Jose van Beckhoven

• Manager at Plant Research International

The bacterium Xanthomonas fragariae is the causative agent of angular leaf spot (ALS) of strawberry, a Regulated Non-Quarantine Pest in Europe (EPPO A2) for plant propagation material. Field experiments were conducted to explore if X. fragariae is dispersed through the air during trimming ALS-diseased strawberry plants with dry or wetted leaves. Tr...

A potential cause of the dissemination of the potato ring rot bacterium Clavibacter sepedonicus (Cs) is the use of automated seed potato cutters. The present study focuses on the question of whether disinfection practices are sufficient to prevent the transmission of Cs from contaminated machine parts to a new tuber lot. The disinfection efficacy w...

The objective of the present study was to establish whether exposure to temperatures of 55-70 °C results in eradication of the pathogen Clavibacter sepedonicus (Cs) in colonised potato tissue, in order to evaluate the efficiency of composting for disinfection of Cs-infected potato waste. Pre-sprouted potato tubers were inoculated and planted to pro...

In two glasshouse experiments, colonization of potato (Solanum tuberosum L.) plants by the bacterial pathogens Dickeya solani and Pectobacterium parmentieri was studied after leaf infection. Leaves, whether or not artificially wounded, were spray-inoculated with various densities of green-fluorescent protein tagged strains of the pathogens, avoidin...

The spread of Xanthomonas fragariae in a strawberry field by mowing and runner cutting machinery was studied in the Netherlands during autumn. The blades of a rotary mower became heavily contaminated after trimming the leaves of symptomatic plants. Use of a contaminated mower resulted in infection of plants that were initially free of X. fragariae,...

Xanthomonas fragariae is the causative agent of angular leaf spot of strawberry, a quarantine organism in plant propagation material in the European Union. Field experiments were conducted to assess the risks for infection of strawberry plants through dispersal of an aerosolized inoculum. In practice, pathogen aerosols can be formed during mowing o...

The survival in drain water of two strains of Ralstonia solanacearum and three strains of Ralstonia pseudosolanacearum , including two strains able to cause wilt in roses, was determined. Water draining from drip‐irrigated rock wool mats on which roses were grown was supplemented with the pathogen and survival was monitored at 4, 12, 20 and 28°C fo...

p>Four commercially available disinfection products were tested for their efficacy against Clavibacter michiganensis subsp. sepedonicus (Cms), causative agent of bacterial ring rot, on wooden potato storage crates. Each of these products represented a different class of biocide, i.e. organic acids (benzoic acid), peroxygenous compounds (potassium p...

In Western-Europe, Pectobacterium carotovorum subsp. brasiliense is emerging as blackleg causing agent. In field experiments in the Netherlands, the virulence of this pathogen was compared with strains of other Dickeya and Pectobacterium strains. In 2013 and 2014, seed potato tubers were vacuum-infiltrated with high densities of bacteria (106 cfu m...

Risico tijdens CATT op overdracht van Xanthomonas zeer gering De belangrijkste conclusie uit het onderzoek naar de overdracht van Xanthomonas van plant naar plant tijdens CATT is dat dit risico uitermate klein is. Bij latent geïnfecteerd plantmateriaal werden geen aanwijzingen gevonden voor de verspreiding van Xanthomonas tijdens CATT. Dit suggeree...

The soft rot Enterobacteriaceae (SRE) Pectobacterium and Dickeya species (formerly classified as pectinolytic Erwinia spp.) cause important diseases on potato and other arable and horticultural crops. They may affect the growing potato plant causing blackleg and are responsible for tuber soft rot in storage thereby reducing yield and quality. Effic...

n 2011, two polytunnel greenhouse experiments were conducted on seed production farms, one under conventional conditions in the South-West (Rilland) and the other under organic conditions in the East (Voorst) of the Netherlands, to study transmission routes of Xanthomonas campestris pv. campestris (Xcc), that can result in seed infections of caulif...

Potato blackleg and stem rot are caused by various soft rot coliforms belonging to the genera Pectobacterium and Dickeya (van der Wolf & De Boer, 2007). In Europe, D. solani, D. dianthicola, P. atrosepticum and P. wasabiae are the main…

As an alternative to MeBr fumigation a 48h Controlled Atmosphere Temperature Treatment (CATT) was developed and scaled up by Wageningen UR in cooperation with the Dutch plant propagating association Plantum. This results in an excellent de-infestation and 99.8 % mortality of the strawberry tarsonemid mite (Phytonemus pallidus). This non-chemical an...

