced the area of myocardial infarction and increased the left ventricular ejection fraction, by regulating autophagy-related proteins in the mTOR pathway to inhibit cell apoptosis.Previous studies have shown that the level of autophagy affected the repair of ischemia-reperfusion injury (Zhang et al. Autophagy initiation is regulated by multiple signaling pathways (Dikic and Elazar As a conserved serine/threonine kinase, GSK-3β exerts biological effects by controlling mTOR (Urbanska et al. The important active components of TGC have a role in protecting the heart by regulating autophagy. Quercetin alleviates oxidative stress-induced damage and restores mitochondrial functions by enhancing autophagy (Daw and Law Molecular docking results indicated that the important compound had a high affinity for the target, suggesting that TGC may protect the heart through autophagy proteins associated with the mTOR pathway. Based on autophagy, exploring the mechanism of TGC in the treatment of MIRI could further clarify the drug target, which was conducive to the research and development of novel medicine for coronary artery disease.Based on network pharmacology and experimental studies, we found that TGC exerted a cardioprotective effect against MIRI, by upregulating autophagy-related proteins through mTOR pathway . These d


Occupational stress and its health effects on occupational populations have attracted extensive attention from researchers in public health. The stressors faced by employees of power grid enterprises are increasing progressively, which is easy to cause occupational stress. The balance of the body’s oxidative-antioxidant levels plays an essential role in maintaining the body’s health status. This study aims to explore occupational stress and its correlation with oxidative-antioxidant levels in employees of a power grid enterprise.A cluster random sampling method was used to investigate the basic information of 528 employees in a power grid enterprise and investigate the two occupational stress models of employees by using the Job Content Questionnaire based on the job demand-control-support (JDC) model, and the Effort-Reward Imbalance Questionnaire based on the effort-reward imbalance (ERI) model, respectively. Peripheral blood samples were collected from the employees to measure the levels of malondialdehyde (MDA), total antioxidant capacity (TAC), and superoxide dismutase (SOD). The correlation between different models of occupational stress level and the body’s oxidation-antioxidation level was further explored.P < 0.05). The ERI model occupational stress was significantly associated with visual display terminal operation . The results of the generalized additive model showed that SOD levels had a non-linear relationship with the D/C ratio as well as the E/R ratio. With the D/C ratio close to 1, SOD levels raised rapidly. When the E/R ratio exceeded 1, the SOD level raised rapidly  . TAC levels were negatively associated with the E/R ratio (P < 0.05).The detection rate of high JDC model occupational stress was 50.6% and the detection rate of high ERI model occupational stress was 50.9%. The JDC model occupational stress was significantly associated with high-temperature and high-altitude operation, visual display terminal operation, monthly income, and exercise (all The detection rates of occupational stress in both models among employees in a power grid enterprise are higher. ERI model occupational stress was associated with body TAC and SOD levels, and JDC model occupational stress was associated with body SOD levels.The online version contains supplementary material available at 10.1186/s12888-022-04226-1. In recent years, occupational stress, characterized by increased job strain and effort-reward imbalance, has been detected more frequently due to changes in working conditions and organizational forms caused by globalization, technological innovation, and digitalization . As a siThere was moderate level evidence from previous studies that high job demands, low job control, high effort-reward imbalance, low relational justice, low procedural justice, role stress, bullying, low social support in the workplace, and other occupational stressors are associated with mental health . IntenseThe job demand-control (JDC) model and the effort-reward imbalance (ERI) model are two assessment models that have an excellent explanatory effect on occupational stress. The combination of the JDC and ERI models can explain more differences in health outcomes and provide a basis for the formulation of occupational stress interventions . The JDCWith the rapid development of the social economy, grid enterprises’ workload of power generation, transmission, and distribution has gradually increased . Power gOxidative stress (OS) is the imbalance of oxidative and antioxidant levels caused by the excessive synthesis of reactive oxygen species (ROS) or the disturbance of the body’s antioxidant system, which eventually leads to adverse reactions such as apoptosis and inflammation . OxidatiStudies have shown that various stressors can increase glucocorticoids by activating the hypothalamic-pituitary-adrenal (HPA) axis and that the inflammatory responses caused by stress can be dynamically regulated with oxidative stress processes to help the body adapt to different environments and maintain homeostasis in the body, which may have protective or detrimental effects . There iA cluster random sampling method was used to select 528 workers from a power grid enterprise in Guangdong as the study population. Inclusion criteria for the study participants included age ≥ 18 years, working years≥1 year, no diagnosed psychiatric disorders and no family history of psychiatric disorders. The questionnaires were investigated face-to-face by trained investigators. Informed consent was obtained from participants. The study was reviewed and approved by the Committee for Medical Ethics of Guangdong Province Hospital for Occupational Disease Prevention and Treatment.The survey includes personnel number, general demographic characteristics , occupational characteristics , occupational hazards  and lifestyle . Smoking was defined as cumulative smoking > 100 cigarettes , drinkinThe Chinese version of the Job Content Questionnaire (JCQ) and the Effort-Reward Imbalance Questionnaire (ERIQ) were used to assess the JDC model and the ERI model occupational stress, respectively. Two kinds of questionnaires both used the 4-point Likert scale. Grading was scored on a scale from 1 to 4, which corresponded from ‘disagree entirely’ to ‘agree entirely’ in sequence. The JCQ contains three dimensions of job demand (5 items), job control (9 items), and social support (8 items), with a total of 22 items, The Cronbach’s alpha coefficients of three dimensions and the overall questionnaire were 0.767, 0.724, 0.912, and 0.898, respectively. The ERI contains three dimensions of effort (6 items), reward (11 items), and over-commitment (6 items), with a total of 23 items. The Cronbach’s alpha coefficients of three dimensions and the overall questionnaire were 0.841, 0.710, 0.728, and 0.863, respectively. The D/C ratio was calculated using the formula “job demand/(job control × 5/9)” . The D/CTwo weeks before the survey, the subjects were advised to avoid excessive alcohol consumption, a high-fat diet, and medication. All subjects have fasted for 12 hours before blood sample collection. Five milliliters peripheral venous blood was drawn from all subjects when the investigation was performed. Then plasma was collected by centrifugation at 4 °C, 2000 r/min for 5 min. The levels of MDA, TAC, and SOD in plasma were measured according to the instructions for the Lipid Oxidation (MDA) Assay Kit, Total SOD Activity Assay Kit (WST-8 method), and Total Antioxidant Capacity Assay Kit (ABTS method), respectively . The levels of MDA, TAC and SOD were measured at 535 nm, 734 nm, and 450 nm respectively using an enzyme marker. The levels of each oxidative-antioxidant index were calculated based on the standard curves. MDA, TAC, and SOD levels were measured in μM, mM, and units, respectively.Before the survey, the investigators are trained uniformly and introduced to the survey content and precautions for subjects. After a detailed explanation of the purpose and content of the survey by the surveyor to the respondents before the survey, face-to-face questioning is used to collect information from the study participants. The questionnaires were checked and collected on-site. Questionnaires with more than 10% of entries missing were rejected as invalid. Epidata double entry and consistency checks were carried out to ensure that the data were authentic and reliable.2 test), we identified any significant variables on occupational stress. To further made summary models, binary logistic regression analysis was used to examine the association between individual characteristics and occupational stress . The logistic regression models took into account all individual characteristic variables with P < 0.05 in the Pearson χ2 test and took the occupational stress group as the dichotomous outcome variable. Dummy variables were created for multi-categorical covariates (monthly income and sleeping time). Odds ratios (ORs) and 95% confidence intervals (95% CIs) for each variable were reported.EpiData 3.1 was used to build the database, and SPSS 25.0 software as well as R version 4.0.5 was used for statistical analysis. Firstly, we examined the association between individual characteristics and the detection rate of occupational stress. By comparing rates of count data  for each variable. The test level α = 0.05 (two-sided).Measurement data of oxidative-antioxidant levels were described as mean ± standard deviation. The comparison of means between two groups was performed using a t-test for two independent samples of data. The comparison of means among multiple groups was performed using ANOVA, and a two-by-two comparison between groups was performed using the Bonferroni test. To further examine the relationship between occupational stress and oxidative-antioxidant levels, we used generalized additive models (GAM) to conduct smooth curve fitting. The R package adopted in generalized additive models is the mgcv package. MDA, TAC, and SOD levels were analyzed as dependent variables, and E/R ratio and D/C ratio as independent variables respectively. The generalized cross-validation (GCV) method was used to select the number of nodes, and the cubic spline function was used to fit the curve. For the oxidative indexes that did not show a non-linear relationship with occupational stress, multiple linear regression was used to further explore variables significantly related to oxidative-antioxidant levels . Dependent variables were oxidative-antioxidant levels and independent variables with Among the 528 study subjects, 463 (87.7%) were male, and 65 (12.3%) were female. The average age was (39.04 ± 9.94) years old, and the average working years was (17.26 ± 11.21) years. More people were married, accounting for 76.1%. And 52.3% of those with an educational level of undergraduate. The work categories were mainly in substation operation and maintenance, accounting for 44.5 and 35.4%, respectively. There were 373 shift workers, accounting for 70.6%. Employees’ monthly income was mainly 5000 ~ and 7000 ~ yuan, accounting for 39 and 33%, respectively. Electromagnetic radiation exposure and high-temperature and high-altitude operation were more numerous, accounting for 62.5 and 62.7%, respectively. In comparison, noise exposure and visual display terminal operation accounted for 33.9 and 33.3%, respectively. The proportion of employees who smoke, drink and exercise is higher, accounting for 30.3, 65.5, and 75.6%, respectively. Most of the employees’ sleeping time was 6 ~ hours, accounting for 46.4%.P < 0.05). In the ERI model, the differences in the detection rates of occupational stress between the groups of gender, age, marital status, working years, noise, electromagnetic radiation, high-temperature and high-altitude operation and, visual display terminal operation were all statistically significant .As shown in Table P < 0.05) in Table To examine the association between sociodemographic variables and occupational stress in two different models, binary logistic regression analysis was performed by taking JDC and ERI occupational stress as dependent variables and the variables with the statistical difference (P < 0.05). The differences in TAC levels among workers of different age, smoking, and drinking were statistically significant .The mean level of MDA, TAC, and SOD of the 528 participants was 1.907 ± 1.231 μM, 1.288 ± 0.175 mM, and 0.354 ± 0. 