Biocontrol 1

APLICACIONES BIOTECNOLÓGICAS
DE LAS RIZOBACTERIAS
BIOCONTROL DE FITOPATÓGENOS
TEMA 6. AGENTES DE BIOCONTROL I

6.0. Introducción. Suelos supresores.
6.1. Características deseables en un buen agente de biocontrol
6.2. Mecanismos de biocontrol:
6.2.1. Antagonismo: antibiosis, sideróforos, enzimas líticos.
6.2.2. Interferencia de moléculas señalizadoras
6.2.3. Predación y parasitismo
6.3. Presentación de la Práctica de Laboratorio
TEMA 7. AGENTES DE BIOCONTROL II

7.1. Mecanismos de biocontrol (continuación)
7.1.1. Resistencia sistémica inducida (ISR)
7.1.2. Competencia por nutrientes y nichos (CNN)
7.1.3. Interferencia con otras actividades del patógeno (germinación, esporulación)
7.2. Cepas de biocontrol de interés comercial
7.2.1. Selección de cepas para Biocontrol
7.2.1. Productos para Biocontrol
7.2.2. Biocontrol en post-cosecha
7.3. Perspectivas de futuro (BioAg alliance)
II. BIOCONTROL DE FITOPATÓGENOS

6.3. Presentación de la Práctica de Laboratorio - II

7.3. Perspectivas de futuro.
Suppressive soils
Roeland L. Berendsen, Corné M.J. Pieterse, Peter A.H.M. Bakker
http://dx.doi.org/10.1016/j.tplants.2012.04.001
Suppressive soils
Ø Concept and current relevance
Ø Types of soil suppressiveness (SS):
• General: collective activity and general targets

not transferable
reduced by steaming
• Specific: Active against specific pathogens

one (or few) responsible microorganisms
transferable (0.1-10% dillution)
eliminated by pasteurization
Increased after repeated monoculture
Ø SS described against different diseases:

potato scab disease (Streptomyces scabies)
Fusarium wilt disease of several plant species,
Rhizoctonia damping-off of sugar beet
Take-all disease of wheat Gaeumannomyces graminis var. Tritici
Ralstonia solanacearum
Meloidogyne incognita (nematode)
(Suppressive) Strawberry fields (last) forever
Disease: Fusarium oxysporum
1st year
3rd year
2nd year
Cha et al., 2015

Biocontrol activity in suppressive
soils: an early hypothesis (1963)
confirmed at 80’s.
Take-all decline
(TDA)
Ø Induced specific suppression of Take all (Gaeumannomyces graminis)(wheat-barley

Ø Following monoculture after strong initail incidence
Ø TDA present in different soils (0.8 Mha in USA NW)
Ø Different rates of reactivation by re-installing crop
- Microbial basis of TAD biocontrol

- Fluorescent Pseudomonas strains producing DAPG
- High numbers (>105/g root)
- DAPG-minus mutants do not control TA
- Induction of DAPG synthesis by pathogen
- No significant resistance developed
- other targets for DAPG-

Some biocontrol examples
Biocontrol of Fusarium with Bacillus strains on plates

Biocontrol of Fusarium
oxysporum by Paenibacillus
alvei on wheat
Pythium ultimum
on sorghum
Biocontrol of Fusarium with Bacillus strains on sorghum

So, … apparently Biocontrol Agents (BCA) do exist!
Are they worthy to work with?
Growing market …
… with compromised chemical alternatives
Glare et al., 2012

The BioAg Alliance
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How to get advantage of BCA?
- Isolation and characterization
- Strain selection
- Use for disease control
- Improving characteristics
- Design treatment conditions
Efficient use of BCA requires good colonization ability (remind previous classes!!) …
…. and what else?

Traits for a good Biocontrol Agent
(i) rapidly utilize seed and root exudates

(ii) colonize and multiply in the rhizosphere and spermosphere environments
and in the interior of the plant
(iii) produce a wide spectrum of bioactive metabolites (i.e., antibiotics,
siderophores, volatiles, and growth-promoting substances)
(iv) compete aggressively with other microorganisms
(v) adapt to environmental stresses.
(vi) Production of resting spores (Bacillus)
+ some commercial traits:
• grow rapidly in vitro and to be mass produced

• Genetically stable
• Effective at low concentration
• Grow on inexpensive media M. Wisniewski webpage
• Long shelf life
• Easy to dispense
• Resistant and compatible to other agrochemicals and processing procedures
• Not detrimental to human health
Main Biocontrol agents
Process of development of microorganisms as useful BCA
Walsh et al., 2001

Some Biocontrol agents
Bacteria
Pseudomonas
Ps. fluorescens CHAO (Keel et al, 1996)
Ps. fluorescens Pf5 (Bender et al., 1999)
Ps. fluorescens F113
Ps. fluorescens WCS365
Ps. chlororaphis
Bacillus
B. cereus UW85 (Handelsmann & Stabb, 1996)
B. subtilis
Paenibacillus polimixa
Fungi
Trichoderma harzianum T-22

