Luca Sella

Fusarium graminearum , the main causal agent of Fusarium head blight of wheat, secretes endoxylanases to hydrolyse xylan, a major component of hemicellulose. Mutants of the highly expressed FGSG_03624 ( xylB ) gene previously showed a disease severity level similar to wild‐type (WT) in wheat spikes. In this study, in contrast, three independent deletion mutants of FGSG_10999 ( xylA ), a second highly expressed endoxylanase gene, were significantly impaired in disease severity. Surprisingly, a knock‐out mutant of xylA in the ΔxylB genetic background (ΔΔxylB+A) exhibited virulence comparable to that of ΔxylB and WT. The ΔxylA mutant shared with ΔΔxylB+A a marked reduction in xylanase activity during in vitro growth, and also with ΔxylB a decreased fungal biomass and deoxynivalenol (DON) accumulation in infected spikes. In contrast to ΔΔxylB+A and ΔxylB, ΔxylA revealed down‐regulation of some endoxylanase and effector genes during spike infection. In addition, when grown on a wheat spike medium, the culture filtrate of ΔxylA showed a significant reduction in total protein secretion and a more severe impairment in necrotizing activity in Nicotiana benthamiana infiltrated leaves compared with ΔxylB and ΔΔxylB+A culture filtrates. When a deletion mutant of xylB was generated in the ΔxylA genetic background, the ΔΔxylA+B mutant displayed severely reduced virulence, similar to that of the parental ΔxylA. Altogether, these results demonstrate that xylA plays a critical role in F. graminearum pathogenicity through unexpected pleiotropic, network‐level alterations, as also supported by the partial restoration observed by complementing the xylA gene in ΔxylA, highlighting the strong influence of genetic background on phenotypic outcomes.

Pomegranate dieback is a disease whose etiology remains only partially understood. In this study, surveys were carried out in orchards located in the Apulia, Basilicata, and Veneto regions from 2016 to 2020 with the objective to identify pathogens involved in pomegranate dieback. Six fungal species were isolated from symptomatic trees and identified through morphological and molecular analyses. In addition to the known pomegranate pathogens Neofusicoccum parvum, Diaporthe eres and D. foeniculina, new fungal species, including Neopestalotiopsis rosae, Sporothrix stenoceras, and one belonging to the Xenoacremonium genus, were identified. This study represents the first report of their association with pomegranate plants exhibiting dieback symptoms. When artificially inoculated on pomegranate trees, these fungi caused wood browning, proving their pathogenicity. All fungal species exhibited optimal growth in the temperature range 25–30 °C, although D. eres and N. roseae showed a good adaptability in the range 5–10 °C. Since some of the identified pathogens were isolated from the same trees, cross-pairing assays were conducted, revealing that these fungi can coexist within the same ecological niche while maintaining their viability. Given the need for sustainable management options against these co-occurring pathogens, biological control strategies were evaluated. In vitro experiments demonstrated that both Bacillus and Trichoderma biological control agents (BCAs) inhibit the investigated pomegranate pathogens, highlighting their potential inclusion in integrated management strategies targeting these newly identified fungal pathogens.

Cultivation of pomegranate ( Punica granatum ) is threatened by the emerging pathogens Coniella granati and Phytophthora palmivora . Whether these microorganisms increase their damage due to simultaneous infection and abiotic stress in pomegranate is unknown. Amylo‐X LC is an effective biofungicide based on Bacillus amyloliquefaciens but its efficacy to protect pomegranate from C . granati and P. palmivora if plants are under abiotic stress has not been tested. Therefore, the effects of drought, waterlogging and co‐infection on the aggressiveness of these two pathogens and the efficacy of Amylo‐X LC were evaluated. Pomegranate trees were subjected to regular watering, drought and waterlogging conditions, treated with Amylo‐X LC, and individually or simultaneously inoculated in the stem by C . granati and P. palmivora . Drought experienced by trees limited P. palmivora , while waterlogging favoured C . granati . In combined infections, the presence of P. palmivora amplified the damage caused by C . granati in trees subjected to regular watering and drought. Amylo‐X LC proved effective in preventing P. palmivora damage and did not allow the synergistic effect of P. palmivora on C . granati to occur in trees under regular watering and drought. Drought further reduced P. palmivora lesions in plants pretreated with Amylo‐X LC compared to those under regular watering. The impact of abiotic stress in pomegranate during C . granati and P. palmivora infections and the efficacy of B. amyloliquefaciens as a potential agent against pomegranate dieback were discussed. We conclude that further efforts to evaluate the efficacy of biological control agents on crops exposed to different environmental scenarios are needed.

