Characterization of Coniella granati Isolates Causing Pomegranate Decline in Italy.

Coniella granati, the causal agent of pomegranate crown rot, twig blight, and fruit decay, is an emerging worldwide pathogen with a heavy impact on pomegranate cultivation. In this study, we report the rapid spread of the fungus in Italian pomegranate orchards associated with crown rot symptoms and provide new results on fungal development, baseline sensitivity to different fungicides, and intraspecific variability by analyzing 11 isolates, representative of populations of the pathogen from comparable pomegranate orchards in different regions of Italy. In vitro assays showed that 25 to 30°C was the optimal range for both colony growth and conidial germination, corroborating the results previously obtained for Californian and Greek isolates. According to the baseline sensitivity assay on the response of colony growth and conidial germination to 10 fungicides, fludioxonil, thiophanate-methyl, tebuconazole, and cyprodinil were the most effective. Random amplified polymorphic DNA (RAPD) analysis, carried out using fourteen 10-mer primers, showed very low intraspecific variability (similarity coefficient >0.95), probably as a result of the recent spread of the pathogen in Italy and the uncommon occurrence of the sexual process as a source of genetic variability. In summary, this study provides new knowledge on C. granati that will be helpful for improving pomegranate crown rot management.

A Large-Scale Validation of an Improved Embryo-Rescue Protocol for the Obtainment of New Table-Grape Seedless Genotypes.

The new trends in the consumption of table grapes and the growing interest in the environmental impact of this crop have pushed breeders toward the development of seedless cultivars endowed with resistance, through crossbreeding programs. To obtain seedless grapes, the use of embryo-rescue techniques is fundamental. In this research, a grape embryo-culture protocol was optimized and validated by using 39 cultivars and 41 cross-combinations carried out in the framework of a large private table grape program of the private network Italian Variety Club in the period 2017-2021 evaluating several factors, such as the improvement in embryo formation, germination and growth, and plantlet development. The embryo culture attitude of crosses between different combinations of seedless parents was assessed, and the rates of embryo development from the extracted ovules mostly ranged from 3.5 to 35.5% with 5 out of 43 genotypes outliers. Experiments conducted at different sampling times, in a range of 43-62 days after pollination (DAP), did not show significant differences between the samples analyzed, while the rate of embryos developed with the applied protocol proved its employability on multiple genotypes, although the grapevine genotype significantly influenced the technique efficiency.

The Mycovirome in a Worldwide Collection of the Brown Rot Fungus Monilinia fructicola.

The fungus Monilinia fructicola is responsible for brown rot on stone and pome fruit and causes heavy yield losses both pre- and post-harvest. Several mycoviruses are known to infect fungal plant pathogens. In this study, a metagenomic approach was applied to obtain a comprehensive characterization of the mycovirome in a worldwide collection of 58 M. fructicola strains. Deep sequencing of double-stranded (ds)RNA extracts revealed a great abundance and variety of mycoviruses. A total of 32 phylogenetically distinct positive-sense (+) single-stranded (ss)RNA viruses were identified. They included twelve mitoviruses, one in the proposed family Splipalmiviridae, and twelve botourmiaviruses (phylum Lenarviricota), eleven of which were novel viral species; two hypoviruses, three in the proposed family Fusariviridae, and one barnavirus (phylum Pisuviricota); as well as one novel beny-like virus (phylum Kitrinoviricota), the first one identified in Ascomycetes. A partial sequence of a new putative ssDNA mycovirus related to viruses within the Parvoviridae family was detected in a M. fructicola isolate from Serbia. The availability of genomic sequences of mycoviruses will serve as a solid basis for further research aimed at deepening the knowledge on virus-host and virus-virus interactions and to explore their potential as biocontrol agents against brown rot disease.

Tracking of Diversity and Evolution in the Brown Rot Fungi Monilinia fructicola, Monilinia fructigena, and Monilinia laxa.

