Chemical and Electrophysiological Characterisation of Headspace Volatiles from Yeasts Attractive to Drosophila suzukii.

Chemical control of Drosophila suzukii (Diptera: Drosophilidae) based on the use of insecticides is particularly challenging as the insect attacks ripening fruits shortly before harvest. An alternative strategy may rely on the use of yeasts as phagostimulants and baits, applied on canopy as attract-and-kill formulations. The aim of this research was to identify the most attractive among six yeast species for D. suzukii: Saccharomyces cerevisiae, Hanseniaspora uvarum, Clavispora santaluciae, Saccharomycopsis vini, Issatchenkia terricola, and Metschnikowia pulcherrima. The volatile profile of C. santaluciae was described for the first time. Behavioural experiments identified H. uvarum and S. vini as the most attractive yeasts. The characterization of yeast headspace volatiles using direct headspace (DHS) and solid-phase microextraction (SPME) revealed several strain-specific compounds. With DHS injection, 19 volatiles were characterised, while SPME revealed 71 compounds constituting the yeast headspace. Both analyses revealed terpenoids including ß-ocimene, citronellol, (Z)-geraniol (nerol), and geranial as distinct constituents of S. vini. H. uvarum and S. vini were further investigated using closed-loop stripping analysis (CSLA) and electroantennography. Out of 14 compounds quantified by CSLA, ethyl acetate, isoamyl acetate, ß-myrcene, benzaldehyde and linalool were detected by D. suzukii antennae and might generate the strong attractiveness of S. vini and H. uvarum. Our results highlight a strong attraction of D. suzukii to various yeasts associated with both the flies and their habitat and demonstrate how different sampling methods can impact the results of volatile compound characterization. It remains to be demonstrated whether the distinct attraction is based on special adaptations to certain yeasts and to what extent the metabolites causing attraction are interchangeable.

Establishing authenticity of hay milk: Detection of silage feeding through cyclopropane fatty acids analysis using 1H NMR spectroscopy.

Cyclopropane fatty acids (CPFAs) serve as indicators of silage feeding, verifying the authenticity of hay milk where silage feeding is forbidden. In this study, the authenticity of hay milk was determined by detecting CPFAs using proton nuclear magnetic resonance (1H NMR) spectroscopy. 245 milk samples were collected in South Tyrol (Italy), categorized as follows: 98 from grass silage-fed cows, 98 from maize silage-fed cows, and 49 authentic hay milk. The limit of detection of CPFAs was 12 µM, corresponding to 70 mg/kg of freeze-dried milk. The CPFAs were absent in all of the hay milk samples, verifying their authenticity. In contrast, 97 % of maize silage and 77 % of grass silage samples exhibited distinct CPFAs signals. These findings were further corroborated by gas chromatography-mass detector (GC-MS) analysis. The study highlights 1H NMR as a robust, and rapid technique for hay milk authentication, supporting alpine dairy production and increasing consumer trust in food authenticity.

Behavioral manipulation of Drosophila suzukii for pest control: high attraction to yeast enhances insecticide efficacy when applied on leaves.

BACKGROUND: The invasive pest, Drosophila suzukii attacks fresh soft-skinned fruit. Broad-spectrum insecticides are implemented for control but there is a need to reduce environmental risks and insecticide residues on fruits. Hanseniaspora uvarum is a yeast frequently found on ripe fruits and associated with D. suzukii. We aim to exploit the ecological association and attraction of D. suzukii to H. uvarum by developing an attract-and-kill strategy, with spray-application on canopy but not fruit. We therefore investigated D. suzukii attraction, egg-laying and mortality when exposed to insecticidal yeast-based formulations. RESULTS: Hanseniaspora uvarum strongly attracted D. suzukii when applied on leaves of grapevine, Vitis vinifera. Notably, this attractiveness was competitive to ripe grape berries that were susceptible to D. suzukii infestation. Moreover, adding H. uvarum enhanced the efficacy of insecticidal formulations against D. suzukii. Flies exposed to leaves treated with yeast-insecticide formulations showed higher mortality and laid a lower number of eggs compared to flies exposed to insecticide alone. In a wind tunnel, all treatments containing H. uvarum alone or in combination with insecticides, caused similar upwind flight and landing at the odor source, which provides evidence that the addition of insecticide did not reduce D. suzukii attraction to yeast. CONCLUSION: Hanseniaspora uvarum can be used to manipulate the behavior of D. suzukii by attracting flies to insecticide formulations. Yeast attraction is competitive to grape berries and improves insecticide effectiveness, suggesting that sprays covering canopy only, could reduce residues on fruit without compromising management efficacy.

