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.

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.

Volatile compounds from Escherichia coli O157:H7 and their absorption by strawberry fruit.

Volatile compounds emitted by cultures of two strains of the pathogenic bacterium Escherichia coliO157:H7 and a nonpathogenic strain of E. coli were trapped on Super-Q porous polymer and identified by GC-MS. The predominant compound produced by all three strains was indole with lesser amounts of other components including methyl ketones, 2-heptanone, 2-nonanone, 2-undecanone, and 2-tridecanone. The vapor-phase profiles of these strains were similar for most chemicals identified but differed with regard to ketones. Strawberry fruit was shown to be a suitable host for E. coli O157:H7 with the population of the bacterium either increasing or remaining stable after 3 days depending on inoculation level. Headspace analysis of the volatile compounds from inoculated fruit yielded no detectable quantity of indole. Strawberry fruit readily absorbed indole and other volatile compounds produced by the bacteria and in some cases metabolized the compounds to new volatile products. Thus, headspace "marker" compounds indicating possible bacterial contamination of fruit were largely removed from the vapor phase by the strawberries.

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.

The impact of alteration of polyunsaturated fatty acid levels on C6-aldehyde formation of Arabidopsis thaliana leaves.

C6-aldehydes are synthesized via lipoxygenase/hydroperoxide lyase action on polyunsaturated fatty acid (PUFA) substrates in plant leaves. The source pools and subcellular location of the processes are unknown. A close relationship is found between the composition of PUFA and the composition of C6-aldehydes. In the current study, this relationship was tested using the Arabidopsis PUFA mutant lines act1, fad2, fad3, fad5, fad6, and fad7. The results indicate that C6-aldehyde formation is influenced by the alteration of C18 PUFA levels. Mutants act1 and fad5, which are deficient in C16 unsaturated fatty acids, had wild-type levels of C6-aldehyde production. Mutants deficient in the chloroplast hexadecenoic acid/oleic acid desaturase (fad6) or hexadecadienoic acid/linoleic acid desaturase (fad7) had altered C6-aldehyde formation in a pattern similar to the changes in the PUFA. Mutations that impair phosphatidylcholine desaturase activity, such as fad2 and fad3, however, resulted in increased E-2-hexenal formation. The enzymes involved in C6-aldehyde production were partially characterized, including measurement of pH optima. The differences in C6-aldehyde formation among the fatty acid mutants of Arabidopsis appeared not to result from alteration of lipoxygenase/hydroperoxide lyase pathway enzymes. Investigation of the fatty acid composition in leaf phospholipids, glycolipids, and neutral lipids and analysis of the fatty acid composition of chloroplast and extrachloroplast lipids indicate that chloroplasts and glycolipids of chloroplasts may be the source or major source of C6-aldehyde formation in Arabidopsis leaves.

Volatile compounds from crabapple (Malus spp.) cultivars differing in susceptibility to the Japanese beetle (Popillia japonica Newman).

The volatile compounds emitted by leaves of four crabapple cultivars susceptible to damage by Japanese beetles and four relatively resistant cultivars were examined. Twelve compounds, mostly terpene hydrocarbons, were identified from intact leaves. The terpenes (E)-ß-ocimene, caryophylene, germacrene D and (E,E)-α-farnesene occurred in significantly higher levels in susceptible cultivars, whereas resistant cultivars produced greater amounts of (E)-4,8-dimethyl 1,3,7-nonatriene and linalool. The relative attractiveness of the cultivars as determined in a pitfall bioassay, however, was not related to their susceptibility to the Japanese beetle as previously determined by defoliation sustained in the field. The attractiveness of individual cultivars was found to be positiviely correlated with linalool as a percent of the total volatile blend emitted by leaves. This study and previous work suggest that variation in susceptibility of crabapple cultivars to defoliation by Japanese beetles is not due to the attractiveness of the individual cultivars but rather to nonvolatile components of susceptibility and/or resistance. A scenario for host location by the Japanese beetle is presented.

Effect of volatile methyl jasmonate on the oxylipin pathway in tobacco, cucumber, and arabidopsis.

The effect of atmospheric methyl jasmonate on the oxylipin pathway was investigated in leaves of tobacco (Nicotiana tabacum L.), cucumber (Cucumis sativa L.), and Arabidopsis thaliana (L.). Differential sensitivities of test plants to methyl jasmonate were observed. Thus, different concentrations of methyl jasmonate were required for induction of changes in the oxylipin pathway. Arabidopsis was the least and cucumber the most sensitive to methyl jasmonate. Methyl jasmonate induced the accumulation of lipoxygenase protein and a corresponding increase in extractable lipoxygenase activity. Atmospheric methyl jasmonate additionally induced hydroperoxide lyase activity and the enhanced production of several volatile six-carbon products. It is interesting that lipid hydroperoxidase activity, which is a measure of hydroperoxide lyase plus allene oxide synthase plus possibly other lipid hydroperoxide-metabolizing activities, was not changed by methyl jasmonate treatment. Methyl jasmonate selectively altered the activity of certain enzymes of the oxylipin pathway (lipoxygenase and hydroperoxide lyase) and increased the potential of leaves for greatly enhanced six-carbon-volatile production.