Monilinia species are among the most devastating fungi worldwide as they cause brown rot and blossom blight on fruit trees. To understand the molecular bases of their pathogenic lifestyles, we compared the newly assembled genomes of single strains of Monilinia fructicola, M. fructigena and M. laxa, with those of Botrytis cinerea and Sclerotinia sclerotiorum, as the closest species within Sclerotiniaceae. Phylogenomic analysis of orthologous proteins and syntenic investigation suggest that M. laxa is closer to M. fructigena than M. fructicola, and is closest to the other investigated Sclerotiniaceae species. This indicates that M. laxa was the earliest result of the speciation process. Distinct evolutionary profiles were observed for transposable elements (TEs). M. fructicola and M. laxa showed older bursts of TE insertions, which were affected (mainly in M. fructicola) by repeat-induced point (RIP) mutation gene silencing mechanisms. These suggested frequent occurrence of the sexual process in M. fructicola. More recent TE expansion linked with low RIP action was observed in M. fructigena, with very little in S. sclerotiorum and B. cinerea. The detection of active non-syntenic TEs is indicative of horizontal gene transfer and has resulted in alterations in specific gene functions. Analysis of candidate effectors, biosynthetic gene clusters for secondary metabolites and carbohydrate-active enzymes, indicated that Monilinia genus has multiple virulence mechanisms to infect host plants, including toxins, cell-death elicitor, putative virulence factors and cell-wall-degrading enzymes. Some species-specific pathogenic factors might explain differences in terms of host plant and organ preferences between M. fructigena and the other two Monilinia species.

A Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits.

Brown rot, caused by different Monilinia species, is a most economically important disease of pome and stone fruits worldwide. In Europe and in Italy, the quarantine pathogen M. fructicola was recently introduced and rapidly spread and, by competing with the main indigenous species Monilinia fructigena and Monilinia laxa, caused relevant changes in Monilinia populations. As a result, in most areas, the pathogen almost replaced M. fructigena and now coexists with M. laxa. The availability of specific and easy-of-use quantification methods is essential to study the population dynamics, and in this work, a new method for the simultaneous quantification of M. fructicola and M. laxa based on droplet digital PCR (ddPCR) technique was established. Under the optimized reaction conditions, consisting of 250/500 nM of primers/probe sets concentration, 58°C as annealing temperature and 50 PCR cycles, the duplex-ddPCR assay was 200-fold more sensitive than duplex-real-time quantitative PCR (qPCR) assay, quantifying < 1 copy µL-1 of target DNA in the PCR mixture. The results obtained with the validation assay performed on apricot and peach fruits, artificially inoculated with conidial suspensions containing different ratios of M. fructicola and M. laxa, showed a high correlation (R 2 = 0.98) between the relative quantity of DNA of the two species quantified by ddPCR and qPCR and a more accurate quantification by ddPCR compared to qPCR at higher concentrations of M. fructicola. The herein described method represents a useful tool for the early detection of Monilinia spp. on stone fruits and for the improving knowledge on the epidemiology of brow rot and interactions between the two prevalent Monilinia species.

Functional Role of Aspergillus carbonariusAcOTAbZIP Gene, a bZIP Transcription Factor within the OTA Gene Cluster.

