Modeling malic acid dynamics to ensure quality, aroma and freshness of Pinot blanc wines in South Tyrol (Italy)

Pinot blanc is a leading grapevine variety in South Tyrol (Italy) for wine production. The high quality of its wines derives from a typical aroma of elegant apple notes and lively acidity. The typicity of the final wine depends on the origin of the vine, the soil, the oenological practices and time of harvest. The South Tyrolean mountainous areas meet the cold climatic requirements of Pinot blanc, which guarantee its sweet-acidic harmony obtained when organic acids are in balance with the other components of the wine. However, increasing temperatures in valley sites during the berry development period boost the activity of malic acid (MA) enzymes, which negatively affect the final sugar/acid ratio. Researchers are currently focused on understanding acid dynamics in wines, and there are no references for the best sugar/acid ratio for Pinot blanc. Moreover, the contribution of individual acids to the sensory profile of this wine has not yet been studied. In this study we address the effect of different climate conditions and site elevations on the sugar/acid ratio in developmental grapevine berries, and we evaluate the effect on wine bouquet. Even if different models and indices have been proposed for predicting sugar content, no predictive models exist for MA in white grapes. In a three-year study (2017, 2018 and 2019) that involved eight vineyards in four different location in South Tyrol at various elevations ranging from 223 to 730 m a.s.l., the relationships between bioclimatic indices, such as growing-degree day (GDD) and grapevine sugar ripeness (GSR) and grapevine berry content were investigated. The analysis reveals that GDD may potentially predict MA dynamics in Pinot blanc; hence, a GDD-based model was used to determine the GDD to reach target MA concentrations (3.5, 3.0, 2.5, 2.0 g/L). This simple model was improved with additional temperature-based parameters by feature selection, and the best three advanced models were selected and evaluated by 5-fold cross-validation. These models could be used to support location and harvest date choice to produce high-quality Pinot blanc wines

Detection, identification and functional characterisation of plant and microbial volatile organic compounds with inhibitory activity against two plant pathogens

