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Sclerotinia sclerotiorum infects host plant tissues by inducing necrosis to source nutrients needed for its establishment. Tissue necrosis results from an enhanced generation of reactive oxygen species (ROS) at the site of infection and apoptosis. Pathogens have evolved ROS scavenging mechanisms to withstand host‐induced oxidative damage. However, the genes associated with ROS scavenging pathways are yet to be fully investigated in S. sclerotiorum. We selected the S. sclerotiorum Thioredoxin1 gene (SsTrx1) for our investigations as its expression is significantly induced during S. sclerotiorum infection. RNA interference‐induced silencing of SsTrx1 in S. sclerotiorum affected the hyphal growth rate, mycelial morphology, and sclerotial development under in vitro conditions. These outcomes confirmed the involvement of SsTrx1 in promoting pathogenicity and oxidative stress tolerance of S. sclerotiorum. We next constructed an SsTrx1‐based host‐induced gene silencing (HIGS) vector and mobilized it into Arabidopsis thaliana (HIGS‐A) and Nicotiana benthamiana (HIGS‐N). The disease resistance analysis revealed significantly reduced pathogenicity and disease progression in the transformed genotypes as compared to the nontransformed and empty vector controls. The relative gene expression of SsTrx1 increased under oxidative stress. Taken together, our results show that normal expression of SsTrx1 is crucial for pathogenicity and oxidative stress tolerance of S. sclerotiorum., Normal expression of SsTrx1 is crucial for pathogenicity, sclerotial development, and oxidative stress tolerance of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating necrotrophic fungal pathogen and has a substantial economic impact on crop production worldwide. Magnaporthe appressoria-specific (MAS) proteins have been suggested to be involved in the appressorium formation in Magnaporthe oryzae. Sscnd1, an MAS homolog gene, is highly induced at the early infection stage of S. sclerotiorum. Knock-down the expression of Sscnd1 gene severely reduced the virulence of S. sclerotiorum on intact rapeseed leaves, and their virulence was partially restored on wounded leaves. The Sscnd1 gene-silenced strains exhibited a defect in compound appressorium formation and cell integrity. The instantaneous silencing of Sscnd1 by tobacco rattle virus (TRV)-mediated host-induced gene silencing (HIGS) resulted in a significant reduction in disease development in tobacco. Three transgenic HIGS Arabidopsis lines displayed high levels of resistance to S. sclerotiorum and decreased Sscnd1 expression. Production of specific Sscnd1 siRNA in transgenic HIGS Arabidopsis lines was confirmed by stem-loop qRT-PCR. This study revealed that the compound appressorium-related gene Sscnd1 is required for cell integrity and full virulence in S. sclerotiorum and that Sclerotinia stem rot can be controlled by expressing the silencing constructs of Sscnd1 in host plants.

The effects of thermosonication (TS) on microbial safety and quality of red pitaya juice during storage were assessed in this study. Freshly prepared red pitaya juices were thermosonicated at 475 W and 56 °C for 20 min. Upon TS processing, native microbiota including aerobic bacteria, yeasts, and molds reduced to less than 10 CFU/mL. Their growth during storage were slow and equal to thermal-processed (83 °C, 1.5 min) samples. During storage at 4 °C for 28 days, soluble solid content, pH, activities of polyphenol oxidase and peroxidase, and browning degree remained unchanged. A visible color decay was observed in TS-processed samples at day 10, mainly resulting from decomposition of betacyanins and the growth of residual native microbiota. Compared to thermal-treated juices, better color retention was obtained by TS treatment. Therefore, TS is a promising alternative technology of thermal methods of juice processing, with equal shelf life and better quality retention effects.

Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a devastating necrotrophic pathogen which causes severe yield losses to oilseed production worldwide. Most of efforts at the genetic mitigation of the disease have not been successful. Present investigation was conducted to functionally characterize the effect of down-regulating Ssoah1 during host infection and explore the possibility of boosting host resistance by silencing this gene. We utilized host-induced gene silencing (HIGS) to silence Ssoah1 gene in the S. sclerotiorum fungus. A HIGS based vector was constructed and transformed into Arabidopsis thaliana. The pathogenicity assays in the transgenic A. thaliana lines revealed three T

The viable but non-culturable (VBNC) state in bacteria is gaining more and more attention around the world, mainly due to it is not only an important strategy for adaption to a stressful environment but also may possibly pose a threat to food safety and public health. It was found that a small subpopulation of Salmonella Typhimurium in pure culture was induced into a VBNC state during thermosonication (TS) processing in our previous study, though few known about the situation in real food and how bacteria were induced into that special state. Base on the speculation that free radicals generated during TS affected induction of VBNC, the relationship between them was investigated preliminarily. It was observed that higher intensity of TS treatment, such as higher power, elevated temperature and prolonged duration resulted in more viable S. Typhimurium cells in carrot juice been inducted into VBNC state. The observed results showed that VBNC incidence indexes were 1000 as TS treatments at 57–62 °C and 380 W for 6 min, and 53 °C and 380 W for 8–10 min were applied, indicating a 100% VBNC state of viable cells in those cases. The ESR spectra revealed three kinds of free radicals, including carbon centered (ethanol) radicals, hydroxyl radical and hydrogen protons were generated in carrot juice during TS processing. The intensity of free radicals was tied to the TS processing parameters and also influenced the occurrence of VBNC. A nonlinear sigmoidal curve of the intensity of free radicals VS the VBNC incidence index in three stages, including a slow phase (with free radicals intensity of 0–0.10), a rapid growth phase (with free radicals intensity ranged 0.10–0.14) and a final equilibrium phase (with free radicals intensity greater than 0.14), was observed and well fitted with the Boltzmann model. Moreover, the significance of free radicals generated during TS processing for induction of VBNC state was verified and confirmed with 0–200 mM sodium pyruvate. The obtained results may contribute to understand the complicated phenomenon and guide the application of TS as a decontamination technique.

The objective of this work was to analyze the effects of thermosonication (TS) on induction of a viable but non-culturable (VBNC) state in Salmonella Typhimurium and to examine incubation factors affecting subsequent resuscitation of cells. A TS treatment of 380 W at 53°C for 30 min induced the VBNC state in S. Typhimurium cells in beef peptone yeast (BPY) broth, apple/carrot juice, physiological saline and phosphate buffer solution. The logarithmic and decline phases of growth were more sensitive to the TS treatment compared to stationary phase cells. Meanwhile, VBNC S. Typhimurium could be resuscitated back to culturable cells by using suitable incubation temperatures and media. Addition of Tween 20 hindered resuscitation compared to the use of BPY medium alone. The optimal growth temperature (i.e. 37°C) was the most suitable temperature to resuscitate cells from the VBNC state. The VBNC incidence index decreased with the addition of sodium pyruvate during TS treatment, as it accelerated resuscitation. The results demonstrated that free radicals produced during TS processing and the growth phase of cells affected induction of the VBNC state in S. Typhimurium. Hence, the kinds and amounts of free radicals generated during TS treatment should be analyzed in the future.

Cellular damage of Escherichia coli (E. coli) induced by high pressure carbon dioxide (HPCD) at 37-57°C and 10-30 MPa for 5-75 min was investigated using scanning electronic microscopy (SEM), transmission electronic microscopy (TEM), confocal laser scanning microscopy (CLSM), and fluorospectrophotometer (FSM). HPCD-induced alterations in the morphology and the intracellular organization of E. coli cells was more susceptible to HPCD. A vast majority of HPCD-treated E. coli cells with seemingly intact morphology sustained severe damage in their intracellular organization. CLSM suggested that initial disruption of the outer membrane and later permeabilization of the cytoplasmic membrane of HPCD-treated E. coli cells was a consecutive and progressive process. These results were confirmed by FSM with the probes PI and SYTO 9. The membrane fluidity of HPCD-treated E. coli cells decreased as suggested by increased fluorescence polarization using FSM with the probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The temperatures of 37, 42 and 47°C alone showed no impact on the outer membrane and membrane fluidity of E. coli cells whereas 57°C alone had greater impact on them. Combined with HPCD, the temperatures of 37, 42 and 47°C disrupted the outer membrane of E. coli cells without damage to the cytoplasmic membrane and of 57°C damaged the cytoplasmic membrane, but all these temperatures decreased the membrane fluidity of E. coli cells. Higher temperature increased HPCD-induced outer membrane disruption and the cytoplasmic membrane damage and decreased the membrane fluidity.

