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1

Microbiología aplicada al sector agroproductivo:Bacterias promotoras del desarrollo vegetal (PGPRs)

Claudio ValverdePrograma Interacciones Biológicas

Departamento de Ciencia y Tecnología

Universidad Nacional de Quilmes

Outline

1. Introduction

2. Bacteria in the soil, rhizosphere, rhizoplane and endophytes

3. Plant growth promoting rhizobacteria – mecanisms

4. Nitrogen fixing plant-microbe symbioses (legumes & actinorhizae)

5. Biocontrol pseudomonads – mecanisms, diversity in soil & rhizosphere

2

Context & perspectives

Food for everybody.Sustain (or increase) yield.

Long term preservation of soil resources.Increasing cost of chemicals and pollution concerns.

Opportunity: to exploit natural biological processes and/or the organisms living in soils and plants.

Agrobiotechnology

3

Microbial-plant interactions in the rhizosphere

Beneficial bacteria

Mycorrhiza

Pathogenicfungi

Nematodes

Protozoans

Pathogenicbacteria

Commensalbacteria

Endophyticbacteria

Viruses

Archaea (?)

Outline

1. Introduction





4

Bacteria in the soil

How many bacteria there are?How diverse they are?How do they organize?Can we grow them in the laboratory?

~108-109 / grThe “great plate count anomaly”: 95–99% of themicrobial community present in the environment is not

readily accessible by traditional culture techniques (Nichols, 2007).

0

0.2

0.4

0.6

0.8

1

N LR HR N LR HR N LR HR N LR HR

Bengolea Monte Buey Pergamino Viale

Actinobacteria Alphaproteobacteria Acidobacteria Firmicutes Bacteroidetes Betaproteobacteria Verrucomicrobia GemmatimonadetesGerman grassland soil (Will et al 2010)

Argentinian agricultural soils under no tillmanagement (unpublished)

Accesible bacteria for bioproducts

May be functionallyimportant. Not(yet) accesible

5

The rhizosphere : the volume of soil overwhich plant roots exert an influence (Hiltner, 1904).

The rhizosphere effect

0

2

4

6

8

10

NA BP MP

log CFU/grSOIL RHIZOSPHERE

Pseudomonads counts in plots under no-tillmanagement in Argentina (Agaras et al;

unpublished work)

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Bacteria in the rhizosphere

Bacteria in the rhizosphere

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Bacteria in the rhizoplane

Barley + Pseudomonas

Rice + Pantoea

Sorghum + Burkholderia

Endophytic bacteria

8

Bacteria within seedsA B

C

Rice varieties cultivated in Argentina (Ruiz et al; unpublished work)

16S rDNA seq

Pantoea sp., Acinetobacter sp., Pseudomonas sp., Sphingomonas sp., Rhizobium sp.

Curtobacterium sp., Microbacterium sp., Staphylococcus sp., Paenibacillus sp.,

Bacteria within seeds

- Curvularia sp.

Huskedseeds

+ Curvularia sp.

Dehuskedseeds

Rice seed flora prevents Curvularia attack

(Ruiz et al; unpublished work)

9

Outline

1. Introduction





The PGPR concept

Plant growth promoting rhizobacteria (PGPR)are a group of free-living bacteria that colonize

the rhizosphere and contribute toincreased growth and yield of crop plants

(Kloepper and Schroth, 1978).

10

PGPR mechanisms ?

Direct : plant-growth-promoting rhizobacteria enhance plant growth in theabsence of pathogens.

• biofertilization (N2 fixation; P mineralization and solubilization)• rhizoremediation• phytostimulation (auxins, volatiles)• stress control (ethylene reduction)

Indirect : plant-growth-promoting rhizobacteria enhance plant growth in the presence of pathogens �� biocontrol.

• antagonism• signal interference• induced systemic resistance• competition for Fe+3

• competition for niche and nutrients

Examples of PGPR

Rhizobia and close relatives (legume symbioses)Frankia (actinorhizal symbioses)

Azospirillum spp.Pseudomonads

Bacillus spp.…

Microbial cooperation – consortia …

Unculturable? (management practices)

11

Outline

1. Introduction





N2 fixing symbioses

soybean

Discaria

N2

NH4+

Norg

Corg

CO2

12

Biological nitrogen fixation in the lab… …and, in the field

N2 fixing symbioses

• a distinctive feature of N-fixing symbiotic plant-microbe

interactions : symbiotic recognition � specificity

• over 150 genes from plant & bacteria are required for

development of functional legume root nodules.

