Jerry A. Bartz

Internal Colonization of Salmonella enterica Serovar Typhimurium in Tomato Plants

Several Salmonella enterica outbreaks have been traced back to contaminated tomatoes. In this study, the internalization of S. enterica Typhimurium via tomato leaves was investigated as affected by surfactants and bacterial rdar morphotype, which was reported to be important for the environmental persistence and attachment of Salmonella to plants. Surfactants, especially Silwet L-77, promoted ingress and survival of S. enterica Typhimurium in tomato leaves. In each of two experiments, 84 tomato plants were inoculated two to four times before fruiting with GFP-labeled S. enterica Typhimurium strain MAE110 (with rdar morphotype) or MAE119 (without rdar). For each inoculation, single leaflets were dipped in 109 CFU/ml Salmonella suspension with Silwet L-77. Inoculated and adjacent leaflets were tested for Salmonella survival for 3 weeks after each inoculation. The surface and pulp of ripe fruits produced on these plants were also examined for Salmonella. Populations of both Salmonella strains in inoculated leaflets decreased during 2 weeks after inoculation but remained unchanged (at about 104 CFU/g) in week 3. Populations of MAE110 were significantly higher (P<0.05) than those of MAE119 from day 3 after inoculation. In the first year, nine fruits collected from one of the 42 MAE119 inoculated plants were positive for S. enterica Typhimurium. In the second year, Salmonella was detected in adjacent non-inoculated leaves of eight tomato plants (five inoculated with strain MAE110). The pulp of 12 fruits from two plants inoculated with MAE110 was Salmonella positive (about 106 CFU/g). Internalization was confirmed by fluorescence and confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can move inside tomato plants grown in natural field soil and colonize fruits at high levels without inducing any symptoms, except for a slight reduction in plant growth.

Untangling metabolic and communication networks: interactions of enterics with phytobacteria and their implications in produce safety

Recent outbreaks of vegetable-borne gastrointestinal illnesses across the globe demonstrate that human enteric pathogens can contaminate produce at any stage of production. Interactions of enterics with native plant-associated microbiota influence the microbiological safety of produce by affecting the attachment, persistence and proliferation of human pathogens on plants. Supermarket surveys have revealed that bacteria, but not fungi or mechanical damage, promote the growth of Salmonella enterica on produce. Field and laboratory studies have indicated that some plant pathogenic bacteria and fungi facilitate the entry and internalization of human pathogens in plants. Conversely, some phytobacteria, including those involved in biocontrol of plant diseases, significantly inhibit attachment and plant colonization by non-typhoidal Salmonella and enterovirulent Escherichia coli by producing antibiotics or competing for nutrients in the phyllosphere. In this review, we attempt to elucidate the mechanisms of interactions between human enteric pathogens and plant-associated microbiota, and describe how these interactions affect produce safety.

Oxidation-reduction potential of chlorine solutions and their toxicity to Erwinia carotovora subsp. carotovora and Geotrichum candidum

The toxicity of chlorine solutions to cells of Erwinia carotovora subsp. carotovora and conidia of Geotrichum candidum suspended in water at pH 6.0, 7.0, or 8.0 was correlated with the free chlorine concentration and oxidation-reduction potential of the solutions. The oxidation-reduction potential was directly correlated with the Log 10 of the chlorine concentration at each pH. Cells of E. c. carotovora were 50 times more sensitive to chlorine than were conidia of G, candidum, with populations of 1 × 10 7 cfu/ml and 1 × 10 7 conidia per milliliter, respectively, Populations of E. c, carotovora were reduced below detectable levels (< 10 2 cfu/ml) by approximately 0,5, 0,5, or 0,75 mg of free chlorine per liter at pH 6.0, 7.0, or 8.0, respectively. In contrast, with conidia of G. candidum, 25, 25, and greater than 30 mg/L, respectively, were required to produce a similar level of efficacy. With both organisms, population reductions were associated with higher initial oxidation-reduction potentials at pH 6.0 than at pH 8.0

