Volatilomics of raspberry fruit germplasm by combining chromatographic and direct-injection mass spectrometric techniques.

The application of direct-injection mass spectrometric (DI-MS) techniques, like Proton Transfer Reaction Time of Flight Mass Spectrometry (PTR-ToF-MS) has been suggested as a reliable phenotyping tool for fruit volatilome assessment in both genetic and quality-related studies. In this study the complexity of raspberry aroma was investigated by a comprehensive untargeted VOC analysis, done by combining SPME-GC-MS and PTR-ToF-MS assessments with multi-block discriminant analysis using the DIABLO mixOmics framework. The aim was to acquire an exhaustive characterization of the raspberry volatilome according to different fruit ripening stages (pink, ripe, and overripe) and genetic variances (50 accessions), as well as to investigate the potential of PTR-ToF-MS as a rapid and high throughput VOC phenotyping tool to address issues related to raspberry fruit quality. Results of this study demonstrated the complementarity between SPME-GC-MS and PTR-ToF-MS techniques to evaluate the raspberry aroma composition. PTR-ToF-MS generates reliable raspberry VOC fingerprints mainly due to a reduced compound fragmentation and precise content estimation. In addition, the high collinearity between isomers of monoterpenes and norisoprenoids, discovered by GC analysis, reduces the main analytic limitation of PTR-ToF-MS of not being able to separate isomeric molecules. The high similarity between the VOC matrices obtained by applying PTR-ToF-MS and SPME-GC-MS confirmed the possibility of using PTR-ToF-MS as a reliable high throughput phenotyping tool for raspberry volatiolome assessment. In addition, results provided by the germplasm collection investigation enabled to distinguish the best performing accessions, based on VOCs composition, to be used as superior parental lines for future breeding programs.

Monitoring alkyl pyrazines in roasted hazelnuts by SHS-GC-IMS: IMS response assessment and standardization.

Gas chromatography coupled with ion mobility spectrometry (IMS) is an analytical tool which is rapidly becoming widespread in the analysis of food volatiles. Despite this increasing popularity, an assessment of the IMS response for several flavor compound classes is not yet available. This study focuses on alkyl pyrazines and their determination in roasted hazelnut pastes. These Maillard reaction products are crucial to determine the aromatic profile of roasted foods and are suitable markers for industrial roasting monitoring. The instrumental response of 8 alkyl pyrazines was studied using a model matrix and a matrix matching approach. The results showed a relevant effect of the pyrazine ring substitution pattern on the concentration-response curve trends, highlighting that an external standardization of the IMS response is required to make possible relative abundance comparisons between analytes. A response standardization was therefore developed and applied to determine alkyl pyrazines in samples with different roasting intensity and geographical and botanical origin.

Taxonomical and functional composition of strawberry microbiome is genotype-dependent.

INTRODUCTION: Specific microbial communities are associated to host plants, influencing their phenotype and fitness.Despite the rising interest in plant microbiome, the role of microbial communities associated with perennial fruit plants remains overlooked. OBJECTIVES: This work provides the first comprehensive descriptionof the taxonomical and functional bacterial and fungal microbiota of below- and above-ground organsof three commercially important strawberry genotypes under cultural conditions. METHODS: Strawberry-associatedfungal and bacterial microbiomes were characterised by Next-Generation Sequencing and the potential functions expressed by the bacterial microbiome were analysed by both in silico and in vitro characterisation of plant growth-promoting abilities of native bacteria. Additionally, the association between the strawberry microbiome, plant disease tolerance, plant mineral nutrient content, and fruit quality was investigated. RESULTS: Results showed that thestrawberry core microbiome included 24 bacteria and 15 fungal operational taxonomicunits (OTUs).However, plant organ and genotype had a significant role in determining the taxonomical and functional composition of microbial communities. Interestingly, the cultivar with the highesttolerance against powdery mildew and leaf spot and the highest fruit productivity was the only one able to ubiquitously recruit the beneficial bacterium, Pseudomonasfluorescens, and to establish a mutualistic symbiosis with the arbuscular mycorrhizaRhizophagus irregularis. CONCLUSION: This work sheds light on the interaction of cultivated strawberry genotypes with a variety of microbes and highlights the importance of their applications to increase the sustainability of fruit crop production.