• rhizobial nodulation genes in mobile elements (megaplasmids

or symbiotic islands) (Methylobacterium & Burkholderia).

• specificity is manifested at different levels of the symbiotic

interaction � “molecular dialogs”

N2 fixing symbioses

13

Long 1996 Cell 8:1885-1898

Gage 2004 Microbiol Mol Biol Rev 2:280–300

• rhizobial flavonoid receptor : NodD

• NodD-induced genes: nodABD (common) and other strain specific nod genes + nol

and noe genes

Nod factors

Early interactions in legume-rhizobia symbioses

Gage 2004 Microbiol Mol Biol Rev 2:280–300

• root hairs respond to Nod factors withcurling, bacteria trapped, infection threaddeveloped, nodule formation induced in cortex.

• bacterial surface polysaccharidesimportant for infection progression

Early interactions in legume-rhizobia symbioses

14

http://www.inoculantespalaversich.com/home.html

Frankia – actinorhizal symbioses

15

Frankia spp.

• Gram-positive bacteria, high G+C (∼∼∼∼68-72%) = actinomycete

• closest to Acidothermus cellulolyticum (rrs, recA, hopanoid

profile) than to Geodermatophilus spp.

• ubiquitous, free-living or root symbiont

• fixes atmospheric N2

• multicellular, filamentous bacteria

• 3 morphotypes: filaments, vesicles and sporangia (spores)

• non motile

Frankia spp.

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Biological nitrogen fixation

N2 + 10H+ + 16 ATP = 2 NH4+ + H2 + 16 ADP

• catalyzed by the enzyme nitrogenase (only present in

a reduced group of bacteria)

• energy demanding process

• but extremely sensitive to O2

The diffusion barrier of vesicles

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Frankia growth

• chemo-organotrophic

• aerobic to microaerophilic

• mesophilic

Valverde & Wall 1999 Can. J. Bot. 77:1302-1310

Frankia genetics

?

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• No genetic tools for the moment…

• plasmids, Tn916 and IS’s

• 3 genomes published (8.7-12 gbp), many more

to come soon

• Agrobacterium transforms Streptomyces…

Frankia genetics

Actinorhizal plants

• 8 families

• ∼∼∼∼250 spp of shrubs or trees (except Datisca spp.)

• prefer temperate regions (everywhere except in the

poles).

• pioneer plants, ecologically important

• N2 fixation

• diverse uses (timber, ornamental, fuel, forage, wind

breaking, etc.)

• representatives from 6 families in Argentina

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Actinorhizal plants

Casuarina cunninghamiana

Alnus acuminata

Discaria trinervis

Elaeagnus angustifolia

=

The actinorhizal symbiosis

N2

NH4+

Norg

Corg

CO2

+

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Specificity

(IV) MMyyrriiccaacceeaaee MMyyrriiccaa

CCoommppttoonniiaa

BBeettuullaacceeaaee AAllnnuuss

CCaassuuaarriinnaacceeaaee GGyymmnnoossttoommaa

CCaassuuaarriinnaa

AAllllooccaassuuaarriinnaa

CCeeuutthhoossttoommaa

IP

sV

EEllaaeeaaggnnaacceeaaee EEllaaeeaaggnnuuss

HHiippppoopphhaaee

SShheepphheerrddiiaa

RRhhaammnnaacceeaaee CCoolllleettiiaa

DDiissccaarriiaa

KKeennttrrootthhaammnnuuss

RReettaanniillllaa

TTeellgguueenneeaa

TTrreevvooaa

CCeeaannootthhuuss

RRoossaasseeaaee DDrryyaass

PPuurrsshhiiaa

CCoowwaanniiaannaa

CCeerrccooccaarrppuuss

CChhaammaaeebbaattiiaa

CCoorriiaarriiaacceeaaee CCoorriiaarriiaa

DDaattiissccaacceeaaee DDaattiissccaa

FFrraannkkiiaa

CCllaaddee II ((iissoollaatteess))

FFrraannkkiiaa

CCllaaddee IIII ((iissoollaatteess))

FFrraannkkiiaa

CCllaaddee IIIIII ((nnoo iissoollaatteess))

A

D

C

(I)

(II)

(III)

Frankia -like

RH

sV

IP

nsV

100 0

Fossil Record (Myr)

Actinorhizal Plants

Family Genus

Wall 2000 J Plant Growth Reg 19:167-182

Infection pathways

Franche et al 1998 Crit Rev Plant Sci 17:1-28

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The root nodule

Valverde & Wall 1999 New Phytol 141:345-354

Why must nodulation be controlled?