095 units, respectively. As shown in Table P < 0.05). In the ERI model, the serum TAC levels of employees in the high occupational stress group were lower than those in the low occupational stress group, and the differences were statistically significant (P < 0.05). See Table In both JDC and ERI models, the serum SOD levels of employees in the high occupational stress group were higher than those in the low occupational stress group, and the differences were statistically significant P < 0.0. In the P < 0.05). The non-linear relationships between TAC with E/R ratio are approximately statistically significant (P = 0.04)  .The results of generalized additive models showed there were non-linear relationships between SOD with E/R ratio as well as D/C ratio, and the difference was statistically significant 4) Fig. S. However) Figs. S and S4.  Fig. S. P < 0.05 in Table P < 0.05 in Table The results of multiple linear regression analysis were shown in Fig. Among occupational populations, general demographic characteristics, occupational characteristics, occupational hazards, and lifestyle all have potential impacts on occupational health. We conduct research on occupational stress as well as oxidative-antioxidant levels in specific occupational groups, and various variables on these aspects were looked at. In this case, a significant result may be obtained by chance by multiple comparisons. To avoid the statistical bias caused by the difference of variables in the multivariate analysis, we used the conservative Bonferroni test for a two-by-two comparison of means among multiple groups, performed smooth curve fitting by the generalized additive model, and only included significant variables in the univariate analysis as covariates to adjust the multivariate model.The study result reveals that the detection rate of JDC and ERI model occupational stress among employees in a power grid enterprise was 50.6 and 50.9%, respectively. Our results were higher than Li et al. , who repMartinez et al.  used theOur study found that MDA levels were significantly associated with marital status and electromagnetic radiation exposure and TAC levels were significantly associated with drinking. However, there were no statistically significant differences between the two models of occupational stress and MDA levels among employees in a power grid enterprise. Salem et al.  surveyedP = 0.04). Multiple linear regression analysis found a negative association between the ERI model occupational stress and TAC levels (P = 0.043), but this was much less significant than the correlation between the E/R ratio and SOD. This may be because our questionnaires did not contain dietary status factors to adjust for the relationship between occupational stress and TAC levels, leading to underestimation or overestimation of the association between exposure and outcome. Although non-enzymatic antioxidants are involved in maintaining redox homeostasis, the TAC levels may be reduced due to the dietary habits and composition of employees who were unable to supplement with antioxidants in a timely manner [Our results showed an approximately statistically significant non-linear relationship between the ERI model occupational stress and TAC levels(y manner .In addition to non-enzymatic antioxidants such as endogenous substances like bilirubin and nutritional compounds such as tocopherols, antioxidant enzymes such as SOD, as important antioxidant indicators, can enhance cellular defense by effectively neutralizing increased levels of oxidation in the body . And anoThere are some limitations. For example, this study is a cross-sectional study of a power grid enterprise, and it is difficult to determine the causal association between occupational stress and oxidative stress levels. In addition, this study did not include a survey on diet, which may affect the analysis of the results. And this information needs to be considered in subsequent studies on occupational stress and oxidative-antioxidant levels in large samples.In summary, monthly income, exercise, high-temperature, and high-altitude operation, and video display terminal operation are factors associated with the occupational stress of employees in power grid enterprises. JDC model occupational stress, ERI model occupational stress, drinking, EMR, and marital status are correlated with oxidative-antioxidant levels. With the development of power grid enterprises, the impact of changes in the working environment and job content on the mental health of workers may lead to changes in TAC and SOD levels. Power grid enterprises should introduce advanced equipment, improve the level of automation, reduce the difficulty of operation, and at the same time provide and ensure the supply to employees with cooling, anti-electromagnetic radiation, visual fatigue relief, and other labor protection appliance. It is also vital for enterprises to focus on employees’ work difficulties and mental health, especially high-income earners, which need to arrange regular medical check-ups and training on mental health for employees. Employees should also maintain moderate exercise, combine work and rest, reduce alcohol consumption, eat a balanced diet, and take timely supplements of foods rich in antioxidants.There are few studies on the correlation between occupational stress and oxidative stress levels. Based on the occupational populations of power grid enterprise employees, our study comprehensively analyzes the correlation between two models occupational stress and oxidative stress from demographic characteristics, occupational characteristics, and lifestyle, especially in occupational hazardous. It could provide a scientific basis for early intervention and health risk assessment research of occupational stress in the occupational populations in power grid industry.Additional file 1: Fig. S1. Smooth curve fitting by the generalized additive model of TAC level and E/R ratio.Additional file 2: Fig. S2. Smooth curve fitting by the generalized additive model of TAC level and D/C ratio.Additional file 3: Fig. S3. Smooth curve fitting by the generalized additive model of MDA level and E/R ratio.Additional file 4: Fig. S4. Smooth curve fitting by the generalized additive model of MDA level and D/C ratio.


Diabetes is associated with an increased risk of cancer. Prostasin is an epithelial sodium channel stimulator that has been associated with suppression of tumours, glucose metabolism and hyperglycaemia-associated tumour pathology. However, the association between prostasin, diabetes and cancer mortality has not been well investigated in humans. We aim to investigate the associations between plasma prostasin and diabetes, and to explore whether prostasin has an effect on cancer mortality risk in individuals with hyperglycaemia.Plasma prostasin was measured using samples from the Malmö Diet and Cancer Study Cardiovascular Cohort, and statistical analysis was performed from both sex-specific quartiles and per 1 SD. The cross-sectional association between plasma prostasin and diabetes was first studied in 4658 participants . After excluding 361 with prevalent diabetes, the associations of prostasin with incident diabetes and cancer mortality risk were assessed using Cox regression analysis. The interactions between prostasin and blood glucose levels as well as other covariates were tested.p for trend <0.0001). During mean follow-up periods of 21.9 ± 7.0 and 23.5 ± 6.1 years, respectively, 702 participants developed diabetes and 651 died from cancer. Prostasin was significantly associated with the incidence of diabetes. The adjusted HR for diabetes in the 4th vs 1st quartile of prostasin concentrations was 1.76  (p for trend <0.0001). Prostasin was also associated with cancer mortality There was a significant interaction between prostasin and fasting blood glucose for cancer mortality risk (p for interaction =0.022), with a stronger association observed in individuals with impaired fasting blood glucose levels at baseline .The adjusted OR for prevalent diabetes in the 4th vs 1st quartile of prostasin concentrations was 1.95   contains peer-reviewed but unedited supplementary material. Prostasin, also known as protease serine S1 family member 8, is an extracellular serine protease with a trypsin-like cleavage specificity . ProstasIn addition to regulating ENaCs, accumulating evidence from expression, cell culture and animal studies supports a suppressive effect of prostasin on tumours at various sites including head, neck and oral squamous cells, breast, colorectum and prostate , 7. ConsCurrent evidence regarding the role of prostasin in the development of diabetes is limited. Two experimental studies have reported a protective effect of hepatic prostasin against glucose and lipid metabolism dysfunction , 17. In To our knowledge, no epidemiological study has yet investigated the association of prostasin levels with diabetes or cancer mortality in humans. In this study, based on a large Swedish population, we aim to investigate the cross-sectional and longitudinal associations between plasma prostasin levels and diabetes, as well as the association between prostasin and cancer mortality. We also explore whether prostasin and its interaction with baseline hyperglycaemia have an effect on cancer mortality risk.n=6103) of participants from the Malmö Diet and Cancer Study were invited to participate in a sub-cohort study, the Malmö Diet and Cancer Study Cardiovascular Cohort [The Malmö Diet and Cancer Study is a large population-based prospective study from Malmö, a city in southern Sweden . During r Cohort . Of thesr Cohort ). TherefAll participants provided written informed consent. The study conformed to the ethical guidelines of the 1975 Declaration of Helsinki, and was approved by the Lund University Ethical Committee .www.ScilifeLab.se), and the same batches of reagents were used for sample assays. Olink NPX Manager Software was used for quality control of the samples and technical performance of the assays. During this process, four internal controls were added for all samples, and external controls were used in every analysis. The lower and upper limits of quantification of prostasin were approximately 0.24 and 7800 pg/ml, respectively. The potential impact of haemolysis on prostasin quantification was evaluated by using serial dilutions of haemolysate (0.25–15 mg/ml) in EDTA-treated plasma. The highest concentration of haemolysate (15 mg/ml) had no impact on assay performance. The intra-assay (within a run) and inter-assay (between runs) coefficients of variation were 5% and 19%, respectively. A pre-processing normalisation procedure was used to normalise technical variation within one run and between runs, by subtracting the Cq value for the extension control