Harnessing the rhizosphere microbiome to enhance plant

productivity

CNPq (2013/456420-4) – NWO (729.004.013)December 2013 - January 2018

Adriana P. D. Silveiraa & Eiko E. Kuramaeb

aAgronomic Institute of Campinas, Dept Microbiology and Soil Quality, IACbNetherlands Institute of Ecology, Dept of Microbial Ecology, NIOO-KNAW, Wageningen

Vacheron et al. 2013

Soilstructure

• Phytohormones • Lytic enzymes • Secondary metabolites

• N fixation, • P solubilization • Siderophore production

Pes cidesFer lizers

Handling

Fungi

Symbio cbacteria

Na vemicrobiota

NamatodesPathogenicbacteria

Pathogenicfungi

Oomycetes

PGPM – Bacteria and fungiUse in sugarcane and why?

Introduction

Objectives

(i) determine the effect of different soil microbial community composition on sugarcane growth inoculated with PGPB

(ii) identify the PGPB traits and genes involved in plant growth promotion

(iii) identify the plant traits and genes involved in plant growth promotion induced by PGPB

• Comparative genomics of nine sugarcane growth- promoting bacterial strains. Dimitrov ,

PostDoc NWO

• Plant growth-promoting bacteria and strigolactones interactions. Gutierrez, MSc, NIOO

• Sugarcane - endophytic bacteria interactions: resistance induction to Colletotrichumfalcatum and changes in the endophytic bacterial community. Rossetto, MSc

• Beneficial microorganisms inoculation in sugarcane plantlets production in "MPB" system. Pierangeli, UG, CNPq/PIBIT

• Co-inoculation of endophytic bacteria and arbuscular mycorrhizal fungi for sugarcane plantlets in "MBP" system. Vicente, UG, FUNDAG/ CNPq-PIBIT

• Modulation of Al toxicity by endophytic bacteria in sugarc: plant response, genetic and functional diversity of rhizosferic and endophytic bacterial communitie. Blanca, PhD, CAPES

• Endophytic bacteria and sugarcane interaction under soil Altoxicity: liming effect. Mazzolani, CNPq/PIBIC

• Beneficial microorganisms associated to sugarcane under different tillage systems. Kisch,

UG, FUNDAG

• Evaluation of soil microbial attributes in tillage systems for sugarcane cultivation. Lanzi,

MSc

• Soil microbial attributes in sugarcane cultivated with straw application. Silva, UG FUNDAG

• PGPB-native microbiota interaction. Cipriano – PostDoc PNPD/CAPES

• Endophytic bacteria and sugarcane interactions: effects on plant physiology and bacterial communities of rhizosphere and endosphere. Freitas – PhD, CAPES

• Effects of bacterial inoculation on sugarcane growth, physiology and assembly of rhizoand endosphere prokaryotic community. Dimitrov - PostDoc NWO

Effects of bacterial inoculation on sugarcane growth, physiology and assembly of rhizo and endosphere

prokaryotic community

Mauricio R. Dimitrov1, Raquel de Paula Freitas2, Sara Adrian Andrade3,Adriana Parada Dias da Silveira2, Eiko E. Kuramae1

1Netherlands Institute of Ecology (NIOO-KNAW), the Netherlands2Instituto Agronômico (IAC -Campinas), Brazil3Universidade de Campinas (UNICAMP), Brazil

Questions

1. Unravel sugarcane (IACSP 95-5000) rhizosphere and root prokaryotic

microbiome.

High Throughput Sequencing of the V3-V4 regions of the 16S rRNA gene (MiSeq).

2. Do different PGPB genome strains influence sugarcane growth and

selecte physiological endpoints?

Shoot and Root dry mass, accumulation of macro and micronutrients, amino acid profile,

enzymatic activity (NR, NiR, GS) and qRT-PCR (GS1, GS2 and SHR5).

3. Do different PGPB genome strains influence assembly of prokaryotic

community in the sugarcane rhizosphere and endosphere?

High Throughput Sequencing of the V3-V4 regions of the 16S rRNA gene (MiSeq).