The objective of this study was to develop a multiplex detection and identification protocol for bacterial soft rot coliforms, namely Pectobacterium wasabiae (Pw), Pectobacterium atrosepticum (Pba) and Dickeya spp., responsible for potato blackleg and tuber soft rot. The procedures were derived from the phylogenetic relationships of these and other...

Treatments of tubers with chemical disinfectants (70 % ethanol, 1 % sodium hypochlorite, 2 % copper sulphate, 5 % peracetic acid, 10 % hydrogen peroxide, 1 % MennoClean (benzoic acid), 5 % trisodium phosphate and 0.2 % caffeine) were evaluated for control of blackleg caused by ‘D. solani’. All disinfectants effectively killed bacteria in axenic cul...

TaqMan assays were developed for the detection of seven Dickeya species, namely D. dianthicola, D. dadantii, D. paradisiaca, D. chrysanthemi, D. zeae, D. dieffenbachiae and D. solani. Sequences of the gene coding for dnaX were used for the design of primers and probes. In studies with axenic cultures of bacteria, the assays were highly specific and...

Vanuit de glastuinbouw zijn een groot aantal technieken bekend om dit water te ontsmetten zoals: langzame zandfiltratie, UV ontsmetting. verhitting, waterstofperoxide en ozon. In de boomteelt worden ook moerasfilters gebruikt. Elk systeem heeft zijn voor- en nadelen waardoor de keuze lastig is. De geschiktheid van de systemen voor de aardbeienteelt...

Het Deltaplan Erwinia (Deel C – Pootaardappelen) is een vervolg op het project 'Bacterievrije pootgoedteelt - een uitdaging!'. Het doel was om meer inzicht te krijgen in de Erwinia-problemen in de pootgoedteelt en uiteindelijk te komen tot een reductie van de economische schade met 50%. Uit het onderzoek (2009-2012) blijkt dat er bij vrijwel elk on...

During seed production, Brassica seed may become infected with Xanthomonas campestris pv. campestris after systemic colonization of plants upon leaf infection, or alternatively, after flower infection. Polytunnel experiments were conducted in 2007 and 2008 to study the relative importance of these colonization routes resulting in seed infection. Ca...

Van 2009 tot 2011 werden kasproeven uitgevoerd om de kolonisatie van tomatenplanten met Clavibacter michiganensis subsp. michiganensis (Cmm) vanuit besmet zaad en de secundaire verspreiding van Cmm in een gewas te bestuderen.

This paper briefly reviews research on the causative agents of blackleg and soft rot diseases of potato, namely Pectobacterium and Dickeya species, and the disease syndrome, including epidemiological and aetiological aspects. It critically evaluates control methods used in practice based on the avoidance of the contamination of plants, in particula...

Interactions between Serratia plymuthica A30 and a blackleg-causing biovar 3 Dickeya sp. were examined. In a potato slice assay, S. plymuthica A30 inhibited tissue maceration caused by Dickeya sp. IPO2222 when co-inoculated at a density at least 10 times greater than that of the pathogen. In glasshouse experiments, population dynamics of the antago...

Possibilities for biocontrol of biovar 3 Dickeya sp. in potato were investigated, using bacteria from rotting potato tissue isolated by dilution plating on nonselective agar media. In a plate assay, 649 isolates were screened for antibiosis against Dickeya sp. IPO2222 and for the production of siderophores. Forty-one isolates (6·4%) produced antibi...

Dickeya species (formerly Erwinia chrysanthemi) cause diseases on numerous crop and ornamental plants world-wide. Dickeya spp. (probably D. dianthicola) were first reported on potato in the Netherlands in the 1970s and have since been detected in many other European countries. However, since 2004–5 a new pathogen, with the proposed name ‘D. solani’...

Mycosphaerella brassicicola (ringspot), Alternaria brassicicola and A. brassicae (dark leaf spot) and Xanthomonas campestris pv. campestris (black spot) can infect leaves of Brussels sprouts resulting in yield losses. Infections of outer leaves of sprouts cause severe losses in quality. Crop residues can be a major primary inoculum source of the pa...

Prime Diagnostics uit Wageningen heeft een methode ontwikkeld waarmee kwekers eenvoudig kunnen bepalen of Prunus is aangetast door de bacterie Xanthomonas arboricola pv pruni. Voor een officiële uitslag is nog steeds wel een test uitgevoerd door de nVWA of Naktuinbouw nodig.

