Intraspecific genetic diversity of Paracidovorax citrulli as an emerging threat to watermelon production in Serbia

Bacterial fruit blotch, caused by Paracidovorax citrulli , is a destructive disease affecting the foliage and fruits of a wide range of cucurbit crops. Because of its seed‐borne nature and high potential for rapid spread, P. citrulli has been recognised as a quarantine organism in Europe and beyond. In Serbia, the first occurrence was recorded in 2014, with subsequent outbreaks reported in 2018, 2021 and 2022 at several localities. Multilocus sequence analysis (MLSA) ( gltA , gmc , pilT , ugpB , gyrB , Aave_1548 ), together with rep‐ (GTG 5 ‐) and arbitrarily primed PCR, indicated the presence of two described groups among Serbian populations: Group I, comprising strains isolated in 2021 and 2022, and Group II, including strains from the first outbreak in 2014 and from 2018. Additionally, analysis of the Aave_1548 gene clearly separated strains within Group I into a third, subtle subcluster, based on a single nucleotide polymorphism. In contrast, the lepA , phaC and trpB genes in MLSA, as well as BOX‐PCR, showed the lowest discriminatory power, resulting in highly similar profiles across all strains. Virulence assays on mature fruits showed the highest level of watery flesh rot on watermelon, followed by cucumber, squash and melon. Inoculated tomato fruits (non‐cucurbit) developed water‐soaked lesions localised at the inoculation site. Generalised linear mixed model analysis segregated the strains into two groups similarly to MLSA: I—more virulent on melon, and II—more virulent on watermelon, cucumber and squash. These findings enhance understanding of the genetic diversity and pathogenic variability of P. citrulli in Serbia and support improved monitoring and disease management strategies.