ORIGINAL RESEARCH published: 15 March 2017 doi: 10.3389/fmicb.2017.00413 Classification of Isolates from the Pseudomonas fluorescens Complex into Phylogenomic Groups Based in Group-Specific Markers Daniel Garrido-Sanz, Eva Arrebola, Francisco Martínez-Granero, Sonia García-Méndez, Candela Muriel, Esther Blanco-Romero, Marta Martín, Rafael Rivilla and Miguel Redondo-Nieto* Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain The Pseudomonas fluorescens complex of species includes plant-associated bacteria with potential biotechnological applications in agriculture and environmental protection. Many of these bacteria can promote plant growth by different means, including modification of plant hormonal balance and biocontrol. The P. fluorescens group is Edited by: currently divided into eight major subgroups in which these properties and many other Martha E. Trujillo, ecophysiological traits are phylogenetically distributed. Therefore, a rapid phylogroup University of Salamanca, Spain assignment for a particular isolate could be useful to simplify the screening of putative Reviewed by: Youn-Sig Kwak, inoculants. By using comparative genomics on 71 P. fluorescens genomes, we have Gyeongsang National University, identified nine markers which allow classification of any isolate into these eight South Korea subgroups, by a presence/absence PCR test. Nine primer pairs were developed for David Dowling, Institute of Technology Carlow, Ireland the amplification of these markers. The specificity and sensitivity of these primer pairs *Correspondence: were assessed on 28 field isolates, environmental samples from soil and rhizosphere and Miguel Redondo-Nieto tested by in silico PCR on 421 genomes. Phylogenomic analysis validated the results: [email protected] the PCR-based system for classification of P. fluorescens isolates has a 98.34% of Specialty section: accuracy and it could be used as a rapid and simple assay to evaluate the potential This article was submitted to of any P. fluorescens complex strain. Evolutionary and Genomic Microbiology, Keywords: Pseudomonas fluroescens complex, PCR, phylogroups, classification a section of the journal Frontiers in Microbiology Received: 02 December 2016 INTRODUCTION Accepted: 27 February 2017 Published: 15 March 2017 The Pseudomonas fluorescens complex of species is one of the most diverse groups within the Citation: Pseudomonas genus, comprising more than fifty validly named species and many unclassified Garrido-Sanz D, Arrebola E, isolates. Members of this group have been isolated from diverse habitats, including water (Mirand Martínez-Granero F, García-Méndez S, and Zemelman, 2002), soil (Andersen et al., 2000), plant tissues (Brown et al., 2012), fungi (Rainey Muriel C, Blanco-Romero E, Martín M, et al., 1993), animals (Vela et al., 2006), and humans (Scales et al., 2015). Many P. fluorescens strains Rivilla R and Redondo-Nieto M (2017) that have been isolated from plant-related environments are described as plant growth-promoting Classification of Isolates from the rhizobacteria (PGPR) due to their ability to influence plant hormonal balance (Kang et al., 2006) Pseudomonas fluorescens Complex into Phylogenomic Groups Based in and improve plant fitness by minimizing the effects of phytopathogens (Raaijmakers et al., 2009), Group-Specific Markers. for which they are of great biotechnological interest. Pseudomonads are also known for the Front. Microbiol. 8:413. utilization of diverse organic compounds as energy and carbon sources (Lessie and Phibbs, 1984), doi: 10.3389/fmicb.2017.00413 making them also suited for bioremediation of polluted environments (Wasi et al., 2013). Despite Frontiers in Microbiology | www.frontiersin.org 1 March 2017 | Volume 8 | Article 413 Garrido-Sanz et al. PCR System for Pseudomonas Classification their beneficial role as PGPR, certain species within the P. complex is composed by more of 50 named species and that fluorescens complex are pathogens, including Pseudomonas most of the phylogroups contain several species (Garrido-Sanz corrugata and Pseudomonas mediterranea, the causal agents of et al., 2016) it is necessary to develop a system that allows the pith necrosis in tomato (Catara, 2007; Trantas et al., 2015) and identification of these mayor P. fluorescens complex phylogroups, Pseudomonas tolaasii, which causes brown blotch disease on as group adscription can provide insights into the potential cultivated mushrooms (Rainey et al., 1993). biotechnological uses of a particular isolate. An analysis of the P. fluorescens complex carried out The present study aims to develop a rapid PCR-based system 25 years ago identified several biotypes by using different assay for routinely classification of P. fluorescens isolates into taxonomic criteria (Stanier et