Aspergillus carbonarius is the principal fungal species responsible for ochratoxin A (OTA) contamination of grapes and derived products in the main viticultural regions worldwide. In recent years, co-expressed genes representing a putative-OTA gene cluster were identified, and the deletion of a few of them allowed the partial elucidation of the biosynthetic pathway in the fungus. In the putative OTA-gene cluster is additionally present a bZIP transcription factor (AcOTAbZIP), and with this work, A. carbonarius ΔAcOTAbZIP strains were generated to study its functional role. According to phylogenetic analysis, the gene is conserved in the OTA-producing fungi. A Saccharomyces cerevisiae transcription factor binding motif (TFBM) homolog, associated with bZIP transcription factors was present in the A. carbonarius OTA-gene cluster no-coding regions. AcOTAbZIP deletion results in the loss of OTA and the intermediates OTB and OTß. Additionally, in ΔAcOTAbZIP strains, a down-regulation of AcOTApks, AcOTAnrps, AcOTAp450, and AcOTAhal genes was observed compared to wild type (WT). These results provide evidence of the direct involvement of the AcOTAbZIP gene in the OTA biosynthetic pathway by regulating the involved genes. The loss of OTA biosynthesis ability does not affect fungal development as demonstrated by the comparison of ΔAcOTAbZIP strains and WT strains in terms of vegetative growth and asexual sporulation on three different media. Finally, no statistically significant differences in virulence were observed among ΔAcOTAbZIP strains and WT strains on artificially inoculated grape berries, demonstrating that OTA is not required by A. carbonarius for the pathogenicity process.

Surface Dielectric Barrier Discharge plasma: a suitable measure against fungal plant pathogens.

Fungal diseases seriously affect agricultural production and the food industry. Crop protection is usually achieved by synthetic fungicides, therefore more sustainable and innovative technologies are increasingly required. The atmospheric pressure low-temperature plasma is a novel suitable measure. We report on the effect of plasma treatment on phytopathogenic fungi causing quantitative and qualitative losses of products both in the field and postharvest. We focus our attention on the in vitro direct inhibitory effect of non-contact Surface Dielectric Barrier Discharge on conidia germination of Botrytis cinerea, Monilinia fructicola, Aspergillus carbonarius and Alternaria alternata. A few minutes of treatment was required to completely inactivate the fungi on an artificial medium. Morphological analysis of spores by Scanning Electron Microscopy suggests that the main mechanism is plasma etching due to Reactive Oxygen Species or UV radiation. Spectroscopic analysis of plasma generated in humid air gives the hint that the rotational temperature of gas should not play a relevant role being very close to room temperature. In vivo experiments on artificially inoculated cherry fruits demonstrated that inactivation of fungal spores by the direct inhibitory effect of plasma extend their shelf life. Pre-treatment of fruits before inoculation improve the resistance to infections maybe by activating defense responses in plant tissues.

Identification of Arthrinium marii as Causal Agent of Olive Tree Dieback in Apulia (Southern Italy).

Olive (Olea europaea L. var. sativa) is one of the most economically important tree crops grown in the Mediterranean basin. Arthrinium Kunze ex Fr. (teleomorph: Apiospora Sacc.) is a widespread fungal genus, and Arthrinium marii Larrondo & Calvo is a ubiquitous species, found in algae, soil, plants, and agricultural communities. A. marii was isolated from olive trees showing dieback from orchards located in Andria and in Fasano, Brindisi (Apulia, southern Italy) and identified based on morphological features and molecular analysis of four genomic regions (ITS, TUB2, TEF1, and LSU). Two-year-old olive plants artificially inoculated with three representative A. marii isolates showed complete dieback within 6 months, and the fungus was reisolated, satisfying Koch's postulates. This is the first report of A. marii causing dieback on olive trees that could represent an important threat for olive cultivation.

Draft Genomic Resources for the Brown Rot Fungal Pathogen Monilinia laxa.

Monilinia laxa is the causal agent of brown rot on stone fruit, and it can cause heavy yield losses during field production and postharvest storage. This article reports the draft genome assembly of the M. laxa Mlax316 strain, obtained using a hybrid genome assembly with both Illumina short-reads and PacBio long-reads sequencing technologies. The complete draft genome consists of 49 scaffolds with total size of 42.81 Mb, and scaffold N50 of 2,449.4 kb. Annotation of the M. laxa assembly identified 11,163 genes and 12,424 proteins which were functionally annotated. This new genome draft improves current genomic resources available for M. laxa and represents a useful tool for further research into its interactions with host plants and into evolution in the Monilinia genus.

