Dottorato in BIOLOGIA AMBIENTALE ED EVOLUZIONISTICA

Titolo della tesi: Metagenomics applied to the study of “Kiwifruit Vine Decline Syndrome” (KVDS) and management of affected plants

Kiwifruit Vine Decline Syndrome (KVDS) is a major emerging threat to kiwifruit orchards, particularly in Italy. First reported in Veneto in 2012, KVDS has since spread to other major kiwifruit-producing regions, causing substantial economic losses due to its severe impact on plant health, yield reduction, and vine death. Despite extensive research, the exact aetiology remains unclear, with a complex interplay of abiotic and biotic factors likely driving disease progression. This PhD project aimed to investigate the microbial community composition associated with KVDS, assess potential pathogenic agents, and explore sustainable disease management strategies, including biofumigation. To achieve these objectives, a combination of metabarcoding, microbiological isolation, pathogenicity assays, and soil health assessments was employed across multiple symptomatic and asymptomatic kiwifruit orchards in the Lazio region over three years (2020-2023). Survey and Microbial Community Assessment: A systematic survey of 20 orchards in the Lazio region was conducted, collecting root and rhizosphere soil samples in spring and autumn. Using culture-dependent and culture-independent approaches, bacterial and fungal/oomycete communities were analysed through Illumina amplicon sequencing of the 16S rRNA gene and ITS2 region, respectively. Results showed that KVDS-affected orchards exhibited significant shifts in microbiota community composition. Pathogen Identification and Pathogenicity Test: Direct isolations from symptomatic roots identified a recurrent presence of Phytopythium vexans, which was consistently associated with diseased plants across multiple years and locations. To confirm pathogenicity, greenhouse trials were conducted under controlled conditions, where Pp. vexans and other putative pathogens were inoculated onto kiwifruit plants under both standard and waterlogging conditions. Inoculated plants displayed increased symptom severity, particularly under excessive irrigation, reinforcing the hypothesis that KVDS is linked to root stress. Soil Health and Enzymatic Activity in KVDS-Affected Orchards: This study demonstrated how KVDS and seasonality influence soil properties, enzymatic activities, and microbial diversity in KVDS-affected orchards. Symptomatic areas exhibited notable shifts in microbial communities, with decreased diversity and increased abundance of opportunistic pathogens. Enzymatic activity patterns, particularly β-glucosidase, suggest potential disruptions in plant-soil interactions. To control the effects of KVDS, maintaining appropriate irrigation levels and employing soil conservation practices are essential to preserving soil structure and microbial stability. The intricate relationship between KVDS, soil health, and microbial communities highlighted the necessity of a comprehensive management approach. Future research should incorporate long-term monitoring and environmental variables to refine disease mitigation strategies and support sustainable kiwifruit production. Rebalancing microbial communities remains a priority in mitigating the impact of KVDS on orchard ecosystems. Biofumigation as a Potential Management Strategy: A preliminary open-field trial was conducted using Biofence FL, a biofumigant derived from Brassica carinata. Soil microbial composition was monitored before and after treatment to assess its impact on soil health and pathogen suppression. Although no significant changes were observed in overall microbial diversity, targeted reductions or increases in oomycetes were noted, suggesting that biofumigation may be a promising avenue for KVDS management when integrated into broader soil health restoration strategies. Fusarium Identification and its Role in KVDS: Although Fusarium species were only sporadically isolated from symptomatic kiwifruit roots, this study provided an opportunity to further refine and test the FusaHelp platform, an online dichotomous key designed for the accurate morphological identification of Fusarium species. Available at www.fusahelp.com, this tool complements molecular approaches, making fungal diagnostics more accessible to researchers and practitioners. With over 20,000 accesses, FusaHelp has demonstrated a significant impact on the scientific community, supporting the identification and management of Fusarium-related diseases across multiple economically important crops. Conclusions and Implications: This research shed light on the multifactorial nature of KVDS, highlighting the role of soil microbiota in plant health. The combination of metabarcoding and traditional microbiological techniques provided novel insights into the microbial ecology of KVDS-affected orchards. Findings underscored the need for integrated disease management approaches that combine improved soil health practices and sustainable agronomic strategies. Future research should focus on refining biofumigation protocols, exploring treatments for disease suppression, and further elucidating the interactions between environmental stressors and microbial community shifts in KVDS development. By advancing our understanding of KVDS and its microbiota dynamic, this study will contribute to the development of more resilient kiwifruit production systems and provide a framework for managing emerging plant diseases linked to microbial dysbiosis.