Stress response, amino acid biosynthesis and pathogenesis genes expressed in Salmonella enterica colonizing tomato shoot and root surfaces.

Salmonella enterica can colonize all parts of the tomato plant. Tomatoes have been frequently implicated in salmonellosis outbreaks. In agricultural settings, Salmonella must overcome stress, nutritional and competition barriers to become established on plant surfaces. Knowledge of the genetic mechanisms underlying Salmonella-plant associations is limited, especially when growing epiphytically. A genome-wide transcriptomic analysis of Salmonella Typhimurium (SeT) was conducted with RNA-Seq to elucidate strategies for epiphytic growth on live, intact tomato shoot and root surfaces. Six plasmid-encoded and 123 chromosomal genes were significantly (using Benjamini-Hochberg adjusted p-values) up-regulated; 54 and 110 detected in SeT on shoots and roots, respectively, with 35 common to both. Key signals included NsrR regulon genes needed to mitigate nitrosative stress, oxidative stress genes and host adaptation genes, including environmental stress, heat shock and acid-inducible genes. Several amino acid biosynthesis genes and genes indicative of sulphur metabolism and anaerobic respiration were up-regulated. Some Type III secretion system (T3SS) effector protein genes and their chaperones from pathogenicity island-2 were expressed mostly in SeT on roots. Gene expression in SeT was validated against SeT and also the tomato outbreak strain Salmonella Newport with a high correlation (R 2 = 0.813 and 0.874, respectively; both p < 0.001). Oxidative and nitrosative stress response genes, T3SS2 genes and amino acid biosynthesis may be needed for Salmonella to successfully colonize tomato shoot and root surfaces.

Salmonella enterica Elicits and Is Restricted by Nitric Oxide and Reactive Oxygen Species on Tomato.

The enteric pathogen Salmonella enterica can interact with parts of the plant immune system despite not being a phytopathogen. Previous transcriptomic profiling of S. enterica associating with tomato suggested that Salmonella was responding to oxidative and nitrosative stress in the plant niche. We aimed to investigate whether Salmonella was eliciting generation of reactive oxygen species (ROS) and nitric oxide (NO), two components of the microbe-associated molecular pattern (MAMP)-triggered immunity (MTI) of plants. We also sought to determine whether this interaction had any measurable effects on Salmonella colonization of plants. Biochemical, gene expression and on-plant challenge assays of tomato vegetative and fruit organs were conducted to assess the elicitation of ROS and NO in response to Salmonella Newport association. The counter bacterial response and the effect of NO and ROS on Salmonella colonization was also investigated. We detected H2O2 in leaves and fruit following challenge with live S. Newport (p < 0.05). Conversely, NO was detected on leaves but not on fruit in response to S. Newport (p < 0.05). We found no evidence of plant defense attenuation by live S. Newport. Bacterial gene expression of S. Newport associating with leaves and fruit were indicative of adaptation to biotic stress in the plant niche. The nitrosative stress response genes hmpA and yoaG were significantly up-regulated in S. Newport on leaves and fruit tissue compared to tissue scavenged of NO or ROS (p < 0.05). Chemical modulation of these molecules in the plant had a restrictive effect on bacterial populations. Significantly higher S. Newport titers were retrieved from H2O2 scavenged leaves and fruit surfaces compared to controls (p < 0.05). Similarly, S. Newport counts recovered from NO-scavenged leaves, but not fruit, were higher compared to control (p < 0.05), and significantly lower on leaves pre-elicited to produce endogenous NO. We present evidence of Salmonella elicitation of ROS and NO in tomato, which appear to have a restricting effect on the pathogen. Moreover, bacterial recognition of ROS and NO stress was detected. This work shows that tomato has mechanisms to restrict Salmonella populations and ROS and NO detoxification may play an important role in Salmonella adaptation to the plant niche.

Metagenomic analysis of bacterial and viral assemblages from a freshwater creek and irrigated field reveals temporal and spatial dynamics.

Lotic surface water sites (e.g. creeks) are important resources for localized agricultural irrigation. However, there is concern that microbial contaminants within untreated surface water may be transferred onto irrigated soil and crops. To evaluate this issue, water samples were collected between January 2017 and August 2018 from a freshwater creek used to irrigate kale and radish plants on a small farm in the Mid-Atlantic, United States. In addition, on one sampling date, a field survey was conducted in which additional water (creek source and point-of-use) and soil samples were collected to assess the viral and bacterial communities pre- and post- irrigation. All samples were processed for DNA extracts and shotgun sequenced on the Illumina HiSeq platform. The resulting metagenomic libraries were assembled de novo and taxonomic and functional features were assigned at the contig and peptide level. From these data, we observed that Betaproteobacteria (e.g. Variovorax) dominated the water, both at the source and point-of-use, and Alphaproteobacteria (e.g. Streptomyces) dominated both pre- and post-irrigated soil. Additionally, in the creek source water there were variations in the abundance of the dominant bacterial genera and functional annotations associated with seasonal characteristics (e.g. water temperature). Antibiotic resistance genes and virulence factors were also identified in the creek water and soil, with the majority specific to their respective habitat. Moreover, an analysis of clustered regularly interspaced short palindromic repeat (CRISPR) arrays showed the persistence of certain spacers through time in the creek water, as well as specific interactions between creek bacteriophages and their hosts. Overall, these findings provide a more holistic picture of bacterial and viral composition, dynamics, and interactions within a freshwater creek that can be utilized to further our knowledge on its suitability and safety for irrigation.

Creek to Table: Tracking fecal indicator bacteria, bacterial pathogens, and total bacterial communities from irrigation water to kale and radish crops.

The impact of microbially contaminated irrigation water on risks to produce safety and public health is a complex issue that is not well understood. This study tracked fecal indicators, pathogenic bacteria, and total bacterial communities from a creek water irrigation source to irrigated produce to assess the impact of irrigation events on soil and produce-associated microbiota. Kale and radishes were drip-irrigated using Mid-Atlantic creek water in October 2017. Plant and soil samples were collected immediately before and after irrigation, and for 3 consecutive days thereafter. All samples (n = 134), including irrigation water, were tested for generic Escherichia coli and total coliforms (TC) using standard membrane filtration or direct plating, and for Salmonella enterica and Listeria monocytogenes by selective enrichment. DNA extracted from all samples was PCR-amplified for the V3-V4 region of the 16S rRNA gene for bacterial community profiling. In soil, TC levels were significantly higher immediately and 3 days post-irrigation compared to pre-irrigation (p < 0.01). E. coli levels in soil increased after irrigation, but the difference was not significant (p = 0.31), and die-off was not observed. No E. coli were detected on kale leaves. TC increased over the study period on radish roots (p < 0.01) but not kale leaves (p = 0.43). Although target pathogens were detected in irrigation water, S. enterica was detected from only one post-irrigation kale sample and L. monocytogenes was not detected in the field. The 16S rRNA gene sequencing data revealed differences in bacterial community structure and composition across sample types and showed that radish soil and root surface bacterial communities were more strongly influenced by irrigation compared to kale samples. This study provides insights into the impact of irrigation water on fresh produce microbiota, revealing that, although irrigation did influence crop-associated microbiota (especially below ground) in the field, bacterial pathogens were not likely transferred to the crop.