Postharvest transfer and survival of Salmonella enterica serotype enteritidis on living lettuce.

UNLABELLED: The potential for postharvest transfer of Salmonella to 'living lettuce' is not well understood. In this study, the transfer of Salmonella enterica Enteritidis (6 log CFU g(-1) ) from worker hands or contaminated roots to leaves of living lettuce was quantified. Transfer rates of Salmonella from contaminated gloves to sequentially handled lettuce heads ranged from 94% to head 1, 82% to head 2 and 69% to head 3. On average, 2.9 ± 0.1 log CFU g(-1) (64%) Salmonella was transferred from inoculated roots to leaves resulting from typical postharvest handling activities for living lettuce. Salmonella persisted on leaves stored at recommended storage temperatures (4°C) and increased 0.5 log CFU g(-1) when stored at temperature abuse conditions (12°C). Salmonella increased 1.6 log CFU g(-1) on roots after 18-day storage at 12°C, emphasizing the need to maintain temperature control to reduce the risk of human illness. SIGNIFICANCE AND IMPACT OF THE STUDY: Hydroponically grown lettuce packaged in plastic clamshells with intact roots, marketed as 'living lettuce', is increasing in popularity due to its extended shelf life. This study demonstrates the transfer of Salmonella from contaminated worker hands and contaminated roots to leaves where it persisted at 4°C for 18 day. Temperature abuse (12°C) increased Salmonella on roots and leaves. These findings suggest that failure to maintain temperatures below 12°C can pose a risk for consumers purchasing living lettuce at markets where recommended storage temperatures are not maintained.

Changes in spinach phylloepiphytic bacteria communities following minimal processing and refrigerated storage described using pyrosequencing of 16S rRNA amplicons.

AIMS: To investigate the changes in bacterial diversity on fresh spinach phyllosphere associated with storage at refrigeration temperatures. METHODS AND RESULTS: Community structure and population dynamics of spinach phylloepiphytic bacteria associated with packaging and refrigeration of ready-to-eat fresh produce were evaluated using pyrosequencing of 16S rRNA gene amplicons. A diverse community responsive to storage at refrigerated temperatures was detected belonging to over 1000 operational taxonomic units, including many diverse members not previously described on the phyllosphere. Of the approx. 8800 unique sequences examined from fresh spinach leaves, 75% were from previously undescribed taxa. The classified sequences from the fresh spinach phyllosphere were assigned to 11 different phyla with the largest number of reads belonging to Proteobacteria and Firmicutes. Packaging and storage of spinach under refrigerated conditions decreased the richness, diversity and evenness of the bacterial community. Refrigeration at 4 and 10°C and storage resulted in a decrease in number of taxa represented from 11 phyla in fresh spinach to only 5 phyla after 1 day of storage. Sequences belonging to γ-Proteobacteria, particularly Pseudomonas spp. and members of the Enterobacteriaceae, were the most numerous after 15 days of storage at both temperatures. Growth inhibition of the genera Escherichia was achieved at 4°C but not at 10°C storage, thus highlighting the importance of temperature in fresh packaged spinach. CONCLUSIONS: The application of pyrosequencing to describe composition and diversity of the phyllosphere on spinach leaves provided a broader outlook of the bacterial composition of this community complementing other phyllosphere studies that have used culture- and nonculture-dependent approaches. SIGNIFICANCE AND IMPACT OF THE STUDY: Pyrosequencing allowed a broader description of the bacterial composition and diversity of the spinach leaf surface than previously obtained using culture-based detection and will be a powerful tool to help ensure the future safety and quality of packaged spinach.

Compartmentation of solutes and water in developing sugarcane stalk tissue.

