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.

Improved Detection and Quantification of Cyclopropane Fatty Acids via Homonuclear Decoupling Double Irradiation NMR Methods.

Over the years, NMR spectroscopy has become a powerful analytical tool for the identification and quantification of a variety of natural compounds in a broad range of food matrices. Furthermore, NMR can be useful for characterizing food matrices in terms of quality and authenticity, also allowing for the identification of counterfeits. Although NMR requires minimal sample preparation, this technique suffers from low intrinsic sensitivity relative to complementary techniques; thus, the detection of adulterants or markers for authenticity at low concentrations remains challenging. Here, we present a strategy to overcome this limitation by the introduction of a simple band-selective homonuclear decoupling sequence that consists of double irradiation on 1H during NMR signal acquisition. The utility of the proposed method is tested on dihydrosterculic acid (DHSA), one of the cyclopropane fatty acids (CPFAs) shown to be a powerful molecular marker for authentication of milk products. A quantitative description of how the proposed NMR scheme allows sensitivity enhancement yet accurate quantification of DHSA is provided.

Applications of Solution NMR Spectroscopy in Quality Assessment and Authentication of Bovine Milk.

Nuclear magnetic resonance (NMR) spectroscopy is emerging as a promising technique for the analysis of bovine milk, primarily due to its non-destructive nature, minimal sample preparation requirements, and comprehensive approach to untargeted milk analysis. These inherent strengths of NMR make it a formidable complementary tool to mass spectrometry-based techniques in milk metabolomic studies. This review aims to provide a comprehensive overview of the applications of NMR techniques in the quality assessment and authentication of bovine milk. It will focus on the experimental setup and data processing techniques that contribute to achieving accurate and highly reproducible results. The review will also highlight key studies that have utilized commonly used NMR methodologies in milk analysis, covering a wide range of application fields. These applications include determining milk animal species and feeding regimes, as well as assessing milk nutritional quality and authenticity. By providing an overview of the diverse applications of NMR in milk analysis, this review aims to demonstrate the versatility and significance of NMR spectroscopy as an invaluable tool for milk and dairy metabolomics research and hence, for assessing the quality and authenticity of bovine milk.

LC-Q-TOF-MS/MS data of phyllobilins in apple peels cv. 'Gala' during ripening under shelf-life conditions.

During senescence and ripening, higher plants degrade the green pigment chlorophyll to linear tetrapyrrols, referred to as phyllobilins (PBs). This dataset provides chromatograms and mass spectral data of PBs acquired from methanolic extracts of cv. Gala apple peels at five different shelf life (SL) stages. Data were obtained using a (ultra-high) pressure liquid chromatograph (UHPLC) coupled to high resolution quadrupole-time of flight-mass spectrometer (HRMS-Q-TOF). A data-dependent inclusion list (IL) ex professo containing all known masses of PBs was applied to address PBs, and fragmentation patterns were studied to confirm their identity operating a MS2 method. Mass accuracy was set to 5 ppm for parent ion peaks, this parameter was adopted as inclusion criterium. The detection of PBs' appearance during ripening can be helpful for assessing the quality and maturity of the apples.

Secondary and primary metabolites reveal putative resistance-associated biomarkers against Erysiphe necator in resistant grapevine genotypes.

Numerous fungicide applications are required to control Erysiphe necator, the causative agent of powdery mildew. This increased demand for cultivars with strong and long-lasting field resistance to diseases and pests. In comparison to the susceptible cultivar 'Teroldego', the current study provides information on some promising disease-resistant varieties (mono-locus) carrying one E. necator-resistant locus: BC4 and 'Kishmish vatkana', as well as resistant genotypes carrying several E. necator resistant loci (pyramided): 'Bianca', F26P92, F13P71, and NY42. A clear picture of the metabolites' alterations in response to the pathogen is shown by profiling the main and secondary metabolism: primary compounds and lipids; volatile organic compounds and phenolic compounds at 0, 12, and 48 hours after pathogen inoculation. We identified several compounds whose metabolic modulation indicated that resistant plants initiate defense upon pathogen inoculation, which, while similar to the susceptible genotype in some cases, did not imply that the plants were not resistant, but rather that their resistance was modulated at different percentages of metabolite accumulation and with different effect sizes. As a result, we discovered ten up-accumulated metabolites that distinguished resistant from susceptible varieties in response to powdery mildew inoculation, three of which have already been proposed as resistance biomarkers due to their role in activating the plant defense response.

