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University of Groningen

The dynamics of root microbiomes along a salt marsh primary successionWang, Miao

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Chapter 5

Evolution of root-associated

Pseudomonas during soil development

Authors: Miao Wang, Michele C. Pereira e Silva, Maryam Chaib de Mares, Monika A.

Chlebowicz, John W. A. Rossen, Joana Falcão Salles

manuscript in preparation for submission to Trends in Microbiology.

Abstract

The genus Pseudomonas comprises a diverse array of species, occupying many different niches, most of which function as plant growth-promoting rhizobacteria (PGPR). In this work, by ap-plying comparative genomics and biochemical analyses of 70 Pseudomonas strains associated with two typical perennial salt marsh plants, Limonium vulgare and Artemisia maritima, isolated both in the rhizosphere and endosphere, we investigated the evolution of Pseudomonas genus along the salt marsh chronosequence from both the genetic and functional perspectives. The core genomes of 70 Pseudomonas strains isolated from rhizosphere and endosphere from the different plants at different successional stages were defined by orthologous protein-coding sequences (CDS) in the draft genomes. Our aim is to identify the core and specific set of genes that may represent major evolutionary events towards niche adaptation and PGP capacity of Pseudomonas genus in different habitats following the development of succession. Furthermore, the bacterial functionality was determined by measuring traits associated with bacterial fit-ness, metabolic potential and plant growth promoting capacity. Our results showed that the Pseudomonas strains were affiliated with three phylogenetic subgroups, from which, the species composition in P. fluorescens subgroup I and II were affected by successional stages, predomi-nating in 65- and 5-year stages. The high gene diversity associated with pathways involved in environmental adaptation, root colonization and biocontrol activity, and nitrogen and phos-phorus metabolisms indicated the multiple potential roles in Pseudomonas genomes. Among these, the strains exhibiting high genetic diversity in adaptation, colonization and biocon-trol were mostly from P. fluorescens subgroup II, indicating that the genomic elements for spe-cific functions were related to the phylogenetic relatedness within the P. fluorescens complex. Whereas no apparent association was found between genomes and the different treatments (soil successional stage, plant species or plant compartment), we pinpointed that the strains with the higher number of genes involved in environmental adaptability were enriched in rhi-zosphere, whereas those with greater potential in phosphorus metabolism were enriched in A. maritma. These results highlight the potential functional requirements for colonization of the specific plant microenvironments or different plant species. Overall, this study provides an overview of the genetic and functional diversity of the root-associated pseudomonads, en-lightening the Pseudomonas evolution along a naturally- developed succession.

Keywords: root-associated, Pseudomonas, evolution, salt marsh, genome

Introduction 135

5

Introduction

A diverse array of bacteria including species of Pseudomonas, Azospirillum, Azotobacter, Bacillus, Klebsiella, Enterobacter, Xanthomonas, and Serratia have been shown to promote plant growth (Bhattacharyya et al., 2012). Among these, the genus Pseudomonas is considered a predominant plant growth-promoting rhizobacteria (PGPR) and has been most frequently reported (Laguerre et al., 1994; Botelho, 2001; Hallmann and Berg, 2006; Lugtenberg and Kamilova 2009; Hayat et al. 2010; Shen et al., 2013b; Jin et al. 2014), impacting plant growth and development by direct or indirect PGP mechanisms (Lugtenberg and Kamilova, 2009; Pliego et al., 2011; Glick, 2012). The direct mecha-nisms include phosphate solubilization, catabolism of molecules related with stress signaling such as bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase, the production of phytohormones, such as auxin, thus stimulating growth, and the production of siderophores (Belimov et al. , 2001; Dodd et al. , 2010; Sharma et al. , 2013; Barea and Richardson, 2015), while the indirect mechanisms are comprised of siderophore-mediated competition for iron, antibiosis, competition for nutrients and niches, or the induction of systemic resistance in the plant host (Weisbeek and Gerrits, 1999; Kamilova et al. , 2005; Nicodème and Grill, 2005; Liu et al. , 2007; Egamberdieva et al. , 2011; Loper et al. , 2012; Shen et al. , 2013b; Haas and Défago, 2015).

The genus Pseudomonas currently comprises more than 100 named spe-cies that have been divided into lineages, groups and subgroups based on multilocus sequence analysis (Yamamoto et al., 2000; Guttman et al., 2008; Mulet et al., 2010), occupying diverse ecological habitats such as water, soil, sediments, and plant surfaces, probably due to their simple nutritional re-quirements and great genetic plasticity (Haas and Défago, 2005; Lugtenberg and Kamilova, 2009; Jain and Das, 2016). The fluorescent Pseudomonads, mainly Pseudomonas fluorescens and Pseudomonas putida, is a major group act-ing as PGPR (Vlassak et al., 1992; Botelho et al., 2006; Bakker et al., 2007; Visca et al., 2007; Jain and Das, 2016). The predominant rhizosphere com-petence of species within this group, especially in the case of P. fluorescens, derives from their ability to rapidly colonize the root surface, in response to plant exudates, thus preventing the post-colonization by plant pathogens by niche occupation. In addition, they often produce an array of secondary me-tabolites that function as biocontrol factors (Lemanceau et al., 1995; Marilley and Aragno, 1999; Botelho, 2001; Siddiqui et al., 2003; Kamilova et al., 2005; Jorquera et al. 2011; Neidig et al., 2011; Loper et al., 2012; Nadeem et al., 2016).

136 CHAPTER 5 Evolution of root-associated Pseudomonas during soil development

Given the spectrum of ecological, metabolic, and biochemical charac-teristics of Pseudomonas genus, it is not surprising that individual plant- associated strains within this group differ at the genomic level (Paulsen et al., 2005; Loper et al., 2012; Redondo-Nieto et al., 2013), as revealed by comparative genomics studies, which provide insights into the genetic ba-sis of diversity and adaptation to specific environmental niches, by identi-fying functionally important genomic elements (Silby et al., 2009; Wu et al., 2011). Comparisons among the genomes of four strains within the P. fluo-rescens group (Pseudomonas protegens Pf-5, P. fluorescens strains SBW25, Pf0-1 and WH6) highlight the tremendous diversity of these bacteria (Paulsen et al., 2005; Silby et al., 2009; Kimbrel et al., 2010; Ramette et al., 2011). For instance, out of the 5741–6009 predicted protein-coding genes iden-tified in each genome, only 3115 were present in all four, composing a core genome representing only 52% to 54% of strain-specific genes, and nearly a third (1488 to 1833 genes) of the predicted proteome was strain-specific, highlighting the heterogeneity of this group of bacteria (Loper et al., 2012). More recently, a comparative genomics analysis of four representative Pseudomonas PGPR strains pinpointed conserved genes among the differ-ent strains that were associated with common characteristics of PGP traits (e.g. rhizosphere competence), while the specific genes differentiated each strain on the basis of its lifestyle, specific ecological adaptations, and phys-iological role in the rhizosphere (Shen et al. 2013b).

We have previously shown that rhizospheric rather than endophytic bacteria associated with salt marsh plants, from which a large percentage belonged to the genus Pseudomonas, followed specific metabolic and bio-chemical patterns along a primary soil succession, such as resistance to salinity stress and antibiotics, and siderophore production (Wang et al., Chapter 4). In this study we used similar setting used in the previous chap-ter to verify whether similar responses were also observed at genome level, by comparing the genomes of isolated fluorescent pseudomonads and ex-ploring the distributions of the key genes or gene complex involved in envi-ronmental adaptation (defense pathways and stress response), rhizosphere colonization (transport, motility and chemotaxis), biocontrol activities (iron acquisition and metabolism) and direct PGP mechanisms (nitrogen and phosphorus metabolism). Furthermore, by comparing the genomes of species isolated from different successional stages along a primary succes-sion, we also explore specific genes that are potentially related to their spec-ificity to soil characteristics (by clustering according to successional stages) or degree of association with the plant (rhizosphere vs endosphere) or plant

Materials and methods 137

5

species, in different soil types along a salt marsh primary succession chro-nosequence (Olff et al., 1997; Dini-Andreote et al., 2014, 2015, Wang et al., 2016). For that, we chose Limonium vulgare and Artemisia maritima, typical pe-rennial salt marsh plants, as our focus species because of their broad dis-tribution along the chronosequence. We hypothesize that the size of core genomes and the functionality of rhizosphere-associated Pseudomonas iso-lates to increase following the increment in the complexity of soil nutrients, organic matter, plant diversity and biomass observed in this system and therefore peaking largely at late stages of soil development. Reversely, we expect the core genome and functional diversity of those Pseudomonas iso-lates associated with root endosphere to remain constant, given the stron-ger plant selectivity and buffering effect previously shown for this system (Dini-Andreote et al., 2014, 2015; Wang et al., 2016).

Materials and methods

Study site and sample collection

The soil development that we investigated is located on the island of Schiermonnikoog, the Netherlands (53°30’N, 6°10’E), and spans more than 100 years of primary succession (Olff et al., 1997) (for detailed information on sampling, see Wang et al. (2016)). For this study, plant samples were col-lected in April in 2016 at locations with successional ages of 5, 15, 35, 65 and 105 years. For the details on the establishment of sampling plots and descriptions on the chronosequence verification, see Olff et al. (1997), Dini-Andreote et al. (2014), and Wang et al. (2016). Briefly, triplicated plots within each of the locations were established at the same base of elevation [verti-cal position relative to mean sea level at the initial elevation gradient on the bare sand flats with a base elevation of 1.16 m ± 2.2 cm (mean ± SE) above Dutch Ordinance Level]. Within each plot, four healthy-looking L. vulgare and A. maritima of similar sizes with attached soil adhering to the intact roots were collected and processed together generating two composite samples per plot. Thirty composite samples in total were collected (5 stages ⨯ 3 plots per stage ⨯ 2 plant species). Each sample was placed in a sterile plastic bag, sealed and transported to the laboratory within 24 h. From each compos-ite sample, we sampled rhizosphere soil and root endosphere (see below).


Pre-treatment of rhizosphere soil and plant root samples

A detailed description for the workflow of the isolation and screening of plant-associated Pseudomonas is illustrated in Figure S1. Rhizosphere soil samples were collected by weighting ten grams of roots with tightly adher-ing soil particles (about 3 g rhizosphere soil). Root samples were transferred into an Erlenmeyer flask containing 47 mL of sterile 1X Phosphate Buffered Saline (PBS buffer) and shaken for 30 min at 200 rpm at room temperature. An aliquot of 1 mL of the suspension with rhizosphere soil was transferred into sterile 1X PBS buffer and serial dilutions (1/10) were prepared.

Plant roots (about 8 g) were thoroughly washed with running tap wa-ter, trimmed to remove adhering soil and dead tissues, followed by sur-face sterilization (immersion in 1.5% NaClO solution (3 min), 70% ethanol (3 min) and sterile distilled water (3×3 min)). The surface-sterilized root samples (5 g) were diced with a sterile scalpel and immersed into 45 mL of 0.9% NaCl solution. After shaking incubation for 1 h at 28 °C, the sus-pension with root pieces was shaken using a horizontal vortex instrument (4×1 min, 30 sec in-between). An aliquot of 1 mL of the suspension contain-ing the released root endophytes was transferred into sterile 1X PBS buffer and serial dilutions (1/10) were prepared. Sterility checks were performed by tissue-blotting surface-sterilized root samples on R2A plates at 28 °C for 2–7 days. Only samples without bacterial growth were considered success-fully sterilized and used further.

Pseudomonas isolation and identification from rhizosphere soil and plant root samples

Gould’s S1 medium, as a selective medium recommended for the isolation of fluorescent pseudomonads (Gould et al., 1985; Johnsen et al., 1996), was used to culture the fluorescent Pseudomonas population. R2A medium, as a non-selective medium for the examination of total heterotrophic bacteria in soil (Ellis et al., 2003), was used to culture the heterotrophic population. Aliquots of 0.1 mL of each dilution of 1 × 10-1, 1 × 10-2, and 1 × 10-3 from rhi-zosphere soil and root samples were respectively spread on S1 and R2A me-dium plates and incubated for 2 days at 25 °C, after which we determined the number of colony forming units (CFU). Thirty-two bacterial colonies per plate with unique morphologies were purified using a streak-plate pro-cedure, transferred onto new S1 and R2A medium plates and further used as


5

templates for BOX-PCR, which is a DNA-based typing method potentially capable of simultaneously screening many DNA regions scattered in the bacterial genome (Brusetti et al., 2008). To improve cell lysis, the colonies were first inoculated into 50 μL of NaOH solution (0.05 M) and then lysed at 95 °C for 15 min in the PCR machine. BOX-PCR was performed by using the BOX-A1R primer (5’-CTACGGCAAGGCGACGCTGACG-3’) (Versalovic et al., 1994). Twenty μL PCR reactions were performed using 0.32 μL 5 U μL-1 Taq DNA Polymerase, 4 μL of 5X Gitschier Buffer [83 mM (NH4)2SO4, 335 mM Tris-HCl (pH 8.8), 32.5 mM MgCl2, 325 mM EDTA (pH 8.8), 1% commercial stock of β-mercaptoethanol, ddH2O], 2 μL 100% DMSO, 1 μL 25 mM of each dNTP in a mixture, 0.32 μL 20 mg mL-1 bovine serum albumin (BSA) (Roche Diagnostics GmbH, Mannheim, Germany), and 0.8 μL of 10 μM BOX-A1R primer and 1 μL of the lysed cell solution. The thermal cycler protocol was 95 °C for 3 min, 35 cycles of 94 °C for 4 sec, 92 °C for 30 sec, 50 °C for 60 sec, 65 °C for 8 min and a final 16 min extension at 65 °C. BOX-PCR profiles were visualized by separation on 2% agarose gel and staining with ethidium bro-mide. Images of the gels were visualized and documented under UV light with Image Master VDS system (Amersham Biosciences, United Kingdom). For the details of the bacterial isolates from rhizosphere soil and root endo-sphere showing unique BOX-PCR profiles, see Table S1. Culture stocks for individual isolates were stored in 25% glycerol at −80 °C.

Molecular characterization of Pseudomonas isolates

A total of 109 bacterial cultures with unique BOX-PCR patterns on Gould’s S1 agar plates (Figure S1) were subjected to total DNA extraction using the MoBio UltraClean Microbial DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA). We followed the instruction manual, except for heat-ing the preparations at 65 °C for 10 minutes with occasional bump vortex-ing for a few seconds every 2-3 minutes. The amount of DNA in each sample was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). All DNA samples were standardized to the equal concentra-tion of 5 ng µL-1 for further analyses.

The 16S rRNA specific region for P. fluorescens amplification was performed using the primer set 16SPSEfluF and 16SPSER (16SPSEfluF 5’-TGCATTCAAAACTGACTG-3’; 16SPSER 5’-AATCACACCGTGGTAACCG-3’) (Scarpellini et al., 2004). PCR was performed in a volume of 50 μL contain-ing 0.2 μL 5 U μL-1 FastStart High Fidelity (FSHF) Taq DNA Polymerase, 5 μL


10X FSHF Reaction buffer without MgCl2, 0.8 μL 50 mM MgCl2 stock solu-tion, 1 μL 10 mM PCR nucleotide mix, 0.5 μL 20 mg mL-1 bovine serum albu-min (BSA) (Roche Diagnostics GmbH, Mannheim, Germany), and 1 μL each of 10 μM primer and 5 ng DNA template. The thermal profile was 94 °C for 2 min, 5 cycles consisting of 94 °C for 45 sec, 55 °C for 1 min, 72 °C for 2 min, 35 cycles consisting of 92 °C for 45 sec, 60 °C for 45 sec, 72 °C for 2 min, and a final extension of 72 °C for 2 min. Amplicons were further checked on 1% (w/v) agarose gel to verify the correct band size, and strains with a single DNA fragment of 850 bp of 16S rRNA were identified as P. fluorescens. For the details of identification of isolates on R2A agar plates, see Wang et al. (Chapter 4). Pseudomonas isolates from the R2A plates were then selected (Wang et al., Chapter 4) and added into analysis in this study.

A total of 70 Pseudomonas unique isolates (41 P. fluorescens strains from S1 agar plates and 30 Pseudomonas strains from R2A agar plates) were sent to whole genome sequencing which was performed at LGC Genomics GmbH (Berlin, Germany) on the Illumina NextSeq 500 V2 platform with a 150-bp paired-end library. Illumina reads were assembled de novo with SPAdes 3.5.0 (http://bioinf.spbau.ru/spades), and all scaffolds larger than 1000 bp were kept. Annotation was performed using the automated online software RAST (Aziz et al., 2008).

The 16S rRNA gene sequences were extracted from the whole genome sequences by using RNAmmer (Lagesen et al., 2007) (http://www.cbs.dtu.dk/services/RNAmmer/), followed by alignment with SINA online (Pruesse et al., 2012) by comparison with the reference sequences available in SILVA databases (Quast et al. 2013; Yilmaz et al. 2014) (https://www.arb-silva.de/aligner/). Closely related strains were identified with the minimum iden-tity with query sequence of 99.9% (Table 1). Phylogenetic and molecular evolutionary analyses with the 16S rRNA gene sequences of Pseudomonas isolates were conducted by using software MEGA 7.0 for bigger datasets (Kumar et al., 2016). The sequences were aligned by using the CLUSTALW (Thompson et al., 1994). Tree constructions were performed using the Maximum Likelihood [ML] method (Cavalli-Sforza and Edwards, 1967; Felsenstein, 1981; 1993). The robustness of the phylogenetic tree was con-firmed by using 1000 bootstrap replications. Additionally, type strains of Serratia fonticola, Kluyvera intermedia, Erwinia rhapontici, Flavobacterium frigi-dimaris, Microbacterium oxydans, Bacillus atrophaeus, Exiguobacterium oxidotol-erans were included as the outgroup, and the validation of the phyloge-netic neighbours of Pseudomonas was carried out by adding the 16S rRNA sequences of type strains obtained from the SILVA rRNA database project.


5

Tab

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6,18

2,27

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533

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2965

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282,

268

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3265

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541

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6115

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375y

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6,20

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360

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5

Phylogenetic tree was visualized and exported using the web-based tool Interactive Tree Of Life (iTol) (Letunic and Bork et al., 2011).

