Efficacy of Pseudomonas graminis CPA-7 against Salmonella spp. and Listeria monocytogenes on fresh-cut pear and setting up of the conditions for its commercial application.

Pseudomonas graminis CPA-7 has been reported to control foodborne pathogens on fresh-cut apple, peach and melon. The first aim of this study was to assess its antagonistic activity against Salmonella spp. and L. monocytogenes on fresh-cut pear. CPA-7 was able to control both pathogens on fresh-cut pear stored in air conditions at 5, 10 and 20 °C. However, when CPA-7 antagonistic effect was tested by simulating commercial application (with antioxidant solution and passive modified atmosphere packaging), its effect decreased and no reductions of foodborne pathogens were reported at 10 °C. Therefore, the second aim was to optimise the antioxidant solution and the packaging in order to retain its antagonistic capacity. The selected antioxidant solution was 2% ascorbic acid +2% sodium citrate +1% CaCl2 according to growth and effect of CPA-7. Film permeability, which affects gas composition inside fruit packages, influenced CPA-7 efficacy. If the biopreservative strain is used, film has to be sufficiently gas permeable to allow CPA-7 function and at the same time to maintain product quality.

Evolutionary processes driving spatial patterns of intraspecific genetic diversity in river ecosystems.

Describing, understanding and predicting the spatial distribution of genetic diversity is a central issue in biological sciences. In river landscapes, it is generally predicted that neutral genetic diversity should increase downstream, but there have been few attempts to test and validate this assumption across taxonomic groups. Moreover, it is still unclear what are the evolutionary processes that may generate this apparent spatial pattern of diversity. Here, we quantitatively synthesized published results from diverse taxa living in river ecosystems, and we performed a meta-analysis to show that a downstream increase in intraspecific genetic diversity (DIGD) actually constitutes a general spatial pattern of biodiversity that is repeatable across taxa. We further demonstrated that DIGD was stronger for strictly waterborne dispersing than for overland dispersing species. However, for a restricted data set focusing on fishes, there was no evidence that DIGD was related to particular species traits. We then searched for general processes underlying DIGD by simulating genetic data in dendritic-like river systems. Simulations revealed that the three processes we considered (downstream-biased dispersal, increase in habitat availability downstream and upstream-directed colonization) might generate DIGD. Using random forest models, we identified from simulations a set of highly informative summary statistics allowing discriminating among the processes causing DIGD. Finally, combining these discriminant statistics and approximate Bayesian computations on a set of twelve empirical case studies, we hypothesized that DIGD were most likely due to the interaction of two of these three processes and that contrary to expectation, they were not solely caused by downstream-biased dispersal.

Dendritic connectivity shapes spatial patterns of genetic diversity: a simulation-based study.

Landscape features notoriously affect spatial patterns of biodiversity. For instance, in dendritic ecological networks (such as river basins), dendritic connectivity has been proposed to create unique spatial patterns of biodiversity. Here, we compared genetic datasets simulated under a lattice-like, a dendritic and a circular landscape to test the influence of dendritic connectivity on neutral genetic diversity. The circular landscape had a level of connectivity similar to that of the dendritic landscape, so as to isolate the influence of dendricity on genetic diversity. We found that genetic diversity and differentiation varied strikingly among the three landscapes. For instance, the dendritic landscape generated higher total number of alleles and higher global Fst than the lattice-like landscape, and these indices also varied between the dendritic and the circular landscapes, suggesting an effect of dendricity. Furthermore, in the dendritic landscape, allelic richness was higher in highly connected demes (e.g. confluences in rivers) than in low-connected demes (e.g. upstream and downstream populations), which was not the case in the circular landscape, hence confirming the major role of dendricity. This led to bell-shaped distributions of allelic richness along an upstream-downstream gradient. Conversely, genetic differentiation (Fst ) was lower in highly than in low-connected demes (which was not observed in circular landscape), and significant patterns of isolation by distance (IBD) were also observed in the dendritic landscape. We conclude that in dendritic networks, the combined influence of dendricity and connectivity generates unique spatial patterns of neutral genetic diversity, which has implications for population geneticists and conservationists.