Biocontrol is in the middle of complex interactions
Discussion papers
Research in Microbiology 168 (2017) 583e593

www.elsevier.com/locate/resmic
Original Article
Characterization of biocontrol bacterial strains isolated from a

suppressiveness-induced soil after amendment with composted almond
shells
Carmen Vida a,b, Francisco M. Cazorla a,b, Antonio de Vicente a,b,*
a
Instituto de Hortofruticultura Subtropical y Mediterr!anea “La Mayora”-Universidad de M!alaga-Consejo Superior de Investigaciones Científicas
(IHSM-UMA-CSIC), 29071 M!alaga, Spain
b
Departamento de Microbiología, Facultad de Ciencias, Universidad de M!alaga, Bulevar Louis Pasteur, 31, 29071 M!alaga, Spain
Received 12 December 2016; accepted 24 March 2017
Available online 1 April 2017
Abstract
The improvement in soil quality of avocado crops through organic amendments with composted almond shells has a positive effect on crop
yield and plant health, and enhances soil suppressiveness against the phytopathogenic fungus Rosellinia necatrix. In previous studies, induced
soil suppressiveness against this pathogen was related to stimulation of Gammaproteobacteria, especially some members of Pseudomonas spp.
with biocontrol-related activities. In this work, we isolated bacteria from this suppressiveness-induced amended soil using a selective medium for
Pseudomonas-like microorganisms. We characterized the obtained bacterial collection to aid in identification, including metabolic profiles,
antagonistic responses, hybridization to biosynthetic genes of antifungal compounds, production of lytic exoenzymatic activities and plant
growth-promotion-related traits, and sequenced and compared amplified 16S rDNA genes from representative bacteria. The final selection of
representative strains mainly belonged to the genus Pseudomonas, but also included the genera Serratia and Stenotrophomonas. Their
biocontrol-related activities were assayed using the experimental avocado model, and results showed that all selected strains protected the
avocado roots against R. necatrix. This work confirmed the biocontrol activity of these Gammaproteobacteria-related members against R.


Mechanisms of Biocontrol: Antagonism
- Production of antimicrobial compounds but also

- Produce in the right microniche
- Compete with other microorganisms
- Escape from predators (protozoa)
Main antibiotics produced by biocontrol agents (Lugtemberg, 2009)
Gram-negative bacteria
HCN
Phenazines (phenazine-1-carboxylic acid, phenazine-1-carboxamine
2,4-DiacetylPhloroglucinol (DAPG)
Pyoluteorin
Pyrrolnitrin
ZwittermycinA
Kanosamine
Volatiles: 2,3 butanediol, sulfur dimetil compounds
Lipopeptide surfactants
Antibiotics in biological control
Relevant determinants of biocontrol

- effect of filtrates
- Analysis of antibiotic-deficient mutants
- enhancement of production à Biocontrol Improvement of biocontrol
- Heterologous expression (plasmid based constructs)
Detection of antibiotic production in situ
reporter genes
analytical techniques: TLC and HPLC (PCA, surfactin, DAPG)
Factors affecting antibiotic production

Internal regulation: GacA/GacS, qurum sensing, sigma factors
external factors:
abiotic: conditions (t, pH, soil mixture) and compounds (glucose,
biotic: Host plant exudates (kanosamine / alfalfa)
genetic determinants for compatibility with BCA
Diversity of antibiotic-producing strains

wide diversity of population of biocontrol agents
differentiation of groups by molecular techniques (RAPD)
selection of superior strains
Raaijmakers et al., 2002
Suppressive soils
Roeland L. Berendsen, Corné M.J. Pieterse, Peter A.H.M. Bakker

one or more severe disease outbreaks (Weller et al., 2007; Cook and Weller, 1987; Hornby, 1979;
Hornby, 1998).
Figure 1. Model of the take-all decline and the role of 2,4-DAPG-producing fluorescent Pseudomonas species.
During monoculture of wheat and after severe outbreaks of take-all, the disease severity declines over the course of
several years (white line). The decline in disease severity is associated with an increase of 2,4-DAPG-producing fluorescent
2,4-Diacetylpholoroglucinol (DAPG)
Pseudomonas antibiotic controlling phytopathogens
Ø Prototype of POLYKETIDE compounds

Ø Phloroglucinol and derivatives are widely used in different fields
> 700 derivatives known
Pharma: muscle relaxant
Explosives (trinitro-PG)
Ø Chemical synthesis established
Ø PG compounds are synthesized as 2ary metabolites
by different organisms through Poliketide synthases.
Ø in Bacteria, synthesized only by Pseudomonas
Ø PG produced in E. coli by heterologous expression
Ø Active against
phytopathogenic fungi, bacteria, anti-helminthic
phytotoxic
Ø Mode of action not fully understood:

Toxicity: eukaryotes (yeast): mitochondrial damage
Bacteria (G+ and G-): cell membrane
inhibits zoospore swimming
responsible of ISR on plants
Table 1. Examples of 2,4-DAPG-producing fluorescent Pseudomonas species with associated biocontrol activities and origin.
Strain Biocontrol activity Origin References
Strains from USA
Ps fluorescens strains 2-79 Wheat (Ggt) Wheat Weller and Cook (1983)
with DAPG-based
Q1-87, Q4-87, Q5-87,Q6-87, Wheat (Ggt) Wheat Keel et al. (1996))
Q13-87, Q37-87, Q86-87,
Biocontrol activity
Q7-87, Q8-87, Q9-87, Q12-87,
Q13-87, Q37-87, Q86-87
Q95-87, Q107-87, Q112-87,
Q139-87
Q2-87 Wheat (Ggt, Pu) Wheat Vincent et al. (1991)
Q65c-80, Q69c-80 , Wheat (Ggt) Wheat Harrison et al. (1993)
Q88-87, Q128-87
PF Wheat (St) Wheat Levy et al. (1992)
Pf-5 Cotton (Pu, Rs), Cotton Howell et al. (1979)
Cucumber (Pu)
Q8r1-96 Wheat (Ggt, Pu) Wheat Raaijmakers and Weller (1997)
Strains from Switzerland
TM1’A4, TM1’A5 Cucumber(Pu, Ps), Tomato Keel et al. (1996)
Cotton (Rs)
Pf1 Tobacco (Tb), cucumber (Pu) Tobacco Keel et al. (1996)
C6-2, C6-9, C6-11, C6-16 Tobacco (Tb) Tobacco Ramette et al. (2003)
C6-23,C10-181, C10-186,
C10-189, C10-190,C10-197,
C10-204, C10-205,
S7-29, S7-42, S7-46, S7-52,
S8-62, S8-110, S8-130, S8-151
CHA0 Tobacco (Tb), wheat (Ggt, Pu), Tobacco Stutz et al. (1986)
cucumber (Pu), Pea (Pu)
CM1ʹ′A2, CΔ1’B2, C*1A1 Cucumber (Pu, Ps), cotton (Rs) Cucumber Fuchs and Défago (1991)
K93.2, K93.3 Cucumber (Pu), tomato (FORL) Tobacco Wang et al. (2001)
P12 Tobacco (Tb), cucumber (Pu) Tobacco Keel et al. (1996)
P97.38, P97.39 Cucumber (Pu), tomato (FORL) Cucumber Wang et al. (2001)
TM1A3, TM1B2 Cucumber (Pu, Ps), cotton (Rs) Tomato Fuchs and Défago (1991)
Strains from Italy
PINR2, PINR3, PILH1 Cucumber (Pu), tomato (FORL) Tobacco Keel et al. (1996)
PITR2, PITR3 Cucumber (Pu), tomato (FORL) Wheat Keel et al. (1996)
K93.52 Cucumber (Pu), tomato (FORL) Tomato Wang et al. (2001
Strains from Ghana
PGNR2, PGNR3, PGNR4 Cucumber (Pu), tomato (FORL) Tobacco Keel et al. (1996)
PGNR1, PGNL1 Cucumber (Pu), tomato (FORL) Tobacco Keel et al. (1996)
Strains from Ireland
F113 Sugar beet (Pu), Potato (Pc) Sugar beet Fenton et al. (1992)
Strains from Slovakia
Troppens et al. 2013).
Biosynthesis of DAPG in fluorescent Pseudomonas
A.
B.
Figure 2. The phl gene cluster and 2,4-DAPG biosynthesis in 2,4-DAPG-producing fluorescent Pseudomonas species.
(a) The phl gene cluster consists of eight genes, presented with their proposed function. (b). The biochemical pathway for
2,4-DAPG synthesis. PhlD catalyzes the synthesis of phloroglucinol (PG) from three molecules of malonyl-CoA. The phlACB
Korterland, 2014
gene products together form a complex, which converts PG to monoacylphloroglucinol (MAPG) and MAPG to 2,4-DAPG.
Yang & Cao, 2012
2,4-DAPG may be degraded to MAPG by the C-C hydrolase Proposed intermediaries
PhlG. Adapted from Troppens et al., 2013.
Regulation of DAPG biosynthesis
Ø Complex regulation
Gac/Rsm postrancritional regulation cascade
PsoR, a LuxR solo regulator responsive to plant compounds
sigma factors rpoS and rpoD

other regulators: PsrA repressor
RNA chaperone Hfq
Ø Synthesis also autoregulated by DAPG (autoinducer)

Ø Biosynthesis controlled by some compounds (salycilate, pyoluorin, fusaric acid)
that antagonize autoregulation
Ø Carbon sources exudated from plants affect DAPG synthesis
Regulation of DAPG synthesis by PsoR in Pseudomonas fluorescens
Effect of host on DAPG-dependent biocontrol
PsoR mutants are impaired in biocontrol of Pythium in wheat but not in cucumber
PsoR enhances expression of phl genes in Ps fluorescens …

wheat
Wild type
psoR mutant cucumber
psoR-overexpressing
In free-living cells
…and in wheat but not in cucumber roots

Cyclic Lipopeptides:
major determinants of biocontrol in Bacillus strains
Cyclic peptides of non-ribosomal origin

Synthesized by NRPS (non-ribosomal peptide synthetases) in Bacillus and Pseudomonas
Five gene clusters for NRPS found in Bacillus amyloliquefaciens FZB42
Highly variable as regarding:
type and sequence of amino acid residues,
nature of the peptide cyclization
nature, length and branching of the fatty acid chain
Chowdhury et al. Biocontrol by root-associated Bacilli
LPs have multiple functions:

surfactants: spreading of cells
direct antagonist against F/B
Biofilm formation
Motility on surfaces (Twitching)
inducing ISR in plants
Three main families

Surfactins
Iturins
Fengycins FIGURE 2 | Effect of FZB42 on Rhizoctonia solani. A clear inhibition
zone indicating growth suppression of the fungal pathogen is visible on
agar plates simultaneously inoculated with both microbes. Bacillomycin D
Desorption/Ionization coupled to time of flight (MALDI TOF) mass
spectrometry of samples taken from the surface of the agar plate within
the inhibition zone (compiled from data obtained by J. Vater, TUB and K.
was detected as the only prominent compound by Matrix-Assisted Laser Dietel, ABiTEP GmbH).
basic set of domains within a module can be extended by substrate- the NRPSs responsible for the incorporat
modifying domains, including domains for substrate epimerization mycA (or ituA), the following four residues
Surfactin family of lipopeptides Heptapeptides with b-fatty acid (12-15C)