Genome comparisons

In order to depict the core and accessory genome in the isolated Pseudomonas strains, we performed a reciprocal best hit search using the OrthoMCL software release five (Li et al. , 2003). For this we downloaded the predicted coding sequences (CDS) of all the 70 strains and performed a blastp search against each other with an E-value cut-off of 10−5 and a sequence coverage higher than 50%, as reported previously (Li et al. , 2003).

We further chose seven RAST subsystems central to our analysis (nitro-gen metabolism, phosphorous metabolism, stress response, membrane transport, iron acquisition and metabolism, motility and chemotaxis, virulence, disease and defense) to build an abundance map, including all genes assigned to the chosen subsystems. Specifically, by clustering the subsystems into four groups according to different mechanisms, respec-tively environmental adaptability (defence pathways and stress response), rhizosphere colonization (transport, motility and chemotaxis), biocon-trol activities (iron acquisition and metabolism) and direct PGP mech-anisms (nitrogen and phosphorus metabolism) (Table 2), we pinpointed the strain-specific gene or gene clusters which determine the evolution of Pseudomonas along the chronosequence.

Biochemical assays for functional traits by using microtiter plate

A detailed description of the functional traits screening for the plant- associated bacteria was given in Wang et al. (Chapter 4). Briefly, we de-tected the resistance to abiotic stress (salinity, osmotic and oxidative stress; growth under different pH), antibiotic resistance to penicillin and streptomycin, metabolic potential (carbon source usage) and plant growth promoting capacity (production of exoprotease, IAA, siderophore; bio-film formation) (Figure S2).


Table 2 Summary of putative genes related to different subsystems in 70 Pseudomonas

genomes

Mechanism System Gene Product Name

Enviromental adaptability

Metal resistance

copB Predicted metal binding proteincopG Copper resistance protein BcopZ Copper chaperonearsB Arsenical resistance operon repressorarsC Arsenate reductase arsR Arsenic efflux pump protein

Osmotic

proV choline ABC transporter, periplasmic binding proteinproX choline ABC transporter, ATP-binding protein [EC:3.6.3.32]betI high-affinity choline uptake protein BetTbetT HTH-type transcriptional regulator BetIsoxR Redox-sensitive transcriptional activator

Antibiotics ampC Beta-lactamase class C and other penicillin binding proteins

Rhizosphere colonization

Chemotaxis

cheA

Chemotaxis protein

cheRcheVcheWcheYcheBcheCcheDcheZmotA

Motility proteinmotBmotY

Twitching motility and type IV pili

tadB Flp pilus assembly protein TadBpilA type IV fimbriae expression regulatory protein PilRpilC type IV pilus assembly protein PilApilE type IV pilus assembly protein PilCpilM type IV pilus biogenesis protein PilEpilN Tfp pilus assembly protein, tip-associated adhesin PilY1pilO type IV pilus assembly protein PilMpilP Tfp pilus assembly protein PilNpilR Tfp pilus assembly protein PilOpilS Tfp pilus assembly protein PilPpilT twitching motility protein PilSpilV twitching motility protein PilTpilY type IV pilus modification protein PilV

Motility

fliD

structure

fliEfliFfliIfliJfliKfliLfliMfliNfliPfliQfliRfliSfliGfliHflgAflgBflgCflgDflgE


5

Data analysis

CFU values were log transformed before statistical analysis. Significant differences in log(CFU) across sample types, plant species, and soil succes-sional stages were identified using three-way analysis of variance (ANOVA).

Mechanism System Gene Product Name

Rhizosphere colonization Motility

flgF

structure

flgGflgKflgLflgHflgMflgNfleN Regulates Flagellar NumberfleS Regulates Flagellar NumberflaA

Flagellin synthesisflaGflhA

BiosynthesisflhBflhF

Biocontrol activity Pyoverdin

pvdA

Pyoverdine synthesis

pvdDpvdEpvdHpvdIpvdMpvdNpvdOpvdPpvdQpvdSpvdY

ABC transporter in pyoverdin gene cluster, ATP-binding component

ABC transporter in pyoverdin gene cluster, periplasmic component

ABC transporter in pyoverdin gene cluster, permease component

Nitrogen and phosphorus mechanism

Phosphorus metabolism

phnA

Phosphonate uptake and degradation

phnKphnLphnNphnBphnFphnGphnHphnIphnJpstA

(Phosphate-specific transport) systempstBpstCpstS

Nitrogen metabolism

nasT Nitrate response regulatorglnK Nitrogen regulatory proteinnarK Nitrate/nitrite transporternirD Nitrite reductase [NAD(P)H] large subunit nrtA nitrate-binding proteinamtB Ammonium transporter


The abundances of genes in each Pseudomonas genome encoding the pro-teins required in each system associated with environmental adaptability, rhizosphere colonization, biocontrol activities, nitrogen and phosphorus mechanisms were depicted with heatmaps (function aheatmap) in NMF package in the R environment (http://www.r-project.org).

The absorbance values indicated for functional traits of abiotic and bi-otic (antibiotics) resistance and metabolic potential of the Pseudomonas iso-lates were first normalized by the absorbance of the bacterial growth at the stationary phase, followed by the standardization of adding the weight cali-brated by the number of BOX-PCR profiles obtained from specific plant com-partment at the certain stage. In terms of the plant growth promoting traits determined by colorimetry, the absorbance values were only pre-treated by the standardization of adding the weight calibrated by the number of BOX-PCR profiles obtained from individual treatment. Specifically, for the meta-bolic potential traits indicated by growth in 14 types of carbon sources, raw data were normalized (by the maximum absorbance value observed across all carbon sources) and used to calculate the niche breadth for each bacterial isolate, by summing all 14 values according to Salles et al. (2009).

In order to test the significance of differences of sample types (i.e., rhi-zosphere and endosphere), plant species (i.e., L. vulgare and A. maritima) and distinct successional stages in the individual functional trait, three-way ANOVA was applied to the log transformed pre-treated absorbance value in the R environment (http://www.r-project.org).

The variation of functional traits along the chronosequence were simu-lated with polynominal regression performed in ggplot2 package, and only the significant regressions were shown. To test for correlations between bac-terial growth under pH and salinity stress and the variations of soil pH and sa-linity along the succession, we applied Spearman’s rank-based correlational analysis. The pairwise comparisons of the functional traits between sample types and between plant species were visualized by boxplots, and the signifi-cance of the influence of either factor was determined by Kruskal-Wallis test. Variation in functional diversity along the chronosequence were calculated with the Bray-Curtis distance matrix, and the pairwise comparisons between two stages were tested by using Post hoc test (function posthoc.kruskal.nemenyi.test) in PMCMR package after Kruskal-Wallis test. The strength of each func-tional trait associated with the Pseudomonas isolates from different sample types, plant species and successional stages were visualized by using heat-map (function aheatmap) in NMF package. Prior to analysis, the pre-treated absorbance values were further standardized by using Z-scores.

Results 149

5

Results

Isolation, screening and characterization of plant-associated bacteria in rhizosphere and root endosphere

The population counts on S1 agar plates of fluorescent pseudomonads iso-lated from the rhizosphere soil and root endosphere of L. vulgare and A. ma-ritima along the chronosequence were significantly influenced by succes-sional stage (F=14.00, P<0.01). Significant differences in population counts were respectively observed between middle (15- and 35- year stages) and late (65- and 105- year stages) successional phases for either rhizosphere or endosphere from both plants (pairwise comparisons, P<0.05). In addi-tion, significant polynomial variations were found for either compartment from both plant species (P<0.05) (Figure S3), respectively decreasing from the initial (5-year stage) to middle phase followed by an increase towards the late phase along the chronosequence.

A total of 109 Pseudomonas strains from S1 agar plates and 95 Pseudomonas strains from R2A agar plates were isolated and screened by genotypic char-acterization (Figure S1; Table S1, number of BOX-PCR patterns; Wang et al., Chapter 4), generating a set of 70 unique strains (41 P. fluorescens strains from S1 agar plates and 30 Pseudomonas strains from R2A agar plates) that were further identified at species level by alignment of the 16S rRNA gene sequences extracted from the whole genome sequences (Table 1).

According to phylogenetic relatedness, the 70 unique Pseudomonas strains were affiliated with three groups (Figure 1A), respectively P. fluorescens sub-group I (35 strains), P. fluorescens subgroup II (27 strains) and P. putida group (8 strains of P. putida species). Among the three groups, P. fluorescens sub-group I was most abundant, and almost all strains were affiliated with P. flu-orescens (except for three strains respectively affiliated with P. poae, P. trivia-lis and P. azotoformans). The dominance of P. fluorescens was also observed in P. fluorescens subgroup II, which accounted for 50% of the total number of strains, while the other half were respectively affiliated with P. brassicace-arum (3), P. anguilliseptica (3), P. protegens (2), P. fulva (2), P. fragi (1), P. psychroph-ila (1) and one unknown strain.

Among the 70 strains, 32.9% and 27.1% were respectively isolated from the 65- and 5-year stage while the remaining ones were almost evenly dis-tributed among other stages (Figure 1B). A slight plant effect on the distribu-tion of the strains was found, with showing more isolates from A. maritima (58.6%) than L. vulgare (41.4%). However, no effect was observed according


to the differences in plant compartments (rhizosphere and endosphere). As the predominant subgroup, the distribution of strains in P. fluorescens sub-group I according to different treatments was similar with that of the to-tal distribution, showing a stronger plant effect under which almost three quarters of the strains (71.4%) were isolated from A. maritima. In terms of P. fluorescens subgroup II, however, the strains were rather evenly distrib-uted over the different treatments, except for a slight predominance of iso-lates from 5-year stage (29.6%). A different scenario was shown for P. putida

Figure 1 Phylogenetic distribution of root-associated Pseudomonas isolates. (A)

Phylogenetic tree based on Maximum Likelihood [ML] method. Green, purple and red

color represent the strains isolated in this study, the Pseudomonas type strains, and

27.1%

17.1%

11.4%

32.9%

11.4%

0

20

40

60 Type

105y

15y35y

5y

65y

Stage

41.4%58.6%

0

20

40

60Type

ArtemisaLimonium

Plant species

50%50%

0

20

40

60Type

EndosphereRhizosphere

Source

Strains from this study Pseudomonas spp. type strains Outgroup type strains

P. �uorescens subgroup II

P. pu

tida

grou

p

P. �uorescens subgroup I

25.7%

22.9%

5.7%

37.1%

8.6%

0

10

20

30

60% 40%

0

10

20

30

71.4%

28.6%

0

10

20

30Type

105y

15y35y

5y

65y

Type

ArtemisaLimonium

TypeEndosphereRhizosphere

29.6%

14.8%

18.5%

22.2%14.8%

0

5

10

15

20

25

48.1%51.9%

0

5

10

15

20

25

48.1%51.9%

0

5

10

15

20

25

Type

105y

15y35y

5y

65y

Type

ArtemisaLimonium


25%

12.5%50%

12.5%

2

4

6

0

12.5%

87.5%

2

4

6

0

37.5% 62.5% 2

4

6

0

Type

105y

15y35y

5y

65y

Type

ArtemisaLimonium


Total

P. �uorescenssubgroup I

P. �uorescenssubgroup II

P. putidasubgroup

(A)(B)

Results 151

5

group, out of the 8 strains, 4 strains were isolated from the rhizosphere of L. vulgare at the 65-year stage, while 2 strains were from the rhizosphere of A. maritima at the 5-year stage, and the remaining 2 strains were respec-tively from the rhizosphere of A. maritima at the 35-year stage and the en-dosphere of L. vulgare at the 105-year stage.

27.1%

17.1%

11.4%

32.9%

11.4%

0

20

40

60 Type

105y

15y35y

5y

65y

Stage

41.4%58.6%

0

20

40

60Type

ArtemisaLimonium

Plant species

50%50%

0

20

40

60Type

EndosphereRhizosphere

Source

Strains from this study Pseudomonas spp. type strains Outgroup type strains

P. �uorescens subgroup II

P. pu

tida

grou

p

P. �uorescens subgroup I

25.7%

22.9%

5.7%

37.1%

8.6%

0

10

20

30

60% 40%

0

10

20

30

71.4%

28.6%

0

10

20

30Type

105y

15y35y

5y

65y

Type

ArtemisaLimonium


29.6%

14.8%

18.5%

22.2%14.8%

0

5

10

15

20

25

48.1%51.9%

0

5

10

15

20

25

48.1%51.9%

0

5

10

15

20

25

Type

105y

15y35y

5y

65y

Type

ArtemisaLimonium


25%

12.5%50%

12.5%

2

4

6

0

12.5%

87.5%

2

4

6

0

37.5% 62.5% 2

4

6

0

Type

105y

15y35y

5y

65y

Type

ArtemisaLimonium


Total

P. �uorescenssubgroup I

P. �uorescenssubgroup II

P. putidasubgroup

(A)(B)

outgroup strains, respectively. (B) Distribution of isolates belonging to individual phyla

along the successional stages, plant compartments and plant species. For successional

stages, pink, blue, navy, cyan and orange color represent 5, 15, 35, 65 and 105-year

stage, respectively. For plant compartments, red refer to rhizosphere isolates whereas

green represent those obtained from the endosphere. Regarding plant species, the

purple refers to L. vulgare isolates and green to A. maritima. The size of each sector rep-

resents the proportion of each treatment.


General genome features and comparative genomics

The general genome features of the Pseudomonas strains is summarized in Table 1. The 70 strains showed a wide range of genome sizes, ranging from 5.2 to 18.7 M, resulting in different numbers of protein-coding genes in subsystems, which was from 3778 to 12963 CDs. Among the strains with large genome sizes in P. fluorescens subgroup I (larger than 7 M), 5 out of the total 8 strains were from the initial stages (5- and 15-year stage) and the other 3 were from the 65-year stage. The strains in this subgroup shared similar GC content, ranging from 59.1% to 61.6%. Within P. fluorescens sub-group II, however, the strains with large genome sizes (10 strains in total) were mostly observed at the 35- and 105-year stages (respectively 4 and 3 strains). The GC content varied largely among the strains in this group, ranging from 50.7% to 62.9%. Differently, the strains belonging to P. putida group shared similar genome sizes and GC content, respectively ranging from 5.7 to 6.4 M and 61.4% to 62.4%.

Genetic diversity

Among the 70 strains, three strains obtained from rhizosphere were found to show high gene abundances in seven subsystems (Figure S7), including two P. protegens strains respectively from 35- and 65-year stages and one P. fluorescens NZ011 strain from 105-year stage. Additionally, another three strains from rhizosphere, respectively P. trivialis from 15-year stage, P. azoto-formans and P. fluorescens PICF7 from 5-year stage, were observed to show high gene abundances in subsystems of membrane transport, motility and chemotaxis, virulence, disease and defense.

Defense pathway and stress response

We tested the abundance of key genes involved in the resistance to penicil-lin, metal (copper and arsenic) and osmotic stress (Figure 2). We observed that except for two P. putida strains from rhizosphere, all other strains had the predicted gene encoding β-lactamases, providing resistant to differ-ent classes of β-lactam antibiotics, such as penicillin. Furthermore, genes encoding for glycine betaine — a major osmoprotectant for many bacte-ria — proline betaine transport system ATP-binding protein (proV) and

Results 153

5

betT

arsC

copB

arsR

copG

ampC

copZ

arsB

proX

betI

proV

85 Pseudomonas putida NBRC 1416463 Pseudomonas putida F1R28 Pseudomonas fragiR95 Pseudomonas fulva 12-XP114 Pseudomonas fluorescens SBW2560 Pseudomonas fulva 12-XP113 Pseudomonas fluorescens PICF7R61 Pseudomonas fluorescensR44 Pseudomonas putida SJTE-1P78 Pseudomonas fluorescens R124P74 Pseudomonas fluorescens R124P56 Pseudomonas fluorescens NZ011P50 Pseudomonas protegens Cab57P49 Pseudomonas putida LS46P48 Pseudomonas putida SJTE-1P33 Pseudomonas putida SJTE-188 Pseudomonas putida F176 Pseudomonas fluorescens54 Pseudomonas fluorescens R12466 Pseudomonas putida SJTE-1R96 Pseudomonas fluorescensR67 Pseudomonas brassicacearum NFM421R5 Pseudomonas anguillisepticaR104 Pseudomonas fluorescensP42 Pseudomonas fluorescens NZ011P36 Pseudomonas fluorescens NCIMB 11764P32 Pseudomonas fluorescens NCIMB 11764P22 Pseudomonas azotoformansP18 Pseudomonas protegens Cab57101 Pseudomonas brassicacearum NFM42173 Pseudomonas brassicacearum NFM42136 Pseudomonas fluorescens70 Pseudomonas fluorescens Pf0-1R69 Pseudomonas anguillisepticaP70 Pseudomonas fluorescens SBW25R29 Pseudomonas fluorescens SBW25P11 Pseudomonas trivialisP8 Pseudomonas fluorescensR50 Pseudomonas fluorescens Pf0-1R32 Pseudomonas fluorescens PICF7R111 Pseudomonas anguillisepticaP40 Pseudomonas fluorescensP39 Pseudomonas fluorescensP37 Pseudomonas fluorescensP34 Pseudomonas fluorescensP24 Pseudomonas fluorescens PICF7P12 Pseudomonas fluorescens PICF7P117 Pseudomonas fluorescens SBW26P110 Pseudomonas fluorescensP104 Pseudomonas fluorescens PICF9P101 Pseudomonas fluorescens PICF769 Pseudomonas psychrophila103 Pseudomonas fluorescens PICF7R98 Pseudomonas fluorescens SBW25R83 Pseudomonas fluorescens PICF7 R54 Pseudomonas sp. L10.10P93 Pseudomonas fluorescens PICF7P90 Pseudomonas poaeP77 Pseudomonas fluorescens SBW25P72 Pseudomonas fluorescens NZ011P41 Pseudomonas fluorescens PICF7P31 Pseudomonas fluorescens PICF7P19 Pseudomonas fluorescens PICF7P130 Pseudomonas fluorescens PICF7P129 Pseudomonas fluorescens SBW26P124 Pseudomonas fluorescens SBW25P120 Pseudomonas fluorescens PICF7P108 Pseudomonas fluorescens PICF10P102 Pseudomonas fluorescens PICF8P106 Pseudomonas fluorescens PICF10

Var1

Var2

Var3

Var1 Successional stage

Var2 Plant speciesArtemisia maritimaLimonium vulgare

Var3 SourceEndosphereRhizosphere

105y

15y35y

5y

65y

0

1

2

Gene abundance

Environmental adaptation

Figure 2 Distribution of genes involved in environmen-

tal adaptability of root-associated Pseudomonas strains.

