improved n retention through plant-microbe interactions
TRANSCRIPT
RESULTS
REFERENCES(1) Davis et al. 2012. PLOS One 7: e47149.(2) Lundberg D. S. et al. 2012. Nature 488: 86-90.(3) Norton J. M. 2008. Nitrogen in agricultural systems. Madison,WI: American Society of Agronomy, Inc.(4) Hofmockel K. et al., 2014. Poster for NIWQP and AFRI PDMeeting.(5) Osterholz W. et al., 2015. PhD Thesis ISU.(6) Kozich J. J. et al. 2013. Applied and EnvironmentalMicrobiology 79: 5112-5120.(7) Caporaso J. G. et al. 2010. Nature Methods 7: 335-336.
(8) Edgar R. C. 2013. Nature Methods 10: 996-998.(9) McMurdie P. J. & Holmes S. 2013. PLOS One 8: e61217.(10) Segata N. et al. 2011. Genome Biology 12: R60.(11) Deng Y. et al. 2012. BMC Bioinformatics 13: 113-132.(12) Quast C. et al. 2013. Nucleic Acids Research 41: D590-D596.
Plant Pathology & Microbiology1 and Ecology, Evolution & Organismal Biology2
NIWQP and AFRI PD MeetingOctober 12-13, 2016
Guillaume Bay1,*, Kirsten Hofmockel2, Chiliang Chen1 and Larry Halverson1
Improved Nitrogen Retention Through Plant-Microbe Interactions
SUMMARY OF FINDINGS• In this poster we highlight the soil and rhizosphere microbial community of the
entire root system of 3-week old maize grown in soil obtained from a conventional2-yr corn/soybean & diversified 4-yr corn/soybean/oats/alfalfa rotation
• Diversified cropping systems modify the structure (abundance and composition)of both the total resident and metabolically active microbial communities (Figures3, 4 & 5)
• Maize strongly influences the structure of the rhizosphere community in acropping system dependent manner (Figures 3, 4 & 5). Likewise the endospherecommunity reflect whether the plant was grown in soil from a specific croppingsystem (Figure 3 and data not shown).
• The richness and evenness of both total and metabolically active microbialcommunities increase as a result of crop diversification (Table 1)
• Crop diversification reduces the abundance of AOB in the soil and AOA & AOBin the rhizosphere (Figure 5)
• Crop diversification tends to increase the level of organization and complexity ofboth soil and rhizosphere microbial networks (Table 2 )
CONCLUSIONS• Soils of diversified system exhibit richer, more even microbial communities whose
structure differs from that of conventional system
• Crop diversification leads to a significant decrease in the AOA/AOB populations,which may contribute to lower N loss from the system
• Crop diversification may enhance substrate use via a better coupling of carbonand N cycles, thanks to a more organized microbial community with a greaterpotential for interactions
• Cropping System Diversification Alters Microbial Community StructureEach datum point represents the qualityfiltered (Q25) relative abundances of allthe OTUs in that sample. OTUs wereclassified against Silva 111 database (12)at 97% similarity. These plots illustratethat although samples from a givenenvironment (bulk soil/rhizosphere/endosphere) clustertogether they are distinct (p ≤ 0.008 andp ≤ 0.018) by cropping system based onAdonis statistical analysis. β-diversity1
community structure (i.e. relativeabundance).1β-diversity: ratio between mean localand mean regional species richness.
p = 0.008 p = 0.018
BA
Conventional DiversifiedRotation Bulk Soil
EndosphereRhizosphereCompartment
Figure 3 | Weighted UniFrac distance PCoA plots showing the effects ofcropping system on bulk soil, and maize rhizosphere and endosphere(A) total and (B) metabolically active bacterial communities
• Cropping System Diversification Influences Microbial Richness and EvennessWe used 3 separate α-diversity indices to assess how croppingsystems influence the composition of the total andmetabolically active microbial community. Observed: countof OTUs in each system; Chao 1: metric for species richness,the count of OTUs in a habitat that does not take into accounttheir abundance; Simpson: metric for assessing how close innumbers is each OTU in a habitat. Values are the average forbulk soil, rhizosphere, and endosphere samples for eachcropping system. In all cases there was greater speciesrichness in the diversified compared to the conventionalcropping system.