Volatile compounds induced by herbivory act as aggregation kairomones for the Japanese beetle (Popillia japonica Newman).

The Japanese beetle is a polyphagous insect that typically aggregates on preferred host plants in the field. We studied the response of Japanese beetles to artificial damage, fresh feeding damage, and overnight feeding damage to test the hypothesis that beetles are attracted to feeding-induced volatiles. Crabapple leaves that had been damaged overnight by Japanese beetles or fall webworms attracted significantly more Japanese beetles than did undamaged leaves. Artificially damaged leaves or leaves freshly damaged by Japanese beetles, however, were not significantly more attractive than undamaged leaves. Leaves that had been damaged overnight by Japanese beetles or fall webworms produced a complex mixture of aliphatic compounds, phenylpropanoid-derived compounds, and terpenoids. In comparison, artificially damaged leaves or leaves with fresh Japanese beetle feeding damage generated a less complex blend of volatiles, mainly consisting of green-leaf odors. Feeding-induced odors may facilitate host location and/or mate finding by the Japanese beetle.

Effects of some leaf-emitted volatile compounds on aphid population increase.

A role of some volatile compounds produced by plant tissues may be as defensive molecules against various pests, including arthropods. Volatile six-carbon compounds derived in plant tissue from polyunsaturated fatty acids via lipoxygenase/hydroperoxide lyase reduced tobacco aphid fecundity at certain concentrations when added to headspace vapor to which aphids were exposed. Both C6 aldehydes and alcohols were effective, with the alcohols having greater activity. (Z)-3-Hexenyl acetate at levels in the headspace similar to those of the alcohols and aldehydes did not reduce aphid fecundity. A 6-hr exposure period to the C6 aldehydes and alcohols was needed for maximum effect on the aphids feeding on tobacco leaves. Analysis of the direct versus indirect effects of these compounds indicates that the volatile aldehydes had both direct effects on aphid fecundity and indirect effects due to induced changes in the leaves upon which the aphids were feeding, while only indirect effects were observed for the alcohols. Tomato leaves have the capacity to produce volatile compounds at levels that impact aphid population increase, with the volatiles produced from crushed leaves having a much larger effect. The C6 aldehydes and alcohols may be components of the fecundity reduction seen with tomato volatiles; however, volatile terpenes showed no effect. These results can be of significance for the genetic alteration of plants for improved aphid resistance.

Developmental change in c(6)-aldehyde formation by soybean leaves.

Damage to plant leaves by wounding or freezing induces the production of large amounts of C(6)-compounds. However, the control of formation of these compounds in leaves is not yet clear. In the current study, C(6)-aldehyde formation by freeze-injured soybean leaves of different ages (based on the leaf positions on the plant) at stage R1 of plant development was investigated. The results demonstrate that C(6)-aldehyde formation by the soybean (Glycine max L.) leaves changes as leaves develop. Younger leaves produce high levels of C(6)-aldehydes, mainly composed of hexanal. Subsequently, as the leaves develop, the level of C(6)-aldehyde formation decreases markedly, followed by an increase with a large shift from hexanal to hexenals. Lipoxygenase and lipolytic acyl hydrolase activity was reduced, and, in contrast, hydroperoxide lyase activity increased. There was little difference in lipoxygenase substrate specificity for linoleic acid and linolenic acid, but hydroperoxide lyase preferentially utilized 13-hydroperoxy-9,11,15-octadecatrienoic acid. In the in vivo lipoxygenase substrate pool, the linoleic acid level declined and the relative level of linolenic acid increased. The change in ratios of linolenic acid to linoleic acid showed a similar trend during soybean leaf development to that of hexenals to hexanal.

Expression of soybean-embryo lipoxygenase 2 in transgenic tobacco tissue.

To assess the role of lipoxygenase (LOX; EC 1.13.11.12) in plants, we increased the expression of LOX in the tissues of Nicotiana tabacum L. cv. 'KY 14' by over-expression of the LOX2 gene from the soybean (Glycine max (L.) Merrill) embryo. The LOX2 cDNA was manipulated by replacing its 5'-untranslated sequence with the translational enhancer of the alfalfa mosaic virus (AMV), and subcloned into a plant expression vector, 3' to a duplicated cauliflower mosaic virus 35S promoter. The AMV-LOX2 construct was transferred into tobacco using Agrobacterium tumefaciens strain A281. The LOX2 was expressed in transgenic tobacco calli, leaves of transgenic plants, and their seed progeny at levels up to 0.1-0.2% of the total extracted protein. The introduced LOX2 affected fatty-acid oxidative metabolism as evidenced by a 50-529% increase in C6-aldehyde production. The impact on C6-aldehyde formation was greater than the effect on production of fatty-acid hydroperoxides. This is consistent with other studies indicating the greater propensity of soybean embryo LOX2 in generating C6-aldehydes than that of other well-characterized LOX isozymes.

Effects of some natural volatile compounds on the pathogenic fungiAlternaria alternata andBotrytis cinerea.

