www.sciencemag.org/cgi/content/full/science.

1203980/DC1

Supporting Online Material for

Deciphering the Rhizosphere Microbiome for Disease-Suppressive Bacteria
Rodrigo Mendes, Marco Kruijt, Irene de Bruijn, Ester Dekkers, Menno van der Voort, Johannes H. M. Schneider, Yvette M. Piceno, Todd Z. DeSantis, Gary L. Andersen, Peter A. H. M. Bakker, Jos M. Raaijmakers* *To whom correspondence should be addressed. E-mail: jos.raaijmakers@wur.nl
Published 5 May 2011 on Science Express DOI: 10.1126/science.1203980

This PDF file includes: Materials and Methods Figs. S1 to S8 Tables S1 to S5 References and Notes

Materials and Methods Soil sample collection and storage The suppressive soil was collected at the end of the 2004 sugar beet growing season from an agricultural field close to the town of Hoeven, the Netherlands (513510N 43444E). Soil was collected at a depth of 0-30 cm from 25 random sites across the field. The conducive soil was harvested from the margin of the sugar beet field; this margin was not cultivated to sugar beet and was covered with grasses and weeds, which were removed prior to soil sampling. Both soils were air dried, sieved (0.5-cm-mesh) to remove plant debris, and stored in buckets at ambient temperatures. Physical-chemical analyses on each soil were performed by BLGG-AgroXpertus (Oosterbeek, The Netherlands). Bioassay to assess disease suppressiveness of soils Sugar beet seeds (cv. Alligator) were sown in square PVC pots (width 6 cm; height 8 cm) containing 250 g of soil with an initial moisture content of 10% (v/w). Plants were grown in a growth chamber at 24C, 70% relative humidity and 16 hour light, and watered weekly with standard Hoagland solution (macronutrients only). Disease suppressiveness was determined for various treatments: i) suppressive soil (S), ii) conducive soil (C), conducive soil amended with 10% (w/w) of suppressive soil (CS), suppressive soil heat-treated at 50C (S50) or 80C (S80) for 1 hour, or gamma-irradiated (60 kGray, Isotron, The Netherlands). For heat treatment, the suppressive soil (moisture content set at 10% v/w) was transferred to a plastic bag and placed in a water bath at 50C or 80C for 1 hour. The bags with soil were made flat (~ up to 4 cm height) to increase the contact area with the surrounding water. For each soil treatment, four replicates were used in a complete randomized experimental design. Four days after seed germination, the number of seedlings was reduced to eight per pot. The fungal pathogen Rhizoctonia solani (anastomosis group AG2-2IIIB) was introduced into the soil by transferring two mycelial agar plugs (5-mm-diameter) of a 1 week-old potato dextrose agar (PDA) culture to two opposite corners of the pots at 1-cm underneath the soil surface. The number of infected sugar beet seedlings was scored every two-three days for a period up to 20 days after pathogen inoculation. The area under the disease progress curve (AUDPC) was determined according to the statistical methods described by Shaner & Finney (15). Rhizosphere DNA isolation and PhyloChip analysis The rhizosphere microbiomes of sugar beet seedlings grown in soils with different levels of disease suppression were subjected to metagenomic-based community analysis. For each of the six soil treatments (identified in Fig. S3), four replicates were used. Metagenomic DNA was isolated in triplicate from each replicate by using the PowerSoil DNA isolation kit (MO BIO Laboratories, Inc.) according to the manufacturers instructions. Microbial profiles for each sample were generated with the PhyloChip assay (Second Genome, CA, USA). All PCR conditions and universal primers used for amplification of 16S rDNA genes from bacteria and archaea were as previously described (7). Fragmentation of the 16S rDNA amplicons, labelling, hybridizations, staining, and scanning of the PhyloChip were performed according to methods described by Hazen et 2