(E-domain), hydroxylation, methylation and heterocyclic ring forma-
tion, which are either inserted at specific locations into the module or
two last residues for mycC (or ituC).
structures of iturin A and mycosubtilin (in
Disolve in membranes,
act in trans as independent catalytic units. A thioesterase domain (Te- inhibited
are inverted) can be by cholesterols
explained by an int
eview domain) is usually present in the last module to ensure Active against
the cleavage B, V, not
mycC and against
Trends in
F (ituD) encodes
ituC. FenFMicrobiology Vol.16 No
a
of the thioester bond between the nascent peptide and the last PCP- (MCT-domain) and the mycA also contain
domain. In several cases, this thioesterase is responsible for the tide synthases. These genes are responsib
cyclization of the peptide. biosynthesis of the fatty acid chain (last el
Three large open reading frames (ORFs) coding for surfactin before its transfer to the first amino ac
synthetases are designated srfA-A, srfA-B and srfA-C [8]. They present [(acyl-CoA ligase (AL-domain)], acyl carrie
a linear array of seven modules (one module per residue). Three keto acyl synthetase (KS-domain), amino
modules are present in the products of srfA-A and srfA-B and the last [78].
Lipopeptides from Bacillus subtilis are synthesized by non ribosomal one in srfA-C. The fatty acid chain is added to the amino
Iturin family of lipopeptides

peptide synthetases (NRPSs) or hybrid polyketide synthases and non in the first module. A first thioesterase fused with the C
ribosomal peptide synthetases (PKSs/NRPSs) (Figure I). These of the last activation PCP domain is responsible for the
modular proteins are responsible for the biosynthesis of several synthesized product from the enzymatic template. A s
hundred bioactive compounds [74]. They are megaenzymes orga- terase/acyltransferase (Te/At-domain) encoded by a four
Heptapeptides with b-fatty acid (14-17C)
nized in iterative functional units called modules that catalyze the D stimulates the initiation of the biosynthesis [75
Pores in membranes,
different reactions leading to polyketide or peptide transformation. plipastatin or fengycin are synthesized by NRPSs en
Each module is subdivided into several catalytic domains responsible operon with five open reading frames ppsA–E (or fenA
for each biochemical reaction. A typical NRPS module usuallyActive against F, poorly against B
first three enzymes contain two modules, the fourth c
comprises !1000 amino acid residues and is responsible for one modules and the last enzyme consists of one module. Un
reaction cycle of selective substrate recognition and activation as an and fengycin, iturin derivatives are synthesized by
adenylate (A-domain), tethering of a covalent intermediate as an hybrid complex [3,6,77]. The operon consists of four
enzyme-bound thioester (Peptidyl-Carrier-Protein, or PCP-domain), frames called fenF, mycA, mycB and mycC or ituD, ituA,
and peptide bond formation (Condensation, or C-domain) [12]. The for mycosubtilin or iturin, respectively. The last three g
basic set of domains within a module can be extended by substrate- the NRPSs responsible for the incorporation of the fir
modifying domains, including domains for substrate epimerization mycA (or ituA), the following four residues for mycB (or
(E-domain), hydroxylation, methylation and heterocyclic ring forma- two last residues for mycC (or ituC). The differe
tion, which are either inserted at specific locations into the module or structures of iturin A and mycosubtilin (in which the las
act in trans as independent catalytic units. A thioesterase domain (Te- are inverted) can be explained by an intragenic doma
domain) is usually present in the last module to ensure the cleavage mycC and ituC. FenF (ituD) encodes a malonyl-CoA
of the thioester bond between the nascent peptide and the last PCP- (MCT-domain) and the mycA also contains genes relat
domain. In several cases, this thioesterase is responsible for the tide synthases. These genes are responsible for the las
cyclization of the peptide. biosynthesis of the fatty acid chain (last elongation and
Three large open reading frames (ORFs) coding for surfactin before its transfer to the first amino acid of the pe
synthetases are designated srfA-A, srfA-B and srfA-C [8]. They present [(acyl-CoA ligase (AL-domain)], acyl carrier protein (AC
a linear array of seven modules (one module per residue). Three keto acyl synthetase (KS-domain), amino transferase (A
modules are present in the products of srfA-A and srfA-B and the last [78].
Fengycin family of lipopeptides
Decapeptides with b-fatty acid (14-18C)

Interaction with membranes,
Active against F
gure 1. Structures of representative members and diversity within the three lipopeptide families synthesized by Bacillus species. Boxed structural groups are those tha
ere shown to be particularly involved in interaction with membranes and/or are supposed to be important for biological activity in addition to the cyclic nature of the
olecule [5,8,17]. To the best of our knowledge, no clear data are available to date for fengycins in this context. Boxed blue, type of branching (linear, iso, anteiso); boxed
ange, acyl chain length; boxed red, ionisable or polar groups; boxed green, hydrophobicity of residue in position 4; boxed yellow, L-Asx(1)–D-Tyr(2)–D-Asn(3) sequence
Iturin A and C, bacillomycin D, F, L and LC and 17 carbons. Though they are also strongly haemolytic, the
ycosubtilin were described as the seven main variants biological activity of iturins is different to surfactins: they
Antifungal activity of Pseudomonas lipopeptide viscosinamide
in Rhizoctonia solani
Viscosinamide - +
Micellium
Zoospore
FEMS Microbiology Reviews

Volume 34, Issue 6, pages 1037-1062, 23 MAR 2010 DOI: 10.1111/j.1574-6976.2010.00221.x
http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2010.00221.x/full#f2
Natural functions of lipopeptides from Bacillus and Pseudomonas:
Swarming
Pseudomonas W.T. LP-deficient
Bacillus Surf+ Surf- Surf-

