Deciphering the Molecular Mechanisms Underlying Avocado Dieback Disease Caused by Neofusicoccum luteum

Background Neofusicoccum luteum is a major causal agent of avocado dieback disease, causing significant economic losses in major producing regions including California, Italy, and Spain. Despite its global importance, the molecular mechanisms underlying N. luteum pathogenicity and tissue-specific infection strategies remain poorly understood. To address this knowledge gap, we sequenced and assembled the complete genome of the virulent N. luteum strain UMAF A1963 and performed transcriptomic analysis of the infection of avocado branches and fruits compared to in vitro growth on potato dextrose agar. Results The high-quality genome assembly (40.82 Mb, N50 = 2.10 Mb) achieved 95% completeness based on Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis and revealed 664 carbohydrate-active enzyme genes (CAZyme), indicating robust plant cell wall degradation capacity. Comparative genomic analysis with related Botryosphaeriaceae species showed extensive conservation, with 7,300 of 10,842 gene clusters (67%) forming a shared core genome, while species-specific clusters reflect lineage-specific adaptations. RNA-seq analysis revealed extensive transcriptional reprogramming during infection, with 2,875 and 2,419 differentially expressed genes identified in branch and fruit infections, respectively, compared to in vitro culture. Functional enrichment analysis revealed tissue-specific strategies: branch infection was characterized by protein refolding, cellulose metabolism, and siderophore processes, while fruit infection showed enhanced carbohydrate transport, glycosphingolipid metabolism, and mitochondrial functions. Among the 235 candidate virulence factors identified, cell wall-degrading enzymes (CWDEs), proteases, and fungal toxins were the most prevalent functions, followed by others such as hormone metabolism, transport proteins, stress response factors, detoxification enzymes, and transcriptional regulators. Genome-wide effector prediction identified 2,745 candidates, with 38 high-confidence effectors showing homology to characterized pathogenicity factors and exhibiting both apoplastic and cytoplasmic targeting patterns. Notably, a putative pectate lyase ( NelutCtg15g0740.1 ) achieved exceptional upregulation (FC = 2,129.81 in branches; FC = 19,186.08 in fruits) and was predicted as a dual-localization effector with 54% cytoplasmic and 76% apoplastic probabilities. Conclusions This study provides the first comprehensive molecular characterization of N. luteum pathogenicity, revealing sophisticated tissue-specific virulence strategies mediated by coordinated expression of cell wall-degrading enzymes, transport systems, detoxification mechanisms, hormone metabolism and effector proteins. The genomic resources and mechanistic insights generated establish a foundation for developing targeted management strategies against avocado dieback disease and advance our understanding of Botryosphaeriaceae pathogenesis.