ABSTRACT Functional characterisation of fungal pathogen effectors is critical in designing durable resistance strategies in wheat ( Triticum aestivum ). Here, we explore the use of Ustilago hordei , a basidiomycete fungal pathogen of barley, as a surrogate system for delivery and functional analysis of effectors from other fungal pathogen species in wheat. Both the haploid wild‐type strain Uh364 and its modified solopathogenic derivative UhDS200 can grow in wheat without eliciting obvious nonhost reactions when infiltrated. We demonstrate that U. hordei can express and secrete effectors SnTOX1 and SnTOX3 from Parastagonospora nodorum into wheat and trigger within 48 h visible necrosis in cultivars having corresponding receptors, which are susceptibility factors in this inverse gene‐for‐gene pathosystem. At the molecular level, this reaction is accompanied by production of reactive oxygen species and induced expression of the wheat PR1b gene, a marker of hypersensitive cell death. However, the system showed limitations in the delivery into wheat of rust fungus effectors. When testing proven Puccinia graminis f. sp. tritici avirulence effectors PgtAVRSr35 or PgtAVRSr50, though transcripts were produced, no protein products could be detected or cell death reactions observed in cultivars harbouring the corresponding resistance genes Sr35 or Sr50 . With previous confirmation of secretion by U. hordei of effectors from Cladosporium fulvum and Fusarium species, our results show that the U. hordei secretion system provides an alternative platform for functional studies of heterologous effectors in wheat to help advance our understanding of plant–pathogen interactions and assist with breeding efforts to achieve resistance to various pathogens.

ABSTRACT In sustainable agriculture, conservation soil tillage methods and organic fertilisation improve soil conditions, impacting the soil microbial population. The present work aimed to evaluate the effects of the combination of these practices on rhizosphere pathobiome distribution and biodiversity of durum wheat rotated with potato during a 2‐year field trial in central Italy. Two conservation tillage techniques (subsoiling and spading) versus ploughing, and organic compost‐based versus mineral fertilisation, were compared across three durum wheat growth stages. Alpha and beta diversity tests indicated that the compost treatment increased rhizosphere microbial diversity and altered community composition during tillering and ripening across both years. Pathogenic and mycotoxigenic fungi were prevalent in the wheat rhizosphere. Alternaria alternata and A. infectoria , potential causal agents of ‘black points’ of wheat grains, were more abundant in plots with mineral fertilisation at the heading stage across the 2 years. Fusarium equiseti and F. brachygibbosum , causal agents of Fusarium head blight, were more abundant in 2022 under spading and mineral fertilisation at tillering, and Fusarium verticillioides under ploughing and mineral fertilisation at heading. In contrast, organic fertilisation promoted the presence of beneficial and potentially antagonistic fungi. Species including Vishniacozyma victoriae , Trichoderma amazonicum , Botryotrichum atrogriseum and Humicola sardiniae were observed in greater relative abundance following compost fertilisation, though their abundance varied by year and growth stage. These findings show that sustainable soil management practices influence the rhizosphere pathobiome, potentially contributing to microbial colonisation of the phyllosphere and carposphere of wheat and rotated crops, highlighting the interconnectedness of soil and plant health.