• formation of new organs

• C sinks

• parasitism by innefective Frankia strains

• more expensive than NO3-/NH4

+ assimilation

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Nodule formation in Dt

Growth system

• root infection [2-3 dai]

• nodule primordia [5-6 dai]

• nodule cell infection [7-9 dai]

• vesicle differentiation [10-12 dai]

• N2 fixation [14-15 dai]

• nodulation stops ∼∼∼∼6-8 wai

Valverde & Wall 1999 New Phytol 141:345-354

0 2 4 6 8 10 12

0

3

6

9

12

15

18

21

24

0

1

2

3

4

5Tap rootLateral rootsLeaf N

Nodule formation in Dt

Valverde et al 2000 Symbiosis 28:49-62

Weeks after inoculation

Numberofnodules

% (w/w)

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RT

-100

-50

0

50

100

150

Nodulation pattern


-100

-50

0

50

100

150

0481216

0481216

-100-80-60-40-20020406080100120

0481216

RT1

RT2

0 days

3 days

6 days


The phenomenon of autoregulation of nodulation

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Systemic nature of autoregulation of nodulation

0 5 10 15 20 25

5

10

15

20

25

Days after inoculationNumberofnodules


Nódulos por planta

0

4

8

12

16

RT1

RT2

C +Fr -nod -nod+Fr

Numberofnodules

Mature nodules locally inhibit nodule development


25

Is N2 fixation activity involved in nodule developmental arrest?

Wall et al 2003 J Exp Bot 54:1253-1258

Air (+N2)

Air Ar

Ar (-N2) Nodule biomass

0

1

2

3

4

5

6

7

ArAir ArAir

-NO3 +NO3

% ofplant DM

Wall et al 2003 J Exp Bot 54:1253-1258

Is N2 fixation activity involved in development arrest?

26

S1

S2

NiNi

Signals controlling nodule formation

S3

Plant hormones and nodulation

+IAA +BAPctrl

27

Frankia growth and nodulation


Outline

1. Introduction





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Biocontrol

Suppresive soils

In a wide sense, it refers to the use ofnatural enemies to reduce the damage causedby natural pests.

• Soils in which plants do not suffer certain diseases, orthe disease incident is reduced, although the pathogen ispresent and the host plant is susceptible.

• Supressiveness can be transferred to conducive soils, and can be eliminated by soil pasteurization orirradiation.

Haas & Défago 2005 Nature Reviews Microbiol. 3:307-319

Described supressive soils

Pathogen Reference

Heterodera spp.(nematode) Kerry 1988; Westphal & Becker 1999

Streptomyces scabies Menzies 1959

Fusarium oxysporum Stotzky & Martin 1963; Scher & Baker

1980

Gaeumannomyces graminis Cook & Rovira 1976

Phytophthora cinnamomi Broadbent & Baker 1974

Plasmodiophora brassicae Murakami et al.2000

Pythium spp. Hancock 1977

Rhizoctonia solani Henis et al. 1978, 1979

¿suppresive soils in Argentina?

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Microbial groups with biocontrol activities

Rhizoctonia solani, Fusarium

moliniforme

Trichoderma spp.

(fungus)

Xanthomonas albilineans, Botrytis

cinerea, Penicillium sp.

Pantoea spp.

Pythium sp., Sclerotinia sclerotiorum,

Botrytis sp. Rhizoctonia sp.

Burkholderia spp.

InsectsBacillus spp. /

Paenibacillus spp.

Fusarium oxysporum, Gaeumannomyces

gramini, Pythium sp., Meloidogyne

incognita (nemátodo), Thielaviopsis

basicola, …

Pseudomonas spp.

PhytopathogensAntagonistic species

El género Pseudomonas

• género heterogéneo de γγγγ-proteobacterias, alto %GC• ampliamente distribuido en la naturaleza• bacilos G(-), poco exigentes• fácil aislamiento y cultivo en el laboratorio• comprende especies promotoras de desarrollo vegetal (PGPRs); en general, no diazotróficas (excepto P. stutzeri).

