Douglas D. Archbold

Survival of Escherichia coli O157:H7 on Strawberry Fruit and Reduction of the Pathogen Population by Chemical Agents

Survival of Escherichia coli O157:H7 was studied on strawberry, a fruit that is not usually washed during production, harvest, or postharvest handling. Two strains of the bacteria were tested separately on the fruit surface or injected into the fruit. Both strains of E. coli O157:H7 survived externally and internally at 23 degrees C for 24 h and at 10, 5, and -20 degrees C for 3 days. The largest reduction in bacterial population occurred at -20 degrees C and on the fruit surface during refrigeration. In all experiments, the bacteria inside the fruit either survived as well as or better than bacteria on the surface, and ATCC 43895 frequently exhibited greater survival than did ATCC 35150. Two strains of E. coli also survived at 23 degrees C on the surface and particularly inside strawberry fruit. Chemical agents in aqueous solution comprising NaOCl (100 and 200 ppm), Tween 80 (100 and 200 ppm), acetic acid (2 and 5%), Na3PO4 (2 and 5%), and H2O2 (1 and 3%) were studied for their effects on reduction of surface-inoculated (10(8) CFU/ml) E. coli O157:H7 populations on strawberry fruit. Dipping the inoculated fruit in water alone reduced the pathogen population about 0.8 log unit. None of the compounds with the exception of H2O2 exhibited more than a 2-log CFU/g reduction of the bacteria on the fruit surface. Three percent H202, the most effective chemical treatment, reduced the bacterial population on strawberries by about 2.2 log CFU/g.

Production of the long-chain alcohols octanol, decanol, and dodecanol by Escherichia coli

As a follow-up to earlier studies on the emission of long-chain alcohols from broth cultures of Gram-negative enteric bacteria, E. coli was examined for the production of 1-octanol, 1-decanol, and 1-dodecanol. Ten strains of E. coli cultured in tryptic soy broth were assayed for volatile metabolites using solid-phase microextraction. Long-chain alcohols were produced by all strains with 1-decanol predominating with production ranging from 23.6 ng mL−1 to 148 ng mL−1. The production of long-chain alcohols followed the onset of the exponential growth phase of the broth culture. Doubling the concentration of glucose (5 g L−1) in the broth had no effect on the concentration of long-chain alcohols produced. Addition of octanoic, decanoic, or dodecanoic acids (as K+ salts) to the broth (100 mg L−1) markedly increased the production of the corresponding alcohols by E. coli, ranging from a 13-fold increase for decanol to a 51-fold increase for dodecanol. However, decanol remained the predominant alcohol detected in all assays. These neutral volatile alcohols may have application as vapor-phase indicators for certain classes of bacteria, particularly, Gram-negative enteric bacteria.

Comparison of long-chain alcohols and other volatile compounds emitted from food-borne and related Gram positive and Gram negative bacteria.

Numerous reports have been published on the antimicrobial activity of synthetic volatile long chain alcohols, such as 1‐decanol and 1‐dodecanol, against bacteria and fungi. The objective of the present study was to survey microorganisms for emission patterns of naturally occurring long chain alcohols and other volatile components to determine if these compounds are associated with certain groups of bacteria. Cultures were grown in trypticase soy broth overnight and volatile compounds were trapped on a porous polymer and identified by mass spectrometry. Subsequently, volatile compounds were collected from 26 strains of food associated bacteria using solid‐phase microextraction and analyzed by gas chromatography. Alcohols comprising 1‐octanol, 1‐decanol, and 1‐dodecanol occurred as products from enteric Gram negative bacteria, which included Citrobacter, Enterobacter, Klebsiella, Salmonella, and Shigella. However, the long chain alcohols were not detected as products from the nonenteric Gram negative species studied which included Acinetobacter, Pseudomonas, and Shewanella. Among Gram positive bacteria, including Bacillus, Enterococcus, Lactococcus, Leuconostoc, Listeria, Staphylococcus, and Streptococcus, the only long chain alcohol detected was 1‐decanol and, if present, it occurred in relatively small amounts. Other classes of compounds emitted by bacteria included methylketones and sulfides. The methylketones were found as products from Gram positive and Gram negative bacteria, whereas the sulfides were closely associated with Gram positive bacteria. In summary, the emission patterns of volatile compounds from bacteria showed many trends including the association of long chain alcohols with enteric Gram negative bacteria. The results provide a basis for future in vivo studies to determine if volatile compounds such as natural long chain alcohols function in the ecology of food‐borne Gram negative bacterial pathogens.

