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 (

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.

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.

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.

Biopreservative methods to control the growth of foodborne pathogens on fresh-cut lettuce.

Fruits and vegetables can become contaminated by foodborne pathogens such as Escherichia coli O157:H7, Salmonella and Listeria monocytogenes, and it has been demonstrated that current industrial sanitizing treatments do not eliminate the pathogens when present. Chemical control is widely used, but biological control appears to be a better solution, mainly using the native microbiota present on fresh produce. The first objective of this study was to isolate native microbiota from whole and fresh-cut produce and to determine whether these bacteria were antagonistic toward foodborne pathogens. A total of 112 putative antagonist isolates were screened for their ability to inhibit the growth of Salmonella enterica on lettuce disks. Five different genera reduced S. enterica growth more than 1-log unit at 20°C at the end of 3 days. When tested against L. monocytogenes 230/3, only Pseudomonas sp. strain M309 (M309) was able to reduce pathogen counts by more than 1-log unit. Therefore, M309 strain was selected to be tested on lettuce disks at 10°C against S. enterica, E. coli O157:H7 and L. monocytogenes. M309 strain was only able to reduce S. enterica and E. coli O157:H7 populations. The second objective was to test different biopreservative methods including M309 strain, Pseudomonas graminis CPA-7 (CPA-7), bacteriophages (Listex P100 and Salmonelex) and nisin at conditions simulating commercial applications against Salmonella and L. monocytogenes on fresh-cut lettuce. The addition of the biopreservative agents did not result in a significant reduction of Salmonella population. However, CPA-7 strain together with nisin reduced L. monocytogenes numbers after 6 days of storage at 10°C. The cocktail of Salmonella and L. monocytogenes was not markedly inactivated by their respective bacteriophage solutions. This study highlighted the potential of biocontrol, but the combination with other technologies may be required to improve their application on fresh-cut lettuce.

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.

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.

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.

Acidification of apple and orange hosts by Penicillium digitatum and Penicillium expansum.

New information about virulence mechanisms of Penicillium digitatum and Penicillium expansum could be an important avenue to control fungal diseases. In this study, the ability of P. digitatum and P. expansum to enhance their virulence by locally modulating the pH of oranges and apples was evaluated. For each host, pH changes with a compatible pathogen and a non-host pathogen were recorded, and the levels of different organic acids were evaluated to establish possible relationships with host pH modifications. Moreover, fruits were harvested at three maturity stages to determine whether fruit maturity could affect the pathogens' virulence. The pH of oranges and apples decreased when the compatible pathogens (P. digitatum and P. expansum, respectively) decayed the fruit. The main organic acid detected in P. digitatum-decayed oranges was galacturonic acid produced as a consequence of host maceration in the rot development process. However, the obtained results showed that this acid was not responsible for the pH decrease in decayed orange tissue. The mixture of malic and citric acids could at least contribute to the acidification of P. digitatum-decayed oranges. The pH decrease in P. expansum decayed apples is related to the accumulation of gluconic and fumaric acids. The pH of oranges and apples was not affected when the non-host pathogen was not able to macerate the tissues. However, different organic acid contents were detected in comparison to healthy tissues. The main organic acids detected in P. expansum-oranges were oxalic and gluconic and in P. digitatum-apples were citric, gluconic and galacturonic. Further research is needed to identify the pathogenicity factors of both fungi because the contribution of organic acids has profound implications.

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.

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.

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.

Use of GFP-tagged strains of Penicillium digitatum and Penicillium expansum to study host-pathogen interactions in oranges and apples.

Penicillium digitatum and Penicillium expansum are responsible for green and blue molds in citrus and pome fruits, respectively, which result in major monetary losses worldwide. In order to study their infection process in fruits, we successfully introduced a green fluorescent protein (GFP) encoding gene into wild type P. digitatum and P. expansum isolates, using Agrobacterium tumefaciens-mediated transformation (ATMT), with hygromycin B resistance as the selectable marker. To our knowledge, this is the first report describing the transformation of these two important postharvest pathogens with GFP and the use of transformed strains to study compatible and non-host pathogen interactions. Transformation did not affect the pathogenicity or the ecophysiology of either species compared to their respective wild type strains. The GFP-tagged strains were used for in situ analysis of compatible and non-host pathogen interactions on oranges and apples. Knowledge of the infection process of apples and oranges by these pathogens will facilitate the design of novel strategies to control these postharvest diseases and the use of the GFP-tagged strains will help to determine the response of P. digitatum and P. expansum on/in plant surface and tissues to different postharvest treatments.

Influence of diluent and sample processing methods on the recovery of the biocontrol agent Pantoea agglomerans CPA-2 from different fruit surfaces.