New High-Quality Draft Genome of the Brown Rot Fungal Pathogen Monilinia fructicola.

Brown rot is a worldwide fungal disease of stone and pome fruit that is caused by several Monilinia species. Among these, Monilinia fructicola can cause severe preharvest and postharvest losses, especially for stone fruit. Here, we present a high-quality draft genome assembly of M. fructicola Mfrc123 strain obtained using both Illumina and PacBio sequencing technologies. The genome assembly comprised 20 scaffolds, including 29 telomere sequences at both ends of 10 scaffolds, and at a single end of 9 scaffolds. The total length was 44.05 Mb, with a scaffold N50 of 2,592 kb. Annotation of the M. fructicola assembly identified a total of 12,118 genes and 13,749 proteins that were functionally annotated. This newly generated reference genome is expected to significantly contribute to comparative analysis of genome biology and evolution within Monilinia species.

Transcriptome sequence resource for the cucurbit powdery mildew pathogen Podosphaera xanthii.

Podosphaera xanthii is the main causal agent of cucurbit powdery mildew in Southern Italy. Illumina sequencing of mRNA from two P. xanthii isolates of opposite mating types (MAT1-1 and MAT1-2) and their sexual cross was used to obtain a detailed de novo Trinity-based assembly of the transcriptome of the fungus. Over 60 million of high-quality paired-end reads were obtained and assembled into 71,095 contigs corresponding to putative transcripts that were functionally annotated. More than 55% of the assembled transcripts (40,221 contigs) had a significant hit in BLASTx search and included sequences related to sexual compatibility and reproduction, as well as several classes of transposable elements and putative mycoviruses. The availability of these new transcriptomic data and investigations on potential source of genetic variation in P. xanthii will promote new insights on the pathogen and its interactions with host plants and associated microbiome.

Global transcriptome analysis and differentially expressed genes in grapevine after application of the yeast-derived defense inducer cerevisane.

BACKGROUND: Cerevisane, made up of cell wall derivatives from the Saccharomyces cerevisiae strain LAS117, is proposed as a resistance inducer in plants. The mode of action of cerevisane was investigated through transcriptome analysis (RNA-Seq) carried out on leaves of potted vines cv. Italia grown in the greenhouse and sprayed at 1-week intervals with cerevisane. Analyses were performed at three time points after one and three sprays as well as on vines challenged with artificial inoculation with Plasmopara viticola, Erysiphe necator and Botrytis cinerea. RESULTS: Cerevisane proved effective against downy mildew and caused an increase in expression levels of several genes related to defense responses to fungal pathogens and other stresses and down-regulation of genes involved in several processes related to plant growth and development. Up-regulated genes included genes encoding (i) enzymes involved in hormone metabolism (i.e. salicylic acid, jasmonate, ethylene) and related plant responses, (ii) defense compounds (i.e. pathogenesis-related proteins, phenylalanine ammonia-lyase, stilbene synthases, lipoxygenase, leucine-rich repeat receptor-like protein kinases, non-specific plant lipid transfer proteins, serine-threonine protein kinases involved in signal transduction, superoxide dismutase and glutathione S-transferase involved in response to oxidative stress), (iii) secondary metabolites (i.e. phenylpropanoids, terpenoids, lignin), and (iv) photosynthetic processes (light harvesting chlorophyll A/B-binding proteins and components of the photosystems). CONCLUSION: Cerevisane can be a useful tool in protection schedules against downy mildew on grapevine aimed at reducing the usage of synthetic fungicides and preventing fungicide resistance. The results provide the first basic knowledge on the mode of action of yeast-derived elicitors effective against P. viticola on grapevine. © 2019 Society of Chemical Industry.

Genome sequence of the brown rot fungal pathogen Monilinia fructigena.