ABSTRACT Phomopsis capsici has recently been identified as the fungal pathogen responsible for walnut ( Juglans regia ) branch blight, a disease that significantly reduces yield and causes substantial economic losses. Despite the known role of secondary metabolites in fungal pathogenicity, the specific toxic components of P. capsici and their mechanisms of action remain poorly understood. This study aimed to isolate and characterise the toxic substances produced by this pathogen to elucidate its phytotoxic effects. Two non‐protein toxins, PC‐1 and PC‐2, were isolated and purified from P. capsici using HPLC, mass spectrometry and NMR. Structural analysis identified PC‐1 as 5‐methylmellein (C 11 H 12 O 3 ), a known phytotoxin, and PC‐2 as tyrosol (C 8 H 10 O 2 ), a compound with antibacterial and antioxidant properties. Biological assays revealed that PC‐1 exhibited strong toxicity, causing severe necrosis and cell death in walnut branches, while PC‐2 showed minimal toxic effects. Further investigation demonstrated that PC‐1 disrupts cellular homeostasis by inducing excessive accumulation of reactive oxygen species (ROS), leading to membrane damage and cell death. This study also observed significant increases in the activities of defence enzymes, superoxide dismutase and peroxidase, in response to PC‐1, highlighting the role of oxidative stress in disease progression. This research represents the first isolation and characterisation of toxic components from P. capsici , providing critical insights into its virulence mechanisms. The findings contribute to a broader understanding of Phomopsis species as both endophytes and pathogens, offering a foundation for developing targeted control strategies for walnut branch blight and other fungal diseases in agriculture, with implications for ensuring global food security.

ABSTRACT Brown‐spot needle blight (BSNB) and Dothistroma needle blight (DNB), caused by Lecanosticta acicola and Dothistroma septosporum , respectively, are foliar fungal diseases threatening pine forests worldwide. They impact natural ecosystems, plantations and nurseries, with risks expected to intensify under climate change and global plant trade. Although caused by different pathogens, BSNB and DNB have similar symptoms, complicating diagnosis. To understand host–pathogen interactions, we investigated physiological and biochemical host responses that may help differentiate these diseases. We studied three ecologically and economically important pine species— Pinus radiata , P. pinaster and P. pinea —after needle inoculation with both pathogens under controlled conditions. At 115 days post‐inoculation, symptomless needles from infected plants and controls were analysed for systemic response, including growth, gas exchange, primary and secondary metabolites profiles and malondialdehyde (MDA), as a stress biomarker. While both pathogens induced similar visible symptoms, host responses were distinct. In P. pinea , L. acicola reduced photosynthetic pigment content (PHP) and photosynthesis, whereas D. septosporum only affected PHP. Both pathogens triggered defence‐related metabolic adjustments, including reduced growth, increased total phenolic compounds (TPC) and MDA. In P. pinaster , D. septosporum lowered TPC and MDA, while L. acicola decreased PHP. P. radiata maintained growth, while reducing MDA levels. These findings highlight that similar symptoms can conceal contrasting, species‐specific host responses. Understanding these differences is essential and highlights the need for molecular studies to identify early detection biomarkers that can differentiate BSNB from DNB, as host metabolic responses offer promising yet underexplored potential for advancing sustainable disease management in forest protection.

ABSTRACT Virus yellows (VY), caused by beet chlorosis virus (BChV), beet mild yellowing virus (BMYV) and beet yellows virus (BYV), is a serious disease affecting sugar beet, leading to significant yield losses. The main vector is the green peach aphid Myzus persicae . Beet mosaic virus (BtMV) is often associated with VY due to the shared vector. Different host plants of VY‐associated viruses can play an important role in the aphid's life cycle and the VY‐epidemiology. This study evaluated the susceptibility of 25 cover crops and flower strip plants to VY‐associated viruses as well as their attractiveness to M. persicae . None of the tested plants were susceptible to BYV or BtMV, while Gypsophila elegans tested positive for BChV and 13 species tested positive for BMYV. Seven of these, including important cover crops and flower strip plants such as Linum usitatissimum , Trifolium resupinatum , T. incarnatum and Centaurea cyanus , were identified as new BMYV hosts. Plant attractiveness to M. persicae varied widely. Under field conditions, only a few species, such as Sinapis alba or Raphanus sativus var. oleiferus , showed high levels of natural infestation by M. persicae . However, a field choice experiment revealed that BMYV infection can occur in different plants, even at low levels of M. persicae infestation. Field trials also confirmed that M. persicae can transmit BMYV from infected cover crops to sugar beet. The findings emphasise the potential importance of cover crops and flower strip plants as green bridges in VY‐epidemiology and highlight the need for research in large‐scale field studies.