Cross-protection or enhanced symptom display in greenhouse tomato co-infected with different Pepino mosaic virus isolates
Hanssen, I.M. ; Gutiérrez-Aguirre, I. ; Paeleman, A. ; Goen, K. ; Wittemans, L. ; Lievens, B. ; Vanachter, A.C.R.C. ; Ravnikar, M. ; Thomma, B.P.H.J. - \ 2010
Plant Pathology 59 (2010)1. - ISSN 0032-0862 - p. 13 - 21.
genomic rna - sequence - protein - population - cucumber - strains - squash - time - uk
The potential of three mild Pepino mosaic virus (PepMV) isolates, belonging to the CH2, EU and LP genotypes, to protect a tomato (Solanum lycopersicum) crop against an aggressive challenge isolate of the CH2 genotype was assessed in greenhouse trials and PepMV symptoms were rated at regular time points. After challenge infection, enhanced symptom display was recorded in plants that were pre-inoculated with a protector isolate belonging to a different genotype (EU, LP) from the challenge isolate. A quantitative genotype-specific TaqMan assay revealed that in these plants, the accumulation of the challenge isolate only temporarily slowed down. By contrast, efficient cross-protection was obtained using the mild isolate of the CH2 genotype, and in this case the challenge isolate was barely detectable in the pre-inoculated plants. These results suggest that the interaction between PepMV isolates largely depends on RNA sequence homology and that post-transcriptional gene silencing plays an important role in cross-protection
Pepino mosaic virus isolates and differential symptomatology in tomato
Hanssen, I.M. ; Paeleman, A. ; Vandewoestijne, E. ; Bergen, L. Van; Bragard, C. ; Lievens, B. ; Vanachter, A.C.R.C. ; Thomma, B.P.H.J. - \ 2009
Plant Pathology 58 (2009)3. - ISSN 0032-0862 - p. 450 - 460.
genomic rna - genetic-structure - sequence - protein - replication - variability - population - distinct - quality - strain
Based on a survey conducted in commercial tomato production in Belgium in 2006, four Pepino mosaic virus (PepMV) isolates that differed in symptom expression in the crop of origin were selected for greenhouse trials. The selected isolates were inoculated onto tomato plants grown in four separate plastic tunnels. PepMV symptom development was assessed regularly and extensive sampling followed by ELISA analyses, genotyping and sequencing was performed to study viral presence and variation in PepMV sequences throughout the trial period. Two isolates (EU-mild and CH2-mild) that were selected based on mild symptom expression in the crop of origin caused only mild symptoms in the trial, while two other isolates (CH2-aggressive and EU + CH2) that were selected for severe symptom display, caused considerably more severe symptoms. Sequence homology between CH2-mild and CH2-aggressive was as high as 99·4%. Results of this study show that differential symptom expression can, at least partially, be attributed to the PepMV isolate, which may be related to minor differences at the nucleotide level between isolates.
Survey on symptom expression and damage caused by Pepino mosaic virus in commercial tomato production in Belgium
Hanssen, I.M. ; Paeleman, A. ; Bergen, L. Van; Vandewoestijne, E. ; Wittemans, L.P.F. ; Goen, K. ; Vanachter, A.C.R.C. ; Thomma, B.P.H.J. - \ 2008
Acta Horticulturae 2008 (2008)808. - ISSN 0567-7572 - p. 185 - 192.
Pepino Mosaic Virus (PepMV) was first reported on greenhouse tomato (Solanum lycopersicum) in the Netherlands and the UK in 1999. Since then PepMV is worldwide established in the tomato industry and has become a significant problem. An extensive survey among Belgian tomato growers was conducted, assessing their experiences with PepMV in the 2005 and 2006 growing season, and their strategies to cope with this relatively new viral threat. Symptom expression and the economical damage caused by PepMV were investigated. An increase in PepMV infected tomato greenhouses was seen during the 2004 and 2005 growing seasons. The infection risk appeared to be significantly higher in dense tomato cultivation areas. It was shown that a PepMV infection early in the growing season does not guarantee fewer symptoms as compared to a late infection. The number of tomato growers that deliberately inoculated their crops early in the growing season as an immunization strategy decreased significantly after 2005, as results were not satisfying
A sensitive RT-PCR detection method reveals the occurrence of latent PepMV infections that are not detectable by ELISA
Hanssen, I.M. ; Paeleman, A. ; Bergen, L. Van; Vanachter, A.C.R.C. ; Lievens, B. ; Thomma, B.P.H.J. - \ 2008
In: Book of Abstracts 3rd international conference of the international working group on legume and vegetable viruses, Ljubljana, Slovenia, 20 - 23 August 2008. - - p. 47 - 47.