al. (6). OTU selection for data analysis differed slightly from Hazen et al. (6) as follows. All OTUs passing PhyCA analysis Stage 1 criteria in this data set were considered for further analyses, allowing the inclusion of Unclassified OTU, which would be excluded by Stage 2 analysis. Additionally, the cut-off values for an OTU to pass Stage 1 were rQ1 0.646, rQ2 0.884, and rQ3 0.945. Data analyses were performed with Primer-E software (version 6.1.13, Plymouth Marine Laboratory). Bacterial isolation from suppressive soil For the functional analyses, the -Proteobacteria were isolated from sugar beet rhizosphere on semi-selective media. Sixteen sugar beet seeds were sown in pots filled with 250 g of suppressive or conducive soils and grown for 20 days (N=3). Rhizosphere (roots with tightly adhering soil) suspensions were prepared, serially diluted and plated onto i) 1/10th strength Tryptic Soy Agar (TSA) supplemented with 100 g ml-1 Delvocid to prevent fungal growth, and ii) Pseudomonas Agar (PSA) supplemented with 40 g ml-1 ampicillin, 12.5 g ml-1 chloramphenicol (16), and 100 g ml-1 Delvocid. TSA plates were incubated at 25C for 5 days and PSA plates for 3 days. From each of the replicate samples and growth media (TSA and PSA), 96 randomly selected bacterial colonies were purified and screened for in vitro antagonism towards R. solani. In these in vitro inhibition assays, the bacterial isolates were point-inoculated at the periphery of 1/5th strength PDA (pH 7.0) plates and incubated for two days at 25C, after which a fresh mycelial PDA agar plug of R. solani was transferred to the centre of the plate. After an additional three days of incubation at 25C, inhibition of hyphal growth of R. solani was scored. Out of 576 bacterial isolates randomly collected from the suppressive soil, 19.3% showed antifungal activity, whereas only 3.3% of 421 isolates randomly collected from the conducive soil inhibited growth of R. solani. 16S rDNA sequencing and phylogeny 16S rDNA was sequenced by Macrogen Inc. (Seoul, South Korea). Sequences were trimmed and submitted to the Ribosomal Database Project for species identification (17). MEGA 3.1 (18) was used to align 16S rDNA sequences and to construct a phylogenetic tree (UPGMA with 10,000 bootstraps). Coupling PhyloChip-based metagenomics with culture-based analysis Two strategies were used to couple the metagenomics-based PhyloChip data with the results of the culture-based analysis. For the first strategy, using Basic Local Alignment Search Tool (http://blast.ncbi.nlm.nih.gov), we aligned the 16S rDNA sequences of representative strains of haplotype clusters I-III (Fig. 4A) with the 16S rDNA sequences of the five Pseudomonadaceae identified by the PhyloChip approach as the top 10% dynamic taxa associated with disease suppression (see Table S3). For the second strategy, 16S rDNA sequences from strains representing the three haplotype clusters (Fig. 4A) were used for BLAST searches in the GreenGenes database (http://greengenes.lbl.gov) used for the PhyloChip array design. Subsequently, the best hits were traced back in our experimental data set revealing that these haplotypes were indeed more abundant in suppressive soils than in conducive soils (Fig. 4B). Their respective abundances were calculated according to the conditions described above in the Rhizosphere DNA isolation and PhyloChip analysis section. 3

Random transposon mutagenesis Plasposon mutagenesis of Pseudomonas sp. strain SH-C52 was performed using pTnMod-OTc (19). From an initial screen of approximately 1,500 random mutants, two mutants of strain SH-C52 were obtained that had lost in vitro activity against R. solani. Single plasposon insertions were confirmed by Southern blot analysis with a probe directed against the tetracycline resistance gene on the plasposon. Plasposon rescue using BamHI or PstI was performed as previously described (19). Cloning and sequencing of the thaABCD genes A fosmid library (7 X genome coverage) with 30-40 kb fragments of genomic DNA of Pseudomonas sp. strain SH-C52 was constructed according to the protocols of the manufacturer (Fosmid Library Production kit, Epicentre). Library clones were blotted onto Hybond N+ membranes (Amersham) and hybridized with dig-labeled probes directed against specific sequences in the thaB gene. Hybridizations were performed under stringent conditions (65C with 0.1xSSC (75 mM NaCl, 7.5 mM sodium citrate, 0.1% sodium dodecyl sulfate). Hybridization-positive clones were subjected to restriction analyses. Contigs were constructed by cluster analysis of these experimental data by the unweighted-pair group method using average linkages. Clones 5.1 and 10.1 were sent for shotgun sequencing (Macrogen, Seoul, Korea). Sequence gaps were closed by primer walking. Bioinformatic analyses Operons and genes were predicted by the Softberry FGENESB program (Softberry, Inc., Mount Kisco, NY), and the identified open reading frames (ORFs) were analyzed using BlastX in the NCBI database and PseudoDB (http://xbase.bham.ac.uk). Putative promoter sequences were identified by the Softberry BPROM program, and putative terminator sequences were identified by the RNA secondary structure prediction program of Genebee (http://www.genebee.msu.su/). Specific domains in the deduced protein sequences of the NRPS genes thaA, thaB and thaC1 were analyzed with PFAM (http://pfam.sanger.ac.uk/search?tabsearchSequenceBlock). Protein sequences of specific domains were aligned in ClustalX (version 1.81). Trees were inferred by neighbor joining using 1,000 bootstrap replicates. Identification of the genes flanking the NRPS genes was performed by BlastX analysis in NCBI, Pseudomonas.com (http://v2.pseudomonas.com/), or PseudoDB and by comparison with genes in the biosynthesis cluster for syringomycin. The C1-domain of thaA as well as the TE domain of the ninth module of thaB (GenBank HQ888764) were used in BlastP comparisons with whole genome sequences of Pseudomonas species available in the databases Pseudomonas.com and PseudoDB. Adenylation (A), thiolation (T), condensation (C) and thioesterase (TE) domains of the NRPS genes were identified by PFAM (http://www.sanger.ac.uk/Software/Pfam/). For phylogenetic analyses of the different domains, alignments were made with ClustalX (version 1.81) and software available at http://www.ebi.ac.uk/clustalw/. Trees were inferred by Neighbor Joining in ClustalX using 1,000 bootstrap replicates.