and the interplate control for each assay, respectively. The data were set relative to a correction factor, and are expressed as normalised protein expression (NPX) values on a log2 scale. The Olink webpage (http://www.olink.se) provides detailed information regarding proteomic panels, proteomics extension assay technology, assay performance, quality control and validation.Information regarding smoking habits, alcohol consumption, current medication, leisure-time physical activity and educational level was obtained from the questionnaire. Participants were classified as smokers or non-smokers (including former smokers and never smoker). A daily alcohol intake >40 g for men and >30 g for women was considered to be a high level of alcohol consumption. Physical activity was represented by an overall leisure-time physical activity score calculated by multiplying the duration of specific activities by the corresponding intensity coefficient. Education was categorised as elementary or less, primary, secondary, upper secondary and further education without a degree and with a university degree. Waist circumference (cm) was measured midway between the iliac crest and the lowest rib margin. BP (mmHg) was measured using a mercury-column sphygmomanometer after a 10 min rest in a supine position. Fasting blood glucose levels (mmol/l) were measured from fresh plasma samples collected from the cubital vein after an overnight fast, according to standardised procedures, at the Department of Clinical Chemistry, Skåne University Hospital. LDL-cholesterol levels (mmol/l) were estimated using Friedewald’s formula. Fasting plasma insulin (mU/l) was measured using radioimmunoassay, and values were converted into pmol/l using the conversion factor 6.945. HOMA2-IR was calculated using a HOMA2-IR calculator . C-react1c Register). In the Swedish National Diabetes Register and the Regional Diabetes 2000 Register, diabetes was diagnosed according to established criteria (fasting plasma glucose concentration ≥7.0 mmol/l with two repeated tests on separate occasions). In the nationwide Swedish drug prescription register, a filled prescription of insulin or glucose-lowering medications (ATC code A10) was required for diagnosis of diabetes. In the Swedish inpatient and outpatient registers, diabetes was diagnosed by a senior physician according to the established criteria. In the Malmö HbA1c Register, individuals were considered to have developed diabetes if they had at least two HbA1c recordings ≥42 mmol/mol (≥6.0%) using the Swedish Mono-S standardisation system .For analyses using diabetes as the endpoint, participants free of diabetes at baseline were followed until incident diabetes, emigration from Sweden, death or the end of follow-up (31 December 2019), whichever came first. Incident cases of diabetes were identified by linkages to both local and national registers, and have been described in detail previously . In briehttp://www.icd9data.com/2007/Volume1/default.htm) or ICD-10 codes C or D00–D48 (http://apps.who.int/classifications/icd10/browse/2016/en), and cardiovascular mortality was defined as ICD-9 codes 390–459 or ICD-10 codes in chapter I, as underlying cause of death.The Swedish cause of death register was used to monitor cause-specific mortality. Cancer mortality was defined as ICD-9 codes 140–239  and were natural-logarithmically transformed before analyses, otherwise results are presented as means ± SD. Categorical variables are presented as numbers and percentages. Differences across quartiles were tested by linear regression for continuous variables and by logistic regression for categorical variables.p values <0.05 (two-sided). All analyses were performed using SAS version 9.3 for Windows .The cross-sectional association between prostasin and diabetes was analysed by multivariable logistic regression using three models. Model 1 assessed the crude association. Model 2 was adjusted for age, sex and waist circumference. Model 3 was additionally adjusted for smoking and drinking habits, LDL-cholesterol, systolic BP and anti-hypertensive medication. After excluding prevalent diabetes, the associations between prostasin (as the dependent variable) and baseline fasting blood glucose levels, plasma insulin levels and HOMA2-IR were assessed by multivariable linear regression after adjusting for the above-mentioned confounders. The association between baseline prostasin and incident diabetes was estimated using a Cox proportional hazards regression model, with time-on-study as the timescale. HRs and 95% CIs were calculated. Prostasin was analysed both as a continuous variable (per 1 SD) and grouped variable (quartiles). Potential covariates taken into account were age, sex, waist circumference, smoking and drinking habits, LDL-cholesterol, systolic BP and anti-hypertensive medication. Fasting blood glucose levels or HOMA2-IR were further adjusted for in a separate model, that may be considered as an overadjusted model. Other sensitivity analyses were performed, including additionally adjusting for CRP, eGFR, physical activity and educational level or lipid-lowering drugs, or adjusting for BMI instead of waist circumference in the final model. Possible interactions between prostasin and covariates in relation to diabetes risk were tested by incorporating interaction terms in the multivariable model. The proportional hazard assumption was tested by evaluating the time-dependent effects of prostasin on diabetes risk, and was also visualised using Kaplan–Meier curves. The linearity assumption of the association was examined using restricted cubic spline functions, with knots placed at 20%, 40%, 60% and 80% of prostasin concentration. We also analysed the association between prostasin (both by sex-specific quartiles and by SD) and mortality  after adjusting for confounders including prior cardiovascular diseases or cancer, where appropriate. Sensitivity analyses were then performed after excluding prior cardiovascular diseases or cancer. The interaction between prostasin and fasting blood glucose levels was evaluated in relation to mortality risk. If an interaction was detected post hoc, analyses were separately performed after dividing participants according to their blood glucose level. Possible effect modifications by competing risk of death were explored using the Fine–Gray proportional sub-distribution hazards models method . C-statip for trend <0.0001). The corresponding OR per 1 SD increase in prostasin was 1.32 .The study population for the cross-sectional analysis included 4658 participants , of whom 361 (7.75%) had prevalent diabetes. The distribution plot of prostasin concentration is shown in ESM Fig. n=1654) and 8.18 ± 0.48 NPX for women (n=2643) (mean ± SD). Baseline characteristics for this group, by quartiles of prostasin, are shown in Table p<0.0001; ESM Table After excluding participants with prevalent diabetes, the study population included 4297 participants (age 57.3 ± 5.9 years). The prostasin concentration was 8.44 ± 0.44 NPX for men n=654 and 8p for trend <0.0001) and that per 1 SD increase in prostasin was 1.23 . The association was attenuated after further adjusting for fasting blood glucose levels or HOMA2-IR in sensitivity analyses, or correcting for competing risk of death, but remained significant. The results were essentially unchanged in all other sensitivity analyses . The association between prostasin and diabetes was more evident among younger participants, in those without impaired fasting blood glucose levels  (Table p for non-linearity =0.30), and there was no evidence that the association was time-dependent  and that per 1 SD increase in prostasin was 1.13  (Table p for interaction =0.022). The HR for cancer mortality were 1.52  and 1.11  per 1 SD increase in prostasin, respectively, among participants with (n=377) and without (n=3920) impaired fasting blood glucose levels at baseline (data not shown).Over a mean follow-up period of 23.5 ± 6.1 years, 651 participants died from cancer. Prostasin was significantly associated with cancer mortality. The cumulative cancer mortality in relation to prostasin quartiles is illustrated in ESM Fig. p for change in C-statistic =0.29 and 0.36, respectively) (data not shown). In addition, prostasin was found to be significantly and linearly associated with all-cause mortality (p for interaction =0.16), but no significant association was found for cardiovascular mortality , and there was no control for confounding in that study, which may explain why the results were different. To our knowledge, no previous evidence has been provided regarding the association between prostasin levels and the incidence of diabetes. The present results were generated from a large study population and with potential confounding factors taken into account. Moreover, the positive cross-sectional associations were confirmed by the prospective analyses. We have therefore addressed this knowledge gap by showing that plasma prostasin is positively associated with diabetes.Prostasin is usually described as a glycosylphos-phatidylinositol-anchored protein, but it also circulates in blood , 17, 28,Our current understanding of the role of prostasin in glucose metabolism comes from two experimental studies , 17. TheThere are several other possible explanations for the association between prostasin and diabetes that have not yet been tested. Recently, α-ENaC knockout in a murine pancreatic beta cell line has been found to increase cell viability and insulin content . We assuPRSS8, a gene encoding prostasin, was causally associated with Alzheimer’s disease [A recent Mendelian randomisation study by Pietzner et al suggested that  disease , possibl disease . Current disease , 17, inc disease . However disease , normal In this study, plasma prostasin levels were associated with cancer mortality in the general population. This association has not been investigated previously. Serum prostasin concentration has been proposed as a prognostic marker for ovarian cancer , 11 but The strengths of our study include the large sample size, the prospective design and the long-term follow-up. However, there are several limitations to this study. One is that prostasin levels were measured in arbitrary units , and thus could not be compared directly with absolute values. However, since the study cohort was taken from the general population, the overall prostasin levels in this study may be regarded as normal by definition. The prostasin concentration was measured using a Proseek Multiplex Oncology panel. This method uses paired DNA-labelled antibodies and has high specificity. We did not make any Bonferroni adjustments for biomarkers not analysed in this study. However, even with a conservative Bonferroni approach, the relationships between prostasin and incidence of diabetes would still be statistically significant. Prostasin levels were measured using frozen blood samples stored at −80°C for over a decade, and its stability during long-term storage is unclear. Nevertheless, the fact that a predictive ability of prostasin was observed in this study suggests that the analytes are sufficiently stable to preserve clinical signals, even though the identified values may not be exactly the same as if measured in fresh samples. In addition, serial measurements of prostasin and risk factors over time are lacking in this study. Another limitation is that diabetes types were not determined in this study. As all the participants in cohort analysis were middle-aged adults without pre-existing diabetes, it is very likely that the incident cases were type 2 diabetes. Incident diabetes cases were identified by multiple registers , which iIn conclusion, plasma prostasin is a new potential risk marker for development of diabetes and for cancer mortality risk, especially in individuals with high blood glucose levels, which may shed new light on the relationship between diabetes and cancer.ESM(PDF 515 kb)