Semi-Targeted Profiling of the Lipidome Changes Induced by Erysiphe Necator in Disease-Resistant and Vitis vinifera L. Varieties.

The ascomycete Erysiphe necator is a serious pathogen in viticulture. Despite the fact that some grapevine genotypes exhibit mono-locus or pyramided resistance to this fungus, the lipidomics basis of these genotypes' defense mechanisms remains unknown. Lipid molecules have critical functions in plant defenses, acting as structural barriers in the cell wall that limit pathogen access or as signaling molecules after stress responses that may regulate innate plant immunity. To unravel and better understand their involvement in plant defense, we used a novel approach of ultra-high performance liquid chromatography (UHPLC)-MS/MS to study how E. necator infection changes the lipid profile of genotypes with different sources of resistance, including BC4 (Run1), "Kishmish vatkhana" (Ren1), F26P92 (Ren3; Ren9), and "Teroldego" (a susceptible genotype), at 0, 24, and 48 hpi. The lipidome alterations were most visible at 24 hpi for BC4 and F26P92, and at 48 hpi for "Kishmish vatkhana". Among the most abundant lipids in grapevine leaves were the extra-plastidial lipids: glycerophosphocholine (PCs), glycerophosphoethanolamine (PEs) and the signaling lipids: glycerophosphates (Pas) and glycerophosphoinositols (PIs), followed by the plastid lipids: glycerophosphoglycerols (PGs), monogalactosyldiacylglycerols (MGDGs), and digalactosyldiacylglycerols (DGDGs) and, in lower amounts lyso-glycerophosphocholines (LPCs), lyso-glycerophosphoglycerols (LPGs), lyso-glycerophosphoinositols (LPIs), and lyso-glycerophosphoethanolamine (LPEs). Furthermore, the three resistant genotypes had the most prevalent down-accumulated lipid classes, while the susceptible genotype had the most prevalent up-accumulated lipid classes.

Chlorophyll breakdown during fruit ripening: Qualitative analysis of phyllobilins in the peel of apples (Malus domestica Borkh.) cv. 'Gala' during different shelf life stages.

Chlorophyll (Chl) degradation is an important process of fruit ripening, mediated by the pheophorbide a oxygenase/phyllobilin (PaO/PB) pathway. Chl is catabolized to linear tetrapyrrolic phyllobilins that resemble the bile pigments. Determining the optimal harvest date is of pivotal importance for the fruit industry. Among the physiological changes at the onset of ripening and during shelf life, the breakdown of Chl appears as a promising indicator for fruit maturity. In addition, PBs are a frequently overlooked component of the human diet with antioxidant and antiproliferative activities. However, their relevance for a healthy diet and their potential as ripening indicator in fruit cannot be evaluated yet, as details on emergence and composition of PBs in fruit are yet elusive. Using apple (Malus × domestica Borkh.) cv. 'Gala' as a model fruit, the Chl content and the emergence and identity of PBs were analyzed during a controlled shelf life period using UV/Vis spectroscopy and ultra-high pressure liquid chromatography coupled to high resolution quadrupole-time of flight-mass spectrometry (UHPLC-Q-TOF-MS). An in-house database with chromatographic and MS data from 51 PBs, revealed ten chlorophyll catabolites, including five NCCs, one YCC, and four DNCCs (including a previously unknown one). PBs were identified with increasing abundance and diversity from the onset of Chl degradation, suggesting a potential role as ripening indicators.

Metabolomic Characterization of Pigmented and Non-Pigmented Potato Cultivars Using a Joint and Individual Variation Explained (JIVE).

Potatoes (Solanum tuberosum L.) are one of the most valuable agricultural crops, and the flesh of these tubers provides various classes of healthy compounds important for human nutrition. This work presents the results of a joint analysis of different chemical classes of compounds which provided insights on the metabolic characterization of pigmented and non-pigmented potato varieties collected from Italy. The identification of common or individual metabolic characteristics across the omic datasets (antioxidants, total polyphenolic content, polyphenols, and sugars) is conducted by Joint and Individual Variation Explained (JIVE), a data fusion multivariate approach. The common part of the multivariate model allowed the separation between non-pigmented and pigmented samples. Polyphenolic compounds were mainly responsible for the separation between purple-fleshed and red-skinned potatoes. An additional detailed analysis of the anthocyanin composition, including the acylated anthocyanins, allowed to pinpoint the diversities between the pigmented potato groups. Furthermore, the presence of an appreciable amount of hydroxycinnamic acids and anthocyanins in the purple-fleshed varieties, which are also characterized by a lower content of sugars, is found. Our results provide scientific evidence for the promotion of promising potato cultivars, which are characterized by a remarkable amount of various health benefit compounds.