Optimization of packaging and storage conditions of a freeze-dried Pantoea agglomerans formulation for controlling postharvest diseases in fruit.

AIMS: This study aimed to evaluate different packaging strategies to extend the shelf life of a freeze-dried formulation of the biocontrol agent Pantoea agglomerans strain CPA-2. METHODS AND RESULTS: Different materials and atmosphere packaging conditions (vacuum and air) were analysed on formulated P. agglomerans cells stored at 25, 5 and -20°C. Results showed the viability of CPA-2 cells stored at 5 or -20°C was significantly higher than when stored at 25°C. The highest viabilities were observed with the plastic material designated as Bottle 1, in nonvacuum packaging in all storage temperatures: 50% after 3 months at 25°C, 100% after 8 months at 5°C and 100 and 74% after 12 and 18 months, respectively, at -20°C; the final concentration was 10(12) CFU g(-1), a good concentration for a commercial product. The efficacy to control blue and green mould on apples and oranges, respectively, of these packed and stored cells was similar to fresh CPA-2 cells. CONCLUSIONS: This work showed a suitable packaging strategy for a freeze-dried formulation of the CPA-2, providing a good shelf life and efficacy against the major postharvest diseases of apples and citrus based on a plastic bottle stored at cold or frozen storage conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The last phase of the commercial development process for biocontrol agents is presented in this work. A bacterium-based product that ensures the efficacy, stability and easy application of the antagonist to control postharvest fungal diseases on fruit was successfully obtained.

Survival of the biological control agent Candida sake CPA-1 on grapes under the influence of abiotic factors.

AIMS: As reliability of preharvest applications of biological control agents (BCAs) to control fruit pathogens is highly dependent on the survival of the selected organism, this study aimed to describe the population dynamics of the yeast-BCA Candida sake (Saito & Ota) CPA-1 on grape berries under the effect of abiotic factors such as temperature, relative humidity, sunlight and rainfall. METHODS AND RESULTS: Candida sake (5 × 10(7) CFU ml(-1)), combined with different concentrations of the food additive Fungicover(®), was applied on grape berry clusters. Treated clusters were then exposed to abiotic factors in field or laboratory conditions, recovering populations to describe C. sake population dynamics. The addition of Fungicover significantly increased C. sake multiplication under optimal growth conditions and improved survival under fluctuating abiotic factors. After field applications, significant differences in populations on grape bunches exposed or covered by fine foliage were detected. Simulated rainfall washed off C. sake populations by 0·6-0·9 log units after 20 mm of rain volume. Allowing populations to establish for 24 h or more, prior to a rain event, persistence on grape berries significantly increased and the effect of rain intensity was not observable. CONCLUSIONS: Candida sake demonstrated high survival ability under unfavourable environmental conditions and persistence under intense rain. The study evidenced the importance of the first period just after application for C. sake survival on grape tissues and also the protective effect of the additive Fungicover. SIGNIFICANCE AND IMPACT OF THE STUDY: This research provides new information on the survival of C. sake under field conditions and its practical implications for recommending timing of spray with this antagonist. Our results could be useful for other yeast antagonists applied before harvest. This work, for the first time, defines population dynamics of a yeast BCA using simulated rainfall.

Mode of action of a fatty acid-based natural product to control Botrytis cinerea in grapes.