|Differential symptom expression in tomato caused by different isolates of Pepino Mosaic Virus
Hanssen, I.M. ; Bergen, L. Van; Vandewoestijne, E. ; Paeleman, A. ; Wittemans, L.P.F. ; Goen, K. ; Bragard, C. ; Lievens, B. ; Vanachter, A.C.R.C. ; Thomma, B.P.H.J. - \ 2008
In: Book of Abstracts 9th International Congress of Plant Pathology, Torino, Italy, 24-29 August 2008 / Porta-Puglia, A., Gonthier, A., - p. S2.378 - S2.378.
Genetic characterization of Pepino mosaic virus isolates from Belgian greenhouse tomatoes reveals genetic recombination
Hanssen, I.M. ; Paeleman, A. ; Wittemans, L.P.F. ; Goen, K. ; Lievens, B. ; Bragard, C. ; Vanachter, A.C.R.C. ; Thomma, B.P.H.J. - \ 2008
European Journal of Plant Pathology 121 (2008)2. - ISSN 0929-1873 - p. 131 - 146.
rna recombination - genomic rna - sequence - pepmv - variability - population - resistance - quality - protein - uk
Over a period of a few years, Pepino mosaic virus (PepMV) has become one of the most important viral diseases in tomato production worldwide. Infection by PepMV can cause a broad range of symptoms on tomato plants, often leading to significant financial losses. At present, five PepMV genotypes (EU, LP, CH2, US1 and US2) have been described, three of which (EU, LP and US2) have been reported in Europe. Thus far, no correlation has been found between different PepMV genotypes and the symptoms expressed in infected plants. In this paper, the genetic diversity of the PepMV population in Belgian greenhouses is studied and related to symptom development in tomato crops. A novel assay based on restriction fragment length polymorphism (RFLP) was developed to discriminate between the different PepMV genotypes. Both RFLP and sequence analysis revealed the occurrence of two genotypes, the EU genotype and the CH2 genotype, within tomato production in Belgium. Whereas no differences were observed in symptom expression between plants infected by one of the two genotypes, co-infection with both genotypes resulted in more severe PepMV symptoms. Furthermore, our study revealed that PepMV recombinants frequently occur in mixed infections under natural conditions. This may possibly result in the generation of viral variants with increased aggressiveness
A robust identification and detection assay to discriminate the cucumber pathogens Fusarium oxysporum f. sp. cucumerinum and f. sp. radicis-cucumerinum
Lievens, B. ; Claes, L. ; Vakalounakis, D.J. ; Vanachter, A.C.R.C. ; Thomma, B.P.H.J. - \ 2007
Environmental Microbiology 9 (2007)9. - ISSN 1462-2912 - p. 2145 - 2161.
vegetative compatibility groups - fungal plant-pathogens - real-time pcr - genetic diversity - formae speciales - rot disease - sp ciceris - stem rot - markers - wilt
The fungal species Fusarium oxysporum is a ubiquitous inhabitant of soils worldwide that includes pathogenic as well as non-pathogenic or even beneficial strains. Pathogenic strains are characterized by a high degree of host specificity and strains that infect the same host range are organized in so-called formae speciales. Strains for which no host plant has been identified are believed to be non-pathogenic strains. Therefore, identification below the species level is highly desired. However, the genetic basis of host specificity and virulence in F. oxysporum is so far unknown. In this study, a robust random-amplified polymorphic DNA (RAPD) marker-based assay was developed to specifically detect and identify the economically important cucumber pathogens F. oxysporum f. sp. cucumerinum and F. oxysporum f. sp. radicis-cucumerinum. While the F. oxysporum radicis-cucumerinum strains were found to cluster in a separate clade based on elongation factor-1¿ phylogeny, strains belonging to F. oxysporum f. sp. cucumerinum were found to be genetically more diverse. This is reflected in the observation that specificity testing of the identified markers using a broad collection of F. oxysporum strains with all known vegetative compatibility groups of the target formae speciales, as well as representative strains belonging to other formae speciales, resulted in two cross-reactions for the F. oxysporum f. sp. cucumerimum marker. However, no cross-reactions were observed for the F. oxysporum f. sp. radicis-cucumerimum marker. This F. oxysporum f. sp. radicis-cucumerimum marker shows homology to Folyt1, a transposable element identified in the tomato pathogen F. oxysporum f. sp. lycopersici and may possibly play a role in host-range specificity in the target forma specialis. The markers were implemented in a DNA array that enabled parallel and sensitive detection and identification of the pathogens in complex samples from diverse origins.