Biogenic volatile organic compounds in the grapevine response to pathogens, beneficial microorganisms, resistance inducers, and abiotic factors.

The synthesis of volatile organic compounds (VOCs) in plants is triggered in response to external stimuli, and these compounds can migrate to distal tissues and neighbouring receivers. Although grapevine VOCs responsible for wine aroma and plant-insect communications are well characterized, functional properties of VOCs produced in response to phytopathogens, beneficial microorganisms, resistance inducers, and abiotic factors have been less studied. In this review, we focused on the emission patterns and potential biological functions of VOCs produced by grapevines in response to stimuli. Specific grapevine VOCs are emitted in response to the exogenous stimulus, suggesting their precise involvement in plant defence response. VOCs with inhibitory activities against pathogens and responsible for plant resistance induction are reported, and some of them can also be used as biomarkers of grapevine resistance. Likewise, VOCs produced in response to beneficial microorganisms and environmental factors are possible mediators of grapevine-microbe communications and abiotic stress tolerance. Although further functional studies may improve our knowledge, the existing literature suggests that VOCs have an underestimated potential application as pathogen inhibitors, resistance inducers against biotic or abiotic stresses, signalling molecules, membrane stabilizers, and modulators of reactive oxygen species. VOC patterns could also be used to screen for resistant traits or to monitor the plant physiological status.

(3ξ,4ξ,5ξ,6ξ,7ξ,11ξ)-3,6-Dihydroxy-8-oxo-9-eremophilene-12-oic Acid, a New Phytotoxin of Alternaria alternata ssp. tenuissima Isolates Associated with Fruit Spots on Apple (Malus × domestica Borkh.).

Alternaria sp. infections on apple (Malus × domestica Borkh.) lead to impaired fruit quality and yield losses by leaf blotches and fruit spots, caused by host-specific toxins (HSTs) of the Alternaria apple pathotype like AM-toxins. Fungal isolates were obtained during severe outbreaks on cv. Gala, Golden Delicious, and Cripps Pink(cov)/Rosy Glow(cov) in South Tyrol and other regions in northern Italy. The isolates were tested for pathogenicity using in vitro assays with detached apple leaves. Conidial suspensions of pathogenic isolates were shown to provoke necrotic lesions also in apple seedlings and on fruits. Detached-leaf assay-guided fractionation of the isolates' culture supernatant and a high-resolution liquid chromatography-mass spectrometry (LC-MS) analysis tentatively identified 27 known Alternaria phytotoxins and a new putative toxin, (3ξ,4ξ,5ξ,6ξ,7ξ,11ξ)-3,6-dihydroxy-8-oxo-9-eremophilene-12-oic acid (1). The constitution and the relative configuration of the ring stereocenters of 1 were elucidated by NMR spectroscopy, revealing unique structural features among Alternaria phytotoxins. Indeed, molecular analysis revealed the lack of the toxin-related genes AMT1, AMT4, and AMT14 in all isolates from the region, suggesting that Alternaria apple blotch in the area was associated with another metabolite (1).

Mono-Locus and Pyramided Resistant Grapevine Cultivars Reveal Early Putative Biomarkers Upon Artificial Inoculation With Plasmopara viticola.

One of the most economically important grapevine diseases is Downy mildew (DM) caused by the oomycete Plasmopara viticola. A strategy to reduce the use of fungicides to compensate for the high susceptibility of V. vinifera is the selection of grapevine varieties showing pathogen-specific resistance. We applied a metabolomics approach to evaluate the metabolic modulation in mono-locus resistant genotypes carrying one locus associated with P. viticola resistance (Rpv) (BC4- Rpv1, Bianca- Rpv3-1, F12P160- Rpv12, Solaris- Rpv10), as well as in pyramided resistant genotypes carrying more than one Rpv (F12P60- Rpv3-1; Rpv12 and F12P127- Rpv3-1, Rpv3-3; Rpv10) taking as a reference the susceptible genotype Pinot Noir. In order to understand if different sources of resistance are associated with different degrees of resistance and, implicitly, with different responses to the pathogen, we considered the most important classes of plant metabolite primary compounds, lipids, phenols and volatile organic compounds at 0, 12, 48, and 96 h post-artificial inoculation (hpi). We identified 264 modulated compounds; among these, 22 metabolites were found accumulated in significant quantities in the resistant cultivars compared to Pinot Noir. In mono-locus genotypes, the highest modulation of the metabolites was noticed at 48 and 96 hpi, except for Solaris, that showed a behavior similar to the pyramided genotypes in which the changes started to occur as early as 12 hpi. Bianca, Solaris and F12P60 showed the highest number of interesting compounds accumulated after the artificial infection and with a putative effect against the pathogen. In contrast, Pinot Noir showed a less effective defense response in containing DM growth.

