Project description:Aspergillus carbonarius causes severe decays on berries in vineyards and is among the main fungal species responsible for grape contamination by ochratoxin A (OTA), which is the foremost mycotoxin produced by this fungus. The main goal of this study was to investigate at the transcriptome level the comparative profiles between two table grape varieties (Victoria and Fraoula, the white and red variety, respectively) after their inoculation with a virulent OTA-producing A. carbonarius strain. The two varieties revealed quite different transcriptomic signatures and the expression profiles of the differential expressed genes (DEGs) highlighted distinct and variety-specific responses during the infection period. The significant enrichment of pathways related to the modulation of transcriptional dynamics towards the activation of defence responses, the triggering of the metabolic shunt for the biosynthesis of secondary metabolites, mainly phenylpropanoids, and the upregulation of DEGs encoding phytoalexins, transcription factors, and genes involved in plant-pathogen interaction and immune signaling transduction was revealed in an early time point in Fraoula, whereas, in Victoria, any transcriptional reprogramming was observed after a delay. However, both varieties, to some extent, also showed common expression dynamics for specific DEG families, such as those encoding for laccases and stilbene synthases. Jasmonate (JA) may play a critical modulator role in the defence machinery as various JA-biosynthetic DEGs were upregulated. Along with the broader modulation of the transcriptome that was observed in white grape, expression profiles of specific A. carbonarius genes related to pathogenesis, fungal sporulation, and conidiation highlight the higher susceptibility of Victoria. Furthermore, the A. carbonarius transcriptional patterns directly associated with the regulation of the pathogen OTA-biosynthesis gene cluster were more highly induced in Victoria than in Fraoula. The latter was less contaminated by OTA and showed substantially lower sporulation. These findings contribute to uncovering the interplay beyond this plant-microbe interaction.
Project description:Grapevine bunch rot, caused by Botrytis cinerea and Aspergillus carbonarius, causes important economic losses every year in grape production. In the present study, we examined the plant protective activity of the biological control agents, Paenibacillus alvei K165, Blastobotrys sp. FP12 and Arthrobacter sp. FP15 against B. cinerea and A. carbonarius on grapes. The in vitro experiments showed that strain K165 significantly reduced the growth of both fungi, while FP15 restricted the growth of A. carbonarius and FP12 was ineffective. Following the in vitro experiments, we conducted in planta experiments on grape berries. It was shown that K165, FP12 and FP15 reduced A. carbonarius rot severity by 81%, 57% and 37%, respectively, compared to the control, whereas, in the case of B. cinerea, the only protective treatment was that with K165, which reduced rot by 75%. The transcriptomic analysis of the genes encoding the pathogenesis-related proteins PR2, PR3, PR4 and PR5 indicates the activation of multiple defense responses involved in the biocontrol activity of the examined biocontrol agents.
Project description:Mycotoxins represent a serious risk for human and animal health. Οchratoxin A (OTA) is a carcinogenic mycotoxin produced by A. carbonarius that constitutes a severe problem for viticulture. In this study, we investigate the development of novel detection and on-line monitoring approaches for the detection of OTA in the field (i.e. out of the chemical laboratory) using advanced molecular sensing. Both stand-alone and hyphenated mass spectrometry (MS) based systems (e.g. Time-of-Flight ToF-MS and gas chromatography GC combined with MS) and compact portable membrane inlet MS (MIMS) have been employed for the first time to detect and monitor volatile emissions of grape berries infected by the fungus Aspergillus carbonarius. In vacuo (electron impact-EI) and ambient ionisation (electrospray ionisation-ESI) techniques were also examined. On-line measurements of the volatile emissions of grape berries, infected by various strains of A. carbonarius with different toxicity levels, were performed resulting in different olfactory chemical profiles with a common core of characteristic mass fragments, which could be eventually used for on-site detection and monitoring allowing consequent improvement in food security.
Project description:Besides signalling to soil organisms, strigolactones (SLs) control above- and below-ground morphology, in particular shoot branching. Furthermore, SLs interact with stress responses, possibly thanks to a crosstalk with the abscisic acid (ABA) signal. In grapevine (Vitis vinifera L.), ABA drives the accumulation of anthocyanins over the ripening season. In this study, we investigated the effects of treatment with a synthetic strigolactone analogue, GR24, on anthocyanin accumulation in grape berries, in the presence or absence of exogenous ABA treatment. Experiments were performed both on severed, incubated berries, and on berries attached to the vine. Furthermore, we analysed the corresponding transcript concentrations of genes involved in anthocyanin biosynthesis, and in ABA biosynthesis, metabolism, and membrane transport. During the experiment time courses, berries showed the expected increase in soluble sugars and anthocyanins. GR24 treatment had no or little effect on anthocyanin accumulation, or on gene expression levels. Exogenous ABA treatment activated soluble sugar and anthocyanin accumulation, and enhanced expression of anthocyanin and ABA biosynthetic genes, and that of genes involved in ABA hydroxylation and membrane transport. Co-treatment of GR24 with ABA delayed anthocyanin accumulation, decreased expression of anthocyanin biosynthetic genes, and negatively affected ABA concentration. GR24 also enhanced the ABA-induced activation of ABA hydroxylase genes, while it down-regulated the ABA-induced activation of ABA transport genes. Our results show that GR24 affects the ABA-induced activation of anthocyanin biosynthesis in this non-climacteric fruit. We discuss possible mechanisms underlying this effect, and the potential role of SLs in ripening of non-ABA-treated berries.
Project description:An increasing number of field studies that focus on grapevine berry development and ripening implement systems biology approaches; the results are highlighting not only the intricacies of the developmental programming/reprogramming that occurs, but also the complexity of how profoundly the microclimate influences the metabolism of the berry throughout the different stages of development. In a previous study we confirmed that a leaf removal treatment to Sauvignon Blanc grapes, grown in a highly characterized vineyard, primarily affected the level of light exposure to the berries throughout their development. A full transcriptomic analysis of berries from this model vineyard details the underlying molecular responses of the berries in reaction to the exposure and show how the berries acclimated to the imposing light stress. Gene expression involved in the protection of the photosynthetic machinery through rapid protein-turnover and the expression of photoprotective flavonoid compounds were most significantly affected in green berries. Overall, the transcriptome analysis showed that the berries implemented multiple stress-mitigation strategies in parallel and metabolite analysis was used to support the main findings. Combining the transcriptome data and amino acid profiling provided evidence that amino acid catabolism probably contributed to the mitigation of a likely energetic deficit created by the upregulation of (energetically) costly stress defense mechanisms. Furthermore, the rapid turnover of essential proteins involved in the maintenance of primary metabolism and growth in the photosynthetically active grapes appeared to provide precursors for the production of protective secondary metabolites such as apocarotenoids and flavonols in the ripening stages of the berries. Taken together, these results confirmed that the green grape berries responded to light stress much like other vegetative organs and were able to acclimate to the increased exposure, managing their metabolism and energy requirements to sustain the developmental cycle toward ripening. The typical metabolic consequences of leaf removal on grape berries can therefore now be linked to increased light exposure through mechanisms of photoprotection in green berries that leads toward acclimation responses that remain intact until ripening.
Project description:Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated in Penicillium species. In Aspergillus species, only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producing A. carbonarius ITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in an Aspergillus species. The absence of OTA and ochratoxin α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β, the dechloro analog of ochratoxin α, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.