Christopher S. Walsh

Bacterial community diversity and variation in spray water sources and the tomato fruit surface

BackgroundTomato (Solanum lycopersicum) consumption has been one of the most common causes of produce-associated salmonellosis in the United States. Contamination may originate from animal waste, insects, soil or water. Current guidelines for fresh tomato production recommend the use of potable water for applications coming in direct contact with the fruit, but due to high demand, water from other sources is frequently used. We sought to describe the overall bacterial diversity on the surface of tomato fruit and the effect of two different water sources (ground and surface water) when used for direct crop applications by generating a 454-pyrosequencing 16S rRNA dataset of these different environments. This study represents the first in depth characterization of bacterial communities in the tomato fruit surface and the water sources commonly used in commercial vegetable production.ResultsThe two water sources tested had a significantly different bacterial composition. Proteobacteria was predominant in groundwater samples, whereas in the significantly more diverse surface water, abundant phyla also included Firmicutes, Actinobacteria and Verrucomicrobia. The fruit surface bacterial communities on tomatoes sprayed with both water sources could not be differentiated using various statistical methods. Both fruit surface environments had a high representation of Gammaproteobacteria, and within this class the genera Pantoea and Enterobacter were the most abundant.ConclusionsDespite the major differences observed in the bacterial composition of ground and surface water, the season long use of these very different water sources did not have a significant impact on the bacterial composition of the tomato fruit surface. This study has provided the first next-generation sequencing database describing the bacterial communities living in the fruit surface of a tomato crop under two different spray water regimes, and therefore represents an important step forward towards the development of science-based metrics for Good Agricultural Practices.

Impact of Organic and Conventional Management on the Phyllosphere Microbial Ecology of an Apple Crop

Bacterial communities associated with the phyllosphere of apple trees (Malus domestica cv. Enterprise) grown under organic and conventional management were assessed to determine if increased biological food safety risks might be linked with the bacterial communities associated with either treatment. Libraries of 16S rRNA genes were generated from phyllosphere DNA extracted from a wash made from the surfaces of leaves and apples from replicated organic and conventional treatments. 16S rRNA gene libraries were analyzed with software designed to identify statistically significant differences between bacterial communities as well as shared and unique phylotypes. The identified diversity spanned eight bacterial phyla and 14 classes in the pooled organic and conventional libraries. Significant differences between organic and conventional communities were observed at four of six time points (P < 0.05). Despite the identification of significantly diverse microfloras associated with organic and conventional treatments, no detectable differences in the presence of potential enteric pathogens could be associated with either organic or conventional management. Neither of the bacterial genera most commonly associated with produce-related illness outbreaks (Salmonella and Escherichia) was observed in any of the libraries. The impressive bacterial diversity that was documented in this study provides a valuable contribution to our developing understanding of the total microbial ecology associated with the preharvest phyllospheres of food crops. The fact that organic and conventional phyllosphere bacterial communities were significantly different at numerous time points suggests that crop management methods may influence the bacterial consortia associated with the surfaces of fruits and vegetables.

Assessment of region, farming system, irrigation source and sampling time as food safety risk factors for tomatoes

In the mid-Atlantic region of the United States, small- and medium-sized farmers use varied farm management methods and water sources to produce tomatoes. It is unclear whether these practices affect the food safety risk for tomatoes. This study was conducted to determine the prevalence, and assess risk factors for Salmonella enterica, Shiga toxin-producing Escherichia coli (STEC) and bacterial indicators in pre-harvest tomatoes and their production areas. A total of 24 organic and conventional, small- to medium-sized farms were sampled for six weeks in Maryland (MD), Delaware (DE) and New Jersey (NJ) between July and September 2012, and analyzed for indicator bacteria, Salmonella and STEC. A total of 422 samples--tomato fruit, irrigation water, compost, field soil and pond sediment samples--were collected, 259 of which were tomato samples. A low level of Salmonella-specific invA and Shiga toxin genes (stx1 or stx2) were detected, but no Salmonella or STEC isolates were recovered. Of the 422 samples analyzed, 9.5% were positive for generic E. coli, found in 5.4% (n=259) of tomato fruits, 22.5% (n=102) of irrigation water, 8.9% (n=45) of soil, 3/9 of pond sediment and 0/7 of compost samples. For tomato fruit, farming system (organic versus conventional) was not a significant factor for levels of indicator bacteria. However, the total number of organic tomato samples positive for generic E. coli (1.6%; 2/129) was significantly lower than for conventional tomatoes (6.9% (9/130); (χ(2) (1)=4.60, p=0.032)). Region was a significant factor for levels of Total Coliforms (TC) (p=0.046), although differences were marginal, with western MD having the highest TC counts (2.6 log CFU/g) and NJ having the lowest (2.0 log CFU/g). Tomatoes touching the ground or plastic mulch harbored significantly higher levels of TC compared to vine tomatoes, signaling a potential risk factor. Source of irrigation water was a significant factor for all indicator bacteria (p<0.0001), and groundwater had lower bacterial levels than surface water. End of line surface water samples were not significantly different from source water samples, but end of line groundwater samples had significantly higher bacterial counts than source (p<0.0001), suggesting that Good Agricultural Practices that focus on irrigation line maintenance might be beneficial. In general, local effects other than cropping practices, including topography, land use and adjacent industries, might be important factors contributing to microbiological inputs on small- and medium-sized farms in the mid-Atlantic region.