W.T. Fen- Fen+ Fen-
Itu- Itu- Itu+

Effect of lipopeptides on biofilm formation
Pseudomonas
Surf+ Surf- Surf-

W.T. Fen- Fen+ Fen- Bacillus
Itu- Itu- Itu+ Microcolony formation on
Pseudomonas

Bacillus lipopeptide interactions in biological control of plant diseases
Ongena & Jacques, 2008

antibacterial compounds including bacillibactin is stimulated in
Lipopeptides Direct Antifungal Activity presence of plant pathogens under laboratory conditions (Li et al.,
Diversity in antagonistic2014). compounds:
Lipopeptides are non-ribosomally synthesized by peptide We determined expression of the corresponding secondary
synthetases (NRPS). NRPS are giant enzymes composed of metabolites by Matrix-Assisted Laser Desorption/Ionization
Synthesis of biocontrol metabolites in the genome of B. amyloliquefaciens FZB42
TABLE 1 | Genes and gene cluster encoding for biocontrol metabolites in Bacillus amyloliquefaciens plantarum FZB42.
Metabolite Genes and gene cluster Size Function Expression in situ Effect against
Sfp-dependent non-ribosomal synthesis of lipopeptides

Surfactin srfABCD 32.0 kb Biofilm, ISR Strong, during root colonization Virus
Bacillomycin D bmyCBAD 39.7 kb Direct suppression, ISR Weak, during root colonization Vungi
Fengycin fenABCDE 38.2 kb Direct suppression, ISR Weak, during root colonization Fungi
Bacillibactin dhbABCDEF 12.8 kb Siderophore During iron deficiency in soil Microbial competitors
Unknown nrsABCDEF 17.5 kb Unknown Unknown Unknown
Sfp-dependent non-ribosomal synthesis of polyketides
Macrolactin mlnABCDEFGHI 53.9 kb Direct suppression Not shown Bacteria
Bacillaene baeBCDE,acpK, baeGHIJLMNRS 74.3 kb Direct suppression Not shown Bacteria
Difficidin dfnAYXBCDEFGHIJKLM 71.1 kb Direct suppression Not shown Bacteria
Sfp-independent non-ribosomal synthesis
Bacilysin bacABCDE,ywfG 6.9 kb Direct suppression Not shown Bacteria, cyanobacteria
Ribosomal synthesis of processed and modified peptides (bacteriocins)
Plantazolicin pznFKGHIAJC DBEL 9.96 kb Direct suppression Unknown B. anthrax, nematodes
Amylocyclicin acnBACDEF 4.49 kb Direct suppression Unknown Closely related bacteria
Synthesis of volatiles
Acetoin/2,3-butandiol bdh,alsDRS 3.6 kb ISR During root colonization Plant pathogens
Frontiers in Microbiology | www.frontiersin.org 3 July 2015 | Volume 6 | Article 780

Antibiotics in Biocontrol Pseudomonas fluorescens
Diversity in antagonistic compounds:
Antibiotic compounds produced by fluorescent Pseudomonads

relevant for biocontrol
Volatile
Biocontrol through volatile compounds
Biocontrol through volatile compounds
Antifungal effect of co-

inoculation of Ps. fluorescens
strains with Botrytis cinerea
Agar-diffusible compounds Volatile compounds

Biocontrol of Botrytis by Pseudomonas on Medicago plants
Growth promoting effects of

volatile organic compounds (VOCs)
from P. Fluorescens
S-containing compounds identified:

dimethyl sulfide
dimethyl disulfide (DMDS),
dimethyl trisulfide
methanethiol
Diversity of volatile compounds
in Ps. fluorescens
Not all volatiles are healthy for plants
Plant growth inhibition and cyanogenesis by P. fluorescens
Control
CHA0, Hcn+
CHA77, Hcn-
HCN-deficient
Dirk Blom et al. Appl. Environ. Microbiol. 2011;77:1000-1008

Mechanisms of Biocontrol: Antagonism
- Production of antimicrobial compounds but also
- Produce in the right microniche

Ø In situ detection techniques
- Compete with other microorganisms

- Escape from predators (protozoa)


Siderophores and biocontrol
Fe: abundant on earth, but highly insoluble under oxic conditions (10-18 M)
Siderophores: Low MW (400-1500 Da) with high affinity (Kd <10-20 M)

quite diverse (>500 known, 270 structures resolved)
Require membrane receptors for uptake
Some chemical groups involved in Fe chelation

Types of siderophores
Complex siderophores from Pseudomonas
Pyoverdin
Detection of siderophore on plate (Blue agar-ChromeAzurol)
Halo produced by
Fe-scavenging due to
siderophore
Transport of siderophores
Chu et al., 2010

Transport of siderophores
Gram-negative Gram-positive
Siderophore-based competence R E V I E W S
a Species 2
Low-iron-affinity
Iron siderophore
producer
Species 1* Species 3
Takes up
Social cheater Iron Iron
heterologous
(mutant of species 1) Iron siderophores
Siderophore production
machinery
Siderophore
High-iron-affinity
siderophore producer Siderophore uptake
Iron machinery
Species 1
b c d
1 1 1
Species 1* Species 1
Species 3
Prevalence
Prevalence
Prevalence
0.5 0.5 0.5
Species 1
Species 1 Species 2
0 * 0 0
Time Time Time
Figure 3 | Simplified models of siderophore-mediated bacterial competition. a | Three competitive

Nature Reviews |scenarios in
Microbiology
Hibbing et al., 2010
iron-limiting conditions in which the use of a siderophore is necessary for iron acquisition. Species 1* represents a
Burkholderia siderophore participates in biocontrol of Colletotrichum
Siderophore-related
inhibition
Siderophore assay on CAS medium
Determination of MIC of siderophore
Production of siderophore by
Burkholderia
Effect of Burkholderia siderophore in biocontrol of Colletotrichum
Siderophore-related
inhibition
Sierophore assay on CAS medium

Discussion paper
Determination of MIC of siderophore
Production of siderophore by
Burkholderia
Role of lytic enzymes in biocontrol
Elad et al., 1983
Viterbo et al., 2002