Experimental set up

F = Phosphorus, Potassium, Magnesiumand micronutrients (Cooper, Zinc, Boronand Molybdenum)

qRT-PCR + enzymatic activity + aa profile

IACSP 95-5000

108

CFU/mL

Sterile substrat

e3L of soil F

3L of soil F+N (350 mg of N/kg soil)

shoot

root

rhizosphere

Macro and Micronutrient

Prokaryotic community (16S rRNA)

108

CFU/mL

108

CFU/mL

Dry mass

Prokaryotic community (16S rRNA)

Dry mass

Time points:0 d30 d45 d

Iso

late

s

Au

xin

P

Solu

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on

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ero

ph

ore

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Cyt

oki

nin

Ch

itin

ase

Glu

can

ase

HC

N

Antagonism

Bip

ola

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i

Fusa

riu

mve

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illio

ides

Cer

ato

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is

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ado

xa

IAC/BECa-088 ND ND ND ND ND ND ND ND ND ND ND

IAC/BECa-090 - - + - - ND + - + + +

IAC/BECa-095 - - + - - ND - + + + +

IAC/BECa-135 - - - - - - - - - + +

IAC/BECa-141 - - - - - + - - + + +

IAC/BECa-152 - + + + - + - - + + +

IAC/BECa 99 + - + - - ND - - - + +

IAC/BECa 128 - - - - - + - - - + +

IAC/BECa 112 - - - - - + - - + + +

Name given to the Isolate Species description/complete genome sequence

IAC/BECa-088 Burkholderia caribensis

IAC/BECa-090 Pectobacterium sp.

IAC/BECa-095 Kosakonia oryzae

IAC/BECa-135 Burkholderia tropica

IAC/BECa-141 Pseudomonas fluorescens

IAC/BECa-152 Herbaspirillum huttiense

Bacterial Genomes Biochemicaly Characterized and Sequenced

ResultsShoot and root biomass

Effect of PGPB increase of shoot > root

ResultsNutrient accumulation

c a b a a b b

d

a

cb

cc c

c

a

b

a

ab b

c

a

b

b

bb

bc

a

b

b

a

ba

c

a

b

a

a

ba

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

Controle 88 90 95 135 141 152

g/kg

treatments

Macronutrients

Sulphur

Phosphorus

Nitrogen

Magnesium

Potassium

Calcium

- Inoculated plants accumulated more macronutrient than non-inoculated plants. - Potassium is involved in regulating the uptake of water, translocation of sugar, stomatal opening and closing, chlorophyll development.

Therefore is associated to photosynthesis.- Rubisco, an enzyme involved in carbon fixation, is very important for photosynthesis. An obligatory co-factor of this enzyme is

Magnesium, which also increased.

ca b a a b b

d

ac

b ac b

b

aa

a a

ba

c

a

ba

a

b

bc

a

b

aa

b

b

0,00

0,10

0,20

0,30

0,40

0,50

0,60

0,70

0,80

0,90

1,00

Controle 88 90 95 135 141 152

mg/

kg

treatments

Micronutrients

Zinc

Manganese

Iron

Copper

Boron

ResultsNutrient accumulation

- Micronutrient significantly higher in inoculated plants. Almost all micronutrients shown here are essential for photosynthesis, and if you remember from a previous slides, at D45, all inoculated plants had a significant increase in dry mass compared to non inoculated plants.

Amino acid profile

ResultSugarcane gene SRH5 expression 45 Days

SHR5 encodes a receptor-like kinase participate in signal transduction involved in the establishment of plant–endophytic bacteria interaction is described for the first time

Decrease of the expression of SHR5 gene in plants inoculated with some strains in absence of N fertilization, suggesting an efficient and beneficial association between the strains and sugarcane.

Capital letters compare means of different treatments with bacteria in the same fertilizer.Small letters compare means of the same bacteria treatment in different fertilizer condition

ResultsTaxonomic composition of the prokaryotic community

Rhizosphere

Endosphere

Results – prokaryotic community assembly

ResultsWhich specific bacterial isolatecaused a significant different?

