Identification of Semiochemicals from Cowpea, Vigna unguiculata, for Low-input Management of the Legume Pod Borer, Maruca vitrata.

Cowpea, Vigna unguiculata L. Walp. (Fabaceae), is one of the most important food legumes grown on the African continent, as it provides an affordable source of dietary protein. Yields of cowpea are significantly reduced through damage by legume pod-borer, Maruca vitrata (Lepidoptera: Crambidae), caterpillars to flowers, tender leaves and pods. Semiochemical-based strategies are considered as environmentally benign and affordable for pest management, particularly on smallholder farms. In this study, we investigated the importance of cowpea flower volatiles as host location cues for egg-laying M. vitrata, and herbivore-induced plant volatiles (HIPVs) as M. vitrata repellents and natural enemy (Apanteles taragamae and Phanerotoma syleptae parasitoid) attractants. In oviposition choice assays, M. vitrata laid more eggs on flowering cowpea plants than non-flowering plants. Coupled gas chromatography-electrophysiology (GC-EAG) analysis using the antennae of female M. vitrata and an extract of flower volatiles collected by dynamic headspace collection revealed the presence of five EAG-active components that were identified by coupled GC-mass spectrometry (GC-MS) analysis as benzaldehyde, benzyl alcohol, acetophenone, a vinylbenzaldehyde isomer and (E)-cinnamaldehyde. A synthetic blend of the identified compounds, prepared using 3-vinylbenzaldehyde, induced M. vitrata to lay as many eggs on non-flowering cowpea as on flowering plants. The moths also preferred laying eggs on intact plants compared to M. vitrata-infested plants. As the emission of EAG-active floral compounds was determined to be lower in the headspace of infested cowpea flowers, the role of HIPVs emitted by M. vitrata-damaged leaves was also investigated. Of the compounds induced by larval damage, (E)-DMNT, indole, n-hexyl acetate, 1-octen-3-ol and linalool were shown by GC-EAG to possess electrophysiological activity. A synthetic blend of the EAG-active compounds, using racemic 1-octen-3-ol and linalool, significantly reduced egg numbers on flowering cowpea. Larval and egg parasitoids, i.e. A. taragamae and Ph. syleptae, respectively, of M. vitrata both preferred the Y-tube olfactometer arm treated with synthetic (E)-DMNT, whereas preference for racemic linalool and (E)-nerolidol was dose-dependent in A. taragamae. Our results provide the platform for the development of future semiochemical-based pest management strategies against M. vitrata on smallholder farms in West Africa.

(2R,5S)-Theaspirane Identified as the Kairomone for the Banana Weevil, Cosmopolites sordidus, from Attractive Senesced Leaves of the Host Banana, Musa spp.

The principal active component produced by highly attractive senesced host banana leaves, Musa spp., for the banana weevil, Cosmopolites sordidus, is shown by coupled gas chromatography-electroantennography (GC-EAG), coupled GC-mass spectrometry (GC-MS), chemical synthesis and coupled enantioselective (chiral) GC-EAG to be (2R,5S)-theaspirane. In laboratory behaviour tests, the synthetic compound is as attractive as natural host leaf material and presents a new opportunity for pest control.

Drought-tolerant Desmodium species effectively suppress parasitic striga weed and improve cereal grain yields in western Kenya.

The parasitic weed Striga hermonthica Benth. (Orobanchaceae), commonly known as striga, is an increasingly important constraint to cereal production in sub-Saharan Africa (SSA), often resulting in total yield losses in maize (Zea mays L.) and substantial losses in sorghum (Sorghum bicolor (L.) Moench). This is further aggravated by soil degradation and drought conditions that are gradually becoming widespread in SSA. Forage legumes in the genus Desmodium (Fabaceae), mainly D. uncinatum and D. intortum, effectively control striga and improve crop productivity in SSA. However, negative effects of climate change such as drought stress is affecting the functioning of these systems. There is thus a need to identify and characterize new plants possessing the required ecological chemistry to protect crops against the biotic stress of striga under such environmental conditions. 17 accessions comprising 10 species of Desmodium were screened for their drought stress tolerance and ability to suppress striga. Desmodium incanum and D. ramosissimum were selected as the most promising species as they retained their leaves and maintained leaf function for longer periods during their exposure to drought stress conditions. They also had desirable phenotypes with more above ground biomass. The two species suppressed striga infestation, both under controlled and field conditions, and resulted in significant grain yield increases, demonstrating the incremental capability of Desmodium species in striga suppression. These results demonstrate beneficial effects of Desmodium species in enhancing cereal productivity in dry areas.