The annotation of treatments — successional stages, plant

species and plant compartments were respectively re-

ferred as Var1, Var2 and Var3. For successional stages, pink,

blue, navy, cyan and orange color represent 5, 15, 35, 65

and 105-year stage, respectively. For plant species, pur-

ple refers to L. vulgare and green to A. maritima. For plant

compartments, red refer to rhizosphere and green to

endosphere isolates. For gene abundance, blue, yellow

and red represent 0, 1, and 2 gene copies, respectively.

betT

arsC

copB

arsR

copG

ampC

copZ

arsB

proX

betI

proV

85 Pseudomonas putida NBRC 1416463 Pseudomonas putida F1R28 Pseudomonas fragiR95 Pseudomonas fulva 12-XP114 Pseudomonas fluorescens SBW2560 Pseudomonas fulva 12-XP113 Pseudomonas fluorescens PICF7R61 Pseudomonas fluorescensR44 Pseudomonas putida SJTE-1P78 Pseudomonas fluorescens R124P74 Pseudomonas fluorescens R124P56 Pseudomonas fluorescens NZ011P50 Pseudomonas protegens Cab57P49 Pseudomonas putida LS46P48 Pseudomonas putida SJTE-1P33 Pseudomonas putida SJTE-188 Pseudomonas putida F176 Pseudomonas fluorescens54 Pseudomonas fluorescens R12466 Pseudomonas putida SJTE-1R96 Pseudomonas fluorescensR67 Pseudomonas brassicacearum NFM421R5 Pseudomonas anguillisepticaR104 Pseudomonas fluorescensP42 Pseudomonas fluorescens NZ011P36 Pseudomonas fluorescens NCIMB 11764P32 Pseudomonas fluorescens NCIMB 11764P22 Pseudomonas azotoformansP18 Pseudomonas protegens Cab57101 Pseudomonas brassicacearum NFM42173 Pseudomonas brassicacearum NFM42136 Pseudomonas fluorescens70 Pseudomonas fluorescens Pf0-1R69 Pseudomonas anguillisepticaP70 Pseudomonas fluorescens SBW25R29 Pseudomonas fluorescens SBW25P11 Pseudomonas trivialisP8 Pseudomonas fluorescensR50 Pseudomonas fluorescens Pf0-1R32 Pseudomonas fluorescens PICF7R111 Pseudomonas anguillisepticaP40 Pseudomonas fluorescensP39 Pseudomonas fluorescensP37 Pseudomonas fluorescensP34 Pseudomonas fluorescensP24 Pseudomonas fluorescens PICF7P12 Pseudomonas fluorescens PICF7P117 Pseudomonas fluorescens SBW26P110 Pseudomonas fluorescensP104 Pseudomonas fluorescens PICF9P101 Pseudomonas fluorescens PICF769 Pseudomonas psychrophila103 Pseudomonas fluorescens PICF7R98 Pseudomonas fluorescens SBW25R83 Pseudomonas fluorescens PICF7 R54 Pseudomonas sp. L10.10P93 Pseudomonas fluorescens PICF7P90 Pseudomonas poaeP77 Pseudomonas fluorescens SBW25P72 Pseudomonas fluorescens NZ011P41 Pseudomonas fluorescens PICF7P31 Pseudomonas fluorescens PICF7P19 Pseudomonas fluorescens PICF7P130 Pseudomonas fluorescens PICF7P129 Pseudomonas fluorescens SBW26P124 Pseudomonas fluorescens SBW25P120 Pseudomonas fluorescens PICF7P108 Pseudomonas fluorescens PICF10P102 Pseudomonas fluorescens PICF8P106 Pseudomonas fluorescens PICF10

Var1

Var2

Var3

Var1 Successional stage



105y

15y35y

5y

65y

0

1

2

Gene abundance

Environmental adaptation


glycine betaine/proline betaine-binding periplasmic protein (proX) were found among all genomes, similar with the genes involving the system for oxidation of choline to GB (betT and betL). However, half of the strains lack copB, which encodes the uncharacterized protein involved in copper resis-tance. Among the strains showing the highest abundance and diversity of genes in the resistance to antibiotics, metal and osmotic stress (a total of 13 strains), 9 were isolated from rhizosphere. Moreover, half of the strains were affiliated with P. fluorescens species. While the other half included three P. brassicacearum strains and three other strains which respectively belonged to P. protegens, P. anguilliseptica and P. azotoformans.

Root colonization

As expected for a rhizobacterium that exhibits strong competitive coloni-zation ability of plant roots, we identified the genes required for chemo-taxis, motility, and adhesion (Figure 3). For chemotaxis, all genes required for protein synthesis were present except for cheC and cheD. For motility, the genes involved in the regulation (fle genes), biosynthesis (fliP, fliQ , fliR and flh genes), structure (flg and fli genes), and motor (motA and motB) components of flagella were found in all genomes. However, flaA gene encoding flagel-lin — the subunit protein, which polymerizes to form the filaments of bac-terial flagella, was only found in certain genomes. In terms of the genes for type IV pilus system, pilB, pilR and pilS were only found in a small propor-tion of genomes, while other pil genes were present for most of the genomes. One P. brassicacearum strain isolated from rhizosphere of L. vulgare at 105-year stage exhibited the highest gene abundance in root colonization systems. In addition, another two P. brassicacearum strains respectively obtained from rhizosphere and endosphere and one P. fluorescens strain from rhizosphere were detected to have all the required genes for root colonization.

Biocontrol activities

The genes associated with Pyoverdin (Pvd) — a fluorescent siderophore produced by fluorescent pseudomonads under low-iron conditions to improve their biocontrol activity — in the Pvd biosynthetic gene clus-ter were all strain-specific, appearing only in less than half of the 70 ge-nomes (Figure 4). Only three strains were found to include all the genes in

Results 155

5

flaA

ch

eC

pilR

pilY

1p

ilSp

ilBch

eD

pilV

pilA

pilC

pilQ

pilP

pilO

pilN

pilM

pilT

pilE

tad

Bfla

Gm

otY

ch

eW

flgM

fleS

fleQ

fliJflg

Jflg

IfliFfliKfliDflg

Lflg

Kflg

EfliSfliQfliPfliLflg

Nflg

Gflg

Fflg

AfliHch

eB

ch

eR

ch

eZ

fliMfliNch

eA

fliIfle

NfliGm

otB

mo

tAflg

HfliEfliRflh

Fflh

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85 Pseudomonas putida NBRC 1416463 Pseudomonas putida F1P22 Pseudomonas azotoformansP113 Pseudomonas fluorescens PICF7P114 Pseudomonas fluorescens SBW25R29 Pseudomonas fluorescens SBW25P70 Pseudomonas fluorescens SBW25P40 Pseudomonas fluorescensP34 Pseudomonas fluorescensP37 Pseudomonas fluorescens101 Pseudomonas brassicacearum NFM421R111 Pseudomonas anguillisepticaR54 Pseudomonas sp. L10.1076 Pseudomonas fluorescens60 Pseudomonas fulva 12-XR95 Pseudomonas fulva 12-XR69 Pseudomonas anguillisepticaR96 Pseudomonas fluorescensR67 Pseudomonas brassicacearum NFM42173 Pseudomonas brassicacearum NFM421R104 Pseudomonas fluorescensP56 Pseudomonas fluorescens NZ011P18 Pseudomonas protegens Cab57P42 Pseudomonas fluorescens NZ011R5 Pseudomonas anguillisepticaP78 Pseudomonas fluorescens R124P74 Pseudomonas fluorescens R124P72 Pseudomonas fluorescens NZ011P50 Pseudomonas protegens Cab5770 Pseudomonas fluorescens Pf0-136 Pseudomonas fluorescens54 Pseudomonas fluorescens R124R50 Pseudomonas fluorescens Pf0-169 Pseudomonas psychrophilaR28 Pseudomonas fragiP24 Pseudomonas fluorescens PICF7P101 Pseudomonas fluorescens PICF7P117 Pseudomonas fluorescens SBW26R61 Pseudomonas fluorescensP77 Pseudomonas fluorescens SBW25R98 Pseudomonas fluorescens SBW25P120 Pseudomonas fluorescens PICF7P124 Pseudomonas fluorescens SBW25P33 Pseudomonas putida SJTE-188 Pseudomonas putida F1R44 Pseudomonas putida SJTE-1P49 Pseudomonas putida LS4666 Pseudomonas putida SJTE-1P48 Pseudomonas putida SJTE-1P32 Pseudomonas fluorescens NCIMB 11764P36 Pseudomonas fluorescens NCIMB 11764P90 Pseudomonas poaeP8 Pseudomonas fluorescensP39 Pseudomonas fluorescensP31 Pseudomonas fluorescens PICF7P11 Pseudomonas triv ialisP19 Pseudomonas fluorescens PICF7R83 Pseudomonas fluorescens PICF7 R32 Pseudomonas fluorescens PICF7P93 Pseudomonas fluorescens PICF7P41 Pseudomonas fluorescens PICF7P130 Pseudomonas fluorescens PICF7P129 Pseudomonas fluorescens SBW26P12 Pseudomonas fluorescens PICF7P110 Pseudomonas fluorescensP108 Pseudomonas fluorescens PICF10P106 Pseudomonas fluorescens PICF10P104 Pseudomonas fluorescens PICF9103 Pseudomonas fluorescens PICF7P102 Pseudomonas fluorescens PICF8 Var1 Successional stage



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Figure 3 Gene clusters required for root colonization of

root-associated Pseudomonas strains. The annotation

and color of treatments — successional stages, plant spe-

cies and plant compartments are the same as in Figure 2.

For gene abundance, blue, light blue, yellow and red rep-

resent 0, 1, 2 and 3 gene copies, respectively.

flaA

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85 Pseudomonas putida NBRC 1416463 Pseudomonas putida F1P22 Pseudomonas azotoformansP113 Pseudomonas fluorescens PICF7P114 Pseudomonas fluorescens SBW25R29 Pseudomonas fluorescens SBW25P70 Pseudomonas fluorescens SBW25P40 Pseudomonas fluorescensP34 Pseudomonas fluorescensP37 Pseudomonas fluorescens101 Pseudomonas brassicacearum NFM421R111 Pseudomonas anguillisepticaR54 Pseudomonas sp. L10.1076 Pseudomonas fluorescens60 Pseudomonas fulva 12-XR95 Pseudomonas fulva 12-XR69 Pseudomonas anguillisepticaR96 Pseudomonas fluorescensR67 Pseudomonas brassicacearum NFM42173 Pseudomonas brassicacearum NFM421R104 Pseudomonas fluorescensP56 Pseudomonas fluorescens NZ011P18 Pseudomonas protegens Cab57P42 Pseudomonas fluorescens NZ011R5 Pseudomonas anguillisepticaP78 Pseudomonas fluorescens R124P74 Pseudomonas fluorescens R124P72 Pseudomonas fluorescens NZ011P50 Pseudomonas protegens Cab5770 Pseudomonas fluorescens Pf0-136 Pseudomonas fluorescens54 Pseudomonas fluorescens R124R50 Pseudomonas fluorescens Pf0-169 Pseudomonas psychrophilaR28 Pseudomonas fragiP24 Pseudomonas fluorescens PICF7P101 Pseudomonas fluorescens PICF7P117 Pseudomonas fluorescens SBW26R61 Pseudomonas fluorescensP77 Pseudomonas fluorescens SBW25R98 Pseudomonas fluorescens SBW25P120 Pseudomonas fluorescens PICF7P124 Pseudomonas fluorescens SBW25P33 Pseudomonas putida SJTE-188 Pseudomonas putida F1R44 Pseudomonas putida SJTE-1P49 Pseudomonas putida LS4666 Pseudomonas putida SJTE-1P48 Pseudomonas putida SJTE-1P32 Pseudomonas fluorescens NCIMB 11764P36 Pseudomonas fluorescens NCIMB 11764P90 Pseudomonas poaeP8 Pseudomonas fluorescensP39 Pseudomonas fluorescensP31 Pseudomonas fluorescens PICF7P11 Pseudomonas triv ialisP19 Pseudomonas fluorescens PICF7R83 Pseudomonas fluorescens PICF7 R32 Pseudomonas fluorescens PICF7P93 Pseudomonas fluorescens PICF7P41 Pseudomonas fluorescens PICF7P130 Pseudomonas fluorescens PICF7P129 Pseudomonas fluorescens SBW26P12 Pseudomonas fluorescens PICF7P110 Pseudomonas fluorescensP108 Pseudomonas fluorescens PICF10P106 Pseudomonas fluorescens PICF10P104 Pseudomonas fluorescens PICF9103 Pseudomonas fluorescens PICF7P102 Pseudomonas fluorescens PICF8 Var1 Successional stage



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pvdH

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R95 Pseudomonas fulva 12-XR69 Pseudomonas anguillisepticaR54 Pseudomonas sp. L10.10R50 Pseudomonas fluorescens Pf0-1R28 Pseudomonas fragiR111 Pseudomonas anguillisepticaP93 Pseudomonas fluorescens PICF7P8 Pseudomonas fluorescensP78 Pseudomonas fluorescens R124P74 Pseudomonas fluorescens R124P72 Pseudomonas fluorescens NZ011P42 Pseudomonas fluorescens NZ011P40 Pseudomonas fluorescensP39 Pseudomonas fluorescensP37 Pseudomonas fluorescensP36 Pseudomonas fluorescens NCIMB 11764P34 Pseudomonas fluorescensP32 Pseudomonas fluorescens NCIMB 11764P24 Pseudomonas fluorescens PICF7P22 Pseudomonas azotoformansP130 Pseudomonas fluorescens PICF7P129 Pseudomonas fluorescens SBW26P12 Pseudomonas fluorescens PICF7P117 Pseudomonas fluorescens SBW26P110 Pseudomonas fluorescensP11 Pseudomonas trivialisP108 Pseudomonas fluorescens PICF10P106 Pseudomonas fluorescens PICF10P104 Pseudomonas fluorescens PICF9P102 Pseudomonas fluorescens PICF8P101 Pseudomonas fluorescens PICF7103 Pseudomonas fluorescens PICF788 Pseudomonas putida F169 Pseudomonas psychrophila54 Pseudomonas fluorescens R12460 Pseudomonas fulva 12-XR44 Pseudomonas putida SJTE-1P90 Pseudomonas poaeP49 Pseudomonas putida LS46P48 Pseudomonas putida SJTE-1P33 Pseudomonas putida SJTE-166 Pseudomonas putida SJTE-1P19 Pseudomonas fluorescens PICF7R67 Pseudomonas brassicacearum NFM421P56 Pseudomonas fluorescens NZ011P18 Pseudomonas protegens Cab57P50 Pseudomonas protegens Cab57P31 Pseudomonas fluorescens PICF7R83 Pseudomonas fluorescens PICF7 P113 Pseudomonas fluorescens PICF7P114 Pseudomonas fluorescens SBW25R98 Pseudomonas fluorescens SBW25R32 Pseudomonas fluorescens PICF7P77 Pseudomonas fluorescens SBW25P41 Pseudomonas fluorescens PICF7P120 Pseudomonas fluorescens PICF7P124 Pseudomonas fluorescens SBW2563 Pseudomonas putida F185 Pseudomonas putida NBRC 1416476 Pseudomonas fluorescens101 Pseudomonas brassicacearum NFM421P70 Pseudomonas fluorescens SBW25R29 Pseudomonas fluorescens SBW25R61 Pseudomonas fluorescens70 Pseudomonas fluorescens Pf0-1R96 Pseudomonas fluorescensR5 Pseudomonas anguillisepticaR104 Pseudomonas fluorescens36 Pseudomonas fluorescens73 Pseudomonas brassicacearum NFM421 Var1 Successional stage



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2

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Var1

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Figure 4 Pyoverdin (Pvd) biosynthetic gene clusters in

biocontrol activity of root- associated Pseudomonas

strains. The annotation and color of treatments — succes-

sional stages, plant species and plant compartments are

the same as in Figure 2. For gene abundance, blue, yellow

and red represent 0, 1, and 2 gene copies, respectively.