D0 D30 and D45

Treatments

Rhizosphere All 88, 95, 135 141, 152

Endosphere 90, 141 and 152 88, 90, 95, 135, 152

Name given to the Isolate Species description

IAC/BECa-088 Burkholderia caribensis

IAC/BECa-090 Pectobacterium sp.

IAC/BECa-095 Kosakonia oryzae

IAC/BECa-135 Burkholderia tropica

IAC/BECa-141 Pseudomonas fluorescens

IAC/BECa-152 Herbaspirillum huttiense

Plant PGPB inoculation:

• Increase of plant growth

• Increase of macro and micronutrients

• Change in aminoacid profile

• Change the composition of rhyzosphere and endosphere microbial communities but not the diversity

• There is synergetic effect of PGPB+AMF in sugarcane

• Induce aclimation to aluminium stress by stimulating the antioxidantplant system

Conclusion

Next

There is a benefic effect of PGPB but we still need studies to fundamental understanding these interactions, in particular with soil microbiome.

The synergetic efect of PGPB + AMF

Omics (metagenome, metatranscriptome, metabolomics, metatranscriptomics) combined with environmetal factors (soil chemistry, GHGs, microbial processes, etc...) and bioinformatics approaches – tools . But we need to develop these tools.

Undergrade students: 5

MSc students: 3

PhD students: 2

Post Doctorate: 2

Publication:

Freitas-Ióris RP, Dimitrovi MR, Andrade SAL; Kuramae EE, Silveira APD. Endophytic bacteria promote sugarcane growth and alter mineral nutrition and foliar free amino acid and polyamines profile. J Plant Nut Soil Sci.

Freitas-Iório RP, Cipriano MAP, Marcos FCC, Sala VMR, Kuramae EE, Souza SAC, Cantarella H, SilveiraAPD. Endophytic bacteria traits and their ability to promote sugarcane growth and nitrogen nutrition. Ann Microbiol.

Cipriano MAP, Lupatini MLopes-Santos L, Silva M, Roesch L, Desteefano S, Freitas S, Kuramae EE. 2016. FEMS Microbiol Ecol.

Dimitrov MR, Silveira APD, Kuramae EE. Comparative genomics of nine sugarcane growth- promoting bacterial strains (in prep).

Dimitrovi MR, Freitas-Ióris RP, Andrade SAL, Silveira APD, Kuramae EE. Effects of bacterial inoculation on sugarcane growth, physiology and assembly of rhizo and endosphere prokaryotic community (in prep).

… many to write

NWO / CNPq output

Course and workshop:

1. Practical course: Soil nucleic acids isolation for Microbial Ecology studiesAgronomic Institute of Campinas, Brazil – Post Graduate Program10-13 December 2013, BrazilAdriana Silveira, Matheus Cipriano, Roos Keiser, Anna Kielak, Eiko Kuramae

2. Workshop: Sustainability and MicrobiomeNWO/Fapesp and NWO/CNPq21-23 November 2017, IAC, Campinas, BrazilHeitor Cantarella, Adriana Silveira and Eiko Kuramae

Prizes:Gabrielle PierangeliPIBITI – IAC Innovation Award – 2016Beneficial microorganisms inoculation in sugarcane plantlets production in "MPB" system

Scientific meetings:

FEMS Microbiology, 2015, Germany

BBEST- October, 2015, Brazil

ISME, 2016, Montreal, Canada

Soil Metagenomics, Dec 2016 Brauschweig

B

D

ThanksThe Silveira group

The Kuramae group

Heitor Cantarella (IAC)Mattias de Holander (NIOO-KNAW) Sara Andrade (UNICAMP )Marcio Leite (NIOO-KNAW) Janaina do Carmo (UFSCar)Raffaella Rossetto (IAC)Agata Pijl (NIOO-KNAW)Flávia Patrício (IB)Hans van Veen (NIOO-KNAW, Leiden Univ)Rosana Giertz (IAC)Harro Bouwemeester (UVA)Silvana Creste Souza (IAC)Juan Pérez Jaramillo (NIOO-KNAW) Mauro Xavier (IAC)Victor J. Carreon (NIOO-KNAW) ....