Usefulness of single copy genes containing introns in Phytophthora for the development of detection tools for the regulated species P. ramorum and P. fragariae

Ioos, Renaud; Laugustin, L.; Schenck, N.; Rose, Sylvie; Husson, Claude; Frey, Pascal

doi:10.1007/s10658-006-9051-2

Cited by 30 publications

(18 citation statements)

References 11 publications

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“…Improved specificity would allow undiluted extracts from cultures to be tested without the risk of false-positive results. One approach to improving the discrimination of similar species is to design a new assay for a different region of the genome (2,13,29). However, modifications to existing assays can also be made to improve specificity without the need to completely redesign the primers and/or probe.…”

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confidence: 99%

Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field

Tomlinson¹,

Barker²,

Boonham³

2007

Appl Environ Microbiol

139

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Phytophthora ramorum is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of P. ramorum in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of P. ramorum in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.

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confidence: 99%

Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field

Tomlinson¹,

Barker²,

Boonham³

2007

Appl Environ Microbiol

139

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“…Identical Phytophthora genotypes were assigned using a cut-off genotype confidence percentage (GCP) value of 90%. The HRM profiles of all the other Phytophthora species were compared to this P. nicotianae normalised reference genotype and were assigned as P. nicotianae genotypes if respective GCP values were higher than 90% or as variations if lower than 90% (Ioos et al 2006;Meng & Wang 2010). The different species tested generated distinctive HRM profiles, thus permitting an accurate identification and discrimination of each Phytophthora species.…”

Section: Discussionmentioning

confidence: 99%

“…These HRM curves were highly informative even if compared with simple melting curves and gave enhanced information, which allowed the efficient differentiation of the tested Phytophthora species. A stunning aspect of our HRM analysis is that we successfully adapted degenerated primers for the Ypt1 marker which was previously employed successfully under standard PCR conditions to discriminate Phytophthora species by sequencing approaches (Ioos et al 2006;Meng & Wang 2010) with a more accurate and reliable molecular analysis. This was entirely confirmed by the successful genotyping of all the 10 Phytophthora species tested.…”

Section: Discussionmentioning

confidence: 99%

“…Another disadvantage is that these regions are too long to enable a perfect optimisation of HMR analyses. However, the ras-related Ypt1 gene is a promising candidate locus by means of phylogenetic studies and for the establishment of accurate molecular diagnostics in order to differentiate Phytophthora species (Ioos et al 2006;Schena & Cooke 2006;Meng & Wang 2010). The high differentiation power of the Ypt1 gene upon Phytophthora species is a consequence of the high variation in the non-coding regions of this gene (Schena & Cooke 2006).…”

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Identification of Phytophthora species by a high resolution melting analysis: an innovative tool for rapid differentiation

Zambounis¹,

Samaras²,

Xanthopoulou³

et al. 2016

Plant Prot. Sci.

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Zambounis A., Samaras A., Xanthopoulou A., Osathanunkul M., Schena L., Tsaftaris A., Madesis P. (2016): Identification of Phytophthora species by a high resolution melting analysis: an innovative tool for rapid differentiation. Plant Protect. Sci., 52: 176-181.A new molecular method via the high resolution melting (HRM) analysis of the Ypt1 gene non-coding regions was validated for ten Phytophthora species with a broad host range from forest trees to crop species. The melting curve analysis of the amplicons specifically grouped all species into 10 respective unique and distinct HRM curve profiles. The analysis of the normalised HRM melting curves, assigning P. nicotianae as a normalised reference genotype, revealed that the genotype similarities among all the species were adequately low, indicating that Ypt1 marker was sufficient to identify and differentiate the tested species. This HRM method is rapid and reproducible allowing the identification of Phytophthora species and the screening of eventual variants eliminating the separate steps and reducing the risk of contamination.

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“…Schena and colleagues (2008) used the variable intron sequences of the Ypt1 gene to develop primers for 15 Phytophthora species. Also Lpv storage protein (Kong et al 2003b), GPA1, TRP, RAS-like (Ioos et al 2006), ß-tubulin (Bilodeau et al 2007) and elicitin genes (Bilodeau et al 2007, Lacourt & Duncan 1997 have been used to design primers for species-specific detection. Generally, detection of single-copy genes is less sensitive than multicopy genes.…”

Section: Detecting and Identifying Phytophthoramentioning

confidence: 99%

Phytophthora in Finnish nurseries

Rytkönen¹

2011

Diss. For.

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International trade and travel have facilitated the spread of harmful organisms around the world. Human-mediated movement of plants and plant products is now generally accepted to be the primary mode of introduction of pathogens and pests. Species of Phytophthora, a genus of plant pathogens, are commonly spread in this way and have caused severe epidemics in silviculture, horticulture and natural systems all over the world.The objective of the study was to gather information on the occurrence of Phytophthora spp. in Finnish nurseries. Furthermore, the aim was to produce information for risk assessments for these Phytophthora spp. and establish molecular means for their detection. Phytophthora cactorum was found to persist in natural waterbodies and results suggest that irrigation water might be a source of inoculum for the disease in nurseries. In addition to P. cactorum, isolates from ornamental Rhododendron in nurseries yielded three species new to Finland: P. ramorum, P. plurivora and P. pini. The only species with quarantine status, P. ramorum, was able to persist in the nursery in spite of an annual European Commission sanitation protocol. Phytophthora plurivora and the closely related P. pini had more hosts among Nordic tree species and other plants abundant in Finnish nurseries and forest ecosystems. They were also found to have higher infectivity rates compared to P. ramorum and P. cactorum. All four species survived two weeks in -5 °C , and thus soil survival of these Phytophthoras in Finland is likely under current climatic conditions.The most common tree species in Finnish nurseries, Picea abies, was highly susceptible to P. plurivora and P. pini in pathogenicity trials. In a histological examination of P. abies shoot tissues inoculated with P. plurivora zoospores, fast necrotrophic growth was observed in nearly all tissues of the fresh shoot. The production of propagules in P. abies shoot tissue was only weakly indicated.In this study, a PCR-DGGE technique was developed for simultaneous detection and identification of Phytophthora spp. It reliably detected Phytophthora in plant tissues and could discriminate most test species as well as indicate instances of multiple-species infections. It proved to be a useful detection and identification tool either applied alone or in concert with traditional isolation culture techniques. The limitations of the method are also discussed.In conclusion, all of the introduced species of Phytophthora had properties that promote a high risk of establishment and spread in Finland. The efficient transport of Phytophthora via commercial traffic and favorable conditions due to expected climate change will increase the incidence and establishment of Phytophthora in new areas. Thus, it is probable that pathogens of this genus will be introduced and become established in Finland and other Nordic countries unless efficient phytosanitary control becomes standard practice in the international plant trade.Keywords: Phytophthora, pathogenicity, survival, molecular detectio...

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Usefulness of single copy genes containing introns in Phytophthora for the development of detection tools for the regulated species P. ramorum and P. fragariae

Cited by 30 publications

References 11 publications

Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field

Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field

Identification of Phytophthora species by a high resolution melting analysis: an innovative tool for rapid differentiation

Phytophthora in Finnish nurseries

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