Zucchini (Cucurbita pepo) is worldwide affected by Pseudomonas syringae, inducing vein clearing, stunting, and necroses during plantlet development. A collection of 58 P. syringae strains isolated from diseased zucchini plantlets was characterized by multilocus sequence analysis (MLSA). A subset of 23 strains responsible for vein clearing of zucchini (VCZ) was evaluated for pathogenicity on zucchini, and their genomes were sequenced. The host range of six VCZ strains was evaluated on 11 cucurbit species. Most VCZ strains belong to clades 2a and 2b-a within phylogroup 2 of P. syringae species complex and are closely related to other strains previously isolated from cucurbits. Genome analyses revealed diversity among VCZ strains within each clade. One main cluster, once referred to by the invalid pathovar name (peponis), gathers VCZ strains presenting a narrow host range including zucchini and squashes. Other VCZ strains present a large host range including zucchini, squashes, cucumber, melons, and in some cases watermelon. The VCZ strain pathogenic features are strongly associated with type III effector repertoires. The presence of avrRpt2 and absence of hopZ5 are associated with a narrow host range, whereas the presence of hopZ5 and absence of avrRpt2 are most generally associated with a large host range. To better detect the different clusters identified with whole genome sequence and pathogenicity analyses, we used a specific-k-mers approach to refine the MLSA scheme. Using this novel MLSA scheme to type P. syringae isolates from diseased cucurbits would give insight into distribution of worldwide strains and origin of epidemics.

Vein clearing of zucchini (VCZ) is a seed-borne bacterial disease that affects young plants of Cucurbita pepo subsp. pepo. VCZ agents are distributed into four phylogenetic clusters within the clades 2a and 2ba of the phylogroup 2 of Pseudomonas syringae species complex. Strains belonging to clades 2b and 2d are sometimes isolated from zucchini seeds but have not been associated with VCZ epidemics. Development of tools able to identify VCZ agents is important to better control the disease. Primers were designed to implement a seven-gene MLSA scheme on a collection of strains isolated from zucchini seeds. A clear predominance of strains with a host range on cucurbits limited to the genus Cucurbita (cluster 2ba-A) and the presence of VCZ strains in a fifth cluster (2ba-C) were evidenced. PCR tests were designed to characterize VCZ clusters and a multiplex qPCR test was proposed to distinguish strains having narrow cucurbit host range, associated to the presence of avrRpt2 and sylC, from broad host range strains associated to the presence of hopZ5 and sylC. Additional qPCR tests targeting clade 2b and 2d were also designed to gain insights on P. syringae strains that could be isolated from cucurbits. Specificity of these tools was evaluated in silico on the whole NCBI database and in vitro on a strain collection, showing a 100% inclusivity, except for the test dedicated to clade-2b strains, and an exclusivity ranging from to 96.7% to 100%. These different tools are intended to serve phylogenetic studies, epidemiological monitoring and seed testing.