A
Disease incidence (%)

80
S C CS S50 S80

70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 18 20

dpi
60 50 40

AB BC

AUDPC

30 20 10 0 S C CS

BC

S50

S80

Fig. S1. Disease suppressiveness of soils and its microbiological nature. (A) Progress of Rhizoctonia damping-off disease of sugar beet seedlings in disease suppressive soil (S), conducive soil (C), conducive soil amended with 10% (w/w) of suppressive soil (CS), suppressive soil heat-treated at 50C (S50) or 80C (S80) for 1 hour. Disease incidence represents the percentage of sugar beet seedlings with damping-off symptoms (mean values SEM, N=4). For each replicate, eight sugar beet seedlings were used. (B) Area under disease progress curve (AUDPC) for each of the five different treatments (mean values SEM, N=4). Different letters above the bars indicate statistically significant differences (P<0.05, Student-Newman-Keuls test).

B
Disease Index

2.5

1.5

0.5

0 S SG

Fig. S2. Effect of -irradiation on soil disease suppressiveness. (A) Disease index ranging from 0 (healthy plant) to 3 (dead plant). (B) Suppressive soil (S) and -irradiated (60 kGray, Isotron, The Netherlands) suppressive soil (SG) were cultivated with sugar beet. Number of infected plants (mean values SEM, N=12) were scored 14 days after germination. Asterisk indicates a statistically significant difference (P<0.05, Students ttest).

I - Experimental design

1 Sugar beet grown in the presence of R. solani

(N=4). grown in 2 Sugar beet R. solani the absence of (N=4).

* Treatments selected
for PhyloChip analysis.

II - PhyloChip analysis

1 Rhizosphere DNA isolation with PowerSoil MO BIO kit.

rDNA amplification; pool of 2 16SPCR (temperature gradient 8X for annealing from 52 to 62 for each replicate. C) to 200 3 Fractionate (50biotin. bp) and end-label with stain, wash and scan; 4 Hybridize, chips (6 treatments; total of 24 N=4).

5 Data analyses using software Primer-E.

of the target 6 Selection three criteria.groups based on Pairwise comparison of top 10% dynamic taxa.

Richness > Evenness > Hierarchical clustering (Bray Curtis Similarity) > > Ordination (MDS) > Microbial Communities Dynamics (SIMPER analysis).

i. more abundant in suppressive than in conducive soil (S>C); ii. more abundant in the 'transplantation soil' than in conducive soil (CS>C); iii. more abundant in the suppressive soil when the pathogen is present (Sr>S).

III Isolation of specific bacterial taxa targeted by PhyloChip analysis

from 1 Bacterial isolationconducive suppressive and soils (~1000 random isolates). General aerobic growth medium (TSA) and Pseudomonadaceae semi-selective medium (PSA).

