TaqMan qPCR assays improve Pseudomonas syringae pv. actinidiae biovar 3 and P. viridiflava (PG07) detection within the Pseudomonas sp. community of kiwifruit

Abstract

Kiwifruit is inhabited by a heterogeneous community of bacteria belonging to the Pseudomonas syringae species complex (Pssc). Only a few of its members, such as the specialist Pseudomonas syringae pv. actinidiae biovar 3 (Psa3), are known as pathogens, but for most of the species, such as P. viridiflava (Pv), a generalist with high intraspecific variation, the nature of their relationship with kiwifruit is unclear. Currently, no culture independent molecular diagnostic assay is available for Pv. In this study we validated two TaqMan qPCR diagnostic assays adopting a strategy that for the first time widely focuses on the Pseudomonas sp. community associated to kiwifruit in Tuscany (Italy). Primers and probes were designed based on the sequence of the lscγ gene of Psa3 (qPCRPsa3) and the rpoD gene of Pv phylogroup 7 (qPCRPv7). Both qPCR assays have a LOD of 60 fg of DNA. By using reference strains along with 240 strains isolated from kiwifruit and characterized ad hoc as Pseudomonas sp., specificity was proven for members of six of the 13 Pssc phylogroups. Moreover, to evaluate the possible effects of seasonal variations in the Pseudomonas sp. community composition on assay specificity, the assays were tested on naturally infected leaves and canes sampled from different orchards throughout a growing season. At last, by proving qPCR’s capacity to detect latent infections in artificially inoculated leaves, their potential usefulness in surveillance programs and for epidemiological studies was verified.