Novel use of PDMS tubing for in-soil capture of plant natural products.

The extraction of small lipophilic molecules (SLMs) in the soil-root interface that play a role in belowground ecological interactions between plants and insect herbivores was investigated. Polydimethylsiloxane (PDMS) microtubing has been shown to absorb root SLMs selectively in low-disturbance setups, where analytes were extracted from the polymer with methanol. This technique was adapted to isolate SLMs that diffuse in the vapour phase in soil and sand and under various experimental parameters, extracting with a plug of diethyl ether pushed through the length of the silicon tubing. Moisture level had a substrate-dependent effect on the recovery rate of analytes that were applied as synthetic blends of known belowground SLM semiochemicals in the media. Higher amounts of two selected SLMs, (E)-caryophyllene and (-)-thujopsene, were extracted from sand, and increased polymer and solvent volume, as well as sampling duration, resulted in more of these two SLMs recovered by extraction. It was also shown that PDMS tubes lose no extraction capacity after repeated use. The signature compound (E)-caryophyllene was successfully isolated from the rhizosphere of maize plants infested with Diabrotica v. virgifera larvae by extracting the silicon tubing with diethyl ether. Because the tubes are preconditioned to reduce the presence of contaminants, such extracts can be directly analysed by GC and GC-MS and used in electrophysiological and behavioural assays. After further modifications, non-invasive, in situ PDMS probes can be developed that extract SLMs from plant rhizosphere for the study of belowground chemical ecology processes.

Electrophysiologically and behaviourally active semiochemicals identified from bed bug refuge substrate.

Bed bugs are pests of public health importance due to their relentless biting habits that can lead to allergies, secondary infections and mental health issues. When not feeding on human blood bed bugs aggregate in refuges close to human hosts. This aggregation behaviour could be exploited to lure bed bugs into traps for surveillance, treatment efficacy monitoring and mass trapping efforts, if the responsible cues are identified. The aim of this study was to identify and quantify the bed bug aggregation pheromone. Volatile chemicals were collected from bed bug-exposed papers, which are known to induce aggregation behaviour, by air entrainment. This extract was tested for behavioural and electrophysiological activity using a still-air olfactometer and electroantennography, respectively. Coupled gas chromatography-electroantennography (GC-EAG) was used to screen the extract and the GC-EAG-active chemicals, benzaldehyde, hexanal, (E)-2-octenal, octanal, nonanal, decanal, heptanal, (R,S)-1-octen-3-ol, 3-carene, ß-phellandrene, (3E,5E)-octadien-2-one, (E)-2-nonenal, 2-decanone, dodecane, nonanoic acid, 2-(2-butoxyethoxy)ethyl acetate, (E)-2-undecanal and (S)-germacrene D, were identified by GC-mass spectrometry and quantified by GC. Synthetic blends, comprising 6, 16, and 18 compounds, at natural ratios, were then tested in the still-air olfactometer to determine behavioural activity. These aggregation chemicals can be manufactured into a lure that could be used to improve bed bug management.

Biosynthesis of natural and novel C-glycosylflavones utilising recombinant Oryza sativa C-glycosyltransferase (OsCGT) and Desmodium incanum root proteins.

The rice C-glycosyltransferase (OsCGT) is one of only a small number of characterised plant C-glycosyltransferases (CGT) known. The enzyme C-glucosylates a 2-hydroxyflavanone substrate with UDP-glucose as the sugar donor to produce C-glucosyl-2-hydroxyflavanones. We tested substrate specificity of the enzyme, using synthetic 2-hydroxyflavanones, and showed it has the potential to generate known natural CGFs that have been isolated from rice and also other plants. In addition, we synthesised novel, unnatural 2-hydroxyflavanone substrates to test the B-ring chemical space of substrate accepted by the OsCGT and demonstrated the OsCGT capacity as a synthetic reagent to generate significant quantities of known and novel CGFs. Many B-ring analogues are tolerated within a confined steric limit. Finally the OsCGT was used to generate novel mono-C-glucosyl-2-hydroxyflavanones as putative biosynthetic intermediates to examine the potential of Desmodium incanum biosynthetic CGTs to produce novel di-C-glycosylflavones, compounds implicated in the allelopathic biological activity of Desmodium against parasitic weeds from the Striga genus.

Isolation and identification of Desmodium root exudates from drought tolerant species used as intercrops against Striga hermonthica.

Plants from the genus Desmodium, in particular D. uncinatum, are used on sub-Saharan small-holder farms as intercrops to inhibit parasitism of cereal crops by Striga hermonthica and Striga asiatica via an allelopathic mechanism. The search for Desmodium species which are adapted to more arid conditions, and which show resilience to increased drought stress, previously identified D. intortum, D. incanum and D. ramosissimum as potential drought tolerant intercrops. Their potential as intercrops was assessed for resource poor areas of rain-fed cereal production where drought conditions can persist through normal meteorological activity, or where drought may have increasing impact through climate change. The chemical composition of the root exudates were characterised and the whole exudate biological activity was shown to be active in pot experiments for inhibition of Striga parasitism on maize. Furthermore, rain fed plot experiments showed the drought tolerant Desmodium intercrops to be effective for Striga inhibition. This work demonstrates the allelopathic nature of the new drought tolerant intercrops through activity of root exudates and the major compounds seen in the exudates are characterised as being C-glycosylflavonoid. In young plants, the exudates show large qualitative differences but as the plants mature, there is a high degree of convergence of the C-glycosylflavonoid exudate chemical profile amongst active Desmodium intercrops that confers biological activity. This defines the material for examining the mechanism for Striga inhibition.

Semiochemicals from herbivory induced cotton plants enhance the foraging behavior of the cotton boll weevil, Anthonomus grandis.

The boll weevil, Anthonomus grandis, has been monitored through deployment of traps baited with aggregation pheromone components. However, field studies have shown that the number of insects caught in these traps is significantly reduced during cotton squaring, suggesting that volatiles produced by plants at this phenological stage may be involved in attraction. Here, we evaluated the chemical profile of volatile organic compounds (VOCs) emitted by undamaged or damaged cotton plants at different phenological stages, under different infestation conditions, and determined the attractiveness of these VOCs to adults of A. grandis. In addition, we investigated whether or not VOCs released by cotton plants enhanced the attractiveness of the aggregation pheromone emitted by male boll weevils. Behavioral responses of A. grandis to VOCs from conspecific-damaged, heterospecific-damaged (Spodoptera frugiperda and Euschistus heros) and undamaged cotton plants, at different phenological stages, were assessed in Y-tube olfactometers. The results showed that volatiles emitted from reproductive cotton plants damaged by conspecifics were attractive to adults boll weevils, whereas volatiles induced by heterospecific herbivores were not as attractive. Additionally, addition of boll weevil-induced volatiles from reproductive cotton plants to aggregation pheromone gave increased attraction, relative to the pheromone alone. The VOC profiles of undamaged and mechanically damaged cotton plants, in both phenological stages, were not different. Chemical analysis showed that cotton plants produced qualitatively similar volatile profiles regardless of damage type, but the quantities produced differed according to the plant's phenological stage and the herbivore species. Notably, vegetative cotton plants released higher amounts of VOCs compared to reproductive plants. At both stages, the highest rate of VOC release was observed in A. grandis-damaged plants. Results show that A. grandis uses conspecific herbivore-induced volatiles in host location, and that homoterpene compounds, such as (E)-4,8-dimethylnona-1,3,7-triene and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene and the monoterpene (E)-ocimene, may be involved in preference for host plants at the reproductive stage.