Diversity Down Below: Does
Fertility Impact the
Rhizosphere Microbial
Community in Poa annua
turf?
Lisa A. Beirn1, Charles J. Schmid1, James W. Hempfling1, James A. Murphy1,
Bruce B. Clarke1, and Jo Anne Crouch2
1Dept.
of Plant Biology & Pathology, Rutgers University, New Brunswick, NJ
2Systematic Mycology and Microbiology Lab, USDA-ARS, Beltsville, MD
Introduction

Nitrogen (N) and potassium (K) are vital for
maintaining healthy turfgrass

Fertilizers in turf are under scrutiny for
potential adverse environmental effects

As a result, fertilizers are becoming
increasingly regulated in the turfgrass system
Introduction

How are these nutrients impacting the microbial
community structure in the turfgrass
rhizosphere?

N has been reported to reduce microbial
biomass and diversity in boreal and forest
ecosystems (Treseder 2008, Allison et al. 2007, Wallenstein et al. 2006)

May not be comparable to a man-made putting
green
Objectives

Determine the composition and distribution of
rhizosphere microorganisms in Poa annua
turf maintained as a putting green

Evaluate if microbial diversity is impacted in
this highly-maintained system
Materials & Methods: Site

Poa annua turf grown on
Nixon sandy loam and
maintained as putting green
turf in North Brunswick, NJ