al., 1966; Palleroni, 1991). More the eight phylogroups in which it is currently divided (Garrido- recently multilocus sequence analysis (MLSA) has divided the P. Sanz et al., 2016). For this purpose, we have used comparative fluorescens complex into a varying number of groups depending genomics to identify specific markers of these groups and develop on the number and identity of the genomes included in the study sets of primers for their amplification. We have tested this system (Mulet et al., 2010; Gomila et al., 2015; Garrido-Sanz et al., 2016). on classified and unclassified Pseudomonas strains along with These MLSA-based analysis have shown good concordance with environmental and rhizosphere samples. Finally, we performed phylogenomics and comparative genomics (Garrido-Sanz et al., in silico PCR (isPCR) and phylogenomic analysis to theoretically 2016), in which eight phylogroups have been identified: P. test and validate the system in all the sequenced genomes mandelii, P. jessenii, P. koreensis, P. corrugata, P. fluorescens, P. available. gessardii, P. chlororaphis, and Pseudomonas protegens. We have previously shown that many ecophysiological traits that are important for biocontrol, plant growth-promotion MATERIALS AND METHODS and bioremediation are distributed phylogenetically among the Datasets, Markers Identification, and main groups within the P. fluorescens complex (Garrido-Sanz Primer Design et al., 2016). For instance, strains within the P. corrugata, Seventy-one genomes and proteomes previously reported P. chlororaphis, and P. protegens groups produce an array of of belonging to the P. fluorescens complex (Garrido-Sanz secondary metabolites with antifungal properties, such as 2,4- et al., 2016) were downloaded from the NCBI FTP server diacetylphloroglucinol, 2-hexyl, 5-propyl resorcinol, phenazines, (ftp.ncbi.nih.gov) in February 2015 (Supplementary Table 1). and other siderophore-based antibiotics such as pyrrolnitrin and Orthologous groups were identified by comparing all-against- pyoluteorin (Nowak-Thompson et al., 1999; Raaijmakers and all using BLASTP (Camacho et al., 2009) and processed with Weller, 2001; Mavrodi et al., 2006; Ramette et al., 2011; Loper OrthoMCL v4 pipeline (Li et al., 2003), using default settings, et al., 2012; Calderón et al., 2013). It has also been reported that 50% alignment coverage cut-off and 1e-5 e-value. The results insecticidal activity of P. fluorescens complex strains also follows were stored in a relational database for further analysis and a phylogenetic distribution, being present in P. protegens and filtered with own designed SQL queries and Python scripts to P. chlororaphis phylogroups (Flury et al., 2016). On the other obtain the CDSs from the protein entries that appeared in all the hand, strains from P. chlororaphis and P. koreensis also carry genomes of each group but not in the remaining genomes. These the biosynthetic gene cluster for indole-3-acetic acid metabolism CDSs were additionally filtered by 500 pb minimum length and (Loper et al., 2012; Garrido-Sanz et al., 2016), which enhances blasted against all the genomes. The sequences with no hits across plant root system and thus increase the nutrient uptake by the genomes outside the groups and high homology within genomes plant (Spaepen et al., 2007). Therefore, routinely phylogroup of the group were selected. The selected markers sequences identification of isolates from the P. fluorescens complex could were then retrieved from all the group’s genomes and aligned be a preliminary step toward obtaining strains with potential using Clustal Omega (Sievers and Higgins, 2014). Conserved biotechnological applications. regions were used to design primers. Degenerated bases were Scarpellini et al. (2004) first developed a 16S rRNA PCR-based introduced to guarantee annealing in all the target genomes. assay to identify P. fluorescens isolates and their corresponding Melting temperature of the primers, absence of dimerization biotype. Several genotipes on DAPG-producing fluorescent and hairpin formation and lack of secondary priming sites pseudomonads were also identified by PCR using the BOX1AR were assessed with OligoAnalyzer 3.1 (https://eu.idtdna.com/ primer (BOX-PCR; Weller et al., 2007). Presently, the increase calc/analyzer). in the number of full sequenced genomes allows the use of phylogenetic and comparative genomic methods, which have provided a more robust insight regarding the delineation of In silico PCR (isPCR) and Phylogenomic groups within the P. fluorescens complex of species (Mulet et al., Analysis 2010; Gomila et al., 2015; Garrido-Sanz et al., 2016). Different Sequences of the nine selected markers plus 100 nts from each PCR-based systems have been developed for the identification end were