Current and Past Project Descriptions
Complete genome sequences of the fire blight pathogens Erwinia amylovora and Erwinia
pyrifoliae
Fire blight, caused by the enterobacteria of the genus Erwinia ,
is a devastating disease of rosaceous plants that has global economic importance for apple
and pear production and trade. The complete genomes of E. amylovora and E.
pyrifoliae were sequenced, annotated, and compared with the genomes of non-pathogenic E. tasmaniensis spp. Several singleton and shared features of the E. amylovora and E. pyrifoliae genomes were identified that
offer a first view into evolutionary aspects within the genus. Comparative genomics
identified or clarified virulence and fitness determinants and secretion systems. Novel
insights revealed in the genome of E. amylovora and E.
pyrifoliae hold potential for exploitation to improve the design of more effective
fire blight control strategies.
MPMI
(2010) 23: 384-393 Complete
genome sequence of thefire blight pathogen Erwinia
amylovora CFBP 1430 and comparison to other Erwinia spp. 
BMC Genomics (2010)
11:2 Complete genome sequence of the fire blight
pathogen Erwinia pyrifoliae DSM 12163T and
comparative genomic insights into plant pathogenicity
Investigators: Dr. Fabio Rezzonico, Dr. Theo H. M. Smits, Tim Kamber, Jochen Blom, Alexander
Goesmann, Sebastian Jaenicke, Dr. Jürg Frey, Dr. Brion Duffy
Analysis of
plant agricultural streptomycin formulations for the presence of antibiotic resistance
genes
Streptomycin is used
in plant agriculture for bacterial disease control, particularly against fire blight
inpome fruit orchards. Concerns that this may increase environmental antibiotic resistance
have led to bans or restrictions on use. Experience with antibiotic use in animal feeds
raises the possible influence of formulation delivered resistance genes. We demonstrate
that agricultural streptomycin formulations do not carry producer organism resistance
genes. By using an optimized extraction procedure, Streptomyces 16S rRNA genes and the
streptomycin resistance gene strA were not detected in agricultural streptomycin
formulations. This diminishes the likelihood for one potential factor in resistance
development due to streptomycin use.
AAC
(2009) 53: 3173-3177 Plant agricultural streptomycin formulations do
not carry antibiotic resistance genes
Investigators: Dr. Fabio Rezzonico, Dr. Virginia
Stockwell, Dr. Brion Duffy.
Search for
pathogenicity markers in clinical and biocontrol Pantoea agglomerans strains
Pantoea
agglomerans strains have recently been registered for agricultural application in the
United States and in New Zealand for biological control of fire blight. However,
registration in Europe is hindered because P. agglomerans is currently listed as a
class 2 pathogen due to reports of some isolates being opportunistic human pathogens.
Since many clinical isolates are clearly from plant origin, e.g. being associated with
thorn wounds, a method for the reliable discrimination of human pathogenic isolates from
biocontrol and/or harmless environmental isolates is needed. We performed a multilocus
phylogenetic analysis using the 16S-rRNA gene (rrs), housekeeping gyrB-gene
(encoding for gyrase B) and two genes relevant to the biocontrol performance of P.
agglomerans, pagRI and paaA encoding the acylhomoserine lactone receptor
and synthase and the pantocine-synthase respectively. Sequencing analysis revealed
incorrect taxonomic designation of a few strains. For genuine P. agglomerans
strains phylogenetic trees for all four loci were congruent with each other and no
separate clustering was found between biocontrol and human pathogenic strains. This
indicates that there was no distinct evolution of a group of P. agglomerans strains
towards human pathogenicity. It appears possible that some isolates became human pathogens
by acquiring pathogenicity factors on plasmids or on other mobile elements as in the case
of plant pathogenic P. agglomerans pathovars betae and gypsophilae.
We performed fAFLP on our P. agglomerans collection to identify genome-wide markers
that may be distinctive for human pathogenic strains, but none were found thusfar.