Site was initiated to
investigate the impacts of
cultural management
practices on anthracnose
severity, caused by
Colletotrichum cereale
Study 1: K Study
Study 2: N Study
Materials & Methods: Sampling
•
Three soil cores (15.9 mm x 50.8 mm) were
sampled from four replicated plots receiving:
•
Study 1: K study
– (1) an intermediate N rate (132 kg N ha-1 yr-1);
– (2) K (200 kg K2O ha-1 yr-1);
– (3) N+K (1:1; 132 kg N ha-1 yr-1 and 200 kg K2O ha-1 yr-1)
•
•
N was applied as urea
K was applied as potassium chloride
•
36 samples total (12 per treatment)
Materials & Methods: Sampling
•
Three soil cores (15.9 mm x 50.8 mm) soil cores
were sampled from four replicated plots receiving:
•
Study 2: N study
– (1) a low N rate, (100 kg N ha-1 yr-1);
– (2) a high N rate, (200 kg N ha-1 yr-1)
•
N was applied as urea
•
24 samples total (12 per treatment)
Materials & Methods: Sampling
5 cm
Materials & Methods: DNA Manipulations
1. Extract DNA
gDNA
16s or
ITS
Overhang
Adapter Primer region
2. PCR amplify region of
interest:
16s archaea & bacteria
ITS1 & ITS2 fungi
3. Index Addition for
Sample Pooling
Overhang
Adapter
Index
16s or
ITS
Primer region
Materials & Methods: DNA Manipulations
4. Sequence on Illumina’s MiSeq
Forward & Reverse Reads,
300 bp in each direction
5. Data Analyzed via QIIME
(Quantitative Insights Into
Microbial Ecology)
6. Taxonomy Assigned Using
Greengenes (16s) and UNITE +
INSDC (ITS) databases
Results: Sequencing Data
•
Generated 2.3 x 107 reads
•
On average:
– 1.38 x 105 seq/per sample
– 253 bp length
•
14 Gb data!
•
8.3 x 105 Operational Taxonomic Units
(OTUs) at 97% identity threshold
Results: Community Structure
Results: Community Structure
Results: Microbial Diversity
Archaea/Bacteria
Fungi
Treatment
Shannon
Diversity Index
N Only
6.38
K Only
N:K
6.48
6.38
Low N
High N
N Only
K Only
N:K
Low N
High N
6.30
6.40
4.98
4.91
5.03
4.93
5.03
Comparing 16s Diversity from P. annua
turf to other biomes*
Biome
Shannon Diversity
Index
Arctic Tundra
6.84
Tropical Forest (Peru)
6.70
Tropical Forest (Argentina)
6.51
Temperate Grassland
6.72
Temperate Coniferous Forest
6.81
Temperate Deciduous Forest
6.81
Boreal Forest
6.79
Hot Desert (New Mexico)
6.62
Hot Desert (California)
6.60
Polar Desert (Antarctica)
6.42
Our Study:
6.30 – 6.48
*Fierer N et al.(2012) Cross-biome metagenomics analyses of soil microbial communities and their functional
attributes PNAS 109: 21390-21395
Results: DCA of Microbial Populations
DCA Analysis of Archaea &
Bacterial Populations
2.5
F07
N Study
F05
DCA1-3
2
F10
F03
E09 1.5
C12
B09
C05 A09
A07
F08
F11 F12
F09E02
E11 1
E10
F06
C07
0.5
F02 E07
C10
F04
A11
C03 C06
E03
A08
0 A10C01
F01
E04 E12 B11
E08
-2
-1 E05
0
2
B08 1
E06 -0.5
E01
C11
B02C08
A05
B05
B04
A06
C02
B12
-1
B07
B01
B06
A12 A03
B10
C09
-1.5
C04
B03
A01
-2A04
K Study
3
A02
-2.5
-3
DCA2
Green = high N, Purple = low N, Red = K only, Orange = N:K, Blue=
intermediate N
Archaea & Bacteria Composition >1% Across All
Samples
Archaeal and Bacterial Composition >1% Across All Samples
Red = low
abundance; Yellow = high abundance
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Other, Other
Rhizobiales
Bacteria, Proteobacteria, Alphaproteobacteria
Alphaproteobacteria
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Hyphomicrobiaceae
Hyphomicrobiaceae
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Bradyrhizobiaceae, Bradyrhizobium
Bradyrhizobium
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Bradyrhizobiaceae, Other
Bradyrhizobiaceae
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Bradyrhizobiaceae, Other
Bradyrhizobiaceae
Bacteria, Proteobacteria, Deltaproteobacteria, Myxococcales
Myxococcales
Bacteria, Bacteroidetes, Saprospirae, Saprospirales, Chitinophagaceae