The biosynthesis of allelopathic di-C-glycosylflavones from the roots of Desmodium incanum (G. Mey.) DC.

The allelopathic root exudate of the drought-tolerant subsistence cereal intercrop D. incanum, protecting against the parasitic weed Striga hermonthica, comprises a number of di-C-glycosylflavones specifically containing C-glucosyl, C-galactosyl and C-arabinosyl moieties. Here we demonstrate that the biosynthesis of all compounds containing a C-glucose involves C-glucosylation of 2-hydroxynaringenin with subsequent C-galactosylation, C-glucosylation or C-arabinosylation. In addition, the crude soluble enzyme extract converts two fluorinated 2-hydroxyflavanone analogues to corresponding mono- and di-C-glycosylflavones demonstrating that some differences in C-ring substitution can be tolerated by the plant enzymes. Elucidating the biosynthesis of these C-glycosylflavones (CGFs) has the potential to open up opportunities for transferring the enzymic and genetic basis for the S. hermonthica inhibiting allelopathic trait to food crop plants.

Delivering sustainable crop protection systems via the seed: exploiting natural constitutive and inducible defence pathways.

To reduce the need for seasonal inputs, crop protection will have to be delivered via the seed and other planting material. Plant secondary metabolism can be harnessed for this purpose by new breeding technologies, genetic modification and companion cropping, the latter already on-farm in sub-Saharan Africa. Secondary metabolites offer the prospect of pest management as robust as that provided by current pesticides, for which many lead compounds were, or are currently deployed as, natural products. Evidence of success and promise is given for pest management in industrial and developing agriculture. Additionally, opportunities for solving wider problems of sustainable crop protection, and also production, are discussed.

Specificity determinants of the silkworm moth sex pheromone.

The insect olfactory system, particularly the peripheral sensory system for sex pheromone reception in male moths, is highly selective, but specificity determinants at the receptor level are hitherto unknown. Using the Xenopus oocyte recording system, we conducted a thorough structure-activity relationship study with the sex pheromone receptor of the silkworm moth, Bombyx mori, BmorOR1. When co-expressed with the obligatory odorant receptor co-receptor (BmorOrco), BmorOR1 responded in a dose-dependent fashion to both bombykol and its related aldehyde, bombykal, but the threshold of the latter was about one order of magnitude higher. Solubilizing these ligands with a pheromone-binding protein (BmorPBP1) did not enhance selectivity. By contrast, both ligands were trapped by BmorPBP1 leading to dramatically reduced responses. The silkworm moth pheromone receptor was highly selective towards the stereochemistry of the conjugated diene, with robust response to the natural (10E,12Z)-isomer and very little or no response to the other three isomers. Shifting the conjugated diene towards the functional group or elongating the carbon chain rendered these molecules completely inactive. In contrast, an analogue shortened by two omega carbons elicited the same or slightly higher responses than bombykol. Flexibility of the saturated C1-C9 moiety is important for function as addition of a double or triple bond in position 4 led to reduced responses. The ligand is hypothesized to be accommodated by a large hydrophobic cavity within the helical bundle of transmembrane domains.

Elucidation of the biosynthesis of the di-C-glycosylflavone isoschaftoside, an allelopathic component from Desmodium spp. that inhibits Striga spp. development.

Isoschaftoside, an allelopathic di-C-glycosylflavone from Desmodium spp. root exudates, is biosynthesised through sequential glucosylation and arabinosylation of 2-hydroxynaringenin with UDP-glucose and UDP-arabinose. Complete conversion to the flavone requires chemical dehydration implying a dehydratase enzyme has a role in vivo to complete the biosynthesis. The C-glucosyltransferase has been partially characterised and its activity demonstrated in highly purified fractions.

Pheromone gland development and pheromone production in lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae).

The sand fly Lutzomyia longipalpis (Lutz & Neiva) (Diptera: Psychodidae: Phlebotominae) is the main vector of American visceral leishmaniasis. Adult males produce a terpenoid sex pheromone that in some cases also acts as male aggregation pheromone. We have analyzed the correlation between male pheromone production levels and pheromone gland cell morphogenesis after adult emergence from pupae. The abdominal tergites of L. longipalpis males were dissected and fixed in glutaraldehyde for transmission electron microscopy, or the pheromone was extracted in analytical grade hexane. Pheromone chemical analysis was carried out at 3- to 6-h intervals during the first 24 h after emergence and continued daily until the seventh day. All extracts were analyzed by gas chromatography. For the morphological analysis, we used insects collected at 0-6, 9-12, 12-14, and 96 h after emergence. Ultrastructural data from 0- to 6-h-old adult males revealed smaller pheromone gland cells with small microvilli at the end apparatus. Lipid droplets and peroxisomes were absent or very rare, but a large number of mitochondria could be seen. Lipid droplets started to appear in the gland cells cytoplasm approximately 9 h after adult emergence, and their number and size increased with age, together with the presence of several peroxisomes, suggesting a role for these organelles in pheromone biosynthesis. At 12-15 h after emergence, the lipid droplets were mainly distributed near the microvilli but were smaller than those in mature older males (4 d old). Pheromone biosynthesis started around 12 h after emergence and increased continuously during the first 3 d, stabilizing thereafter, coinciding with the period when males are more able to attract females.