Chlorine Concentration and the Inoculation of Tomato Fruit in Packinghouse Dump Tanks

Chlorine concentrations (pH 6 to 7 and 22 to 27°C) that killed arthrospores (spores) of Geotrichum candidum or sporangioles (spores) of Rhizopus stolonifer, causal agents of sour rot and Rhizopus rot, respectively, in moving water within 30 to 45 s did not prevent these pathogens from inoculating wounded tomatoes (Lycopersicon esculentum) in a water flume containing chlorine and spores. Free chlorine concentrations of 20 or 25 mg/liter were lethal to spores of G. candidum within 30 s in most in vitro tests, whereas spores of R. stolonifer were slightly less sensitive. Wounded tomatoes placed in a flume with free chlorine at 30 mg/liter and then exposed to spores for 1 min developed about 50% less decay incidence during storage at 24°C for 6 days than did fruit exposed to spores and water alone. In the absence of chlorine, incidence averaged 57% (range, 15 to 95%) for R. stolonifer and 38% (range, 17 to 58%) for G. candidum. Sporadic sour rot lesions were observed among fruit that had been treated with free chlorine at 75 mg/liter, whereas chlorine at up to 180 mg/liter failed to completely protect fruit from Rhizopus rot. A water-soluble dye rapidly penetrated wounds on tomato fruit. The dye framed the outlines of cells at the wound surface and appeared to penetrate into a few intercel-lular spaces. Application of 1% sodium hypochlorite decolorized the dye on the wound surface, whereas deposits located below the wound surface remained blue. Thus, spores suspended in moving water can escape the action of chlorine if carried into intercellular spaces by diffusion or by capillary movement of cell sap and water.

Postharvest Decay Risk Associated with Hydrocooling Tomatoes

Tomatoes (breaker stage) hydrocooled with a cell suspension of Erwinia carotovora subsp. carotovora containing 50 to 200 mg of free chlorine per liter (ppm) (10°C, pH 7) remained decay free during a 10-day storage at 20°C. Sporadic disease appeared during storage of tomatoes similarly cooled with chlorinated water containing spores of Rhizopus stolonifer. In contrast, when chlorine was omitted from the pathogen suspensions, 50 to 100% of the fruit became diseased. A laboratory-scale shower hydrocooler reduced fruit temperatures from 35 to 15°C within 13.3 min, whereas a flume cooler produced the same temperature reduction in 10.5 min. In both systems, tomatoes increased in weight during cooling, evidence for water uptake. Larger weight increases occurred among tomatoes cooled in the shower than in the flume. An upward instead of downward orientation of stem scars under the shower streams led to significantly larger weight increases, presumably because pores in the stem scar were continuously flooded with water. Tomatoes intermittently submerged in cold water (10 2-min immersions followed by 30-s pauses) absorbed significantly less water than those continuously submerged for 20 min. Hydrocooling appears to be a viable method for rapid cooling of tomatoes. Technical refinements in the hydrocooling process that prevent continuous coverage of fruit surfaces by water should reduce water uptake and the associated risk of pathogen internalization. Maintenance of free chlorine at up to 200 ppm in the cooling water and prevention of direct water pressure on fruit should minimize decay risks. No evidence of phytotoxicity was observed among fruit infiltrated with 200 ppm of chlorine. These tomatoes ripened similarly to those that were not cooled or were cooled in tap water.

