Steve Baeyen

Publications

• 4.41

  Impact points

  Sequence Diversity in the Dickeya fliC Gene: Phylogeny of the Dickeya Genus and TaqMan® PCR for 'D. solani', New Biovar 3 Variant on Potato in Europe.

  Johan Van Vaerenbergh, Steve Baeyen, Paul De Vos, Martine Maes

  PloS one. 01/2012; 7(5):e35738.

  Worldwide, Dickeya (formerly Erwinia chrysanthemi) is causing soft rot diseases on a large diversity of crops and ornamental plants. Strains affecting potato are mainly found in D. dadantii, D. dianthicola and D. zeae, which appear to have a marked geographical distribution. Furthermore, a few Dicke... [more] Worldwide, Dickeya (formerly Erwinia chrysanthemi) is causing soft rot diseases on a large diversity of crops and ornamental plants. Strains affecting potato are mainly found in D. dadantii, D. dianthicola and D. zeae, which appear to have a marked geographical distribution. Furthermore, a few Dickeya isolates from potato are attributed to D. chrysanthemi and D. dieffenbachiae. In Europe, isolates of Erwinia chrysanthemi biovar 1 and biovar 7 from potato are now classified in D. dianthicola. However, in the past few years, a new Dickeya biovar 3 variant, tentatively named 'Dickeya solani', has emerged as a common major threat, in particular in seed potatoes. Sequences of a fliC gene fragment were used to generate a phylogeny of Dickeya reference strains from culture collections and with this reference backbone, to classify pectinolytic isolates, i.e. Dickeya spp. from potato and ornamental plants. The reference strains of the currently recognized Dickeya species and 'D. solani' were unambiguously delineated in the fliC phylogram. D. dadantii, D. dianthicola and 'D. solani' displayed unbranched clades, while D. chrysanthemi, D. zeae and D. dieffenbachiae branched into subclades and lineages. Moreover, Dickeya isolates from diagnostic samples, in particular biovar 3 isolates from greenhouse ornamentals, formed several new lineages. Most of these isolates were positioned between the clade of 'D. solani' and D. dadantii as transition variants. New lineages also appeared in D. dieffenbachiae and in D. zeae. The strains and isolates of D. dianthicola and 'D. solani' were differentiated by a fliC sequence useful for barcode identification. A fliC TaqMan®real-time PCR was developed for 'D. solani' and the assay was provisionally evaluated in direct analysis of diagnostic potato samples. This molecular tool can support the efforts to control this particular phytopathogen in seed potato certification.

• UTILIZATION OF FTA (R) CARDS COMBINED WITH ONE-STEP REAL-TIME PCR FOR RAPID DETECTION OF QUARANTINE VIRUSES AND PHYTOPLASMAS

  De Jonghe, Kris, Tahzima, Rachid, Scheerlinck, Dries, Baeyen, Steve, Maes, Martine

  4th International qPCR Event TUM, Weihenstephan, Freising, Germany; 03/2009

  Smooth international trade of planting material often depends on efficient inspections at the port of entry. Therefore, reliable and fast screening for regulated plant pathogens is an important challenge. This study evaluates the use of Whatman FTA (R) card technology (a commercially available filte... [more] Smooth international trade of planting material often depends on efficient inspections at the port of entry. Therefore, reliable and fast screening for regulated plant pathogens is an important challenge. This study evaluates the use of Whatman FTA (R) card technology (a commercially available filter paper impregnated with patented chemicals) as RNA and DNA extraction method compared to commercial plant extraction kits (DNeasy, RNeasy Plant – Qiagen; Invisorb DNA and RNA Plant – Invitek), combined by a one-step qPCR detection for a selection of regulated pathogens in host plants. The method was tested for 2 viruses ( Pepino mosaic virus , Potexvirus, Flexiviridae; Tomato spotted wilt virus , Tospovirus, Bunyavirudae) and a phytoplasma ( Apple proliferation phytoplasm , AP Phytoplasma). A dilution series of respective in vitro transcripts of the target RNA fragment served as standard for quantification of the RNA viruses in the infected leaf samples. Quantification of phytoplasma DNA was done by means of plasmids containing a 16S rDNA target fragment. Both were added to a mock-RNA/DNA extraction of healthy control plants. With the commercial RNA and DNA extraction kits detection was more sensitive and quantification more reliable than with the FTA card method. However, the FTA card technology followed by a one-step qPCR detection enabled reliable screening of leaf tissue for infection with our target regulated pathogens in less than 4 hours. For the viruses, the RNA fixation on the filter paper was most efficient when the leaf samples were homogenised in a standard ELISA extraction buffer. For AP Phytoplasma DNA, a direct leaf punch on the FTA cards was sufficient for a reliable detection of this pathogen in infected leaf tissue. In conclusion we can state that the FTA card technology followed by one-step qPCR could offer a fast and reliable screening method for sanitary control on the regulated pathogens