Determining the populations of biocontrol agents applied as a postharvest treatment on fruit surfaces is fundamental to the assessment of the microorganisms' ability to colonise and persist on fruit. To obtain maximum recovery, we must develop a methodology that involves both diluent and processing methods and that does not affect the viability of the microorganisms. The effect of diluent composition was evaluated using three diluents: phosphate buffer, peptone saline and buffered peptone saline. An additional study was performed to compare three processing methods (shaking plus sonication, stomaching and shaking plus centrifugation) on the recovery efficiency of Pantoea agglomerans strain CPA-2 from apples, oranges, nectarines and peaches treated with this biocontrol agent. Overall, slight differences occurred among diluents, although the phosphate buffer maintained the most ideal pH for CPA-2 growth (between 5.2 and 6.2). Stomaching, using the phosphate buffer as diluent, was the best procedure for recovering and enumerating the biocontrol agent; this fact suggested that no lethal effects from naturally occurring antimicrobial compounds present on the fruit skins and/or produced when the tissues were disrupted affected the recovery of the CPA-2 cells, regardless of fruit type. The growth pattern of CPA-2 on fruits maintained at 20°C and under cold conditions was similar to that obtained in previous studies, which confirms the excellent adaptation of this strain to conditions commonly used for fruit storage.

The infection capacity of P. expansum and P. digitatum on apples and histochemical analysis of host response.

Fruit ripening is a complex process that involves a variety of biochemical changes and is also associated with increased susceptibility to pathogens. The present study determined the effects of fruit maturity and storage conditions on the infection capacity of a host (P. expansum) and non-host (P. digitatum) pathogen on apple. A range of inoculum concentrations and two different storage temperatures were utilized. Exposure to P. expansum at 20 °C resulted in significant differences in rot dynamics in apples collected at the earliest harvest date compared to all later harvest dates and inoculum concentrations assayed. Greater differences in infection capacity between harvests were obtained when fruit was stored at low temperature (0 °C). In contrast, P. digitatum was able to infect apples only under specific conditions and disease symptoms were limited to the initial wound inoculation site. When apples were resistant to P. digitatum, a visible browning reaction around the infection site was observed. Histochemical analyses of tissues surrounding the wound site were conducted. A positive reaction for lignin was observed in immature apples as early as 1 day after inoculation with either pathogen. Experiments conducted with the non-host pathogen indicated that lignification was an essential component of resistance in apples harvested prior to maturity or at commercial maturity. Apples harvested at an over-mature stage and inoculated with P. digitatum did not show evidence of staining for lignin until 7 days post-inoculation. Control samples only showed positive reaction in immature harvest. Results demonstrated that the maturity stage of fruit is an important factor in apple resistance to both P. expansum and P. digitatum and that lignin accumulation seems to play an important role when resistance is observed. Moreover, this is the first report demonstrating that P. digitatum, a non-host pathogen, has a limited capacity to infect apples.

Presence and survival of Escherichia coli O157:H7 on lettuce leaves and in soil treated with contaminated compost and irrigation water.

Escherichia coli O157:H7 outbreaks associated with produce consumption have brought attention to contaminated compost manure, and polluted irrigation water as potential sources of pathogens for the contamination of these crops. The aim of this study was to determine the potential transfer of E. coli O157:H7 from soil fertilized with contaminated compost or irrigated with contaminated water to edible parts of lettuce together with its persistence in soil under field conditions in two different seasons (fall and spring). Moreover, its survival on lettuce sprinkled with contaminated irrigation water was evaluated, as well as the prevalence of aerobic mesophilic, Enterobacteriaceae and Pseudomonadaceae in control lettuce samples. Four treatments, contaminated compost, surface and sprinkle irrigation with contaminated water and uninoculated pots, were used in this work. Contaminated compost was applied to soil in the pots before lettuce was transplanted and contaminated irrigation water was applied twice and three times on the plants after the seedlings were transplanted, for sprinkle and surface irrigation, respectively. E. coli O157:H7 survived in soil samples for 9 weeks at levels, 4.50 log cfu gdw(-1) (dw, dry weight) in fall and 1.50 log cfu gdw(-1) in spring. The pathogen survives better in fall, indicating an important influence of environmental factors. E. coli O157:H7 population in lettuce leaves after sprinkle irrigation was very high (between 10(3) and 10(6) cfu g(-1)), but decreased to undetectable levels at field conditions. There was also transfer of E. coli O157:H7 from soil contaminated with compost or irrigated with contaminated water to lettuce leaves, mainly to the outer ones. The mean counts for aerobic mesophilic, Enterobacteriaceae and Pseudomonadaceae populations were also influenced by environmental conditions; higher levels were observed under fall conditions than in spring conditions. Contamination of lettuce plants in the field can occur through both contaminated composted manure and irrigation water and persist for several months.