Metabolomic Characterization of Commercial, Old, and Red-Fleshed Apple Varieties.

In this study, a metabolomic investigation was presented to correlate single polyphenolic compounds in apple pulp with quality characteristics such as antioxidant activity and content of phenolic compounds and anthocyanins in apple skin. Since the concentration of these compounds is influenced by environmental factors, the twenty-two apple cultivars originate from the same site. The polyphenolic compounds were analyzed by ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS). The antioxidant activity, phenolic content, and anthocyanins were evaluated on the sunny and the shady sides of apple skin by spectrometric assays. In old apple varieties, the measured parameters were higher than in the commercial and red-fleshed varieties. By contrast, the profile of flavan-3-ols and anthocyanins was variable amongst commercial and red-fleshed varieties. The partial least square (PLS) method was applied to investigate the association between the skin proprieties and the metabolic profile of the pulp. The highest coefficients of determination in prediction (Q2) were obtained for compounds quantified in old cultivars. These results provided information to define the old apple varieties as a reliable group based on the pathway of the antioxidant compounds and anthocyanins content. Our results show the possibility to find cultivars with promising health features based on their content of polyphenols suitable for commercialization or breeding.

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.

Novel Types of Hypermodified Fluorescent Phyllobilins from Breakdown of Chlorophyll in Senescent Leaves of Grapevine (Vitis vinifera).

The tetrapyrrolic chlorophyll catabolites (or phyllobilins, PBs) were analyzed in yellow fall leaves of the grape Chardonnay, a common Vitis vinifera white wine cultivar. The major fractions in leaf extracts of V. vinifera, tentatively assigned to PBs, were isolated and their structures elucidated. The dominant fraction is a dioxobilin-type non-fluorescent Chl-catabolite of a previously observed type. Two less polar fluorescent PBs were characterized as a novel dioxobilin-type fluorescent Chl-catabolite with a bicyclo-1',6'-glycosyl architecture, and its new fluorescent formyloxobilin-type analogue. The discovery of persistent hypermodified fluorescent PBs with the architecture of bicyclo-[17.3.1]-PBs (bcPBs), suggests the activity of an unknown enzyme that forges the 20-membered macroring at the tetrapyrrolic core of a fluorescent PB. bcPBs may play specific physiological roles in grapevine plants and represent endogenous anti-infective agents, as found similarly for other organic bicyclo-[n.3.1]-1',6'-glycosyl derivatives.

Specific phosphorothioate substitution within domain 6 of a group II intron ribozyme leads to changes in local structure and metal ion binding.

Group II introns are large self-splicing ribozymes that require high amounts of monovalent and divalent metal ions for folding and catalysis under in vitro conditions. Domain 6 of these ribozymes contains a highly conserved adenosine whose 2'-OH acts as a nucleophile during self-cleavage via the branching pathway. We have previously suggested a divalent metal ion that binds to the major groove at the GU wobble pair above the branch-A in a minimal, but active branch domain construct (D6-27) from the yeast mitochondrial intron Sc.ai5γ. Here we characterize metal ion binding to the phosphate oxygens at the branch site. In vitro transcription yielded a D6-27 construct where all R P oxygens of the uridine phosphate groups are replaced by sulfur (α-thio-D6-27). We determined its NMR structure, the second RNA-only structure containing thiophosphate groups. [31P] resonances were assigned and chemical shift changes monitored upon titration with Cd2+. In addition, the two uridines flanking the branch-point, U19 and U21 were specifically thioated by chemical synthesis (thio-U19-D6-27 and thio-U19/U21-D6-27), enabling us to study Cd2+ binding at the R P-, as well as the S P- position of the corresponding phosphate oxygens. Our studies reveal that both non-bridging phosphate oxygens of U19 are involved in metal ion coordination, whereas only the major groove phosphate oxygen of U21 is influenced. Together with NOE data of a hexaamminecobalt(III) titration, this suggests a single metal ion binding site at the GU wobble pair above the branch point in the major groove of D6 of this group II intron ribozyme.