Conversely, a fAFLP band which may be characteristic for biocontrol strains was
identified, although subsequent PCR analysis showed that the corresponding gene fragment
was present also in human pathogenic strains. Supporting the phylogenetic data obtained
with gene sequencing, clustering of strains based on UPGMA analysis of the fAFLP pattern
showed no distinction between biocontrol and human pathogenic strains. If confirmed, the
results obtained so far suggests that multiple, strain-specific mechanisms may be
responsible for the different cases of human pathogenicity.
BMC Microbiology
(2009) 9:204 Genotypic comparison of Pantoea agglomerans plant
and clinical strains
Investigators: Dr. Fabio Rezzonico, Dr. Theo Smits, Dr.
Cosima Pelludat, Dr. Jürg Frei, Dr. Brion Duffy.
Characterization of the
role of luxS in the fire blight pathogen Erwinia amylovora
Fire blight,
caused by E. amylovora, is among the most serious diseases of pome fruits and
related Rosaceae. Biocontrol is being explored as an alternative to current control
options of prevention and sanitation measures and, in the U.S.A., to antibiotic
applications (mostly prohibited in Europe). Current biocontrol strains inhibit pathogen
growth through antibiosis or competition. We are seeking novel approaches for biocontrol
through reduction in virulence and environmental fitness by targeting gene regulatory
systems. We investigated the role of LuxS in E. amylovora as a putative
autoinducer signal (AI-2), metabolic compound in the activated methyl cycle (AMC), and
virulence factor on apple. AI-2 is described in animal pathogenic bacteria as crucial for
the transition to pathogenic existence inside the host, but its role in plant pathogens is
largely unknown. In some bacteria, LuxS has been linked to the disruption of the AMC with
involvement in growth and environmental fitness. Elucidating the role of LuxS in E.
amylovora is critical to determining how it can best be targeted for the design of
novel disease control strategies. Our data show that the production of virulence factors,
pathogenicity and the ecological competence of E. amylovora is in fact reduced in luxS
mutants. Lowered AI-2 detectable levels, absence of known receptors for AI-2 signals,
lowered competitiveness performance of mutants in minimal media and apple flowers, and
altered expression of methionine pathway genes lead us to hypothesize that the importance
of LuxS for E. amylovora lies more in its metabolic role in the AMC rather than in
autoinduction and quorum sensing.
MPMI (2007)
20: 1284-1297 The role of luxS in the fire blight pathogen Erwinia
amylovora is limited to metabolism and does not involve quorum sensing.
BMC Microbiology
(2008) 8:154 Lack of genomic evidence of AI-2 receptors suggests a
non-quorum sensing role for luxS in most bacteria.
Investigators: Dr. Fabio Rezzonico, Dr. Brion Duffy.
Project HiDRAS: High-quality
Disease Resistant Apples for a Sustainable Agriculture
The project HiDRAS aims at the
identification of the genetic factors controlling apple fruit quality with the objective
of increasing the acceptability of disease resistant apples and therefore their diffusion,
leading to a remarkable reduction in the use of fungicides.One of HiDRAS objectives is to
supply breeders with new and powerful tools, such as molecular markers linked to fruit
quality and pathogen resistance QTLs, to improve selection methods, implementing
innovative techniques and exploiting the advantages offered by the most recent
biotechnological tools. The proposed research will provide the scientific and
technological basis for the development of new or improved products, corresponding to
consumer requirements.
TGG (2009)
5: 211-223 (S1- S2) Development and test of 21 multiplex PCRs
composed of SSRs spanning most of the apple genome
Investigators: A. Patocchi, F.
Fernández-Fernández, K. Evans, D. Gobbin, F. Rezzonico, A. Boudichevskaia, F. Dunemann,
M. Stankiewicz-Kosyl, F. Mathis-Jeanneteau, C. E. Durel, L. Gianfranceschi, F. Costa, C.
Toller, V. Cova, D. Mott, M. Komjanc, E. Barbaro, L. Kodde, E. Rikkerink, C.
Gessler, W. E. van de Weg.