pvdH

pvdI

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R95 Pseudomonas fulva 12-XR69 Pseudomonas anguillisepticaR54 Pseudomonas sp. L10.10R50 Pseudomonas fluorescens Pf0-1R28 Pseudomonas fragiR111 Pseudomonas anguillisepticaP93 Pseudomonas fluorescens PICF7P8 Pseudomonas fluorescensP78 Pseudomonas fluorescens R124P74 Pseudomonas fluorescens R124P72 Pseudomonas fluorescens NZ011P42 Pseudomonas fluorescens NZ011P40 Pseudomonas fluorescensP39 Pseudomonas fluorescensP37 Pseudomonas fluorescensP36 Pseudomonas fluorescens NCIMB 11764P34 Pseudomonas fluorescensP32 Pseudomonas fluorescens NCIMB 11764P24 Pseudomonas fluorescens PICF7P22 Pseudomonas azotoformansP130 Pseudomonas fluorescens PICF7P129 Pseudomonas fluorescens SBW26P12 Pseudomonas fluorescens PICF7P117 Pseudomonas fluorescens SBW26P110 Pseudomonas fluorescensP11 Pseudomonas trivialisP108 Pseudomonas fluorescens PICF10P106 Pseudomonas fluorescens PICF10P104 Pseudomonas fluorescens PICF9P102 Pseudomonas fluorescens PICF8P101 Pseudomonas fluorescens PICF7103 Pseudomonas fluorescens PICF788 Pseudomonas putida F169 Pseudomonas psychrophila54 Pseudomonas fluorescens R12460 Pseudomonas fulva 12-XR44 Pseudomonas putida SJTE-1P90 Pseudomonas poaeP49 Pseudomonas putida LS46P48 Pseudomonas putida SJTE-1P33 Pseudomonas putida SJTE-166 Pseudomonas putida SJTE-1P19 Pseudomonas fluorescens PICF7R67 Pseudomonas brassicacearum NFM421P56 Pseudomonas fluorescens NZ011P18 Pseudomonas protegens Cab57P50 Pseudomonas protegens Cab57P31 Pseudomonas fluorescens PICF7R83 Pseudomonas fluorescens PICF7 P113 Pseudomonas fluorescens PICF7P114 Pseudomonas fluorescens SBW25R98 Pseudomonas fluorescens SBW25R32 Pseudomonas fluorescens PICF7P77 Pseudomonas fluorescens SBW25P41 Pseudomonas fluorescens PICF7P120 Pseudomonas fluorescens PICF7P124 Pseudomonas fluorescens SBW2563 Pseudomonas putida F185 Pseudomonas putida NBRC 1416476 Pseudomonas fluorescens101 Pseudomonas brassicacearum NFM421P70 Pseudomonas fluorescens SBW25R29 Pseudomonas fluorescens SBW25R61 Pseudomonas fluorescens70 Pseudomonas fluorescens Pf0-1R96 Pseudomonas fluorescensR5 Pseudomonas anguillisepticaR104 Pseudomonas fluorescens36 Pseudomonas fluorescens73 Pseudomonas brassicacearum NFM421 Var1 Successional stage



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pstCpstApstSpstBphnHphnFphnIphnJphnLphnGphnKphnBphnNnarKnasTnrtAnirDphnAam

tBglnK

88 Pseudomonas putida F1P90 Pseudomonas poaeR69 Pseudomonas anguillisepticaR54 Pseudomonas sp. L10.10R111 Pseudomonas anguillisepticaP19 Pseudomonas fluorescens PICF7P31 Pseudomonas fluorescens PICF7R28 Pseudomonas fragi60 Pseudomonas fulva 12-XR95 Pseudomonas fulva 12-XR83 Pseudomonas fluorescens PICF7 R50 Pseudomonas fluorescens Pf0-1R32 Pseudomonas fluorescens PICF7R29 Pseudomonas fluorescens SBW25P93 Pseudomonas fluorescens PICF7P70 Pseudomonas fluorescens SBW25P41 Pseudomonas fluorescens PICF7P24 Pseudomonas fluorescens PICF7P130 Pseudomonas fluorescens PICF7P129 Pseudomonas fluorescens SBW26P12 Pseudomonas fluorescens PICF7P114 Pseudomonas fluorescens SBW25P113 Pseudomonas fluorescens PICF7P110 Pseudomonas fluorescensP108 Pseudomonas fluorescens PICF10P106 Pseudomonas fluorescens PICF10P104 Pseudomonas fluorescens PICF9101 Pseudomonas brassicacearum NFM421P102 Pseudomonas fluorescens PICF869 Pseudomonas psychrophilaR67 Pseudomonas brassicacearum NFM421R5 Pseudomonas anguillisepticaR104 Pseudomonas fluorescens36 Pseudomonas fluorescens73 Pseudomonas brassicacearum NFM421R98 Pseudomonas fluorescens SBW25R96 Pseudomonas fluorescensR61 Pseudomonas fluorescensR44 Pseudomonas putida SJTE-1P8 Pseudomonas fluorescensP78 Pseudomonas fluorescens R124P77 Pseudomonas fluorescens SBW25P74 Pseudomonas fluorescens R124P72 Pseudomonas fluorescens NZ011P56 Pseudomonas fluorescens NZ011P50 Pseudomonas protegens Cab57P49 Pseudomonas putida LS46P48 Pseudomonas putida SJTE-1P42 Pseudomonas fluorescens NZ011P40 Pseudomonas fluorescensP39 Pseudomonas fluorescensP37 Pseudomonas fluorescensP36 Pseudomonas fluorescens NCIMB 11764P34 Pseudomonas fluorescensP33 Pseudomonas putida SJTE-1P32 Pseudomonas fluorescens NCIMB 11764P22 Pseudomonas azotoformansP18 Pseudomonas protegens Cab57P124 Pseudomonas fluorescens SBW25P120 Pseudomonas fluorescens PICF7P117 Pseudomonas fluorescens SBW26P11 Pseudomonas trivialisP101 Pseudomonas fluorescens PICF7103 Pseudomonas fluorescens PICF785 Pseudomonas putida NBRC 1416476 Pseudomonas fluorescens70 Pseudomonas fluorescens Pf0-166 Pseudomonas putida SJTE-154 Pseudomonas fluorescens R12463 Pseudomonas putida F1 Var1 Successional stage



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Nitrogen and Phosphorus metabolism

Figure 5 Distribution of genes involved in nitrogen and phos-

phorus metabolisms of the root-associated Pseudomonas

strains. The annotation and color of treatments — succes-

sional stages, plant species and plant compartments are

the same as in Figure 2. For gene abundance, blue, yel-

low and red represent 0, 1, and 2 gene copies, respectively.

pstCpstApstSpstBphnHphnFphnIphnJphnLphnGphnKphnBphnNnarKnasTnrtAnirDphnAam

tBglnK

88 Pseudomonas putida F1P90 Pseudomonas poaeR69 Pseudomonas anguillisepticaR54 Pseudomonas sp. L10.10R111 Pseudomonas anguillisepticaP19 Pseudomonas fluorescens PICF7P31 Pseudomonas fluorescens PICF7R28 Pseudomonas fragi60 Pseudomonas fulva 12-XR95 Pseudomonas fulva 12-XR83 Pseudomonas fluorescens PICF7 R50 Pseudomonas fluorescens Pf0-1R32 Pseudomonas fluorescens PICF7R29 Pseudomonas fluorescens SBW25P93 Pseudomonas fluorescens PICF7P70 Pseudomonas fluorescens SBW25P41 Pseudomonas fluorescens PICF7P24 Pseudomonas fluorescens PICF7P130 Pseudomonas fluorescens PICF7P129 Pseudomonas fluorescens SBW26P12 Pseudomonas fluorescens PICF7P114 Pseudomonas fluorescens SBW25P113 Pseudomonas fluorescens PICF7P110 Pseudomonas fluorescensP108 Pseudomonas fluorescens PICF10P106 Pseudomonas fluorescens PICF10P104 Pseudomonas fluorescens PICF9101 Pseudomonas brassicacearum NFM421P102 Pseudomonas fluorescens PICF869 Pseudomonas psychrophilaR67 Pseudomonas brassicacearum NFM421R5 Pseudomonas anguillisepticaR104 Pseudomonas fluorescens36 Pseudomonas fluorescens73 Pseudomonas brassicacearum NFM421R98 Pseudomonas fluorescens SBW25R96 Pseudomonas fluorescensR61 Pseudomonas fluorescensR44 Pseudomonas putida SJTE-1P8 Pseudomonas fluorescensP78 Pseudomonas fluorescens R124P77 Pseudomonas fluorescens SBW25P74 Pseudomonas fluorescens R124P72 Pseudomonas fluorescens NZ011P56 Pseudomonas fluorescens NZ011P50 Pseudomonas protegens Cab57P49 Pseudomonas putida LS46P48 Pseudomonas putida SJTE-1P42 Pseudomonas fluorescens NZ011P40 Pseudomonas fluorescensP39 Pseudomonas fluorescensP37 Pseudomonas fluorescensP36 Pseudomonas fluorescens NCIMB 11764P34 Pseudomonas fluorescensP33 Pseudomonas putida SJTE-1P32 Pseudomonas fluorescens NCIMB 11764P22 Pseudomonas azotoformansP18 Pseudomonas protegens Cab57P124 Pseudomonas fluorescens SBW25P120 Pseudomonas fluorescens PICF7P117 Pseudomonas fluorescens SBW26P11 Pseudomonas trivialisP101 Pseudomonas fluorescens PICF7103 Pseudomonas fluorescens PICF785 Pseudomonas putida NBRC 1416476 Pseudomonas fluorescens70 Pseudomonas fluorescens Pf0-166 Pseudomonas putida SJTE-154 Pseudomonas fluorescens R12463 Pseudomonas putida F1 Var1 Successional stage



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the cluster, respectively two P. protegens strains from rhizosphere and one P. brassicacearum strain from endosphere. In addition, the strain-specific pvdL gene encoding pyoverdine chromophore synthetase was only found in these three genomes.

Nitrogen and phosphorus metabolism

Genes related to nitrate availability (nrt and nirB genes) existed in all ge-nomes except for P. fragi strain from endosphere of L. vulgare at the 65-year stage (Figure 5). For phosphate metabolism, however, except for phnA gene, other phn genes involved in phosphonate uptake and degradation were all strain-specific, only observed in five strains, whereas the genes required for the Pst (phosphate-specific transport) system were generalized among all genomes. Therefore, the strain-specific phn genes differentiated the 70 genomes in terms of nitrogen and phosphorus metabolism. Eventually, two P. fluorescens strains and two P. anguilliseptica strains respectively ob-tained from rhizosphere and endosphere of A. maritima, together with one unknown Pseudomonas strain from rhizosphere of L. vulgare were found to have all the genes in these two systems.

Biochemical properties of plant-associated Pseudomonas

For the biochemical properties of the 70 unique Pseudomonas strains, anal-yses of variance revealed that successional stage, plant compartment and plant species respectively affected the antibiotic resistance (penicillin, F=3.505, P<0.05; streptomycin, F=3.212, P<0.05), siderophore production (F=4.104, P<0.05), and bacterial fitness (osmotic stress resistance, F=9.121, P<0.01; growth under different pH, F=4.114, P<0.05) (Table S3). Moreover, penicillin resistance was found to be influenced by the interactive effect of successional stage and plant compartments, and the interaction of plant species and plant compartments (respectively, F=2.905, P<0.05; F=4.050, P<0.05). The latter one was also observed to affect siderophore production (F=4.626, P<0.05). The interaction among the three factors was observed to exert significant influence on growth under different pH (F=4.142, P<0.05).

Furthermore, when analyzed along the chronosequence, only isolates ob-tained from the endosphere showed significant patterns for resistance to sa-linity and osmotic stress along the chronosequence (Figure S4), respectively

Discussion 159

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exhibiting a reverse hump-shaped trend (r2=0.277, P<0.01; r2=0.372, P<0.001) with a slight decrease from the initial stage to 65-year stage followed by a sharp increase towards 105-year stage, which was also observed for the growth un-der different pH (P<0.01) (Figure S5). While other traits of endophytes were relatively constant across the succession. In terms of Pseudomonas isolates from rhizosphere, however, no significant trend was shown for the biochem-ical properties along the chronosequence (Figure S4, S5).

In addition, distributions of bacterial fitness traits according to differ-ent plant compartments was respectively shown for resistance against sa-linity and osmotic stress, with significant enrichment in rhizosphere at both 15- and 65-year stages (P<0.05) (Figure S6). These two traits also dif-fered in plant species, respectively showing enrichment in L. vulgare at the 65- and 5-year stage (P<0.05). Moreover, for streptomycin resistance, en-richment in endosphere was observed at 5-year stage (P<0.01). While for other biochemical traits, no enrichment was found either in different plant compartment or plant species (P>0.05).

Specifically, regarding the biochemical properties including bacterial fit-ness, plant growth promoting capacity and metabolic potential, two strains from rhizosphere consistently exhibited high profiles, respectively P. fluo-rescens from 105-year stage and P. azotoformans from 5-year stage (FigureS8–S11). Moreover, one endophytic strain P. putida SJTE-1 from 105-year stage was also found to show high activities in bacterial fitness (Figure S8, S9).

Discussion

The genus Pseudomonas is one of the most diverse bacterial genera, occu-pying many different niches and exhibiting versatile metabolic capacities (Haas et al., 2005; Gross and Loper, 2009; Mulet et al., 2010; Jun et al., 2015). A number of pseudomonad strains function as plant growth-promoting rhizobacteria (PGPR), which can protect plants from various soilborne pathogens and/or stimulate plant growth (Haas et al., 2005; Berendsen et al., 2015). Comparative genomics analyses of different PGPR have gener-ated information about the genetic basis of diversity and adaptation in this genus (Silby et al., 2009; Shen et al., 2013b; Jun et al., 2015; Garrido-Sanz et al., 2016), providing functional information on the genomic elements that allow these species to cope with different habitats or microenviron-ments. In this study, we investigated the specific genes or gene clusters in-volved in the evolution of Pseudomonas from both the genetic and functional


perspectives, by comparing 70 Pseudomonas genomes obtained from the rhi-zosphere and endosphere of two salt marsh plants, L. vulgare and A. mari-tima, growing on a natural gradient of soil physicochemical constraints (Olff et al., 1997; Dini-Andreote et al., 2014, 2015; Wang et al., 2016).

Taxonomic distribution and genomic diversity of root-associated Pseudomonas isolates

Among the 70 Pseudomonas genomes, P. fluorescens was the predominant spe-cies spreading in different subgroups, encompassing large breadth of phy-logenetic distance. The enormous phylogenetic heterogeneity shown by the strains belonging to P. fluorescens group has been reported by many stud-ies (Silby et al., 2009; Loper et al., 2012; Jun et al., 2015). The heterogeneity in P. fluorescens group was also revealed when analyzing the distribution of strains belonging to different subgroups of P. fluorescens according to differ-ent successional stages, plant compartments and plant species. Different from the even distribution of P. fluorescens subgroup II between different plant compartments of different plant species, most of the strains in P. flu-orescens subgroup I were unique to A. maritima, indicating a stronger selec-tion from this plant, probably in response to specific root exudates, which resulted in a unique pseudomonad composition (Hartmann et al., 2009; Berendsen et al., 2012). The stronger plant effect exerted on P. fluorescens subgroup I could also be explained by the predominance of P. fluorescens species in this group, which are known for their PGPR traits. However, this selective force was not found for P. fluorescens subgroup II, which could be explained by the diverse species composition in this group.

In addition, the absolute predominance of P. fluorescens subgroup I and II in 65- and 5-year stages correlates well with the high population density of Pseudomonas observed in these two stages, which has been attributed to the various niches provided by the enrichment of soil nutrients and plant bio-mass (65-year stage) or from a constant influx of different microbes deriv-ing from marine input (5-year stage; Dini-Andreote et al., 2014; Wang et al., 2016, Chapter 4). This was also confirmed in our previous study on the rhizo-sphere of L. vulgare where unique OTUs belonging to Pseudomonas were found at 5- and 65-year stages (Wang et al., 2016). Similar distribution according to stages was also found for the non-dominant group in this study, P. putida.

Despite the plant specificity observed, especially for P. fluorescens subgroup I, we observed an even stronger response to different plant compartments

Discussion 161

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(rhizosphere and endosphere), where different biochemical environment determines the degree of association with plant host, eventually lead-ing to the recruitment of unique species (Gaiero et al., 2013; Timm et al., 2015). The apparent selectivity according to plant compartment was also confirmed in our previous study on the functionality of bacterial isolates (Wang et al., chapter 4), although these results might be driven by the low number of isolated species.

Despite the similar isolation conditions and relative taxonomic close-ness of these isolates, we observed significant differences in the genetic di-versity among the genomes, highlighting the considerable functional di-versity within Pseudomonas genus in the plant microbiome. However, we did not observe the presence of gene or gene clusters that uniquely discrimi-nated isolates between plant compartments (rhizosphere and endosphere), or plant species (L. vulgare and A. maritima), nor among different stages. According to Timm et al. (2015), this could be attributed to the: (1) wide range of potential mechanisms for plant- bacteria interactions, (2) misidentifica-tion of pathways, (3) actual expression of these pathways on plant, or (4) in-ability to predict function for all genes. However, we found certain genomic elements involved in specific mechanisms, which may serve as an reference for species adaptation in specific habitats and for potential interactions be-tween root and PGP Pseudomonas strains, as discussed below.

Environmental adaptability

Rhizosphere bacteria usually survive in a highly variable environment, therefore, they have evolved several traits related to adaptation (Ramos et al. , 2001). Consistently, most of the strains showing the highest abun-dance and diversity of genes in the resistance to antibiotics, metal and os-motic stress were P. fluorescens species isolated from rhizosphere. In addi-tion, among these P. fluorescens strains with high gene profiles associated with adaptability, an absolute large proportion were affiliated with sub-group II, indicating a great genetic diversity within a single species (P. flu-orescens) and that the distribution of these genomic elements is dependent on the P. fluorescens complex groups, being higher in subgroup II.

Heavy metals and antibiotic resistance: Most heavy metals are toxic at higher concentrations. For example, copper ions can damage the cy-toplasmic membrane of E. coli by catalyzing harmful redox reactions (Hoshino et al. , 1999). Consequently, certain soil bacteria have developed


resistance to toxic metals, either via active efflux mechanisms to pump the toxic metals out (Nies, 2003), or by enzymatic detoxification to con-vert a toxic ion into a harmless one (Mejare and Bulow, 2001; Vuilleumier and Pagni, 2002). Our genomic analysis revealed that all the genes related to copper and arsenic resistance (except for copB) were generalized among the 70 genomes. The lack of copB gene in half of the genomes could be explained by the complementary effect from copA, which is an effective copper pump at low and high copper concentrations. While copB ap-peared to be a low- affinity copper export ATPase, which was only rele-vant if the media copper concentration was exceedingly high (Völlmecke et al. , 2012). The results on copper and arsenic resistance were surpris-ing, given that these pseudomonads were isolated from undisturbed soils from a natural reserve area. However, it has been demonstrated that bac-terial exposure to heavy metals precede anthropogenic-derived sources (Baker-Austin et al. , 2006). Thus, our findings suggest either a previ-ous selection for these genes or that these metals are present in this soil. Interestingly, these genes are often found in association with antibiotic resistance (Baker-Austin et al. , 2006; Seiler and Berendonk, 2012; Pal et al. , 2015; Mcarthur et al. , 2017), which is supported by our data, as all the Pseudomonas genomes (except for two P. putida strains, respectively P. putida NBRC 14164 and P. putida F1, from rhizosphere) in our study con-tained the predicted gene encoding for β-lactamases, confirming the po-tential antibiotic resistance. This activity has been associated with differ-ent species in this genus, which encompass a broad spectrum of putative multidrug resistance proteins, such as penicillin-binding protein- mediated resistance from P. aeruginosa (Georgopapadakou et al. , 1993; Davies et al. , 2008; Smith et al. , 2013). Therefore, the antibiotic resistance observed for P. putida NBRC 14164 strain from the result of our biochemical tests could be explained by the complementary effect from other genes encoding for penicillin- binding proteins instead of the gene detected in this study. In addition, the presence of the detected gene involved β-lactamase biosyn-thesis did not necessarily lead to the antibiotic resistance which was only found for a certain amount of strains, suggesting that more genes related to these pathways of antibiotic resistance and the actual gene expression need to be explored. It is interesting to highlight that higher abundance of antibiotic resistance genes have been previously detected in a metagenomics survey in bulk soil samples collected from the same area, whose presence have been attributed to the bacterial ability to colonize terres-trial environments (Dini-Andreote, submitted).

