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