Applications of Chlorine Dioxide Gas for Control of Bacterial Soft Rot in Tomatoes

Chlorine dioxide (ClO2) gas was generated from a mixture of sodium chlorite and ferric chloride plus water (impregnated into zeolite) in a Tyvek sachet over a 2- or 24-h period. The gas was distributed by a fan over wound-inoculated tomato fruit (Lycopersicon esculentum) enclosed in a sealed aluminum pressure cooker. Within 24 h of inoculation with 6 log10 CFU of Erwinia carotovora subsp. carotovora per wound and storage at 22 to 24°C, bacterial soft rot was observed on >80% of the nontreated wounds (10 wounds/fruit and 4 or 6 fruit/treatment). By contrast, wounds that had been exposed to an atmosphere containing up to 99 mg ClO2 during a 2- or 24-h period remained firm and dry with no evidence of bacterial activity or soft rot. After 72 h of incubation, wounds exposed to 88 mg ClO2 produced over 24 h or 99 mg ClO2 produced over 2 h were free of decay, whereas bacterial soft rot was observed in ca. 12% and less than 5% of wounds treated with 0.75 mg or 7.5 mg, respectively, for either 2 or 24 h. Wounds that had not been inoculated remained free of bacterial soft rot throughout the entire storage period. Wounds exposed to the highest doses of ClO2, 88 mg/24 h or 99 mg/2 h, became bleached and sunken. Additionally, the stem scars on these fruit became cracked, sunken, and bleached. The intact cuticle was not visibly affected, and there was no observed change in overall fruit color. ClO2 gas may be effective for controlling postharvest decays of fruit that have been inoculated prior to or during harvest.

An assessment of the decay hazard associated with hydrocooling strawberries

The decay hazard associated with hydrocooling strawberries was related to whether they became inoculated with postharvest pathogens during the procedure. Storage of hydrocooled berries at warmer temperatures or for longer periods than recommended allowed inoculation to be expressed into disease. Cooling strawberries by immersion hydrocooling did not consistently lead to increased postharvest decays when compared with conventional forced-air cooling in two separate trials. No differences in decay incidence were found in berries that were hydrocooled versus forced-air cooled and then stored 7 days at 1°C plus 1 day at 20°C. With a 15-day storage regime (14 days at ≤7°C plus 1 day at 20°C), hydrocooled fruit developed less decay than forced-air-cooled fruit in one trial but more decay in a second. Wrapping baskets of cooled berries with plastic film promoted disease development and slowed the moisture loss from both hydrocooled and forced-air-cooled berries. The wrap did not promote disease more when applied to wet, hydrocooled berries as compared with dry, forced-air-cooled berries. Residual moisture left on the berries by the hydrocool treatment did not predispose the fruit to postharvest decays. In contrast, wounds and abrasions on hydrocooled fruit were temporarily water soaked and berries typically increased in weight as they were hydrocooled. Berries cooled in water containing spores of Botrytis cinerea or Rhizopus stolonifer developed nearly 100% decay incidence during a storage regime that favored specific development of gray mold (11 days at 7°C) or Rhizopus rot (2 days at 24°C). Chlorinating the hydrocooler water (120 mg of free chlorine per liter at pH 6.5) before adding the berries and spores reduced the incidence of gray mold to 43%; in contrast, berries hydrocooled in clean water developed 61% gray mold. Chlorination of the clean water led to significant reductions in the incidence of gray mold (44%) but did not affect the incidence of Rhizopus rot. The hydrocool method for cooling strawberries with the addition of proper chlorination has promise as a rapid method for cooling and cleaning berries and reducing gray mold inoculum on berry surfaces.

Calcium and potassium fertilization of potatoes grown in North Florida I. Effects on potato yield and tissue Ca and K concentrations

