|Fabio "Rex" Rezzonico|
Forschungsanstalt ACW, Agroscope
of Research Interests
Current and Past Project Descriptions
The prokaryotic CRISPR/Cas defense system: function and practical applications in population genetics of Erwinia amylovora.
The CRISPR/Cas system confers acquired
heritable immunity against mobile nucleic acid elements in
prokaryotes, limiting phage infection and horizontal gene transfer of
plasmids. In CRISPR arrays characteristic repeats are interspersed with
similarly-sized non-repetitive spacers derived from transmissible
genetic elements and acquired when the cell is challenged with
foreign DNA. New spacers are added sequentially and the number and type
of CRISPR units can differ among strains, providing a record of
phage/plasmid exposure within a species and a valuable typing tool. The
aim of this work was to investigate CRISPR diversity in the highly
homogeneous species Erwinia amylovora,
the causal agent of fire blight. A total of 18 CRISPR genotypes were
defined within a collection of 37 cosmopolitan strains. Strains from
Spiraeoideae clustered in three major groups: both group II and III
were composed exclusively from bacteria originating from the United
States, whereas group I generally contained strains of more recent
dissemination in Europe, New Zealand and the Middle East. Strains from
Rosoideae and Indian hawthorn (Rhaphiolepis
clustered separately and displayed a higher intrinsic diversity
compared to isolates from Spiraeoideae. Reciprocal exclusion was
generally observed between plasmid content and cognate spacer
sequences supporting the role of the CRISPR/Cas system in protecting
against foreign DNA elements. However, in several group III strains
retention of plasmid pEU30 is inconsistent with a functional
Dr. Fabio Rezzonico, Dr.
Theo H. M. Smits, Dr. Brion Duffy.
Complete genome sequences of the fire blight pathogens Erwinia amylovora, Erwinia pyrifoliae and biocontrol strain Pantoea vagans C9-1
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.
SAM, doi: j.syapm.2013.04.00392 Phylogenetic position and virulence apparatus of the pear flower necrosis pathogen Erwinia piriflorinigrans CFBP 5888T as assessed by comparative genomics
JMM (2013), 92: 332-339 Erwinia amylovora loop-mediated isothermal amplification (LAMP) assay for rapid pathogen detection and on-site diagnosis of fire blight
MPP (2012), 13: 975-984 Lipopolysaccharide biosynthesis genes discriminate between Rubus- and Spiraeoideae-infective genotypes of Erwinia amylovora
J Bacteriology (2012), 194: 1615-1616 Complete genome sequence of clinical isolate Pantoea ananatis LMG 5342
Trees (2012), 26: 227-238 Genomics and current genetic understanding of Erwinia amylovora and the fire blight antagonist Pantoea vagans
PLoS ONE (2011), 6(7): e22247 Metabolic versatility and antibacterial metabolite biosynthesis are distinguishing genomic features of the fire blight antagonist Pantoea vagans C9-1
J Biotech (2010), 155:34-39 Evolutionary insights from Erwinia amylovora genomics
JB (2010) 192: 6486-6487 Genome sequence of the biocontrol agent Pantoea vagans strain C9-1
MPMI (2010) 23: 384-393 Complete
genome sequence of the fire blight pathogen Erwinia amylovora CFBP 1430 and comparison to other Erwinia spp.
FEMS Microbiol Lett (2010) 308: 48-54 Genomic and phenotypic characterization of a nonpigmented variant of Pantoea vagans biocontrol strain C9-1 lacking the 530-kb megaplasmid pPag3
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
Dr. Fabio Rezzonico, Dr. Virginia Stockwell, Dr. Brion Duffy.
Search for pathogenicity markers in clinical and biocontrol Pantoea agglomerans strains
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
(2012), 90: 315-320 Development of species-, strain- and antibiotic
biosynthesis-specific quantitative PCR assays for Pantoea agglomerans as tools for
(2012), 81: 137-139
Misidentification slanders Pantoea
agglomerans as a serial killer
TID (2012), 14: 220-221 Pantoea clinical isolates cannot be accurately assigned to species based on metabolic profiling
AEM (2010) 76: 4497-4509 Application of whole-cell Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for rapid identification and clustering analysis of Pantoea species
BMC Microbiology (2009) 9: 204 Genotypic comparison of Pantoea agglomerans plant and clinical strains
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.
Sensors (2012), 12: 6645-6665 Detection of AI-2 receptors in genomes of Enterobacteriaceae suggests a role of type-2 quorum sensing in closed ecosystems
BMC Microbiology (2008) 8: 154 Lack of genomic evidence of AI-2 receptors suggests a non-quorum sensing role for luxS in most bacteria.
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.
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.
Mol Breeding (2010), 28: 535-547 Genotyping of pedigreed apple breeding material with a genome-covering set of SSRs: trueness-to-type of cultivars and their parentages
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.
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.
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.
AEM (2004) 70: 5119-5131 Comparison of ATPase-encoding type III secretion system gene hrcN in biocontrol fluorescent pseudomonads and phytopathogenic proteobacteria.
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.
ACW Wädenswil, Switzerland: Gruppe
Istituto Agrario San Michele all'Adige, Italy: SafeCrop
Swiss Federal Institute of Technology Zürich (ETH), Switzerland: Plant Pathology Group