Chitinophagaceae
Bacteria, Proteobacteria, Gammaproteobacteria, Xanthomonadales, Xanthomonadaceae
Xanthomonadaceae
Bacteria, Acidobacteria, DA052, Ellin6513
Acidobacteria
Bacteria, TM7, TM7-1
Bacteria
Bacteria, Acidobacteria, Acidobacteriia, Acidobacteriales, Acidobacteriaceae
Acidobacteriaceae
Bacteria, Planctomycetes, Planctomycetia, Pirellulales, Pirellulaceae
Pirellulaceae
Bacteria, OD1, ZB2
Bacteria
Bacteria, Proteobacteria, Gammaproteobacteria, Legionellales, Coxiellaceae, Aquicella
Aquicella
Bacteria, Proteobacteria, Gammaproteobacteria, Xanthomonadales, Sinobacteraceae
Sinobacteraceae
Bacteria, Proteobacteria, Alphaproteobacteria, Ellin329
Alphaproteobacteria
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Methylocystaceae
Methylocystaceae
Bacteria, OD1, SM2F11
Bacteria
Bacteria, Gemmatimonadetes, Gemm-1
Gemmatimonadetes
Bacteria, Acidobacteria, Chloracidobacteria, RB41, Ellin6075
Chloracidobacteria
Bacteria, Planctomycetes, Phycisphaerae, WD2101
Phyciosphaerae
Bacteria, Verrucomicrobia, Spartobacteria, Chthoniobacterales, Chthoniobacteraceae, DA101
Chthoniobacteraceae
Bacteria, Proteobacteria, Gammaproteobacteria, Legionellales, Coxiellaceae
Coxlellaceae
Bacteria, Chlorobi, SJA-28
Chlorobi
Archaea, Crenarchaeota, Thaumarchaeota, Nitrososphaerales, Nitrososphaeraceae, Candidatus Nitrososph
Candidatus
Nitrosphaera
Archaea, Crenarchaeota, Thaumarchaeota, Cenarchaeales, SAGMA-X, Other
Crenarchaeales
Bacteria, Acidobacteria, Acidobacteriia,
Acidobacteriales, Koribacteraceae, Candidatus Koribacter
Candidatus
Koribacter
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Hyphomicrobiaceae, Rhodoplanes
Rhodoplanes
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales
Rhizobiales
Bacteria, Proteobacteria, Alphaproteobacteria, Rhodospirillales, Rhodospirillaceae
Rhodospirillaceae
Bacteria, Acidobacteria, Solibacteres,
Solibacterales, Solibacteraceae, Candidatus Solibacter
Candidatus
Solibacter
Bacteria, Acidobacteria, Solibacteres, Solibacterales
Solibacterales
Bacteria, Chloroflexi, Anaerolineae, SBR1031, A4b
Chloroflexi
Bacteria, OP11, WCHB1-64, d153
Bacteria
d
Archaea, Crenarchaeota, Thaumarchaeota, Cenarchaeales, SAGMA-X
Crenarchaeales
Koribacteraceae
Intermediate N
K Only
N:K
Low N
High N
F12
F11
F10
F09
F08
F07
F06
F05
F04
F03
F02
F01
E12
E11
E10
E09
E08
E07
E06
E05
E04
E03
E02
E01
C12
C11
C10
C09
C08
C07
C06
C05
C04
C03
B12
C02
C01
B11
B10
B09
B08
B07
B06
B05
B04
B03
B02
B01
A12
A11
A10
A09
A08
A07
A06
A05
A04
A03
A02
A01
Bacteria, Acidobacteria, Acidobacteriia, Acidobacteriales, Koribacteraceae
N fixing species in the Rhizobiales more
abundant in low N plots than high N plots
Archaeal and Bacterial Composition >1% Across All Samples
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Other, Other
Bacteria, Proteobacteria, Alphaproteobacteria
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Hyphomicrobiaceae
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Bradyrhizobiaceae, Bradyrhizobium
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Bradyrhizobiaceae, Other
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Bradyrhizobiaceae, Other
Bacteria, Proteobacteria, Deltaproteobacteria, Myxococcales
Intermediate N
K Only
N:K
Low N
High N
Bacteria, Bacteroidetes, Saprospirae, Saprospirales, Chitinophagaceae
Bacteria, Proteobacteria, Gammaproteobacteria, Xanthomonadales, Xanthomonadaceae
Bacteria, Acidobacteria, DA052, Ellin6513
Bacteria, TM7, TM7-1
Bacteria, Acidobacteria, Acidobacteriia, Acidobacteriales, Acidobacteriaceae
Bacteria, Planctomycetes, Planctomycetia, Pirellulales, Pirellulaceae
Red = low abundance; Yellow = high abundance
Bacteria, OD1, ZB2