Valorization of Traditional Italian Walnut (Juglans regia L.) Production: Genetic, Nutritional and Sensory Characterization of Locally Grown Varieties in the Trentino Region.

Juglans regia (L.) is cultivated worldwide for its nutrient-rich nuts. In Italy, despite the growing demand, walnut cultivation has gone through a strong decline in recent decades, which led to Italy being among the top five net importing countries. To promote the development of local high-quality Italian walnut production, we devised a multidisciplinary project to highlight the distinctive traits of three varieties grown in the mountainous region Trentino (northeast of Italy): the heirloom 'Bleggiana', a second local accession called local Franquette and the French cultivar 'Lara', recently introduced in the local production to increase yield. The genetic characterization confirmed the uniqueness of 'Bleggiana' and revealed local Franquette as a newly described autochthonous variety, thus named 'Blegette'. The metabolic profiles highlighted a valuable nutritional composition of the local varieties, richer in polyphenols and with a lower ω-6/ω-3 ratio than the commercial 'Lara'. 'Blegette' obtained the highest preference scores from consumers for both the visual aspect and tasting; however, the volatile organic compound profiles did not discriminate among the characterized cultivars. The described local varieties represent an interesting reservoir of walnut genetic diversity and quality properties, which deserve future investigation on agronomically useful traits (e.g., local adaptation and water usage) for a high-quality and sustainable production.

Ethylene Production Affects Blueberry Fruit Texture and Storability.

Ethylene, produced endogenously by plants and their organs, can induce a wide array of physiological responses even at very low concentrations. Nevertheless, the role of ethylene in regulating blueberry (Vaccinium spp.) ripening and storability is still unclear although an increase in ethylene production has been observed in several studies during blueberry ripening. To overcome this issue, we evaluated the endogenous ethylene production of a Vaccinium germplasm selection at different fruit ripening stages and after cold storage, considering also textural modifications. Ethylene and texture were further assessed also on a bi-parental full-sib population of 124 accessions obtained by the crossing between "Draper" and "Biloxi", two cultivars characterized by a different chilling requirement and storability performances. Our results were compared with an extensive literature research, carried out to collect all accessible information on published works related to Vaccinium ethylene production and sensitivity. Results of this study illustrate a likely role of ethylene in regulating blueberry shelf life. However, a generalisation valid for all Vaccinium species is not attainable because of the high variability in ethylene production between genotypes, which is strictly genotype-specific. These differences in ethylene production are related with blueberry fruit storage performances based on textural alterations. Specifically, blueberry accessions characterized by the highest ethylene production had a more severe texture decay during storage. Our results support the possibility of tailoring ad hoc preharvest and postharvest strategies to extend blueberry shelf life and quality according with the endogenous ethylene production level of each cultivar.

Does Organic Farming Increase Raspberry Quality, Aroma and Beneficial Bacterial Biodiversity?

Plant-associated microbes can shape plant phenotype, performance, and productivity. Cultivation methods can influence the plant microbiome structure and differences observed in the nutritional quality of differently grown fruits might be due to variations in the microbiome taxonomic and functional composition. Here, the influence of organic and integrated pest management (IPM) cultivation on quality, aroma and microbiome of raspberry (Rubus idaeus L.) fruits was evaluated. Differences in the fruit microbiome of organic and IPM raspberry were examined by next-generation sequencing and bacterial isolates characterization to highlight the potential contribution of the resident-microflora to fruit characteristics and aroma. The cultivation method strongly influenced fruit nutraceutical traits, aroma and epiphytic bacterial biocoenosis. Organic cultivation resulted in smaller fruits with a higher anthocyanidins content and lower titratable acidity content in comparison to IPM berries. Management practices also influenced the amounts of acids, ketones, aldehydes and monoterpenes, emitted by fruits. Our results suggest that the effects on fruit quality could be related to differences in the population of Gluconobacter, Sphingomonas, Rosenbergiella, Brevibacillus and Methylobacterium on fruit. Finally, changes in fruit aroma can be partly explained by volatile organic compounds (VOCs) emitted by key bacterial genera characterizing organic and IPM raspberry fruits.