The inactivation of Salmonella typhimurium inoculated into acidified carrot juice subjected to dense phase carbon dioxide (DPCD) was investigated. The pressures in the study were 10, 20 and 30 MPa, the temperatures were 32, 37 and 42 degrees C, and the treatment time was 5-90 min. The inactivation effect of DPCD was enhanced by increasing pressure and temperature. The sigmoid inactivation curves were characterized with the lag phase, exponential inactivation phase, and resistant phase. The inactivation curves were fitted to the modified Gompertz equation and the modified Logistic equation, the modified Gompertz equation was superior since its lowest residual sum of squares (RSS) was lower although there was no significant difference of goodness-of-fit between both models as indicated by F-test. The lambda (the duration of the lag phase) and t(4-D) (the time necessary to achieve 4-log cycles reduction) decreased with increasing pressure or temperature. The k(dm) (the maximum specific value of the inactivation rate, min(-1)) increased with increasing temperatures, and decreased with increasing pressures. The activation energy (Ea) and the activation volume (Va) necessary for inactivating S. typhimurium by DPCD were 19.06-29.39 kJ mol(-1) and 18.89-58.27 cm(3) mol(-1).

Apple juice was pasteurized by high pressure carbon dioxide (HPCD) at 20MPa with CO(2) concentration of 4.5-5.3% and mild heat (MH) at atmospheric pressure. Microbial inactivation and stability of natural microorganisms in apple juice were investigated. The temperatures were 37, 42, 47, 52, 57 and 62 degrees C, treatment time was 30min, and storage temperatures of pasteurized apple juice were 2 and 28 degrees C. The aerobic bacteria (AB) treated by MH at 62 degrees C and by HPCD ator =52 degrees C were almost totally inactivated, the microbial counts were10CFU/mL. The yeasts and moulds (YM) treated by MH ator =57 degrees C and by HPCD ator =42 degrees C were totally inactivated. HPCD increased the susceptibility of these natural microorganisms to temperature and enhanced their microbial inactivation. The AB in apple juice treated by HPCD ator =52 degrees C and the YM treated by HPCD ator =57 degrees C, the AB and the YM treated by MH at 62 degrees C showed a better stability during storage at 2 and 28 degrees C, but apple juice treated by HPCD ator =47 degrees C was characterized with high microbial counts of the ABor =2.75x10(3)CFU/ml. A viable but non-culturable (VBNC) state of the YM treated by MH at 57 degrees C and by HPCD at 42, 47 and 52 degrees C was observed during storage at 28 degrees C. Apparently the proper temperature of HPCD treatment of apple juice at 20MPa and stored at 2 and 28 degrees C was greater than or equal to 52 degrees C, while for MH treatment the proper temperature increased to 62 degrees C in this study.

