Development of Tools to Detect and Identify Strains Belonging to the Pseudomonas syringae Species Complex Responsible for Vein Clearing of Zucchini

Abstract

Vein clearing of zucchini (VCZ) is a disease caused by seedborne bacteria that affects young plants of Cucurbita pepo subsp. pepo. VCZ agents are distributed into four phylogenetic clusters within clades 2a and 2ba of phylogroup 2 of the Pseudomonas syringae species complex. All these strains are pathogenic to squash, but only certain strains can also attack melon and cucumber. Strains belonging to clades 2b and 2d are sometimes isolated from zucchini seeds but have not been associated with VCZ epidemics. Identification tools for VCZ agents are required to improve disease control. Primers were designed to implement a seven-gene multilocus sequence analysis (MLSA) scheme for a collection of 60 strains isolated from zucchini seeds. The MLSA showed a clear predominance of strains of cluster 2ba-A and the presence of VCZ strains in a fifth cluster (2ba-C). PCR tests were designed to characterize strains in the VCZ clusters, and a multiplex qPCR test was proposed to distinguish strains with a cucurbit host range extended to melon and cucumber, harboring hopZ5 and sylC, from other strains harboring avrRpt2 and sylC. Additional qPCR tests were also designed to gain insights into clade-2b and -2d strains that can be isolated from cucurbits. These tools evaluated in silico with the NCBI database and experimentally with a collection of 112 strains detected all target strains, except for the test dedicated to clade-2b strains, and excluded 96.7 to 100% of nontarget strains. These tools are intended to serve phylogenetic studies, epidemiological monitoring, and seed testing. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .