A Genomic Redefinition of Pseudomonas avellanae species

Marco Scortichini,Simone Marcelletti,Patrizia Ferrante,Giuseppe Firrao,Dawn Arnold

doi:10.1371/journal.pone.0075794

Marco Scortichini, Simone Marcelletti + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0075794

Copy DOI

Abstract

The circumscription of bacterial species is a complex task. So far, DNA-DNA hybridization (DDH), 16S rRNA gene sequencing, and multiocus sequence typing analysis (MLSA) are currently the preferred techniques for their genetic determination. However, the average nucleotide identity (ANI) analysis of conserved and shared genes between two bacterial strains based on the pair-wise genome comparisons, with support of the tetranucleotide frequency correlation coefficients (TETRA) value, has recently been proposed as a reliable substitute for DDH. The species demarcation boundary has been set to a value of 95-96% of the ANI identity, with further confirmation through the assessment of the corresponding TETRA value. In this study, we performed a genome-wide MLSA of 14 phytopathogenic pseudomonads genomes, and assessed the ANI and TETRA values of 27 genomes, representing seven out of the nine genomospecies of Pseudomonas spp. sensu Gardan et alii, and their phylogenetic relationships using maximum likelihood and Bayesian approaches. The results demonstrate the existence of a well demarcated genomic cluster that includes strains classified as P. avellanae, P. syringae pv. theae, P. s. pv. actinidiae and one P. s. pv. morsprunorum strain all belonging to the single species P. avellanae. In addition, when compared with P. avellanae, five strains of P. s. pv. tomato, including the model strain DC3000, and one P. s. pv. lachrymans strain, appear as very closely related to P. avellanae, with ANI values of nearly 96% as confirmed by the TETRA analysis. Conversely, one representative strain, previously classified as P. avellanae and isolated in central Italy, is a genuine member of the P. syringae species complex and can be defined as P. s. pv. avellanae. Currently. The core and pan genomes of P. avellanae species consist of 3,995 and 5,410 putative protein-coding genes, respectively.

Highlights

A rapid and destructive decline of cultivated hazelnut (Corylus avellana L.) was first observed in northern Greece during the 1970s
We revealed the existence of a well-demarcated P. avellanae species that includes strains classified as P. syringae pv. theae, P. s. pv. actinidiae and one P. s. pv. morsprunorum strain
We found that the strains of P. avellanae, P. s. pv. theae, and P. s. pv. actinidiae displayed average nucleotide identity (ANI) values that were consistently higher than 97.5% in any reciprocal comparison

Summary

Introduction

A rapid and destructive decline of cultivated hazelnut (Corylus avellana L.) was first observed in northern Greece during the 1970s. Avellanae and the disease was defined as bacterial canker of hazelnut [1]. A similar hazelnut disease was observed in central Italy, and the causal agent was identified as P. s. Based on 16S rRNA gene sequence and fatty acid analyses, the causal agent of the bacterial canker of hazelnut in Greece and Italy was subsequently elevated to the species level and named P. avellanae [4]. Molecular fingerprinting analyses using repetitive-sequence PCR [8] and classical tests such as the production of fluorescent pigments onto culture media [9] showed some clear differences between the P. avellanae populations found in Greece and Italy, and these differences were considered to be representative of the variability of the species. Two different lineages belonging to the same species were recognized and retained as originating separately [10] but evolving to infect cultivated hazelnut trees [11]

Methods

Results

Conclusion