Exploiting phytochemicals for developing a 'push-pull' crop protection strategy for cereal farmers in Africa.

Lepidopteran stemborers and parasitic weeds in the genus Striga are major constraints to efficient production of cereals, the most important staple food crops in Africa. Smallholder farmers are resource constrained and unable to afford expensive chemicals for crop protection. Development of a push-pull approach for integrated pest and weed management is reviewed here. Appropriate plants were discovered that naturally emit signalling chemicals (semiochemicals). Plants highly attractive for egg laying by stemborer pests were selected and employed as trap crops (pull), to draw pests away from the main crop. Of these, Napier grass, Pennisetum purpureum (Schumach), despite its attractiveness, supported minimal survival of the pests' immature stages. Plants that repelled stemborer pests, notably molasses grass, Melinis minutiflora P. Beauv., and forage legumes in the genus Desmodium, were selected as intercrops (push). Desmodium intercrops suppress Striga hermonthica (Del.) Benth. through an allelopathic mechanism. Their root exudates contain novel flavonoid compounds, which stimulate suicidal germination of S. hermonthica seeds and dramatically inhibit its attachment to host roots. The companion crops provide valuable forage for farm animals while the leguminous intercrops also improve soil fertility and moisture retention. The system is appropriate as it is based on locally available plants, not expensive external inputs, and fits well with traditional mixed cropping systems in Africa. To date it has been adopted by more than 30,000 smallholder farmers in East Africa where maize yields have increased from ∼1 t ha(-1) to 3.5 t ha(-1). Future directions for semiochemical delivery by plants including biotechnological opportunities are discussed.

Companion cropping to manage parasitic plants.

Parasitic plants, through a range of infestation strategies, can attack crop plants and thereby require management. Because such problems often occur in resource-poor farming systems, companion cropping to manage parasitic plants is an appropriate approach. Many examples of companion cropping for this purpose have been reported, but the use of cattle forage legumes in the genus Desmodium as intercrops has been shown to be particularly successful in controlling the parasitic witchweeds (Striga spp.) that afflict approximately one quarter of sub-Saharan African cereal production. Through the use of this example, the development of effective companion crops is described, together with developments toward widespread adoption and understanding the underlying mechanisms, both for sustainability and ensuring food security, and also for exploitation beyond the cropping systems described here.

Isoschaftoside, a C-glycosylflavonoid from Desmodium uncinatum root exudate, is an allelochemical against the development of Striga.

In East African small-holder farming of maize, the cattle forage legume, Desmodium uncinatum is used as an intercrop due to its allelopathic inhibition of parasitism by Striga hermonthica, an obligate parasitic weed that can devastate the maize crop. Bioassay-guided fractionation of the root extract of D. uncinatum revealed isoschaftoside to be the main compound in the most potent fraction inhibiting growth of germinated S. hermonthica radicles. Bioassays repeated with isoschaftoside isolated from a different plant source, Passiflora incarnata, proved it to be a biologically active component. Analysis of the root exudates produced by hydroponically grown D. uncinatum showed isoschaftoside to be present in the hydroponic media at biologically active concentrations of 10-100 nM.

High-throughput ESI-MS analysis of binding between the Bombyx mori pheromone-binding protein BmorPBP1, its pheromone components and some analogues.

Chip-assisted high-throughput ESI-MS analysis of the pheromone-binding protein of the silkworm moth Bombyx mori, BmorPBP1, incubated with its pheromone components bombykol, bombykal and analogues was developed. The protein bound to bombykol ((10E,12Z)-hexadecadien-1-ol) and all 3 of its geometric isomers to a lesser extent, and showed relaxed specificity toward different chain lengths possessing unsaturation. BmorPBP1 did not bind to bombykal ((10E,12Z)-hexadecadienal), demonstrating molecular recognition of the insect pheromone components.