• 17 genomas secuenciados disponibles(P. aeruginosa, P. putida, P. fluorescens, P. syringae, P. entomophila)

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Características de género (Bergey’s)

• bacilos rectos• 0.5-1.0 µµµµm x 1.5-5.0 µµµµm• flagelación polar • no esporulan (pero VBNC)• metabolismo oxidativo (oxidasa +) (O2, NO3

-)• utilizan una amplia variedad de fuentes de C• no requieren vitaminas• no crecen a pH < 5.0• pigmentos fluorescentes hidrosolubles

Taxonomía molecular (204 spp. en DMSZ)

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Aislamiento: medios y condiciones • medios minerales con diferentes fuentes de C• aerobiosis, 25-37 °C• King’s B / Pseud. ágar F o P / Castric / Cetrimide / Gould’s S1

King’s B (~P. ágar F)

Peptona cas/car 20 g/LMgSO4.7H2O 1.5 g/LK2HPO4.3H2O 1.5 g/LGlicerol 10 g/L

Castric

L-glutámico 5.9 g/LGlicina 0.76 g/LL-metionina 1.5 g/LNaH2PO4.H2O 0.9 g/LK2HPO4 0.87 g/L+ 10 ml/L 0.2M MgSO4.7H2O + 10 ml/L 2 mM FeCl3.6 H2O

P. ágar P

Peptona gelatina 20 g/LK2SO4 10 g/LMgCl2 1.4 g/LGlicerol 10 g/L

Cetrimide

Peptona gelatina 20 g/LK2SO4 10 g/LMgCl2 1.4 g/LGlicerol 10 g/LCTAB (cetrimide) 0.3 g/LÁc. nalidíxico 15 µµµµg/ml

Gould’s S1

Casaminoácidos 5 g/LNaHCO3 1 g/LMgSO4.7H2O 1 g/LK2HPO4 2.3 g/LSacarosa 10 g/LGlicerol 10 g/LSarcosil 1.2 g/LTrimetoprima 20 µµµµg/ml

No selectivo - indicador

Selectivo - indicador

Herramientas para el estudio

• Genómica comparativa (17 genomas secuenciados, >50 en proceso)• Electro-transformación• Conjugación• Transducción• Transposición (mutagénesis Tn5; attTn7)• Intercambio alélico (doble recombinación) ��mutantes sin rastros• Fusiones reporteras (lacZ, gfp, lux)• Vectores de expresión (pME6032)• Expresión de proteínas fluorescentes para microscopía• Oligos específicos para FISH

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Pseudomonas probióticas (Haas & Keel 2003)

Kumar et al., 2007Ensayo a campo(n.i.)Maíz(n.i.)P. corrugata

Han et al., 2006, Kang et al., 2006Chin-A-Woeng et al., 2001aMaddula et al., 2008Johnsson et al., 1998Carlier et al., 2008

---Ensayo a campoEnsayo a campo

RSI, antibiosis, AIAAntibiosis AntibiosisAntibiosisAntibiosis, solub. fosfatos

Pepino, tabacoTomateTrigoCebada, avenaTrigo

O6PCL139130-84MA342SR1

P. chlororaphis

Belimov et al 2007.Reducción de etileno

TomateAm3P. brassicacearum

Kumar et al., 2005-Antibiosis, AIA(antagonismo fúngico in vitro)

PUPa3P. aeruginosa

ReferenciasEnsayos a campo / Producto comercial

Actividad PGPR1Cultivo testeadoAislamientoEspecie

(después de >45 años de los estudios pioneros)

Pseudomonas probióticas (Haas & Keel 2003)

Koch et al., 2002Huang et al., 2004Van den Broek et al., 2003

---

Antibiosis Antibiosis Antibiosis

RemolachaSandíaTrigo

DSS73M18PCL1171

P. spp.

Cheng et al., 2007-Reducción de etileno

ColzaUW4P. putida

Haas & Keel, 2003Raaijmakers et al., 2002Moenne-Loccoz et al., 1998Raaijmakers & Weller, 2001De Leij et al., 1995; Naseby et al., 2001 www.rizobacter.com.arShaharoona et al., 2008de la Fuente et al., 2004

--Ensayo a campo-Ensayo a campoRizofos®Ensayo a campo-

Antibiosis, ¿RSI?AntibiosisAntibiosis Antibiosis, ¿competencia?Competencia Solubilización de fosfatos, Control de patógenosReducción de etilenoAntibiosis

Tomate, pepino, tabaco, trigo Algodón, trigo, pepinoRemolacha, arvejaTrigoArveja, trigo Trigo, MaízTrigoLotus, poroto, tomate

CHA0Pf-5F113Q8r1-96SBW25(n.i.)ACC50UP61

P. fluorescens

ReferenciasEnsayos a campoy/o producto comercial

Actividad PGPR1Cultivo testeadoAislamientoEspecie

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¿Cuáles son los mecanismos probióticos?

