In Situ Evaluation of Paenibacillus alvei in Reducing Carriage of Salmonella enterica Serovar Newport on Whole Tomato Plants

Isolation and screening of antagonistic bacteria.The native microflora of various plant organs (including leaves, shoots, roots, and blossoms) and soil from various Eastern Shore tomato-growing locations were examined. Simply, 3 g of plant material or soil was mixed for 5 min in 1 ml of phosphate-buffered saline (PBS). An aliquot (100 μl) was plated onto nutrient yeast glucose agar (NYGA). Ten colonies with unique morphologies that developed within 48 h at 30°C under aerobic conditions were picked for further purification, and a 3% KOH test was done to differentiate the Gram status without staining (13). The pure cultures were then tested for antagonistic activity in vitro by using an agar plug method (14). Briefly, pour plates of each test organism were prepared by mixing a 4-ml suspension of a plate culture grown overnight with sterile water in ca. 20 ml of warm tryptic soy agar (TSA). After incubation overnight at 35°C, agar plugs were punched from the agar with a sterile 10-mm stainless steel borer. Plugs were placed onto TSA agar containing a lawn of 10⁶ cells of S. enterica serovar Newport (15) and incubated at 35°C. Clear zones surrounding the plugs were measured at incubation periods of 24, 48, and 96 h.

Bacterial cultures.Isolates of potential bacterial antagonists and indicator strains (Table 1) were propagated on TSA at 35°C. Stock cultures grown overnight at 35°C on TSA were then resuspended in brain heart infusion (BHI) broth with 25% glycerol and stored at −80°C. Three tomato plant-associated bacterial pathogens, Erwinia carotovora subsp. carotovora, Pseudomonas syringae pv. tomato strain dc3000, and Ralstonia solanacearum race 5, were grown on TSA at 25°C (Table 1).

Phenotypic and biochemical characterizations of potential bacterial antagonists.The morphological characteristics of potential bacterial antagonists were observed by Gram and spore stains. These isolates were further tested with the Vitek 2 compact biochemical identification system (bioMérieux, Inc., Durham, NC) and the Biolog Omnilog microbial identification system (Biolog, Hayward, CA) with Gen III MicroPlates for biochemical properties, according to the manufacturers' instructions.

16S rRNA gene amplification and sequencing.Genomic DNA of potential bacterial antagonists was extracted by using the Wizard genomic DNA purification kit (Promega, Madison, WI). A pair of universal primers specific for bacterial 16S rRNA, Eubac27 and R1492 (16), were used to amplify the corresponding gene. PCR amplification of the 16S rRNA gene was performed with a Hotstart Taq Plus DNA polymerase kit (Qiagen, Valencia, CA) under the following conditions: after an initial 5-min incubation at 95°C, the mixture was subjected to 30 cycles, each including 1 min at 95°C, 1 min at 58°C, and 1 min at 72°C. A final extension step was performed at 72°C for 10 min. Primers 4F, 27F, 357F, 578F, 1000R, and 1492R were used for sequencing (16). The BLAST algorithm was used for a homology search against GenBank. Only results from the highest-score queries were considered for phylotype identification, with 99% minimum similarity (17).

Determination of mode of action and spectrum of antimicrobial activities.To determine the mode of action and antimicrobial spectrum of the bacterial antagonists, both an agar plug assay (using bacterial cultures) and a Bioscreen assay (using culture supernatants) were performed against a broad spectrum of major food-borne pathogens and bacterial phytopathogens (Table 1). In the agar plug assay, bactericidal effects against pathogenic bacterial strains in the zone of inhibition were confirmed when no viable cells were recovered on TSA plates. In the Bioscreen assay, the antagonist supernatant from a culture grown overnight was filter sterilized with a 0.22-μm-pore-size cellulose acetate (CA) membrane filter. Each 3-ml TS-15 cell-free culture supernatant (CFCS) was inoculated with 3 μl of 10⁸ CFU/ml bacterial culture (Table 1). Aliquots (200 μl) were then dispensed into sterile Bioscreen C microwell plates (Growth Curves USA, Piscataway, NJ) and incubated as described above for the respective bacterial strains. Bacterial growth was determined in five replicates by measuring the optical density at 600 nm (OD₆₀₀) at 20-min intervals for 24 h.

Tomato fruit assay.Red, round, ripe tomato fruits (130 ± 20 g each) were purchased from a local supermarket and refrigerated for no more than 3 days. Tomatoes were equilibrated to room temperature (RT) before testing and washed with 75% ethanol for surface sterilization and to remove any waxy residue if present. After air drying in a laminar flow hood, tomatoes were aseptically placed onto sterile metal trays with the stem scars facing down. A 20-μl drop (18) of a suspension of an S. Newport culture grown overnight (washed twice with PBS and resuspended in 5 ml of PBS) was placed within a 3-cm-diameter circle on the side of the tomato, equidistant from both ends of the tomato. The Salmonella inoculum was allowed to dry before antagonist inoculation. A 40-μl drop of the antagonist culture suspension (washed twice with PBS and resuspended in 5 ml of fresh tryptic soy broth [TSB]) or 40 μl of TSB only was then placed on top of the Salmonella inoculum. After 1.5 h in the hood, completely air-dried samples were placed into a humidity chamber (i.e., a closed container filled with 1.5 liters of water in a 30°C incubator). After 24 h of incubation, each tomato was placed into a sterile Whirl-Pak filter bag containing 30 ml of PBS and hand rubbed for 5 min to dislodge surface-inoculated Salmonella. The wash suspension was diluted 10-fold in PBS, and 0.1-ml aliquots of the appropriate dilutions were spread onto XLD agar (Becton, Dickinson and Company, Sparks, MD) to determine the surviving Salmonella populations.

