Research in this field focuses on the development of techniques for the reliable identification and detection of plant-related organisms. Our tests enable the detection of viruses, bacteria, phytoplasms, fungi, nematodes and insects in soil, water, air, plant material, inocula, etc.

Because agrosystems are becoming less and less dependent on chemical control, there is an increasing demand for fast and reliable methods for detecting plant-related organisms. Such methods can be used for quality-monitoring of substrates (i.e. to ensure that compost, potting soil and water are free from plant (or even human!) pathogens and do contain plant-health-promoting organisms), product quality control (i.e. monitoring of crop pests or diseases that may cause problems during the post-harvest phase (for example mycotoxins)), compliance with export regulations (detection of Q-organisms), prevention (i.e. test for absence of plant-parasitic organisms in starting material, soil, recirculation water, etc.). We develop tests for these purposes. 

• Technology
• Developed detection methods
• Current projects

• ELISA (see example 1 and 2)
• immuno fluorescence (IF) and immuno fluorescence colony staining (IFC) (example)

DNA/RNA based techniques, such as:

• AFLP  (example)
• RAPD-PCR and SCAR-PCR (example)
• REP-PCR
• ISSR (example)
• SSR
• RFLP
• (RT-) PCR
• NASBA (example1 and 2)
• Molecular Beacon probes (example 1 and 2)
• AmpliDet RNA (example)
• TaqMan (example 1 and 2)
• DGGE (example 1 and 2)
• Micro-array based multiplex detection (example)
• pUMA (example)

More information: Carolien Zijlstra - carolien.zijlstra@wur.nl

• Multiplex detection(enabling the simultaneous specific detection of many different organisms in one test, i.e. using DNA/RNA microarrays or immunoarrays).
• Development of generic amplification methods for (groups of) pathogens.
• Quantitative detection (in combination with good sampling methods) providing data for risk analysis and decision support systems for several pathogens.
• Detection of human pathogens in food (raw vegetables) and feed.
• PortCheck  
• Ecogenomics
• Phage display (a way to produce antibodies to any kind of organism without the need of immunisation)
  

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Multiplex detection
Detecting the presence of harmful organisms in food, feed, water, soil, air or any other substrate is important for health and safety management. Many different molecular techniques for the detection of pathogens have been described, each with their own protocol, as well as equipment, chemical reagents and, above all, expertise. If different pathogens need to be detected simultaneously, this approach is costly. The multiplicity of assays available for a specific pathogen leads to a lack of consistency among the various testing agencies and hampers standardization.

The newest development in analysis of nucleic acids is the micro-array technology, in which thousands of different oligos can be spotted on little more than one square cm. Micro-array technology  provides the next generation of DNA diagnostics to measure different pathogens on a single chip. For each pathogen many different targets can be detected in parallel. This will improve specificity, allows detection of pathogenic variants of the target and avoid laborious confirmation procedures. Several micro-array systems are available.

For sensitive detection requirements, amplification of the target DNA of the pathogen to be detected is a prerequisite. PCR is a common method of creating copies of specific fragments of DNA. In a simplex PCR assay where one set of PCR primers is used, small samples of DNA can produce sufficient copies to be visualized on micro-arrays. In order to amplify multiple targets in one PCR assay, multiple primer sets can be used. However, the drawbacks of such multiplex PCR assays are that the sensitivity is decreased enormously and the number of different targets to be amplified in one assay is limited. Moreover, the dynamic range of the targets present in the sample to be tested is not always reflected in the outcome of the test: those targets that are present in very low amounts will most of the time not be amplified in contrast to those that are abundantly present.

To overcome such problems the pUMA technique was developed. pUMA stands for “padlock based Universal Multiplex detection Array”. DNA extracted from a sample to be investigated is mixed with specific padlock-probes that bind to the targets to be detected. These specific padlock-probes are subsequently ligated, amplified and detected on a micro-array. Amplification occurs using one generic PCR primer set. Several micro-array systems can be used. Results with our prototype pUMA that enables simultaneous detection of eight pathogens using this array system look very promising. Combination of micro-arrays with 96 wells plates will enable the analysis of 96 samples at the same time.

Application of the pUMA technique is universal and opens possibilities to monitor health hazard problems in such a way that in a single test numerous harmful organisms can be detected in a sensitive, reliable and fast manner.

