Almond decline and dieback have become significant challenges in newly established orchards, with symptoms including internal necrosis, canker, and external gummosis. This work aims to explore the potential fungal and bacterial causative agents through metabarcoding and traditional culture plate isolation across six almond cultivars. Our results emphasize the multifactorial nature of almond decline and dieback, with possible co-infections by opportunistic fungi and bacteria playing a central role. Classical isolation identified 47 fungal species or genera, including Diaporthe amygdali, Diplodia corticola, Phytophthora sp., and several Fusarium species. Almond metabarcoding revealed a more diverse microbial community, highlighting the prevalence of soilborne pathogens such as Neocosmospora rubicola, Dactylonectria estremocensis, and Plectosphaerella niemeijerarum. Soil metabarcoding suggested that these pathogens likely originate from nursery substrates or soils shared with other crops, such as olives and vineyards, that serve as a source of inoculum. ‘Soleta’ generally presented lower richness when compared to the other tested cultivars, suggesting a higher degree of biotic stress and decreased plant resilience. This study highlights the value of integrating NGS approaches to comprehensively study complex diseases and the need for further research on pathogen interactions and cultivar susceptibility for the future development of new sustainable, targeted management strategies in almond orchards.

With the rising prominence of organic farming systems in European Union countries, motivated by agricultural policies, there is pressure for effective disease management strategies. To address this challenge, the use of plant resistance inducers (PRIs) and elicitors has emerged as a promising approach. In this study, we compared the impact of integrated production with organic agriculture farming practices, specifically applying PRIs and elicitors in the latter, on the expression levels of stress-responsive genes in two grapevine cultivars, ‘Alicante Bouschet’ and ‘Trincadeira’. Our findings revealed that the organic farming system led to upregulation of eight of the 12 studied genes in at least one cultivar, indicating a significant influence of production mode. The upregulated genes were associated with plant stress-responsive genes (PR1, PR2, PR4, and TLP9), sugar metabolism (HT5), phenylpropanoids (STS1), enzymes related to jasmonic acid synthesis and response to biotic stresses, respectively (LOX, PER42). Also, the ‘Alicante Bouschet’ cultivar consistently displayed significantly higher levels of transcript accumulation on most of the stress-related genes compared to the ‘Trincadeira’ cultivar in both production modes. Our study provides valuable insights into the effectiveness of PRIs and elicitors in increasing plant expression levels of stress-responsive genes, leading to greater resilience to pathogen attacks and emphasizing their position in organic agriculture.

Antimicrobial peptides (AMPs) are key components of plant innate immunity, offering broad spectrum protection against pathogens and represent promising alternatives to chemical pesticides for sustainable crop protection. Despite their broad range antimicrobial activity and low potential for resistance development, the deployment of AMPs in agriculture has been severely limited by instability, poor bioavailability and the lack of efficient, field-compatible delivery strategies. Harnessing viral vectors as platforms for AMP expression in plants represents a powerful strategy to enhance plant innate immunity. This review provides an overview of the potential of viral vectors for transient gene expression, functional genomics and genome editing. We discuss the design, construction and delivery of viral vectors, as well as the main challenges associated with AMP expression, including cytotoxicity and stability. Finally, inspired by adeno-associated virus (AAV) mediated AMP delivery strategies in mammals, we propose a vaccine-like strategy for plant protection, in which viral vectors enable endogenous AMP production Although plants lack adaptive immunity, virus-mediated AMP expression may function as a biochemical analog, reinforcing basal defence layers and enhancing tolerance to pathogen infection. By integrating viral biotechnology with plant defence mechanisms, this approach could redefine the future of sustainable agriculture.

Plant viruses pose serious threats to global crop production, and members of the genus Tobamovirus are particularly problematic due to their environmental stability, efficient mechanical transmission and rapid global spread. Tomato brown rugose fruit virus (ToBRFV) has emerged as one of the most damaging tobamovirus affecting tomato, a crop of major economic importance worldwide. ToBRFV has been reported in more than 45 countries, including Portugal. However, to date, no peer-reviewed molecular characterization of local isolates has been published, and official records classify its presence in Portugal as transient. This study confirms the occurrence of ToBRFV and provides the first comprehensive genomic and phylogenetic characterization of local virus isolates in Portugal. RNA-seq generated 192,852,438 reads, of which 103,882,115 (58.9%) mapped to ToBRFV, allowing reconstruction of a complete 6393 nt viral genome. A second full-length consensus sequence was independently obtained from the same composite sample using an overlapping Sanger sequencing strategy, differing by only two SNPs. Comparative genomic, functional, structural, and phylogenetic analysis revealed low diversity, with most variation located in replicase-coding regions, while movement and coat protein genes remained highly conserved. Nucleotide-based phylogenies resolved geographically structured clades, although the Portuguese sequences formed a strongly supported subclade with a Chinese isolate. These findings support recent global dissemination of ToBRFV and reinforce the importance of integrated surveillance and genomic monitoring for effective virus management.