OBJECTIVES: Monilinia fructigena (phylum Ascomycota, family Sclerotiniaceae) is a plant pathogen that causes brown rot and blossom blight in pome fruit and stone fruit of the Rosaceae family, which can cause significant losses in the field and mainly postharvest. The aim of this study was to create a high-quality draft of the M. fructigena genome assembly and annotation that provides better understanding of the epidemiology of the pathogen and its interactions with the host(s) and will thus improve brown rot management. DATA DESCRIPTION: We report here on the genome sequence of M. fructigena strain Mfrg269 that was collected from plum in southern Italy. This is assembled into 131 scaffolds, with a total size of 43.125 Mb, with 9960 unique protein-coding genes. The novel genomic resources allow improved genomic comparisons among the most important pathogens belonging to the Monilinia genus, with the aim being to improve the knowledge of their plant-pathogen interactions, population biology, and control.

A Ready-to-Use Single- and Duplex-TaqMan-qPCR Assay to Detect and Quantify the Biocontrol Agents Trichoderma asperellum and Trichoderma gamsii.

Trichoderma asperellum strain icc012 and Trichoderma gamsii strain icc080, the microbial active ingredients of RemedierTM (ISAGRO, Novara, Italy), are biocontrol agents (BCAs) employable for crop protection against a wide range of fungal pathogens, including soil-borne pathogens and fungi involved in grapevine trunk disease. In this study, single and duplex real-time quantitative PCR (qPCR) methods to detect and quantify T. asperellum and T. gamsii were developed. Primers/probe sets were designed on the T. asperellum and T. gamsii rpb2 genes and tested for specificity on a panel of microorganisms commonly associated with grape wood and soil. No differences were observed comparing single- and duplex-qPCR assays on different BCAs, 1 pg of target DNA was detected approximately at Cq = 34. R2-values and the efficiency were always equal to 0.99 and >80%, respectively. The detection limit of the duplex-qPCR assay on artificially inoculated samples was 2 × 103 and 4 × 104 conidia g-1 of grape wood tissue and soil, respectively. The methods will be useful to better schedule BCA application in the field and in grapevine nurseries, as well as for investigating the dynamic of BCA populations.

De novo assembly and comparative transcriptome analysis of Monilinia fructicola, Monilinia laxa and Monilinia fructigena, the causal agents of brown rot on stone fruits.

BACKGROUND: Brown rots are important fungal diseases of stone and pome fruits. They are caused by several Monilinia species but M. fructicola, M. laxa and M. fructigena are the most common all over the world. Although they have been intensively studied, the availability of genomic and transcriptomic data in public databases is still scant. We sequenced, assembled and annotated the transcriptomes of the three pathogens using mRNA from germinating conidia and actively growing mycelia of two isolates of opposite mating types per each species for comparative transcriptome analyses. RESULTS: Illumina sequencing was used to generate about 70 million of paired-end reads per species, that were de novo assembled in 33,861 contigs for M. fructicola, 31,103 for M. laxa and 28,890 for M. fructigena. Approximately, 50% of the assembled contigs had significant hits when blasted against the NCBI non-redundant protein database and top-hits results were represented by Botrytis cinerea, Sclerotinia sclerotiorum and Sclerotinia borealis proteins. More than 90% of the obtained sequences were complete, the percentage of duplications was always less than 14% and fragmented and missing transcripts less than 5%. Orthologous transcripts were identified by tBLASTn analysis using the B. cinerea proteome as reference. Comparative transcriptome analyses revealed 65 transcripts over-expressed (FC ≥ 8 and FDR ≤ 0.05) or unique in M. fructicola, 30 in M. laxa and 31 in M. fructigena. Transcripts were involved in processes affecting fungal development, diversity and host-pathogen interactions, such as plant cell wall-degrading and detoxifying enzymes, zinc finger transcription factors, MFS transporters, cell surface proteins, key enzymes in biosynthesis and metabolism of antibiotics and toxins, and transposable elements. CONCLUSIONS: This is the first large-scale reconstruction and annotation of the complete transcriptomes of M. fructicola, M. laxa and M. fructigena and the first comparative transcriptome analysis among the three pathogens revealing differentially expressed genes with potential important roles in metabolic and physiological processes related to fungal morphogenesis and development, diversity and pathogenesis which need further investigations. We believe that the data obtained represent a cornerstone for research aimed at improving knowledge on the population biology, physiology and plant-pathogen interactions of these important phytopathogenic fungi.