Potatoes (Solanum tuberosum L.) were grown during 3 seasons on an Ellzey fine sandy soil to evaluate the effects of Ca and K rate, Ca placement, and cultivar on tuber yield and Ca and K concentration. Mehlich I soil extractable Ca concentration ranged from 436 to 860 mg·kgt-1. Application of Ca at 0, 450 and 900 kg·hat-1 from gypsum increased soil Ca in each season, petiole Ca in 2 of 3 seasons, and tuber medulla and periderm Ca concentrations in one of three seasons. Tuber yields were increased from 29.8 to 31.6 t·hat-1 with an increase from 0 to 450 kg Ca·hat-1 in one of three seasons. Tuber specific gravity was not affected by Ca rate. With an increase in application of K from 225 to 450 kg·hat-1, Ca concentrations were decreased in the petiole and medulla in 2 of 3 seasons and in the periderm in 1 of 3 seasons. Petiole, medulla, and periderm K concentrations were increased in 2 of 3 seasons with an increase in K rate. Yields were not affected by K rate, but specific gravities of tubers were lower in 2 of 3 seasons with an increase in K rate. Broadcast placement of Ca resulted in higher petiole, medulla, and periderm Ca and higher tuber yields in 1 of 2 seasons than with band placement (the placement effect on yield occurred in the season that placement had no effect on tissue Ca). Periderm tissue of Superior was higher in Ca than that of La Chipper, and petiole tissues of Atlantic were higher than that of Superior, but medulla tissue in 1 of 2 seasons was higher in Ca with Superior than Atlantic. Tuber yields and specific gravities were higher with La Chipper and Atlantic than with Superior.CompendioSe cultivó papas (Solanum tuberosum L.) durante tres tremporadas en un suelo Ellzey arenoso fino para evaluar los efectos de las dosis de Ca y K, de la localización del Ca y del cultivar sobre el rendimiento en tubérculos y la concentración de Ca y K. La concentración Mehlich I del Ca extraíble del suelo varió de 436 a 860 mgkg·hat-1 La aplicación de Ca a razón de 0, 450 y 900 kg·hat-1 de sulfato de calcio hidratado (gypsum) incrementó el Ca del suelo en cada temporada, el Ca del peciolo en dos de tres temporadas, y la concentración del Ca de la médula del tubérculo y del peridermo en una de tres temporadas. Los rendimientos en tubérculos fueron incrementados de 29,8 a 31,6 t·hat-1 con un incremento de 0 a 450 kg Ca·hat-1 en una de tres temporadas. La gravedad específica del tubérculo no fue afectada por la dosis de Ca. Con un incremento en la aplicación de K de 225 a 450 kg·hat-1 las concentraciones de Ca disminuyeron en el peciolo y en la médula en dos de tres temporadas y en el peridermo en una de tres temporadas. Con un incremento en la dosis de K las concentraciones de K en el peciolo, médula y peridermo se incrementaron en dos de tres temporadas. Los rendimientos no fueron afectados con la dosis de K, pero las gravedades específicas de los tubérculos fueron menores en dos de tres temporadas cuando se incrementó la dosis de K. La aplicación de Ca al voleo dio por resultado una mayor concentración de Ca en el peciolo, médula y peridermo y a mayores rendimientos en tubérculos en una de dos temporadas en comparación con la aplicación en banda (el efecto del lugar de la aplicación sobre el rendimiento occurrió en la temporada en que esta variable no tuvo efecto sobre el Ca de los tejidos). El tejido del peridermo de Superior mostró un contenido de Ca más alto que el de La Chipper y los tejidos de los peciolos de Atlantic tuvieron igualmente un contenido más alto que los de Superior, pero el tejido de la médula, en una de dos temporadas, tuvo un contenido más alto de Ca en Superior que en Atlantic. Los rendimientos en tubérculos y las gravedades específicas fueron mayores para La Chipper y para Atlantic de lo que fueron para Superior.