Alternaria is a genus that contains several important plant pathogens affecting nearly 400 plant species worldwide, including economically important crops such as grapes, citrus, and ornamental plants. Rapid, scalable, and efficient methods of pathogen detection are crucial for managing plant diseases and ensuring agricultural productivity. Current amplicon sequencing protocols for Alternaria detection often require the enzymatic barcoding of amplicons, increasing hands-on time, cost, and contamination risk. We present a proof-of-concept study using custom barcoded primers, combining universal primers targeting ITS1 and ITS2 regions (600 bp) coupled with Oxford Nanopore Technologies (ONT) barcode sequences. Sequencing was performed on infected grapevine, mandarin orange, thuja, and maple tree samples. In total, we analyzed 38 samples using qPCR; 8 tested positive for Alternaria, which were sequenced using a newly developed protocol. As a result, we could identify Alternaria in every positive sample, and besides the pathogen of interest, we could identify the associated mycobiome. This protocol reduces hands-on time and cost, making a significant advancement over current sequencing methods. Future work will focus on optimizing our approach for high-throughput sequencing of up to 96 samples and determining the method’s applicability for large-scale mycobiome analysis.

In recent years, a new phenomenon of early olive drop is causing production losses in olive groves throughout northern Italy. To analyze the possible causes, field and laboratory trials were performed to assess the involvement of fungal pathogens and insect pests in this disease. External and internal symptoms of fungal infections or insect-feeding activities were researched. Fungi present in healthy and dislodged olives were investigated. The relationship between olives that fell and Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) infestation was assessed in a controlled infestation trial, and the effectiveness of an insecticidal strategy in reducing early olive drop was tested in open field conditions. A comparable number of fungi, mostly endophytes, were isolated and identified from both healthy and dislodged olives. The damage observed on dislodged olives was primarily ascribed to pentatomids feeding activity. Six stink bugs species were found in olive canopies, that is, the invasive H. halys, which was by far the most abundant, and Acrosternum heegeri Fieber, Nezara viridula (Linnaeus), Palomena prasina (Linnaeus), Piezodorus lituratus (Fabricious), and Rhaphigaster nebulosa (Poda). Halyomorpha halys caused intense fruit drop in the controlled infestation trial, and its infestation level significantly correlated with the number of olives that fell. Native stink bugs, present in much lower population compared to H. halys, could also partially contribute to early drop of olives. Insect proof net significantly reduced the early olive drop disease, while insecticide applications only partially reduced the stink bugs population density and, proportionally, early olive drop.

Botrytis cinerea is a well-known plant pathogen responsible for grey mould disease infecting more than 500 plant species. It is listed as the second most important plant pathogen scientifically and economically. Its impact is particularly severe in grapes since it affects both the yield of grape berries and the quality of wines. While various methods for detecting B. cinerea have been investigated, the application of Oxford Nanopore Technology (ONT) for complete ribosomal operon sequencing, which has proven effective in human and animal fungal research and diagnostics, has not yet been explored in grapevine (Vitis vinifera) disease research. In this study, we sequenced complete ribosomal operons (∼5.5 kb amplicons), which encompass the 18S, ITS1, 5.8S, ITS2, and 28S regions, from both pure cultures of B. cinerea and infected grapevine leaf samples. Minimap2, a sequence alignment tool integrated into the EPI2ME software, served as a taxonomy classifier, utilizing the custom reference database FRODO. The results demonstrate that B. cinerea was detectable when this pathogen was not the dominant fungal species in leaf samples. Additionally, the method facilitates host DNA-free sequencing and might have a good potential to distinguish other pathogenic and non-pathogenic fungal species hosted within grapevine’s infected leaves, such as Alternaria alternata, Saccharomyces cerevisiae, Saccharomyces boulardii, Mucor racemosus, and Ascochyta rabie. The sequences were uploaded to the NCBI database. Long amplicon sequencing method has the capacity to be broadened to other susceptible crops and pathogens, as a valuable tool for early grey rot detection and mycobiome research. Future large-scale studies are needed to overcome challenges, such as comprehensive reference databases for complete fungal ribosomal operons for grape mycobiome studies.

Plasmopara viticola, the agent of grapevine downy mildew, causes enormous economic damage, and its control is primarily based on the use of synthetic fungicides. The European Union policies promote reducing reliance on synthetic plant protection products. Biocontrol agents such as Trichoderma spp. constitute a resource for the development of biopesticides. Trichoderma spp. produce secondary metabolites such as peptaibols, but the poor water solubility of peptaibols limits their practical use as agrochemicals. To identify new potential bio-inspired molecules effective against P. viticola, various water-soluble peptide analogs of the peptaibol trichogin were synthesized. In grapevine leaf disk assays, the peptides analogs at a concentration of 50 μM completely prevented P. viticola infection after zoosporangia inoculation. Microscopic observations of one of the most effective peptides showed that it causes membrane lysis and cytoplasmic granulation in both zoosporangia and zoospores. Among the effective peptides, 4r was selected for a 2-year field trial experiment. In the vineyard, the peptide administered at 100 μM (equivalent to 129.3 g/ha) significantly reduced the disease incidence and severity on both leaves and bunches, with protection levels similar to those obtained using a cupric fungicide. In the second-year field trial, reduced dosages of the peptide were also tested, and even at the peptide concentration reduced by 50 or 75%, a significant decrease in the disease incidence and severity was obtained at the end of the trial. The peptide did not show any phytotoxic effect. Previously, peptide 4r had been demonstrated to be active against other fungal pathogens, including the grapevine fungus Botrytis cinerea. Thus, this peptide may be a candidate for a broad-spectrum fungicide whose biological properties deserve further investigation.