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Saline stress: The high adaptability of P. fluorescens in stressed environment, especially saline soils, has been reported by other studies (Egamberdieva et al., 2011; Cho et al., 2015). All Pseudomonas strains in our study were ob-served to contain genes involved in glycine betaine (GB) catabolism, indi-cating the wide distribution of genomic elements associated with resis-tance to osmotic stress among this genus. The genomic analysis by Timm et al. (2015) showed that the genomes of P. chlororaphis GP72, P. fluorescens Pf-5, and P. aeruginosa M18 contained at least one complete gene set required for conversion of GB to glycine. According to our results of the biochemi-cal tests, the majority of Pseudomonas strains were capable to resist salin-ity and osmotic stress, although at different levels. The GB catabolism of Pseudomonas was also confirmed by phenotypic tests by other studies (Liu et al., 2007; Wargo et al., 2008). An overall presence of genes involved in saline stress has been previously observed in metagenomics data from this area, where the higher abundance was detected in the later stages of suc-cession (Dini-Anreote et al. submitted).

Root colonization and biocontrol activity

Competitive colonization ability of plant roots is essential for a competent rhizobacterium (Kamilova et al., 2005). Pseudomonad PGPR show certain competitive colonization traits, such as motility and the ability to attach to the root surface (de Weert et al., 2002; Bolwerk et al., 2003; Neidig et al., 2011; Loper et al., 2012; Nadeem et al., 2016). The high genetic diversity in chemotaxis, motility and adhesion exhibited by the three P. brassicacearum strains and one P. fluorescens strain belonging to P. fluorescens subgroup II may indicate the high colonization power of these strains (Achouak et al., 2000; Belimov et al., 2001; Redondo-Nieto et al., 2013). By screening the colonization pattern by two variants of P. brassicacearum during Arabidopsis thaliana root colonization, Achouak et al. (2004) confirmed that the com-petitive colonization ability was largely resulted from the phenotypic vari-ation, which was verified by Lalaouna et al. (2012), by showing the naturally occurring mutations in the gacS-gacA two-component system. The distinct strain-specific genes, cheC and cheD, indicated their importance role in dif-ferentiating the genetic diversity in chemotaxis among these genomes. The copies of the ‘core’ genes, e.g. cheAWY, are clustered in multiple distinct loca-tions and additional genes are present (cheC, cheD, cheV and cheX) that gener-ate greater mechanistic diversity (Szurmant and Ordal, 2004). Our results


suggest that it might be worth exploring the system involving cheC and cheD for these competent root colonizers, i.e. the three P. brassicacearum strains and one P. fluorescens strain found in this study.

Genes involved in siderophore production are often associated with root colonizing capacity in the rhizosphere. Pyoverdines are a diverse group of fluorescent siderophores produced by pseudomonads, which facilitate iron uptake of these bacteria to improve their biocontrol activity (Ravel and Cornelis, 2003; Visca et al., 2007). In our study, all the detected genes for the siderophore (pyoverdine) biosynthesis pathways were strain-specific, especially for pvdL, which was found in its completion only in two P. prote-gens strains from rhizosphere and one P. brassicacearum strain from endo-sphere. This suggests that these species from P. fluorescens subgroup II could have a competitive advantage in iron-limited environments, which was consistent with the findings by Garrido-Sanz et al. (2016). They revealed that a hemophore-dependent heme acquisition biosynthetic and trans-port clusters were present in all the strains from the P. protegens group, by identifying different clusters of orthologous CDSs involved in the biosyn-thesis of several iron-siderophores among different subgroups within the P. fluorescens complex (Garrido-Sanz et al. 2016). Hartney et al. (2013) also demonstrated that, in addition to producing its own siderophores, P. prote-gens Pf-5 also utilized ferric complexes of some pyoverdines produced by other strains of Pseudomonas spp. as sources of iron, therefore providing a competitive advantage in the rhizosphere, where biologically available iron is limited. In addition, the presence of several genes involved in siderophore biosynthesis from other P. fluorescens and P. putida strains also highlighted the rhizo- competence properties of different subgroups in the Pseudomonas ge-nus (Cho et al., 2015; Timm et al., 2015; Garrido-Sanz et al., 2016). However, from the results of biochemical test, these three strains containing all the detected genes for the pyoverdine biosynthesis pathway were not observed with high siderophore production, which could be explained by the lack of the appropriate conditions in which gene expression takes place. In addition, the two strains exhibited highest siderophore production (respectively P. flu-orescens and P. azotoformans strain from the rhizosphere of L. vulgare) lack of pvd genes, indicating other siderophore biosynthesis pathways may exist in these strains, such as pyochelin (Pch) and pyrroloquinoline quinone (PQQ) (Phoebe Jr. et al., 2001; Brandel et al., 2012; Cho et al., 2015).

Discussion 165

5

Nitrogen and phophorus metabolisms

Certain rhizobacteria are able to solubilize insoluble or poorly soluble min-eral phosphates by producing acid phosphatases and organic acids (mainly gluconic acid), which are then made available to plants (Achal et al., 2007; Gupta et al., 2012). Several Pseudomonas spp. have been described as good phosphate solubilizers (de Werra et al., 2009; Rocha et al., 2016; Xie et al., 2016). The two systems required for the phosphonate uptake and degra-dation were quite different in terms of the gene distributions among the 70 genomes. The phn gene cluster was found only in five strains, while the pst gene cluster was observed in each genome. In addition, the strain- specific phn genes were also specific to plant species, only found associated with strains isolated from A. maritima. This results indicated the high phos-phate solubilizing ability of the two P. fluorescens strains and two P. anguilli-septica strains, which respectively belonged to P. fluorescens subgroup I and II.

The nitrate-assimilation process begins with the transport of nitrate into the cell. Nitrate is further reduced to nitrite in a two-electron reaction by a cytoplasmic molybdenum-containing nitrate reductase followed by a six-electron nitrite reduction to produce ammonia by a siroheme-nitrite reductase (Moreno-Vivián et al., 1999; Richardson et al., 2001). Studies of nitrate assimilation in heterotrophic bacterial species are scarce, however, examination of available genomes suggests that assimilatory nitrate reduc-tases (Nas) are phylogenetically widespread in bacterial and archaeal het-erotrophs (Richardson et al., 2001; Luque-Almagro et al., 2011). According to our results, genes involved in nitrate assimilation were found in all ge-nomes (except for one endophytic P. fragi strain), contrary to those in phos-phorus metabolism, indicating a wide range of Pseudomonas associated with plant root with potential nitrate assimilation capacity.

Biochemical characteristics of root-associated Pseudomonas isolates along the chronosequence

Contrary to our hypothesis that the functionality of rhizosphere Pseudomonas isolates will increase as the increment of soil nutrients and plant biomass along the chronosequence, all the biochemical traits tested for the isolates were relatively constant without showing a clear pattern across the succession (Figure S4). These results were inconsistent with the findings that functional properties of Pseudomonas, such as antagonism


against the soil-borne pathogen and gene expression related to organic compound degradation, were influenced by soil types (sandy, loamy sand and loam) (Garbeva et al., 2004a; Afzal et al., 2011). Thus, besides the poten-tial effect of soil types, rhizosphere effect also play an important role on the community structure and functions of rhizosphere microbiome (Schmidt et al., 2004; Costa et al., 2006b, 2007; Schreiter et al., 2014). Besides the P. fluorescens and P. putida strains observed in this study with widely recog-nized PGP traits (Meziane et al., 2005; Bakker et al., 2007; Yao et al., 2010; Loper et al., 2012; Jain and Das, 2016; Nadeem et al., 2016), one particular rhizosphere isolate P. azotoformans was also found to show high functional profiles in biochemical properties, which confirmed the high gene abun-dance in the systems related to environmental adaptability, which was in accordance with the wide range of biocontrol activity performed by this species (Sang et al., 2014; Fang et al., 2016).

Similar to the rhizosphere isolates, the majority of traits associated with endophytic Pseudomonas strains, except for resistance to salinity and osmotic stress, remained constant along the chronosequence, confirm-ing our hypothesis. Due to the plant selectivity and buffering effect exert-ing on the endophytes (Hallmann et al., 1997; Rosenblueth and Martínez-Romero, 2006; Schulz and Boyle, 2006), the community structure and functional profile of the root endophytes could remain stable (Wang et al., 2016, Chapter 4). Interestingly, the dramatic increase in resistance against salinity and osmotic stress at the 105-year stage was in line with the distri-butions of biochemical characteristics tested for all the endophytic isolates along the chronosequence (Wang et al., Chapter 4). These patterns in func-tionality might be explained by the community turnover of endophytes observed for L. vulgare-associated bacterial communities in our previous studies (Wang et al., 2016). This drastic variation in endophytic community composition and functionality could be derived from the reduced plant control on the endophytes, resulting from the stress experienced by both L. ulgare and A. maritima, given the higher salinity level and severe competi-tion with other dominant plants (specially Elytrigia atherica) at the late stage (Schrama et al., 2012).

Conclusion

Similar to previous studies of the P. fluorescens group (Silby et al., 2009; Loper et al., 2012; Timm et al., 2015), we also observed two subgroups within this

Conflict of interest 167

5

complex, supporting the segregation of the P. fluorescens group into multiple species. P. fluorescens subgroup I was associated with plant species. Moreover, the predominance of P. fluorescens subgroup I and II from 65- and 5-year stages indicated some soil-specific effect on the bacterial composition, but further studies are necessary to pinpoint the soil physicochemical condi-tions they respond to. The high gene diversity displayed by the isolates ob-tained in this study suggests that the genus Pseudomonas can fill multiple roles in the microbiome — a fact that is overlooked in studies focusing on the genetic variability of the 16S rRNA gene. Although we did not observe an association between genome and the different treatments (soil succes-sional stage, plant species or plant compartment), we did observe that the strains with the higher number of genes involved in environmental adapt-ability were enriched in rhizosphere, whereas those with greater potential in phosphorus metabolism were enriched in A. maritma. These results high-light the potential functional requirements for colonization of the specific plant microenvironments or different plant species. In addition, the strains exhibiting high genetic diversity in adaptation, colonization and biocontrol were mostly from P. fluorescens subgroup II. These results indicated that the genomic elements for specific functions were related to the phylogenetic re-latedness within the P. fluorescens complex, which could serve as a genetic ba-sis for the Pseudomonas evolution along a series of environmental changes.

Conflict of interest

The authors declare of no conflict of interest.

Acknowledgements

We thank Han Olff, Matty Berg, Chris Smit, Maarten Schrama and Ruth Howison for information on sampling locations and plant species. We are grateful to Jolanda K Brons and Armando Cavalcante Franco Dias for sampling expeditions. We thank the ‘Nederlandse Vereniging voor Natuurmonumenten’ for granting us access to the salt marsh. This work was supported by Chinese Scholarship Council, on a personal grant to MW.


Supplementary materials

Table S1 Summary of P. fluorescens isolates from different plants, sources and succes-

sional stages

Sample ID Stage Plant Source Replicates weight To

tal n

um

ber

of

iso

-la

tes

for

each

sta

ge

Tota

l nu

mb

er o

f se

-le

cted

co

lon

ies

wit

h

un

iqu

e m

orp

ho

log

ies

fro

m S

1 ag

ar p

late

Nu

mb

er o

f B

ox

pat

tern

s

P93 1D ART 5y Artemisia maritima Endosphere 4 0.531252 32 8

P90 1D ART 5y Artemisia maritima Endosphere 4 0.46875P106 2C ART 15y Artemisia maritima Endosphere 1 0.375

4 24 5P101 2B ART 15y Artemisia maritima Endosphere 1 0.291667P102 2B ART 15y Artemisia maritima Endosphere 2 0.208333P104 2A ART 15y Artemisia maritima Endosphere 1 0.125P108 3A ART 35y Artemisia maritima Endosphere 3 1 1 25 3P74 6C ART 65y Artemisia maritima Endosphere 2 0.376812

9 69 16

P129 6A ART 65y Artemisia maritima Endosphere 2 0.101449P130 6A ART 65y Artemisia maritima Endosphere 2 0.101449P77 6C ART 65y Artemisia maritima Endosphere 1 0.057971P78 6C ART 65y Artemisia maritima Endosphere 2 0.130435P113 6B ART 65y Artemisia maritima Endosphere 2 0.072464P120 6B ART 65y Artemisia maritima Endosphere 1 0.028986P114 6B ART 65y Artemisia maritima Endosphere 2 0.057971P117 6B ART 65y Artemisia maritima Endosphere 2 0.072464P34 1G ART 5y Artemisia maritima Rhizosphere 2 0.285714

6 42 10

P36 1G ART 5y Artemisia maritima Rhizosphere 2 0.309524P37 1G ART 5y Artemisia maritima Rhizosphere 2 0.166667P39 1G ART 5y Artemisia maritima Rhizosphere 1 0.047619P40 1G ART 5y Artemisia maritima Rhizosphere 2 0.142857P24 1F ART 5y Artemisia maritima Rhizosphere 1 0.047619P19 2C ART 15y Artemisia maritima Rhizosphere 2 0.354839

3 25 6P11 2A ART 15y Artemisia maritima Rhizosphere 2 0.516129P12 2A ART 15y Artemisia maritima Rhizosphere 2 0.129032P31 3A ART 35y Artemisia maritima Rhizosphere 2 0.269231

4 26 6P32 3A ART 35y Artemisia maritima Rhizosphere 1 0.192308P33 3A ART 35y Artemisia maritima Rhizosphere 1 0.269231P18 3C ART 35y Artemisia maritima Rhizosphere 2 0.269231P41 8A ART 105y Artemisia maritima Rhizosphere 3 0.219178

2 73 7P56 8C ART 105y Artemisia maritima Rhizosphere 4 0.780822P110 1G LIM 5y Limmonium vulgare Endosphere 5 0.393443

2 122 11P72 1G LIM 5y Limmonium vulgare Endosphere 6 0.606557P70 6B LIM 65y Limmonium vulgare Endosphere 2 0.56

2 25 4P124 6C LIM 65y Limmonium vulgare Endosphere 2 0.44P22 1F LIM 5y Limmonium vulgare Rhizosphere 6 1 1 28 6P42 3A LIM 35y Limmonium vulgare Rhizosphere 7 1 1 39 7P49 6A LIM 65y Limmonium vulgare Rhizosphere 4 0.283951

3 81 12P50 6A LIM 65y Limmonium vulgare Rhizosphere 3 0.160494P48 6A LIM 65y Limmonium vulgare Rhizosphere 5 0.555556P8 8A LIM 105y Limmonium vulgare Rhizosphere 8 1 1 55 8

Supplementary materials 169

5

Table S2 Screening procedure for the fluorescent pseudomonads isolates from S1 agar

plate

Plate ID Source Plant species Stage

Dilution (log10)

Total number

of colonies on each

plate

Total number of selected colonies

with unique morphol-

ogies from each plate

Number of Box

patterns

BOX pat-tern ID

Number of

colonies

1 rhizosphere Limonium vulgare 5y -2 25 14 2 1 62 8

2 rhizosphere Limonium vulgare 5y -2 147 17 4

3 44 35 36 4


7 38 49 6

10 311 4



14 415 816 617 518 419 620 3

6 rhizosphere Limonium vulgare 65y -2 27 15 1 21 15


22 823 624 725 9




11 rhizosphere Artemisia maritima 5y -2 96 7 1 34 7

12 rhizosphere Artemisia maritima 5y -2 118 23 4

35 536 737 638 5


39 240 241 242 443 2


44 645 546 547 11

15 rhizosphere Artemisia maritima 15y -1 15 4 2 48 249 2


50 451 352 553 754 4



Plate ID Source Plant species Stage

Dilution (log10)

Total number

of colonies on each

plate

Total number of selected colonies

with unique morphol-

ogies from each plate

Number of Box

patterns

BOX pat-tern ID

Number of

colonies




21 endosphere Limonium vulgare 5y -2 300 16 1 63 16

22 endosphere Limonium vulgare 5y -1 207 32 4

64 865 866 767 9

23 endosphere Limonium vulgare 5y -1 133 32 368 1769 1570 10


25 endosphere Limonium vulgare 65y -2 193 9 1 74 9


27 endosphere Artemisia maritima 5y -2 78 32 8

78 379 380 581 682 383 384 485 5

28 endosphere Artemisia maritima 15y -1 72 16 2 86 987 7


30 endosphere Artemisia maritima 35y -1 8 5 1 91 5




96 397 498 399 4

100 4101 4102 5


103 3104 2105 2106 2107 2108 2109 3


5

Table S3 Three-way anova to test the influence of successional stages, plant species

and sources on different biochemical traits St

age

Pla

nt

spec

ies

Sou

rce

Stag

e:P

lan

t sp

ecie

s

Stag

e:So

urc

e

Pla

nt

spec

ies:

Sou

rce

Stag

e:P

lan

t sp

ecie

s:So

urc

e

Carbon source usage

F value 0.085 0.028 0.822 0.072 0.679 2.438 1.028

Pr(>F) 0.772 0.868 0.368 0.789 0.413 0.123 0.315

Oxidative stress resistance

F value 2.770 1.260 0.027 0.294 0.864 1.900 3.779

Pr(>F) 0.101 0.266 0.870 0.589 0.356 0.173 0.056

Salinity tolerance

F value 0.832 2.588 2.407 0.008 0.051 2.899 0.417

Pr(>F) 0.365 0.112 0.125 0.929 0.822 0.093 0.520

PEG stress resistance

F value 1.980 9.121 3.891 1.566 0.227 0.038 0.297

Pr(>F) 0.164 0.003** 0.053 0.215 0.635 0.845 0.587

Growth under different pH

F value 0.029 4.114 3.493 0.843 0.509 0.471 4.142

Pr(>F) 0.863 0.043* 0.062 0.359 0.476 0.493 0.042*

Growth under penicillin

F value 3.505 0.122 2.034 0.806 2.905 4.050 0.046

Pr(>F) 0.013* 0.728 0.159 0.526 0.030* 0.049* 0.954

Growth under streptomycin

F value 3.212 1.095 0.041 0.933 2.451 0.339 0.728

Pr(>F) 0.019* 0.300 0.839 0.452 0.057 0.562 0.487

Exoprotease produciton

F value 0.001 0.606 0.777 0.185 3.643 0.776 2.151

Pr(>F) 0.972 0.439 0.381 0.668 0.060 0.381 0.147

Biofilm formation

F value 1.087 2.773 0.001 1.134 0.167 1.393 0.128

Pr(>F) 0.301 0.101 0.970 0.291 0.684 0.242 0.722

Consuming iron ion

F value 0.025 0.907 4.104 0.849 0.970 4.626 2.031

Pr(>F) 0.874 0.344 0.047* 0.360 0.328 0.035* 0.159


Table S4 Two-way anova to test the influence of successional stages and plant species

on different biochemical traits

Stage Plant species Stage:Plant species

Carbon source usage

RhiosphereF value 0.180 1.498 0.110

Pr(>F) 0.674 0.231 0.743

EndosphereF value 0.464 0.965 0.940

Pr(>F) 0.501 0.333 0.340

Oxidative stress resistance


Pr(>F) 0.541 0.789 0.165


Pr(>F) 0.058 0.070 0.183

Salinity tolerance


Pr(>F) 0.258 0.836 0.831


Pr(>F) 0.687 0.046* 0.535

PEG stress resistance


Pr(>F) 0.091 0.054 0.507


Pr(>F) 0.601 0.052 0.242

Growth under different pH


Pr(>F) 0.744 0.382 0.601


Pr(>F) 0.589 0.062 0.024*

Growth under penicillin


Pr(>F) 0.170 0.209 0.876


Pr(>F) 0.010* 0.354 0.145

Growth under streptomycin


Pr(>F) 0.123 0.894 0.626


Pr(>F) 0.013* 0.366 0.315

Exoprotease produciton


Pr(>F) 0.301 0.278 0.554


Pr(>F) 0.081 0.936 0.096

Biofilm formation


Pr(>F) 0.325 0.113 0.564


Pr(>F) 0.787 0.598 0.225

Consuming iron ion


Pr(>F) 0.491 0.302 0.187


Pr(>F) 0.420 0.037* 0.478


5

Table S5 Functional diversity indices for different successional stages

Traits Source Stage Functional diversity P-value (K-W test)

Total

Rhizosphere

5y 0.63±0.21

0.001***15y 0.5±0.1235y 0.9±0.1265y 0.46±0.14105y 0.68±0.24

Endosphere

5y 0.39±0.15

0.001***15y 0.74±0.2235y 0.98±0.0265y 0.68±0.25105y 0.68±0

Plant associate traits

Rhizosphere

5y 0.65±0.33

0.5915y 0.81±0.2935y 0.79±0.2565y 0.66±0.31105y 0.73±0.29

Endosphere

5y 0.77±0.35

0.6515y 0.75±0.3235y 0.76±0.0565y 0.8±0.27105y 0.88±0

Abiotic stress resistance

Rhizosphere

5y 0.6±0.2

0.05415y 0.49±0.0935y 0.88±0.165y 0.39±0.15105y 0.6±0.17

Endosphere

5y 0.51±0.23

0.10315y 0.68±0.3135y 0.95±065y 0.58±0.2105y 0.54±0

Antibiotic resistance

Rhizosphere

5y 0.57±0.26

0.017*15y 0.36±0.2135y 0.77±0.2665y 0.33±0.2105y 0.46±0.2

Endosphere

5y 0.27±0.16

0.009**15y 0.52±0.4135y N.A.65y 0.56±0.3105y 0.52±0

Metabolic potential

Rhizosphere

5y 0.64±0.22

0.05615y 0.56±0.2235y 0.79±0.165y 0.47±0.13105y 0.68±0.26

Endosphere

5y 0.37±0.17

0.012*15y 0.59±0.1735y 0.95±065y 0.57±0.25105y 0.87±0


Fig

ure

S1

Wo

rk fl

ow

of

iso

lati

ng

an

d m

ole

cula

r ch

arat

eriz

atio

n o

f ro

ot-

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ed P

seu

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mo

nas

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late

s

Wor

k flo

w

S1 m

edia

1ml

100μ

l

10-2

10-3

10-4

10-5

10-6

10-1

1ml

rhiz

osph

ere

endo

sphe

re

100μ

l

BOX-

PCR

PCR

of

Pseu

dom

onas

fluo

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ens

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SEflu

& 1

6SPS

ER

Mic

rotit

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atter

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as st

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er 1

6S rR

NA

gene

se

quen

cing

with

prim

ers

B8F

& U

1406

RR2

A

+


5

Figure S2 Biochemical assays for functional traits screening of root-associated

Pseudomonas isolates

Figure S3 Variation of colony forming units (CFU) of root-associated Pseudomonas

isolates along the chronosequence. For plant species, solid and dashed line represent

A. maritima and L. vulgare, respectively. For plant compartments, black refer to rhizo-

sphere isolates whereas green represent those obtained from the endosphere

Bacterial culture in stationary phase

Centrifuge

Culture supernatant

Carbon source usage (14 carbon sources)

Antibiotic resistance (streptomycin , tetracycline and penicillin)


Salinity tolerance(7% and 10%)

Different pH (5, 6, 8, 9)

H2O2 tolerance(0,00025% and0,0005%)

PEG (polyethylene glycolMn6000) stress

Exoprotease activity

IAA production

Siderophore production

biofilm formation

Functional traits screening

�x3,��r2�

0.0

2.5

5.0

7.5

10.0

0 25 50 75 100Stage (years)

log

CFU

/ g

fresh

soi

l

SourceEndosphere

Rhizosphere

PlantArtemisia

Limonium

y=4+2.4x+0.25x2-2.8x3, r2=0.69, P-value=0.019Artemisia

y=3.7+1.3x-1.9x2-2x3, r2=0.587, P-value=0.026Artemisia

y=3.2+0.76x+0.35x2-2.5x3, r2=0.655, P-value=0.011Limonium


Figure S4 Variation of functional traits of Pseudomonas isolates from rhizosphere and

endosphere along the chronosequence. (A) Metabolic potential, (B) Salinity stress re-

sistance, (C) Oxidative stress resistance, (D) Osmotic stress resistance, (E) Penicillin resis-

tance, (F) Streptomycin resistance, (G) Exoprotease production, (H) Biofilm production

and (I) Siderophore production. Grey color refer to rhizosphere isolates whereas green

represent those obtained from the endosphere

0.0

2.5

5.0

7.5

10.0

12.5

0 25 50 75 100

Gro

wth

und

er 1

4 ca

rbon

sou

rces

(%)

0

50

100

150

200

0 25 50 75 100

Gro

wth

und

er s

alin

ity s

tress

(%)

Endosphere y=28+29x+111x2, r2=0.277, P-value=0.004

0

25

50

75

100

0 25 50 75 100

Gro

wth

und

er o

xida

ttive

stre

ss (%

)

0

50

100

150

0 25 50 75 100

Endosphere y=16+33x+97x2, r2=0.372, P-value<0.001

Gro

wth

und

er P

EG s

tress

(%)

.

0

50

100

150

200

0 25 50 75 100

Gro

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)

0

50

100

150

200

0 25 50 75 100

Gro

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(%)

0.0

0.1

0.2

0.3

0.4

0.5

0 25 50 75 100

Abso

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Stage (years)

0.000

0.025

0.050

0.075

0.100

0 25 50 75 100

Abso

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film

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n

Stage (years)

0

20

40

60

0 25 50 75 100

Abso

rban

ce o

f con

sum

ed ir

on

Stage (years)

(A) (B) (C)

(D) (E) (F)

(G) (H) (I)

SourceEndosphereRhizosphere


5Fi

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re S

5 V

aria

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n o

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olid

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es r

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and

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een

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nt

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ts. S

olid

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nes

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er t

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e, re

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tive

ly

0

306090

025

5075

100

Stag

e (y

ears

)

Growth under different pH (%)

pHpH

5

pH6

pH8

pH9

Plan

t spe

cies

Arte

mis

ia m

ariti

ma

Lim

oniu

m v

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re

0306090

025

5075

100

Stag

e (y

ears

)

Growth under different pH (%)

pHpH

5

pH6

pH8

pH9

Sour

ce Endo

sphe

re

Rhi

zosp

here

pH6,

A.m

ariti

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0075

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-val

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pH5,

end

osph

ere,

y=2

.3+1

4x+1

2x2 , r

2 =0.2

66, P

-val

ue=0

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pH6,

end

osph

ere,

y=2

2+1.

3x+1

00x2 , r

2 =0.4

22, P

-val

ue<0

.001

pH8,

end

osph

ere,

y=1

9+15

x+93

x2 , r2 =0

.378

, P-v

alue

<0.0

01pH

9, e

ndos

pher

e, y

=98+

17x+

85x2 , r

2 =0.5

76, P

-val

ue<0

.001

(A)

(B)


Fig

ure

S6

Co

mp

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on

s o

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s in

Fig

ure

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ile b

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nt

L. v

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are

036912

515

3565

105

036912

515

3565

105

Growth under 14 carbon sources (%) Growth under 14 carbon sources (%)

Stag

e (y

ears

)

0255075

515

3565

105

0255075

515

3565

105

Growth under oxidative stress (%) Growth under oxidative stress (%)

050100

150

515

3565

105

050100

150

515

3565

105

Growth udner salinity stress (%) Growth udner salinity stress (%)

050100

150

515

3565

105

050100

150

515

3565

105

Growth under PEG stress (%) Growth under PEG stress (%)

fact

or(S

ourc

e)En

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here

Rhi

zosp

here

fact

or(P

lant

_spe

cies

)Ar

tem

isia

mar

itim

aLi

mon

ium

vul

gare

Stag

e (y

ears

)St

age

(yea

rs)

Stag

e (y

ears

)

050100

150

515

3565

105

050100

150

515

3565

105

Growth under penicillin stress (%) Growth under penicillin stress (%)

050100

150

515

3565

105

050100

150

515

3565

105

Growth under streptomycin stress (%) Growth under streptomycin stress (%)

0.0

0.2

0.4

0.6

515

3565

105

0.0

0.2

0.4

0.6

515

3565

105

Absorbance of exoprotease Absorbance of exoprotease

0.00

0.04

0.08

0.12

515

3565

105

0.00

0.04

0.08

0.12

515

3565

105

Absorbance of biofilm formation Absorbance of biofilm formation

0204060

515

3565

105

0204060

515

3565

105

Absorbance of consumed iron ion Absorbance of consumed iron ion

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

(I)


5Fi

gu

re S

6 —

CO

NTI

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(A) –

(I)

resp

ecti

vely

rep

rese

nts

th

e sa

me

fun

ctio

n a

s in

Fig

ure

S4.

Gre

y b

oxes

ref

er t

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nt

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036912

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036912

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3565

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Growth under 14 carbon sources (%) Growth under 14 carbon sources (%)

Stag

e (y

ears

)

0255075

515

3565

105

0255075

515

3565

105

Growth under oxidative stress (%) Growth under oxidative stress (%)

050100

150

515

3565

105

050100

150

515

3565

105

Growth udner salinity stress (%) Growth udner salinity stress (%)

050100

150

515

3565

105

050100

150

515

3565

105

Growth under PEG stress (%) Growth under PEG stress (%)

fact

or(S

ourc

e)En

dosp

here

Rhi

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fact

or(P

lant

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cies

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mon

ium

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e (y

ears

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age

(yea

rs)

Stag

e (y

ears

)

050100

150

515

3565

105

050100

150

515

3565

105

Growth under penicillin stress (%) Growth under penicillin stress (%)

050100

150

515

3565

105

050100

150

515

3565

105

Growth under streptomycin stress (%) Growth under streptomycin stress (%)

0.0

0.2

0.4

0.6

515

3565

105

0.0

0.2

0.4

0.6

515

3565

105

Absorbance of exoprotease Absorbance of exoprotease

0.00

0.04

0.08

0.12

515

3565

105

0.00

0.04

0.08

0.12

515

3565

105

Absorbance of biofilm formation Absorbance of biofilm formation

0204060

515

3565

105

0204060

515

3565

105

Absorbance of consumed iron ion Absorbance of consumed iron ion

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

(I)


Nitrogen M

etabolism

Phosphorus M

etabolism

Stress R

esponse

Mem

brane Transport

Motility and C

hemotaxis

Virulence D

isease and Defense

Iron acquisition and metabolism

66 Pseudomonas putida SJTE-1P48 Pseudomonas putida SJTE-1P49 Pseudomonas putida LS46P33 Pseudomonas putida SJTE-1R44 Pseudomonas putida SJTE-1P32 Pseudomonas fluorescens NCIMB 11764P36 Pseudomonas fluorescens NCIMB 1176488 Pseudomonas putida F1P90 Pseudomonas poaeP102 Pseudomonas fluorescens PICF8P113 Pseudomonas fluorescens PICF7P108 Pseudomonas fluorescens PICF10103 Pseudomonas fluorescens PICF7P12 Pseudomonas fluorescens PICF7P104 Pseudomonas fluorescens PICF9P72 Pseudomonas fluorescens NZ011R5 Pseudomonas anguillisepticaR96 Pseudomonas fluorescensR54 Pseudomonas sp. L10.10R83 Pseudomonas fluorescens PICF7 P41 Pseudomonas fluorescens PICF7R32 Pseudomonas fluorescens PICF7P70 Pseudomonas fluorescens SBW25R29 Pseudomonas fluorescens SBW25R50 Pseudomonas fluorescens Pf0-1P114 Pseudomonas fluorescens SBW2570 Pseudomonas fluorescens Pf0-1P31 Pseudomonas fluorescens PICF763 Pseudomonas putida F185 Pseudomonas putida NBRC 14164P106 Pseudomonas fluorescens PICF10P93 Pseudomonas fluorescens PICF7P130 Pseudomonas fluorescens PICF7P120 Pseudomonas fluorescens PICF7P74 Pseudomonas fluorescens R124P19 Pseudomonas fluorescens PICF7R28 Pseudomonas fragi69 Pseudomonas psychrophilaP78 Pseudomonas fluorescens R12454 Pseudomonas fluorescens R12460 Pseudomonas fulva 12-XR95 Pseudomonas fulva 12-XP34 Pseudomonas fluorescensP40 Pseudomonas fluorescensP37 Pseudomonas fluorescensP8 Pseudomonas fluorescensP110 Pseudomonas fluorescensP39 Pseudomonas fluorescensR61 Pseudomonas fluorescens76 Pseudomonas fluorescensP124 Pseudomonas fluorescens SBW25R98 Pseudomonas fluorescens SBW25P77 Pseudomonas fluorescens SBW25P129 Pseudomonas fluorescens SBW2636 Pseudomonas fluorescens73 Pseudomonas brassicacearum NFM421R104 Pseudomonas fluorescensP42 Pseudomonas fluorescens NZ011R69 Pseudomonas anguillisepticaP117 Pseudomonas fluorescens SBW26R111 Pseudomonas anguillisepticaP101 Pseudomonas fluorescens PICF7R67 Pseudomonas brassicacearum NFM421101 Pseudomonas brassicacearum NFM421P24 Pseudomonas fluorescens PICF7P22 Pseudomonas azotoformansP56 Pseudomonas fluorescens NZ011P50 Pseudomonas protegens Cab57P18 Pseudomonas protegens Cab57