Objectives
To develop a multiplex amplification and detection system using padlock probes and micro-arrays

Principle investigators
Peter Bonants, Carolien Zijlstra, Leo van Overbeek, Ronald van Doorn, Arjen Speksnijder and Cor Schoen

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Development of generic amplification methods for (groups of) pathogens.
Many PCR-based techniques have been developed for the detection of plant pathogens. These PCR tests are frequently based on information obtained via the specific sequences (ITS, 16S, mtDNA, etc.) or REP, BOX and ERIC fingerprint patterns (for bacteria), RAPD or AFLP analysis, species-, pathotype- or fysio-specific PCR primers have been developed; using such primers DNA fragments of a specific characterized amplified region (SCAR) can be obtained by PCR. Using gel electrophoresis, the length can be determined of the acquired fragment with which the organism in question is detected. As a result, the detection of multiple species in a single test (multiplex) is possible in some cases. SCAR primers have been developed for the detection of phytoplasm (Stolbur), fungi ((Phytophthora fragariae, P. cactorum, P. cryptogea, P. nicotianae, Pythium aphanidermatum, Rhizoctonia solani subgroups and Synchitrium endobioticum in soil, Olpidium brassicae, O. radicale in roots, water and soil, Alternaria alternata, A. raphani, A. dauci, Nectria galigena, Guignardia citricarpa, Phoma clematidina and Fusarium foetens in plant material), root-knot nematodes (M. chitwoodi, M. fallax, M. hapla, M. minor, M. naasi, M. incognita, M. arenaria, M. javanica), bacteria (Xantomonas fragariae, Xantomonas axonopodus pv dieffenbachiae, Clavibacter michiganensis subsp. sepedonicus) and insects (Trips palmi).

Principle investigators
Carolien Zijlstra, Peter Bonants, Ronald van Doorn, Michel Klerks, Arjen Speksnijder and Cor Schoen

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Quantitative detection
For the sensitive and specific detection of plant pathogens TaqMan PCR methods are being developed. TaqMan PCR is an amplification method in which amplification can be followed real time using a so-called TaqMan probe. In that way quantitative data can be obtained.

Pathogens
Phytophthora ramorum, Meloidogyne chitwoodi, M. fallax, Globodera pallida, G. rostochiensis, Guignardia citricarpa, Synchitrium endobioticum, Fusarium foetens, Phytophthora sp., Verticilium fungicola, Liriomyza sativae, L. huidobrensis, L. trifolii, L. bryoniae

Objectives
To develop specific and sensitive TaqMan PCR methods, including primers and probes for a great number of plant pathogens.

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PORTCHECK
Link Portcheck website :  http://www.portcheck.eu.com/

PORT CHECK is a combined RTD and demonstration activity aimed to deliver the tools and procedures to allow EU Member State Plant Health competent laboratories and inspection services to perform molecular diagnostic assays “on-site” and at points of entry. The project will develop and evaluate real-time PCR (TaqMan) assays for a number of key harmful organisms, including Phytophthora ramorum (sudden oak death) and pinewood nematode; and transfer these assays to field portable real-time PCR platforms which were originally developed for bio-warfare and bio-terrorism applications. Particular attention will be paid to the problems associated with sampling and nucleic acid extraction in field conditions. The proposal mobilizes an impressive consortium, including both academic and SME research partners, along with probably the most comprehensive and representative network of official Plant Health competent laboratory and inspection service organizations ever assembled for an EU RTD proposal. The consortium also includes partners with a proven track record of developing and successfully deploying on-site diagnostic kits to official Plant Health inspection services. Consultation with the principle stakeholders of the technology (inspection services, competent laboratories, DG SANCO, trade representatives etc.) at the outset and throughout the life of the project is given paramount importance. Adoption of the new technology should represent a step change in the way Plant Health services carry out inspections in support of Council Directive 2000/29/EC and will contribute to the reduced risk of the importation (and export) and establishment of harmful organisms. This in turn will help to prevent any consequential negative impacts on the sustainability of European agricultural systems, protect critically important natural forest ecosystems and other negative economic, social and environmental effects associated with the establishment of non-indigenous plant pests and diseases.

