Direct Contact - Sorptive Tape Extraction coupled with Gas Chromatography - Mass Spectrometry to reveal volatile topographical dynamics of lima bean (Phaseolus lunatus L.) upon herbivory by Spodoptera littoralis Boisd.

The dynamics of plant volatile (PV) emission, and the relationship between damaged area and biosynthesis of bioactive molecules in plant-insect interactions, remain open questions. Direct Contact-Sorptive Tape Extraction (DC-STE) is a sorption sampling technique employing non adhesive polydimethylsiloxane tapes, which are placed in direct contact with a biologically-active surface. DC-STE coupled to Gas Chromatography - Mass Spectrometry (GC-MS) is a non-destructive, high concentration-capacity sampling technique able to detect and allow identification of PVs involved in plant responses to biotic and abiotic stresses. Here we investigated the leaf topographical dynamics of herbivory-induced PV (HIPV) produced by Phaseolus lunatus L. (lima bean) in response to herbivory by larvae of the Mediterranean climbing cutworm (Spodoptera littoralis Boisd.) and mechanical wounding by DC-STE-GC-MS. Time-course experiments on herbivory wounding caused by larvae (HW), mechanical damage by a pattern wheel (MD), and MD combined with the larvae oral secretions (OS) showed that green leaf volatiles (GLVs) [(E)-2-hexenal, (Z)-3-hexen-1-ol, 1-octen-3-ol, (Z)-3-hexenyl acetate, (Z)-3-hexenyl butyrate] were associated with both MD and HW, whereas monoterpenoids [(E)-β-ocimene], sesquiterpenoids [(E)-nerolidol] and homoterpenes (DMNT and TMTT) were specifically associated with HW. Up-regulation of genes coding for HIPV-related enzymes (Farnesyl Pyrophosphate Synthase, Lipoxygenase, Ocimene Synthase and Terpene Synthase 2) was consistent with HIPV results. GLVs and sesquiterpenoids were produced locally and found to influence their own gene expression in distant tissues, whereas (E)-β-ocimene, TMTT, and DMNT gene expression was limited to wounded areas. DC-STE-GC-MS was found to be a reliable method for the topographical evaluation of plant responses to biotic and abiotic stresses, by revealing the differential distribution of different classes of HIPVs. The main advantages of this technique include: a) in vivo sampling; b) reproducible sampling; c) ease of execution; d) simultaneous assays of different leaf portions, and e) preservation of plant material for further "omic" studies. DC-STE-GC-MS is also a low-impact innovative method for in situ PV detection that finds potential applications in sustainable crop management.