AIMS: To investigate the efficacy and mode of action of the fatty acid-based product Foodcoat(®) (FC) against Botrytis cinerea. METHODS AND RESULTS: In vitro, in vivo and field experiments were carried out to investigate the effect of different concentrations of FC on B. cinerea germination and infection of grape leaves and berries, using three selected isolates and comparing results with those achieved by the commercialized product Protector(HML) (PRT). Furthermore, the effect of field applications of FC on the grape berry microbiota was investigated. FC reduced B. cinerea germination and grape berry severity by up to 54 and 96%, respectively, compared with the untreated controls. CONCLUSIONS: Foodcoat demonstrated efficacy that was equal or greater than the registered product, PRT. A multiple mode of action was hypothesized for FC suppression of B. cinerea, including: inhibition of germination and germ tube alteration, protection of host green tissues and enhancement of the natural yeast populations on the berry surface. SIGNIFICANCE AND IMPACT OF THE STUDY: The efficacy of both products has been quantified and their modes of action described, suggesting them for field applications against B. cinerea, alone or in combined strategies. This is also the first report of a fatty acid-based product stimulating natural yeast populations on grape berries.

Effectiveness of a bacteriophage in reducing Listeria monocytogenes on fresh-cut fruits and fruit juices.

Listeria monocytogenes is a serious foodborne pathogen and new strategies to control it in food are needed. Among them, bacteriophages hold attributes that appear to be attractive. The objective of this study was to investigate the efficacy of the bacteriophage Listex P100 to control L. monocytogenes growth on melon, pear and apple products (juices and slices) stored at 10 °C. L. monocytogenes grew well in untreated fruit slices. In juices, the pathogen grew in untreated melon, survived in untreated pear and decreased in untreated apple. Phage treatment was more effective on melon followed by pear, but no effect on apple products was observed. Reductions of about 1.50 and 1.00 log cfu plug(-1) for melon and pear slices were found, respectively. In juices, higher reductions were obtained in melon (8.00 log cfu mL(-1)) followed by pear (2.10 log cfu mL(-1)) after 8 days of storage. L. monocytogenes in apple juice was unaffected by phage treatment in which the phage decreased to almost undetectable numbers. These results highlight that Listex P100 could avoid pathogen growth on fresh-cut and in fruit juices with high pH during storage at 10 °C. The combination with other technologies may be required to improve the phage application on high acidity fruits.

The demographic history of populations experiencing asymmetric gene flow: combining simulated and empirical data.

Population structure can significantly affect genetic-based demographic inferences, generating spurious bottleneck-like signals. Previous studies have typically assumed island or stepping-stone models, which are characterized by symmetric gene flow. However, many organisms are characterized by asymmetric gene flow. Here, we combined simulated and empirical data to test whether asymmetric gene flow affects the inference of past demographic changes. Through the analysis of simulated genetic data with three methods (i.e. bottleneck, M-ratio and msvar), we demonstrated that asymmetric gene flow biases past demographic changes. Most biases were towards spurious signals of expansion, albeit their strength depended on values of effective population size and migration rate. It is noteworthy that the spurious signals of demographic changes also depended on the statistical approach underlying each of the three methods. For one of the three methods, biases induced by asymmetric gene flow were confirmed in an empirical multispecific data set involving four freshwater fish species (Squalius cephalus, Leuciscus burdigalensis, Gobio gobio and Phoxinus phoxinus). However, for the two other methods, strong signals of bottlenecks were detected for all species and across two rivers. This suggests that, although potentially biased by asymmetric gene flow, some of these methods were able to bypass this bias when a bottleneck actually occurred. Our results show that population structure and dispersal patterns have to be considered for proper inference of demographic changes from genetic data.

Candida sake CPA-1 and other biologically based products as potential control strategies to reduce sour rot of grapes.