Observed Simpson
8820 9423 ± 53 0.99619153 9747 ± 45 0.9946Diversified
Total Community Chao 1
Conventional
Observed Simpson
7551 8377 ± 0.99768948 9449 ± 32 0.9971
ConventionalDiversified
Active Community Chao 1
130
Table 1 | Bacterial Diversity Indices
• Cropping System Diversification Alters Microbial Community StructureWe used linear discriminant analysis effect size(LEfSe) (10) to assess how cropping systemsaffect the composition of microbial communities.Taxa that are significantly different (p = 0.05)are mapped onto the cladogram at the taxonomiclevel they differ between cropping system. Nodesand branches correspond to discriminant taxonand are colored according to the highest-rankedgroup (i.e. conventional or diversified) for thattaxon. When the relative abundance for aspecific taxon is not significantly different, thecor-responding node is colored yellow. Circlesrepresent phylogenetic levels from Kingdom toFamily from the inside outwards. Taxa withknown nitrifying/denitrifying members as well asphyla of interest are represented by a uniquenumerical identifier.
Bulk Soil Rhizosphere
Tota
l Com
mun
ityAc
tive
Com
mun
ity
1 21a1b
3
4
5
67
8
9
1 101a
1c3
4
5
6
14
9
11
12
13
15
1 21a
3
6
9
16
17
14
151a
1c
10
16
6
18
14
8
9
ConventionalDiversified
1: α-Proteobacteriab
1a: Sphingomonadaceaed
& Erythrobacteraceaed
1b: Burkholderialesc
1c: Rhizobiaceaed
2: β-Proteobacteriab
3: Pseudomonadales 4: Thaumarchaeotaa
5: Actinobacteriaa
6: Rubrobacteriab
7: Bacteroidetesa
8: Cyanobacteriaa
9: Nitrospiraceaed
10: Nitrosomonadaceaed
11: δ-Proteobacteriab
12: Euryarchaeotaa
13: Acidobacteriaa
14: Elusimicrobiaa
15: Planctomycetesa
16: Verrucomicrobiaa
17: Holophagaeb
18: Chloroflexia
where a: Phylum, b: Class,c: Order, d: Family.
Figure 4 | Cladograms of cropping system effects ontaxonomic enrichments in the total and metabolicallyactive bulk soil and rhizosphere communities
• Diversified Cropping Systems Exhibit Distinct Microbial Community Networks
The interactions amongmicrobial communities wereassessed using a randommatrix theory-based approachimplemented in the MolecularEcological Network Analysispipeline (11). Bold valuesindicate which croppingsystem possesses attributes formore resilient and moreinteractive networks.
Table 2 | Major topological properties of the molecular ecological co-occurrence networksof microbial communities from conventional and diversified cropping systems
R² of Network Total links Average Average Average Modularitypower size (% pos/neg connectivity path length clustering (number of
law interactions) coefficient modules)
Total CommunityBulk Soil
Conventional 0.91 988 4127 (85/15) 8.354 5.31 0.288 0.579 (87)Diversified 0.85 863 6386 (82.5/17.5) 14.8 4.352 0.362 0.452 (60)
RhizosphereConventional 0.90 737 1934 (96.5/3.5) 5.248 6.732 0.263 0.745 (78)Diversified 0.88 904 3540 (98.2/1.8) 7.832 6.803 0.357 0.713 (43)
Active CommunityBulk Soil
Conventional 0.89 2176 6946 (77/23) 6.384 6.874 0.249 0.628 (142)Diversified 0.89 2331 9632 (77.4/22.6) 8.264 6.351 0.267 0.655 (121)
RhizosphereConventional 0.72 1366 10566 (78.3/21.7) 15.47 5.23 0.41 0.598 (14)Diversified 0.52 854 9928 (99.4/0.6) 23.251 8.353 0.416 0.317 (58)
We used qPCR to measure the abundance ofAOA and AOB. While there was no significanteffect of cropping system on soil AOAabundance there were fewer AOB in soilsfrom the diversified system (A). In contrast,there was a dramatic decrease in abundanceof AOA and AOB on maize roots grown in soilfrom the diversified cropping system (B).Values are means ± SE; n = 8. Values within acompartment with different letters arestatistically different.