Bacteria, Proteobacteria, Gammaproteobacteria, Legionellales, Coxiellaceae, Aquicella
Bacteria, Proteobacteria, Gammaproteobacteria, Xanthomonadales, Sinobacteraceae
Bacteria, Proteobacteria, Alphaproteobacteria, Ellin329
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Methylocystaceae
Bacteria, OD1, SM2F11
Bacteria, Gemmatimonadetes, Gemm-1
Bacteria, Acidobacteria, Chloracidobacteria, RB41, Ellin6075
Bacteria, Planctomycetes, Phycisphaerae, WD2101
Bacteria, Verrucomicrobia, Spartobacteria, Chthoniobacterales, Chthoniobacteraceae, DA101
Bacteria, Proteobacteria, Gammaproteobacteria, Legionellales, Coxiellaceae
Bacteria, Chlorobi, SJA-28
Archaea, Crenarchaeota, Thaumarchaeota, Nitrososphaerales, Nitrososphaeraceae, Candidatus Nitrosos
Archaea, Crenarchaeota, Thaumarchaeota, Cenarchaeales, SAGMA-X, Other
Bacteria, Acidobacteria, Acidobacteriia, Acidobacteriales, Koribacteraceae, Candidatus Koribacter
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Hyphomicrobiaceae, Rhodoplanes
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales
Bacteria, Proteobacteria, Alphaproteobacteria, Rhodospirillales, Rhodospirillaceae
Bacteria, Acidobacteria, Solibacteres, Solibacterales, Solibacteraceae, Candidatus Solibacter
Bacteria, Proteobacteria, Gammaproteobacteria, Xanthomonadales, Sinobacteraceae
Low pH loving ammonium oxidizing archaea at
least 3 times more abundant in lower pH K study
Bacteria, Proteobacteria, Alphaproteobacteria, Ellin329
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Methylocystaceae
Bacteria, OD1, SM2F11
Bacteria, Gemmatimonadetes, Gemm-1
Bacteria, Acidobacteria, Chloracidobacteria, RB41, Ellin6075
Bacteria, Planctomycetes, Phycisphaerae, WD2101
Bacteria, Verrucomicrobia, Spartobacteria, Chthoniobacterales, Chthoniobacteraceae, DA101
Bacteria, Proteobacteria, Gammaproteobacteria, Legionellales, Coxiellaceae
Bacteria, Chlorobi, SJA-28
Archaea, Crenarchaeota, Thaumarchaeota, Nitrososphaerales, Nitrososphaeraceae, Candidatu
Archaea, Crenarchaeota, Thaumarchaeota, Cenarchaeales, SAGMA-X, Other
Bacteria, Acidobacteria, Acidobacteriia, Acidobacteriales, Koribacteraceae, Candidatus Koribac
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Hyphomicrobiaceae, Rhodoplanes
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales
Bacteria, Proteobacteria, Alphaproteobacteria, Rhodospirillales, Rhodospirillaceae
Bacteria, Acidobacteria, Solibacteres, Solibacterales, Solibacteraceae, Candidatus Solibacter
Bacteria, Acidobacteria, Solibacteres, Solibacterales
Bacteria, Chloroflexi, Anaerolineae, SBR1031, A4b
Bacteria, OP11, WCHB1-64, d153
Archaea, Crenarchaeota, Thaumarchaeota, Cenarchaeales, SAGMA-X
Intermediate N
K Only
Intermediate N
K Only
N:K
Low N
F12
F11
F10
F09
F08
F07
F06
F05
F04
F03
F02
F01
E12
E11
E10
E09
E08
E07
E06
E05
E04
E03
E02
E01
C12
C11
C10
C09
C08
C07
C06
C05
C04
C03
B12
C02
C01
B11
B10
B09
B08
B07
B06
B05
B04
B03
B02
B01
A12
A11
A10
A09
A08
A07
A06
A05
A04
A03
A02
A01
Bacteria, Acidobacteria, Acidobacteriia, Acidobacteriales, Koribacteraceae
High N
Low N
N:K
High N
Red = low abundance; Yellow = high abundance
Low pH loving acidobacteria more abundant in
higher pH N study
Archaeal and Bacterial Composition >1% Across All Samples
Intermediate
IntermediateNN
K Only
N:K
Low N
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Other, Other
Rhizobiales
Bacteria, Proteobacteria, Alphaproteobacteria
Alphaproteobacteria
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Hyphomicrobiaceae
Hyphomicrobiaceae
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Bradyrhizobiaceae, Bradyrhizobium
Bradyrhizobium
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Bradyrhizobiaceae, Other
Bradyrhizobiaceae
