Pseudomonas Species Associated with Leaf Spot of Spinach in Serbia

The aim of this study was to identify and characterize the bacterial pathogen causing leaf spot of spinach using phenotypic and molecular analysis. Isolation on NSA resulted in the formation of numerous morphologically uniform Pseudomonas-like colonies, from which 12 representative isolates were selected for further identification. All isolates caused disease symptoms on spinach plants and leaves inoculated under controlled conditions. The LOPAT test results indicated that the spinach isolates could not be assigned to any of the previously described groups of fluorescent Pseudomonas. Based on multi-locus sequence analysis (MLSA) of the gltA, gapA, gyrB, and rpoD genes, the spinach isolates were assigned to distinct phylogenetic lineages within the broad Pseudomonas fluorescens species complex. Nine isolates (SP12, SP21, SP22, SP31, SP32, SP33, SP34, SPR1, SPR4) formed a core clade (Group I) clustering with unclassified Pseudomonas sp. reference strains, while SP14 and SP24 formed well-supported basal branches leading to this group. Isolate SPR3 represented an early-diverging lineage branching between the P. marginalis reference group and the remaining spinach isolates. These findings provide the first characterization of Pseudomonas strains associated with leaf spot of spinach in Serbia and demonstrate diversity within the P. fluorescens species complex on this host, though whole-genome sequencing will be required for definitive species-level identification. Host range assessment revealed significant differences in AUDPC values among the tested host types (Holm-adjusted p < 0.01). Beet exhibited the highest disease intensity, followed by sugar beet, whereas Swiss chard showed the lowest disease intensity under the experimental conditions. These findings demonstrate the considerable diversity of the Pseudomonas population associated with spinach and represent the first report of spinach-associated strains belonging to the P. fluorescens species complex in Serbia (Vojvodina), where such investigations have not previously been conducted or reported.