Field and greenhouse application of an attract-and-kill formulation based on the yeast Hanseniaspora uvarum and the insecticide spinosad to control Drosophila suzukii in grapes.

BACKGROUND: The invasive insect Drosophila suzukii (Matsumura) is an important pest of several red grape varieties. The yeast Hanseniaspora uvarum (Niehaus), which is associated with D. suzukii, strongly attracts flies and stimulates them to feed on yeast-laden food. In the present study, a formulation based on H. uvarum culture with spinosad insecticide was applied to the foliage of vineyards and control of D. suzukii was compared to applying spinosad to the whole plant. After successful H. uvarum and insecticide application in the vineyard, we tested additional H. uvarum-based formulations with spinosad in a greenhouse to determine their capacity to control D. suzukii. RESULTS: Application of the H. uvarum-spinosad formulation at 36.4 g of spinosad per hectare reduced the D. suzukii field infestation at the same rate as applying 120 g of spinosad per hectare and prevented spinosad residues on grapes. Leaves treated with H. uvarum and spinosad in the field and transferred to a laboratory assay caused high mortality to flies and reduced the number of eggs laid on fruits. Formulations with spinosad applied in the greenhouse showed that both H. uvarum culture and the yeast cell-free supernatant of a centrifuged culture increased fly mortality and reduced the number of eggs laid compared to the unsprayed control. CONCLUSION: In comparison to typical spinosad spray applications, the use of H. uvarum in combination with spinosad as an attract-and-kill formulation against D. suzukii reduces pesticide residues on the fruits by targeting the treatment to the canopy and decreasing the amount of insecticide per hectare without compromising control efficacy.

Authenticity of Hay Milk vs. Milk from Maize or Grass Silage by Lipid Analysis.

Hay milk is a traditional dairy product recently launched on the market. It is protected as "traditional specialty guaranteed" (TSG) and subjected to strict regulations. One of the most important restrictions is that the cow's feed ration must be free from silage. There is the need for analytical methods that can discriminate milk obtained from a feeding regime including silage. This study proposes two analytical approaches to assess the authenticity of hay milk. Hay milk and milk from cows fed either with maize or grass silage were analyzed by targeted GC-MS for cyclopropane fatty acid (dihydrosterculic acid, DHSA) detection, since this fatty acid is strictly related to the bacterial strains found in silage, and by HPLC-HRMS. The presence of DHSA was correlated to the presence of maize silage in the feed, whereas it was ambiguous with grass silage. HPLC-HRMS analysis resulted in the identification of 14 triacylglycerol biomarkers in milk. With the use of these biomarkers and multivariate statistical analysis, we were able to predict the use of maize and grass silage in the cow's diet with 100% recognition. Our findings suggest that the use of analytical approaches based on HRMS is a viable authentication method for hay milk.

Chemosensory Profile of South Tyrolean Pinot Blanc Wines: A Multivariate Regression Approach.

A multivariate regression approach based on sensory data and chemical compositions has been applied to study the correlation between the sensory and chemical properties of Pinot Blanc wines from South Tyrol. The sensory properties were identified by descriptive analysis and the chemical profile was obtained by HS-SPME-GC/MS and HPLC. The profiles of the most influencing (positively or negatively) chemical components have been presented for each sensory descriptor. Partial Least Square Regression (PLS) and Principal Component Regression (PCR) models have been tested and applied. Visual (clarity, yellow colour), gustatory (sweetness, sourness, saltiness, bitterness, astringency, and warmness) and olfactory (overall intensity, floral, apple, pear, tropical fruit, dried fruit, fresh vegetative, spicy, cleanness, and off-odours) descriptors have been correlated with the volatile and phenolic profiles, respectively. Each olfactory descriptor was correlated via a PCR model to the volatile compounds, whereas a comprehensive PLS2 regression model was built for the correlation between visual/gustatory descriptors and the phenolic fingerprint. "Apple" was the olfactory descriptor best modelled by PCR, with an adjusted R2 of 0.72, with only 20% of the validation samples falling out of the confidence interval (α = 95%). A PLS2 with 6 factors was chosen as the best model for gustatory and visual descriptors related to the phenolic compounds. Finally, the overall quality judgment could be explained by a combination of the calibrated sensory descriptors through a PLS model. This allowed the identification of sensory descriptors such as "olfactory intensity", "warmness", "apple", "saltiness", "astringency", "cleanness", "clarity" and "pear", which relevantly contributed to the overall quality of Pinot Blanc wines from South Tyrol, obtained with two different winemaking processes and aged in bottle for 18 months.