Volatile organic compounds (VOCs) play crucial ecological roles in interactions among organisms. For example, plant VOCs can act as a powerful deterrent of herbivore insects and pathogens or they can act as resistance inducers to stimulate plant defences. Likewise, bioactive VOCs can be emitted by beneficial microorganisms and they may potentially act as key molecules in the microbe-microbe and plant-microbe communications. However, scarce information is available concerning the role of VOCs produced by grapevine (Vitis vinifera) plants and beneficial bacteria belonging to the Lysobacter genus in defence mechanisms against two important phytopathogenic oomycetes, namely Plasmopara viticola and Phytophthora infestans, which are the causal agents of grapevine downy mildew and potato late blight, respectively. The major objectives of this PhD thesis were the detection, identification and the functional characterization of VOCs from Vitis spp. and Lysobacter spp., in order to better understand their role in plant-microbe and microbe-microbe communications and to identify new active molecules from natural origin to control phytopathogens. In particular, VOCs from resistant and susceptible grapevine genotypes were identified following P. viticola inoculation and their effect as toxic molecules against downy mildew was explored (publications 1 and 2). Likewise, VOCs produced by Lysobacter spp. were identified and characterised, in order to identify microbial VOCs able to inhibit P. infestans growth (publication 3). In order to reach these goals, a headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME/GC-MS) and proton transfer reaction time of flight-mass spectrometry (PTR-ToF-MS) have been used. Two downy mildew resistant hybrids (SO4 and Kober 5BB) and the susceptible V. vinifera cultivar Pinot noir were analysed in vitro using PTR-ToF-MS. We found that P. viticola inoculation resulted in a significant increase monoterpene and sesquiterpene emission by resistant genotypes (SO4 and Kober 5BB) and not by the susceptible cultivar (Vitis vinifera Pinot noir; publication 1). Grapevine VOCs were further identified by HS-SPME/GC-MS using greenhouse-grown plants. The four resistant genotypes tested (BC4, Kober 5BB, SO4 and Solaris) showed significantly increased production of VOCs after P. viticola inoculation under greenhouse conditions. Conversely, no significant emission of volatile terpenes was detected from Pinot noir plants after P. viticola inoculation, suggesting that VOCs of resistant genotypes could play an important role in grapevine resistance against downy mildew. The chemical structures of P. viticola-induced VOCs were identified by retention index and the GC-MS spectrum evaluation and VOCs potentially involved in the grapevine resistance were selected according to their emission profiles. Pure compounds were tested against P. viticola by leaf disk assays and different experiments were set up, in order to elucidate the efficacy of pure VOCs both in a liquid suspension of P. viticola sporangia and after application via the gas phase. These experiments revealed six (2-phenylethanol, β-caryophyllene, β-selinene, trans-2-pentenal, 2-ethylfuran, and β-cyclocitral) and four VOCs (2-phenylethanol, trans-2-pentenal, 2-ethylfuran, and β-cyclocitral) which impaired downy mildew symptoms after direct application of liquid suspension and after treatment with VOC enriched air (without direct contact with the leaf tissue), respectively. With these results we demonstrated that VOCs produced by resistant grapevine genotypes are related to post-infection mechanisms and may contribute to grapevine resistance against P. viticola by inhibition of pathogen development (publication 2). In the second part of the PhD project, the volatilome of Lysobacter spp. was characterised for its inhibitory activity against the soil pathogen P. infestans (publication 3). The effect of VOCs emitted by Lysobacter strains was demonstrated in vitro by dual-culture assay and profiles were characterised by HS-SPME/GC-MS and PTR-ToF-MS analysis. Interestingly, the biocontrol activity and VOC profiles of Lysobacter spp. depended on the bacterial growth media. In particular, VOCs with inhibitory properties (pyrazines, pyrrole and decanal) were mainly emitted by Lysobacter type strains grown on a protein-rich medium, demonstrating the importance of the culture medium composition to optimise the biocontrol efficacy of Lysobacter spp. against plant pathogens. In summary, the presented thesis showed that both analytical chemistry techniques used (PTR-ToF-MS and HS-SPME/GC-MS) can be employed synergistically to detect and identify VOCs from different biological matrixes such as leaf tissue or bacterial cultures. The presented thesis also suggested that VOCs contribute to grapevine resistance and they can effectively be used to control economically important plant pathogens such as P. viticola. Furthermore, results generated in this work indicate that nutrient availability may affect the aggressiveness of Lysobacter spp. in the soil to maximise biocontrol efficacy against P. infestans. However, further metabolomic and transcriptomic analyses are required to investigate the VOC-mediated plant defence mechanisms and to characterize metabolic changes and VOC emissions of Lysobacter spp. grown in soil condition

Cellular regulations of grapevine resistance induced by Trichoderma spp.

Trichoderma-induced resistance is mediated by transcriptional, translational and post-translational regulations. Trichoderma-induced resistance is affected by the plant genotype, environment and fungal strai

Techno-economic performance of HCl and SO2 removal in waste-to-energy plants by furnace direct sorbent injection

With the impending release of Best Available Techniques (BAT) conclusions on waste incineration, existing European waste-to-energy (WtE) plants will be required to achieve a higher efficiency in the removal of several target pollutants, such as acid gases (above all, HCl and SO2). The direct injection of a sorbent in the furnace as a primary deacidification stage may be a cost-effective option to achieve the required performances. The present study investigated the furnace injection of a specific dolomitic sorbent, with the aim of identifying the techno-economic optimum for the sorbent feed rate considering different scenarios of flue gas composition. A full-scale test run campaign was carried out on two WtE plants and a phenomenological model linking HCl and SO2 conversion to reactant feed rate was developed. The model allowed assessing the cost-effectiveness of dolomitic sorbent furnace injection for WtE plants equipped with either a single or a two-stage acid gas treatment system. The addition of dolomitic sorbent resulted particularly suitable for WtE plants equipped with a single stage treatment coping with a high SO2 concentration in the flue gas, where the injection of dolomitic sorbent can increase the redundancy of flue gas treatment with a reduction of the total operating costs

