Identification of Trichoderma spp. volatile organic compounds (VOCs) by HS-SPME/GC-MS analysis

Introduction Trichoderma spp. are among the most widespread soil microorganisms and have been widely used as biological control agents against numerous phytopathogens [1]. Trichoderma biological control mechanisms are based on the production of antimicrobial substances, lytic enzymes, competition with other microorganisms for nutrients and/or space, mycoparasitism, and plant resistance induction [2]. Moreover, Trichoderma spp. are known to produce numerous volatile organic compounds (VOCs) [3] that play crucial roles in the inter-kingdom communications and biological control mechanisms [4]. Three Trichoderma strains, such as T. asperellum T34 (T34), T. atroviride SC1 (SC1), T. harzianum T39 (T39) are well documented for their ability to cope with a broad spectrum of phytopathogens and are commercialised as biofungicides. Several studies highlighted the importance of Trichoderma spp. as an alternative strategy against grapevine pathogens [5], but no information is available on the possible biological control mechanism mediated by Trichoderma VOCs against grapevine downy mildew (caused by Plasmopara viticola). The aim of this study was to identify VOCs produced by T34, SC1 and T39 using headspace-solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME/GC-MS) analysis. Materials and Methods Volatile-mediated effects of Trichoderma strains were assessed on grapevine leaf disks incubated with uninoculated potato dextrose agar (PDA) Petri dishes (Control) and dishes with PDA-grown T34, SC1 or T39 colonies without contact with leaf tissues. Leaf disks were inoculated with a suspension of P. viticola sporangia and downy mildew severity was assessed at six days post inoculation (dpi). For headspace analysis of Trichoderma spp. VOCs, 20 µL of the conidial suspension of the respective Trichoderma strain were inoculated on PDA poured into sterile 20 mL-HS vials. VOCs produced by Trichoderma strains were measured using HS-SPME/GC-MS analysis at 48 or 72 h of incubation at 25°C. For compound identification, deconvoluted mass spectra were compared with an in-house library obtained with authentic reference standards. Compound annotation was achieved imposing a relative deviation of retention index (RI) value lower than 2% from the reference value published in the NIST14 database, and according to the mass spectrum similarity score set higher than 70%. Two independent experiments were carried out. Five of the identified VOCs were selected according to their emission profiles and pure compounds were tested against P. viticola by leaf disk assay. Results VOCs produced by T34, SC1 or T39 reduced downy mildew symptoms on grapevine leaf disks. HS-SPME/GC-MS analysis revealed a total of 26 and 21 Trichoderma VOCs found in the first and second experiments, respectively. Trichoderma VOCs included alkenes, ketones, pyrones, furanes and terpenes. Terpenes were successfully identified using manual inspection of extracted ion current (EIC) chromatograms at m/z 136 for monoterpenes, m/z 202 and 204 for sesquiterpenes, and m/z 272 for diterpenes. VOC profiles differed according to the Trichoderma strains and T39 was the highest producer of VOCs and terpenes compared to T34 and SC1. Five metabolite groups were identified according to changes in abundance among the three Trichoderma strains: VOCs with consistent changes in both experiments at both (Group 1), or at one time point (Group 2); VOCs with different changes in abundance in both experiments (Group 3); VOCs detected only in the first (Group 4) or second (Group 5) experiment. Five VOCs (two sesquiterpenes, one alkene, one furan and one pyrone) were tested against P. viticola and each VOC was applied to a filter paper disk on the lid of the Petri dish without contact with leaf tissues. Three VOCs slightly reduced the development of downy mildew symptoms at dosages of 50 mg/L in air volume. Two pure VOCs significantly reduced downy mildew symptoms at the dosage of 10 mg/L in air volume. Discussion/Conclusions HS-SPME/GC-MS analysis is a powerful tool to detect and identify Trichoderma VOCs. VOC profiles differed in the three Trichoderma strains and T39 was the highest producer of terpenes, followed by T34 and SC1, indicating genotypic determinants of VOC production. Five VOCs (two sesquiterpenes, one alkene, one furan and one pyrone) reduced downy mildew symptoms on grapevine leaf disks. Preliminary results suggest that two most effective VOCs enhanced the grapevine defence responses against P. viticola. These results indicate that Trichoderma VOCs are promising molecules that could be further developed to naturally control grapevine downy mildew. Further transcriptomic and metabolomic studies are required to shed light on the grapevine defence mechanisms activated by Trichoderma VOCs against downy mildew.