Mating System in the Brown Rot Pathogens Monilinia fructicola, M. laxa, and M. fructigena.

Monilinia fructicola, M. laxa, and M. fructigena are the most important pathogens responsible for brown rot disease of stone and pome fruits. Information on their mating system and sexual behavior is scant. A mating-type-specific PCR-based assay was developed and applied to 155 Monilinia isolates from 10 countries and 10 different host plants. We showed that single isolates carry only one of two opposite idiomorphs at the MAT1 locus consistent with a heterothallic mating system for all three species. MAT1-1 and MAT1-2 mating types were detected in similar proportions in samples of isolates of each species and hence there do not appear to be genetic obstacles to the occurrence of sexual reproduction in their populations. Inter simple sequence repeat markers suggested that asexual reproduction is prevalent, but that sexual recombination occurs in M. fructicola populations in Italy. The genetic architectures of the MAT1 loci of the three pathogens were analyzed. MAT1-1 and MAT1-2 idiomorphs are flanked upstream and downstream by the APN2 and SLA2 genes and resemble those of Botrytis cinerea and other heterothallic fungi in the family Sclerotiniaceae. Each idiomorph contains a specific couple of genes, MAT1-1-1 (with alpha-box domain) and MAT1-1-5 in MAT1-1, and MAT1-2-1 (with HMG-box domain) and MAT1-2-10 in MAT1-2. Small gene fragments (dMAT1-1-1 and dMAT1-2-1) from the opposite idiomorph were detected close to their flanking regions. Constitutive expression of the four MAT1 genes during vegetative growth was ascertained by transcriptomic analysis (RNA-Seq). Antisense transcription of the MAT1-1-1 and MAT1-2-1 genes and intergenic transcribed regions of the MAT1 locus were detected. These results represent new insights into the mating systems of these three economically-important pathogens which could contribute to improve the knowledge on their population biology.

Functional Characterization of the alb1 Orthologue Gene in the Ochratoxigenic Fungus Aspergillus carbonarius (AC49 strain).

Aspergillus carbonarius, belonging to the group Nigri, is the main species responsible for contamination by ochratoxin A (OTA) in grapes and derivative products. OTA can accumulate in the mycelium and in black conidia of the fungus and released into the matrix. Here, we have deleted in A. carbonarius the alb1 orthologue gene of A. fumigatus, involved in melanin biosynthesis. Three A. carbonarius Δalb1 mutants were characterized for morphologic traits and OTA production on different media and temperatures. Δalb1 mutants showed a fawn color of conidia associated with a significant reduction of the conidiogenesis and a statistically significant increase (p ≤ 0.01) of total OTA production as compared to the wild type (WT) strain. The alb1 gene somehow affected OTA partitioning since in Δalb1 mutants OTA amount was lower in conidia and was more abundantly secreted into the medium as compared to the WT. On grape berries the Δalb1 mutants and the WT caused lesions with similar sizes but OTA amount in berry tissues was higher for the mutants. These results demonstrate that A. carbonarius conidia pigmentation is largely dependent on polyketide biosynthesis. The gene is not directly involved in virulence and its deletion affects morphological features and OTA production in the fungus.

Phenotypic and Molecular Investigations on Hypovirulent Cryphonectria parasitica in Italy.

