Intraspecies competition among Salmonella enterica isolates in the lettuce leaf apoplast.

Multiple Salmonella enterica serovars and strains have been reported to be able to persist inside the foliar tissue of lettuce (Lactuca sativa L.), potentially resisting washing steps and reaching the consumer. Intraspecies variation of the bacterial pathogen and of the plant host can both significantly affect the outcome of foliar colonization. However, current understanding of the mechanisms underlying this phenomenon is still very limited. In this study, we evaluated the foliar fitness of 14 genetically barcoded S. enterica isolates from 10 different serovars, collected from plant and animal sources. The S. enterica isolates were vacuum-infiltrated individually or in pools into the leaves of three- to four-week-old lettuce plants. To estimate the survival capacity of individual isolates, we enumerated the bacterial populations at 0- and 10- days post-inoculation (DPI) and calculated their net growth. The competition of isolates in the lettuce apoplast was assessed through the determination of the relative abundance change of barcode counts of each isolate within pools during the 10 DPI experimental period. Isolates exhibiting varying apoplast fitness phenotypes were used to evaluate their capacity to grow in metabolites extracted from the lettuce apoplast and to elicit the reactive oxygen species burst immune response. Our study revealed that strains of S. enterica can substantially differ in their ability to survive and compete in a co-inhabited lettuce leaf apoplast. The differential foliar fitness observed among these S. enterica isolates might be explained, in part, by their ability to utilize nutrients available in the apoplast and to evade plant immune responses in this niche.

Dual transcriptomic analysis reveals metabolic changes associated with differential persistence of human pathogenic bacteria in leaves of Arabidopsis and lettuce.

Understanding the molecular determinants underlying the interaction between the leaf and human pathogenic bacteria is key to provide the foundation to develop science-based strategies to prevent or decrease the pathogen contamination of leafy greens. In this study, we conducted a dual RNA-sequencing analysis to simultaneously define changes in the transcriptomic profiles of the plant and the bacterium when they come in contact. We used an economically relevant vegetable crop, lettuce (Lactuca sativa L. cultivar Salinas), and a model plant, Arabidopsis thaliana Col-0, as well as two pathogenic bacterial strains that cause disease outbreaks associated with fresh produce, Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium 14028s (STm 14028s). We observed commonalities and specificities in the modulation of biological processes between Arabidopsis and lettuce and between O157:H7 and STm 14028s during early stages of the interaction. We detected a larger alteration of gene expression at the whole transcriptome level in lettuce and Arabidopsis at 24 h post inoculation with STm 14028s compared to that with O157:H7. In addition, bacterial transcriptomic adjustments were substantially larger in Arabidopsis than in lettuce. Bacterial transcriptome was affected at a larger extent in the first 4 h compared to the subsequent 20 h after inoculation. Overall, we gained valuable knowledge about the responses and counter-responses of both bacterial pathogen and plant host when these bacteria are residing in the leaf intercellular space. These findings and the public genomic resources generated in this study are valuable for additional data mining.

Breeding Crops for Enhanced Food Safety.

An increasing global population demands a continuous supply of nutritious and safe food. Edible products can be contaminated with biological (e.g., bacteria, virus, protozoa), chemical (e.g., heavy metals, mycotoxins), and physical hazards during production, storage, transport, processing, and/or meal preparation. The substantial impact of foodborne disease outbreaks on public health and the economy has led to multidisciplinary research aimed to understand the biology underlying the different contamination processes and how to mitigate food hazards. Here we review the knowledge, opportunities, and challenges of plant breeding as a tool to enhance the food safety of plant-based food products. First, we discuss the significant effect of plant genotypic and phenotypic variation in the contamination of plants by heavy metals, mycotoxin-producing fungi, and human pathogenic bacteria. In addition, we discuss the various factors (i.e., temperature, relative humidity, soil, microbiota, cultural practices, and plant developmental stage) that can influence the interaction between plant genetic diversity and contaminant. This exposes the necessity of a multidisciplinary approach to understand plant genotype × environment × microbe × management interactions. Moreover, we show that the numerous possibilities of crop/hazard combinations make the definition and identification of high-risk pairs, such as Salmonella-tomato and Escherichia coli-lettuce, imperative for breeding programs geared toward improving microbial safety of produce. Finally, we discuss research on developing effective assays and approaches for selecting desirable breeding germplasm. Overall, it is recognized that although breeding programs for some human pathogen/toxin systems are ongoing (e.g., Fusarium in wheat), it would be premature to start breeding when targets and testing systems are not well defined. Nevertheless, current research is paving the way toward this goal and this review highlights advances in the field and critical points for the success of this initiative that were discussed during the Breeding Crops for Enhanced Food Safety workshop held 5-6 June 2019 at University of California, Davis.

Human Pathogen Colonization of Lettuce Dependent Upon Plant Genotype and Defense Response Activation.

Fresh produce contaminated with human pathogens may result in foodborne disease outbreaks that cause a significant number of illnesses, hospitalizations, and death episodes affecting both public health and the agribusiness every year. The ability of these pathogens to survive throughout the food production chain is remarkable. Using a genetic approach, we observed that leaf colonization by Salmonella enterica serovar Typhimurium 14028s (S. Typhimurium 14028s) and Escherichia coli O157:H7 was significantly affected by genetic diversity of lettuce (Lactuca sativa L. and L. serriola L.). In particular, there was a significant variation among 11 lettuce genotypes in bacterial attachment, internalization, and apoplastic persistence after surface- and syringe-inoculation methods. We observed a significant correlation of the bacterial leaf internalization rate with stomatal pore traits (width and area). Moreover, bacterial apoplastic populations significantly decreased in 9 out of 11 lettuce genotypes after 10 days of surface inoculation. However, after syringe infiltration, populations of E. coli O157:H7 and S. Typhimurium 14028s showed positive, neutral, or negative net growth in a 10-day experimental period among seedlings of different lettuce types. The relative ability of the bacteria to persist in the apoplast of lettuce genotypes after syringe inoculation was minimally altered when assessed during a longer period (20 days) using 3.5- to 4-week-old plants. Interestingly, contrasting bacterial persistence in the lettuce genotypes Red Tide and Lollo Rossa was positively correlated with significant differences in the level of reactive oxygen species burst and callose deposition against S. Typhimurium 14028s and E. coli O157:H7 which are related to plant defense responses. Overall, we characterized the genetic diversity in the interaction between lettuce genotypes and enterobacteria S. Typhimurium 14028s and E. coli O157:H7 and discovered that this genetic diversity is linked to variations in plant immune responses towards these bacteria. These results provide opportunities to capitalize on plant genetics to reduce pathogen contamination of leaves.

Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast.

Bacterial pathogens must enter the plant tissue in order to cause a successful infection. Foliar bacterial pathogens that are not able to directly penetrate the plant epidermis rely on wounds or natural openings to internalize leaves. This protocol describes a procedure to estimate the population size of Pseudomonas syringae in the leaf apoplast after surface inoculation of Arabidopsis rosettes.