Pathogen-Induced Leaf Chlorosis: Products of Chlorophyll Breakdown Found in Degreened Leaves of Phytoplasma-Infected Apple (Malus × domestica Borkh.) and Apricot (Prunus armeniaca L.) Trees Relate to the Pheophorbide a Oxygenase/Phyllobilin Pathway.

Phytoplasmoses such as apple proliferation (AP) and European stone fruit yellows (ESFY) cause severe economic losses in fruit production. A common symptom of both phytoplasma diseases is early yellowing or leaf chlorosis. Even though chlorosis is a well-studied symptom of biotic and abiotic stresses, its biochemical pathways are hardly known. In particular, in this context, a potential role of the senescence-related pheophorbide a oxygenase/phyllobilin (PaO/PB) pathway is elusive, which degrades chlorophyll (Chl) to phyllobilins (PBs), most notably to colorless nonfluorescent Chl catabolites (NCCs). In this work, we identified the Chl catabolites in extracts of healthy senescent apple and apricot leaves. In extracts of apple tree leaves, a total of 12 Chl catabolites were detected, and in extracts of leaves of the apricot tree 16 Chl catabolites were found. The seven major NCC fractions in the leaves of both fruit tree species were identical and displayed known structures. All of the major Chl catabolites were also found in leaf extracts from AP- or ESFY-infected trees, providing the first evidence that the PaO/PB pathway is relevant also for pathogen-induced chlorosis. This work supports the hypothesis that Chl breakdown in senescence and phytoplasma infection proceeds via a common pathway in some members of the Rosaceae family.

Chlorophyll Catabolites in Senescent Leaves of the Plum Tree (Prunus domestica).

In cold extracts of senescent leaves of the plum tree (Prunus domestica ssp. domestica), six colorless non-fluorescent chlorophyll catabolites (NCCs) were characterized, named Pd-NCCs. In addition, several minor NCC fractions were tentatively classified. The structure of the most polar one of the NCCs, named Pd-NCC-32, featured an unprecedented twofold glycosidation pattern. Three of the NCCs are also functionalized at their 32 -position by a glucopyranosyl group. In addition, two of these glycosidated NCCs carry a dihydroxyethyl group at their 18-position. In the polar Pd-NCC-32, the latter group is further glycosidated at the terminal 182 -position. Four other major Pd-NCCs and one minor Pd-NCC were identified with five NCCs from higher plants known to belong to the 'epi'-series. In addition, tentative structures were derived for two minor fractions, classified as yellow chlorophyll catabolites, which represented (formal) oxidation products of two of the observed Pd-NCCs. The chlorophyll catabolites in leaves of plum feature the same basic structural pattern as those found in leaves of apple and pear trees.

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.

Chemodiversity in the fingerprint analysis of volatile organic compounds (VOCs) of 35 old and 7 modern apple cultivars determined by proton-transfer-reaction mass spectrometry (PTR-MS) in two different seasons.

Volatile organic compounds (VOCs) are chemical species that play an important role in determining the characteristic aroma and flavor of fruits. Apple (Malus × domestica Borkh.) cultivars differ in their aroma and composition of VOCs. To determine varietal differences in the aroma profiles, VOCs emitted by 7 modern and 35 old apple cultivars were analyzed using Proton Transfer Reaction Mass Spectrometry (PTR-MS). PTR-MS is a rapid, reproducible, and non-destructive spectrometric technique for VOC analysis of single fruits, developed for direct injection analysis. In the present study, we analyzed the differences in the emission of VOCs from single fruits at harvest and after a storage period of 60±10 days, followed by 3 d of shelf life. Our results show that VOC profile differences among apple cultivars were more pronounced after storage than at harvest. Furthermore, chemodiversity was higher in old cultivars compared to modern cultivars, probably due to their greater genetic variability. Our data highlight the importance of storage and shelf life are crucial for the development of the typical aroma and flavor of several apple cultivars. The validity of the method is demonstrated by comparison of two different harvest years.

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.