Multi-year surveillance of race structure and pathogenic variation in Fusarium oxysporum f. sp. lactucae populations in Spain

Fusarium wilt of lettuce, caused by Fusarium oxysporum forma specialis lactucae (FOLac), is an increasing constraint to lettuce production worldwide, particularly because changes in race structure and the emergence of isolates able to overcome host resistance can compromise cultivar-based management. However, information on FOLac race composition and pathogenic variation in Spain remains limited. This study characterized the race structure, molecular diversity, and pathogenic behavior of FOLac isolates collected in Spain between 2019 and 2025. A total of 81 isolates recovered from symptomatic lettuce tissues and soil samples from major lettuce-producing regions were analyzed. Race assignment was performed using race-specific PCR and detection of SIX effector genes, and phylogenetic relationships were inferred from concatenated tef-1α and rpb2 sequences. Pathogenicity assays were conducted with selected race 1 on differential lettuce cultivars to assess aggressiveness and resistance responses. Race 1 was predominant, representing 75.3% of the isolates, and showed broad geographic distribution and temporal persistence. Race 4 was less frequent, representing 9.9% of the isolates, but was detected in southeastern Spain, indicating localized occurrence in a major production area. The remaining 14.8% of the isolates, all recovered from soil samples, were not assigned to FOLac. Phylogenetic analysis separated isolates molecularly assigned to FOLac from non-assigned F. oxysporum isolates but did not resolve races 1 and 4 as distinct groups. Pathogenicity assays revealed variation in aggressiveness among race 1 isolates, including resistance-breaking behavior on `Costa Rica No. 4´ and `Palmos´. These results show that FOLac populations in Spain are epidemiologically and biologically heterogeneous and highlight the need to integrate molecular tools and pathogenicity assays for race surveillance, breeding programs, and disease management.