Assessing populations of a disease suppressive microorganism and a plant pathogen using DNA arrays
Lievens, B. ; Claes, L. ; Krause, M.S. ; Vanachter, A.C.R.C. ; Cammue, B.P.A. ; Thomma, B.P.H.J. - \ 2007
Plant Science 172 (2007)3. - ISSN 0168-9452 - p. 505 - 514.
rhizoctonia damping-off - pythium root-rot - polymerase-chain-reaction - composted hardwood bark - amended potting mixes - trichoderma-hamatum - biological-control - microbial activity - container medium - identification
Understanding the relationships between disease suppressive microbial populations and plant pathogens is essential to develop procedures for effective and consistent disease control. Currently, DNA array technology is the most suitable technique to simultaneously detect multiple microorganisms. Although this technology has been successfully applied for diagnostic purposes, its utility to assess different microbial populations, as a basis for further study of population dynamics and their potential interactions, has not yet been investigated. In this study, a DNA macroarray with multiple levels of phylogenetic specificity was developed to measure population densities of a specific disease suppressive microorganism, Trichoderma hamatum isolate 382, and the plant pathogen Rhizoctonia solani. Amongst others, the DNA array contained genus-, species- and isolate-specific detector oligonucleotides and was optimized for sensitive detection and reliable quantification of the target organisms in potting mix samples. Furthermore, this DNA array was used to quantify disease severity as well as incidence of severe disease based on pathogen population densities in the growing medium. Taking into account the unlimited expanding possibilities of DNA arrays to include detector oligonucleotides for other and more microorganisms, this technique has the potential for studying the population dynamics and ecology of several target populations in a single assay
|Detecting single nucleotide polymorphisms using DNA arrays for plant pathogen diagnosis
Lievens, B. ; Claes, L. ; Vanachter, A.C.R.C. ; Cammue, B.P.A. ; Thomma, B.P.H.J. - \ 2006
In: Book of Abstracts Cost Action 853 meeting, Wageningen, The Netherlands, 19-20 June 2006 - p. 21 - 21.
Real-time PCR for detection and quantification of fungal and oomycete tomato pathogens in plant and soil samples
Lievens, B. ; Brouwer, M. ; Vanachter, A.C.R.C. ; Cammue, B.P.A. ; Thomma, B.P.H.J. - \ 2006
Plant Science 171 (2006)1. - ISSN 0168-9452 - p. 155 - 165.
polymerase-chain-reaction - verticillium-dahliae - quantitative pcr - pythium - identification - roots - dna - microsclerotia - phytophthora - diagnostics
Although new, rapid detection and identification technologies are becoming available more and more for various plant pathogens, pathogen quantification remains one of the main challenges in the disease management of many crops. Currently, real-time polymerase chain reaction (PCR) is the most straightforward technique to quantify pathogen presence. This manuscript describes the use of real-time PCR to quantitatively assess the presence of a number of economically important fungal and oomycete tomato pathogens in biological samples. We demonstrate that pathogen DNA can be accurately quantified over at least four orders of magnitude. Additionally, we demonstrate the feasibility of the technique to quantify pathogen biomass in complex biological samples.
Detecting single nucleotide polymorphisms using DNA arrays for plant pathogen diagnosis
Lievens, B. ; Claes, L. ; Vanachter, A.C.R.C. ; Cammue, B.P.A. ; Thomma, B.P.H.J. - \ 2006
FEMS Microbiology Letters 255 (2006)1. - ISSN 0378-1097 - p. 129 - 139.
dot-blot hybridization - wilt pathogens - identification - differentiation - microarray - phytophthora - potato
The lack of a rapid and reliable means for routine pathogen identification has been one of the main limitations in plant disease management, and has pushed the development of culture-independent, molecular approaches. Currently, DNA array technology is the most suitable technique for high-throughput detection and identification, as well as quantification, of multiple pathogens in a single assay. Closely related pathogens that may have completely different host ranges or pathogenicity often differ in only a single to a few base pairs in genes that may be targeted for identification. Therefore, the ability to discriminate single nucleotide polymorphisms (SNPs) should be pursued in any diagnostic assay. In this paper, we demonstrate the utility of DNA array technology to detect SNPs while accounting for specific criteria such as the position of the mismatch, the sequence of the oligonucleotide, and the length and amount of labeled amplicons that are hybridized. When disregarding mismatches at the extreme ends of the oligonucleotides, cross hybridization to single mismatch oligonucleotides is rare when processing environmental samples that contain genetic material from unknown sources. In addition to plant pathology, this study is relevant for any field of research where DNA arrays are used to detect mutations or polymorphisms, ranging from human diagnostics to environmental microbiology and microbial ecology.