Biosynthesis of trans-2-Hexenal in Response to Wounding in Strawberry Fruit

Wounded strawberry fruit produces a diverse group of volatile compounds including aldehydes, alcohols, and esters derived from the lipoxygenase (LOX) and hydroperoxide lyase (HPL) pathways. Because the wound volatiles may play an important role in plant-fungal interaction, the goal of this study was to develop a greater understanding about the biosynthesis of the major wound volatile, trans-2-hexenal (t-2-H), produced by strawberry fruit upon wounding. To that end, composition and quantity of total and free fatty acids of control and wounded strawberry fruit were analyzed. In addition, activities of the key enzymes, LOX and HPL, and production of C6 aldehydes were determined. Intact strawberry fruit did not produce detectable t-2-H which is derived from alpha-linolenic acid (18:3). However, in response to wounding by bruising, strawberry fruit emitted t-2-H and its precursor cis-3-hexenal (c-3-H). The level of total lipid 18:3 in the fruit increased 2-fold in response to wounding, whereas free 18:3 declined slightly ( approximately 30%). At 10 min following wounding, fruit exhibited a 25% increase in LOX activity, which leads to the production of 13-hydroperoxyoctadecatrienoic acid (13-HPOT) from 18:3. The activity of HPL, which catalyzes formation of cis-3-hexenal from 13-HPOT, increased 2-fold by 10 min after wounding. Thus, during a 15 min period after wounding, free 18:3 substrate availability and the activity of two key enzymes, LOX and HPL, changed in a manner consistent with increased c-3-H and t-2-H biosynthesis.

The role of SORBITOL DEHYDROGENASE in Arabidopsis thaliana

SORBITOL DEHYDROGENASE (SDH, EC 1.1.1.14) catalyses the interconversion of polyols and ketoses (e.g. sorbitol ⟷ fructose). Using two independent Arabidopsis thaliana (L.) Heynh. sdh knockout mutants, we show that SDH (At5g51970) plays a primary role in sorbitol metabolism as well as an unexpected role in ribitol metabolism. Sorbitol content increased in both wild-type (WT) and mutant plant leaves during drought stress, but mutants showed a dramatically different phenotype, dying even if rewatered. The lack of functional SDH in mutant plants was accompanied by accumulation of foliar sorbitol and at least 10-fold more ribitol, neither of which decreased in mutant plants after rewatering. In addition, mutant plants were uniquely sensitive to ribitol in a concentration-dependent manner, which either prevented them from completing seed germination or inhibited seedling development, effects not observed with other polyols or with ribitol-treated WT plants. Ribitol catabolism may occur solely through SDH in A. thaliana, though at only 30% the rate of that for sorbitol. The results indicate a role for SDH in metabolism of sorbitol to fructose and in ribitol conversion to ribulose in A. thaliana during recovery from drought stress.

Strawberry resistance toTetranychus urticae Koch: Effects of flower, fruit, and foliage removal—comparisons of air- vs. nitrogen-entrained volatile compounds

An increase in resistance to the two-spotted spider mite (TSSM),Tetranychus urticae Koch, is observed in field-grown strawberry plants during the period from flowering to postharvest. This seasonal phenomenon was investigated to determine the influence of the metabolic sink, that is, fruiting in the plant. Removal of flowers and fruit and partial removal of foliage did not alter the pattern of resistance of the strawberry plant to TSSM. Bioassays were conducted in concert with chemical analyses. Headspace chemicals emitted from foliage samples were entrained in air and trapped on Tenax, identified, and compared with those entrained in nitrogen and trapped. Terpenes were among the major compounds entrained in air, whereas alcohols were obtained with nitrogen. The air-entrained headspace compounds did not appear to correlate quantitatively with the development of mite resistance in the control plants or those subjected to metabolic sink (flower and fruit) removal. Evidence was obtained for the presence of heretofore unreported strawberry foliage headspace components, namely, (Z)-3-hexenyl 2-meth-ylbutyrate, (Z)-3-hexenyl tiglate, (E)-β-ocimene, (Z)-β-ocimene, α-farnesene, and germacrene D.

Loss of ripening capacity of pawpaw fruit with extended cold storage.

The fruit ripening traits of pawpaw [ Asimina triloba (L.) Dunal] were examined after harvest and after cold storage at -2, 2, 4, and 6 degrees C for up to 12 weeks. Generally, fruits stored at 2-4 degrees C for 4 weeks ripened normally, but those stored at -2 degrees C did not ripen normally, those stored at 6 degrees C were overripe, and by 6-8 weeks those stored at 2-4 degrees C had a lower respiration rate and ethylene production, lower firmness, and lower pH than fruit cold-stored for 4 weeks or less. These changes, and the occasional development of brown discoloration in the pulp once the fruits were moved back to room temperature, were evidence of chilling injury by 6 weeks. After harvest and through 4 weeks of cold storage, the main volatile compounds produced by fruit were methyl and ethyl octanoates and hexanoates. Volatile production significantly increased >5-fold in fruit ripened for 72 h after harvest or after removal from up to 4 weeks of cold storage. Fruit cold-stored for 6 weeks or more produced fewer total volatiles and esters but increased levels of such off-flavor compounds as ethyl acetate, ethyl propionate, and hexanoic and decanoic acids. Alcohol acyltransferase (AAT) activity declined in cold-stored fruit but was not correlated with either total volatile production or total ester production. Alcohol dehydrogenase activity did not change during ripening after harvest or cold storage. Lipoxygenase activity was highest just after harvest or after 2 weeks of cold storage, but was low by 4 weeks. Thus, ripening of pawpaw fruit seems to be limited to 4 weeks at 2-4 degrees C with loss of ability to continue ripening and chilling injury symptoms evident at colder temperatures and after longer periods of cold storage.