The inactivation of Staphylococcus aureus exposed to dense-phase carbon dioxide (DPCD) was investigated, and the kinetics of come-up time (CUT) in pressurization was monitored with come-down time (CDT) and temperature fluctuation in depressurization. CUT was about 2.5, 3.5, 4.0 and 4.0 min; CDT was 3.4, 3.7, 4.5 and 4.5 min; lowest temperature of samples in depressurization was 4, −1, −15 and −22C, corresponding to 10, 20, 30 and 40 MPa at 37C. The inactivation behavior of S. aureus was closely related to the variables of process pressure, holding-pressure time (HPT), process temperature and process cycling. The log reduction of S. aureus at 40 MPa for 30-min HPT was significantly greater (P

Protein changes in Escherichia coli, when subjected to high-pressure carbon dioxide (HPCD) at 10 MPa and 3 °C for 5–75 min, were assessed using the Bradford method, 2D electrophoresis (2-DE) and liquid chromatography-electrospray ionization-MS-MS (LC-ESI-MS-MS). The changes in DNA in E. coli under the same conditions were also investigated by using flow cytometry with propidium iodide and acridine orange, agarose gel electrophoresis (AGE) and the comet assay. The results showed that HPCD induced leakage loss of the proteins and DNA of E. coli as a function of treatment time. With regard to the protein changes, 182 proteins in the 2-DE profile were not found in the HPCD-treated E. coli. Among 20 selected protein spots exhibiting significant changes in intensity, 18 protein spots were identified as 15 known proteins and two as hypothetical proteins. These proteins were involved in cell composition, energy metabolism pathways, nucleic acid metabolism, global stress regulation and general metabolism. The DNA denaturation of E. coli induced by HPCD was demonstrated in this study for the first time to our knowledge, and the denaturation was enhanced by increasing treatment time. However, HPCD did not cause DNA degradation, as suggested by both AGE analysis and the comet assay.

Inactivation of Escherichia coli in cloudy apple juice by dense phase carbon dioxide (DPCD) was investigated. The pressures were 10, 20 and 30 MPa, the temperatures were 32, 37 and 42 degrees C. The inactivation kinetic behavior of E. coli conformed to a sigmoid curve with a shoulder and a tail, which was closely related with temperature or pressure. With the increase of temperature or pressure, the shoulder became unclear or even disappeared. The experimental data were well fitted to a model proposed by Xiong et al. [Xiong, R., Xie, G., Edmondson, A.E., Sheard, M.A., 1999. A mathematical model for bacterial inactivation. International Journal of Food Microbiology 46, 45-55], the kinetic parameters of t(lag) (the lag time length), f (the initial proportion of less resistant population), k(1) (the inactivation rate constant of less resistant fraction) and k(2) (the inactivation rate constant of resistant fraction), and t(4)(-)(D) (the time required for an 4-log-cycle reduction of bacteria under a given condition) were obtained from this model. The t(lag) declined from 4.032 to 0.890 min and t(4)(-)(D) from 54.955 to 18.840 min, k(1) was 1.74-4.4 times of k(2). Moreover, the model was validated by more experimental data, the accuracy factor (Af), bias factor (Bf), root mean square error (RMSE), sum of squares (SS), and correlation coefficient (R(2)) were used to evaluate this model performance, indicating that the model could provide a good fitting to the experimental data.

The inactivation of Escherichia coli in cloudy apple juice by dense phase carbon dioxide (DPCD) was investigated. With CO2 at 20 MPa and 37 degrees C or at 30 MPa and 42 degrees C, the inactivation of E. coli significantly increased (p0.05) when increasing the exposure time, which conformed to a fast-to-slow two-stage kinetics. The two stages were well fitted to first-order reactions. Higher temperature or pressure significantly enhanced the bactericidal effect of DPCD (p0.05), the maximum reduction was 7.66 log CFU at 45 MPa and 52 degrees C for 30 min. The survival curves against temperature or pressure were fitted using a linear equation with high regression coefficients (R20.94). The temperature inactivation rate (kT) and pressure inactivation rate (kP) were obtained. Higher kT or kP indicated higher susceptibility of E. coli to temperature or pressure. Moreover, there was good linear correlation of kT with pressure (R2=1.00). Also, kP increased with increasing temperature except for 37 degrees C. Greater inactivation of E. coli was obtained with 99.9% CO2 than with 99.5% CO2 or with the initial number of 10(5) CFU/mL than with that of 10(8) CFU/mL at 20 MPa and 37 degrees C.