Identification of volatile organic compounds emitted by different grapevine genotypes in response to downy mildew infection

Vitis vinifera is susceptible to several pathogens including Plasmopara viticola, the causal agent of downy mildew. American grapevine species are resistant or tolerant to P. viticola and breeding programs have introduced resistance traits to susceptible cultivars. Although grapevine resistance to P. viticola has been widely characterized in resistant genotypes, the possible contribute of volatile organic compounds (VOCs) was not yet investigated. The aim of this work was the characterization of VOCs produced by resistant and susceptible genotypes in response to P. viticola inoculation, in order to identify VOCs associated to grapevine resistance. The susceptible V. vinifera cultivar Pinot noir, and the resistant genotypes Kober 5BB, SO4, BC4 and Solaris were grown under greenhouse conditions and they were subsequently inoculated with P. viticola. Leaves were harvested immediately before (0 dpi) and six days (6 dpi) after inoculation, and the lower disease severity in resistant genotypes as compared with Pinot noir was confirmed. A solid-phase microextraction-gas chromatography-mass spectrometry approach (SPME/GC-MS) was used to analyze VOCs emitted by the five genotypes studied. GC-MS chromatograms showed specific VOC emission profiles of the four resistant genotypes as compared with Pinot noir at 6 dpi. VOCs specifically emitted by resistant genotypes were then selected, and pure compounds were tested against P. viticola by leaf discs assays. Particularly, three sesquiterpenes, two aldehydes and one heterocyclic compound significantly reduced downy mildew severity on Pinot noir, demonstrating that VOCs could play an important role in the resistance against downy mildew by direct toxicity against P. viticola

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/identifed 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 efcacy 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

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

Identification and activity testing of volatile organic compounds (VOCs) found in different grapevine genotypes in response to downy mildew infection

Vitis vinifera is susceptible to several pathogens including Plasmopara viticola, the causal agent of downy mildew (1). American grapevine species are resistant or tolerant to P. viticola and breeding programs have introduced resistance traits to susceptible cultivars. Although grapevine resistance mechanisms against downy mildew have been widely characterized in resistant genotypes (1), the possible contribution of volatile organic compounds (VOCs) was not yet investigated. The aim of this work was the characterization of VOCs produced by resistant and susceptible grapevine genotypes in response to P. viticola inoculation, in order to identify VOCs associated to grapevine resistance against downy mildew. The susceptible V. vinifera cultivar Pinot noir, and the resistant genotypes Kober 5BB, SO4, BC4 and Solaris were grown under greenhouse conditions and they were subsequently inoculated with P. viticola. Leaves were harvested immediately before (T0) and six days (T1) after inoculation, and the lower disease severity in resistant genotypes as compared with Pinot noir was confirmed. A headspace-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME/GC-MS) approach was used to analyze VOCs from the five studied genotypes. GC-MS chromatograms showed specific VOC emission profiles of the four resistant genotypes as compared with Pinot noir at T1. VOCs specifically found in resistant genotypes were selected, and pure compounds were tested against P. viticola sporangia by leaf disk assays. Particularly, four sesquiterpenes, one C5 aldehyde, one terpenoid, one alcohol and one heterocyclic compound were tested in liquid suspension with P. viticola sporangia and significantly reduced downy mildew symptoms on Pinot noir leaf disks. Moreover, four of these VOCs were tested in air volume and displayed significant reduction of downy mildew symptoms demonstrating that VOCs could play an important role in the resistance against downy mildew by direct toxicity against P. viticola sporangia

Identification of volatile organic compounds in different grapevine genotypes after inoculation with Plasmopara viticola

