A robust and specific real-time PCR tool for the detection of Phytophthora lateralis in plant tissues

Schenck, N.; Fourrier‐Jeandel, Céline; Ioos, Renaud

doi:10.1007/s10658-016-0909-7

Cited by 7 publications

(4 citation statements)

References 19 publications

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“…Similarly, isolation tests of Phytophthora species from bleeding bark lesions are only reliable at the active advancing lesion fronts whereas slightly older parts of the lesions are quickly colonised by secondary pathogenic fungi preventing the isolation of the primary Phytophthora pathogen ( Erwin & Ribeiro 1996 , Jung & Blaschke 2004 , Jung 2009 ). In recent years, an array of highly sensitive, high-throughput, species-specific molecular detection methods have been developed, which facilitate the diagnosis of Phytophthora diseases significantly, and are particularly useful for routine screening of high numbers of samples for harmful and emerging Phytophthora pathogens ( Schubert et al 1999 , Nechwatal et al 2001 , Schena et al 2006 , Martin et al 2012 , Scibetta et al 2012 , Sikora et al 2012 , Than et al 2013 , King et al 2015 , Schenck et al 2016 ). Recent metagenomic approaches provide an efficient tool for large-scale surveys of Phytophthora diversity ( Vettraino et al 2012 , Català et al 2015 , Sapkota & Nicolaisen 2015 , Burgess et al 2017 ).…”

Section: Introductionmentioning

confidence: 99%

Canker and decline diseases caused by soil- and airborne Phytophthora species in forests and woodlands

Jung

Pérez‐Sierra

Durán³

et al. 2018

persoonia

145

208

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Most members of the oomycete genus Phytophthora are primary plant pathogens. Both soil- and airborne Phytophthora species are able to survive adverse environmental conditions with enduring resting structures, mainly sexual oospores, vegetative chlamydospores and hyphal aggregations. Soilborne Phytophthora species infect fine roots and the bark of suberized roots and the collar region with motile biflagellate zoospores released from sporangia during wet soil conditions. Airborne Phytophthora species infect leaves, shoots, fruits and bark of branches and stems with caducous sporangia produced during humid conditions on infected plant tissues and dispersed by rain and wind splash. During the past six decades, the number of previously unknown Phytophthora declines and diebacks of natural and semi-natural forests and woodlands has increased exponentially, and the vast majority of them are driven by introduced invasive Phytophthora species. Nurseries in Europe, North America and Australia show high infestation rates with a wide range of mostly exotic Phytophthora species. Planting of infested nursery stock has proven to be the main pathway of Phytophthora species between and within continents. This review provides insights into the history, distribution, aetiology, symptomatology, dynamics and impact of the most important canker, decline and dieback diseases caused by soil- and airborne Phytophthora species in forests and natural ecosystems of Europe, Australia and the Americas.

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Section: Introductionmentioning

confidence: 99%

Canker and decline diseases caused by soil- and airborne Phytophthora species in forests and woodlands

Jung

Pérez‐Sierra

Durán³

et al. 2018

persoonia

145

208

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“…The real-time PCR assay amplified an endogenous plant DNA sequence present in the sample (5.8S rDNA) as a co-extracted and co-amplified internal control. It reveals any flaws in DNA extraction and the presence of PCR inhibitors [ 32 – 34 ]. These protocols were further validated using naturally infected citrus material collected in the field.…”

