The Distribution of Onion Virulence Gene Clusters Among Pantoea spp.

Shaun P Stice,Paul M Severns,Gi Yoon Shin,Bhabesh Dutta,María Inés Siri,Guillermo A Galván,Brian H Kvitko,Santosh Koirala,Stefanie De Armas,Teresa Coutinho

doi:10.3389/fpls.2021.643787

Abstract

Pantoea ananatis 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 alt, 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 alt gene cluster aids in tolerance to thiosulfinates generated during onion tissue damage. Whole genome sequencing of other Pantoea species suggests that these gene clusters are present outside of P. ananatis. To assess the distribution of these gene clusters, two PCR primer sets were designed to detect the presence of HiVir and alt. Two hundred fifty-two strains of Pantoea 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 Pantoea species, 41 isolation sources, and 23 countries, collected from 1946–2019, was tested. There is a significant association between the alt PCR assay and Pantoea strains recovered from symptomatic onion (P < 0.001). There is also a significant association of a positive HiVir PCR and RSN assay among P. ananatis strains but not among Pantoea spp., congeners. This may indicate a divergent HiVir cluster or different pathogenicity and virulence mechanisms. Last, we describe natural alt positive [RSN+/HiVir+/alt+] P. ananatis strains, which cause extensive bulb necrosis in a neck-to-bulb infection assay compared to alt negative [RSN+/HiVir+/alt–] P. ananatis strains. A combination of assays that include PCR of virulence genes [HiVir and alt] and an RSN assay can potentially aid in identification of onion-bulb-rotting pathogenic P. ananatis strains.

Highlights

Onion center rot is an economically impactful disease that routinely results in significant losses to the yield and marketability of onion (Allium cepa L.)
An objective of this study was to assess the utility of two PCR assays and the phenotypic red scale necrosis (RSN) assay for identifying Pantoea onion virulence genes
The assays were originally developed for use with P. ananatis strains; to investigate the breadth of their utility and potential limitations, we included a wider selection of Pantoea spp

Summary

Introduction

Onion center rot is an economically impactful disease that routinely results in significant losses to the yield and marketability of onion (Allium cepa L.). Recent comparative and functional genetic studies have determined that the HiVir and alt gene clusters function together to drive necrotrophic infection of onion by virulent P. ananatis strains (Stice et al, 2020). Deletion of the first gene in the HiVir cluster pepM, a phosphoenolpyruvate mutase gene, renders strains unable to induce necrotic lesions on onion foliage and tobacco leaves. The mutant strains were unable to induce necrosis on detached red onion scales three days after inoculation (red onion scale necrosis, RSN), compared to the wild-type (WT) strain (Asselin et al, 2018; Stice et al, 2020). P. ananatis strains reported as non-pathogenic on onion lacked the HiVir cluster (Asselin et al, 2018)

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Discussion

Conclusion