Principle investigators
Peter Bonants, Carolien Zijlstra and Cor Schoen

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Detection of human pathogens in food (raw vegetables) and feed
Problem description and objectives
In recent years organic farming has received increased attention. In organic farming no chemicals or pesticides are used, the soil is fertilized with organically obtained manure or compost, and the crops are not genetically modified. Organic manure is commonly used for fertilization. However, organic manure might contain human pathogens like E. coli O157:H7, Campylobacter and Salmonella spp., introducing a risk of contamination of crops. Contamination of the crops can occur due to rainfall (soil splashes on crops), but might also occur via the roots or naturally present openings in epidermal tissue. In this case the pathogens can spread throughout the plant endophytically, potentially resulting in very high concentrations of the pathogen in the plant. Since the pathogen is present in the plant, the pathogens cannot be removed by washing during processing. At this point the product is a potential threat for consumer health, especially in the case of freshly consumed vegetables.

To control and prevent contamination of the crops with human pathogens, more insight is needed in the sources of contamination, the possible routes of contamination, and identification of preventive factors controlling the extent of contamination. To study this, reliable detection methods are needed. Moreover, knowledge about the pathogen-plant interaction will generate insight in the risks associated with the presence of particular strains of human pathogens in particular cultivars of vegetables (lettuce) produced in a particular manner (organic or conventional).

The objectives of this research are to provide suitable qualitative and quantitative molecular-based methods for detection of E. coli O157:H7 and Salmonella spp. present in compost, soil, manure, plant material and water. Transmission (through time) of human pathogens from contaminated manure into plants is investigated, enabling risk analysis. In addition, human pathogen-plant interactions is studied at the genome level to elucidate the possible pathways of contamination, eventually leading towards tools to reduce risk of contamination.

Thus, we investigate the transmission of human pathogens from manure/compost to soil into plants, and develop molecular biology-based detection methods enabling the qualitative and quantitative detection of E. coli O157:H7 and Salmonella spp. during transmission from manure to compost, soil, and plants. To investigate potential molecular mechanisms that enable the pathogen to colonize the plant, pathogen-plant interactions, contamination-related gene-expression in plants, and gene-expression of the pathogen in contaminated plants during the contamination process are studied at the molecular level. Model gene-expression mechanisms are described on the basis of this research, providing insight in possible routes of preventing contamination of crops with human pathogens via manure, possibly leading towards approaches that lower human health threats.

History
In recent years the awareness of food poisoning by human pathogens in fresh vegetables has increased, as shown by a strong increase of reported incidents for E. coli O157:H7 and Salmonella spp. (CCD reports over 1995-1999). Most outbreaks related to consumption of fresh vegetables are reported for E. coli O157:H7 and Salmonella spp. Therefore, food quality and food safety are of major concern nowadays. Until now it is not clear what the actual risks are for introducing health threatening organisms like E. coli O157:H7 and Salmonella spp. in the production chain, and how they will affect the safety of the product for the consumer. One major critical point of infection of plants with these pathogens is by contact with manure, insufficiently heated compost, or contaminated water. For example, E. coli O157:H7 can enter lettuce seedlings internally through the roots. Lettuce plants grown in soil amended with contaminated irrigation water or manure slurry became internally contaminated in the edible part under conditions where external contamination of the edible part was prevented. An Indian study on naturally contaminated raw vegetables (seven types, eight replicates) revealed an incidence of 33% for Salmonella, 57% for Staphylococcus aureus, 56% for P. aerogunosa, 56% for Enterobacter, 32% for faecal E. coli. However, quantitative relationships between contamination levels in manure with those in soil and plant tissues have not been published.

Moreover, the pathogen-plant interaction is thought to play a crucial role in infection and colonisation of the plant. For example, roots of hydroponically grown tomato plants preferentially took up a certain serotype of Salmonella spp. After 9 days of exposure, all tested samples of hypocotyls plus cotyledons, stems and true leaves were positive for one serotype of Salmonella spp. at between log 3.6 and 4.0 CFU per g fresh plant material. Other serotypes were found at much lower concentration or were not detected. Artificial cutting of the roots did not clearly result in a higher uptake compared to non-damaged roots. These findings suggest the presence of an active and controlled interaction between pathogen and plant, based on presence or absence of certain genes/proteins within the plant and/or pathogen. Comparison of healthy plant gene-expression profiles with infected plant profiles, might show differences in expression patterns and identify the genes involved with the plant response of colonisation by the pathogen. Also, a study of genes expressed by the pathogen when infecting the plant, will contribute to insight in the infection mechanism used by the pathogen, and may lead to a prediction of the risk of plant invasion based on the genetic make-up of plant and pathogen strain. Thus, investigating the interaction of the pathogen with the plant might elucidate the mechanism behind infection and colonization, subsequently leading to a better understanding of plant infection by human pathogens. It will give rise to suggestions for preventing contaminations of freshly consumed vegetables, reducing the health threat for consumers.

Problem definition
Contamination of plants with human pathogens is gaining increased attention due to recent outbreaks related to consumption of fresh vegetables. Critical points for introduction of human pathogens in the production chain are mainly thought to be the use of manure or compost for fertilization and the use of contaminated water on crop fields. To understand the route of infection of crops, and identifying factors influencing the infection rate, the transmission of the pathogens from manure or compost to soil into the plants needs to be identified. Highly sensitive and specific quantitative detection methods are needed since these pathogens are surrounded by a great variety of microorganisms in the substrates of interest (manure, compost and soil). In addition, the interaction between pathogen and plant is thought to play a major role in infectivity and colonization of the plant. The basis of the interaction is believed to be embedded in the genome of both organisms, mainly the expression of so-called virulence-genes of the pathogens, and defence mechanisms of the plant. It is still uncertain whether the human pathogen behaves like a plant pathogen, parasite, or acts as a symbiont in the plant. Each lifestyle is dependent on presence or absence of genes being expressed or suppressed by either the pathogen or plant. Insight in the fundamental pathways and interactions between human pathogen and plant might eventually lead towards the prevention of human pathogens to be introduced in the production chain of freshly consumed vegetables.

Research objectives
Development of methods for extraction of nucleic acids from soil, manure, compost and plant, as well as methods for qualitative and quantitative detection of E. coli O157:H7 and Salmonella spp.
Studying the transmission of human pathogens from soil/ manure/ compost to plant
Studying pathogen/plant interaction at the molecular level
Studying contamination-related gene-expression in plants
Studying gene-expression of the pathogen in a contaminated plant during contamination process
Studying molecular mechanisms that enable the pathogen spread in plant

Principle investigators
Michel Klerks and Carolien Zijlstra

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Ecogenomics
Microbial communities in soil determine to a great extent plant health. Known functions related to micro-organisms that improve plant health are: antagonism against pathogens, regulators of plant resistance, plant stress reduction and nutrient cycling and acquisition. As about 99% of the bacteria in soil are nonculturable, the exact role of soil microbial communities in disease suppression is largely unknown. Present knowledge about sustainability of soils is only derived from the culturable microflora. Soil metagenomics, by means of cloning of large DNA fragments from the total soil community is an option to explore novel functions from the nonculturable soil microflora. These novel functions in combination with the existing knowledge from the culturable microflora may result in better knowledge and predictability of the disease suppressive status of various soils.

Objectives
Identification of the most important bacterial groups involved in disease suppression in bulk and rhizosphere soil, and inside plants growing in suppressive soils. Identification of novel traits in the nonculturable fractions of bulk and rhizosphere soils and endophyte communities, which are responsible for disease suppression and improvement of plant health. To establish agronomic measures responsible for shifts in antagonistic populations in bulk and rhizosphere soil, and inside plants.
To predict soil suppressiveness by screening for selected target genes responsible for disease suppression and improvement of plant health.

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Phage display
Recent advances in gene technology have greatly facilitated the genetic manipulation, production, identification and conjugation of recombinant antibody fragments.
The application of bioengineered antibodies can be widespread. Within the core business of Plant Research International, recombinant antisera can be produced for the detection of plant pathogens.

Objectives
Early serological detection and identification of most fungi is problematic due to cross-reactivity of conventional polyclonal sera whilst DNA based detection methods are laborious. Recombinant antibodies targeting single antigens are more specific. Species-specific single chain recombinant antibodies will be constructed from phage-display libraries and can than be used for rigid serological detection of fungi. This will also circumvent the need of animals for antibody production.