John H. Loughrin

Suppression of a P450 hydroxylase gene in plant trichome glands enhances natural-product-based aphid resistance

Trichome glands on the surface of many higher plants produce and secrete exudates affecting insects, microbes, and herbivores. Metabolic engineering of gland exudation has potential for improving pest/disease resistance, and for facilitating molecular farming. We identified a cytochrome P450 hydroxylase gene specific to the trichome gland and used both antisense and sense co-suppression strategies to investigate its function. P450-suppressed transgenic tobacco plants showed a ≥41% decrease in the predominant exudate component, cembratriene-diol (CBT-diol), and a ≥19-fold increase in its precursor, cembratriene-ol (CBT-ol). Thus, the level of CBT-ol was raised from 0.2 to ≥4.3% of leaf dry weight. Exudate from antisense-expressing plants had higher aphidicidal activity, and transgenic plants with exudate containing high concentrations of CBT-ol showed greatly diminished aphid colonization responses. Our results demonstrate the feasibility of significantly modifying the natural-product chemical composition and aphid-interactive properties of gland exudates using metabolic engineering. The results also have implications for molecular farming.

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 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.

Glycosidically bound volatile components of Nicotiana sylvestris and N. Suaveolens flowers

Abstract Glycosidically bound volatile components were extracted from flowers of Nicotiana sylvestris and N. suaveolens and isolated using liquid column chromatography with an Amberlite XAD-2 resin. These glycosides, which were putative precursors of fragrance compounds, were hydrolysed enzymatically and a number of the volatiles released were subsequently identified by GC-MS including compounds not previously identified in the floral headspace of these species. Average yields of volatiles released from glycosides were ca 230 and 1050 μg g −1 for N. sylvestris and N. suaveolens flowers, respectively. Compounds in the glycosidically bound fraction were all phenylpropanoid-derived volatiles (e.g. benzyl alcohol, benzaldehyde, E -cinnamyl alcohol, benzyl salicylate) with the exception of the monoterpene α-terpineol. No pronounced diurnal changes in the levels of glycosidically bound volatile compounds could be noted from flowers of either species. However, pronounced differences were obtained in the concentrations of glycosidically bound volatiles at different floral maturity stages.

Diurnal emission of volatile compounds by Japanese beetle-damaged grape leaves

Abstract The volatile compounds liberated by Japanese beetle ( Popillia japonica Newman) feeding on grape leaves ( Vitis labrusca L.) were studied. Ten consecutive collections of 3 hr duration were performed on live vines starting at 09:00 and continuing until 15:00 the following day. Release of most compounds followed a diurnal pattern, with the period of peak emission from 12:00 to 15:00 and the period of least emission from 00:00 to 03:00. Nineteen compounds were identified from the beetle-damaged vines, most of which were aliphatic aldehydes, alcohols and esters as well as terpene hydrocarbons. During periods of peak emission, volatile production from beetle-damaged vines was about 50-times higher than that of undamaged vines.

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.

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)-β-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 diurnal sampling on the headspace composition of detached Nicotiana suaveolens flowers

Abstract Compounds emitted over a 24 hour sampling period by detached flowers of Nicotiana suaveolens were examined after collecting flowers at 12:00 and 24

Identification of some volatile compounds from strawberry flowers

Abstract Volatile compounds from strawberry flowers were entrained in air, trapped on Tenax, and eluted with solvent. Headspace components were analysed by GC and GC-MS and included aromatic compounds not detected in strawberry leaf headspace.

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.