Quantitative assessment of phytopathogenic fungi in various substrates using a DNA macroarray
Lievens, B. ; Brouwer, M. ; Vanachter, A.C.R.C. ; Lévesque, C.A. ; Cammue, B.P.A. ; Thomma, B.P.H.J. - \ 2005
Environmental Microbiology 7 (2005)11. - ISSN 1462-2912 - p. 1698 - 1710.
dot-blot hybridization - real-time pcr - verticillium-dahliae - oligonucleotide array - genetic-analysis - rapid detection - amplified dna - identification - soil - pathogens
Detection, identification and quantification of plant pathogens are the cornerstones of preventive plant disease management. To detect multiple pathogens in a single assay, DNA array technology currently is the most suitable technique. However, for sensitive detection, polymerase chain reaction (PCR) amplification before array hybridization is required. To evaluate whether DNA array technology can be used to simultaneously detect and quantify multiple pathogens, a DNA macroarray was designed and optimized for accurate quantification over at least three orders of magnitude of the economically important vascular wilt pathogens Verticillium albo-atrum and Verticillium dahliae. A strong correlation was observed between hybridization signals and pathogen concentrations for standard DNA added to DNA from different origins and for infested samples. While accounting for specific criteria like amount of immobilized detector oligonucleotide and controls for PCR kinetics, accurate quantification of pathogens was achieved in concentration ranges typically encountered in horticultural practice. Subsequently, quantitative assessment of other tomato pathogens (Fusarium oxysporum, Fusarium solani, Pythium ultimum and Rhizoctonia solani) in environmental samples was performed using DNA array technology and correlated to measurements obtained using real-time PCR. As both methods of quantification showed a very high degree of correlation, the reliability and robustness of the DNA array technology is shown
Disease Notes: Root and foot rot on tomato caused by Phytophthora infestans detected in Belgium
Lievens, B. ; Hanssen, I.R.M. ; Vanachter, A.C.R.C. ; Cammue, B.P.A. ; Thomma, B.P.H.J. - \ 2004
Plant Disease 88 (2004)1. - ISSN 0191-2917 - p. 86 - 86.
Design and development of a DNA array for rapid detection and identification of tomato vascular wilt pathogens
Lievens, B. ; Brouwer, M. ; Vanachter, A.C.R.C. ; Lévesque, C.A. ; Cammue, B.P.A. ; Thomma, B.P.H.J. - \ 2003
FEMS Microbiology Letters 223 (2003)1. - ISSN 0378-1097 - p. 113 - 122.
verticillium-albo-atrum - dot-blot hybridization - f-sp lycopersici - nonpathogenic fusarium-oxysporum - biological-control - genetic-analysis - dahliae - microsclerotia - combination - resistance
Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici, and Verticillium wilt, caused by either Verticillium albo-atrum or Verticillium dahliae, are devastating diseases of tomato (Lycopersicon esculentum) found worldwide. Monitoring is the cornerstone of integrated pest management of any disease. The lack of rapid, accurate, and reliable means by which plant pathogens can be detected and identified is one of the main limitations in integrated disease management. In this paper, we describe the development of a molecular detection system, based on DNA array technology, for rapid and efficient detection of these vascular wilt pathogens. We show the utility of this array for the sensitive detection of these pathogens from complex substrates like soil, plant tissues and irrigation water, and samples that are collected by tomato growers in their greenhouses.
|Design and development of a DNA array for early detection and rapid identification of multiple tomato vascular wilt pathogens
Lievens, B. ; Krause, M.S. ; Brouwer, M. ; Vanachter, A.C.R.C. ; Lévesque, C.A. ; Cammue, B.P.A. ; Thomma, B.P.H.J. - \ 2003
In: The American Phytopathological Society Annual Meeting, Charlotte, North Carolina, USA, 9-13 August 2003 - p. S52 - S52.
|Development of a DNA array for early detection and rapid identification of tomato vascular wilt pathogens
Lievens, B. ; Brouwer, M. ; Cammue, B.P.A. ; Ceuster, T.J.J. De; Vanachter, A.C.R.C. ; Thomma, B.P.H.J. - \ 2003
In: Book of Abstracts 55th International Symposium on Crop Protection, Gent, Belgium, 6 May 2003 [S.l] : S.n. - p. 81 - 81.