Site-directed
mutagenesis of Erwinia amylovora autoinducer biosynthetic gene for AHL
E.
amylovora causes fire blight, a devastating disease of apple and pear introduced from
North America that is currently spreading in Europe. Antibiotics are the most effective
controls used in the U.S., but they are not permitted in Europe due to concern about
resistance. Developing new strategies for control this disease is urgenly needed. One
attractive option is biological control using natural antagonistic microorganisms. We have
recently discovered that E. amylovora produces an acyl-homoserine lactone (AHL)
type of autoinducer signal protein, that coordinates the expression of critical virulence
genes in a cell-density sensitive manner (quorum-sensing). The importance of AHL in fire
blight is currently unknown, but interferring with quorum-sensing could be an effective
strategy to suppress plant disease. The aim of this project is to construct an AHL mutant
that can be evaluated for its ability to produce quorum-sending factors and to cause fire
blight.
JB (2005) 187: 3206-3213 Autoinduction
in Erwinia amylovora: evidence of an acyl-homoserine lactone signal in the fire
blight pathogen.
Investigators: Dr. Lazaro Molina, Dr. Fabio Rezzonico,
Prof. Dr. Geneviève Defago, Dr. Brion Duffy.
Importance of
2,4-diacetylphloroglucinol and hydrogen cyanide in plant protection by Pseudomonas
fluorescens
Many
biocontrol fluorescent pseudomonads protect plants from soil-borne diseases by the
production of antimicrobial secondary metabolites. The importance of
2,4-diacetylphloroglucinol (Phl) and hydrogen cyanide (HCN) in biocontrol was evidenced
for few strains with mutant analysis and with population studies of root-colonizing Pseudomonas
in disease-suppressive and conducive soils. However, many pseudomonads with biocontrol
ability can produce several biocontrol compounds simultaneously and comparison of
wild-types and mutants showed that inactivation of one biocontrol trait could modify the
expression of the others, making the assessment of the relative importance of each
compound not a straightforward task. Therefore a statistically-unbiased comparison of
wild-type biocontrol pseudomonads with or without the ability to produce HCN and/or Phl
would greatly improve the understanding of the role of these metabolites in plant
protection. Here we show an in planta comparison of 230 biocontrol pseudomonads
selected from a screening of 3132 bacterial isolates obtained from 63 soils collected
worldwide and demonstrate that HCN- and especially Phl-producers are indeed associated
with superior biocontrol in both the Pythium ultimum-cucumber and Fusarium
oxysporum-tomato pathosystems. Some of the bait plants used to isolate the bacteria
from the soil showed the ability to select for populations with higher fraction of Phl+
strains, which in turn resulted to better biocontrol performance. Interestingly, all 76
Phl+ isolates were genotypically HCN+ suggesting that Phl production
is restricted to HCN producers. Therefore Phl and HCN could be used in combination as
biocontrol markers for streamlining selection of new biocontrol isolates. PCR protocols
are available to identify phl in Pseudomonas. Since HCN is much simpler to
detect, the search for additional Phl+ strains may be preceeded by a rapid
phenotypic screen for HCN+ isolates.
NP (2007) 173: 861-872 Is
the ability of biocontrol fluorescent pseudomonads to produce the antifungal metabolite
2,4-diacetylphloroglucinol really synonymous with higher plant protection?
Investigators: Dr. Fabio Rezzonico, Marcello Zala, Dr.
Christoph Keel, Dr. Brion Duffy, Prof. Dr. Yvan Moënne-Loccoz, Prof. Dr. Geneviève
Défago.
