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BACKGROUND: Field-grown leafy vegetables can be damaged by biotic and abiotic factors, or mechanically damaged by farming practices. Available methods to evaluate leaf tissue damage mainly rely on colour differentiation between healthy and damaged tissues. Alternatively, sophisticated equipment such as microscopy and hyperspectral cameras can be employed. Depending on the causal factor, colour change in the wounded area is not always induced and, by the time symptoms become visible, a plant can already be severely affected. To accurately detect and quantify damage on leaf scale, including microlesions, reliable differentiation between healthy and damaged tissue is essential. We stained whole leaves with trypan blue dye, which traverses compromised cell membranes but is not absorbed in viable cells, followed by automated quantification of damage on leaf scale. RESULTS: We present a robust, fast and sensitive method for leaf-scale visualisation, accurate automated extraction and measurement of damaged area on leaves of leafy vegetables. The image analysis pipeline we developed automatically identifies leaf area and individual stained (lesion) areas down to cell level. As proof of principle, we tested the methodology for damage detection and quantification on two field-grown leafy vegetable species, spinach and Swiss chard. CONCLUSIONS: Our novel lesion quantification method can be used for detection of large (macro) or single-cell (micro) lesions on leaf scale, enabling quantification of lesions at any stage and without requiring symptoms to be in the visible spectrum. Quantifying the wounded area on leaf scale is necessary for generating prediction models for economic losses and produce shelf-life. In addition, risk assessments are based on accurate prediction of the relationship between leaf damage and infection rates by opportunistic pathogens and our method helps determine the severity of leaf damage at fine resolution.
RESUMO
This study examined the biological and food safety relevance of leaf lesions for potential invasion of food pathogens into the plant tissue (internalization). This was done by determining the role of artificial leaf damage in terms of damaged leaf area on proliferation of E. coli O157:H7 gfp+. In a two-factorial experiment, unwashed fresh baby leaf spinach (Spinacia oleracea L.) was subjected to four damage levels (undamaged, low, moderate, high damage; factor 1) and three incubation intervals (0, 1, 2 days post-inoculation; factor 2). Individual leaves were immersed for 15 s in a suspension loaded with E. coli O157:H7 gfp+ (106 CFU × mL-1). The leaves were analyzed individually using image analysis tools to quantify leaf area and number and size of lesions, and using confocal laser scanning and scanning electron microscopy to visualize leaf lesions and presence of the introduced E. coli strain on and within the leaf tissue. Prevalence of E. coli O157:H7 gfp+ was assessed using a culture-dependent technique. The results showed that size of individual lesions and damaged leaf area affected depth of invasion into plant tissue, dispersal to adjacent areas, and number of culturable E. coli O157:H7 gfp+ directly after inoculation. Differences in numbers of the inoculant retrieved from leaf macerate evened out from 2 days post-inoculation, indicating rapid proliferation during the first day post-inoculation. Leaf weight was a crucial factor, as lighter spinach leaves (most likely younger leaves) were more prone to harbor E. coli O157:H7 gfp+, irrespective of damage level. At the high inoculum density used, the risk of consumers' infection was almost 100%, irrespective of incubation duration or damage level. Even macroscopically intact leaves showed a high risk for infection. These results suggest that the risk to consumers is correlated with how early in the food chain the leaves are contaminated, and the degree of leaf damage. These findings should be taken into account in different steps of leafy green processing. Further attention should be paid to the fate of viable, but non-culturable, shiga-toxigenic E. coli on and in ready-to-eat leafy vegetables.
RESUMO
Consumers appreciate leafy green vegetables such as baby leaves for their convenience and wholesomeness and for adding a variety of tastes and colors to their plate. In Western cuisine, leafy green vegetables are usually eaten fresh and raw, with no step in the long chain from seed to consumption where potentially harmful microorganisms could be completely eliminated, e.g., through heating. A concerning trend in recent years is disease outbreaks caused by various leafy vegetable crops and one of the most important foodborne pathogens in this context is Shiga toxin-producing Escherichia coli (STEC). Other pathogens such as Salmonella, Shigella, Yersinia enterocolitica and Listeria monocytogenes should also be considered in disease risk analysis, as they have been implicated in outbreaks associated with leafy greens. These pathogens may enter the horticultural value network during primary production in field or greenhouse via irrigation, at harvest, during processing and distribution or in the home kitchen/restaurant. The hurdle approach involves combining several mitigating approaches, each of which is insufficient on its own, to control or even eliminate pathogens in food products. Since the food chain system for leafy green vegetables contains no absolute kill step for pathogens, use of hurdles at critical points could enable control of pathogens that pose a human health risk. Hurdles should be combined so as to decrease the risk due to pathogenic microbes and also to improve microbial stability, shelf-life, nutritional properties and sensory quality of leafy vegetables. The hurdle toolbox includes different options, such as physical, physiochemical and microbial hurdles. The goal for leafy green vegetables is multi-target preservation through intelligently applied hurdles. This review describes hurdles that could be used for leafy green vegetables and their biological basis, and identifies prospective hurdles that need attention in future research.