UNLABELLED: Sour rot of grapes is becoming increasingly important disease in many wine-growing regions, while consistent chemical or biological control has not been reported. Authors evaluated relative incidence and severity of sour rot in untreated grapevines and the effect of different biologically based treatments on sour rot at harvest. Applications of Candida sake CPA-1 plus Fungicover® , Ulocladium oudemansii and chitosan were carried out in an organic vineyard in Lleida area, Spain, during the 2009 and 2010 growing seasons. At harvest, incidence and severity of sour rot were assessed. Significantly higher incidence and severity of sour rot were observed in untreated plots in 2009, when meteorological conditions after veraison were warmer. All treatments including C. sake CPA-1 significantly reduced (P < 0·05) severity of sour rot in both seasons, ranging from 40 to 67% compared with the untreated control. Incidence of sour rot was not significantly reduced by any treatment. This study helps to characterize development of sour rot in the dry Mediterranean climate conditions of the experiment, whereas also represents the first report of biological control of sour rot. Treatments with the tested biologically based products are a promising strategy to control sour rot. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies on sour rot of grapes are scarce in literature, and this is the first work specifically evaluating sour rot in Spanish vineyards. Sour rot control in field conditions through applications of antagonistic micro-organisms is reported for first time in this study, showing elevated severity reductions (40-67% compared with control). As there are no options available for sour rot control in vineyards, results point Candida sake CPA-1 as an interesting control strategy against grape bunch rots.

Formulation development of the biocontrol agent Bacillus subtilis strain CPA-8 by spray-drying.

AIMS: To prepare commercially acceptable formulations of Bacillus subtilis CPA-8 by spray-drying with long storage life and retained efficacy to control peach and nectarine brown rot caused by Monilinia spp. METHODS AND RESULTS: CPA-8 24-h- and 72-h-old cultures were spray dried using 10% skimmed milk, 10% skimmed milk plus 10% MgSO(4) , 10% MgSO(4) and 20% MgSO(4) as carriers/protectants. All carriers/protectants gave good percentages of powder recovery (28-38%) and moisture content (7-13%). CPA-8 survival varied considerably among spray-dried 24-h- and 72-h-old cultures. Seventy-two hours culture spray dried formulations showed the highest survival (28-32%) with final concentration products of 1·6-3·3 × 10(9) CFU g(-1) , while viability of 24-h-old formulations was lower than 1%. Spray-dried 72-h-old formulations were selected to subsequent evaluation. Rehydration of cells with water provided a good recovery of CPA-8 dried cells, similar to other complex rehydration media tested. Spray-dried formulations stored at 4 ± 1 and 20 ± 1°C showed good shelf life during 6 months, and viability was maintained or slightly decreased by 0·2-0·3-log. CPA-8 formulations after 4- and 6 months storage were effective in controlling brown rot caused by Monilinia spp. on nectarines and peaches resulting in a 90-100% reduction in disease incidence. CONCLUSIONS: Stable and effective formulations of biocontrol agent B. subtilis CPA-8 could be obtained by spray-drying. SIGNIFICANCE AND IMPACT OF THE STUDY: New shelf-stable and effective formulations of a biocontrol agent have been obtained by spray-drying to control brown rot on peach.

Endospore production allows using spray-drying as a possible formulation system of the biocontrol agent Bacillus subtilis CPA-8.

The role of endospore production by Bacillus subtilis CPA-8 on survival during spray-drying was investigated by comparison with a non-spore-forming biocontrol agent Pantoea agglomerans CPA-2. Endospore formation promoted heat resistance in CPA-8 depending on growth time (72 h cultures were more resistant than 24 h ones). The survival of CPA-8 and CPA-2 after spray-drying was determined after being grown in optimised media for 24 and 72 h. Spray-dried 72 h CPA-8 had the best survival (32%), while CPA-2 viability was less than 2%. CPA-8 survival directly related with its ability to produce endospores. Spray-dried CPA-8 reduced Monilinia fructicola conidia germination similarly to fresh cells, demonstrating that spray-drying did not adversely affect biocontrol efficacy. Endospore production thus improves CPA-8 resistance to spray-drying. These results can provide a reliable basis for optimising of the spray-drying formulation process for CPA-8 and other microorganisms.

Infection capacities in the orange-pathogen relationship: compatible (Penicillium digitatum) and incompatible (Penicillium expansum) interactions.