Linda Thomashow lab

Troxler et al 1997 Plant Pathol 46:62-71

Colonización radicular (biofilms / ¿carácter endofítico?)

¿Especificidad?

¿Cuáles son los mecanismos probióticos?Resistencia sistémica inducida

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¿Cuáles son los mecanismos probióticos?Fitohormonas / volátiles

¿Cuáles son los mecanismos probióticos?Disponibilidad de fosfato

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¿Cuáles son los mecanismos probióticos?Disponibilidad de fosfato

0.0

50.0

100.0

150.0

200.0

250.0

300.0

Ctrl 1 2 3

18.2 5 6

14.1 8

µµµµg/ml P

oprF PCR-RFLP

Pseudomonas fluorescens CHA0 : a biocontrol model microorganism

M.C. Álvarez Crespo & C. Valverde 2006

36

• isolated from a soil suppresive of the tobaco pathogenThielaviopsis basicola (Morens, Switzerland).

• protects several plants against diverse phytopathogens

• it has become a model strain to study mecanisms andregulation of biocontrol properties.

Pseudomonas fluorescens CHA0 : a biocontrol model microorganism

Biocontrol de fitopatógenos

Valverde et al 2003 Mol Microbiol 50:1361-1379l

Pepino – Pythium ultimumTrigo – Gaeumannomyces

gramini var. tritici

Flaishman et al 1990 Curr Microbiol 20:121-124

¿Cuáles son los mecanismos probióticos?

37

Factors that contribute to biocontrol activity of Pseudomonas fluorescens strain CHA0

• extracellular secondarymetabolites (antibiotics and HCN)

• Extracellular enzymes (protease AprA and lipase LipA)

• Induced systemic resistance (partly via DAPG)

• ¿unidentified factor X?

Biocontrol activity in strain CHA0 is regulated by

• Carbon source (plant host)

• Availabilty of micronutrients (e.g., Zn+2)

• Phosphate availability (high phosphorus inhibit ATB production)

• Temperature (optimal 12-26 °C)

• Signals from other organisms (fusaric acid, plant exudates)

• cell density (quorum sensing)

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Influencia de factores ambientales en la síntesis de antibióticos

Shanahan et al 1992 AEM 58:353-358

Fuente de C

Shanahan et al 1992 AEM 58:353-358

Temperatura

Duffy & Defago 1999 AEM 65:2429-2438

Fosfato

Duffy & Defago 1999 AEM 65:2429-2438

Micronutrientes

Maurhofer et al 2004 AEM 70:1990-1998

Inducción genes phl en cocultivo Pf in vitro

DAPG-

DAPG- / DAPG+

Inducción genes phl en cocultivo Pf in planta

Maurhofer et al 2004 AEM 70:1990-1998

Regulación de genes phl por ác. fusárico in vitro

Notz et al 2002 AEM 68:2229-2235

Regulación de genes phl por ác. fusárico in planta

Notz et al 2002 AEM 68:2229-2235

Influencia de factores bióticos en la síntesis de antibióticos

39

Efecto “rizosfera”

Expresión de genes phl Colonización

Notz et al 2001 Phytopathol 91:873-881

Influencia de factores bióticos en la síntesis de antibióticos

Regulación genética de la síntesis de antibióticos

ADN

ARNm

polipéptidos

enzimas

metabolito

40

Regulación transcripcional

RNAP

DAPG

PLT

PhlF

Ac. fusárico

Haas & Keel 2003 Annu Rev Phytoptahol 41:117-153

RNAP

ANR�O2

RNAPlux box

AHLs

PhzI

PhzA

Regulación global post-transcripcional

ADN operones “biocontrol”

ARNm

enzimas paraproductos

extracelulares

biocontrol

SISTEMAREGULADOR GLOBALGac / Rsm

41

Regulación global post-transcripcional :El sistema Gac/Rsm en P. fluorescens CHA0

Valverde et al, 2003, Mol Microbiol 50: 1361-1379Valverde, C. & Haas, D. 2008. “Small RNAs controlled by two-component systems”. In: “Bacterial Signal

Transduction: network and drug targets” (R. Utsumi, ed.). ISBN 978-0-387-78884-5. pp 54-79..