The Growing Season, but Not the Farming System, Is a Food Safety Risk Determinant for Leafy Greens in the Mid-Atlantic Region of the United States

ABSTRACT Small- and medium-size farms in the mid-Atlantic region of the United States use varied agricultural practices to produce leafy greens during spring and fall, but the impact of preharvest practices on food safety risk remains unclear. To assess farm-level risk factors, bacterial indicators, Salmonella enterica, and Shiga toxin-producing Escherichia coli (STEC) from 32 organic and conventional farms were analyzed. A total of 577 leafy greens, irrigation water, compost, field soil, and pond sediment samples were collected. Salmonella was recovered from 2.2% of leafy greens (n = 369) and 7.7% of sediment (n = 13) samples. There was an association between Salmonella recovery and growing season (fall versus spring) (P = 0.006) but not farming system (organic or conventional) (P = 0.920) or region (P = 0.991). No STEC was isolated. In all, 10% of samples were positive for E. coli: 6% of leafy greens, 18% of irrigation water, 10% of soil, 38% of sediment, and 27% of compost samples. Farming system was not a significant factor for levels of E. coli or aerobic mesophiles on leafy greens but was a significant factor for total coliforms (TC) (P < 0.001), with higher counts from organic farm samples. Growing season was a factor for aerobic mesophiles on leafy greens (P = 0.004), with higher levels in fall than in spring. Water source was a factor for all indicator bacteria (P < 0.001), and end-of-line groundwater had marginally higher TC counts than source samples (P = 0.059). Overall, the data suggest that seasonal events, weather conditions, and proximity of compost piles might be important factors contributing to microbial contamination on farms growing leafy greens.

Indole-3-acetic acid concentration and ethylene evolution during early fruit development in peach

Ethylene evolution was measured from greenhouse-grown ‘Jerseyglo’ peach fruits beginning 29 days after anthesis. Indole-3-acetic acid (IAA) levels were measured in the pericarp and seed tissues of individual fruits on a single shoot when variable ethylene evolution was noted. Despite hand-pollinating all flowers on the same day, variability within the shoot existed in fruit fresh weight, IAA levels, and ethylene evolution. Seed IAA concentration increased as fruit and seed fresh weight increased and ranged from 106 to 1572 ng. g−1. As pericarp fresh weight increased, IAA levels in this tissue decreased. Ethylene evolution rates ranged from 0.21 to 1.07 nl. g.−1 h−1 and were not correlated with IAA concentration in seed, pericarp, or the whole fruit. High rates of ethylene evolution from the whole fruit occurred prior to increased IAA concentration in the seed.Fruits were excised from field-grown ‘Redskin’ peach trees beginning 40 days after full bloom. Fruits from field sampled shoots appeared to be more physiologically advanced than the greenhouse-grown ‘Jerseyglo’ fruits. Pericarp IAA concentration was low, ranging from 2.8 to 6.5 ng. g−1. Seed concentrations accounted for 75% of the IAA found in the fruit and ranged from 239 to 1042 ng. g−1. As with greenhouse-grown samples, whole fruit IAA concentration tended to decrease as fruits increased in fresh weight.

Ethylene evolution by apple fruits during their early-season growth and development

Abstract Ethylene (C 2 H 4 ) evolution from developing apple ( Malus domestica , Borkh.) fruits declined rapidly during the 8-week period after petal fall (PF). No difference in C 2 H 4 evolution was measured between developing and senescing fruits during the third, fourth and fifth weeks after PF. High levels of C 2 H 4 evolution were detected, however, in some senescing fruits at 7 and 8 weeks after PF. C 2 H 4 evolution could not be used to separate developing and senescing fruits during the period of “June drop”. In the first 4 weeks after PF in ‘Stayman’ fruits, and up to 22 days after anthesis in ‘Golden Delicious’ fruits, measured C 2 H 4 among replicates was extremely variable. This variability did not appear to be caused by tissue wounding during sampling. Wounding did increase fruitlet C 2 H 4 , but reduced variability. Artificially-induced parthenocarpic fruits evolved low levels of C 2 H 4 . Parthenocarpic fruits obtained by spraying with GA 3 evolved a greater level of C 2 H 4 than did unsprayed, parthenocarpic fruits. We suggest that the high variability measured early in the growing season may be caused by rapid changes in fruit and seed development which stimulate surges in C 2 H 4 production.