siu mui tsai - fapesp.brpartners –national and international •siu mui tsai •carlos cerri...
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Monitoring the microbial diversity and functional activities inresponse to land-use changes and deforestation undersoybean and sugarcane cultivation
Siu Mui Tsaitsai @ cena.usp.br
CENA
Av. Centenário – 303
13.416-000 Piracicaba - SP
Programa FAPESP de Pesquisa em Mudanças Climáticas Globais 2016 (PFPMCG – Proc. 2008/58114-3)
Partners – National and International
• Siu Mui Tsai• Carlos Cerri
• Brigitte Feigl
• Acacio Navarrete, Fabiana Cannavan
• Lucas Mendes, Clovis Borges, Dennis Goss
• Caio Yoshiura, Marcela Arnaldo, Andressa Venturini
• Heitor Cantarella
• Raffaella Rossetto
State University of Sao Paulo
• Eliana Lemos
• Newton La Scala Jr.
• Jackson de Souza
• Eliamar Pedrinho
• Eiko Kuramae
• Hans van Veen
• Wim van der Putten
• Paul Bodelier
Instituto Agronomico de Campinas
University of Sao Paulo
•Karoline Faust
• Brendan Bohannan – UO
• Klaus Nüsslein – UMAS
• Jorge Rodrigues – UTA
• J. Zhou - OSU
USA (Proc. 2014/50320-
EndophytesMycorrhiza
Sustainable Management
(Sugarcane)
• Non-burning–raw cane
• Improvement of 2nd
generation biofuel
production
• Below-ground studies
Sugarcane
BradyrhizobiaEndophytesMycorrhizaSoybean
Sustainable Management
(Soybean)
• Minimum tillage system
• Genetic improvement
for stress tolerance and
better root development
THE CONTRIBUTION FROM THE BIOLOGICAL PROCESSES IS
HIGHLY SIGNIFICANT IN THE TROPICS
Metzker (2010)
Molecular Analyses are decreasing rapidly!
• Análises filogenéticas• Sequenciamento do 16S rRNA• Análise de imagens comunidades
Potencial GenéticoEm teoria, o que fazem?
• Metagenômica• Funcionalidade dos genes• Rede neural de genes
NEW METHODS ARE AVAILABLE FOR MICROBIAL DIVERSITY STUDIES
Cardenas & Tiedje (2008 )Current Opinion in Biotechnology 2008, 19:544–549
Community StructureWho and how are they organized?
• Taxonomy and Phylogeny• 16S rRNA Sequencing• Community Imaging Analysis
Genetic PotentialIn theory, what do they do?
• Metagenomics• Functional genes• Neural network of genes
Gene ExpressionNa prática, o que fazem?
• Metatranscriptomics
• Metaproteomics
Functional CharacterizationWho are the real functional ones?
• New cultivation methods• Stable isotope probing (SIP)• NanoSIMS• Card FISH
Análises Multivariadas:
PCA; RDA; CA
CCA;NMDS
Riqueza
Diagramas de Venn
UTO’s dominantes
Gráficos de
Abundância
Análises Multivariadas:PCA; NMDS
Análise de Similaridade:ANOSIM
Análises de Diversidade:Diversidade, Riqueza, Equitabilidade
Análises univariadas:
ANOVA
The Ex Ante Carbon-balance Tool
PREDIÇÃO de Balanço de CO2
equivalente
Análise de bibliotecas de clones
Univariada;
Multivariada;
ModelosGLM; etc.
Análise de sequênciasgeradas em largaescala (NGS)
Integração de dados de redes neurais
Análise de perfismetagenômicos
ARCHAEALand-use systems in Western Amazon
DQ
53
47
03
-SO
DQ148640-SM
0.05
Reference Sequences- Francis et al., 2005- Park et al., 2006
Method – Neighbor-JoiningDistance – Jukes-CantorMatrix PAM (Dayhoff)
Primary Forest
Traditional cropping system
Pasture
Secundary Forest
7Navarrete et al. (2011)
Alfa-Diversity = Pasture > Forest
Beta-Diversity = Pasture < Forest
Jan 2013
Cultivated Soil Forest Soil
Lemos et al. 2008BACTERIAL ABUNDANCE IN CULTIVATED SOIL
Brazilian Amazon
Sampling sites
Forest
Cropland
October 2009
April 2010
Forest Cropland Forest Cropland
Soybean rhizosphere
Soybean rhizosphere
Porto dos Gaúchos municipality
Ipiranga do Norte municipality
BS-F1
BS-F2 BS-C2
BS-C1 SR-I
SR-II BS-F3
BS-F4 BS-C4
BS-C3 SR-III
SR-IV
2013
11
The Neural Network Investigating the microbial
co-occurrence profiling
Barberán et al., 2011
Co-occurrence among microbial communities and soil chemical factors
H
K
T
pH
P
OM
B
Fe
H+Al
Al
Cu
Plan
B.Bur
Ac.3
Ac.1
B.Oth
D.Oth
Cy
A.Oth
Ver.3 G.Xan
Ver.Spa
Nit
F.Clo
Ac.6A.Rho
Ac.17
OthGem
G.Oth
A.Rhi
Kte
Ac.7
Ac.16
G.Leg
A.Sph
F.Bac
B.Sph Ver.Opi
ChlaP.Oth
Ac.4
Ac.2
Ac.4
H+Al
B
Fe
Al
Acidobacteriasub-group 4
Navarrete et al. (2013)
P
CEC
Cu
Ca
SB
MnH
Zn
Al