Penicillium digitatum and Penicillium expansum are the most devastating pathogens of citrus and pome fruits, respectively. Whereas P. digitatum is a very specific pathogen that only infects Citrus fruits, P. expansum has a broader host range but has not been reported to be infectious in Citrus. To determine the responses of fruits and the infection capacities of both moulds, two varieties of oranges at different maturity stages, different inoculum concentrations and two different storage temperatures were studied. In compatible interactions, no significant differences in rot dynamics among harvests were found with a 10(7) conidia mL(-1) inoculum concentration at both temperatures tested (20 °C and 4 °C). However, at other inoculum concentrations, significant differences in rot dynamics were found, especially in immature fruits. Incompatible interactions showed that P. expansum could infect oranges at commercial maturity in both tested varieties. Decay incidence and severity were higher at 4 °C than at 20 °C. In addition to infection capacity studies, histochemical tests were performed to detect wound-healing compounds for both pathogens. A positive reaction for lignin was detected for both pathogens in immature oranges over a short period (48 h). In all cases, no reactions were found in control samples. Our results indicate that pathogen concentration, host maturity and storage temperature can play important roles in the defence mechanisms of fruit. Furthermore, to our knowledge, this is the first work that demonstrates that P. expansum can infect oranges under favourable conditions.

Pathogenic potential of the surviving Salmonella Enteritidis on strawberries after disinfection treatments based on ultraviolet-C light and peracetic acid.

Fresh fruits, especially strawberries, are usually consumed raw without any step to ensure their food safety. Salmonella enterica is one of the most important etiologic agents for foodborne diseases throughout the world and its ability to respond to some stress responses makes it even more dangerous. In the present investigation, we study the survival of S. Enteritidis (CECT-4300) on strawberries after 2-min of various disinfection steps (NaClO (200 ppm), peracetic acid (PAA; 40 ppm), water-assisted UV-C (WUV-C), and the combination WUV-C and 40 ppm of PAA (WUV-C + PAA)) and after 5 days of cold storage (4 °C). Moreover, the pathogenic potential of the surviving bacteria, such as the ability to survive throughout the gastrointestinal tract (GI) and subsequently the capability to adhere to and invade Caco-2 cells, was tested at each sampling point. After 2-min of washing procedures, reductions of S. Enteritidis on strawberries were ≥1.2 log, with no significant differences among treatments. However, the use of WUV-C + PAA treatment achieved the highest reductions in washing water, in which S. Enteritidis was not detected (

Transfer of Listeria innocua from contaminated compost and irrigation water to lettuce leaves.

Many foodborne outbreaks of some pathogens such as Escherichia coli O157:H7, Salmonella or Listeria have been associated with the consumption of contaminated vegetables. Contaminated manure and polluted irrigation water are probable vehicles for the pathogens. The aim of this study was to determine the potential transfer of Listeria innocua from soil fertilized with contaminated compost or irrigated with contaminated water to the edible parts of lettuce grown on these soils together with its survival in lettuce and in soil under field conditions during two different seasons. Moreover, its survival on lettuce sprinkled with contaminated irrigation water was evaluated. L. innocua survived in soil samples for 9 weeks at high concentrations, 10(5) cfu gdw(-1) in fall and 10(3) cfu gdw(-1) in spring. Pathogen survived better in fall, indicating an important influence of temperature and humidity. L. innocua population in lettuce leaves was very high on lettuce leaves after sprinkling, but decreased to undetectable levels at field conditions. There was also transfer of L. innocua from soil contaminated with compost or irrigated with contaminated water to lettuce leaves, mainly to the outer ones. Survival profiles of L. innocua on lettuce and soil samples contaminated either by application of contaminated compost or surface irrigation water was similar. Our results indicated that contaminated compost and contaminated irrigation water can play an important role in the presence of foodborne pathogens on vegetables.