A Breach in Plant Defences: Pseudomonas syringae pv. actinidiae Targets Ethylene Signalling to Overcome Actinidia chinensis Pathogen Responses.

Ethylene interacts with other plant hormones to modulate many aspects of plant metabolism, including defence and stomata regulation. Therefore, its manipulation may allow plant pathogens to overcome the host's immune responses. This work investigates the role of ethylene as a virulence factor for Pseudomonas syringae pv. actinidiae (Psa), the aetiological agent of the bacterial canker of kiwifruit. The pandemic, highly virulent biovar of this pathogen produces ethylene, whereas the biovars isolated in Japan and Korea do not. Ethylene production is modulated in planta by light/dark cycle. Exogenous ethylene application stimulates bacterial virulence, and restricts or increases host colonisation if performed before or after inoculation, respectively. The deletion of a gene, unrelated to known bacterial biosynthetic pathways and putatively encoding for an oxidoreductase, abolishes ethylene production and reduces the pathogen growth rate in planta. Ethylene production by Psa may be a recently and independently evolved virulence trait in the arms race against the host. Plant- and pathogen-derived ethylene may concur in the activation/suppression of immune responses, in the chemotaxis toward a suitable entry point, or in the endophytic colonisation.

Development of a Novel Phenotypic Roadmap to Improve Blueberry Quality and Storability.

Improved fruit quality and prolonged storage capability are key breeding traits for blueberry (Vaccinium spp.) fruit. Until now, breeding selection was mostly oriented on the amelioration of agronomic traits, such as flowering time, chilling requirement, or plant structure. Up until now, however, the storage effect on fruit quality has not been extensively studied, mostly because objective and handy phenotyping tools to evaluate quality traits were not available. In this study we are proposing a novel phenotyping protocol to support breeding selection and quality control within the entire blueberry production chain. Volatile organic compounds (VOCs) and texture traits, were measured by Proton Transfer Reaction- Time of Flight- Mass Spectrometry (PTR-ToF-MS) and a texture analyzer respectively, taking into consideration the influence of prolonged storage. The exploitation of the genetic variability existing within the investigated blueberry germplasm collection (including both southern and northern highbush, hybrids, and rabbiteyes) allowed the identification of the best performing cultivars, based on texture and VOCs variability, to be used as superior parental lines for future breeding programs. The comprehensive characterization of blueberry aroma allowed the identification of a wide array of spectrometric features, mostly related to aldehydes, alcohols, terpenoids, and esters, that can be used as putative biomarkers to rapidly evaluate the blueberry aroma variations related to genetic differences and storability. In addition, this study revealed a lack of straightforward relationship between harvest and postharvest quality features, that might be genotype-dependent.

Investigation of the transcriptomic and metabolic changes associated with superficial scald physiology impaired by lovastatin and 1-methylcyclopropene in pear fruit (cv. "Blanquilla").

To elucidate the physiology underlying the development of superficial scald in pears, susceptible "Blanquilla" fruit was treated with different compounds that either promoted (ethylene) or repressed (1-methylcyclopropene and lovastatin) the incidence of this disorder after 4 months of cold storage. Our data show that scald was negligible for the fruit treated with 1-methylcyclopropene or lovastatin, but highly manifested in untreated (78% incidence) or ethylene-treated fruit (97% incidence). The comparison between the fruit metabolomic profile and transcriptome evidenced a distinct reprogramming associated with each treatment. In all treated samples, cold storage led to an activation of a cold-acclimation-resistance mechanism, including the biosynthesis of very-long-chain fatty acids, which was especially evident in 1-methylcyclopropane-treated fruit. Among the treatments applied, only 1-methylcyclopropene inhibited ethylene production, hence supporting the involvement of this hormone in the development of scald. However, a common repression effect on the PPO gene combined with higher sorbitol content was found for both lovastatin and 1-methylcyclopropene-treated samples, suggesting also a non-ethylene-mediated process preventing the development of this disorder. The results presented in this work represent a step forward to better understand the physiological mechanisms governing the etiology of superficial scald in pears.