Simultaneous tracking of the position of the ball and player locations and activities places high demands on visual attention in team ball sport athletes. Owing to their extensive sports training, these athletes may be expected to exhibit visual attention skills superior to non-athletes; however, the results of studies examining this are inconsistent. Thus, the first aim of this study was to assess the impact of participating in a team ball sport on visual attention. There is limited empirical evidence indicating a sex difference in visual attention, and few studies have reported on visual attention in male and female athletes. Thus, the second aim of this study was to determine whether team ball sport experience affected any sex differences in visual attention. In total, 44 highly skilled basketball players—22 men (mean age: 21.86 ± 2.15 years) with a mean (SD) of 8.46 (2.92) years training experience and 22 women (mean age: 21.32 ± 1.58 years) with a mean (SD) of 8.22 (2.44) years of training experience—and 44 non-athletic undergraduate college students—22 men (mean age: 21.62 ± 1.88 years) and 22 women (mean age: 21.55 ± 1.72 years)—were recruited and completed this study. Visual attention was measured by using the multiple object tracking (MOT) task. Skilled basketball players showed superior tracking accuracy to non-athletic college students on the MOT task. A significant sex difference was found only among the non-athletic college students, with better tracking accuracy for men than for women on the MOT task. By contrast, no significant sex difference was observed among the skilled basketball players for tracking accuracy on the MOT task. These findings indicated that team ball sport training may enhance visual attention as assessed by tracking accuracy. Given that the male and female basketball players in this study had similar training experience and game performance demands, long-term team ball sport experience appeared to minimize the sex difference in visual attention found among non-athletic students. Visual attention plays a crucial role in every task involving perceiving and acting, especially participating in sports. Visual attention underlies perceptual skills in sports , it may A number of studies have examined the relationship between team ball sport participation and visual attention. Many studies have demonstrated that compared with non-athletes, athletes participating in team ball sports exhibit superior visual attention . It is wA common finding in the general population is that males perform better than females on tasks measuring visuospatial skills owing to sociocultural and biological factors . HoweverAn important study demonstrated that sport experience reduce sex effects in visual attention , which sOne well-established paradigm used to investigate visual attention is multiple object tracking (MOT), which was originated by Therefore, the present study had two objectives. The first aim was to assess the impact of participating in a team ball sport on the development of visual attention. Derived from this aim, the second but main goal of this study was to investigate whether team ball sport experience eliminated sex differences in visual attention. Our hypotheses were twofold. First, we hypothesized that athletes involved in team ball sports would exhibit tracking performance superior to that of non-athletes in the MOT task. Second, we hypothesized that sex differences for tracking performance in the MOT task would be observed only among non-athletes, and that team ball sport activities would minimize these sex differences in athletes.Calculation of the required sample size was performed using G*Power 3.1.9. With an effect size of 0.35, a desired power of 0.90, and an alpha level of 0.05, we determined that the sample size would need to be 22 per group. Thus, a total of 44 basketball players were recruited from six China University Basketball Association (CUBA) teams. The players comprised 22 men (age: 21.86 ± 2.15 years) with 8.46 (SD = 2.92) years of training experience, and 12.70 (SD = 2.36) training hours per week as well as 22 women (age: 21.32 ± 1.58 years), with 8.22 (SD = 2.44) years of training experience and 12.62 (SD = 2.15) training hours per week. All basketball players were first- and second-level national athletes. The non-athlete group—composed of 22 men (age: 21.62 ± 1.88 years) and 22 women (age: 21.55 ± 1.72 years)—were undergraduate students who had never participated in basketball training or any other team ball sports. All participants reported normal or corrected-to-normal levels of visual function and were right-handed. The participants were asked to avoid staying up late  at night to ensure good sleep quality. All recruited basketball players and college students completed the study. The study protocol was approved by the ethics committee of Shanghai University of Sport (No. 2015003SUS). Informed consent  was obtained in writing prior to the test session. Each participant who completed the research was paid $10 in return for their time. None of the participants had solved a MOT test before or had been trained in MOT.The experiment was conducted using a Dell G5 15 laptop computer with MATLAB R2016a and Psychtoolbox 3 software. Visual stimuli were presented on a 15.6-inch (39.62 cm) monitor with a 1,920 × 1,080 resolution and a 60-Hz refresh rate. The participants were seated at a distance of 55 cm from the screen and tested individually in a quiet room and with low lighting. At the beginning of the task, the preparation screen was presented, which said “press the left mouse button to start the test” see . A whiteThe experimental procedure consisted of 40 trials divided into two blocks, with a rest period of 5 min between the blocks. The task took approximately 15 min to complete. Participants received five practice trials to ensure that they were familiar with the task procedure before the formal testing session.Data were recorded and collected by MATLAB R2016a software. IBM SPSS, version 23.0, software was used to calculate the tracking accuracy and to conduct statistical analyses. A univariate general linear model (UNIANOVA) analysis was carried out, with sex  and group  as independent variables, and tracking accuracy on the MOT task as a dependent variable. Further analyses were conducted using the simple effects test for significant interactions. Simple effects were analyzed by adding the following to the software analysis:/EMMEANS = TABLES (sex*group) COMPARE (group) ADJ (LSD),/EMMEANS = TABLES (group*sex) COMPARE (gender) ADJ (LSD). Effect size calculations were performed using Cohen’s d and the classifications as follows: 0.2, 0.5, and 0.8 for small, medium, and large effect sizes. An alpha level of 0.05 was considered statistically significant. We calculated tracking accuracy by determining the number of correctly selected targets across all 40 trails for each participant.F = 18.928, p < 0.001, ηP2 = 0.184, and indicated that males  had significantly better tracking performance than females . There was also a significant main effect of group, F = 83.494, p < 0.001, ηP2 = 0.498, indicating that basketball players  had significantly better tracking performance than students . In addition, a significant interaction was observed between sex and group, F = 83.494, p < 0.001, ηP2 = 0.498  had significantly better tracking performance than female students , p < 0.01, d = 3.02. An analysis by group showed that male students  had significantly better tracking performance than female students , p < 0.01, d = 1.94. However, there was no significant difference between male basketball players  and female basketball players   had significantly better tracking performance than male students , .09) see .The current study was designed (1) to investigate the differences in visual attention between team ball sport athletes and non-athletic college students and (2) to examine whether team ball sport experience affects sex differences in visual attention. Our results provided evidence to support our hypothesis that basketball athletes would show superior performance to non-athletes for tracking accuracy in a MOT task. Our results also showed a significant sex difference in target tracking accuracy on the MOT task in the non-athlete group. By contrast, no sex difference in target tracking accuracy on the MOT task was detected among the basketball players. Our results suggested that team ball sport experience was associated with the development of visual attention, which minimized the sex difference.Our results demonstrating that the tracking accuracy of basketball players was markedly better than that of non-athletic college students in the MOT task indicated that the effects of team ball sport experience transferred from a sport-specific task to the MOT task. This finding is consistent with those from other studies that found that basketball players exhibit better visual attention than non-athletic university students . In teamWe also found a significant sex difference in performance in the MOT task among the non-athletic college students, with men showing higher tracking accuracy than women. Our finding is consistent with a previous study showing an effect of sex in a MOT task, with men showing superior tracking performance . SimilarOf primary interest to us were our results showing a sex by group interaction. The college students showed a significant sex difference in tracking accuracy on the MOT task. However, as hypothesized, we found no statistically significant sex difference among the team ball sport athletes. To our knowledge, only a few studies have explored whether engaging in team sports minimizes sex differences in visual attention. One study reported that female volleyball athletes and male volleyball athletes exhibited similar visual selective attention capacity . AnotherThe current study has a few limitations. One major shortcoming was that the absence of examining whether discriminates less-skilled vs. expert athletes. These types of studies will provide a greater understanding of whether visual attention in non-domain specific tasks, such as the MOT task used in this study, results in fine-grained differences between levels of expertise. Furthermore, we didn’t investigate whether superior performance in non-domain specific MOT tasks is associated with superior performance in domain-specific decision-making tasks, such as the recognition paradigms and pattern recall , 2013. FIn conclusion, the results of the present research demonstrate that team ball sport players showed higher tracking accuracy than non-athletic college students in a MOT task. In addition, in the non-athlete college group, male students exhibited tracking performance superior to female students; by contrast, there was no such difference between male and female basketball athletes. The findings of this study indicate that the effects of long-term training in team ball sports transferred to improved visual attention and minimized sex differences in visual attention as assessed by tracking accuracy.The original contributions presented in this study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of the Shanghai University of Sport. The patients/participants provided their written informed consent to participate in this study.PJ and ZG contributed to the design of the study, data collection, and writing—original draft preparation. TF contributed to methodology and manuscript preparation. All authors read and agreed with the submitted version of the manuscript.