Effect of temperature and water activity on in vitro germination of Monilinia spp.

AIMS: This study evaluated the effect of temperature (0-38 degrees C) and water activity (a(w): 0.87-0.99) on the lag phase prior to germination and the percentage of germination over time for Monilinia laxa, Monilinia fructicola and Monilinia fructigena. METHODS AND RESULTS: More than 80% of viable conidia germinated at 25 degrees C and 0.99 a(w) within 2 h for M. fructicola and M. fructigena and 4 h for M. laxa. There was no germination at 38 degrees C, and all three Monilinia spp. germinated at 0 degrees C. At the lowest a(w) (0.87), none of the Monilinia spp. was able to germinate at any of the incubation temperatures studied. Whereas at 0.90 a(w), conidia were only able to germinate at 15, 25 and 30 degrees C for the three species studied, except for M. fructicola at 15 degrees C. In contrast, at 0.95, 0.97 and 0.99 a(w), germination occurred at all studied temperatures less 38 degrees C. Generally, the lag phase was longer at low levels of a(w) (0.90-095), and differences were more evident as temperatures were far from the optimum (0-5 degrees C). CONCLUSIONS: Germination and lag phase period were markedly influenced by temperature and a(w), and in general when conditions of temperature and a(w) were suboptimal, the lag phase was longer and the percentage of germination was lower. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of the germination requirements of this fungus is important in order to understand their behaviour in natural situations and to provide baseline data required for the construction of new prediction models. Our study might be used to develop a predictive model to understand and control the disease caused by Monilinia spp.

Effects of packaging type and storage temperature on the growth of foodborne pathogens on shredded 'Romaine' lettuce.

Fresh produce can be a vehicle for the transmission of pathogens capable of causing human illnesses and some of them can grow on fresh-cut vegetables. The survival and growth of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes inoculated onto shredded lettuce was determined under modified atmosphere packaging conditions, at various storage temperatures. We also monitored changes in pH and gas atmospheres within the packages and the growth of psychrotrophic and mesophilic microorganisms. After pathogen inoculation, shredded lettuce was packaged in films of different permeability and stored at 5 and 25 degrees C. After 10 days at 5 degrees C populations of E. coli O157:H7 and Salmonella decreased approximately 1.00 log unit while L. monocytogenes increased about 1.00 log unit, in all package films. Moreover, the pathogens level increased between 2.44 and 4.19 log units after 3 days at 25 degrees C. Psychrotrophic and mesophilic bacteria had similar growth at both temperatures with higher populations in air than in the other atmospheres. The composition of the storage atmosphere within the packaging of lettuce had no significant effect on the survival and growth of the pathogens used in this study at refrigeration temperatures. The results obtained can be considered as a warning indicator, which reinforces the necessity for corrective measures to avoid contamination of vegetables.

Microbiological quality of fresh lettuce from organic and conventional production.