Section: Introductionmentioning

confidence: 99%

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants

et al. 2020

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Background Asiatic Citrus Canker, caused by Xanthomonas citri pv. citri, severely impacts citrus production worldwide and hampers international trade. Considerable regulatory procedures have been implemented to prevent the introduction and establishment of X. citri pv. citri into areas where it is not present. The effectiveness of this surveillance largely relies on the availability of specific and sensitive detection protocols. Although several PCR- or real-time PCR-based methods are available, most of them showed analytical specificity issues. Therefore, we developed new conventional and real-time quantitative PCR assays, which target a region identified by comparative genomic analyses, and compared them to existing protocols. Results Our assays target the X. citri pv. citri XAC1051 gene that encodes for a putative transmembrane protein. The real-time PCR assay includes an internal plant control (5.8S rDNA) for validating the assay in the absence of target amplification. A receiver-operating characteristic approach was used in order to determine a reliable cycle cut-off for providing accurate qualitative results. Repeatability, reproducibility and transferability between real-time devices were demonstrated for this duplex qPCR assay (XAC1051-2qPCR). When challenged with an extensive collection of target and non-target strains, both assays displayed a high analytical sensitivity and specificity performance: LOD95% = 754 CFU ml− 1 (15 cells per reaction), 100% inclusivity, 97.2% exclusivity for XAC1051-2qPCR; LOD95% = 5234 CFU ml− 1 (105 cells per reaction), 100% exclusivity and inclusivity for the conventional PCR. Both assays can detect the target from naturally infected citrus fruit. Interestingly, XAC1051-2qPCR detected X. citri pv. citri from herbarium citrus samples. The new PCR-based assays displayed enhanced analytical sensitivity and specificity when compared with previously published PCR and real-time qPCR assays. Conclusions We developed new valuable detection assays useful for routine diagnostics and surveillance of X. citri pv. citri in citrus material. Their reliability was evidenced through numerous trials on a wide range of bacterial strains and plant samples. Successful detection of the pathogen was achieved from both artificially and naturally infected plants, as well as from citrus herbarium samples, suggesting that these assays will have positive impact both for future applied and academic research on this bacterium.

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“…The species-specific DNA-based molecular tools could be useful for early and accurate detection of plant pathogens (Gachon et al 2004 ; Falacy et al 2007 ). Therefore, PCR and quantitative real-time PCR (qPCR) based identification and detection of the unculturable fungal pathogens using species-specific oligonucleotides has become the most comprehensive, accurate, and rapid technology (Schenck et al 2016 ; Lee et al 2016 ). In recent advancements, qPCR has emerged with high resolution melting (HRM) analysis as an alternative to hydrolysis probe chemistry for detection and quantification of fungal pathogens (Summers et al 2015 ; Lee et al 2016 ; Zambounis et al 2015 ).…”

Section: Introductionmentioning

confidence: 99%

Simultaneous detection of downy mildew and powdery mildew pathogens on Cucumis sativus and other cucurbits using duplex-qPCR and HRM analysis

et al. 2020

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Powdery mildew and downy mildew are two devastating diseases on cucumber and other cucurbit crops caused by Podosphaera xanthii and Pseudoperonospora cubensis, respectively. Identification and detection of these pathogens from field and plant material could be significant for the selection of resistant varieties and formulation of disease management strategies. In the present study, a duplex qPCR assay developed for simultaneous detection and quantification of both pathogens from different samples. Two sets of species-specific primers developed for the detection of P. xanthii and P. cubensis pathogens by targeting the internal transcribed spacer (ITS) region of the rDNA gene cluster. The specificity of designed primers was also evaluated against the different microbial, plant, soil, and environmental samples. Initially, the individual assays for P. cubensis and P. xanthii were validated using their corresponding speciesspecific primers, which amplified the prominent and distinctive products of ~ 705 bp and ~ 290 bp size, respectively. SYBR green-based duplex real-time PCR assay was developed to detect and quantify both mildew pathogens from different field samples. The species-specific oligonucleotide primer sets showed high specificity with melt curve peaks at 85.83 °C and 88.05 °C, for P. xanthii and P. cubensis, respectively. The relative quantification and lowest detection limit of qPCR assays using tenfold diluted plasmid (Csp1 and Csd1) DNA were estimated (0.1 pg/µl) through a standard curve. In this study, the species-specific PCR and qPCR assays in both simplex and duplex formats have been validated successfully. These assays could be useful for efficient detection and quantification of mildew pathogens from the cucumber and other cucurbit crops.

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A robust and specific real-time PCR tool for the detection of Phytophthora lateralis in plant tissues

Cited by 7 publications

References 19 publications

Canker and decline diseases caused by soil- and airborne Phytophthora species in forests and woodlands

Canker and decline diseases caused by soil- and airborne Phytophthora species in forests and woodlands

Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants

Simultaneous detection of downy mildew and powdery mildew pathogens on Cucumis sativus and other cucurbits using duplex-qPCR and HRM analysis

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