Wide transcriptional investigation unravel novel insights of the on-tree maturation and postharvest ripening of 'Abate Fetel' pear fruit.

To decipher the transcriptomic regulation of the on-tree fruit maturation in pear cv. 'Abate Fetel', a RNA-seq transcription analysis identified 8939 genes differentially expressed across four harvesting stages. These genes were grouped into 11 SOTA clusters based on their transcriptional pattern, of which three included genes upregulated while the other four were represented by downregulated genes. Fruit ripening was furthermore investigated after 1 month of postharvest cold storage. The most important variation in fruit firmness, production of ethylene and volatile organic compounds were observed after 5 days of shelf-life at room temperature following cold storage. The role of ethylene in controlling the ripening of 'Abate Fetel' pears was furthermore investigated through the application of 1-methylcyclopropene, which efficiently delayed the progression of ripening by reducing fruit softening and repressing both ethylene and volatile production. The physiological response of the interference at the ethylene receptor level was moreover unraveled investigating the expression pattern of 12 candidate genes, initially selected to validate the RNA-seq profile. This analysis confirmed the effective role of the ethylene competitor in downregulating the expression of cell wall (PG) and ethylene-related genes (ACS, ACO, ERS1, and ERS2), as well as inducing one element involved in the auxin signaling pathway (Aux/IAA), highlighting a possible cross-talk between these two hormones. The expression patterns of these six elements suggest their use as molecular toolkit to monitor at molecular level the progression of the fruit on-tree maturation and postharvest ripening.

Pathogen-induced changes in floral scent may increase honeybee-mediated dispersal of Erwinia amylovora.

Honeybees are well recognised for their key role in plant reproduction as pollinators. On the other hand, their activity may vector some pathogens, such as the bacterium Erwinia amylovora, the causative agent of fire blight disease in pomaceous plants. In this research, we evaluated whether honeybees are able to discriminate between healthy and E. amylovora-infected flowers, thus altering the dispersal of the pathogen. For this reason, honeybees were previously trained to forage either on inoculated or healthy (control) apple flower. After the training, the two honeybee groups were equally exposed to inoculated and control flowering apple plants. To assess their preference, three independent methods were used: (1) direct count of visiting bees per time frame; (2) incidence on apple flowers of a marker bacterium (Pantoea agglomerans, strain P10c) carried by foragers; (3) quantification of E. amylovora populations in the collected pollen loads, proportional to the number of visits to infected flowers. The results show that both honeybee groups preferred control flowers over inoculated ones. The characterisation of volatile compounds released by flowers revealed a different emission of several bioactive compounds, providing an explanation for honeybee preference. As an unexpected ecological consequence, the influence of infection on floral scent increasing the visit rate on healthy flowers may promote a secondary bacterial spread.

Direct Injection Analysis of Fruit VOCs by PTR-ToF-MS: The Apple Case Study.

The instrumental characterization of volatile organic compounds (VOCs) is essential to have a precise, reliable, and reproducible estimation of food aroma and, therefore, of the overall product quality. In this report, we introduce four analytical approaches based on PTR-MS (proton transfer reaction-mass spectrometry) technology suitable to fully investigate the complexity of apple aroma. In our opinion, these proposed methodologies can be applied, with slight modification, to every kind of fruit for destructive and nondestructive rapid VOC fingerprinting.

Apple fruit superficial scald resistance mediated by ethylene inhibition is associated with diverse metabolic processes.

Fruits stored at low temperature can exhibit different types of chilling injury. In apple, one of the most serious physiological disorders is superficial scald, which is characterized by discoloration and brown necrotic patches on the fruit exocarp. Although this phenomenon is widely ascribed to the oxidation of α-farnesene, its physiology is not yet fully understood. To elucidate the mechanism of superficial scald development and possible means of prevention, we performed an integrated metabolite screen, including an analysis of volatiles, phenols and lipids, together with a large-scale transcriptome study. We also determined that prevention of superficial scald, through the use of an ethylene action inhibitor, is associated with the triggering of cold acclimation-related processes. Specifically, the inhibition of ethylene perception stimulated the production of antioxidant compounds to scavenge reactive oxygen species, the synthesis of fatty acids to stabilize plastid and vacuole membranes against cold temperature, and the accumulation of the sorbitol, which can act as a cryoprotectant. The pattern of sorbitol accumulation was consistent with the expression profile of a sorbitol 6-phosphate dehydrogenase, MdS6PDH, the overexpression of which in transgenic Arabidopsis thaliana plants confirmed its involvement in the cold acclimation and freezing tolerance.