Sixty-five potato strains of the soft rot-causing plant pathogenic bacterium Dickeya spp., and two strains from hyacinth, were characterised using biochemical assays, REP-PCR genomic finger printing, 16S rDNA and dnaX sequence analysis. These methods were compared with nineteen strains representing six Dickeya species which included the type strain...

Dickeya zeae is a pectinolytic bacterium responsible for soft rot disease in flower bulb crops. In this study, the possibility of controlling soft rot disease in hyacinth by using antagonistic bacteria isolated from hyacinth bulbs was explored. Bacterial isolates with potential for biocontrol were selected on the basis of antibiosis against D. zeae...

For detection of the plant pathogenic Tulip virus X (TuVX), a panel of six recombinant antibodies was identified. To this end, a repertoire of variable domains from heavy-chain immunoglobulins (VHH) was cloned from an alpaca, which had been immunized with TuVX. Binding domains were selected by phage display and panning on immobilized TuVX particles...

To monitor seed potatoes for potato virus X, Y and PLRV, a multiplex microsphere immunoassay (MIA) was developed based on the Luminex xMAP technology, as an alternative to ELISA. The xMAP technology allowed detection of a number of antigens simultaneously whereas ELISA only allowed simplex detection of antigens. The use of paramagnetic beads in the...

Various essential oils, organic acids, Biosept, (grapefruit extract), Tillecur and extracts of stinging nettle and golden rod were tested for their antimicrobial properties in order to disinfect vegetable seed. In in vitro assays, thyme oil, oregano oil, cinnamon oil, clove oil and Biosept had the highest activity against the seed borne pathogens X...

Suspected Dickeya sp. strains were obtained from potato plants and tubers collected from commercial plots. The disease was observed on crops of various cultivars grown from seed tubers imported from the Netherlands during the spring seasons of 2004–2006, with disease incidence of 2–30% (10% in average). In addition to typical wilting symptoms on th...

To monitor seed potatoes for potato virus X, Y and PLRV, a multiplex microsphere immunoassay (MIA) was developed based on the Luminex xMAP technology, as an alternative to ELISA. The xMAP technology allowed detection of a number of antigens simultaneously whereas ELISA only allowed simplex detection of antigens. The use of paramagnetic beads in the...

In Nederland veroorzaken Erwiniabacteriën veel schade in bloembollen, aardappel, ui, witlof en bloemisterijgewassen. Er heerst nog steeds veel onduidelijkheid over de vraag waar deze Erwinia's vandaan komen. Ook is onduidelijk hoe lang deze kunnen overleven in grond, water of op materialen die in de teelt en verwerking gebruikt worden. Plant Resear...

Quorum sensing plays a role in the regulation of soft rot diseases caused by the plant pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. The signal molecules involved in quorum sensing in P. carotovorum subsp. carotovorum belong to the group of N-acyl homoserine lactones (AHLs). In our study, we screened bacteria isolated from the...

Diagnostiek van plant pathogenen met behulp van serologische diagnostica is in Nederland al vele tientallen jaren een standaard technologie. Het is een van de pijlers onder ons intensieve en hoogstaande keurings- en kwaliteitssysteem en heeft als zodanig duidelijk bijgedragen heeft aan de vooraanstaande positie die Nederland op agrarisch gebied inn...

Rotveroorzakende bacteriën, met name pectinolytische Erwinia spp., zijn verantwoordelijk voor veel schade in de diverse gewassen, vooral in de teelt van aardappelen en de bloembolgewassen hyacint, zantedeschia en iris. In deze literatuurstudie worden de beschikbare gegevens nog eens nader bekeken vanuit de vraag of een praktische en kosteneffectiev...

Crops and weeds were tested for their ability to host Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot in potato. Ten crops grown in rotation with potato in Europe, namely maize, wheat, barley, oat, bush bean, broad bean, rape, pea and onion and five cultivars of sugar beet were tested by stem and root inocul...

An overview is given of rapid methods for the detection of plant-related organ-isms in plant material, soil, compost, water, etc. Protein-based detection assays such as isozyme analysis, ELISA, immunofluorescence colony-staining and future applications of immunological tests are described as well as nucleic acid-based tests. Examples of RNA amplifi...