Role of compounds secreted by antagonistic fungi relevant in biocontrol
Daguerre et al., 2014

one, harzianolide and harzianopyridone have antimicro- use of chitinases, glucanases, and proteas
syringae pv. tomato (Sultana et al., 2009; Kojima et al., 2013)
fects at high doses but are ISR inducers at low concen- sal. However, there is also evidence that s
and P. oligandrum against Ralstonia solanaceraum (Kawamura
ns (Table 5). et al., 2009). A parasitic
number of P. responses depend
oligandrum genes on the host,
encoding
elicitor-like proteins, whether functionally characterized or
triggering by other antagonistic fungi is well described. molecular mechanisms underlying the
identified using transcriptomic data are listed in Table 5
ver, the nature of the elicitor is still poorly known. The
(Picard areBenhamou
et al., 2000; even more et al., complex
2001; Mohamed than originally th
et al.,
Penicillium simplicissimum enhanced resistance of2007;
barley
MasunakaDistribution of fungal
et al., Therefore,
2010). Mycorrhizalmore lytic
fungi isolation
are also power-and chara
enzymes
mentsand other biocontrol
ful ISR elicitors (Cameron et al., 2013): colonization of Oryza
orbiculare by inducing active oxygen species formation, are needed to fully understand wh
sativa roots by Glomus intraradices promoted systemic induc-
cation, salicylic acid accumulation and activation ofofde-
tion related
cive proteins
defense-related andand confered
genes suppressive soils.
resistance to M. The
oryzae (Campos-Soriano et al., 2012).
genes (Shimizu et al., 2013). In addition, F. equiseti and microorganism and gene-by-gene stud
sp. elicited A. thaliana systemic resistance against P. fungi is useful because it inc
7. Conclusion
Soil disease suppression by antagonistic fungi has been

Fig. 4 e Distribution of the functionally characterized genes demonstrated on several occasions. Nevertheless, the best
according to the fungal genus. fungal biocontrol agents described up to now belong to the Tri-
choderma genus: 78 % of the functionally characterized genes
associated with antagonism were found in Trichoderma spe-
harzianum chitinase Chit42 in tobacco and potato plants cies, followed by Coniothyrium, Pythium and Clonostachys with
enhanced resistance to the foliar pathogens A. alternata, A. sol- 6, 5 and 4 %, respectively (Fig. 4). Antagonism depends on
ani, B. cinerea, and to the soil-borne pathogen R. solani (Lorito diverse genes involved in signaling, antibiosis, transport or
et al., 1998) (Table 5). T. longibrachiatum cellulases, T. viride xyla- mycoparasitism as discussed above. Forty-four percent and
nase Xyn2/Eix, T. harzianum endopolygalacturonase ThPG1 or 26 % of the genes identified are related to mycoparasitism
T. asperellum swollenin (expansin-like protein) TasSwo also and antibiosis, respectively (Fig. 5). ISR, signaling/genetic
elicited systemic resistance (Martinez et al., 2001; Rotblat reprogramming and competition represent only 12, 11 and
et al., 2002; Brotman et al., 2008; Mora ! n-Diez et al., 2009) 5 % of the genes, respectively. We now have an idea of what
(Table 5). Non-enzymatic proteins such as T. virens and T. atro- occurs in the presence of pathogenic fungi, from detection
viride cerato-platanins Sm1/Epl1 also trigger plant systemic to elimination, as briefly schematized in Figs 1 and 3. First
resistance (Table 5). Secondary metabolites like 20mer peptai- clues suggest that similar mechanisms are used by biocontrol
bol (alamethicin and trichokinin), 18mer peptaibol, 6-pentyl- agents to antagonize a pathogenic fungus. For example, the
a-pyrone, harzianolide and harzianopyridone have antimicro- use of chitinases, glucanases, and proteases is almost univer-
bial effects at high doses but are ISR inducers at low concen- sal. However, there is also evidence that signaling and myco-
trations (Table 5). parasitic responses depend on the host, suggesting that the
ISR triggering by other antagonistic fungi is well described. molecular mechanisms underlying the antagonistic Daguerre et al., 2014
effect
Tobacco + Th endochitinase / Rhizoctonia
Tobacco + Th endochitinase / Alternaria

- Large repertoire from genome sequencing of 7 Trichoderma sp
- Transgenic plants showed high level of expression of proteins
- Chitinases, glucanases, xylanases, cellobiohydrolases, HSP proteins
- Improved control of pathogens (+/- problems with plant vigor)

- Potential as plant biofactories for enzymes


Signal interference in Biocontrol
Mechanism based on the degradation of the cell-to-cell communication signals (AHL)
Coordination of pathogen behaviour (Quorum sensing) is critical for virulence
Interference with signalling (Quorum Quenching) might decrease efficiency of pathogen

Quorum sensing and gene regulation
for social behaviour in bacteria
Quorum quenching and Quorum

inhibition and gene regulation for
social behaviour in bacteria
Signal interference in Biocontrol
Mechanism based on the degradation of the cell-to-cell communication signals (AHL)
Coordination of pathogen behaviour is critical for virulence

Interference with signalling (Quorum Quenching) might decrease efficiency of pathogen
Enzymes: AHL lactonases

AHL acylases
Plant produce AHL mimics to interfere

Generation of transgenic plants expressing QS or QQ functions
Signals for Quorum sensing in bacteria
Biosynthesis of AHLs via LuxI family proteins
QS signal molecules
are chemically diverse
P. Williams, 2007
Quorum sensing is relevant for global regulation in phytopathogenic bacteria
Mole et al., 2007

QUORUM QUENCHING
Enzymatic degradation of AHL quorum signals as a tool for biocontrol
Quorum quenching contributes to biocontrol
Dong & Zhang, 2003
QS control of virulence genes