pH
H+Al
V
m
Ac.17
Ac.16Ac.7
Ac.6
Ac.5
Ac.2
Ac.10
Ac.Oth
G.Xan
Ver.3 Ac.13
Ac.4
A.Oth
G.Oth
Ver.Oth
Ver.Opi
B.Bur
Ac.1
G.Leg
Nit
KteOth Plan
B.Oth
Myx
D.Oth
A.Rhi
A.Rho
Gem
F.Bac
P.Oth
OM
A.SphChla
B.SphA.Aci
A.Act
Ver.3
H
Navarrete et al. (2015)
Verrucomicrobiasub-group 3
Soil Moisture
Co-occurrence among microbial communities and soil moisture
Functional bio-indicators
Functional bio-indicators
Forest
After Deforestation
Navarrete et al. (2012) ISME14
Amazonian deforestation alters soil bacterial networksTaxonomic
Network
Functional
Network
Navarrete et al. (in preparation)
(A) Two soybean crop fields
(B) Five points in each site
(C) Mesocosm experiment
(D) Rhizosphere and bulk soil
(DNA)
AC
B
D C
Rhizosphere Bulk
THE RHIZOSPHERE EFFECT - SOYBEAN
Mendes et al. (2012) ISME14Mendes et al. (ISME J., accepted)
RHIZOSPHERE X BULK SOIL
Microbial Taxonomy
Year 1 Year 5Mendes et al. (ISME J.,
accepted)
RhizosphereBulk Soil
Rhizosphere and bulk soil network analysis based on taxonomic
and functional abundance
Bulk Soil Rhizosphere
Total Correlations
310 236
Positive Correlations
191 143
Less
Complex
Mendes et al. (2012) ISME14
Mendes et al. (ISME J., accepted)
Functional and taxonomic profiles of bulk soil samples (red) and rhizosphere (green) of
nitrogen metabolism, calculated for metagenomes from bulk soil and rhizosphere and
compared to SEED database using a maximum E-value of 1e-5 and a minimum alignment
length of 50 bp.
RHIZOSPHERE X BULK SOIL
Microbial Taxonomy and Functional Diversity
Mendes et al. (ISME J., accepted)
nifH
narG
amoA
(Dinitrogenase reductase)
(Ammonia monooxygenase)
(Nitrate reductase)NO3
NO2
N2O
NH3
NH2OH
NO2
NO
N2
nirK/nirS
nosZ
norB
hao
nxrA
The Functional Genes of N-Cycle
Javier Izquierdo and Klaus Nüsslein
Treatments
N0: no amendments
N60: 60 Kg ha-1 N (urea)
Straw: 10 t ha-1 sugarcane straw (variety CTC-2)
Vinasse: 120 m3 ha-1
- 150 Kg ha-1 P2O5 and K2O (all treatments)
- Plants: CTC-2 sugarcane variety
Bioindicators forFunctional Microbiota
In Agriculture
GeoChip Analysis
Agilent array 4 x 180K
Agilent array 8 x 60K
GeoChip v. 5.0
167,044 distinct probes, covering
395,894 sequences
Coding ~1500 functional gene
families
Functional Analyses of the Biological Processes
Geochip V.5
27
Navarrete et al. (in preparation)
Non-bold letters were used to identify samples from Serra Azul experimental field.Bold letters were used to identify samples from Assis experimental field.
Carbon cycling (N=9)
Zinc homeostasis (N=5)
Nitrogen cycling (N=2)
Phosphorus cycling (N=2)
Secondary metabolism (N=4)
Stress(N=6)
Virulence (N=17)
0 5 10 20Doses of ZnSO4 applied to the sugarcane-cultivated soils
Gene expression for
virulence
Less at 10 and 20 kg
Zn/ha
Heatmap
The multidimensional focus for prospecting functional diversity of the microbiota
(NSF-FAPESP 2014/50320-4)
Bonhannan, Nüsslein e Rodrigues, 2012
Bonhannan, Nüsslein e Rodrigues, 2012
The multidimensional focus for prospecting functional diversity of the microbiota
Dual-laser trace gas monitor W-QC-TILDAS-SC-D
Dual quantum cascade laser trace gas analyzer configured for simultaneous detection of the 13CH4 and CH3D isotopic ratios
Future Perspectives
narG nirK nirS
nosZ AOB
Crenarchaeota
Bacteria
tota
l
Laurent PhilippotINRA, France
pmoAmcrAmmoX
PUBLICATION - RECORDS
• Papers – 20(PNAS, ISME J., Mol. Ecology, Microb. Ecology, FEMS, GCB, SBB, Ant.van Leeuwenhoek, Frontiers in Microbiology, PLOS One)
• Book Chapters - 3
Group Composition: Ph.D. Thesis – 6M.Sc. Dissertation – 6Pos-Doc -5
• International Conferences Attendance: 12
• Workshop: Meta-omics and Bioinformatics in Microbial Ecology – 124 participants (Nov. 2014)
• Awards: Prêmio CAPES Tese 2014
Menção Honrosa CAPES Tese– 20151st Prize – FertBio 20141st Prize – Humics Substances 20141st Prize – Simpósio CENA 2014
OBRIGADA!
Acacio Navarrete
Fabiana Cannavan
LucasMendes
http://www.brmicrobiome.org/#!organizing-committee/c1dt1
The Brazilian Microbiome Project
US NSF-FAPESP BIOTA Dimensions
FernandoAndreote
JorgeRodrigues
Luiz FernandoRoesch
VictorPylro
RodrigoMendes
PlinioCamargo