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In this work, liposomes loaded with the fungicide, Fludioxonil (FLUD), for the containment of fungal diseases in agriculture were developed. Three types of vesicles with different compositions were compared: (I) plain vesicles, composed of soy phosphatidylcholine and cholesterol; (II) PEG-coated vesicles, with an additional polyethylene glycol coating; and (III) cationic vesicles, containing didodecyldimethylammonium bromide. Nanometric-sized vesicles were obtained both by the micelle-to-vesicle transition method and by the extrusion technique, and encapsulation efficiency, drug loading content, and Zeta potential were determined for all the samples. The extruded and PEGylated liposomes were the most stable over time and together with the cationic ones showed a significant prolonged FLUD release capacity. The liposomes’ biological activity was evaluated on conidial germination, germ tube elongation and colony radial growth of the ascomycete Botrytis cinerea, a phytopathogenic fungus affecting worldwide many important agricultural crops in the field as well as in the postharvest phase. The extruded and PEGylated liposomes showed greater effectiveness in inhibiting germ tube elongation and colony radial growth of the fungal pathogen, even at 0.01 µg·mL−1, the lowest concentration assessed.

Grapevine trunk diseases are a threat to table- and grape-wine cultivation worldwide. Phaeomoniella chlamydospora (Pch) is a vascular fungus recognized as one of the most important pathogens associated with grapevine trunk diseases. The relationships between xylem vessel features and Pch susceptibility of 10 table- and 17 wine-grape genotypes, as well as 3 rootstocks, were investigated by image analysis of 50 µm cross-sections and artificial Pch inoculation on one-year-old vine cuttings. Vessels were grouped in the diameter classes 1–30, 31–60, 61–90, 91–120, and >120 µm. Among the table-grape varieties, ‘Sable’, ‘Timco’, and ‘Red Globe’ showed higher densities of large vessels (>120 μm) than ‘Italia’, ‘Sugar Crisp’, and ‘Sugraone’. Among the wine-grape varieties, ‘Minutolo’, ‘Montepulciano’, ‘Primitivo’ CDTa19, and ‘Verdeca’ showed higher densities of large vessels than ‘Aglianico’, ‘Nero di Troia’, ‘Sangiovese’, and ‘Susumaniello’. In the rootstocks, the vessel diameters were 50.8, 54.0, and 60.9 μm for ‘34 E.M.’, ‘140 Ruggieri’, and ‘1103 Paulsen’, in that order. For table-grape varieties, Pch was re-isolated from 13.3% for ‘Sugar Crisp’ and ‘Sugraone’ cuttings up to 93.3% for ‘Timco’. For wine-grape varieties, Pch re-isolation ranged from 51.1% (‘Bombino nero’, ‘Negroamaro’ D15, and ‘Sangiovese’) to 81.1% (‘Montepulciano’), while for the rootstocks, the values were from 33 to 51%. A principal component analysis (PCA) revealed a positive correlation between the frequencies of large vessels and Pch re-isolation. In addition, in wine grapes and rootstocks, higher Pch re-isolation frequencies in the lateral parts of cuttings were correlated (r = 0.79) to a higher frequency of large vessels. The results highlight relationships between grapevine xylem vessel sizes and susceptibility to P. chlamydospora that are worthy of further research.

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.

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 (R2 = 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.

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.

Grapevine trunk diseases (GTDs) are one of the most economically important diseases of grapevines, causing yield reductions and limiting vineyard lifespans. Fungal pathogens responsible for GTDs primarily infect grapevines through pruning wounds. The lack of systemic fungicides to stop the advance of fungi in vine vascular systems makes the use of pruning wound protection with either synthetic chemicals or biological control agents (BCAs) the main available GTD control strategy. Demands for sustainable grape production and increasing limitations on pesticide usage have raised interest in potential use of BCAs as pruning wound protectants against GTDs. The objectives of this study were to characterize 16 Trichoderma isolates from southern Italy using molecular methods, and to evaluate their potential activity as BCAs in vitro against the GTD canker-causing fungi Diplodia seriata, Eutypa lata and Neofusicoccum parvum. Molecular studies along with phylogenetic analyses identified eight species, including Trichoderma atroviride, T. guizhouense, T. harzianum, T. koningiopsis, T. longibrachiatum, T. paratroviride, T. paraviridescens, T. spirale, and one unidentified Trichoderma sp. All these species had optimum mycelium growth between 25 and 35°C. In vitro dual culture experiments assessed antagonistic capabilities of all the Trichoderma isolates against D. seriata, E. lata and N. parvum, and showed that the isolates inhibited mycelial growth of the pathogens by up to 70%. The most inhibitory isolates were tested in planta for capability to protect pruning wounds against D. seriata and N. parvum in a detached cane assay. All the selected Trichoderma isolates gave >80% control of D. seriata and 60% control of N. parvum, and protected pruning wounds for up to 21 d after treatment.

Abstract 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.

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 OTβ. 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.

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.

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.

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.