Biological control of pathogen communication
in the rhizosphere: A novel approach applied
to potato soft rot due to Pectobacterium atrosepticum
Alexandre Crépin & Corinne Barbey & Amélie Cirou & Mélanie Tannières &
Nicole Orange & Marc Feuilloley & Yves Dessaux & Jean-François Burini &
Denis Faure & Xavier Latour AHL analogs
7–37 31 32 Plant Soil (2012) 358:27–37
atho- Table 1 Structural traits and related function of main molecules studied in biocontrol approach of potato soft rot due to
he Pectobacterium atrosepticum
ad-
uring Name Role Structure Impact on the Impact on the Impact on the Impact on the
age, pathogen antagonist pathogen potato tuber
ti- (Abbreviation)
uce
us-
Received: 21 September 2011 / Accepted: 10 October 2011 / Published online: 9 November 2011 density density communication soft rot
s
-
# Springer Science+Business Media B.V. 2011 N-3-oxo-octanoyl-L-
homoserine lactone
Signaling
Neutral Neutral
Stimulation
Soft rot
on- molecule (quorum sensing) inducer
na (3-oxo-C8-HSL)
se
nals. gamma- Inhibition
teria
he Abstract caprolactone
(GCL)
Methods This emerging strategy
Biostimulant Neutral Stimulation
(quorum quenching)
Control
of
d
he Background and aims Recent basic knowledge on the 6-caprolactone
characterization of the signaling m
s and Biostimulant Neutral Stimulation Neutral No control
the regulation of virulence in pectinolytic bacteria (6CL) the target pathogen, then the
revealed pathogen communication via quorum sens- gamma- structural analogs to stimulate
Biostimulant Neutral Stimulation
Inhibition
Control
heptalactone
ing signalsenvironment
rhizosphere, NAHSL-degrading
as awithout crucial event for the expression of
causing adverse effects induced by
(GHL)
microflora able to degrade both mo (quorum quenching)
virulence theand
% of the total cultivable bacteria
otential resource for developing
the onset of disease symptoms. In this
release of pathogen signals.
To apply this principle, a collection of compounds,
Results The biocontrol method has
paper,Quenchingwewithpresent
Dong et al. 2001; Reimmann et al.
icard et al. 2005). andof NAHSL,
the conserved core discuss were usedadvances assays.
which show some structural or metabolic relations
newincrease of thethe
in Aasignificant first time
applied to cultures of S. tuberosum in microcosm
for the control o
substitution to a hydroxyl on some NAHSL (Uroz et
AHL analogs would enrich rhizosphere

ratio of NAHSL- al. 2005, 2008).
cterium, Bacillus,biocontrol
m the potato rhizosphere belong
approach based on twothe interference
individually as a sole carbon source in a synthetic
was observed in of atrosepticum.
degrading bacteria among total cultivable bacteria
several independent experiments. degradingThisbacteria psychrotrophic
GCL-mediated biostimulation of native NAHSL-
was also evaluated on an
Most of these on quenching, AHL-degrading bacteria
Pseudomonas, medium inoculated with soil samples. After
trum and Rhodococcus genera cycles of enrichment, the resulting bacterial consortia bacteria, the growth of which was industrial-scale plant hydroponic system (PHS),
Cirou et al. 2007).microbial
However, the communication
were compared for their capacity to involved
inactivate in thestimulated
cellular
by GCL amendment, were sizes
also able toN-acyl-homoserine
use
GCL as a sole carbon source. They mainly belong to
lactones
which is used for hydroponic production of the in
certified tubers in greenhouse production of potato
egradation activities appear to be NAHSL. All consortia obtained from lactone enrich-
these quenchingdensity
bacteria do not and
mentsmicroenvironment sensoring. the Rhodococcus erythropolis speciescell
exhibited a better NAHSL-degrading activity communication
suggesting that
that triggers so
plants (Cirou et al. 2011). In these systems, bacterial
10
Enrichment of BCA for Pectobacterium atrosepticum through addition of AHL analogs
32 Plant Soil (2012) 358:27–37
Table 1 Structural traits and related function of main molecules studied in biocontrol approach of potato soft rot due to
Pectobacterium atrosepticum
Name Role Structure Impact on the Impact on the Impact on the Impact on the
(Abbreviation) pathogen antagonist pathogen potato tuber

density density communication soft rot
N-3-oxo-octanoyl-L-
Signaling Stimulation
homoserine lactone Neutral Neutral Soft rot
(3-oxo-C8-HSL)
molecule (quorum sensing) inducer AHL analogs
gamma- Inhibition
caprolactone Biostimulant Neutral Stimulation Control
(GCL) (quorum quenching)
6-caprolactone
Biostimulant Neutral Stimulation Neutral No control
(6CL)
Rhizosphere enriched in quenching bacteria

gamma- - up to 70% of population after GCL
Inhibition
Biostimulant Neutral Stimulation Control
heptalactone enrichment
(quorum quenching)
(GHL)
- Rhodococcus erythropolis
- GCL degrader
- AHL degrader
applied to cultures of S. tuberosum in microcosm substitution to a hydroxyl on- some
Biocontrol of P. atrosepticum
NAHSL (Uroz et
assays. A significant increase of the ratio of NAHSL- al. 2005, 2008).
degrading bacteria among total cultivable bacteria GCL-mediated biostimulation of native NAHSL-
was observed in several independent experiments. degrading bacteria was also evaluated on an
Most of these bacteria, the growth of which was industrial-scale plant hydroponic system (PHS),
32 Plant Soil (2012) 358:27–37
Table 1 Structural traits and related function of main molecules studied in biocontrol approach of potato soft rot due to
Pectobacterium atrosepticum
Name Role Structure Impact on the Impact on the Impact on the Impact on the
(Abbreviation) pathogen antagonist pathogen potato tuber

density density communication soft rot
N-3-oxo-octanoyl-L-
Signaling Stimulation
homoserine lactone Neutral Neutral Soft rot
molecule (quorum sensing) inducer
(3-oxo-C8-HSL)
gamma- Inhibition
caprolactone Biostimulant Neutral Stimulation Control
(GCL) (quorum quenching) AHL analogs
6-caprolactone
Biostimulant Neutral Stimulation Neutral No control
(6CL)
gamma- Inhibition
heptalactone Biostimulant Neutral Stimulation Control
(GHL) (quorum quenching)
Rhizosphere enriched in quenching bacteria