2 Genetic and phenotypic characterization (107 isolates). 3 with PhyloChip analysis.

Coupling culture-based analysis

Screening for antagonistic traits (in vitro tests, PCR) > BOX-PCR > > 16S rDNA sequencing > in vivo bioassays. Alignments and BLAST searches in the GreenGenes database using 16S rDNA sequences of the functional bacterial groups.

IV - Genes and pathways involved in disease suppression

1 Genetic, bioinformatic, and functional analyses

Mutagenesis > Genome library constructions > Sequencing > Signature-sequence-based predictions > in vivo bioassays.

Fig. S3. Overall strategy used to decipher the rhizosphere microbiome of sugar beet seedlings grown in disease suppressive soil. Soils with different levels of disease suppression are designated as: suppressive soil (S); conducive soil (C); conducive soil amended with 10% (w/v) suppressive soil (CS); suppressive soil heat treated at 50C (S50); suppressive soil heat treated at 80C (S80); and suppressive soil inoculated with the fungal pathogen Rhizoctonia solani (Sr). For each replicate of each treatment (N=4), total DNA was isolated and pooled from three independent extractions using 250 mg of rhizosphere soil.

Firmicutes, 20% Cyanobacteria, 1% Chloroflexi, 1% Bacteroidetes, 4% Actinobacteria, 9% Acidobacteria, 2% All others, 4% Verrucomicrobia, 2%

Planctomycetes, 2%

Proteobacteria, 39%

Unclassified, 16%

Fig. S4. Composition of bacterial communities in the rhizosphere microbiome of sugar beet seedlings grown in soils with different levels of disease suppressiveness. The sum of the microbial abundance of all six soil treatments (N=4) is shown.

2D Stress: 0.06

Fig. S5. Non-metric multi-dimensional scaling (MDS) ordination of the rhizosphere microbiomes of sugar beet seedlings grown in soils with different levels of disease suppressiveness. Based on the relative abundance of 33,346 taxa identified in the sugar beet rhizosphere microbiome, a resemblance matrix was generated using Bray Curtis similarity. MDS analysis was performed with Primer-E (version 6.1.13). suppressive soil (S); conducive soil (C); conducive soil amended with 10% (w/v) suppressive soil (CS); suppressive soil heat treated at 50C (S50); suppressive soil heat treated at 80C (S80) for 1 hour; and X suppressive soil inoculated with Rhizoctonia solani (Sr).

10

A
50

Proteobacteria

B
50

Firmicutes

60

60

Bray Curtis Similarity

70

Bray Curtis Similarity S80_1 S80_4 S80_2 S80_3 S50_4 S50_3 S50_1 S50_2 S_2 S_1 S_3 S_4 Sr_1 Sr_3 Sr_2 CS_3 CS_4 CS_1 CS_2 Sr_4 C_1 C_4 C_2 C_3

70

80

80

90

90

100 100 CS CS CS S50 S50 CS Sr Sr Sr Sr S50 S50 S80 S80 S80 S80 S S S C C C C S

C
50

Cyanobacteria

60

Bray Curtis Similarity

70

80

90

100 CS CS CS S50 S50 S50 S50 CS Sr Sr Sr Sr S80 S80 S80 S80 S S S C C C C S

Fig. S6. Clustering analysis of the rhizosphere microbiome for (A) Proteobacteria, (B) Firmicutes, and (C) Cyanobacteria. When separate clustering analyses were performed for the Proteobacteria or Firmicutes, each of these groups allowed discrimination between the six soil treatments as was the case in the overall cluster analysis (see Fig. 2B), reinforcing their association with disease suppressiveness. In contrast, for other phyla such as the Cyanobacteria (C) dissimilar patterns were found.

11

R. solani inoculation point

0
q

10

15

20

B
Control SH-A1 SH-B3 SH-C52 O33 0 5

Distance (cm)

*
10 15 20

Spread of R. solani (cm)