Chestnut blight is caused by the fungus Cryphonectria parasitica. As one of the most ecologically important diseases of Castanea spp., C. parasitica can rapidly kill trees. In Europe, mitigation of disease severity took place spontaneously through colonization of C. parasitica by mycoviruses, which reduced the virulence of the fungus. In the framework of a survey, 138 C. parasitica isolates were identified, and virulent/hypovirulent phenotypes were determined through morphological properties and pathogenicity tests. For a pool of four hypovirulent isolates, dsRNA was extracted, cDNA synthesized, and a library subjected to next-generation sequencing. The bioinformatics analysis allowed detecting and reconstructing the complete genome of Cryphonectria hypovirus 1 (CHV-1), denoted as CHV-1 Marche. When compared with the available genomes of other hypoviruses that affected the virulence of C. parasitica, available in databases, CHV-1 Marche showed some nucleotide diversity. The approach used in this study was effective to explore the virome inside a pool of hypovirulent C. parasitica isolates. Next-generation sequencing allowed us to exclude the presence of any other ssRNA and dsRNA viruses infecting the fungus and determine CHV-1 as the only responsible of hypovirulence of C. parasitica in the analyzed samples.

Use of biocontrol agents and botanicals in integrated management of Botrytis cinerea in table grape vineyards.

BACKGROUND: There is increasing interest in the use of biological control agents (BCAs) and botanicals (BOTs) due to increasing awareness of the environmental and human health risks associated with synthetic plant protection products. The BCAs Bacillus subtilis strain QST713, Bacillus amyloliquefaciens strain D747 and Aureobasidium pullulans strains DSM14940 and DSM14941, and the BOTs Melaleuca alternifolia and terpenic extracts are proposed for the control of grey mould in vineyards. This study was aimed at evaluating their effectiveness in integrated crop management strategies and their outcomes in terms of the management of fungicide resistance and residues. RESULTS: In field trials carried out on table grapes in southern Italy, use of BCAs or BOTs alternately or mixtures of BCAs or BOTs with the succinate dehydrogenase inhibitor fungicide fluopyram showed efficacy of up to 96% against grey mould on bunches, comparable with the chemical reference strategy (up to 87%). By contrast, use of BCAs or BOTs (up to 11 sprays) alone was not effective (< 30%) under high disease pressure. The integrated use of BCAs or BOTs reduced the spread of succinate dehydrogenase inhibitor-resistant conidia, as well as fungicide residues in grapes. CONCLUSIONS: Spray schedules based on integration of BCAs or BOTs with fungicides are effective against grey mould and reduce the risk of fungicide resistance in B. cinerea and fungicide residues in grapes. © 2017 Society of Chemical Industry.

RNA-Seq Reveals OTA-Related Gene Transcriptional Changes in Aspergillus carbonarius.

Ochratoxin A (OTA) is a mycotoxin harmful for animals and humans. Aspergillus carbonarius is the main responsible for OTA contamination of grapes and derived products. Gene transcriptional profiling of 4 A. carbonarius strains was carried out by RNA-Seq analysis to study transcriptome changes associated with OTA production. By comparing OTA inducing (OTAI) vs. non-inducing (OTAN) cultural conditions, a total of 3,705 differentially expressed genes (DEGs) (fold change > |2| and FDR ≤ 0.05) were identified. Several genes involved in primary metabolic processes, with particular regard to carbohydrate and amino acid metabolisms, secondary metabolic processes, transport, response to stress and sporulation were up-regulated by OTAI conditions at all the analysed sampling times (4, 6 and 8 DAI) or starting from 6 DAI. Highly up-regulated DEGs encoding enzymes involved in biosynthesis of secondary metabolites, oxidoreductases, transporters and transcription factors were examined for their potential involvement in OTA biosynthesis and related metabolic pathways. Differential expression of genes encoding polyketide synthases (pks), non-ribosomal peptide synthetases (nrps) and chloroperoxidase (cpo) was validated by RT-qPCR. Among clusters of co-regulated genes involved in SM biosynthesis, one putative OTA-gene cluster, including both pks and nrps genes, was detected in the A. carbonarius genome.