The potential of AmpliDet RNA for specific detection of Ralstonia solanacearum in potato tuber samples and surface water was demonstrated. AmpliDet RNA is a procedure based on nucleic acid sequence based amplification (NASBA) of RNA sequences and homogeneous real time detection of NASBA amplicons with a molecular beacon. The procedure is carried ou...

The survival of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, was studied in water, to assess the risks for dissemination of Cms via surface water and infection of potato crops by irrigation. Cms was able to survive for a maximum period of 7 days in non-sterile surface water at 10°C, a peri...

Studie naar de epidemiologie van de bacterieziekten bruinrot (Ralstonia solacearum) en ringrot (Clavibacter michiganensis subsp. sepedonicus). De nieuwe methode is gebaseerd op amplificatie van 16S rRNA sequenties met behulp van NASBA (nucleic acid sequence based amplification). NASBA is een methode waarmee nucleïnezuren, maar in het bijzonder RNA-...

The addition of an internal control (IC) at the appropriate concentration enables recognize of false negatives in the detection of Agrobacterium tumefaciens and improves the reliability of PCR for crown gall diagnosis. Co-amplification of the IC and target sequence from A. tumefaciens ensures the attainment of at least one amplification product in...

To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNA, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon. Five methods were evaluat...

The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt. The presence of intact 16S rRNA is considered to be a useful indicator for viability, as a rapid degradation of thi...

ABSTRACT After outbreaks of potato brown rot in three different fields in the Netherlands, the fate of the brown rot pathogen, Ralstonia solanacearum biovar 2, was monitored in soil by immunofluorescence colony staining (IFC) supported by R. solanacearum division-2 specific polymerase chain reaction. In selected areas of all fields, the R. solanace...

In the last decade, nucleic acid sequence based amplification methods have been developed for many important plant pathogenic bacteria. Although they possess excellent properties with respect to sensitivity and specificity, the introduction of these techniques for routine detection has been hampered by in particular the lack of robustness. In diffe...

A procedure was developed for specific and sensitive quantitative detection of Ralstonia (Pseudomonas) solanacearum biovar 2 (race 3) in soil. It is based on immunofluorescence colony-staining (IFC) followed by confirmation of the identity of fluorescent colonies by PCR-amplification or dilution plating on a semi-selective medium, SMSA. Addition of...

A number of phenotypic and molecular fingerprinting techniques, including physiological profiling (Biolog), restriction fragment length polymorphism (RFLP), enterobacterial repetitive intergenic consensus (ERIC) and a phage typing system, were evaluated for their ability to differentiate between 60 strains of Erwinia carotovora ssp. atroseptica (Ec...

An expression system (pSKGFP), which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent proteins, was designed. This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selected recombinant antibodies by flow cytometry or fl...

The vector pSKAP/S was constructed to enable overexpression of single-chain variable fragment antibody (scFv)-alkaline phosphatase fusion proteins. In pSKAP/S, the scFv were genetically fused to the mutated Escherichia coli PhoA/S gene that encodes an alkaline phosphatase with increased specific activity. The restriction sites incorporated into pSK...

ABSTRACT Recombinant single-chain antibodies (scFvs) against the lipopolysaccharide of Ralstonia solanacearum (biovar 2, race 3) were successfully selected by phage display from a large combinatorial antibody library. Characterization with regard to cross-reaction and use in routine immunoassays showed that the selected antibodies had improved char...

A PCR-based kit, ProbeliaTM, for the detection ofErwinia carotovora subsp.atroseptica (Eca) on potatoes was evaluated at five laboratories in four countries. The kit is based on DNA-specific PCR amplification followed by detection of amplicons by hybridization to a peroxidase-labelled DNA probe in a microplate. Specificity of the PCR primers for Ec...

Recently, several outbreaks of brown rot caused by Ralstonia solanacearum (Smith) Smith biovar 2, race 3 were reported in Western Europe. Knowledge of the variation in the population of the target pathogen forms the basis of epidemiological studies and control measures. In this study, the genetic diversity of race 3 strains from France, The Netherl...

Recombinant antibodies against the lipopolysaccharide of Ralstonia solanacearum (biovar 2, race 3) were successfully selected by phage display from a large combinatorial antibody library within six weeks. The isolated monoclonal scFv antibodies reacted both in ELISA and in IF with race 3 strains, but with only few strains belonging to other races....