Fig. S7. Suppression of Rhizoctonia damping-off disease by selected strains of the Proteobacteria. (A) Representation of the in vivo bioassay to determine the ability of antagonistic bacterial isolates to suppress damping-off disease of sugar beet seedlings caused by Rhizoctonia solani. A mycelial plug of the fungal pathogen is point-inoculated at 1-cm underneath the soil surface at the edge of the tray (indicated by an arrow). Within a time period of 2-3 weeks, R. solani progressively infects sugar beet seedlings positioned in a 20-cm row with a 1-cm distance between the seedlings. The level of disease suppression is quantified by measuring the disease spread as indicated by seedlings with damping-off symptoms. (B) Spread of damping-off disease of sugar beet seedlings in conducive soil that is untreated (Control), treated with Pseudomonas sp. strain SH-A1 (haplotype A), strain SH-B3 (haplotype B) and strain SH-C52 (haplotype C), and the nonribosomal peptide synthetase mutant of strain SH-C52 (O33). Pseudomonas sp. strains representing haplotypes A, B and C, and mutant O33 were inoculated in soil (105 cells g-1 soil) one day prior to pathogen inoculation (mean values SEM, N=8). An asterisk indicates a statistically significant difference (P<0.05, Dunnett test) between the treatment and the untreated conducive soil (Control).

12

5 kb thaA
module 2 module 1 module 3 module 4 module 5

O33 KO25

KO26

thaC2 thaD

thaC1 thaB
module 6 module 7 module 8 module 9 module 9

C A T C A T C A T C A T C A T C C A T C A T C A T C T TE

A T

Cl

Ser

Asp/Glu

Thr

Thr

Asp/Glu

Thr

B
SH-C52

Cl

O33

KO26

Fig. S8. Schematic representation of the biosynthetic gene clusters responsible for the antifungal activity of Pseudomonas sp. strain SH-C52. (A) Genetic organization of the thaAB (29.7-kb) and thaC1C2D (4.4-kb) gene clusters identified in Pseudomonas sp. strain SH-C52. Underneath the nonribosomal peptide synthetase (NRPS) genes thaA, thaB and thaC1, is the module and domain organization of the encoded proteins. The domains are labeled by: C, condensation; A, adenylation; T, thiolation and TE, thioesterification. The first module is predicted to be an initiation module as it harbours a condensation (C)-domain with structural features that are typical for C-domains involved in N-acylation of the first amino acid of the molecule. Modules 2 through 9 are predicted to elongate the peptide chain via incorporation of one amino acid per module. Together these nine catalytic domains are predicted to generate a peptide which is cleaved at the end of the assembly line by a thioesterase (TE) domain, resulting in the release of a linear product or a cyclic peptide via an intramolecular cyclization reaction. Based on the signature sequences in the adenylation (A)-domains, 6 of the amino acid residues in the peptide moiety could be predicted but 3 could not. Genes thaC1 and thaC2 share similarities with syrB1 and syrB2, respectively, the latter being involved in chlorination of the ninth amino acid residue (Thr) of syringomycin, the lipopeptide antibiotic produced by P. syringae. ThaD has 73% sequence identity to SyrC, an aminoacyltransferase that shuttles threonyl and chlorothreonyl residues to the syr-syp biosynthetic assembly line in P. syringae. Based on these in silico analysis, the encoded compound is predicted to be a chlorinated lipopeptide with nine amino acid residues. Triangles represent the positions of the single disruptions in the thaAB and thaCD gene clusters obtained by either random (white triangle) or site-directed mutagenesis (black triangle). (B) In vitro hyphal growth inhibition of R. solani by Pseudomonas sp. strain 13

SH-C52 and its respective mutants O33 and KO26. The mutant KO25 showed the same lack of in vitro activity as O33. Disruption of the thaB or thaC2 genes largely eliminated the antifungal activity of strain SH-C52, which can be observed by the significantly smaller inhibition zones of these mutants in comparison with the inhibition caused by parental strain SH-C52.

14

Table S1. Physical and chemical properties of the disease suppressive and conducive soils obtained from Hoeven, The Netherlands. Both soils were classified as sandy soils based on analyses performed by BLGG-AgroXpertus (Oosterbeek, The Netherlands).
Suppressive soil Chemical analysis pH Organic matter (%) CaCO3 (%) NH4 NO3 P K Mg Na Mn Cu Co B Zn Particle diameter (m) 0-2 2-16 16-50 50-105 105-150 150-210 210-300 300-420 420-600 600-2000 M50 median particles size 2.5 3.4 7.5 18.9 20.7 25.5 16.3 4.1 0.8 0.4 159 5.8 2.9 < 0.1 ----- mg kg ----4.5 17.6 3.2 93.0 37.0 20.0 <0.25 ----- g kg ----42 <2.5 109 272 ----- % ----1.4 2.0 6.5 17.0 21.4 26.1 17.8 5.3 1.6 0.9 165 38 7.2 97 2446
-1 -1

