Factors Shaping the Root-Associated Microbiome of Populus species

Christopher W. SchadtOak Ridge National Laboratory &University of Tennessee - Knoxville

Natural Systems Components

Aim 1: Define Aim 2: Dissect

plantsfungi

bacteria

Interactions & Signaling

Plant-Microbe Interfaces

Caney Fork River, TN

Yadkin River, NC

PMI Plant-Microbe Interactions: Genotype & Environment Interactions in Structuring the Populus Microbiome

Populus deltoides (2009-2011 Campaigns)

Populus trichocarpa (2012 & Beyond)

Studies focused on broad associations of microbiome patterns within natural riparian populations in the Southeast.

Studies of common-garden GWAS populations that will allow examination of detailed genome-level associations between host & microbiome

Gottel, NR, et al. (2011). Populus deltoides roots harbor distinct microbial communities within the endosphere and rhizosphere across contrasting soil types. Appl. Environ. Microbiol. 77, 5934-5944.

Objective:

Assess the composition and variability within and between, endosphere and rhizosphere communities

Campaign 1: Caney Fork Study of Upland and Bottomland Root Endophyte and Rhizosphere

Communities

N. Gottel H. Castro

Endosphere and rhizosphere communities are distinct regardless of origin from upland or

bottomland sites

Fungi

PCA analysis shows distinct endophyte and rhizosphere communities regardless of upland or bottomland origin

BacteriaEndo|Rhizo

FungiEndo|Rhizo

Hierarchical clustering of OTUs also shows most appear specialized to either the rhizosphere or endosphere niche, and are rarely in abundance in both

Caney Fork River, TN

Yadkin River, NC

Campaign 2: A P. deltoides regional studyObjectives• Verify the separation

between the endosphere and rhizosphere microbial niches

• Examination of seasonal effects on microbiome structure

• Test the effects of soil and host characteristics across a broad range of natural conditions

M. Shakya W. Muchero H. Castro

P. deltoides genotypes and soils from TN and NC are diverse, but covary by region• Soils exhibit a wide range of variation. pH, organic matter, nitrogen,

and soil texture vary widely across the sites• SSR genotyping of trees shows a variation in population structure with

division between samples originating from different regionsSoil texture across sites Plant genotypes across sites

NMDS analyses of Caney Fork and Yadkin datasets show partitioning by sample type and region• Rhizosphere/

Endosphere divide is confirmed

• Variability between endophyte samples is extreme (but diversity low)

• Structure according to regional origin and season is also significant

• Variance partitioning between a suite of host and environmental factors explains only 33% to 56% of community-wide microbiome variation

• Within Phyla & and dominant OTUs more significant trends are observed

Disentangling the drivers of community patterns remain an elusive task

Fungi Bacteria

Common garden / GWAS populations of Populus trichocarpa in the Pacific Northwest• 1100 variants of P.

trichocarpa collected across it’s latitudinal range replicated in four common gardens in BC, OR and CA

• >900 of these have been resequenced to at least 12X depth by JGI.

G. TuskanW. Muchero

2012 CSP project examining associations of host characteristics and the root microbiome • Pathogen Susceptibility

(Meampsora infection rates)• Copy number variation

(Lectin Receptor Kinase)

• Plant Growth (Biomass)

• Secondary Chemistry (Lignin S:G)

Larger scale of analyses facilitated by new JGI collaboration via 2012 CSP program• Combination of large scale rRNA community profiling multiplexed on

MiSeq Illumina system (all samples, all traits)- 8 genotypes selected at extremes for 4 traits (32 genotypes)- 32 genotypes w/ 3 replicates in 2 locations (192 trees)- Endophyte, rhizospshere & bulk soil samples (576 samples)- Bacterial, fungal & archaeal community (1728 rRNA profiles)

• In depth metagenomic profiles using 32 lanes of HiSeq Illumina profiling (select sample/trait)

a-Proteobacteria

b-Proteobacteria

BacilliBacteroidetes

• Isolated 2800+ bacteria & 900+ fungi

• 43 bacterial isolates have been sequenced from P. deltoides with a concentration on Pseudomonas

• 6 fungal isolates underway through various JGI programs

g-Proteobacteria

Conserved among our iso-lates and ref-erence organ-

isms, 1535, 52%Conserved in

our isolate organisms, 969, 33%

Found in some of our isolates only, 426, 15%

2930 predicted KEGG orthologous groups in 21 Pseudomonas isolates and 4 reference spp.

D. Pelletier S. Brown

What is the genomic and functional diversity within Populus isolates?

S. Utturkar

R. Vilgalys G. Bonito T. Karpinets

Pseudomonas spp. show high functional diversity

TREE ECO-UNIT STRAIN Anti QS Sid Prot Phos IAA(ug/ml) NO3 MHBB1B endophyte Pseudomonas GM17 2 high + + - 0.2 + neg

B1B endophyte Pseudomonas GM21 neg neg + - - 2.4 - o

B4A rhizosphere Pseudomonas GM49 1 neg + - - 0.1 - o

B4A rhizosphere Pseudomonas GM48 1 neg + + - 0.1 - +B2B endophyte Pseudomonas GM33 1 neg + - + 1.0 - +

U2A rhizosphere Pseudomonas GM74 1 neg + - - 1.0 - o

B4B endophyte Pseudomonas GM55 neg neg + - + 1.5 - o

B3B endophyte Pseudomonas GM41 1 neg + + + 1.5 + +

B1B endophyte Pseudomonas GM18 1 low + + + 0.6 - +

B4B endophyte Pseudomonas GM50 2 low + + + 1.0 - o

U3B endophyte Pseudomonas GM102 1 neg - + 0.9 - o

U2B endophyte Pseudomonas GM79 2 neg + + - 2.2 + +

B2B endophyte Pseudomonas GM30 3 med + ++ - 0.9 - o

B4B endophyte Pseudomonas GM24 4 neg + ++ - 1.5 - negB1B endophyte Pseudomonas GM16 3 neg + ++ + 1.4 - neg

B2A rhizosphere Pseudomonas GM25 1 neg + + + 0.8 - o

  soil  Pseudomonas fluorescens PfO1 ND ND ND ND ND ND ND ND

U2B endophyte Pseudomonas GM80 1 neg + ++ - 0.9 - o

B4B endophyte Pseudomonas GM60 neg neg + + - 0.9 + o

U1B endophyte Pseudomonas GM67 1 neg + - - 1.0 + o

U2B endophyte Pseudomonas GM78 1 neg + - + 1.3 - o  Cotton root Pseudomonas fluorescens Pf5 2 ND + + + 0.1 neg

U3A rhizosphere Pseudomonas GM84 1 neg + - + 1.7 - o

  endophyte Pseudomonas putida W619 ND ND ND ND ND ND ND ND  pathogen Pseudomonas syringae DC3000 ND ND ND ND ND ND ND ND

ORNL

Often same types of endophytic isolates seen across Populus (ORNL) and Arabidopsis

(Dangle)

UNC

Sur Paredes

AcknowledgementsSchadt Lab at ORNLMigun ShakyaMike RobesonZamin YangNeil GottelHector CastroMarilyn Kerley

ORNL CollaboratorsJerry TuskanMitch DoktyczDale PelletierMircea PodarJessy L. LabbéWellington MucheroTatiana KarpinetsSteven BrownSagar Utturkar

JGISusannah TringeStephanie MalfattiTijana Glavina del Rio

Duke UniversityRytas VillgalysGregory BonitoKhalid HameedJay KrishnanMichael GajdeczkaAndrii Grygansky

INRAFrancis Martin