• Cropping System Diversification Affects Ammonia-Oxidizers’ Abundance
Figure 5 | Effect of diversification on AOA and AOB amoAgene abundance in (A) the bulk soil and (B) the rhizosphere
p < 0.001
AB
B
A
Microbial Type & Cropping SystemAOA
Conventional
Diversified Conventional
DiversifiedAOB
p = 0.02p = 0.31 p < 0.001
B
AA
A
BA
Conventional
DiversifiedAOA
Microbial Type & Cropping System
Conventional
DiversifiedAOB
amoA
gene
cop
y nu
mbe
r g s
oil-1
(×10
6 )
amoA
gene
cop
y nu
mbe
r cm
root
-2(×
106 )
0
15
30
45
60
75
90
105
120
135
150
0
0.25
0.50
0.75
1
1.25
1.50
1.75
2
2.25
*contact: [email protected]
CONTEXT• Microbes play an essential role in soils, being able to shape plant development and nutrient
availability (2).• Ammonia-oxidizing bacteria (AOB) and archaea (AOA) mediate the rate-limiting step of
nitrification, the conversion of NH4+ to NO3
-, contributing to eutrophication of water (3).• Despite similar total %C and N contents and NH4
+ pool sizes, compared to conventionalsystems soil from diversified systems have lower NO3
- pool sizes.• Prior work at the Marsden site also demonstrated increased soil protease activity (4) but
comparable NH3 mineralization rates (5) in the diversified compared to conventionalcropping system.
• Soil microbiome = soil microbes not directly influenced by plant rootsRhizosphere microbiome = microbes infuenced by rhizodeposits
OBSERVATIONSAt the Marsden long-term cropping system site, more diversified 4-year rotations(maize/soybean/oat/alfalfa) managed with lower inorganic nitrogen (N)-inputs and periodicapplication of composted manure (≈ 100 kg N ha-1) can yield comparably to conventionallymanaged 2-year rotations (maize-soybean) receiving normal rates of inorganic N-fertilizer,and result in lower N loss than conventional systems (1).
APPROACHWe used 16S amplicon sequencing and qPCR of ammonia oxidizers to assess the effect ofcropping system on the soil total resident (DNA-based) and metabolically active (rRNA-based)community profiles and whether the maize root harbors distinct communities in a croppingsystem-specific manner.
HYPOTHESISAs compared to simpler cropping systems, soils in diversified cropping systems foster differentmicrobial assemblages resulting in tighter coupling of available N supply and demand, andsmaller inorganic N pools.
MATERIAL & METHODS2. Plants Grown in Rhizotrons
Figure 2 Twenty-one-day old maize plants growing in rhizotrons filledwith soil from conventional and diversified cropping system plots.
1. Experimental Site
3. Sample Collection, Sequencing, qPCR & Data Analysis
Figure 1 Marsden field site in Iowa, USA, established in 2002;randomized complete block (n-4) design where each phase ofeach rotation is present every year. Plots are 18 m × 85 m.
2. DNA & RNA extractions
3a. qPCR on ammonia mono-oxygenase (amoA) gene of AOA/AOB from soil and rhizosphere microbiomes
Phyloseq in (9)
MENAP (Molecular Ecological Network Analysis Pipeline)
(11)
LEfSe (Linear Discriminant Analysis Effect Size)
(10)
1. Rhizotrons:- Bulk soil collection- Sampling of the microbial
communities (washing of roots & sonication) (8)UPARSE
OTU Clustering Pipeline
Quantitative Insights Into Microbial Ecology
(7)
3b. Illumina MiSeq sequencing on bulk soil
and rhizosphere communities (6)