- up to 70% of population after GCL
applied to cultures of S. tuberosum in microcosm
enrichment
substitution to a hydroxyl on some NAHSL (Uroz et
assays. A significant increase of the ratio of NAHSL- al. 2005, 2008). - Rhodococcus erythropolis
degrading bacteria among total cultivable bacteria - GCL degrader
GCL-mediated biostimulation of native NAHSL-
was observed in several independent experiments. degrading bacteria was also evaluated on an
- AHL degrader
Most of these bacteria, the growth of which was industrial-scale plant hydroponic system (PHS),
stimulated by GCL amendment, were also able to use which is used for hydroponic - Biocontrol
production ofof P. atrosepticum
the
GCL as a sole carbon source. They mainly belong to certified tubers in greenhouse production of potato
the Rhodococcus erythropolis species suggesting that plants (Cirou et al. 2011). In these systems, bacterial
GCL stimulates the growth of one of the most populations reach up to 1010 cfu/g fresh weight on the
effective NAHSL-degrading species (Cirou et al. surface of Solanum tuberosum roots and tubers (van
Expression of AHL synthases on transgenic plants
Detection of AHLs on leaves and roots of transgenic plants
Effect of plant-derived AHLs on E. carotovora virulence

P. Williams, 2007
AHL expression in plant lead to increased virulence !!



Mycoparasitism on
phytopathogenic fungi by
Trichoderma/Hypocrea
Druzhinina et al., 2011

Mycoparasitism of Hypocrea/Trichoderma spp.
Druzhinina et al., 2011

Bacterial - Fungus predation/parasitism
Collimonas / Fusarium Pseudomonas / Magnaporthe grisea

Three bacterial strategies to derive nutrition from fungi
Leveau & Preston 2008

Dialog between Collimonas and Aspergillus (F. Mena, 2011)
Biocontrol agents contain combinations of different protection mechanisms
(Pseudomonas fluorescens strains)
Hernández-León et al., 2015.

Biocontrol 1

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APLICACIONES BIOTECNOLÓGICAS

TEMA 6. AGENTES DE BIOCONTROL I

TEMA 7. AGENTES DE BIOCONTROL II

TEMA 6. AGENTES DE BIOCONTROL I

TEMA 7. AGENTES DE BIOCONTROL II

Roeland L. Berendsen, Corné M.J. Pieterse, Peter A.H.M. Bakker

Ø Types of soil suppressiveness (SS):

• General: collective activity and general targets

• Specific: Active against specific pathogens

Ø SS described against different diseases:

Cha et al., 2015

Ø Induced specific suppression of Take all (Gaeumannomyces graminis)(wheat-barley

- Microbial basis of TAD biocontrol

- other targets for DAPG-

Biocontrol of Fusarium with Bacillus strains on plates

Biocontrol of Fusarium with Bacillus strains on sorghum

Are they worthy to work with?

… with compromised chemical alternatives

Glare et al., 2012

The BioAg Alliance:

Novozymes & Monsanto JOINT ANNOUNCEMENT

Multiple components converging to Reducing agricultural inputs with

BREEDING BIOTECHNOLOGY BIOLOGICALS CHEMISTRY AGRONOMICS

- Isolation and characterization

- Use for disease control

- Design treatment conditions

…. and what else?

(i) rapidly utilize seed and root exudates

+ some commercial traits:

• grow rapidly in vitro and to be mass produced

Walsh et al., 2001

Trichoderma harzianum T-22

Research in Microbiology 168 (2017) 583e593

Characterization of biocontrol bacterial strains isolated from a

TEMA 6. AGENTES DE BIOCONTROL I

TEMA 7. AGENTES DE BIOCONTROL II

- Production of antimicrobial compounds but also

Main antibiotics produced by biocontrol agents (Lugtemberg, 2009)

Volatiles: 2,3 butanediol, sulfur dimetil compounds

Relevant determinants of biocontrol

Factors affecting antibiotic production

Diversity of antibiotic-producing strains

Roeland L. Berendsen, Corné M.J. Pieterse, Peter A.H.M. Bakker

Ø Prototype of POLYKETIDE compounds

Ø Mode of action not fully understood:

Biosynthesis of DAPG in fluorescent Pseudomonas

sigma factors rpoS and rpoD

Ø Synthesis also autoregulated by DAPG (autoinducer)

PsoR enhances expression of phl genes in Ps fluorescens …

…and in wheat but not in cucumber roots

Cyclic peptides of non-ribosomal origin

LPs have multiple functions:

Three main families

Surfactin family of lipopeptides Heptapeptides with b-fatty acid (12-15C)

Iturin family of lipopeptides

Decapeptides with b-fatty acid (14-18C)

FEMS Microbiology Reviews

Pseudomonas W.T. LP-deficient

Bacillus Surf+ Surf- Surf-

FEMS Microbiology Reviews

Surf+ Surf- Surf-

FEMS Microbiology Reviews

Ongena & Jacques, 2008