Exploring Blueberry Aroma Complexity by Chromatographic and Direct-Injection Spectrometric Techniques.

Blueberry (Vaccinium spp.) fruit consumption has increased over the last 5 years, becoming the second most important soft fruit species after strawberry. Despite the possible economic and sensory impact, the blueberry volatile organic compound (VOC) composition has been poorly investigated. Thus, the great impact of the aroma on fruit marketability stimulates the need to step forward in the understanding of this quality trait. Beside the strong effect of ripening, blueberry aroma profile also varies due to the broad genetic differences among Vaccinium species that have been differently introgressed in modern commercial cultivars through breeding activity. In the present study, divided into two different activities, the complexity of blueberry aroma was explored by an exhaustive untargeted VOC analysis, performed by two complementary methods: SPME-GC-MS (solid phase microextraction- gas chromatography-mass spectrometry) and PTR-ToF-MS (proton transfer reaction-time of flight-mass spectrometry). The first experiment was aimed at determining the VOC modifications during blueberry ripening for five commercially representative cultivars ("Biloxi," "Brigitta Blue," "Centurion," "Chandler," and "Ozark Blue") harvested at four ripening stages (green, pink, ripe, and over-ripe) to outline VOCs dynamic during fruit development. The objective of the second experiment was to confirm the analytical capability of PTR-ToF-MS to profile blueberry genotypes and to identify the most characterizing VOCs. In this case, 11 accessions belonging to different Vaccinium species were employed: V. corymbosum L. ("Brigitta," "Chandler," "Liberty," and "Ozark Blue"), V. virgatum Aiton ("Centurion," "Powder Blue," and "Sky Blue"), V. myrtillus L. (three wild genotypes of different mountain locations), and one accession of V. cylindraceum Smith. This comprehensive characterization of blueberry aroma allowed the identification of a wide pull of VOCs, for the most aldehydes, alcohols, terpenoids, and esters that can be used as putative biomarkers to rapidly evaluate the blueberry aroma variations related to ripening and/or senescence as well as to genetic background differences. Moreover, the obtained results demonstrated the complementarity between chromatographic and direct-injection mass spectrometric techniques to study the blueberry aroma.

PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis.

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that belongs to the Direct-Injection Mass Spectrometric (DIMS) technologies. These techniques allow the rapid determination of volatile organic compounds (VOCs), assuring high sensitivity and accuracy. In general, PTR-MS requires neither sample preparation nor sample destruction, allowing real time and non-invasive analysis of samples. PTR-MS are exploited in many fields, from environmental and atmospheric chemistry to medical and biological sciences. More recently, we developed a methodology based on coupling PTR-ToF-MS with an automated sampler and tailored data analysis tools, to increase the degree of automation and, consequently, to enhance the potential of the technique. This approach allowed us to monitor bioprocesses (e.g. enzymatic oxidation, alcoholic fermentation), to screen large sample sets (e.g. different origins, entire germoplasms) and to analyze several experimental modes (e.g. different concentrations of a given ingredient, different intensities of a specific technological parameter) in terms of VOC content. Here, we report the experimental protocols exemplifying different possible applications of our methodology: i.e. the detection of VOCs released during lactic acid fermentation of yogurt (on-line bioprocess monitoring), the monitoring of VOCs associated with different apple cultivars (large-scale screening), and the in vivo study of retronasal VOC release during coffee drinking (nosespace analysis).

Genome-wide association study unravels the genetic control of the apple volatilome and its interplay with fruit texture.