-1

0

1

2

3

4

5Var1 Successional stages



105y

15y35y

5y

65y

Var1Var2Var3

Plant associated functional gene abundance

P11 Pseudomonas trivialis

Nitrogen M

etabolism

Phosphorus M

etabolism

Stress R

esponse

Mem

brane Transport

Motility and C

hemotaxis

Virulence D

isease and Defense

Iron acquisition and metabolism

66 Pseudomonas putida SJTE-1P48 Pseudomonas putida SJTE-1P49 Pseudomonas putida LS46P33 Pseudomonas putida SJTE-1R44 Pseudomonas putida SJTE-1P32 Pseudomonas fluorescens NCIMB 11764P36 Pseudomonas fluorescens NCIMB 1176488 Pseudomonas putida F1P90 Pseudomonas poaeP102 Pseudomonas fluorescens PICF8P113 Pseudomonas fluorescens PICF7P108 Pseudomonas fluorescens PICF10103 Pseudomonas fluorescens PICF7P12 Pseudomonas fluorescens PICF7P104 Pseudomonas fluorescens PICF9P72 Pseudomonas fluorescens NZ011R5 Pseudomonas anguillisepticaR96 Pseudomonas fluorescensR54 Pseudomonas sp. L10.10R83 Pseudomonas fluorescens PICF7 P41 Pseudomonas fluorescens PICF7R32 Pseudomonas fluorescens PICF7P70 Pseudomonas fluorescens SBW25R29 Pseudomonas fluorescens SBW25R50 Pseudomonas fluorescens Pf0-1P114 Pseudomonas fluorescens SBW2570 Pseudomonas fluorescens Pf0-1P31 Pseudomonas fluorescens PICF763 Pseudomonas putida F185 Pseudomonas putida NBRC 14164P106 Pseudomonas fluorescens PICF10P93 Pseudomonas fluorescens PICF7P130 Pseudomonas fluorescens PICF7P120 Pseudomonas fluorescens PICF7P74 Pseudomonas fluorescens R124P19 Pseudomonas fluorescens PICF7R28 Pseudomonas fragi69 Pseudomonas psychrophilaP78 Pseudomonas fluorescens R12454 Pseudomonas fluorescens R12460 Pseudomonas fulva 12-XR95 Pseudomonas fulva 12-XP34 Pseudomonas fluorescensP40 Pseudomonas fluorescensP37 Pseudomonas fluorescensP8 Pseudomonas fluorescensP110 Pseudomonas fluorescensP39 Pseudomonas fluorescensR61 Pseudomonas fluorescens76 Pseudomonas fluorescensP124 Pseudomonas fluorescens SBW25R98 Pseudomonas fluorescens SBW25P77 Pseudomonas fluorescens SBW25P129 Pseudomonas fluorescens SBW2636 Pseudomonas fluorescens73 Pseudomonas brassicacearum NFM421R104 Pseudomonas fluorescensP42 Pseudomonas fluorescens NZ011R69 Pseudomonas anguillisepticaP117 Pseudomonas fluorescens SBW26R111 Pseudomonas anguillisepticaP101 Pseudomonas fluorescens PICF7R67 Pseudomonas brassicacearum NFM421101 Pseudomonas brassicacearum NFM421P24 Pseudomonas fluorescens PICF7P22 Pseudomonas azotoformansP56 Pseudomonas fluorescens NZ011P50 Pseudomonas protegens Cab57P18 Pseudomonas protegens Cab57

-1

0

1

2

3

4




105y

15y35y

5y

65y

Var1Var2Var3

Plant associated functional gene abundance

P11 Pseudomonas trivialis

Figure S7 Distribution of functional gene abundances

in seven subsystems of Pseudomonas isolates. The

annotation of treatments — successional stages, plant

species and plant compartments were respectively re-

ferred as Var1, Var2 and Var3. For successional stages,

pink, blue, navy, cyan and orange color represent 5, 15,

35, 65 and 105-year stage, respectively. For plant spe-

cies, purple refers to L. vulgare and green to A. ma-

ritima. For plant compartments, red refer to rhizo-

sphere and green to endosphere isolates


5

NaC

l 10%

pH8

pH9

PE

G

pH5

H2O2

0.0005%

pH6

P101,CP005975 Pseudomonas fluorescens PICF7P120,CP005975 Pseudomonas fluorescens PICF7P32,CP010945 Pseudomonas fluorescens NCIMB 11764P42,AJXJ01000877 Pseudomonas fluorescens NZ011R111,AF439803 Pseudomonas anguillisepticaR83,CP005975 Pseudomonas fluorescens PICF7 R95,CP002727 Pseudomonas fulva 12-XP102,CP005975 Pseudomonas fluorescens PICF8P113,CP005975 Pseudomonas fluorescens PICF7P24,CP005975 Pseudomonas fluorescens PICF7P114,AM181176 Pseudomonas fluorescens SBW25P77,AM181176 Pseudomonas fluorescens SBW25P39,CP008896 Pseudomonas fluorescensR5,AF439803 Pseudomonas anguillisepticaP104,CP005975 Pseudomonas fluorescens PICF970,CP000094 Pseudomonas fluorescens Pf0-1R29,AM181176 Pseudomonas fluorescens SBW25R67,CP002585 Pseudomonas brassicacearum NFM421R96,JYHW01000058 Pseudomonas fluorescens69,JQ782901 Pseudomonas psychrophila63,CP000712 Pseudomonas putida F1P41,CP005975 Pseudomonas fluorescens PICF7P12,CP005975 Pseudomonas fluorescens PICF7P78,ALYL01000006 Pseudomonas fluorescens R124P74,ALYL01000006 Pseudomonas fluorescens R124P50,AP014522 Pseudomonas protegens Cab57P49,ALPV02000017 Pseudomonas putida LS46P33,AKCL01000071 Pseudomonas putida SJTE-1P36,CP010945 Pseudomonas fluorescens NCIMB 11764R32,CP005975 Pseudomonas fluorescens PICF7P129,AM181176 Pseudomonas fluorescens SBW26P117,AM181176 Pseudomonas fluorescens SBW26R98,AM181176 Pseudomonas fluorescens SBW25P130,CP005975 Pseudomonas fluorescens PICF7101,CP002585 Pseudomonas brassicacearum NFM421P37,CP008896 Pseudomonas fluorescensR28,CP013861 Pseudomonas fragiP93,CP005975 Pseudomonas fluorescens PICF736,LACH01000011 Pseudomonas fluorescens88,CP000712 Pseudomonas putida F154,ALYL01000006 Pseudomonas fluorescens R12460,CP002727 Pseudomonas fulva 12-X85,AP013070 Pseudomonas putida NBRC 1416466,AKCL01000071 Pseudomonas putida SJTE-1P40,CP008896 Pseudomonas fluorescensR54,CP012676 Pseudomonas sp. L10.10R104,CP012831 Pseudomonas fluorescensR61,CP008896 Pseudomonas fluorescensP106,CP005975 Pseudomonas fluorescens PICF10P110,CP008896 Pseudomonas fluorescensR50,CP000094 Pseudomonas fluorescens Pf0-1P90,AJ492829 Pseudomonas poaeP124,AM181176 Pseudomonas fluorescens SBW2576, LACH01000011 Pseudomonas fluorescensP11,CP011507 Pseudomonas trivialis103,CP005975 Pseudomonas fluorescens PICF773, CP002585 Pseudomonas brassicacearum NFM421R69,AF439803 Pseudomonas anguillisepticaP19,CP005975 Pseudomonas fluorescens PICF7P34,CP008896 Pseudomonas fluorescensP31,CP005975 Pseudomonas fluorescens PICF7P70,AM181176 Pseudomonas fluorescens SBW25P56,AJXJ01000877 Pseudomonas fluorescens NZ011P108,CP005975 Pseudomonas fluorescens PICF10P72,AJXJ01000877 Pseudomonas fluorescens NZ011P48,AKCL01000071 Pseudomonas putida SJTE-1P18,AP014522 Pseudomonas protegens Cab57P22,CP014546 Pseudomonas azotoformansP8,CP008896 Pseudomonas fluorescensR44,AKCL01000071 Pseudomonas putida SJTE-1

0

2

4

6

Var1 Successional stages



105y

15y35y

5y

65y

Var1Var2Var3


NaC

l 10%

pH8

pH9

PE

G

pH5

H2O2

0.0005%

pH6

P101,CP005975 Pseudomonas fluorescens PICF7P120,CP005975 Pseudomonas fluorescens PICF7P32,CP010945 Pseudomonas fluorescens NCIMB 11764P42,AJXJ01000877 Pseudomonas fluorescens NZ011R111,AF439803 Pseudomonas anguillisepticaR83,CP005975 Pseudomonas fluorescens PICF7 R95,CP002727 Pseudomonas fulva 12-XP102,CP005975 Pseudomonas fluorescens PICF8P113,CP005975 Pseudomonas fluorescens PICF7P24,CP005975 Pseudomonas fluorescens PICF7P114,AM181176 Pseudomonas fluorescens SBW25P77,AM181176 Pseudomonas fluorescens SBW25P39,CP008896 Pseudomonas fluorescensR5,AF439803 Pseudomonas anguillisepticaP104,CP005975 Pseudomonas fluorescens PICF970,CP000094 Pseudomonas fluorescens Pf0-1R29,AM181176 Pseudomonas fluorescens SBW25R67,CP002585 Pseudomonas brassicacearum NFM421R96,JYHW01000058 Pseudomonas fluorescens69,JQ782901 Pseudomonas psychrophila63,CP000712 Pseudomonas putida F1P41,CP005975 Pseudomonas fluorescens PICF7P12,CP005975 Pseudomonas fluorescens PICF7P78,ALYL01000006 Pseudomonas fluorescens R124P74,ALYL01000006 Pseudomonas fluorescens R124P50,AP014522 Pseudomonas protegens Cab57P49,ALPV02000017 Pseudomonas putida LS46P33,AKCL01000071 Pseudomonas putida SJTE-1P36,CP010945 Pseudomonas fluorescens NCIMB 11764R32,CP005975 Pseudomonas fluorescens PICF7P129,AM181176 Pseudomonas fluorescens SBW26P117,AM181176 Pseudomonas fluorescens SBW26R98,AM181176 Pseudomonas fluorescens SBW25P130,CP005975 Pseudomonas fluorescens PICF7101,CP002585 Pseudomonas brassicacearum NFM421P37,CP008896 Pseudomonas fluorescensR28,CP013861 Pseudomonas fragiP93,CP005975 Pseudomonas fluorescens PICF736,LACH01000011 Pseudomonas fluorescens88,CP000712 Pseudomonas putida F154,ALYL01000006 Pseudomonas fluorescens R12460,CP002727 Pseudomonas fulva 12-X85,AP013070 Pseudomonas putida NBRC 1416466,AKCL01000071 Pseudomonas putida SJTE-1P40,CP008896 Pseudomonas fluorescensR54,CP012676 Pseudomonas sp. L10.10R104,CP012831 Pseudomonas fluorescensR61,CP008896 Pseudomonas fluorescensP106,CP005975 Pseudomonas fluorescens PICF10P110,CP008896 Pseudomonas fluorescensR50,CP000094 Pseudomonas fluorescens Pf0-1P90,AJ492829 Pseudomonas poaeP124,AM181176 Pseudomonas fluorescens SBW2576, LACH01000011 Pseudomonas fluorescensP11,CP011507 Pseudomonas trivialis103,CP005975 Pseudomonas fluorescens PICF773, CP002585 Pseudomonas brassicacearum NFM421R69,AF439803 Pseudomonas anguillisepticaP19,CP005975 Pseudomonas fluorescens PICF7P34,CP008896 Pseudomonas fluorescensP31,CP005975 Pseudomonas fluorescens PICF7P70,AM181176 Pseudomonas fluorescens SBW25P56,AJXJ01000877 Pseudomonas fluorescens NZ011P108,CP005975 Pseudomonas fluorescens PICF10P72,AJXJ01000877 Pseudomonas fluorescens NZ011P48,AKCL01000071 Pseudomonas putida SJTE-1P18,AP014522 Pseudomonas protegens Cab57P22,CP014546 Pseudomonas azotoformansP8,CP008896 Pseudomonas fluorescensR44,AKCL01000071 Pseudomonas putida SJTE-1

0

2

4

6

Var1 Successional stages



105y

15y35y

5y

65y

Var1Var2Var3


Figure S8 Distribution of abiotic stress resistance of

root-associated Pseudomonas isolates. The anno-

tation of treatments — successional stages, plant

species and plant compartments and correspond-

ing colors are the same with those in Figure S7


penicillin

streptomycin

103,CP005975 Pseudomonas fluorescens PICF7P101,CP005975 Pseudomonas fluorescens PICF7P102,CP005975 Pseudomonas fluorescens PICF8P104,CP005975 Pseudomonas fluorescens PICF9P113,CP005975 Pseudomonas fluorescens PICF7P120,CP005975 Pseudomonas fluorescens PICF7P124,AM181176 Pseudomonas fluorescens SBW25P32,CP010945 Pseudomonas fluorescens NCIMB 11764P36,CP010945 Pseudomonas fluorescens NCIMB 11764P42,AJXJ01000877 Pseudomonas fluorescens NZ011P49,ALPV02000017 Pseudomonas putida LS46R111,AF439803 Pseudomonas anguillisepticaR5,AF439803 Pseudomonas anguillisepticaR83,CP005975 Pseudomonas fluorescens PICF7 R96,JYHW01000058 Pseudomonas fluorescensP18,AP014522 Pseudomonas protegens Cab57R67,CP002585 Pseudomonas brassicacearum NFM421R95,CP002727 Pseudomonas fulva 12-XR32,CP005975 Pseudomonas fluorescens PICF773, CP002585 Pseudomonas brassicacearum NFM421P77,AM181176 Pseudomonas fluorescens SBW25P24,CP005975 Pseudomonas fluorescens PICF769,JQ782901 Pseudomonas psychrophilaP39,CP008896 Pseudomonas fluorescensP114,AM181176 Pseudomonas fluorescens SBW25R98,AM181176 Pseudomonas fluorescens SBW25P117,AM181176 Pseudomonas fluorescens SBW2663,CP000712 Pseudomonas putida F1P129,AM181176 Pseudomonas fluorescens SBW26P130,CP005975 Pseudomonas fluorescens PICF770,CP000094 Pseudomonas fluorescens Pf0-1R29,AM181176 Pseudomonas fluorescens SBW25P78,ALYL01000006 Pseudomonas fluorescens R124101,CP002585 Pseudomonas brassicacearum NFM42136,LACH01000011 Pseudomonas fluorescensP40,CP008896 Pseudomonas fluorescensP74,ALYL01000006 Pseudomonas fluorescens R124P50,AP014522 Pseudomonas protegens Cab57P12,CP005975 Pseudomonas fluorescens PICF7P41,CP005975 Pseudomonas fluorescens PICF7P37,CP008896 Pseudomonas fluorescensR104,CP012831 Pseudomonas fluorescensP19,CP005975 Pseudomonas fluorescens PICF7P31,CP005975 Pseudomonas fluorescens PICF7P33,AKCL01000071 Pseudomonas putida SJTE-166,AKCL01000071 Pseudomonas putida SJTE-160,CP002727 Pseudomonas fulva 12-XR54,CP012676 Pseudomonas sp. L10.10R61,CP008896 Pseudomonas fluorescensP106,CP005975 Pseudomonas fluorescens PICF10P34,CP008896 Pseudomonas fluorescensP90,AJ492829 Pseudomonas poaeR28,CP013861 Pseudomonas fragiR69,AF439803 Pseudomonas anguillisepticaP110,CP008896 Pseudomonas fluorescens85,AP013070 Pseudomonas putida NBRC 14164R50,CP000094 Pseudomonas fluorescens Pf0-176, LACH01000011 Pseudomonas fluorescens54,ALYL01000006 Pseudomonas fluorescens R124P11,CP011507 Pseudomonas triv ialisP93,CP005975 Pseudomonas fluorescens PICF7P108,CP005975 Pseudomonas fluorescens PICF1088,CP000712 Pseudomonas putida F1P56,AJXJ01000877 Pseudomonas fluorescens NZ011P70,AM181176 Pseudomonas fluorescens SBW25P72,AJXJ01000877 Pseudomonas fluorescens NZ011P48,AKCL01000071 Pseudomonas putida SJTE-1P8,CP008896 Pseudomonas fluorescensR44,AKCL01000071 Pseudomonas putida SJTE-1P22,CP014546 Pseudomonas azotoformans

0

1

2




105y

15y35y

5y

65y

Var1Var2Var3


penicillin

streptomycin

103,CP005975 Pseudomonas fluorescens PICF7P101,CP005975 Pseudomonas fluorescens PICF7P102,CP005975 Pseudomonas fluorescens PICF8P104,CP005975 Pseudomonas fluorescens PICF9P113,CP005975 Pseudomonas fluorescens PICF7P120,CP005975 Pseudomonas fluorescens PICF7P124,AM181176 Pseudomonas fluorescens SBW25P32,CP010945 Pseudomonas fluorescens NCIMB 11764P36,CP010945 Pseudomonas fluorescens NCIMB 11764P42,AJXJ01000877 Pseudomonas fluorescens NZ011P49,ALPV02000017 Pseudomonas putida LS46R111,AF439803 Pseudomonas anguillisepticaR5,AF439803 Pseudomonas anguillisepticaR83,CP005975 Pseudomonas fluorescens PICF7 R96,JYHW01000058 Pseudomonas fluorescensP18,AP014522 Pseudomonas protegens Cab57R67,CP002585 Pseudomonas brassicacearum NFM421R95,CP002727 Pseudomonas fulva 12-XR32,CP005975 Pseudomonas fluorescens PICF773, CP002585 Pseudomonas brassicacearum NFM421P77,AM181176 Pseudomonas fluorescens SBW25P24,CP005975 Pseudomonas fluorescens PICF769,JQ782901 Pseudomonas psychrophilaP39,CP008896 Pseudomonas fluorescensP114,AM181176 Pseudomonas fluorescens SBW25R98,AM181176 Pseudomonas fluorescens SBW25P117,AM181176 Pseudomonas fluorescens SBW2663,CP000712 Pseudomonas putida F1P129,AM181176 Pseudomonas fluorescens SBW26P130,CP005975 Pseudomonas fluorescens PICF770,CP000094 Pseudomonas fluorescens Pf0-1R29,AM181176 Pseudomonas fluorescens SBW25P78,ALYL01000006 Pseudomonas fluorescens R124101,CP002585 Pseudomonas brassicacearum NFM42136,LACH01000011 Pseudomonas fluorescensP40,CP008896 Pseudomonas fluorescensP74,ALYL01000006 Pseudomonas fluorescens R124P50,AP014522 Pseudomonas protegens Cab57P12,CP005975 Pseudomonas fluorescens PICF7P41,CP005975 Pseudomonas fluorescens PICF7P37,CP008896 Pseudomonas fluorescensR104,CP012831 Pseudomonas fluorescensP19,CP005975 Pseudomonas fluorescens PICF7P31,CP005975 Pseudomonas fluorescens PICF7P33,AKCL01000071 Pseudomonas putida SJTE-166,AKCL01000071 Pseudomonas putida SJTE-160,CP002727 Pseudomonas fulva 12-XR54,CP012676 Pseudomonas sp. L10.10R61,CP008896 Pseudomonas fluorescensP106,CP005975 Pseudomonas fluorescens PICF10P34,CP008896 Pseudomonas fluorescensP90,AJ492829 Pseudomonas poaeR28,CP013861 Pseudomonas fragiR69,AF439803 Pseudomonas anguillisepticaP110,CP008896 Pseudomonas fluorescens85,AP013070 Pseudomonas putida NBRC 14164R50,CP000094 Pseudomonas fluorescens Pf0-176, LACH01000011 Pseudomonas fluorescens54,ALYL01000006 Pseudomonas fluorescens R124P11,CP011507 Pseudomonas triv ialisP93,CP005975 Pseudomonas fluorescens PICF7P108,CP005975 Pseudomonas fluorescens PICF1088,CP000712 Pseudomonas putida F1P56,AJXJ01000877 Pseudomonas fluorescens NZ011P70,AM181176 Pseudomonas fluorescens SBW25P72,AJXJ01000877 Pseudomonas fluorescens NZ011P48,AKCL01000071 Pseudomonas putida SJTE-1P8,CP008896 Pseudomonas fluorescensR44,AKCL01000071 Pseudomonas putida SJTE-1P22,CP014546 Pseudomonas azotoformans