Characterisation of Bombyx mori Odorant-binding proteins reveals that a general odorant-binding protein discriminates between sex pheromone components.

In many insect species, odorant-binding proteins (OBPs) are thought to be responsible for the transport of pheromones and other semiochemicals across the sensillum lymph to the olfactory receptors (ORs) within the antennal sensilla. In the silkworm Bombyx mori, the OBPs are subdivided into three main subfamilies; pheromone-binding proteins (PBPs), general odorant-binding proteins (GOBPs) and antennal-binding proteins (ABPs). We used the MotifSearch algorithm to search for genes encoding putative OBPs in B. mori and found 13, many fewer than are found in the genomes of fruit flies and mosquitoes. The 13 genes include seven new ABP-like OBPs as well as the previously identified PBPs (three), GOBPs (two) and ABPx. Quantitative examination of transcript levels showed that BmorPBP1, BmorGOBP1, BmorGOBP2 and BmorABPx are expressed at very high levels in the antennae and so could be involved in olfaction. A new two-phase binding assay, along with other established assays, showed that BmorPBP1, BmorPBP2, BmorGOBP2 and BmorABPx all bind to the B. mori sex pheromone component (10E,12Z)-hexadecadien-1-ol (bombykol). BmorPBP1, BmorPBP2 and BmorABPx also bind the pheromone component (10E,12Z)-hexadecadienal (bombykal) equally well, whereas BmorGOBP2 can discriminate between bombykol and bombykal. X-ray structures show that when bombykol is bound to BmorGOBP2 it adopts a different conformation from that found when it binds to BmorPBP1. Binding to BmorGOBP2 involves hydrogen bonding to Arg110 rather than to Ser56 as found for BmorPBP1.

New genetic opportunities from legume intercrops for controlling Striga spp. parasitic weeds.

In smallholder farming in East Africa, intercropping of maize with the cattle forage legume, Desmodium uncinatum Jacq., prevents parasitism by Striga hermonthica (Del.) Benth. (witchweed) through an allelopathic mechanism. Isoschaftoside, a di-C-glycosylflavone, isolated from the root extract and root exudate of Desmodium, interferes with in vitro radicle development of germinated Striga. The biosynthetic pathway of this class of compound is already mostly present in edible legumes and in cereals, so characterisation of the enzyme and genes that control C-glycosylflavone biosynthesis has the potential to create this protection mechanism in other agriculturally important plants.

Antiectoparasitic activity of the gum resin, gum haggar, from the East African plant, Commiphora holtziana.

The mechanism of ixodid tick (Acari: Ixodidae) repellency by gum haggar, a resin produced by Commiphora holtziana (Burseraceae), was investigated by evaluating activity against the cattle tick, Boophilus microplus. In an arena bioassay, a hexane extract of the resin of C. holtziana exhibited a repellent effect lasting up to 5h. The hydrocarbon fraction of the resin extract was shown to account for the repellent activity, and was analysed by coupled gas chromatography-mass spectrometry (GC-MS). Major sesquiterpene hydrocarbons were tentatively identified as germacrene-D, delta-elemene and beta-bourbonene. The identity and stereochemistry of the former compound was confirmed as the (+)-isomer by peak enhancement using enantioselective GC, whereas the latter 2 compounds, which are most likely degradation products of germacrene-type precursors, were identified through isolation by preparative gas chromatography followed by microprobe-NMR spectroscopy. GC comparison of gum haggar with another resin, C. myrrha, which was inactive in the tick bioassay, showed that the latter contained much lower levels of these hydrocarbons. To assess the suitability of the gum haggar resin as a general acarine repellent, further tests were made on a major acarine pest of European and US animal husbandry systems, the red poultry mite, Dermanyssus gallinae (Acari: Dermanyssidae). Gum haggar extract, and the isolated hydrocarbon fraction, showed strong repellent effects in an olfactometer assay, and again gum myrrh showed no effect. These findings provide a scientific basis for the observed anti-tick properties of gum haggar, and demonstrate the potential for its development as a general acarine repellent for use in animal husbandry systems.

Structure, ratios and patterns of release in the sex pheromone of an aphid, Dysaphis plantaginea.