The grapevine Vitis vinifera cv Pinot noir is susceptible to several pathogens including Plasmopara viticola that is the causal agent of downy mildew [1]. Hybrids of V. berlandieri and V. riparia (SO4 and Kober 5BB) and hybrids of Muscadinia rotundifolia and V. vinifera (BC4) and others such as Solaris are resistant or tolerant to downy mildew. It has been demonstrated recently [2] that resistant in vitro hybrids SO4 and Kober 5BB emit volatile organic compounds (VOCs) in response to P. viticola infection. In particular, the most interesting class of VOCs constitutes terpenoids (mono- and sesquiterpenes) emitted by the resistant cultivars, whereas for Pinot noir no terpenes have been detected under the tested conditions. In the present study we have used gas chromatography coupled with mass spectrometry (GC-MS) to study in more detail the chemical identity of the compounds produced by selected plants of the five genotypes Pinot noir, Kober 5BB, SO4, BC4 and Solaris. All the genotypes were cultured in the greenhouse and leaves were harvested immediately (0 dpi) and six (6 dpi) days after the inoculation with P. viticola. All samples were immediately frozen and homogenized under cooled conditions. VOCs were extracted by using solid phase microextraction (SPME) and analyzed by GC-MS. Mass spectral deconvolution and annotation / identification of volatile compounds was based on comparison of mass spectra and retention indices with reference values and performed by Metabolite Detector software [3]. Preliminary results showed increased levels compared to day zero of sesquiterpenes in resistant cultivars six days after inoculation, demonstrating that terpenes could play an important role in plant resistance against downy mildew in resistant genotypes. [1] C. Gessler, I. Pertot, M. Perazzolli. (2011). Plasmopara viticola: a review of knowledge on downy mildew of grapevine and effective disease management. Phytopathologia Mediterranea, 50, p. 3-44 [2] A. Algarra Alarcon, V. Lazazzara, L. Cappellin, P. L. Bianchedi, R. Schuhmacher, G. Wohlfahrt, I. Pertot, F. Biasioli and M. Perazzolli. (2015). Emission of volatile sesquiterpenes and monoterpenes in grapevine genotypes following Plasmopara viticola inoculation in vitro. Journal of Mass Spectrometry, 50, p. 1013–1022 [3] K. Hiller, J. Hangebrauk, C. Jäger, J. Spura, K. Schreiber, D. Schomburg. (2009) MetaboliteDetector: comprehensive analysis tool for targeted and nontargeted GC/MS based metabolome analysis. Analytical Chemistry, 81(9), p. 3429-343

Identification by HS-SPME/GC-MS and activity testing of Trichoderma spp. volatile organic compounds against grapevine downy mildew

Downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most destructive diseases of the grapevine [1]. Fungi of the genus Trichoderma are economically important biocontrol agents since they play a crucial role in plantgrowth promotion, mycoparasitism of plant pathogens and priming of plant defence. Trichoderma spp. also release a high diversity of volatile organic compounds (VOCs), which play a decisive role against plant pathogens [2]. However, the possible contribution of Trichoderma VOCs in antagonistic processes against grapevine downy mildew has not yet been investigated. In this work, VOC emission profiles of three Trichoderma strains belonging to T. asperellum, T. atroviride and T. harzianum were analysed using headspace-solidphase microextraction gas chromatography-mass spectrometry (HS-SPME/GC-MS). Total ion current chromatograms were processed by an open source software [3], and statistical analysis on the obtained raw data was carried out using an in-house R-script. VOCs emitted by the Trichoderma strains at specific time-points were selected, and pure compounds were tested against downy mildew by leaf disks assays on susceptible grapevines. The development of downy mildew symptoms was reduced on leaf disks exposed to air treated with two sesquiterpenes, one hydrocarbon, or one heterocyclic compound, indicating the efficacy of these VOCs against downy mildew in plant tissues. Our data suggest that VOCs emitted by the Trichoderma strains under study can effectively be detected and identified by HS-SPME/GC-MS, and can inhibit the development of downy mildew symptoms on susceptible grapevine