A bioassay system was developed to test the effects of volatile compounds on the growth of hyphae from germinating fungal spores. Volatiles from crushed tomato leaves inhibited hyphal growth of two fungal pathogens,Alternaria alternata andBotrytis cinerea. Aldehydes, including C6 and C9 compounds formed by the lipoxygenase enzyme pathway upon wounding leaves, inhibited growth of both fungal species. Terpene hydrocarbons, 2-carene and limonene, had no significant effect on hyphal growth. The quantities of volatile compounds in the vapor phase of the bioassay system were measured by direct headspace sampling and GC analysis.

Soybean leaves contain multiple lipoxygenases.

Chromatofocusing of soybean (Glycine max L.) leaf lipoxygenases revealed three distinct peaks of activity. Based on their isoelectric points (pls), pH optima, and mutant analysis it appears that the leaf isozymes are different from those described from mature soybean seed. At least one leaf lipoxygenase appears to differ from those found in hypocotyls. The pls of the main bands of the three leaf lipoxygenase peaks are 6.67, 5.91, and 5.67. The pH optima curves of three active fractions exhibit peaks at pH 6.2, 5.5, and 8.5, respectively. One of the fractions has two polypeptides with slightly different molecular weights, both of which react to soybean seed lipoxygenase antibodies. The other two fractions contain a polypeptide of unit molecular weight reacting with the lipoxygenase antibodies.

Effect of storage conditions on nitrosated, acylated, and oxidized pyridine alkaloid derivatives in smokeless tobacco products.

Very large concentration increases in nitrite (34-fold), nitrosated pyridine alkaloids, and related 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (14- to 33-fold) occurred in moist snuff during storage at 24 degrees C for 52 weeks, whereas, decreases in all parent and some acylated pyridine alkaloids were observed in the same material. Nitrite concentrations in dry snuff decreased up to 90% during storage; increased contents of nitrosated alkaloids and NNK of 30 to 80% were also observed. Storage effects on chewing tobacco included a 75% increase in nitrite and small increases of nitrosated alkaloids and NNK. Sums of parent alkaloids in moist snuff decreased 24 and 54% after storage for 24 weeks at 24 and 32 degrees C, respectively, while sums of alkaloid derivatives increased, up to 36-fold for nitrosated alkaloids and NNK, 92% for acylated, and 133% for oxidized components. Levels of N'-nitrosonornicotine, NNK, and N'-nitrosoanatabine after 52 weeks' storage at 24 degrees C were 547, 41, and 296% higher, respectively, in ambient air-exposed moist snuff than in the nonexposed counterpart. A mathematical model was evaluated and used to calibrate nonlinear gas chromatography alkali bead detector response to the individual components. This permitted the use of a single analysis for all required individual compounds over a wide concentration range.

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)-ß-ocimene, (Z)-ß-ocimene, α-farnesene, and germacrene D.

Strawberry foliage headspace vapor components at periods of susceptibility and resistance toTetranychus urticae Koch.

Headspace components from strawberry foliage have been isolated by nitrogen entrainment and Tenax trapping. Traps were eluted with hexane, and components were analyzed by gas chromatography-mass spectrometry. Fifteen compounds were identified by comparison with authentic standards,trans-2-hexenal, 1-hexanol,trans-2-hexen-1-ol,cis-3-hexen-1-ol, hexyl acetate,cis-3-hexenyl acetate, 6-methyl-5-hepten-2-ol, 1-octanol, 1-octen-3-ol, linalool, α-terpineol, methyl salicylate, ethyl saiicylate, benzyl alcohol, and 2-phenylethanol. The relative amounts of these components were compared at flowering and after fruit harvest when plants were more resistant to the two-spotted spider mite,Tetranychus urticae Koch. The predominant components,cis-3-hexen-1-ol and its acetate, did not change markedly between the sampling periods, but methyl salicylate increased approximately 10-fold after fruit harvest. Methyl salicylate at low concentrations under bioassay conditions did not affect mite behavior. The biosynthetic relationship of this compound to other phenols which have been implicated in plant resistance is discussed.

Effects of air-curing environment on alkaloid-derived nitrosamines in burley tobacco.

Levels of nitrite and pyridine alkaloid-derived total tobacco-specific nitrosamines (TSNA) were significantly higher in tobacco leaf (normal or late harvest) air-cured at 32 degrees C/83% relative humidity (RH) than in more moderate environments, i.e., 15 degrees C/50% RH and 24 degrees C/70% RH. These constituents increased appreciably from day 10 to day 21 of the cure. The near-concurrent appearances of maximal total contents of TSNA [sum of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB) and 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone) (NNK)] and nitrite supports the concept that nitrite is a limiting and proximal precursor of total TSNA during the curing of tobacco. During a long curing period (50 days) at 32 degrees C/83% RH, nicotine and anatabine contents decreased, but TSNA contents increased in leaves of all harvest dates and stalk positions. These results support the view that nicotine and anatabine are precursors of TSNA. Measurement of nitrite and individual alkaloids during post-harvest processing of tobacco leaf may provide an index of the potential accumulation of alkaloid-derived nitrosamines.