Previous studies have suggested that the apoplast solution of sugarcane stalk tissue contains high concentrations of sucrose, but the accuracy of these reports has been questioned because sucrose leakage from damaged cells may have influenced the results. In this study, the solute potential of the apoplast and symplast of the second (immature), tenth, twentieth, thirtieth, and fortieth internodes of field-grown sugarcane (Saccharum spp. hybrid) stalk tissue was determined by two independent methods. Solute potential of the apoplast was measured either directly by osmometry from solution collected by centrifugation, or inferred from the initial water potential of fully hydrated tissue determined by thermocouple psychrometry before the tissue was progressively dehydrated for generation of water potential isotherms. Both methods produced nearly identical values ranging from -0.6 to -1.8 megapascals for immature and mature tissue, respectively. The solute potential of the symplast determined by either method ranged from -1.0 to approximately -2.2 megapascals for immature and mature internodes, respectively. Solute quantitation by HPLC agreed with concentrations inferred from osmometry. Washing thirtieth internode tissue in deionized water increased pressure potential from 0.29 to 1.96 megapascals. The apoplast of mature sugarcane stalk tissue is a significant storage compartment for sucrose containing as much as 25% of the total tissue water volume and as much as 21% of the stored sucrose.

Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : III. Sensitivity of Germination to Water Potential and Abscisic Acid during Development.

Muskmelon (Cucumis melo L.) seeds are germinable 15 to 20 days before fruit maturity and are held at relatively high water content within the fruit, yet little precocious germination is observed. To investigate two possible factors preventing precocious germination, the inhibitory effects of abscisic acid and osmoticum on muskmelon seed germination were determined throughout development. Seeds were harvested at 5-day intervals from 30 to 65 days after anthesis (DAA) and incubated either fresh or after drying on factorial combinations of 0, 1, 3.3, 10, or 33 micromolar abscisic acid (ABA) and 0, -0.2, -0.4, -0.6, or -0.8 megapascals polyethylene glycol 8000 solutions at 30 degrees C. Radicle emergence was scored at 12-hour intervals for 10 days. In the absence of ABA, the water potential (Psi) required to inhibit fresh seed germination by 50% decreased from -0.3 to -0.8 megapascals between 30 and 60 DAA. The Psi inside developing fruits was from 0.4 to 1.4 megapascals lower than that required for germination at all stages of development, indicating that the fruit Psi is sufficiently low to prevent precocious germination. At 0 megapascal, the ABA concentration required to inhibit germination by 50% was approximately 10 micromolar up to 50 DAA and increased to >33 micromolar thereafter. Dehydration improved subsequent germination of immature seeds in ABA or low Psi. There was a linear additive interaction between ABA and Psi such that 10 micromolar ABA or -0.5 megapascal osmotic potential resulted in equivalent, and additive, reductions in germination rate and percentage of mature seeds. Abscisic acid had no effect on embryo solute potential or water content, but increased the apparent minimum turgor required for germination. ABA and osmoticum appear to influence germination rates and percentages by reducing the embryo growth potential (turgor in excess of a minimum threshold turgor) but via different mechanisms. Abscisic acid apparently increases the minimum turgor threshold, while low Psi reduces turgor by reducing seed water content.

Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : IV. Characteristics of the Perisperm during Seed Development.

We previously reported that an apparent water potential disequilibrium is maintained late in muskmelon (Cucumis melo L.) seed development between the embryo and the surrounding fruit tissue (mesocarp). To further investigate the basis of this phenomenon, the permeability characteristics of the tissues surrounding muskmelon embryos (the mucilaginous endocarp, the testa, a 2- to 4-cell-layered perisperm and a single cell layer of endosperm) were examined from 20 to 65 days after anthesis (DAA). Water passes readily through the perisperm envelope (endosperm + perisperm), testa, and endocarp at all stages of development. Electrolyte leakage (conductivity of imbibition solutions) of individual intact seeds, decoated seeds (testa removed), and embryos (testa and perisperm envelope removed) was measured during imbibition of freshly harvested seeds. The testa accounted for up to 80% of the total electrolyte leakage. Leakage from decoated seeds fell by 8- to 10-fold between 25 and 45 DAA. Presence of the perisperm envelope prior to 40 DAA had little effect on leakage, while in more mature seeds, it reduced leakage by 2- to 3-fold. In mature seeds, freezing, soaking in methanol, autoclaving, accelerated aging, and other treatments which killed the embryos had little effect on leakage of intact or decoated seeds, but caused osmotic swelling of the perisperm envelope due to the leakage of solutes from the embryo into the space between the embryo and perisperm. The semipermeability of the perisperm envelope of mature seeds did not depend upon cellular viability or lipid membrane integrity. After maximum seed dry weight is attained (35-40 DAA), the perisperm envelope prevents the diffusion of solutes, but not of water, between the embryo and the surrounding testa, endocarp, and mesocarp tissue.

Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : V. Water Relations of Imbibition and Germination.

The initiation of radicle growth during seed germination may be driven by solute accumulation and increased turgor pressure, by cell wall relaxation, or by weakening of tissues surrounding the embryo. To investigate these possibilities, imbibition kinetics, water contents, and water (Psi) and solute (psi(s)) potentials of intact muskmelon (Cucumis melo L.) seeds, decoated seeds (testa removed, but a thin perisperm/endosperm envelope remains around the embryo), and isolated cotyledons and embryonic axes were measured. Cotyledons and embryonic axes excised and imbibed as isolated tissues attained water contents 25 and 50% greater, respectively, than the same tissues hydrated within intact seeds. The effect of the testa and perisperm on embryo water content was due to mechanical restriction of embryo swelling and not to impermeability to water. The Psi and psi(s) of embryo tissues were measured by psychrometry after excision from imbibed intact seeds. For intact or decoated seeds and excised cotyledons, Psi values were >-0.2 MPa just prior to radicle emergence. The Psi of excised embryonic axes, however, averaged only -0.6 MPa over the same period. The embryonic axis apparently is mechanically constrained within the testa/perisperm, increasing its total pressure potential until axis Psi is in equilibrium with cotyledon Psi, but reducing its water content and resulting in a low Psi when the constraint is removed. There was no evidence of decreasing psi(s) or increasing turgor pressure (Psi-psi(s)) prior to radicle growth for either intact seeds or excised tissues. Given the low relative water content of the axes within intact seeds, cell wall relaxation would be ineffective in creating a Psi gradient for water uptake. Rather, axis growth may be initiated by weakening of the perisperm, thus releasing the external pressure and creating a Psi gradient for water uptake into the axis. The perisperm envelope contains a cap of small, thin-walled endosperm cells adjacent to the radicle tip. We hypothesize that weakening or separation of cells in this region could initiate radicle expansion.

Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : I. Water Relations of Seed and Fruit Development.

Total water potential (psi), solute potential, and turgor potential of field-grown muskmelon (Cucumis melo L.) fruit tissue (pericarp) and seeds were determined by thermocouple psychrometry at 5-day intervals from 10 to 65 days after anthesis (DAA). Fruit maturity occurred between 44 and 49 DAA, and seed germination ability developed between 35 and 45 DAA. Pericarp psi was essentially constant at approximately -0.75 megapascal (MPa) from 10 to 25 DAA, then decreased to a minimum value of -1.89 MPa at 50 DAA before increasing to -1.58 MPa at 65 DAA. Seed psi remained relatively constant at approximately -0.5 MPa from 10 to 30 DAA then decreased to -2.26 MPa at 50 to 60 DAA before increasing to -2.01 MPa at 65 DAA. After a rapid increase to 20 DAA, seed fresh weight declined until 30 DAA due to net water loss, despite continuing dry weight gain. As fruit and seed growth rates decreased, turgor potential initially increased, then declined to small values when growth ceased. A disequilibrium in psi was measured between seeds and pericarp both early and late in development. From 20 to 40 DAA, the psi gradient was from the seed to the tissue, coinciding with water loss from the seeds. From 50 to 65 DAA, seed psi decreased, causing a reversal of the psi gradient and a slight increase in seed water content. The partitioning of solutes between symplast and apoplast may create and maintain psi gradients between the pericarp and seed. The low solute potential within the pericarp due to solute accumulation and loss of cellular compartmentation during ripening and sensecence may be involved in prevention of precocious germination of mature seeds.