Spodoptera frugiperda; J.E. Smith (Lepidoptera: Noctuidae), is now an economically important pest that causes huge losses to maize productivity in sub-Saharan Africa. Variations in sub-population genetics and the processes of rapid adaptation underpinning the invasion remain unclear. For this, the genetic identity and diversity of FAW populations in Uganda were revealed by sequencing 87 samples (collected across the country). Based on the partial mitochondrial cytochrome oxidase I (COI) gene polymorphisms, we further examined the mitochondrial haplotype configuration and compared the FAW in Uganda with sequences from other parts of the world. The molecular target for organophosphate and carbamate resistance, acetylcholinesterase, was also investigated.The fall armyworm (FAW), COI gene sequences showed the presence of both rice (predominant) and corn strain haplotypes, with a haplotype diversity of 0.382. Based on the COI marker, pairwise difference distribution analyses, and neutrality tests, showed that the FAW populations in Uganda and the rest of Africa are evolving neutrally, but those in America and Asia are undergoing expansion. Our findings support observations that invasive FAW populations throughout the rest of Africa and Asia share a common origin. Sequencing of the S. frugiperda ace-1 gene revealed four amino acid substitutions, two of which (A201S and F290V) were previously shown to confer organophosphate resistance in both S. frugiperda and several other insect species. The other two previously reported new variations in positions g-396 and g-768, are presumed to be related to the development of insecticide resistance.Analysis of the partial COI, more evolutionary studies incorporating the Spodoptera frugiperda whole-genome sequence are required to precisely understand the FAW population dynamics, introduction paths, origin, and subsequent spread.This research has increased our knowledge of the genetics of FAW in Uganda, which is critical for pest surveillance and the detection of resistance. However, due to the low gene polymorphism of The online version contains supplementary material available at 10.1186/s12864-022-09093-4. Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a devastating polyphagous and highly migratory herbivorous insect pest, that is native to the Western Hemisphere [https://bfvg.com.au/2020/04/06/when-was-fall-armyworm-detected-in-australia/). The pest feeds on 353 host plant species in 76 plant families [The fall armyworm (FAW), misphere . Since imisphere , FAW hasmisphere , 4 and rfamilies . FAW damfamilies , 10. Thefamilies . The damfamilies .The establishment of FAW in Africa is of special concern. In 2017, 2.5 to 6 million USD maize loss was estimated in Africa . The annSpodoptera frugiperda exists in two morphologically identical but genetically distinct strains: the corn strain designated as “C” strain and the rice strain designated as “R” strain, which are considered to differ in host preference, physiology, and behavior [cytochrome oxidase subunit I (COI) and the nuclear gene triosephosphate isomerase (Tpi) polymorphisms are used to distinguish them [Tpi gene can accurately predict host association [behavior –19. The behavior . The onlbehavior , 20, 21.ish them , 22 and ish them , 24. Thoish them , 23–26, ociation , 28. In ociation , 28. RecBacillus thuringiensis (Bt) toxins [Bt crops have been recorded in multiple FAW populations [The fall armyworm’s remarkable environmental adaptation is reflected in its genetics and polyphagy  and the ) toxins –32. To dulations –35, resuS. frugiperda has been reported in Uganda, there is limited knowledge on its genetic diversity, distribution, and resistance status. The existing information in Uganda indicates the occurrence of two strains, identified using COI markers [COI and Tpi markers [COI, Tpi, and ace-1 partial genes DNA markers. Of key interest, analyses of genetic identity, structure and diversity of FAW populations in Uganda were conducted. We also analyzed whether the invading FAW carry point mutations associated with resistance to organophosphate insecticides within the acetylcholinesterase 1 gene.Losses attributed to FAW damage in maize-dependent smallholder and commercial farms across Africa have raised a need for management interventions to guard against the present and future food insecurity. The use of insecticides in emergency response to reduce FAW spread and damage has been widespread since its introduction into the continent . In Ugan markers . Moreove markers , and few markers . LimitedCOIA, COIB, Tpi, and ace-1 genes of the expected band sizes , 5 sequences  were not considered in downstream analyses. Only 87 sequences were therefore used for genetic diversity and resistance studies.All primer sets used in this study amplified successfully and gave PCR products of mCOI602 strain defining polymorphic locus found in the barcode region (COIA), revealed the “R” strain to be found in 75%  was studied, which contains polymorphic loci mCOI1164D and mCOI1287R, resulting in five haplotypes, four of which belong to the Corn strain (CSh1–4) and one to the Rice strain category. COIB haplotyping was done for 87 samples of FAW from Uganda, out of which three haplotypes (which represent the entire population) were detected. Out of the three haplotypes, 71% . The COI-CSh1 haplotype , denote the COI-RSa1 and COI-CSh4 haplotypes, respectively.Another strain marker segment (Tpi gene for Ugandan populations (n = 85), revealed three haplotypes, the TpiCa1a homozygous , OP185932 (TpiCa2a) and OP185933 (TpiRa1a). The agreement of both COI and Tpi markers would be a stricter criterion for strain identification and host association. In Uganda, however, 99% of the samples had discordant Tpi and COI markers results for both identity and host association prediction. Tpi ‘C’ haplotype was detected in 99% of the samples collected from corn plants. Notably, one Tpi ‘R’ haplotype was also found in a cornfield, making it uncertain if TpiRa1a FAW prefer rice strain host plants or not.Studies on the polymorphisms at strain-defining loci of the fourth exon in the n Africa . The thrCOIA barcode region for the 87 Ugandan samples  studied belonged to a single rice haplotype  which was found across the country. The rest (n = 22) belonged to the ‘C’ strain haplotype , which was the most common. There was no region-specific haplotype in Uganda. They all showed an irregular distribution pattern throughout the country.Our analysis revealed 16 polymorphic sites (16 mutations) with a nucleotide diversity (Pi) of 0.00794 after analysis of a 770-bp The results of Fu’s Fu test and Tajima’s D test statistic were both significantly positive Table . Within COI partial gene region that was common to all of the sequences, revealed 5, 19, and 19 haplotypes from the rest of Africa, America, and Asia, respectively  were identical to Uganda RS and CS, respectively  found in Uganda  were detected together in a single insect and the F290V allele was the most common point mutation detected across the country  which could be related to the development of resistance . The resCOIA gene polymorphisms, with 75% of the samples belonging to the COIA ‘R’ strain and the rest to the COIA “C” strain. Similar findings were also reported by [COI-RS predominance trend was previously documented in India, East Africa, and South Africa [The fall armyworm has become a new economically important pest of maize in sub-Saharan Africa . An earlorted by . Furtherh Africa .COIB) was studied. This segment contains polymorphic loci mCOI1164D and mCOI1287R, resulting in five haplotypes, four of which belong to the Corn strain (CSh1–4) and one to the Rice Strain category. The relative distribution of COIB haplotypes (CSh4 and CSh2) has been used to analyze FAW groups’ ancestry in America. CSh4 haplotype predominates in FAW populations from Florida and the East coast of America, whereas CSh2 haplotype predominates in FAW populations from Texas and most other regions of America [n = 24) of the corn samples belonged to the COI sub-haplotype 4 (COI-CSh4) - which was previously reported in Togo [COI gene segment, more evolutionary studies incorporating the Spodoptera frugiperda whole- genome sequence are required to understand the FAW population dynamics, introduction paths, origin, and subsequent global spread. We also detected the COI-CSh1 sub-haplotype in one of the samples analyzed, marking the first detection of the Western Hemisphere minority “C” strain FAW haplotype in Africa. As a result, it’s unknown if this Western Hemisphere minority haplotype will remain infrequent in Africa, as it is in the Americas. However, favorable agro-ecological conditions of sub-Saharan Africa [S. frugiperda as an endemic pest for several generations and this could potentially favor hybridization between S. frugiperda strains as evolutionary pressures continue to shape and favor their reproduction, adaptation, subsequent survival, and further spread.Another mitochondrial strain marker segment  to be the new hotspot for the expansion of FAW COIA rice haplotypes in addition to America. This observation is in line with recent findings that the FAW population in India is still expanding [COIA gene polymorphisms, and this could support the current neutral evolution, in addition to the observed lower diversity.The population genetic test statistics, based on the xpanding . We founTpi nuclear gene that is known to distinguish between strains of FAW and detect potential cases of hybridization [COI and Tpi markers-a pattern which was also seen in India, South Africa, and East Africa [COI marker as rice or corn strains were classed as Tpi-corn strains (Tpi- CS). The results suggest the better Tpi marker accuracy in determining host association and further demonstrate the possibility of a massive FAW hybridization pattern occurring in the region. However, it is worth noting that one Tpi ‘R’ haplotype was also found in a cornfield in Pallisa district in Eastern Uganda. As a result, it’s critical to figure out if TpiRa1a samples have a distinct feeding behavior that could have major ramifications for pest management in Africa’s crops.We also sequenced a portion of the sex-linked dization , 22, 45,t Africa , 26, 36.t Africa , 46, theAChE family [Our study of six mutations previously identified to be involved in resistance to organophosphates insecticides, revealed the F290V allele as the