Insect communication is primarily via chemicals. In Aphidinae aphids, the structure and ratio of iridoid (monoterpenoid) chemicals are known to be important components of the sex pheromone. However, for enhanced species specificity, it has been suggested that release of sex pheromone might be restricted to a narrow time period within the diel cycle. Here, we determine the structure, ratios and release patterns of iridoid chemicals produced by a serious global pest, the rosy apple aphid, Dysaphis plantaginea. Volatiles were collected from batches of oviparae (sexual females) and chemicals identified by gas chromatography, mass-spectrometry and microscale NMR spectroscopy. (1R,4aS,7S,7aR)-Nepetalactol and (4aS,7S,7aR)-nepetalactone were detected in a 3.7:1 ratio. To investigate timing of release, we constructed a sequential sampling device that allowed volatile chemicals to be captured hourly from 95 same-aged oviparae over 20 consecutive days. Release patterns of the two sex pheromone components show that D. plantaginea oviparae release high levels of the two components during photophase and low levels during scotophase. Release of the two components increased significantly during the first 3 h of photophase and thereafter remained at a high level until the onset of scotophase. The ratio of (1R,4aS,7S,7aR)-nepetalactol to (4aS,7S,7aR)-nepetalactone released did not change significantly between days two to 14 of the adult stadium, but from the 15th day onward there was a significant decrease in the relative amount of (1R,4aS,7S,7aR)-nepetalactol. Pheromone release was greatest on the eighth day of the adult stadium, with up to 8.4 ng of pheromone released per ovipara per hour. This is the first report on the full structural identification and ratios of volatile iridoid components collected from D. plantaginea oviparae and is also the most detailed temporal study on sex pheromone release from any aphid species. The lack of a temporally narrow and distinct period of very high sex pheromone release suggests that alternative mechanisms or factors for species recognition and isolation may be important. Findings are discussed broadly in relation to the biology of the aphid.

C-methylated and C-prenylated isoflavonoids from root extract of Desmodium uncinatum.

A pterocarpan, 1,9-dihydroxy-3-methoxy-2-methylpterocarpan (named uncinacarpan) and two isoflavanones, 5,7-dihydroxy-2',3',4'-trimethoxy-6-(3-methylbut-2-enyl)isoflavanone (named uncinanone D) and 5,4'-dihydroxy-7,2'-dimethoxy-6-methylisoflavanone (named uncinanone E), were isolated from the CH(2)Cl(2) root extract of Desmodium uncinatum (Jacq.) DC and characterised by spectroscopic methods. In addition, a rare pterocarpan edudiol and two known abietane diterpenes, 7-oxo-15-hydroxydehydroabietic acid and 7-hydroxycallitrisic acid were identified. The fraction of the root extract that was analysed induced germination of Striga hermonthica seeds, but none of the isolated compounds showed this activity.

Development of a pheromone trap monitoring system for orange wheat blossom midge, Sitodiplosis mosellana, in the UK.

Field-trapping experiments with synthetic 2,7-nonadiyl dibutyrate, the female-produced sex pheromone of the orange wheat blossom midge, Sitodiplosis mosellana (Géhin), demonstrated that pheromone traps were highly attractive to males and caught very few non-target organisms. Different formulations of pheromone were tested to identify the optimum release rate and dispenser type for use in pheromone traps in the UK. Key findings were that racemic pheromone was as effective as enantiomerically pure (2S,7R)-2,7-nonadiyl dibutyrate, that release rates higher than 0.5 microg day(-1) were not necessary and that the optimal formulation was a 1 mg pheromone loading in a rubber septum. Pheromone traps gave a reliable indication of peak midge emergence, onset of flight and abundance of midges throughout the season. A strong correlation between maximum trap catch and crop infestation levels was obtained.

Response of economically important aphids to components of Hemizygia petiolata essential oil.

The essential oil of Hemizygia petiolata Ashby (Lamiaceae) contains high levels (>70%) of the sesquiterpene (E)-beta-farnesene, the alarm pheromone for many economically important aphid species. In order to test the suitability of H. petiolata oil as a source of (E)-beta-farnesene for use in new integrated aphid control strategies, behavioural responses of pest aphid species were studied in laboratory and field experiments. In alarm pheromone assays the peach-potato aphid, Myzus persicae Sulzer, and the pea aphid, Acyrthosiphon pisum (Harr), showed a lower level of response to the oil than expected given the high levels of (E)-beta-farnesene. It was shown that minor components in the oil, (+)-bicyclogermacrene and (-)-germacrene D, caused inhibition of the alarm response for M. persicae and A. pisum respectively. Nevertheless, in olfactometer studies the oil was directly repellent to A. pisum and the grain aphid, Sitobion avenae F. Sitobion avenae was not only repelled by (E)-beta-farnesene but also by (-)-germacrene D. Furthermore, although it was not directly repellent to M. persicae, the oil interfered with its attraction to host plant stimuli. In field plot experiments, numbers of A. pisum were significantly reduced in plots treated with a slow release formulation of the oil, when compared with control plots.