Field releases of the wild-type plant growth-promoting rhizobacterium Pseudomonas fluorescens 89B-27, its bioluminescent derivative GEM-8 (89B-27::Tn4431), and a spontaneous rifampin-resistant variant estimating the wild-type population. Seed and root samples were taken 0, 7, 14, 21, or 28, 35 or 42, and 70 days after planting in each year and proc...

Immunomagnetic separation (IMS) procedures for the selective separation of Erwinia carotovora subsp. atroseptica from potato peel extract were optimized for the recovery of target and removal of non-target bacteria. A streptomycin-resistant strain of Erw. carotovora subsp. atroseptica was used in combination with a crystal violet pectate (CVP) medi...

The potential of polymerase chain reaction (PCR) for verifying the identity of colonies stained by the immunofluorescence colony-staining (IFC) procedure was investigated. Using primers directed against conserved sequences of the pectate lyase-genes coding for isozymes PLa, PLd and PLe of Erwinia chrysanthemi, the authors confirmed the identity of...

The potential of polymerase chain reaction (PCR) for verifying the identity of colonies stained by the immunofluorescence colony-staining (IFC) procedure was investigated. Using primers directed against conserved sequences of the pectate lyase-genes coding for isozymes PLa, PLd and PLe of Erwinia chrysanthemi, the authors confirmed the identity of...

In this study antisera against Photorhabdus luminescens strains were prepared for the first time. P. luminescens is a bacterial symbiont of entomopathogenic nematodes belonging to the genus Heterorhabditis. To characterize P. luminescens strains and form variants, we produced polyclonal antisera against P. luminescens PE (obtained from nematode str...

Isolation of antigens on immunomagnetic beads and subsequent analysis with SDS-PAGE and Western blotting (immunomagnetic isolation-Western blotting (IMI-WB)) was used to verify positive ELISA results for Erwinia chrysanthemi and Erw. carotovora subsp. atroseptica in potato peel extracts. Direct analysis of highly contaminated extracts by Western bl...

Sixteen bacterial strains, cross-reacting with antibodies againstErwinia chrysanthemi (Ech), were isolated from potato peel extracts, ditch water, and the rhizosphere of wheat, onion, sugar beet and chicory using the immunofluorescence colony-staining procedure. Based on fatty acid profiles, isolates were classified as belonging to thePseudomonas f...

High density lipoproteins (HDL) are responsible for the Reverse Cholesterol Transport (RCT). The role of the composition of the HDL particle in RCT, involving free cholesterol (chol) uptake from cell membranes, is not completely understood. We have therefore studied the uptake capacity from subjects with a wide variety of plasma HDL cholesterol con...

The specificity of two antisera raised to whole cells ofErwinia chrysanthemi (Ech), serogroup O1Ha, was studied in double antibody sandwich (DAS-) ELISA with 100 strains of different plant pathogenic bacteria (PPB), including 39 Ech strains, and of one of these antisera with 900 saprophytic bacteria isolated from extracts of potato peelings of Dutc...

In a previous study we have shown that Chinese hamster ovary (CHO) cells deficient in intact peroxisomes, lack the nonspecific lipid transfer protein (nsL-TP; sterol carrier protein 2) (van Heusden, G.P.H., Bos, K., Raetz, C.R.H. and Wirtz, K.W.A. (1990) J. Biol. Chem. 265, 4105-4110). The consequences of the absence of peroxisomes and of nsL-TP on...

The distribution of the nonspecific lipid transfer protein (i.e., sterol carrier protein 2) over the various subcellular fractions from rat liver and adrenal gland was determined by enzyme immunoassay and immunoblotting. This distribution is very different in each of these two tissues. In liver, 66% of the transfer protein is present in the membran...

A recently developed chiral gas chromatographic method and a diastereomeric high-performance liquid chromatographic method for the analysis of aspartic acid enantiomers in protein hydrolyzates have been evaluated. Although both techniques are fast and convenient, the latter is preferred because of its higher reproducibility and shorter analysis tim...

Racemization of aspartyl residues in proteins is a post-translational process, related to ageing. A method is presented for the detection of aspartic acid enantiomers in protein hydrolysates, based on chiral capillary gas chromatography. It is fast, easy and preferable to the usual diastereomeric dipeptide technique. We present evidence that traces...

Aims: To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNATM, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon. Methods and Resul...

Aims: The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt. The presence of intact 16S rRNA is considered to be a useful indicator for viability, as a rapid degradation...