Detection of apple proliferation disease in Malus × domestica by near infrared reflectance analysis of leaves.

In this study near infrared spectroscopical analysis of dried and ground leaves was performed and combined with a multivariate data analysis to distinguish 'Candidatus Phytoplasma mali' infected from non-infected apple trees (Malus × domestica). The bacterium is the causative agent of Apple Proliferation, one of the most threatening diseases in commercial apple growing regions. In a two-year study, leaves were sampled from three apple orchards, at different sampling events throughout the vegetation period. The spectral data were analyzed with a principal component analysis and classification models were developed. The model performance for the differentiation of Apple Proliferation diseased from non-infected trees increased throughout the vegetation period and gained best results in autumn. Even with asymptomatic leaves from infected trees a correct classification was possible indicating that the spectral-based method provides reliable results even if samples without visible symptoms are analyzed. The wavelength regions that contributed to the differentiation of infected and non-infected trees could be mainly assigned to a reduction of carbohydrates and N-containing organic compounds. Wet chemical analyses confirmed that N-containing compounds are reduced in leaves from infected trees. The results of our study provide a valuable indication that spectral analysis is a promising technique for Apple Proliferation detection in future smart farming approaches.

Persistence of a Yeast-Based (Hanseniaspora uvarum) Attract-and-Kill Formulation against Drosophila suzukii on Grape Leaves.

The production of phagostimulant and attractive volatile organic compounds (VOCs) by yeasts can be exploited to improve the efficacy of attract-and-kill formulations against the spotted wing drosophila (SWD). This study evaluated the persistence over one week of a yeast-based formulation under greenhouse conditions. Potted grape plants were treated with: (i) potato dextrose broth (PDB), (ii) PDB containing spinosad (PDB + S), and (iii) H. uvarum fermentation broth grown on PDB containing spinosad (H. u. + S). Laboratory trials were performed to determine the survival and the oviposition rate of SWD after exposure to treated leaves. Ion-exchange chromatography was performed to measure carbohydrates, sugar alcohols, and organic acids on leaf surfaces, while amino acids were assessed through liquid chromatography-mass-spectrometry. Additionally, the VOCs released by plants treated with H.uvarum were collected via closed-loop-stripping analysis and compared to those emitted by untreated leaves. A higher mortality was observed for adult SWDs in contact with H. uvarum containing spinosad compared to PDB containing spinosad. Generally, a decrease in the amounts of non-volatile compounds was observed over time, though numerous nutrients were still present one week after treatment. The application of the yeast-based formulation induced the emission of VOCs by the treated leaves. The concentration of 2-phenylethanol, one of the main VOCs emitted by yeasts, decreased over time. These findings describe the presence of potential phagostimulants and compounds attractive to SWD in a yeast-based attract-and-kill formulation and demonstrate the efficacy of the formulation over one week.

Comparative Lipidomics of Different Yeast Species Associated to Drosophila suzukii.

Yeasts constitute a dietary source for the spotted wing drosophila (SWD) and produce compounds that attract these flies. The study of the chemical composition of the yeast communities associated with SWD should therefore help to understand the relationship between the biology of the insect and the yeast's metabolism. In the present study, the lipidome of five yeast species isolated from grapes infested by SWD (three Hanseniaspora uvarum strains, Candida sp., Issatchenkia terricola, Metschnikowia pulcherrima and Saccharomycopsis vini) and a laboratory strain of Saccharomyces cerevisiae was explored using an untargeted approach. Additionally, the lipid profile of two species, S. cerevisiae and H. uvarum, which were reported to elicit different responses on SWD flies based on feeding and behavioral trials, was compared with a chemical enrichment approach. Overall, 171 lipids were annotated. The yeast species could be distinguished from each other based on their lipid profile, except for the three strains of H. uvarum, which were very similar to each other. The chemical enrichment analysis emphasized diversities between S. cerevisiae and H. uvarum, that could not be detected based on their global lipid profile. The information concerning differences between species in their lipidome may be of interest to future entomological studies concerning the yeast-insect interaction and could help to explain the responses of SWD to diverse yeast species.