PrsmY-lacZ

mRNAs �� enzimas �� biocontrol

GacS / GacA TCS

Autoinductor

RsmA / RsmE

RsmZ / RsmY / RsmX

Regulación global post-transcripcional :El sistema Gac/Rsm en P. fluorescens CHA0

Heeb et al, 2002, J. Bacteriol. 184: 1046-1056.Valverde et al, 2003, Mol Microbiol 50: 1361-1379. Kay et al 2005. PNAS. 102:17136-17141

42

Diálogos con otras pseudomonas

PrsmY-lacZ

P. fluorescens P. fluorescens P. corrugata P. alcaligenes

Inducción de sistema Gac por exudados radiculares

PamsY-luxAB

+ sugar beet root exudate

43

Interacciones con otros microorganismos

Pseudomonas como mycorrhiza helper bacteria (MHBs)

wt

gacS

wt

gacS

Neobodo designis (flagellate)

Vahlkampfia sp. (amoeba)

Amoebae fed with GFP-tagged P. fluorescens

CHA0gacS

45

Pseudomonas fluorescens CHA0 kill nematodes

Romanowski et al., Microbial Pathogenesis, in press

CHA0

gacS

Pseudomonas fluorescens CHA0 kill nematodes

Romanowski et al., MicrobialPathogenesis, in pressCHA0 gacS

46

HCN is the main toxicity factor in fast paralytic killing

2. Volatile killing assay

C. elegans

on E. coli

Test strain

Test strain Fast paralytic killing

E. coli OP50 -wild type (CHA0) +gacS- -hcn- -

1. No fast killing in plates with open lid

Fast paralytic killing + -

CHA0 lawn + nematodes

Romanowski et al., Microbial Pathogenesis, in press

Biocontrol activity and predation escape are linked by secondary metabolites

C. elegans, protists P. fluorescens CHA0(secondary metabolites)

Phytopathogencontrol

Rhizospherecolonization

Predation

Defence

47

Aislamiento a partir de rizosfera

TSA 1/10 Gould’s S1

S1 (luz blanca) S1 (luz UV)

Diversidad de pseudomonas en suelo y rizosfera

¿Qué factores afectan la diversidad de poblaciones de pseudomonas?

• Ubicación geográfica (clima)

• Características edáficas (física y química del suelo)

• Prácticas de labranza

• Supresividad / conducividad

• Interacciones con hongos micorrícicos y fitopatógenos

• Especie vegetal (efecto rizosfera) / genotipos

• Estado fisiológico del cultivo

• Zona del sistema radicular / profundidad

48

Métodos para estudiar diversidad microbiana

Métodos para estudiar diversidad microbiana basados en ADN

• PCR+RFLP, PCR-SSCP, PCR+DGGE/TGGE, PCR T-RFLP, PCR ARDRA/RISA

• Análisis metagenómico

• FISH y FISH+MAR

• Re-asociación de ADN

49

Soybean / maize intercropping

Maize after wheat

No-till farming in Argentina

Wheat after maize

Soybean + maize after wheat

Wheat after soybean

Maize after wheat

Maize after Vicia

0

2

4

6

8

10

12

14

16

18

20

Millions of hectares

~75% of the present agricultural production(soybean 60%, wheat 15%, maize 15%)

Source: AAPRESID

No-till farming in Argentina (1977- 2006)

50

The benefits

• Water retention, organic matter retention, increased biological activity, better structure, less erosion.

• No till farming combined with crop rotations and rational fertilizer use, increases soil quality and sustain productivity.

The problems

• Market pressure and internal taxes push for soybean monoculture.

• Diseases, nutrient losses.

No-till farming in Argentina

Diversidad molecular de pseudomonas en lotes bajo siembra directa en Argentina

Puesta a punto de protocolos de PCR-RFLP para analizar diversidad total

Porina principal OprF

Alineamiento de secuencias

Diseño de oligonucleótidos

51


Puesta a punto de protocolos de PCR-RFLP para analizar diversidad total

Regulador transcripcional GacA

Alineamiento de secuencias

Diseño de oligonucleótidos

Pool de colonias

Lisado (DNA molde)

oprF PCR-RFLP


52

Pool de colonias

Lisado (DNA molde)

gacA PCR-RFLP


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