Fruit quality represents a fundamental factor guiding consumers' preferences. Among apple quality traits, volatile organic compounds and texture features play a major role. Proton Transfer Reaction-Time of Flight-Mass Spectrometry (PTR-ToF-MS), coupled with an artificial chewing device, was used to profile the entire apple volatilome of 162 apple accessions, while the fruit texture was dissected with a TAXT-AED texture analyzer. The array of volatile compounds was classed into seven major groups and used in a genome-wide association analysis carried out with 9142 single nucleotide polymorphisms (SNPs). Marker-trait associations were identified on seven chromosomes co-locating with important candidate genes for aroma, such as MdAAT1 and MdIGS. The integration of volatilome and fruit texture data conducted with a multiple factor analysis unraveled contrasting behavior, underlying opposite regulation of the two fruit quality aspects. The association analysis using the first two principal components identified two QTLs located on chromosomes 10 and 2, respectively. The distinction of the apple accessions on the basis of the allelic configuration of two functional markers, MdPG1 and MdACO1, shed light on the type of interplay existing between fruit texture and the production of volatile organic compounds.

Interference with ethylene perception at receptor level sheds light on auxin and transcriptional circuits associated with the climacteric ripening of apple fruit (Malus x domestica Borkh.).

Apple (Malus x domestica Borkh.) is a model species for studying the metabolic changes that occur at the onset of ripening in fruit crops, and the physiological mechanisms that are governed by the hormone ethylene. In this study, to dissect the climacteric interplay in apple, a multidisciplinary approach was employed. To this end, a comprehensive analysis of gene expression together with the investigation of several physiological entities (texture, volatilome and content of polyphenolic compounds) was performed throughout fruit development and ripening. The transcriptomic profiling was conducted with two microarray platforms: a dedicated custom array (iRIPE) and a whole genome array specifically enriched with ripening-related genes for apple (WGAA). The transcriptomic and phenotypic changes following the application of 1-methylcyclopropene (1-MCP), an ethylene inhibitor leading to important modifications in overall fruit physiology, were also highlighted. The integrative comparative network analysis showed both negative and positive correlations between ripening-related transcripts and the accumulation of specific metabolites or texture components. The ripening distortion caused by the inhibition of ethylene perception, in addition to affecting the ethylene pathway, stimulated the de-repression of auxin-related genes, transcription factors and photosynthetic genes. Overall, the comprehensive repertoire of results obtained here advances the elucidation of the multi-layered climacteric mechanism of fruit ripening, thus suggesting a possible transcriptional circuit governed by hormones and transcription factors.

Chilling-Induced Changes in Aroma Volatile Profiles in Tomato.

Fruit and vegetables are regularly stored by consumers in the refrigerator at temperatures that may be well below the recommended storage temperatures. Apart from causing visible symptoms such as watery, sunken areas on the skin, chilling may also induce changes in fruit textural properties and flavour. The aim of this research was to investigate the effect of low temperature storage on tomato flavour and off-flavour production. To more closely mimic the real-consumer aroma perception while eating, in addition to the standard solid-phase microextraction gas chromatography-mass spectrometry (SPME/GC-MS) analysis, volatiles were also measured using a chewing device connected to a proton-transfer reaction-mass spectrometer (PTR-MS). Aroma volatiles were assessed in red ripe tomatoes of the cvs Cappricia RZ (round truss) and Amoroso RZ (cocktail truss) stored at refrigerator temperature (4 °C) and at higher temperatures (16 and 22 °C) for 20 days. The changes in aroma production were also monitored when the fruit was brought from room to refrigerator temperature and vice versa. After bringing the fruit from room to refrigerator temperature, the abundance of most volatiles was greatly reduced within 3 to 5 h, closely following the decrease in fruit temperature. When temperature was restored to room temperature following varying times of cold storage, the abundance of most volatiles increased again, but generally not to the original levels. Overall, the effects of low temperature storage on the decrease in volatile abundance were more pronounced in cv Cappricia RZ than in cv Amoroso RZ. On the contrary, the production of off flavours following prolonged cold storage was more pronounced in cv Amoroso RZ than in cv Cappricia RZ. Apart from changes in the overall abundance of the volatiles, marked changes in the volatile profile were observed in fruit stored for longer times in the cold and this may at least in part explain the negative effect of cold storage on overall tomato flavour.