Identification and functional characterization of grapevine volatile organic compounds for the sustainable control of downy mildew

Introduction Grapevine (Vitis vinifera) is one of the most widely cultivated fruit crops and is susceptible to various pathogens, such as Plasmopara viticola that causes downy mildew (Gessler et al., 2011). Wild grapevine species are resistant to P. viticola and breeding programs have introduced resistance traits to susceptible cultivars. Plant defence responses are based on different mechanisms and volatile organic compounds (VOCs) play a crucial role in the communication between plants and other organisms. Although the emission of VOCs upon P. viticola inoculation was shown in resistant grapevine genotypes (Algarra Alarcon et al., 2015), the molecular structure and functional role of these molecules in the grapevine defence was not yet investigated. The aim of this study was to identify and functionally characterize VOCs produced by resistant and susceptible grapevine genotypes in response to P. viticola in order to further develop innovative methods for the sustainable control of downy mildew. Material and methods The susceptible V. vinifera cultivar Pinot noir and four resistant genotypes (Kober 5BB, SO4, BC4 and Solaris) were grown for three months under greenhouse conditions. Plants were inoculated with a suspension of P. viticola sporangia as previously described (Perazzolli et al., 2012). Downy mildew severity was assessed at seven days after inoculation according to the OIV-452 descriptor and scores from 1 (the most susceptible) to 9 (the totally resistant) were assigned (Bellin et al., 2009). Leaf samples were collected before (T0) and six days (T1) after P. viticola inoculation and five replicates (plants) were analysed for each genotype at each time point. The complete experiment was carried out twice. Each sample was frozen in liquid nitrogen and ground to a fine powder. Leaf powder was weighed into 20 mL headspace vials and analysed by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME/GC-MS; Weingart, et al. 2012). Eight of the identified VOCs were selected according to their emission profiles and pure compounds were tested against P. viticola by leaf disk assays. Downy mildew development was assessed on leaf disks at one, two and six days post inoculation (dpi) by aniline blue staining. Results and discussion VOC profiles measured by HS-SPME/GC-MS analysis were consistent between the two experiments. Terpenes, isoprenoids, aldehydes, alcohols, esters and heterocyclic compounds were found in both experiments in all five tested genotypes . In general, after P. viticola treatment (T1) the abundance of the detected VOCs was higher in resistant genotypes as compared with Pinot noir. Interestingly, the relative induction of VOC levels was found to be significantly higher in resistant genotypes compared to only small changes in Pinot noir. Compared to Pinot noir, treatment with P. viticola resulted in two sesquiterpenes being more abundant in all five resistant genotypes, while three other sesquiterpenes showed a higher abundance in three resistant genotypes (BC4, Kober 5BB and Solaris). At T1, Kober 5BB and Solaris showed also a higher abundance of one heterocyclic compound and one isoprenoid as compared with Pinot noir. Finally, the abundance of a C5 aldehyde was higher in Kober 5BB as compared with Pinot noir at T1. These eight pure VOCs were tested against P. viticola in liquid suspension and in air volume. The eight VOCs impaired the development of downy mildew symptoms at dosages that ranged from 0.1 to 10.0 g/L in liquid suspension. However, five of them also showed severe phytotoxic effects on leaf disks at the dosage of 10.0 g/L. Four pure VOCs (one isoprenoid, one alcohol, one C5 aldehyde and one heterocyclic compound) significantly reduced downy mildew symptoms at the dosage of 20.0 mg/L in air volume, when each VOC was applied to a filter paper disk and placed on the lid of the Petri dish. Microscope observations with aniline blue staining revealed marked morphological changes in VOC-treated leaf disks after P. viticola inoculation. The number of pathogen structures was reduced in leaf disks treated with one isoprenoid, one alcohol and one heterocyclic compound as compared to control disks at one, two and six dpi. Moreover, no P. viticola structures were visible on leaf disks treated with the C5 aldehyde. This aldehyde and one isoprenoid were also able to reduce the diameter of P. viticola sporangia. In conclusion, downy mildew increased the production of VOCs (terpenes, isoprenoid, alcohols, aldehydes and heterocyclic compounds) in resistant but not in the susceptible genotype and these molecules are associated to the activation of grapevine defence mechanisms. Moreover, VOCs of resistant genotypes have the porential to contribute to grapevine resistance and significantly reduced downy mildew symptoms on susceptible leaf disks, indicating that they can be further developed as sustainable control molecules.