High-throughput method for detection and quantification of lesions on leaf scale based on trypan blue staining and digital image analysis.

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.

Size Matters: Biological and Food Safety Relevance of Leaf Damage for Colonization of Escherichia coli O157:H7 gfp.

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.

Multi-year dynamics of ranavirus, chytridiomycosis, and co-infections in a temperate host assemblage of amphibians.

Chytridiomycosis and ranavirosis are 2 emerging infectious diseases that have caused significant global amphibian decline. Although both have received much scrutiny, little is known about interactions between the 2 causative agents Batrachochytrium dendrobatidis (Bd) and ranavirus (Rv) at the individual host and population levels. We present the first longitudinal assessment of Bd, Rv, and co-infections of a temperate amphibian assemblage in North America. From 2012 to 2016, we assessed the temporal oscillations of Bd, Rv and co-infection dynamics in a sample of 729 animals representing 13 species. Bd, Rv, and co-infected amphibians were detected during all 5 yr. Bd, Rv, and co-infection prevalence all varied annually, with the lowest instances of each at 2.1% (2013), 7.9% (2016), and 0.6% (2016), respectively. The highest Bd, Rv, and co-infection prevalence were recorded in 2012 (26.8%), 2016 (38.3%), and 2015 (10.3%), respectively. There was no association between Bd or Rv infection prevalence and co-infection, either when assessing the entire amphibian assemblage as a whole (odds ratio 1.32, 95% CI: 0.83-2.1, p = 0.29) or within species for amphibians that were more numerically represented (n > 40, p > 0.05). This suggests neither Bd nor Rv facilitate host co-infections within the sampled host assemblage. Instead, the basis for co-infections is the spatiotemporal distribution of both pathogens. Despite lack of interplay between Bd and Rv in this population, our study highlights the importance of considering numerous pathogens that may be present within amphibian habitats in order to properly anticipate interactions that may have direct bearing on disease outcomes.

Light spectrum modifies the utilization pattern of energy sources in Pseudomonas sp. DR 5-09.

Despite the overruling impact of light in the phyllosphere, little is known regarding the influence of light spectra on non-phototrophic bacteria colonizing the leaf surface. We developed an in vitro method to study phenotypic profile responses of bacterial pure cultures to different bands of the visible light spectrum using monochromatic (blue: 460 nm; red: 660 nm) and polychromatic (white: 350-990 nm) LEDs, by modification and optimization of a protocol for the Phenotype MicroArray™ technique (Biolog Inc., CA, USA). The new protocol revealed high reproducibility of substrate utilization under all conditions tested. Challenging the non-phototrophic bacterium Pseudomonas sp. DR 5-09 with white, blue, and red light demonstrated that all light treatments affected the respiratory profile differently, with blue LED having the most decisive impact on substrate utilization by impairing respiration of 140 substrates. The respiratory activity was decreased on 23 and 42 substrates under red and white LEDs, respectively, while utilization of one, 16, and 20 substrates increased in the presence of red, blue, and white LEDs, respectively. Interestingly, on four substrates contrasting utilization patterns were found when the bacterium was exposed to different light spectra. Although non-phototrophic bacteria do not rely directly on light as an energy source, Pseudomonas sp. DR 5-09 changed its respiratory activity on various substrates differently when exposed to different lights. Thus, ability to sense and distinguish between different wavelengths even within the visible light spectrum must exist, and leads to differential regulation of substrate usage. With these results, we hypothesize that different light spectra might be a hitherto neglected key stimulus for changes in microbial lifestyle and habits of substrate usage by non-phototrophic phyllospheric microbiota, and thus might essentially stratify leaf microbiota composition and diversity.