Previously there was no available information on the levels of indicator bacteria and the prevalence of pathogens in fresh lettuce grown in organic and conventional farms in Spain. A total of 72 lettuce samples (18 farms for 4 repetitions each) for each type of the agriculture were examined in order to assess the bacteriological quality of the lettuces, in particular the prevalence of selected pathogens. The lettuce samples were analyzed for the presence of aerobic mesophilic, psychrotrophic microorganisms, yeasts and moulds, Enterobacteriaceae, mesophilic lactic acid bacteria, Pseudomonas spp. and presumptive Escherichia coli, Salmonella spp. and Listeria monocytogenes. The mean aerobic mesophilic counts (AM) were 6.35 +/- 0.69 log(10) cfu g(-1) and 5.67 +/- 0.80 log(10) cfu g(-1) from organic and conventional lettuce, respectively. The mean counts of psychrotrophic microorganisms were 5.82 +/- 1.01 log(10) cfu g(-1) and 5.41 +/- 0.92 log(10) cfu g(-1) from organic and conventional lettuce, respectively. Yeasts and moulds (YM) mean counts were 4.74 +/- 0.83 log(10) cfu g(-1) and 4.21 +/- 0.96 log(10) cfu g(-1) from organic and conventional lettuce, respectively. Lactic acid bacteria (LAB) were present in low numbers and the mean counts were 2.41 +/- 1.10 log(10) cfu g(-1) and 1.99 +/- 0.91 log(10) cfu g(-1) from organic and conventional lettuce, respectively. Pseudomonas spp. mean counts were 5.49 +/- 1.37 log(10) cfu g(-1) and 4.98 +/- 1.26 log(10) cfu g(-1) in organic and conventional lettuce, respectively. The mean counts for Enterobacteriaceae were 5.16 +/- 1.01 log(10) cfu g(-1) and 3.80 +/- 1.53 log(10) cfu g(-1) in organic and conventional lettuce, respectively. E. coli was detected in 22.2% (16 samples) of organic lettuce and in 12.5% (9 samples) of conventional lettuce. None of the lettuce samples was positive for E. coli O157:H7, L. monocytogenes and Salmonella spp. From the samples analyzed by principal component analysis (PCA) a pattern with two different groups (conventional and organic) can be observed, being the highest difference between both kinds of samples the Enterobacteriaceae count.

Occurrence of selected viral and bacterial pathogens and microbiological quality of fresh and frozen strawberries sold in Spain.

Strawberry production and exports have been increasing in Spain in recent decades. However, little information is available about their microbiological quality. Due to the growing concern about the microbial safety of these fruits, the objective of this investigation was to study the microbiological quality and the prevalence of the main foodborne pathogens on strawberries sold in Spain. Fresh (n = 152) and frozen (n = 31) samples were obtained from marketplaces and fields in 2017 and 2018. The samples were assayed for total aerobic mesophilic microorganisms (TAM), moulds and yeasts (M&Y), total coliforms (TC), Escherichia coli, Salmonella spp., Listeria monocytogenes as well as Norovirus (NoV) GI and GII. The microbiological counts ranged from <1.70 (detection limit, dl) - 5.89 log10 CFU/g (mean 3.78 log10 CFU/g) for TAM; 2.10-5.86 log10 CFU/g (mean 3.80 log10 CFU/g) for M&Y; and <0.70 (dl) - 4.91 log10 CFU/g (mean 2.15 log10 CFU/g) for TC in fresh strawberries. In frozen strawberries, the counts were <1.70 (dl) - 3.66 log10 CFU/g (mean 2.30 log10 CFU/g) for TAM; <1.70 (dl) - 2.76 log10 CFU/g (mean 1.82 log10 CFU/g) for M&Y; and <0.70(dl) - 1.74 log10 CFU/g (mean 0.77 log10 CFU/g) for TC. All the samples in this study tested negative for Salmonella spp., L. monocytogenes. E. coli and NoV GI and GII genome. A global overview of all the data was executed using Principal Component Analysis (PCA), and the results showed that the scores and loadings according to principal components 1 (PC1) and 2 (PC2) accounted for 75.9% of the total variance, allowing a distinction between fresh and frozen samples. The presence of moulds was significantly higher in the supermarket samples whereas the presence of total coliforms was significantly higher in the field samples (p < 0.05). Although pathogenic microorganisms were not found, preventative measures and prerequisites in the strawberry production chain must be considered in order to avoid possible foodborne diseases related to the microbiological quality of the fruit.

Evaluation of a sanitizing washing step with different chemical disinfectants for the strawberry processing industry.