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Bradyrhizobiaceae, Other
Bradyrhizobiaceae
Bacteria, Proteobacteria, Deltaproteobacteria, Myxococcales
Myxococcales
Bacteria, Bacteroidetes, Saprospirae, Saprospirales, Chitinophagaceae
Chitinophagaceae
Bacteria, Proteobacteria, Gammaproteobacteria, Xanthomonadales, Xanthomonadaceae
Xanthomonadaceae
Bacteria, Acidobacteria, DA052, Ellin6513
Acidobacteria
Bacteria, TM7, TM7-1
Bacteria
Bacteria, Acidobacteria, Acidobacteriia, Acidobacteriales, Acidobacteriaceae
Acidobacteriaceae
Bacteria, Planctomycetes, Planctomycetia, Pirellulales, Pirellulaceae
Pirellulaceae
Bacteria, OD1, ZB2
Bacteria
Bacteria, Proteobacteria, Gammaproteobacteria, Legionellales, Coxiellaceae, Aquicella
Aquicella
Bacteria, Proteobacteria, Gammaproteobacteria, Xanthomonadales, Sinobacteraceae
Sinobacteraceae
Bacteria, Proteobacteria, Alphaproteobacteria, Ellin329
Alphaproteobacteria
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Methylocystaceae
Methylocystaceae
Bacteria, OD1, SM2F11
Bacteria
Bacteria, Gemmatimonadetes, Gemm-1
Gemmatimonadetes
Bacteria, Acidobacteria, Chloracidobacteria, RB41, Ellin6075
Chloracidobacteria
Bacteria, Planctomycetes, Phycisphaerae, WD2101
Phyciosphaerae
Bacteria,
Verrucomicrobia, Spartobacteria, Chthoniobacterales, Chthoniobacteraceae, DA101
Chthoniobacteraceae
Bacteria, Proteobacteria, Gammaproteobacteria, Legionellales, Coxiellaceae
Coxlellaceae
Bacteria, Chlorobi, SJA-28
Chlorobi
Archaea, Crenarchaeota, Thaumarchaeota, Nitrososphaerales, Nitrososphaeraceae, Candidatus Nitrososph
Candidatus
Nitrosphaera
Archaea, Crenarchaeota, Thaumarchaeota, Cenarchaeales, SAGMA-X, Other
Crenarchaeales
Bacteria, Acidobacteria, Acidobacteriia,
Acidobacteriales, Koribacteraceae, Candidatus Koribacter
Candidatus
Koribacter
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales, Hyphomicrobiaceae, Rhodoplanes
Rhodoplanes
Bacteria, Proteobacteria, Alphaproteobacteria, Rhizobiales
Rhizobiales
Bacteria, Proteobacteria, Alphaproteobacteria, Rhodospirillales, Rhodospirillaceae
Rhodospirillaceae
Bacteria, Acidobacteria, Solibacteres,
Solibacterales, Solibacteraceae, Candidatus Solibacter
Candidatus
Solibacter
Bacteria, Acidobacteria, Solibacteres, Solibacterales
Solibacterales
Bacteria, Chloroflexi, Anaerolineae, SBR1031, A4b
Chloroflexi
Bacteria, OP11, WCHB1-64, d153
Bacteria
d
Archaea, Crenarchaeota, Thaumarchaeota, Cenarchaeales, SAGMA-X
Crenarchaeales
High N
Red = low abundance; Yellow = high abundance
Koribacteraceae
Intermediate N
K Only
N:K
Low N
High N
F12
F11
F10
F09
F08
F07
F06
F05
F04
F03
F02
F01
E12
E11
E10
E09
E08
E07
E06
E05
E04
E03
E02
E01
C12
C11
C10
C09
C08
C07
C06
C05
C04
C03
B12
C02
C01
B11
B10
B09
B08
B07
B06
B05
B04
B03
B02
B01
A12
A11
A10
A09
A08
A07
A06
A05
A04
A03
A02
A01
Bacteria, Acidobacteria, Acidobacteriia, Acidobacteriales, Koribacteraceae
Conclusions

Fertility does impact microbial communities
within the two studies
 In some cases, this may correlate with
microbial function

Underlying factors are also influencing the
community between the two studies
 Age? Renovation?
 Caution when sampling
Conclusions

Rhizosphere of P. annua turf contains
many organisms yet to be described

Overall, P. annua rhizosphere supports a
diverse, species rich microbial community
 Similar to biomes that have not received
any fertility inputs
Future Directions

Is fertility impacting microbial function?

Pathogen virulence?

Does this change during the growing
season?

In summer 2014, field study initiated to
examine 5 rates of nitrogen on microbial
gene expression in P. annua turf
 RNA-Seq based experiment
Funding
Rutgers Center for
Turfgrass Science
Rutgers
Greg Behringer
Bryan Rabin
USDA-ARS
Dr. Martha Malapi-Wight
Dan Veltri