| Summary: | Aphid resistance against Rhopalosiphum padi and Sitobion avenae in modern wheat is lacking with current aphid control measures relying on the use of insecticides. The current way of insecticide use is unsustainable and new approaches are needed to control aphid pests. Previous studies have identified Triticum monoccocum accessions MDR045 and MDR049 showing promising signs of resistance against both R. padi and S. avenae, however, the mechanisms involved in their resistance remain elusive. The aim of this project is to further characterise the aphid resistance mechanisms in MDR045 and MDR049, focusing on the role of the plant’s secondary metabolites (SMs).
Headspace extracts and leaf material was collected from MDR045 and MDR049 subjected to increasing S. avenae densities from 0, 1, 5, 10 and 25 aphids on the flag leaf for 24hrs. Triticum monococcum MDR037 and Triticum aestivum Solstice were used as aphid susceptible controls. Leaf material was collected from the aphid feeding site (CC), same leaf as the aphid feeding site (SL) and different leaf to the aphid feeding site (DL) to assess any systemic response to aphid feeding. Volatile organic compound (VOC) composition of headspace extracts was assessed via GC and behavioural activity assessed via four-arm olfactometry assays. Tentative identification of active VOCs was carried out via gas chromatography-electoantennography (GC-EAG) and gas chromatography-mass spectrometry (GC-MS) and confirmed by co-injection with authentic standards and using synthetic VOC blends in olfactometry assays. Leaf extract chemistry was assessed using liquid chromatography-mass spectrometry (LC-MS) and bioactivity assessed using aphid artificial feeding assays. Bioassay guided fractionation and active compound identification was carried out using preparative high performance liquid chromatography (HPLC-prep), liquid chromatography-mass spectrometry-mass spectrometry (LC-MS/MS), nuclear magnetic resonance (NMR) and authentic standards, identifying one of the active leaf SMs. The activity of the identified active leaf SMs was confirmed using aphid artificial feeding assays with authentic standards and this activity was compared to similarly structured plant SMs. Transcriptomic analysis was carried out between MDR049 and MDR037 under 0 and 25 S. avenae densities for 24hrs at CC and DL to investigate genes potentially involved in the SM based aphid resistance in MDR049.
VOCs from MDR045 and MDR049 induce an aphid density dependent negative behavioural response against S. avenae with 25 and 1 S. avenae densities required to induce this activity in MDR045 and MDR049 respectively. Twenty-one of 24 EAG active VOCs were identified and confirmed as being responsible for this activity. Leaf extracts from MDR045 and MDR049 possess aphid-density dependent aphid antibiotic activity against S. avenae. A systemic response to aphid feeding was observed in MDR049 confined to SL. Two leaf SMs were isolated as responsible for the aphid antibiotic activity of MDR049 with 1 leaf SM identified. The activity of the identified SM was confirmed and shown to be unique compared to other similarly structured plant SMs. S. avenae feeding induces differential gene expression in MDR049 with minimal expression induced in MDR037. Three of the top 5 differentially expressed genes in MDR049 compared to MDR037 under S. avenae infestation are potential pathogen recognition receptors. Potential candidate genes responsible for the biosynthesis of the active VOCs and the active leaf SM in MDR049 could not be identified.
The project confirmed that SMs play a role in the aphid resistance mechanisms of MDR045 and MDR049 against S. avenae. The VOCs responsible for the negative behavioural response induced by MDR045 and MDR049 against S. avenae have been identified and confirmed, and are potentially indicative of antixenotic resistance mechanisms. One of the leaf SMs involved in the aphid antibiotic activity of MDR045 and MDR049 against S. avenae was identified and confirmed. From the transcriptomic analysis, a potential hypothesis has been proposed surrounding the aphid resistance mechanism induced by MDR049.
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