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Var1Var2Var3


Figure S9 Distribution of antibiotic resistance of

root-associated Pseudomonas isolates. The an-

notation of treatments — successional stages,

plant species and plant compartments and cor-

responding colors are the same with those in

Figure S7


5biofilm

formation

consumed iron ion

exoprotease

P42,AJXJ01000877 Pseudomonas fluorescens NZ011R28,CP013861 Pseudomonas fragiR44,AKCL01000071 Pseudomonas putida SJTE-1P32,CP010945 Pseudomonas fluorescens NCIMB 11764R95,CP002727 Pseudomonas fulva 12-XP120,CP005975 Pseudomonas fluorescens PICF7R54,CP012676 Pseudomonas sp. L10.10P129,AM181176 Pseudomonas fluorescens SBW26P113,CP005975 Pseudomonas fluorescens PICF7P77,AM181176 Pseudomonas fluorescens SBW25P49,ALPV02000017 Pseudomonas putida LS46R111,AF439803 Pseudomonas anguilliseptica36,LACH01000011 Pseudomonas fluorescensR69,AF439803 Pseudomonas anguilliseptica76, LACH01000011 Pseudomonas fluorescens69,JQ782901 Pseudomonas psychrophila73, CP002585 Pseudomonas brassicacearum NFM421P101,CP005975 Pseudomonas fluorescens PICF7P24,CP005975 Pseudomonas fluorescens PICF766,AKCL01000071 Pseudomonas putida SJTE-1R104,CP012831 Pseudomonas fluorescensP130,CP005975 Pseudomonas fluorescens PICF7P39,CP008896 Pseudomonas fluorescensP33,AKCL01000071 Pseudomonas putida SJTE-163,CP000712 Pseudomonas putida F1P114,AM181176 Pseudomonas fluorescens SBW25P117,AM181176 Pseudomonas fluorescens SBW26P124,AM181176 Pseudomonas fluorescens SBW25R67,CP002585 Pseudomonas brassicacearum NFM42160,CP002727 Pseudomonas fulva 12-XR98,AM181176 Pseudomonas fluorescens SBW25101,CP002585 Pseudomonas brassicacearum NFM421P48,AKCL01000071 Pseudomonas putida SJTE-1R96,JYHW01000058 Pseudomonas fluorescensP12,CP005975 Pseudomonas fluorescens PICF7P104,CP005975 Pseudomonas fluorescens PICF9P102,CP005975 Pseudomonas fluorescens PICF8P36,CP010945 Pseudomonas fluorescens NCIMB 11764R61,CP008896 Pseudomonas fluorescensP78,ALYL01000006 Pseudomonas fluorescens R124P41,CP005975 Pseudomonas fluorescens PICF7P40,CP008896 Pseudomonas fluorescensP50,AP014522 Pseudomonas protegens Cab57P37,CP008896 Pseudomonas fluorescens103,CP005975 Pseudomonas fluorescens PICF770,CP000094 Pseudomonas fluorescens Pf0-1P108,CP005975 Pseudomonas fluorescens PICF10P106,CP005975 Pseudomonas fluorescens PICF10P31,CP005975 Pseudomonas fluorescens PICF7P19,CP005975 Pseudomonas fluorescens PICF7P93,CP005975 Pseudomonas fluorescens PICF7P90,AJ492829 Pseudomonas poaeP70,AM181176 Pseudomonas fluorescens SBW2554,ALYL01000006 Pseudomonas fluorescens R12488,CP000712 Pseudomonas putida F1P110,CP008896 Pseudomonas fluorescensP72,AJXJ01000877 Pseudomonas fluorescens NZ011P11,CP011507 Pseudomonas trivialisP18,AP014522 Pseudomonas protegens Cab57R83,CP005975 Pseudomonas fluorescens PICF7 P34,CP008896 Pseudomonas fluorescensR50,CP000094 Pseudomonas fluorescens Pf0-1P74,ALYL01000006 Pseudomonas fluorescens R124R29,AM181176 Pseudomonas fluorescens SBW25R5,AF439803 Pseudomonas anguillisepticaR32,CP005975 Pseudomonas fluorescens PICF7P56,AJXJ01000877 Pseudomonas fluorescens NZ011P22,CP014546 Pseudomonas azotoformansP8,CP008896 Pseudomonas fluorescens85,AP013070 Pseudomonas putida NBRC 14164

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6 Var1 Successional stages



105y

15y35y

5y

65y

Var1Var2Var3

Plant associated traits

biofilm form

ation

consumed iron ion

exoprotease

P42,AJXJ01000877 Pseudomonas fluorescens NZ011R28,CP013861 Pseudomonas fragiR44,AKCL01000071 Pseudomonas putida SJTE-1P32,CP010945 Pseudomonas fluorescens NCIMB 11764R95,CP002727 Pseudomonas fulva 12-XP120,CP005975 Pseudomonas fluorescens PICF7R54,CP012676 Pseudomonas sp. L10.10P129,AM181176 Pseudomonas fluorescens SBW26P113,CP005975 Pseudomonas fluorescens PICF7P77,AM181176 Pseudomonas fluorescens SBW25P49,ALPV02000017 Pseudomonas putida LS46R111,AF439803 Pseudomonas anguilliseptica36,LACH01000011 Pseudomonas fluorescensR69,AF439803 Pseudomonas anguilliseptica76, LACH01000011 Pseudomonas fluorescens69,JQ782901 Pseudomonas psychrophila73, CP002585 Pseudomonas brassicacearum NFM421P101,CP005975 Pseudomonas fluorescens PICF7P24,CP005975 Pseudomonas fluorescens PICF766,AKCL01000071 Pseudomonas putida SJTE-1R104,CP012831 Pseudomonas fluorescensP130,CP005975 Pseudomonas fluorescens PICF7P39,CP008896 Pseudomonas fluorescensP33,AKCL01000071 Pseudomonas putida SJTE-163,CP000712 Pseudomonas putida F1P114,AM181176 Pseudomonas fluorescens SBW25P117,AM181176 Pseudomonas fluorescens SBW26P124,AM181176 Pseudomonas fluorescens SBW25R67,CP002585 Pseudomonas brassicacearum NFM42160,CP002727 Pseudomonas fulva 12-XR98,AM181176 Pseudomonas fluorescens SBW25101,CP002585 Pseudomonas brassicacearum NFM421P48,AKCL01000071 Pseudomonas putida SJTE-1R96,JYHW01000058 Pseudomonas fluorescensP12,CP005975 Pseudomonas fluorescens PICF7P104,CP005975 Pseudomonas fluorescens PICF9P102,CP005975 Pseudomonas fluorescens PICF8P36,CP010945 Pseudomonas fluorescens NCIMB 11764R61,CP008896 Pseudomonas fluorescensP78,ALYL01000006 Pseudomonas fluorescens R124P41,CP005975 Pseudomonas fluorescens PICF7P40,CP008896 Pseudomonas fluorescensP50,AP014522 Pseudomonas protegens Cab57P37,CP008896 Pseudomonas fluorescens103,CP005975 Pseudomonas fluorescens PICF770,CP000094 Pseudomonas fluorescens Pf0-1P108,CP005975 Pseudomonas fluorescens PICF10P106,CP005975 Pseudomonas fluorescens PICF10P31,CP005975 Pseudomonas fluorescens PICF7P19,CP005975 Pseudomonas fluorescens PICF7P93,CP005975 Pseudomonas fluorescens PICF7P90,AJ492829 Pseudomonas poaeP70,AM181176 Pseudomonas fluorescens SBW2554,ALYL01000006 Pseudomonas fluorescens R12488,CP000712 Pseudomonas putida F1P110,CP008896 Pseudomonas fluorescensP72,AJXJ01000877 Pseudomonas fluorescens NZ011P11,CP011507 Pseudomonas trivialisP18,AP014522 Pseudomonas protegens Cab57R83,CP005975 Pseudomonas fluorescens PICF7 P34,CP008896 Pseudomonas fluorescensR50,CP000094 Pseudomonas fluorescens Pf0-1P74,ALYL01000006 Pseudomonas fluorescens R124R29,AM181176 Pseudomonas fluorescens SBW25R5,AF439803 Pseudomonas anguillisepticaR32,CP005975 Pseudomonas fluorescens PICF7P56,AJXJ01000877 Pseudomonas fluorescens NZ011P22,CP014546 Pseudomonas azotoformansP8,CP008896 Pseudomonas fluorescens85,AP013070 Pseudomonas putida NBRC 14164

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15y35y

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65y

Var1Var2Var3

Plant associated traits

Figure S10 Distribution of plant growth promoting

traits of root-associated Pseudomonas isolates. The

annotation of treatments — successional stages,

plant species and plant compartments and corre-

sponding colors are the same with those in Figure S7


Figure S11 Distribution of metabolic potential of

root-associated Pseudomonas isolates. The anno-

tation of treatments — successional stages, plant

species and plant compartments and correspond-

ing colors are the same with those in Figure S7

ArabinoseSerineG

lucoseButyrolactamG

alactoseValinePutrescineSuccin.AcidLactic.AcidAlanineG

lycineG

lycerolThreonineFructose

P101,CP005975 Pseudomonas fluorescens PICF7P120,CP005975 Pseudomonas fluorescens PICF7P32,CP010945 Pseudomonas fluorescens NCIMB 11764P42,AJXJ01000877 Pseudomonas fluorescens NZ011R111,AF439803 Pseudomonas anguillisepticaR83,CP005975 Pseudomonas fluorescens PICF7 P113,CP005975 Pseudomonas fluorescens PICF7P114,AM181176 Pseudomonas fluorescens SBW25P24,CP005975 Pseudomonas fluorescens PICF7P39,CP008896 Pseudomonas fluorescensP77,AM181176 Pseudomonas fluorescens SBW2563,CP000712 Pseudomonas putida F169,JQ782901 Pseudomonas psychrophilaP117,AM181176 Pseudomonas fluorescens SBW2670,CP000094 Pseudomonas fluorescens Pf0-1P50,AP014522 Pseudomonas protegens Cab57P78,ALYL01000006 Pseudomonas fluorescens R124P12,CP005975 Pseudomonas fluorescens PICF7P41,CP005975 Pseudomonas fluorescens PICF7P40,CP008896 Pseudomonas fluorescensP102,CP005975 Pseudomonas fluorescens PICF8R96,JYHW01000058 Pseudomonas fluorescensP33,AKCL01000071 Pseudomonas putida SJTE-160,CP002727 Pseudomonas fulva 12-XP93,CP005975 Pseudomonas fluorescens PICF7P104,CP005975 Pseudomonas fluorescens PICF988,CP000712 Pseudomonas putida F1P74,ALYL01000006 Pseudomonas fluorescens R124P49,ALPV02000017 Pseudomonas putida LS4666,AKCL01000071 Pseudomonas putida SJTE-1R29,AM181176 Pseudomonas fluorescens SBW25P106,CP005975 Pseudomonas fluorescens PICF10R67,CP002585 Pseudomonas brassicacearum NFM421R28,CP013861 Pseudomonas fragiP110,CP008896 Pseudomonas fluorescensP37,CP008896 Pseudomonas fluorescensR98,AM181176 Pseudomonas fluorescens SBW25P130,CP005975 Pseudomonas fluorescens PICF7R61,CP008896 Pseudomonas fluorescensP129,AM181176 Pseudomonas fluorescens SBW26101,CP002585 Pseudomonas brassicacearum NFM421P124,AM181176 Pseudomonas fluorescens SBW25P36,CP010945 Pseudomonas fluorescens NCIMB 1176485,AP013070 Pseudomonas putida NBRC 14164P108,CP005975 Pseudomonas fluorescens PICF10P11,CP011507 Pseudomonas triv ialis36,LACH01000011 Pseudomonas fluorescensP48,AKCL01000071 Pseudomonas putida SJTE-1P31,CP005975 Pseudomonas fluorescens PICF7P19,CP005975 Pseudomonas fluorescens PICF7P90,AJ492829 Pseudomonas poae54,ALYL01000006 Pseudomonas fluorescens R124P72,AJXJ01000877 Pseudomonas fluorescens NZ011P34,CP008896 Pseudomonas fluorescensR54,CP012676 Pseudomonas sp. L10.10R104,CP012831 Pseudomonas fluorescensR69,AF439803 Pseudomonas anguillisepticaR32,CP005975 Pseudomonas fluorescens PICF7P56,AJXJ01000877 Pseudomonas fluorescens NZ011R50,CP000094 Pseudomonas fluorescens Pf0-1R44,AKCL01000071 Pseudomonas putida SJTE-1P18,AP014522 Pseudomonas protegens Cab57P22,CP014546 Pseudomonas azotoformans76, LACH01000011 Pseudomonas fluorescensP70,AM181176 Pseudomonas fluorescens SBW25P8,CP008896 Pseudomonas fluorescensR5,AF439803 Pseudomonas anguillisepticaR95,CP002727 Pseudomonas fulva 12-X73, CP002585 Pseudomonas brassicacearum NFM421103,CP005975 Pseudomonas fluorescens PICF7

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105y

15y35y

5y

65y

Var1Var2Var3

Metabolic potential

ArabinoseSerineG

lucoseButyrolactamG

alactoseValinePutrescineSuccin.AcidLactic.AcidAlanineG

lycineG

lycerolThreonineFructose

P101,CP005975 Pseudomonas fluorescens PICF7P120,CP005975 Pseudomonas fluorescens PICF7P32,CP010945 Pseudomonas fluorescens NCIMB 11764P42,AJXJ01000877 Pseudomonas fluorescens NZ011R111,AF439803 Pseudomonas anguillisepticaR83,CP005975 Pseudomonas fluorescens PICF7 P113,CP005975 Pseudomonas fluorescens PICF7P114,AM181176 Pseudomonas fluorescens SBW25P24,CP005975 Pseudomonas fluorescens PICF7P39,CP008896 Pseudomonas fluorescensP77,AM181176 Pseudomonas fluorescens SBW2563,CP000712 Pseudomonas putida F169,JQ782901 Pseudomonas psychrophilaP117,AM181176 Pseudomonas fluorescens SBW2670,CP000094 Pseudomonas fluorescens Pf0-1P50,AP014522 Pseudomonas protegens Cab57P78,ALYL01000006 Pseudomonas fluorescens R124P12,CP005975 Pseudomonas fluorescens PICF7P41,CP005975 Pseudomonas fluorescens PICF7P40,CP008896 Pseudomonas fluorescensP102,CP005975 Pseudomonas fluorescens PICF8R96,JYHW01000058 Pseudomonas fluorescensP33,AKCL01000071 Pseudomonas putida SJTE-160,CP002727 Pseudomonas fulva 12-XP93,CP005975 Pseudomonas fluorescens PICF7P104,CP005975 Pseudomonas fluorescens PICF988,CP000712 Pseudomonas putida F1P74,ALYL01000006 Pseudomonas fluorescens R124P49,ALPV02000017 Pseudomonas putida LS4666,AKCL01000071 Pseudomonas putida SJTE-1R29,AM181176 Pseudomonas fluorescens SBW25P106,CP005975 Pseudomonas fluorescens PICF10R67,CP002585 Pseudomonas brassicacearum NFM421R28,CP013861 Pseudomonas fragiP110,CP008896 Pseudomonas fluorescensP37,CP008896 Pseudomonas fluorescensR98,AM181176 Pseudomonas fluorescens SBW25P130,CP005975 Pseudomonas fluorescens PICF7R61,CP008896 Pseudomonas fluorescensP129,AM181176 Pseudomonas fluorescens SBW26101,CP002585 Pseudomonas brassicacearum NFM421P124,AM181176 Pseudomonas fluorescens SBW25P36,CP010945 Pseudomonas fluorescens NCIMB 1176485,AP013070 Pseudomonas putida NBRC 14164P108,CP005975 Pseudomonas fluorescens PICF10P11,CP011507 Pseudomonas triv ialis36,LACH01000011 Pseudomonas fluorescensP48,AKCL01000071 Pseudomonas putida SJTE-1P31,CP005975 Pseudomonas fluorescens PICF7P19,CP005975 Pseudomonas fluorescens PICF7P90,AJ492829 Pseudomonas poae54,ALYL01000006 Pseudomonas fluorescens R124P72,AJXJ01000877 Pseudomonas fluorescens NZ011P34,CP008896 Pseudomonas fluorescensR54,CP012676 Pseudomonas sp. L10.10R104,CP012831 Pseudomonas fluorescensR69,AF439803 Pseudomonas anguillisepticaR32,CP005975 Pseudomonas fluorescens PICF7P56,AJXJ01000877 Pseudomonas fluorescens NZ011R50,CP000094 Pseudomonas fluorescens Pf0-1R44,AKCL01000071 Pseudomonas putida SJTE-1P18,AP014522 Pseudomonas protegens Cab57P22,CP014546 Pseudomonas azotoformans76, LACH01000011 Pseudomonas fluorescensP70,AM181176 Pseudomonas fluorescens SBW25P8,CP008896 Pseudomonas fluorescensR5,AF439803 Pseudomonas anguillisepticaR95,CP002727 Pseudomonas fulva 12-X73, CP002585 Pseudomonas brassicacearum NFM421103,CP005975 Pseudomonas fluorescens PICF7

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105y

15y35y

5y

65y

Var1Var2Var3

Metabolic potential

university of groningen the dynamics of root microbiomes ... · abstract the genus pseudomonas...

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