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• Real-time PCR assays based on the multi-copy rDNA ITS region and the single-copy β-tubulin gene for detection and quantification of the strawberry pathogen Colletotrichum acutatum

  Jane Debode, Wendy Van Hemelrijck, Steve Baeyen, Piet Creemers, Kurt Heungens, Martine Maes

  4th international qPCR Symposium, Freising-Weihenstephan, Germany; 03/2009

  C. acutatum is one of the most important fungal pathogens of strawberry worldwide. The disease is responsible for up to 80% plant death in nurseries and yield losses of over 50% in strawberry production fields. Symptoms include fruit rot, crown rot and lesions on stolons. However, C. acutatum may al... [more] C. acutatum is one of the most important fungal pathogens of strawberry worldwide. The disease is responsible for up to 80% plant death in nurseries and yield losses of over 50% in strawberry production fields. Symptoms include fruit rot, crown rot and lesions on stolons. However, C. acutatum may also persist on young strawberry plants without causing visible symptoms. Such latent infections are considered to be the main cause of dissemination of C. acutatum . Detection and quantification of C. acutatum during this latent phase using real-time PCR might aid considerably in the reduction of its spread. This paper describes the development of real-time PCR assays using primers and probes designed to the multi-copy rDNA ITS1 region and the single-copy β-tubulin 2 gene of C. acutatum . The sensitivity of both assays is compared and the data are used to calculate the genome size and ITS copy number of C. acutatum . Finally, detection and quantification of C. acutatum is demonstrated in artificially and naturally infected strawberry leaves. Using TaqMan technology, the ITS-based real-time PCR assay could reliably detect as little as 50 fg genomic DNA, 100 copies target DNA, or 25 conidia. The β-tubulin-based assay was circa 66 times less sensitive, and therefore less suitable for detection purposes. However, by applying the β-tubulin primers together with the ITS primers to both genomic DNA and known numbers of cloned target DNA, they proved very useful in revealing some insights into the genome of C. acutatum . Specifically, we estimated a genome size of 60.0 Mbp for C. acutatum and the presence of circa 20 tandem copies of the ITS region in one genome of C. acutatum . Concerning the detection of C. acutatum in strawberry leaves, we were able to detect C. acutatum in plant tissue mixes containing only 0.001% infected tissue. In addition, real-time PCR analysis of leaf samples taken at various times after inoculation indicated that the assay allows monitoring of early symptomless growth of C. acutatum on strawberry leaves. Finally, the assay allowed detection of C. acutatum in naturally infected and symptomless strawberry leaves collected from production fields and planting material. In conclusion, our results demonstrate that the ITS-based real-time PCR assay developed in the present study, is a highly sensitive technique that can be used in routine quarantine inspections to screen strawberry planting material for C. acutatum contamination.

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• Quantitative detection and monitoring of Colletotrichum acutatum in strawberry leaves using real-time PCR.

  DEBODE J., VAN HEMELRIJCK W., BAEYEN S., CREEMERS P., HEUNGENS K., MAES M.

  Plant Pathology. 01/2009; 58.
• 2.43

  Impact points

  Molecular detection of Puccinia horiana in Chrysanthemum x morifolium through conventional and real-time PCR.

  Hossein Alaei, Steve Baeyen, Martine Maes, Monica Höfte, Kurt Heungens

  Journal of microbiological methods. 11/2008;

  Puccinia horiana Henn. is a quarantine organism and one of the most important fungal pathogens of Chrysanthemum x morifolium cultivars grown for cut flower or potted plant production (florist's chrysanthemum) in several regions of the world. Highly specific primer pairs were identified for conve... [more] Puccinia horiana Henn. is a quarantine organism and one of the most important fungal pathogens of Chrysanthemum x morifolium cultivars grown for cut flower or potted plant production (florist's chrysanthemum) in several regions of the world. Highly specific primer pairs were identified for conventional, nested, and real-time PCR detection of P. horiana based on the specific and sensitive PCR amplification of selected regions in the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA (rDNA). Using these different PCR versions, 10 pg, 10 fg, and 5 fg genomic DNA could be detected, respectively. When using cloned target DNA as template, the detection limits were 5000, 50, and 5 target copies, respectively. These detection limits were not affected by a background of chrysanthemum plant DNA. The DNA extraction method was optimized to maximize the recoverability of the pathogen from infected plant tissue. A CTAB extraction protocol or a selection of commercial DNA extraction methods allowed the use of 10 ng total (plant+pathogen) DNA without interference of PCR inhibitors. Due to the specificity of the primers, SYBR Green I technology enabled reliable real time PCR signal detection. However, an efficient TaqMan probe is available. The lowest proportion of infected plant material that could still be detected when mixed with healthy plant material was 0.001%. The real-time PCR assay could detect as few as eight pure P. horiana basidiospores, demonstrating the potential of the technique for aerial detection of the pathogen. The amount of P. horiana DNA in plant tissue was determined at various time points after basidiospore inoculation. Using the real-time PCR protocol, it was possible to detect the pathogen immediately after the inoculation period, even though the accumulation of pathogen DNA was most pronounced near the end of the latent period. The detection system proved to be accurate and sensitive and could help not only in pathogen diagnosis but also in pathogen monitoring and disease forecasting systems.