Field trials in a high tunnel. (i) Plants.Trials were performed in 2010 (July through September) on tomato cultivar BHN602 in an insect-screened high tunnel at the U.S. Department of Agriculture (USDA) Beltsville Agricultural Research Center (BARC) north farm, Beltsville, MD. Tomato plants were grown from seeds in one of the BARC greenhouses in commercial organic peat mix (Johnny's 512 mix; Johnny's Selected Seeds, Fairfield, ME) and fertilized with Neptune's Harvest Organic Fish/Seaweed Blend fertilizer (Gloucester, MA) before and after transplanting. In the high tunnel, fertilizer was supplied from a single injector through drip tape supplemented with an Organic Materials Review Institute (OMRI)-approved calcium source to prevent blossom end rot. Black plastic mulch was used to cover the 8 planting beds (2 by 20 ft each) over the drip tape. Planting slits were made in the black plastic at 15-in. intervals to accommodate 13 transplants per bed. Plants were staked by using the Florida weave method with nylon support strings when they were 10 in. high. All plants were irrigated immediately after transplanting and at least weekly to achieve 1 to 1.5 in. water and to meet fertility requirements. Soil moisture was monitored by irrometers and digitally on a Hobo weather station that was located in the center of the high tunnel. Temperature, RH (relative humidity), PAR (photosynthetically active radiation), and total SR (solar radiation) were also monitored and recorded during this time.

(ii) Experimental design.A split-plot design was used with two treatments (Salmonella only and Salmonella with an antagonist) as the first-level subplot. Inoculation sites, including leaf, blossom, and tomato fruit, were each assigned a second-level subplot, with each inoculation site as an independent experimental unit and day of harvest postinoculation as a repeated measure. The second level corresponds to harvests used for 0 (2 h after inoculation as a benchmark for percent recovery)-, 1-, 2-, 3-, and 5- or 6-day persistence trials. Thirteen plants were planted in each plot. One plant on each end of each bed served as an uninoculated border plant, leaving 11 replicates per plot.

(iii) Inoculum preparation.Because of concerns about the safe use of pathogens in the field, an attenuated S. Newport 17 ΔtolC::aph strain was constructed for the high-tunnel study. The tolC gene on the S. Newport strain 17 chromosome was replaced by a cassette containing a kanamycin resistance gene by using the one-step inactivation method described previously by Datsenko and Wanner (19). TolC is an outer membrane protein important not only for the efflux of small compounds but also for the export of large proteins. Disruption of tolC abolished the ability of S. Typhimurium to adhere to, invade, and survive in eukaryotic cells (20). An S. Enteritidis tolC mutant was shown to be avirulent in the BALB/c mouse model as well (21). TSB suspensions of an S. Newport 17 ΔtolC::aph strain culture grown overnight were washed twice in PBS and then spot inoculated onto three marked leaves (20 μl each), six to nine blossoms (10 μl each), and three breaker-to-red tomato fruits (20 μl each) for a final concentration of ∼10⁹ CFU/ml per plant. The inoculation spots were allowed to air dry (∼1 h) before the antagonist was applied. Antagonist cell suspensions were made from a bacterial lawn. After two washes with PBS, cells were resuspended in 10 ml of TSB. Forty microliters of the antagonist cell suspension or sterile TSB was applied to the same inoculation spot on leaves and fruits, and 10 μl was applied to Salmonella-inoculated blossoms, of each plant in the “with”- or “without”-antagonist group, respectively. Leaves, blossoms, and fruits were harvested at 0, 1, 2, 3, and 5 days postinoculation (dpi) (for blossoms) or at 6 dpi (for leaves and fruits).

(iv) Sample collection.Inoculated leaves, blossoms, and fruits from each plant were removed with sterile scissors and placed into individual plastic zipper bags, which were sealed and transported in an insulated cooler to the laboratory for analysis within 1 h. For leaves and blossoms, each sample bag was filled with 15 ml and 10 ml of PBS, respectively, and hand rubbed for 3 min to dislodge surface populations of Salmonella. For fruits, each sample bag was filled with 30 ml of PBS and subjected to sonication at 55 Hz/min for 30 s (22), which has been shown to be harmless to the infecting microorganisms. PBS was diluted or concentrated through filtration (at later time points in the experiment) and surface plated (0.1 ml in duplicate) onto TSA with kanamycin (TSA-Kan) (50 μg/ml). Plates were incubated at 35°C overnight, and kanamycin-resistant colonies were counted. Two colonies were randomly picked from each TSA-Kan plate and confirmed by PCR using a set of verification primers (19).

(v) Statistical analysis.Estimates of the rate of reduction in bacterial counts were obtained by fitting a robust linear model of the log-transformed CFU onto days (days after inoculation). The slopes of the fitted lines from antagonist-treated and untreated surfaces were compared for differences in the rates of reduction. The analysis was performed by using the R statistical software package, version 2.11.1, with the robust library. The results were tallied for each combination of plant location, antagonist, plant, and day. Within each plant location, both a regression and a rank test compared the effect of using the antagonist with that of not using it. The sum of the counts on all plates was divided by the sum of the volumes (0.1 ml) of the initial sample. An imputation procedure, discussed previously by Blodgett (23), accounted for the plates for which the bacteria were too numerous to count.