Downy mildew symptoms on grapevines can be reduced by volatile organic compounds of resistant genotypes

Volatile organic compounds (VOCs) play a crucial role in the communication of plants with other organisms and are possible mediators of plant defence against phytopathogens. Although the role of non-volatile secondary metabolites has been largely characterised in resistant genotypes, the contribution of VOCs to grapevine defence mechanisms against downy mildew (caused by Plasmopara viticola) has not yet been investigated. In this study, more than 50 VOCs from grapevine leaves were annotated/identified by headspace-solid-phase microextraction gas chromatography-mass spectrometry analysis. Following P. viticola inoculation, the abundance of most of these VOCs was higher in resistant (BC4, Kober 5BB, SO4 and Solaris) than in susceptible (Pinot noir) genotypes. The post-inoculation mechanism included the accumulation of 2-ethylfuran, 2-phenylethanol, β-caryophyllene, β-cyclocitral, β-selinene and trans-2-pentenal, which all demonstrated inhibitory activities against downy mildew infections in water suspensions. Moreover, the development of downy mildew symptoms was reduced on leaf disks of susceptible grapevines exposed to air treated with 2-ethylfuran, 2-phenylethanol, β-cyclocitral or trans-2-pentenal, indicating the efficacy of these VOCs against P. viticola in receiver plant tissues. Our data suggest that VOCs contribute to the defence mechanisms of resistant grapevines and that they may inhibit the development of downy mildew symptoms on both emitting and receiving tissues.

. Overview of the experimental design. Leaf samples of the susceptible Vitis vinifera cultivar Pinot noir and four resistant Vitis spp. hybrids (BC4, Kober 5BB, SO4 and Solaris) were collected immediately before inoculation (0 dpi) and six days post inoculation (6 dpi) with Plasmopara viticola. Ground leaves were subjected to headspace-solid-phase microextraction gas chromatography-mass spectrometry analysis (HS-SPME/GC-MS) and two independent experimental repetitions were analysed to annotate/identify volatile organic compounds (VOCs). VOCs were selected according to their different levels in resistant and susceptible genotypes after pathogen inoculation and they were tested as single pure compounds in the functional assays. Two protocols were tested to asses the effect of pure VOCs against P. viticola i) in water suspension and ii) in air volume without direct contact with the leaf tissue. Figure S2. Comparison of the measured mass spectra of the volatile organic compounds (VOCs) in grapevine leaf samples with that of the corresponding pure VOC: 2-phenylethanol (A), ɣ-cadinene (B), δ-cadinene (C), β-caryophyllene (D), trans-2-pentenal (E), 2-ethylfuran (F), and β-cyclocitral (G). The mass spectrum similarity score and retention index values are reported for each VOC. Table Legends   Table S1. Volatile organic compounds (VOCs) detected by headspace-solid phase microextraction-gas chromatography-mass spectrometry from five grapevine genotypes in the first experiment. Table S2. Volatile organic compounds (VOCs) detected by headspace-solid phase microextraction-gas chromatography-mass spectrometry from five grapevine genotypes in the in the second experiment.
Column A. VOCs were grouped in six metabolite groups according to their profiles in: VOCs with a higher abundance in all resistant genotypes as compared with Pinot noir in both experiments in at least one time point (Group 1); VOCs with a higher abundance in two or more resistant genotypes as compared with Pinot noir in both experiments in at least one time point (Group 2), VOCs with a higher abundance in only one resistant genotype as compared with Pinot noir in both experiments in at least one time point (Group 3); VOCs with a lower abundance in at least one resistant genotype as compared with Pinot noir in both experiments in at least one time point (Group 4); VOCs with different abundance profiles in the two experiments (Group 5); VOCs only found in the first or in the second experiment (Group 6).
Column B. Names of VOCs found in grapevine leaves using a HS-SPME-GC-MS analysis. Green cells represent VOCs with increased abundance consistent in the two experiments. Orange cells represent VOCs with decreased abundance consistent in the two experiments. White cells represent VOCs with increased or decreased abundance in one of the two experiments.
Column C. CAS Registry Numbers. Source: http://webbook.nist.gov/chemistry/ Column D. Measured retention index (Measured RI). Column E. Retention index measured from an in-house library of authentic reference standards (Reference RI). Column F. Measured retention time (Measured RT).
Columns G, M, W, AG, AQ. Mean of absolute peak area (abundance) expressed as counts per seconds (cps) of five biological replicates (plants) at 0 dpi. Columns H, N, X, AH, AR. Standard error of absolute peak area (abundance) expressed as cps of five biological replicates at 0 dpi.
Columns I, O, Y, AI, AS. Mean of absolute peak area (abundance) expressed as cps of five biological replicates at 6 dpi. Columns J, P, Z, AJ, AT. Standard error of absolute peak area (abundance) expressed as cps of five biological replicates at 6 dpi.
Columns K, Q, AA, AK, AU. Fold change (FC) values between 0 and 6 dpi for each genotype. Values are reported for significant changes (p ≤ 0.05 of Kruskal-Wallis test and FC fold change > 1.5). Coloured cells represent consistent statistical differences in the two experiments (green and orange for VOC with increased or decreased peak area, respectively).