Conducive soil 5.6 2.7 < 0.1 10.2 1.7 2.5 69.0 30.0 <6.0 0.62

15

Table S2. Fresh weight (N=4) of sugar beet seedlings grown in suppressive soil and conducive soil in the absence of the pathogen R. solani. No significant differences were observed between the two treatments (P<0.05, Students t-test).
Soil Suppressive Conducive Fresh weight (mg) (SD) 202 (49) 256 (38)

16

Table S3. The top 10% most dynamic subset of the rhizosphere microbiome that meets all of the following criteria: i) more abundant in suppressive than in conducive soil, ii) more abundant in the transplantation soil (conducive soil + 10% suppressive soil) than in the conducive soil, and iii) more abundant in the suppressive soil when the fungal pathogen R. solani is present.
Representative OTU sequence Phylum Proteobacteria Affiliation Pseudomonadaceae GenBank accession EU538127 EU434358 EU537608 EU434526 EU535118 Burkholderiaceae AY550913 AY439198 AY321306 AY326592 AB299578 AY439195 AY178068 AF408946 AB079372 Xanthomonadales L76222 AY218744 Firmicutes Lactobacillaceae EF096273 Clone / strain antecubital fossa skin clone nbt82e01 Pseudomonas libanensis strain a110 antecubital fossa skin clone nbt74g09 Pseudomonas fluorescens strain b339 antecubital fossa skin clone nbt171b09 Burkholderia sp. FDS-1 soil clone MeBr 20 Burkholderia tropica LM2-37603 Amazon soil clone 141-1 Burkholderia sp. 70-VN5-1W soil clone MeBr 1 Burkholderia sp. UCT 15 Burkholderia sp. Ellin104 Burkholderia sp. S-2 Rhodanobacter lindaniclasticus penguin droppings clone KD5-94 mouse cecum clone obob1_aaa03h11

17

Table S4. Frequency of antagonistic bacteria isolated from the rhizosphere of sugar beet plants grown in disease suppressive or conducive soil. Bacteria were isolated on a general aerobic growth medium (TSA) and on a medium semi-selective for members of the Pseudomonadaceae. For each soil 200 - 300 isolates were randomly selected and tested for their ability to inhibit mycelial growth of the fungal pathogen Rhizoctonia solani in vitro. Isolates that inhibit mycelial growth were classified as antagonistic.
Aerobic medium antagonistic 4/96 3/96 0/96 7/288 4/76 3/38 4/75 11/189

Soil type Suppressive

Replicate 1 2 3 Total

Pseudomonas medium antagonistic 39/96 42/96 23/96 104/288 0/78 1/81 2/73 3/232

% 4.2 3.1 0.0 2.4 5.3 7.9 5.3 5.8

% 40.6 43.8 24.0 36.1 0.0 1.2 2.8 1.3

Conducive

1 2 3 Total

Number of antagonistic isolates / total number of tested isolates.

18

Table S5. Sequence identities of the 16S rDNA genes of the ten bacterial groups (haplotypes SH-A to SH-J) from the disease suppressive soil with the 16S rDNA genes present on the PhyloChip. The data shown represent the best BLAST hits of 16S rDNA sequences present in the PhyloChip database (GreenGenes).
Haplotype* SH-A SH-B SH-C SH-D SH-E SH-F SH-G SH-H SH-I SH-J

Hit in the BLAST search Pseudomonas sp. HNR09 Pseudomonas sp. A1Y13 Pseudomonas sp. HNR09 Pseudomonas sp. HNR09 Pseudomonas sp. HNR09 Pseudomonas sp. BIHB 811 Pseudomonas sp. BIHB 811 Pseudomonas sp. HNR09 Pseudomonas sp. HNR09 Pseudomonas sp. HNR09

GenBank accession EU373356 AY512624 EU373356 EU373356 EU373356 DQ885950 DQ885950 EU373356 EU373356 EU373356

Score 1352 1355 1354 1354 1354 1349 1349 1352 1352 1354

Identity (%) 99.78 99.93 99.85 99.85 99.85 99.71 99.71 99.78 99.78 99.85

Haplotype cluster I II I I I III III I I I

* or BOX-PCR group greengenes.lbl.gov/cgi-bin/nph-blast_interface.cgi

19

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