Pinot Blanc: Impact of the Winemaking Variables on the Evolution of the Phenolic, Volatile and Sensory Profiles.

The impact of two different winemaking practices on the chemical and sensory complexity of Pinot Blanc wines from South Tyrol (Italy), from grape pressing to the bottled wine stored for nine months, was studied. New chemical markers of Pinot blanc were identified: astilbin and trans-caftaric acid differentiated the wines according to the vinification; S-glutathionylcaftaric acid correlated with the temporal trends. Fluorescence analysis displayed strong time-evolution and differentiation of the two wines for gallocatechin and epigallocatechin, respectively. After nine months of storage in bottle, the control wine showed higher amounts of most ethyl esters, acetate esters and octanoic acid, whereas higher alcohols characterized instead the wine obtained with prefermentative cold maceration. The sensory panel found notes of apple and tropical fruit in the control wine and attributed a higher overall quality judgement to it, whereas the cold-macerated wine was described by olfactory intensity, spicy and pear attributes.

3-Chloro-5-trifluoromethylpyridine-2-carboxylic acid, a Metabolite of the Fungicide Fluopyram, Causes Growth Disorder in Vitis vinifera.

The aim of this study was to investigate the effect of 3-chloro-5-trifluoromethylpyridine-2-carboxylic acid (PCA), a metabolite of the fungicide fluopyram, on grapevine. During spring and summer 2015, grapevine growth disorders were observed in several countries in Europe. An unprecedented herbicide-like damage was diagnosed on leaves and flowers, causing significant loss of harvest. This study proposes PCA as the causing agent of the observed growth disorders. PCA was shown to cause leaf epinasty, impaired berry development that leads to crop loss, and root growth anomalies in Vitis vinifera similar to auxin herbicides in a dose-dependent manner. Using both field trials and greenhouse experiments, the present study provides first evidence for a link between the application of fluopyram in vineyards 2014, the formation of PCA, and the emergence of growth anomalies in 2015. Our data could be useful to optimize dosage, application time point, and other conditions for an application of fluopyram without phytotoxic effects.

A Near Infrared Spectroscopy (NIRS) and Chemometric Approach to Improve Apple Fruit Quality Management: A Case Study on the Cultivars "Cripps Pink" and "Braeburn".

The potential of near infrared spectroscopy (NIRS) in the wavelength range of 1000-2500 nm for predicting quality parameters such as total soluble solids (TSS), acidity (TA), firmness, and individual sugars (glucose, fructose, sucrose, and xylose) for two cultivars of apples ("Braeburn" and "Cripps Pink") was studied during the pre- and post-storage periods. Simultaneously, a qualitative investigation on the capability of NIRS to discriminate varieties, harvest dates, storage periods and fruit inhomogeneity was carried out. In order to generate a sample set with high variability within the most relevant apple quality traits, three different harvest time points in combination with five different storage periods were chosen, and the evolution of important quality parameters was followed both with NIRS and wet chemical methods. By applying a principal component analysis (PCA) a differentiation between the two cultivars, freshly harvested vs. long-term stored apples and, notably, between the sun-exposed vs. shaded side of apples could be found. For the determination of quality parameters effective prediction models for titratable acid (TA) and individual sugars such as fructose, glucose and sucrose by using partial least square (PLS) regression have been developed. Our results complement earlier reports, highlighting the versatility of NIRS as a fast, non-invasive method for quantitative and qualitative studies on apples.

Diphenylamine residues in apples caused by contamination in fruit storage facilities.

The potential of fruit storage facilities that are contaminated with the widely used chemical antioxidant diphenylamine to cross-contaminate untreated apples (Malus × domestica Borkh.) was studied. A new sample preparation method identified the storage room paint, contaminated from past treatments, as the major source of cross-contamination in the analyzed facilities. Diphenylamine amounts of up to 917 g were found in a single storage room and were shown to correlate with the extent of cross-contamination on stored apples. Our data support a diffusion-based mechanism where the wall paint releases the antioxidant to the storage room atmosphere even years after the last treatment. Given the extent of cross-contamination found in our model experiments and under commercial storage conditions, we deduce a significant risk of exceeding the potentially upcoming maximum residue level of 0.01 mg kg(-1) on stored fruit in contaminated rooms even years after the last diphenylamine treatment.