• Sensitive real-time PCR detection of Xanthomonas fragariae in strawberry plants.

  VANDROEMME J, BAEYEN S, VAN VAERENBERGH J, DE VOS P, AND M. MAES.

  Plant Pathology. 01/2008; 57.

• Experiences and perspectives for the use of a Paenibacillus strain as a plant protectant.

  M Maes, S Baeyen

  Communications in agricultural and applied biological sciences. 02/2003; 68(4 Pt B):457-62.

  A study on the microbial ecology in an active slow sand filter, used for disinfecting the circulating plant nutrient solutions, showed that spore-forming plant-associated bacteria belonging to the Bacillus-Paenibacillus complex are well adapted for transmission in the solutions and passage through t... [more] A study on the microbial ecology in an active slow sand filter, used for disinfecting the circulating plant nutrient solutions, showed that spore-forming plant-associated bacteria belonging to the Bacillus-Paenibacillus complex are well adapted for transmission in the solutions and passage through the filter. Therefore, strains from this bacterial group were suitable candidates for biological control in irrigated and closed plant growth systems. The spore-forming Paenibacillus polymyxa strain PpDGB was selected in in vitro tests as a potent pathogen-antagonist and was tested as a prophylactic protection agent in the plant rhizosphere, especially for cultures stages that are highly susceptible to stress and disease. Plant cuttings, in vitro plants and seeds of different plant types were bacterized and planted in their typical disease-conducive environment where nutrient solutions or water irrigation was applied and further plant development was monitored. Observed plant parameters were plant survival, weight, chlorophyll concentration in the leaf mesophyl, root health and root hair formation. The PpDGB treatment initially induced stress in the plants, which was observed as a transient stop in plant transpiration. This effect caused some necrosis in the most stress-sensitive in vitro plant species. In the other plants this stress period was followed by a significant enhancement in plant growth. In case of seed treatment, more seeds germinated and seedling growth was faster. In the tested formulation, PpDGB enhanced growth but not disease resistance, probably due to simultaneous activation of the residual plant pathogens. Therefore variant formulations have to be tested. The influence of PpDGB on the composition of the bacterial communities in the rhizosphere was assessed by DGGE profiling. In soilless plant cultures, PpDGB-driven profile changes could be observed from the 5th day after the initial treatment. P. polymyxa bacteria were shown to be widely present in association with plants and specific PpDGB detection in plant and rhizosphere was only possible with newly developed strain-specific PCR primers based on Nif H gene sequences. Quantitative PCR based on SYBR Green fluorescence enabled detection of low PpDGB concentrations in the plant rhizosphere.

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• Sequence Diversity in the Dickeya fliC Gene: Phylogeny of the Dickeya Genus and TaqMan® PCR for 'D. solani' , New Biovar 3 Variant on Potato in Europe

  Johan Van Vaerenbergh, Steve Baeyen, Paul De Vos, Martine Maes

  PLoS ONE. 7(5):e35738.

  Worldwide, Dickeya (formerly Erwinia chrysanthemi ) is causing soft rot diseases on a large diversity of crops and ornamental plants. Strains affecting potato are mainly found in D. dadantii , D. dianthicola and D. zeae , which appear to have a marked geographical distribution. Furthermore, a few Di... [more] Worldwide, Dickeya (formerly Erwinia chrysanthemi ) is causing soft rot diseases on a large diversity of crops and ornamental plants. Strains affecting potato are mainly found in D. dadantii , D. dianthicola and D. zeae , which appear to have a marked geographical distribution. Furthermore, a few Dickeya isolates from potato are attributed to D. chrysanthemi and D. dieffenbachiae . In Europe, isolates of Erwinia chrysanthemi biovar 1 and biovar 7 from potato are now classified in D. dianthicola . However, in the past few years, a new Dickeya biovar 3 variant, tentatively named ‘ Dickeya solani ’, has emerged as a common major threat, in particular in seed potatoes. Sequences of a fli C gene fragment were used to generate a phylogeny of Dickeya reference strains from cul