Effects of Balanced Pruning Severity on Traminette (Vitis spp.) in a Warm Climate

Canopy architecture, yield components, fruit composition, vine size, Ravaz index, and midwinter primary bud cold hardiness of Traminette grapevines were measured in response to balanced pruning treatments of 20, 30, or 40 nodes retained for the first 454 g of dormant pruning weight and an additional 10 nodes for each additional 454 g in 2006 and 2007. As pruning severity increased from 40 + 10 to 20 + 10, shoots per hectare decreased and the distance between shoots increased. Leaf area per vine and leaf layer number showed a linear increase as pruning severity decreased. As the pruning severity decreased, the number of clusters harvested per vine and yield increased. Fruit composition was not affected by pruning severity. Vine size and leaf area per unit of fruit weight were not affected by the treatments, but Ravaz index (weight of fruit per weight of pruned wood) increased linearly as pruning severity decreased in 2007. In 2007, as pruning severity increased, more nodes were mature by late fall and buds were more cold hardy in midwinter. Results suggest that in the absence of any measurable effects of disease incidence or adverse effects on fruit composition, the 40 + 10 treatment should be used in the lower Midwest of the United States, as it optimized yield with only a slight decrease in primary bud cold hardiness.

Microbial Populations of Botrytis cinerea-Inoculated Strawberry Fruit Exposed to Four Volatile Compounds†

Aerobic, microaerophilic, coliform, and mold populations of Botrytis cinerea-inoculated strawberry fruit not exposed (control) or exposed to low and high quantities of four volatile compounds during storage at 2 degrees C were determined after storage for 7 days and after removal of the volatile and transfer to 22 degrees C for 3 days. Fruit harvested at the ripe stage were inoculated with 10(6) conidia B. cinerea per ml and were placed in plastic containers containing no volatile compound (control) or two quantities of (E)-2-hexenal (10 or 100 microliters), (E)-2-hexenal diethyl acetal (30 or 300 microliters), benzaldehyde (30 or 300 microliters), or methyl benzoate (12 or 60 microliters). The fruit containers were overwrapped with a low-density polyethylene film, sealed, stored at 2 degrees C for 7 days, and then transferred to 22 degrees C for 3 days. Aerobic, microaerophilic, and coliform populations of fruit exposed to volatile compounds tended to be lower than the controls after storage at 2 degrees C for 7 days and, depending on the volatile compound, similar, lower, or higher than the controls after transfer and storage at 22 degrees C. However, due to variability in initial aerobic, microaerophilic, and coliform populations of the fruit used in the different trials (P < 0.05), none of the differences between control and treatment and between treatments within a sample time were significant (P > 0.05). Strawberry fruit exposed to 100 microliters of (E)-2-hexenal was the only treatment that did not show a significant increase in mold populations after transfer and storage at 22 degrees C for 3 days. Additional studies are needed to determine if (E)-2-hexenal can be used in combination with other postharvest storage conditions, such as low temperature and controlled/modified atmosphere, to delay mold spoilage and extend the shelf life of the strawberry.

Comparative analyses of polyphenolic composition of Fragaria spp. color mutants

White-fruited mutants of Fragaria vesca, and one of F. x ananassa, were studied to determine the identity and quantity of major flavonols (FVLs), flavan-3-ols (FV3Ls), hydroxycinnamic acids (HCAs), and ellagic acid (EA)-derived compounds, by using HPLC-MS. The content of 22 compounds across the major groups were used to assess the possibility of unique mutations among the mutant gentoypes. Total HCAs were lower in the white than the red cultivars of both species, except for 2 white F. vesca cultivars. Total FVLs were comparable in white fruit of both species, although a red F. x ananassa had more than a red F. vesca. Total FV3Ls were higher in red than white cultivars of both species. Total EA-derived content was generally higher in white than in red F. vesca. Principal component analysis and a combined heatmap and hierarchical cluster analysis clearly discriminated among the five white F. vesca genotypes.