most common (73%) across the country, while the A201S (1%) occurred at a very low frequency detected only in combination with the F290V allele, and was never found as a single mutation in a single insect. The low frequency of the A201S allele in all of the invading populations studied could suggest that the majority of these resistant FAW had only the F290V resistance mutation. The G227A allele was not identified in our samples. This mutation is highly conserved among the E family , 39 and Whereas the allelic frequency distribution pattern in this study is different from that of Brazil , our results are similar to the pattern observed in previous samples from Uganda, China and Malawi . A similAphis gossypii [Chilo suppressalis, sequence analysis found an amino acid mutation which revealed a strong correlation between frequencies of the A201S mutation and phenotypic levels of resistance to triazophos organophosphate insecticide [Musca domestica [The A201S mutation which changes the amino acid alanine for serine have been reported to form hydrogen bonds that interact with the substrate, stabilizing the enzyme-substrate complex, and causing changes in the active site that modify sensitivity to the enzyme inhibiting the bond with organophosphate insecticides, thereby creating resistance , 46. Itsgossypii . In Chilecticide . The preomestica  indicateomestica . In thisAChE enzyme and Ach to increase resistance to 37 times [Cydia pomonella but registered a high level of resistance (130-fold) to the carbamate carbaryl [ace-1 mutations were carried into Uganda and Africa continent at the time of the invasion. A possibility of introduction of these mutations into Africa from the New World where FAW is native was suggested by Guan et al. [acetylcholinesterase gene [S. frugiperda populations underwent rapid selection in favor of the F290V mutant allele in Uganda, and other African nations where a similar allelic frequency pattern occurs, with a few individuals selected in favor of the A201S mutant allele.The mutation F290V  in sistance . This stugiperda , no addiS. frugiperda populations in Uganda. We detected two FAW groups, later identified as corn and rice strains, with the latter being more prevalent based on the COI marker. Spodoptera frugiperda in Uganda had point mutations in the ace-1 partial gene that have previously been documented to confer organophosphate resistance in both S. frugiperda and several other insect species. According to population genetic test results, populations in Uganda and the rest of Africa are still evolving neutrally, while populations in America and Asia are expanding. Both S. frugiperda strains are widely distributed in Uganda, and our study demonstrated that the corn strain FAW populations invading Uganda and other African countries are similar to those found in Florida and the Caribbean. However, due to the low gene polymorphism of COI, additional evolutionary studies incorporating the S. frugiperda whole genome sequence are required to comprehend the FAW population dynamics, introduction paths, origin, and subsequent global dissemination. When the mitochondrial and Tpi marker results were compared, the results indicated that the Tpi marker was more accurate in determining host association. Point mutation analysis of the ace-1 partial gene segment revealed four mutations  that were previously reported as being associated with resistance to organophosphate pesticides. This work has improved our understanding of pest genetics in Uganda, which is important for pest surveillance and detection of resistance. The findings of this study can be used to improve integrated management strategies aimed at the long-term development and deployment of environmentally friendly genetically modified Bt. corn, as well as the selection of effective insecticides for control, in combination with traditional host plant resistance breeding and other biological control methods. Crop protection, enhanced maize production, food security, and improved income for stakeholders, particularly smallholder farmers in Africa and elsewhere, will all result from effective management.This study provides the most fundamental information on the genetic identity and diversity of http://www.fao.org/agriculture/seed/cropcalendar/aezones.do?isocode=UGA). About 8–9 larval and adult samples were collected from each of the 11 agro-ecological zones, resulting in a total sample size of 95. Upon field inspection, samples (both larvae and adults) were handpicked and stored individually in 5 mL screw-cap tubes containing absolute ethanol. The samples were packed in Ziploc® bags and then transported to the National Crops Resources Research Institute (NaCRRI), Namulonge molecular laboratory, and kept in -20 °C before DNA extraction.The larval and adult samples were collected during the country-wide survey which was conducted between August and October 2017 in ninety-five (95) maize-growing districts of Uganda. All agro-ecological zones in the country were represented in the sample collection  was extracted from 95 samples using a modified Chelex-100 method  previousCOI and Tpi were amplified using three different primer sets, previously designed and used by [COIA barcode region, primer pair (COI_101F/ COI_911R), COI_101F, 5′-TTCGAGCTGAATTAGGGACTC-3′ and COI_911R, 5′-GATGTAAAATATGCTCGTGT-3′ that give amplicons length of 811 bp, were used  [Partial gene sequences of  used by . To ampl(indels) .ace-1 partial gene segment was amplified using primers set ace_1F (5′-GAACGCTGTCATGCTGTGG-3′) and ace_1R (5′-CCGAGGGCCATTCATTCACTC C-3′) designed [The designed , to prod2 (2.5 mM), 0.5 μl dNTPs (10 mM), 1 μl of each primer (10 μM), 0.5 μl (2.5 units) of Dream Taq DNA polymerase® (ThermoFisher-Scientific) and 1 μl of the template DNA was used. The PCR thermo-cycling conditions for all the primer sets used in this study consisted of 94 °C (2 minutes), followed by 35 cycles of 94 °C (30s), 56 °C (35 s), and 72 °C (45 s) for denaturation, annealing, and extension, respectively. The PCR products were analyzed using electrophoresis in 1XTAE buffer on a 1.4% (w/v) agarose gel stained with 5 μl of ethidium bromide (0.5 μg/mL) and run at 80 V for 1 hour. The gels were then visualized under a UV light source and photographed using gel documentation system (U: GENIUS3-SYNGENE). The PCR products that gave positive results of the expected band sizes were then sequenced at Macrogen Europe BV. .A PCR mixture of 16.0 μl of nuclease-free water , 2.5 μl PCR buffer, 2.5 μl MgClCOIA samples analyzed) sequences in MEGA-X ver. 10.1.7 [COIA Sequences which were shorter than the selected 542 bp length were omitted from downstream analyses. The final list of 510 sequences was generated to enable DNA polymorphism analysis. To explore the biogeographic patterning of FAW and the relatedness of distinct sub-populations, we compared the diversity of the Ugandan FAW population with those of other geographic locations. For this, we separated the FAW population into four major groupings, each of which could be studied at the population level. The dataset was divided into four broad categories as 1. The rest of Africa; 2. America (which includes FAW from both North and South America); 3. Asian countries ; and 4 Uganda based on the geographical distribution. These groups were analyzed for descriptive statistics such as nucleotide diversity, number of haplotypes (H), haplotype diversity (Hd), genetic neutrality tests and mismatch distribution analysis using DnaSP ver 6.12.03 [n = 99), Asia  (n = 178), and the rest of Africa (n = 146) in addition to the 87 samples from Uganda.ClustalW was used to align trimmed , with five low-quality sequences being excluded from further analysis. As a result, only 87 samples were used for downstream analysis. The generated COI sequences revealed 100% similarity with S. frugiperda and were used to study the diversity of Ugandan populations. Additional 423 Genbank sequences from different parts of the world, and previously used by [S. frugiperda strains, we investigated the Ugandan S. frugiperda COI and Tpi gene polymorphisms using strain defining loci and polymorphic sites which were previously reported [mCOI602 strain defining polymorphic locus is found in the barcode region (COIA)  was studied, which contains polymorphic loci mCOI1164D and mCOI1287R, which is used to determine the five haplotypes, four of which belong to the Corn strain (CSh1–4) and one to the Rice Strain category  or rice- strain (Tpi-R), respectively.Out of the 95  used by  were als used by . For molreported , 23, 24.Ver. 1.7) [Spodoptera species, and S. frugiperda corn and rice strains) from NCBI [ver 2021.2.2.) [COI and Tpi DNA sequences of FAW selected were compared with DNA sequences of the FAW host strains and other Spodoptera species that are available in the GenBank [The popART software (er. 1.7)  was useder. 1.7) . To inferom NCBI , which wrom NCBI , 41, we 21.2.2.) . Both CO GenBank .ver. 6.5 [Analysis of molecular variance was used to investigate the genetic structure of the four geographical groups. We conducted six independent AMOVA analyses, first comparing the four geographical groups and then comparing alternative groupings combinations . These analyses were performed using the GenAlEx software ver. 6.5 , 58 usinTorpedo californica mature enzyme) [ace-1 gene have been reported to confer resistance to organophosphate insecticides in several other insects [We investigated six mutations previously identified to be involved in resistance to organophosphates. Three of these mutations, A201S, G227A, and F290V (numbering corresponding to  enzyme)  in the a insects –57, 60.  insects , 16, 39.The electro-pherograms obtained were visualized, edited, and assembled with Chromas software package (Version 2.6.6). The two reference sequences from the GenBank— KC435024 and KC435023 for resistance and susceptibility, respectively—were aligned alongside the Ugandan nucleotide sequences. MEGA 7.0 Clustal X  was usedS. frugiperda populations in Uganda based on mitochondrial gene COI and Tpi gene markers.Using R software version 3.5.3 , bar plots were created to show the pattern of strain distribution for Additional file 1: Supplementary figure 1. Amplification of Spodoptera frugiperda cytochrome oxidase 1 subunits, Triosephosphate isomerease and acetylcholinesterase partial gene segments. Supplementary figure 2.COI and Tpi gene segments used in this study.