Factors That Affect Proliferation of Salmonella in Tomatoes Post-Harvest: The Roles of Seasonal Effects, Irrigation Regime, Crop and Pathogen Genotype

Main Objectives Fresh fruits and vegetables become increasingly recognized as vehicles of human salmonellosis. Physiological, ecological, and environmental factors are all thought to contribute to the ability of Salmonella to colonize fruits and vegetables pre- and post-harvest. The goal of this study was to test how irrigation levels, fruit water congestion, crop and pathogen genotypes affect the ability of Salmonella to multiply in tomatoes post-harvest. Experimental Design Fruits from three tomato varieties, grown over three production seasons in two Florida locations, were infected with seven strains of Salmonella and their ability to multiply post-harvest in field-grown tomatoes was tested. The field experiments were set up as a two-factor factorial split plot experiment, with the whole-plot treatments arranged in a randomized complete-block design. The irrigation treatment (at three levels) was the whole-plot factor, and the split-plot factor was tomato variety, with three levels. The significance of the main, two-way, and three-way interaction effects was tested using the (type III) F-tests for fixed effects. Mean separation for each significant fixed effect in the model was performed using Tukey’s multiple comparison testing procedure. Most Important Discoveries and Significance The irrigation regime per se did not affect susceptibility of the crop to post-harvest proliferation of Salmonella. However, Salmonella grew significantly better in water-congested tissues of green tomatoes. Tomato maturity and genotype, Salmonella genotype, and inter-seasonal differences were the strongest factors affecting proliferation. Red ripe tomatoes were significantly and consistently more conducive to proliferation of Salmonella. Tomatoes harvested in the driest, sunniest season were the most conducive to post-harvest proliferation of the pathogen. Statistically significant interactions between production conditions affected post-harvest susceptibility of the crop to the pathogen. UV irradiation of tomatoes post-harvest promoted Salmonella growth.

Calcium and potassium fertilization of potatoes grown in North Florida. II. Effect on the bacterial soft rot potential in the tubers

In studies on a sandy soil in Florida over a 3-yr period (1984–86), applications of Ca (CaSO4) inconsistently affected the bacterial soft rot potential of potato tubers (Solanum tuberosum). The potential was estimated from the severity of the disease (percent surface area decayed) in tubers that had been immersed in an aqueous cell suspension ofErwinia carotovora subsp.carotovora and then incubated 4 days in a mist chamber at 24 C. With freshly harvested tubers (tested within 5 days after harvest), disease severity averaged 49, 11, and 4% over all treatments in 1984, 1985, and 1986, respectively, which was roughly proportional to rainfall on the crop,e.g., 37, 18, and 15 cm, respectively. Disease severity decreased, did not change, and increased with increased Ca rate (0, 450, and 900 kg.ha-1) in 1984, 1985, and 1986, respectively. In each season, severity was affected by interactions involving the Ca treatments. Most interactions involved cultivar. The efficacy of Ca applications to potatoes grown in low Ca soils for enhancement of tuber resistance to bacterial soft rot may be limited by factors associated with environment or cultivar that are not completely understood.CompendioEn estudios efectuados en un suelo arenoso de Florida durante un periodo de tres años (1984–1986), las aplicaciones de Ca (CaSO4) afectaron inconsistentemente el potencial bacteriano de la purdrición blanda de los tubérculos de papa (Solanum tuberosum). El potencial fue estimado por la severidad de la enfermedad (porcentaje de área de superficie afectada) en tubérculos que habían sido sumergidos en una suspensión acuosa deErwinia carotovora subsp.carotovora y luego incubada por cuatro días en una cámara nebulizadora a 24°C. Con tubérculos recién cosechados (probados dentro de los cinco días después de la cosecha), la severidad de la enfermedad promedió 49, 11 y 4% sobre todos los tratamientos en 1984, 1985 y 1986, respectivamente. La severidad de la enfermedad disminuyó, no varió y se incrementó con el incremento de la dosis de Ca (0, 450 y 900 kg.ha−1) en 1984, 1985 y 1986, respectivamente. En cada temporada, la severidad fue afectada por interacciones incluyendo a los tratamientos con Ca. La mayoría de las interacciones incluyeron al cultivar. La eficacia de las aplicaciones de Ca a las papas cultivadas en suelos de bajo contenido de Ca para mejorar la resistencia de los tubérculos a la pudricion blanda bacteriana puede ser limitada por factores asociados con el medio ambiente o el cultivar que no están totalmente comprendidos.