Strawberries are often consumed fresh or only receive minimal processing, inducing a significant health risk to the consumer if contamination occurs anywhere from farm to fork. Outbreaks of foodborne illness associated with strawberries often involve a broad range of microbiological agents, from viruses (human norovirus) to bacteria (Salmonella spp. and Listeria monocytogenes). The addition of sanitizers to water washes is one of the most commonly studied strategies to remove or inactivate pathogens on berries as well as avoid cross contamination due to reuse of process wash water. The risk posed with the safety issues of by-products from chlorine disinfection in the fruit industry has led to a search for alternative sanitizers. We evaluated the applicability of different chemical sanitizers (peracetic acid (PA), hydrogen peroxide (H2O2), citric acid (CA), lactic acid (LA) and acetic acid (AA)) for the inactivation of S. enterica, L. monocytogenes and murine norovirus (MNV-1) on strawberries. A control treatment with chlorine (NaClO) (100 ppm) was included. For each sanitizer, different doses (40, 80 and 120 ppm for PA and 1, 2.5 and 5% for H2O2, LA, AA and CA) and time (2 and 5 min) were studied in order to optimize the decontamination washing step. The best concentrations were 80 ppm for PA, 5% for H2O2 and 2.5% for organic acids (LA, AA and CA) after 2 min treatment. Results indicate that the sanitizers selected may be a feasible alternative to chlorine (100 ppm) for removing selected pathogenic microorganisms (P > 0.05), with reductions about ≥2 log for bacterial strains and ≥ 1.7 log for MNV-1. As the washing water may also increase the microbial counts by cross-contamination, we observed that no pathogenic bacteria were found in wash water after 5% H2O2 and 80 ppm PA after 2 min treatment. On the other hand, we also reported reductions about total aerobic mesophyll (TAM) (0.0-1.4 log CFU/g) and molds and yeasts (M&Y) (0.3-1.8 log CFU/g) with all alternative sanitizers tested. Strawberries treated did not shown significant differences about physio-chemical parameters compared to the untreated samples (initial). For this study, the optimal sanitizer selected was PA, due to the low concentration and cost needed and its microbiocidal effect in wash water and fruit. Notwithstanding the results obtained, the effect of PA in combination with other non-thermal technologies such as water-assisted ultraviolet (UV-C) light should be studied in future research to improve the disinfection of strawberries.

Microbial interaction between Salmonella enterica and main postharvest fungal pathogens on strawberry fruit.

The microbial interaction between Salmonella enterica and the main postharvest fungal pathogens of strawberries was evaluated. Inoculation of fungal suspension was done 2 (D2) and 1 (D1) day(s) before and at the same time (D0) as S. enterica. Fruits were stored at 20 °C and 4 °C. At both temperatures, Botrytis cinerea and Rhizopus stolonifer caused a decrease in S. enterica population. Treatments where the mould was inoculated (D2, D1 and D0) achieved a significant logarithmic reduction (P < 0.05) of S. enterica populations after 48 h (20 °C) and 14 days (4 °C) compared to fungal-uninoculated fruits (CK). Regarding temperature, average reductions were significantly higher at 4 °C (3.38 log10 CFU/wound) than at 20 °C (1.16 log10 CFU/wound) (P < 0.05). Average reductions comprising all treatments were 1.91 and 0.41 log10 CFU/wound for B. cinerea and R. stolonifer at 20 °C, and 3.39 and 3.37 log10 CFU/wound for B. cinerea and R. stolonifer at 4 °C. A linear log10 model was fitted in order to predict the inactivation rate (kmax, log10 CFU/h) of S. enterica. Inactivation rates were higher at 20 °C for D2 treatments than at 4 °C throughout the running time. The main inactivation rate was obtained for B. cinerea at 20 °C (0.160 ±â€¯0.027/h), which was found to have stronger inhibitory activity against S. enterica than R. stolonifer. Univariate analysis ANOVA was carried out to evaluate the effect of different external variables on the inhibition of S. enterica. Results found that single effects were significant (P < 0.05) except for the pH. The inhibitory effect caused by the action of moulds in conjunction with some environmental factors could indicate the potential interactions between strawberry fungal pathogens and S. enterica.