The Distribution of Onion Virulence Gene Clusters Among Pantoea spp.

Abstract

<i>Pantoea ananatis</i> is a gram-negative bacterium and the primary causal agent of center rot of onions in Georgia. Previous genomic studies identified two virulence gene clusters, HiVir and <i>alt</i>, associated with center rot. The HiVir gene cluster is required to induce necrosis on onion tissues via synthesis of pantaphos, (2-hydroxy[phosphono-methyl)maleate), a phosphonate phytotoxin. The <i>alt</i> gene cluster aids in tolerance to thiosulfinates generated during onion tissue damage. Whole genome sequencing of other <i>Pantoea</i> species suggests that these gene clusters are present outside of <i>P. ananatis</i>. To assess the distribution of these gene clusters, two PCR primer sets were designed to detect the presence of HiVir and <i>alt</i>. Two hundred fifty-two strains of <i>Pantoea</i> spp. were phenotyped using the red onion scale necrosis (RSN) assay and were genotyped using PCR for the presence of these virulence genes. A diverse panel of strains from three distinct culture collections comprised of 24 <i>Pantoea</i> species, 41 isolation sources, and 23 countries, collected from 1946-2019, was tested. There is a significant association between the <i>alt</i> PCR assay and <i>Pantoea</i> strains recovered from symptomatic onion (<i>P</i> < 0.001). There is also a significant association of a positive HiVir PCR and RSN assay among <i>P. ananatis</i> strains but not among <i>Pantoea</i> spp., congeners. This may indicate a divergent HiVir cluster or different pathogenicity and virulence mechanisms. Last, we describe natural <i>alt</i> positive [RSN⁺/HiVir⁺/<i>alt</i> ⁺] <i>P. ananatis</i> strains, which cause extensive bulb necrosis in a neck-to-bulb infection assay compared to <i>alt</i> negative [RSN⁺/HiVir⁺/<i>alt</i> ^-] <i>P. ananatis</i> strains. A combination of assays that include PCR of virulence genes [HiVir and <i>alt</i>] and an RSN assay can potentially aid in identification of onion-bulb-rotting pathogenic <i>P. ananatis</i> strains.