Biocontrol
implications of type III secretion system in Pseudomonas fluorescens
The type III
protein secretion system (T3SS) is an essential element of pathogenicity in both animal
and plant pathogens, but can also occur in non-pathogenic bacteria such as biocontrol
pseudomonads. Phylogenetic analysis of the ATPase-encoding T3SS gene hrcN indicates
that T3SS is ancient in most biocontrol Pseudomonas strains but not in Pseudomonas
fluorescens KD, which seems to have acquired a T3SS more recently by horizontal gene
transfer. This hypothesis was strengthened by the comparison of the organisation and
sequence of hrc genes. Strain KD is not a phytopathogen being not able to elicit an
hypersensitive response on cucumber or tobacco, but can protect cucumber against
damping-off caused by Pythium ultimum. Inactivation of the T3SS was achieved by
inserting an W-cassette into hrcV (encoding a conserved
membrane-spanning protein of the T3SS). Levels of plant protection by this mutant against P.
ultimum on cucumber were lower than those of the wild type, indicating that a
functional T3SS contributes to the biocontrol activity of strain KD. Expression of hrcV
was assessed in strain KD using a plasmid-based transcriptional inaZ fusion with
the promoter controlling the corresponding hrc operon. In non-sterile soil, inaZ
activity of the pseudomonad was similar in bulk soil and in the cucumber rhizosphere, but
it was higher when P. ultimum was also added, especially in the rhizosphere. In
vitro, an increase in inaZ activity was observed when KD was in the presence of P.
ultimum but not with the wheat pathogen Gaeumannomyces graminis var. tritici,
against which KD shows no biocontrol activity. In addition, pectinase activity levels of P.
ultimum were reduced when KD was present. These results indicate that the expression
of T3SS genes is induced by the presence of the pathogen rather than the root, and they
suggest that the contribution of T3SS to the biocontrol activity of KD targets the
pathogen directly. Research is now being performed to determine the exact mechanisms of
plant protection by KD.
AEM (2004) 70: 5119-5131
Comparison of ATPase-encoding type III secretion system gene hrcN in biocontrol
fluorescent pseudomonads and phytopathogenic proteobacteria.
MPMI (2005) 18: 991-1001
The type III secretion system of biocontrol Pseudomonas fluorescens KD targets the
phytopathogenic Chromista Pythium ultimum and promotes cucumber protection.
Investigators: Fabio Rezzonico, Prof. Dr. Geneviève
Défago, Prof. Dr. Yvan Moënne-Loccoz.
Project reactivation: we
are investigating the distribution of T3SS in a collection of well described biocontrol
P. fluorescens strains in order to understand if the secretion machinery plays a role
in plant association or biocontrol.
Investigators: Dr. Davide Gobbin, Dr. Fabio Rezzonico.
Development of a QCPCR-based method to trace Pseudomonas fluorescens
CHA0 in soil
The
root-colonizing bacterium Pseudomonas fluorescens CHA0 can protect plants from
soil-borne fungal diseases. When introduced into the environment, CHA0 may under certain
conditions persist as mixed populations of culturable and non-culturable cells, which
limits the usefulness of colony counts. Quantitative competitive PCR (QC-PCR) is a method
based on the coamplification, with the same set of primers, of the DNA sequence to be
quantified and a known amount of a similar sequence of slightly different size (the
competitor). It allows quantification of the target, which in turn enables an estimation
of the number of corresponding cells. The objectives of this work were to determine if
QC-PCR can be applied to enumerate P.
fluorescens CHA0 cells in vitro and to assess the effect of stress on the performance
of the assay.
AEM (2003) 69: 686-690
Effect of stress on the performance of a phlA-based quantitative PCR assay to
monitor biocontrol Pseudomonas fluorescens CHA0.
Investigators: Fabio Rezzonico, Dr. Fabio Mascher, Prof.
Dr. Geneviève Défago.
Project reactivation: we
are now in process to refine the protocol with biocontrol strain Pseudomonas
fluorescens Pf153 in order to set up a method which is totally unaffected by
variability in cell-lysis and DNA-extraction efficiency.
SBB (2007) 39:
1609-1619 Quantification of the biocontrol agent Pseudomonas fluorescens
Pf153 in soil using a quantitative competitive PCR assay unaffected by variability in cell
lysis- and DNA-extraction eficiency.
Investigators: Dr. Davide Gobbin, Dr. Fabio Rezzonico.
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