Rhizoctonia spp. growth and to possess biostimulant properties in cereal crops. As a first, the present study investigated its effectiveness as a seed treatment of the dicot species oilseed rape (Brassica napus var. oleifera). For this, seeds were treated with different fungicides: (i) the conventionally used active ingredient Thiram, (ii) Sedaxane, or (iii) Sedaxane in combination with Fludioxonil and Metalaxyl-M, and later sown in soil inoculated with Rhizoctonia solani. The resulting shoot and root growth from the treated seeds were recorded in early growth stages and the presence of Rhizoctonia DNA in the basal stem tissue was quantified. Here we demonstrate that all the fungicide treatments were effective in greatly reducing the presence of Rhizoctonia DNA, with Thiram confirming to have high fungicidal effects. Following seed treatment, shoot and root growth at the 2-leaf stage was reduced regardless of inoculation, indicating that the fungicides became phytotoxic, with particular respect to Thiram. In seedlings grown in inoculated soil, significant biostimulation of the roots was observed at the 4-leaf stage of treatments containing both Sedaxane alone and in a mixture. Leaf area was stimulated in control soil not inoculated with Rhizoctonia, likely due to improved PSII efficiency, stomatal conductance, and CO2 assimilation rate. Young oilseed rape seedlings are thus highly sensitive to seed treatments with these fungicides, and in particular to Thiram. The retardation in growth is quickly overcome by the 4-leaf stage however. We confirm that Sedaxane indeed possesses root biostimulant properties in oilseed rape, which are enhanced in combination with other fungicides. Such biostimulating properties impose its greatest effects under conditions of biotic stress.The SDHI fungicide Sedaxane has shown to efficiently control  Fludioxonil inhibits the transport-associated phosphorylation of glucose and prevents glycerol synthesis  have been made commercially available to implement chemical control of  pathway . Seed apynthesis . The comynthesis . NonetheBesides identifying the highly effective a.i. of fungicides, there has been a growing interest in exploring their plant biostimulant properties as supplementary effects when used in seed or leaf treatments. Fungal compounds conferring biostimulant effects relating to improved tolerance to abiotic stresses have already been discovered and characterised in various fungicides, including the ubiquinol oxidase inhibitors (Qol) strobilurins and azoles . The bioR. solani versus the biostimulant effect of Sedaxane in the dicotyledonous oilseed rape (Brassica napus var. oleifera), in comparison with the conventional fungicide Thiram ; (ii) assess whether the application of Sedaxane in combination with other active ingredients has greater efficacy as compared to Sedaxane alone in both controlling the pathogen and promoting plant growth; and (iii) highlight any physiological response, particularly relating to photosynthesis, to the tested fungicides in young oilseed rape plants. To achieve these goals, Sedaxane was applied as seed treatment on oilseed rape alone or in combination with Fludioxonil and Metalaxyl-M and compared to seeds treated with Thiram. The effects were measured in early growth stages of plants grown in rhizoboxes (2-leaf) and pots (4-leaf) containing soil inoculated with R. solani and compared to controls not inoculated. Root  and shoot  parameters were assessed at plant harvest together with the amount of DNA of R. solani in the stem base tissues of oilseed rape through quantitative PCR (qPCR) analysis.Given this background, the goals of this study were to (i) discriminate the antifungal efficacy against 22.12O5 and 300 kg K2O per hectare was added and carefully mixed with the substrate before rhizobox/pot filling.Plants of the oilseed rape variety SY-Harnas , provided by CETAPP (France), were grown both in transparent-wall rhizoboxes  until 2-leaf stage, and in cylindrical PVC pots  until 4-leaf stage. Plants grown in both types of containers were cultured in a greenhouse at the “Lucio Toniolo” experimental farm of the University of Padua . Rhizoboxes and pots were filled with a sterilized mixture (48 h in an oven at 105°C) of silty-loam soil (pH 8.4) and fine sand (1:1 w/w). A recommended dose of fertilizers, corresponding to 100 kg N, 150 kg PR. solani (+ Rhizoctonia) or not (− Rhizoctonia), and results were compared with controls left untreated with fungicides  and Sdx in combination with Fludioxonil and Metalaxyl-M (Flu+Met+Sdx) were investigated under conditions in which the soil had been inoculated with ngicides Table 1.R. solani strain RS 22 (AG2-1). Four infected barley seeds were applied to each rhizobox and pot as follows: seeds were pestled in a mortar and the resulting flour was mixed with 1 mL of milliQ water. The inoculum solution was equally divided and added to soil at three equally spaced positions, wherein oilseed rape seeds were sown a few days later. In order to avoid artefacts due to organic matter supply, four infected and sterilized (in autoclave at 120°C for 20 min) barley seeds were similarly applied in the treatments not inoculated (− Rhizoctonia) of both rhizoboxes and pots. As such, the treatments constituting non-treated soils contained inactivated pathogens. After soil inoculation, rhizoboxes and pots were kept at a constant temperature of 15°C for 10 days prior sowing to allow the development of the Rhizoctonia mycelium.Soil inoculation was performed before sowing by using barley seeds infected with Three seeds of oilseed rape per rhizobox and pot were sown at 1 cm depth and equally spaced apart, in correspondence with the positions of pathogen inoculation. Plants were kept in the greenhouse at 20°C/15°C (day/night), 12h/12h (light/dark) and 70% air humidity. The whole rhizoboxes were covered with a black film to avoid any interaction of light with root growth through the transparent walls, and the rhizoboxes were placed at a 45° angle to facilitate un-destructive root observations. Oilseed rape plants were grown until 24 Days After Sowing  in rhizoboxes and 28 DAS in the pot trial (4-leaf stage). Plants were regularly watered in both rhizoboxes and pots throughout the experiment.2.2Root growth parameters were revealed in the rhizobox trial only. Root depth was measured at 2-day intervals, from 6 to 17 DAS, by means of a ruler leaned on the lower transparent wall of the rhizoboxes. The dynamics of root deepening (Y) was fit with the Gompertz equation, as follows:where a (asymptote), b , and c (deepening rate) are coefficients and x is time (DAS).At the end of the rhizobox trial, plants were harvested and their shoots and roots were separated. The roots were gently washed, separated from soil particles and stored in a 15% v/v ethanol solution until further processing. Root length, surface area and diameter were measured by analysis of 1-bit 300-DPI TIFF images of the root systems acquired through a flatbed scanner  using the WinRhizo software .The dry weight of the roots was later measured in plants grown both in rhizoboxes and pots, after oven-drying for 48 h at 105°C.2.3Fv′/Fm′), stomatal conductance, electron transport rate (ETR) and CO2 Net Assimilation (A) were determined following Due to their greater development in comparison to plants grown in rhizoboxes, the photosynthetic activity was measured at 27 DAS only in plants grown in pots by means of an infrared gas analyzer LI-6800 . PSII Photosynthetic Efficiency (Fv′/Fm′ ratio was measured as an index of efficiency in energy harvesting by the oxidized (open) reaction centres of photosystem II (PSII) in the last developed leaf, where Fv′ and Fm′ represent variable and maximal fluorescence, respectively. As Fm′ includes minimal fluorescence (0′F) of a dark-adapted leaf, Fv′ is calculated as Fm′ – 0′F. Dark adaptation was set with the use of a far-red light to excite photosystem I (PSI), thus forcing electrons to drain from PSII. Only a few seconds of far-red light are needed to obtain this effect. The fluorimeter provides a “dark pulse” routine used to determine F0′. Five Fv′/Fm′ records were registered for each leaf of each replicate.The Leaf area was assessed after plant harvest at the end of both rhizobox and pot trials, by means of the LI-3100C Area Meter . Shoot dry biomass was determined after oven-drying for 48 h at 105°C.2.4R. solani in oilseed rape plants, 1cm long portions of the stem base were collected from each plant grown in the pot trial. Three biological replicates (n = 3) per treatment were analyzed, each obtained by mixing 3 subsamples (plants) from every pot. Two technical replicates were performed on each biological replicate. Genomic DNA was extracted using the DNeasy Plant Mini Kit , quantified by agarose gel electrophoresis, and then used as template in a qPCR reaction. The qPCR was performed on a Rotor-Gene Q 2plex (Qiagen) using primers specific for Brassica napus ENTH gene .2.5Analysis of variance (ANOVA) was performed using CoStat software  with Student Newmal-Keuls test in order to highlight significant differences among means at p ≤ 0.05, and significance of the main effects “inoculum”, “treatment” and “inoculum × treatment” interaction.Rhizoctonia soil inoculum, in terms of shoot and root growth and photosynthetic parameters. Before analysis, multivariate data normality was verified by the Shapiro test using R 3.0.1 software  and factorial discriminant analysis  with Wilks’ lambda and Pillai’s trace tests  were carsoftware , and dat33.13.1.1R. solani did not significantly affect the initial rooting of oilseed rape plants, which reached a maximum root depth of ~40 cm in absence of the pathogen (− Rhizoctonia) and ~35 cm in presence of the pathogen (+ Rhizoctonia) at 17 DAS (p ≤ 0.05), but similar values to controls at the end of the trial.The inoculum with t 17 DAS Figure 1R. solani inoculated soil (+ Rhizoctonia), only the seed treatment with Flu+Met+Sdx allowed for increased root depth starting from 10 DAS, as compared to all the other treatments. The plants treated with Thiram and Sdx showed substantially similar root deepening pattern to untreated controls (p ≤ 0.05), with Sedaxane alone or in combination with the other two a.i. (Flu+Met+Sdx) significantly improving the average root depth in comparison with Thiram over the investigated period.In controls Figure 13.1.2R. solani inoculum significantly reduced the root surface area (−44% vs. absolute control) and increased root diameter (+27%) of oilseed rape plants grown in rhizoboxes  in explaining root surface area, as well as their interaction (p ≤ 0.05) (p ≤ 0.05). Thiram also induced a significant reduction in root surface area in inoculated soil , Sedaxane a slight reduction (-17%), and Flu+Met+Sdx a slight increase (+16%) but which were both not statistically different from the untreated control.The two main fixed effects treatment and inoculum were statistically significant ( ≤ 0.05) Table S2p ≤ 0.05) and Treatment (p ≤ 0.001) also strongly affected root diameter (p ≤ 0.05) reduced by Thiram and Sdx treatments in both inoculated  and not inoculated soil . A full recovery of root diameter was recorded with the use of Sdx in combination with Fludioxonil+Metalaxyl-M (Flu+Met+Sdx), even leading to slight improvements vs. not inoculated soil (+19% vs. absolute control)  and Inoculum and their interaction (p ≤ 0.05) (R. solani significantly reduced the leaf area (–14% vs. absolute control) and particularly the shoot dry biomass (–34%) of oilseed rape plants cultivated in rhizoboxes (p > 0.05) with all the fungicides in both not inoculated and R. solani inoculated soil however, while Thiram showed marked phytotoxicity in infected soil .The ANOVA revealed that leaf area and shoot biomass were significantly affected by both Treatment ( ≤ 0.05) Table S2As regards shoot biomass, a similar trend as leaf area was observed; a significant decrease by Thiram in both inoculated (–68% vs. untreated control) and non-inoculated soil (–54% vs. absolute control). Under soil infection, Sedaxane alone and in combination with other fungicides (Flu+Met+Sdx) allowed an appreciable recovery of shoot dry biomass, which was statistically similar to the absolute control Figure 33.23.2.1p ≤ 0.05) (R. solani and without any fungicide treatment (untreated control) was still reduced (–20% vs. absolute control)  and non-inoculated soil (+19% vs. absolute control). In inoculated soil (+ Rhizoctonia), also the application of Sedaxane alone (Sdx) significantly increased root dry biomass by +37% (p ≤ 0.05)  Table S33.2.2R. solani DNA was quantified in the stem base tissue of oilseed rape plants of the pot trial. The quantification was carried out in plants grown in inoculated soil (+ Rhizoctonia) and which had received a seed treatment, and compared to untreated plants as well as the absolute control (– Rhizoctonia). All the fungicides drastically reduce the presence of the fungal DNA in the plant tissues  effect on leaf area (p ≤ 0.05) (p > 0.05).The ANOVA disclosed Treatment as being the only fixed effect with a significant (eaf area Table S3p > 0.05).As regards shoot biomass, the ANOVA did not reveal any significant effect from neither Inoculum nor Treatment Table S33.2.4Fv′/Fm′), was significantly affected by Treatment (p ≤ 0.001) and to a lesser extent by Inoculum (p ≤ 0.05) at the 4-leaf stage. Neither Inoculum nor Treatment main effects significantly affected ETR or any other physiological parameter  and from –10% to –16% in inoculated soil (p ≤0.05) for the PSII efficiency and ETR respectively. An increase trend was observed in the PSII efficiency and ETR (p > 0.05) when the Flu+Met+Sdx seed treatment was given to plants grown in non-inoculated soil  in non-inoculated soil (– Rhizoctonia). Here, a +120% (p ≤0.05) and +78% (p ≤ 0.05) increase followed seed treatment with Sedaxane in combination with Fludioxonil and Metalaxyl-M for gsw and CO2 assimilation rate respectively (2 assimilation rate (-57%) in comparison to the absolute control, and lower and not significant variations were observed with Thiram.A similar trend was observed in regards to the variation in stomatal conductance (gsw) and COectively Figure 7R. solani allowed for a relevant increase in both stomatal conductance  and CO2 assimilation rate  as compared to the absolute control. Under soil infection, a decrease of both stomatal conductance and CO2 assimilation was revealed following all the fungicide treatments, in particular with Sedaxane alone and in a mixture (Flu+Met+Sdx). However, these treatments merely permitted stomatal conductance and CO2 assimilation rates to reach comparable levels to those of the absolute control.Noteworthy, soil inoculation by 3.2.52 assimilation rate (A) associated to F2. According to the vector direction of each variable in PCA, there is strong positive correlation among all the parameters related to the photosynthetic activity, and a clear negative correlation between shoot dry biomass and leaf area.Principal Component Analysis (PCA) applied to the pot trial allowed for the identification of two synthetic variables, F1 (64.42%) and F2 (18.59%), which explained an overall variability of 81.02% Figure 8According to the centroids position and cluster separation in MDA, a different plant response was observed under the investigated treatments. Within non-inoculated soil, the effects of seed treatment with Sedaxane in combination with Fludioxonil and Metalaxyl-M (Flu+Met+Sdx) was mostly related to variations in physiological parameters of leaf photosynthesis. Instead, the effects of Thiram and Sedaxane alone were mostly associated to variations in plant biomass (both root and shoot) under soil inoculation, and leaf area in non-inoculated soil.4R. solani and Mycosphaerella reliana . The full recovery of root biomass at the 4-leaf stage in plants that received seed treatment therefore illustrates the effectiveness of the formulations and doses of all tested fungicides.We here demonstrate that all the investigated fungicides efficiently protected against Rhizoctonia, no seed treatment). As such, Sedaxane mixed with Fludioxonil and Metalaxyl-M had ultimately  a clear root biostimulating power, as conveyed by the increased root biomass in both the R. solani inoculated soil and non-inoculated soil. This might be due to a synergistic effect of the three fungicides due to their differing modes of action (MOA) and target sites in pathogens. Indeed, Fludioxonil is a preventive fungicide, while Metalaxyl-M and Sedaxane have systemic activity. Fludioxonil targets the histidine kinase enzyme involved in osmotic signal transduction (MOA E2), Metalaxyl-M the RNA polymerase I enzyme (MOA A1), while Sedaxane is a succinate dehydrogenase inhibitor (SDHI) blocking fungus respiration (MOA C2). When applied alone, Sedaxane showed significant biostimulating effects on root biomass under Rhizoctonia-inoculated soil, but not in non-inoculated soil. Indeed, sedaxane has already been reported to have auxin- and gibberellin-like activities in various cereals crops  with Sedaxane use in either lone use or in mixture, in absence of soil inoculation remain unclear. In previous studies , decreasRhizoctonia DNA from the stem base tissues of oilseed rape, the presence of mycelium in the rhizosphere is also hypothesized to secrete toxic compounds which affect root growth and development in the host plant. However, this requires further specific investigations to be confirmed. Through secondary metabolism, R. solani can produce several compounds that aid the infection process, such as fatty acids, steroids, phenolic compounds and glycoproteins, all of which are also associated to root phytotoxicity (R. solani pressure.Despite efficient exclusion of toxicity . HoweverRhizoctonia soil infection rather than without the pathogen. Actually, we argue that in presence of this pathogen a priming effect probably exists in oilseed rape plants, which involves a physiochemical response capable of corroborating the biostimulant effect of the fungicide. Priming is known to activate systemic defense responses only when the plant is challenged by a pathogen or is exposed to abiotic stress (R. solani could be ascribed to a plant response related to priming. While this hypothesis requires further investigation to clarify the possible involved mechanisms, Sedaxane in combination with Fludioxonil and Metalaxyl-M can be agronomically exploited in a dicot species such as oilseed rape to control Rhizoctonia. A faster growth within the 4-leaf stage would also allow oilseed rape to quickly overcome the initial delicate phases of growth, with possible improved tolerance against abiotic stresses as well. Soil sterilization of rhizoboxes and pot trials was necessary to disentangle the fungal protection properties from the biostimulating effects of Sedaxane, while further investigations in real field conditions will be necessary to ascertain possible interactions with the soil microbiota and the molecular cross-talk of biopriming.The question which arises is why root biostimulation with Sedaxane is better expressed under c stress . Since nc stress , we hypo5R. solani, which can be exploited agronomically. Here we confirm an efficient control of this pathogen also in the dicot oilseed rape by Sedaxane either alone or in combination with Fludioxonil and Metalyl-M. Commercial formulations with low contents of these a.i are good candidates for replacing the over-used conventional fungicide Thiram, which is being withdrawn in various countries worldwide. Although all the fungicides tested here had initial phytotoxicity, Sedaxane exhibited clear root biostimulant properties as a supplementary effect to its antifungal features, like similarly observed in cereal crops. This suggests new opportunities for exploiting the biostimulant value of this fungicide in other Brassicaceae species, such as turnip, cabbage, kale, and leaf rape. The optimal biostimulant effects of Sedaxane are observed in combination with other a.i. and under high Rhizoctonia pressure however. As seed treatments require small amounts of fungicides in the field, the discovery of new a.i. or natural compounds with double effects at low doses is expected to reduce the impact of agrochemicals on the environment. This is advantageous both for the agricultural industry and for improving sustainable practices.Seed treatment with Sedaxane is consolidating in cereal crops, showing successful control of a wide spectrum of fungal pathogens, particularly Rhizoctonia, further investigations will be necessary to clarify the physiological mechanisms sustaining the maximal stimulating effect under high pathogen pressure and whether these results are confirmed in the open field.Our preliminary results suggest that young oilseed rape seedlings are highly sensitive to seed treatments with fungicides, particularly Thiram, but the growth retardation is quickly overcome within the 4-leaf stage. At this developmental stage, the biostimulant properties could be observed aboveground as well. While fungicide biostimulation is associated with improved photosynthetic efficiency and stomatal conductance in absence of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Conceptualization, TV: Methodology, TV and LS: Investigation, FC, ADS: Resources, TV: Data curation, FC, ADS. and GB: Writing—original draft preparation, FC, ADS and AP: Writing-review and editing, AP, SSM, LS and TV: Supervision, TV: Project administration, TV: Funding acquisition, TV. All authors contributed to the article and approved the submitted version.