Development of a real-time PCR assay for detection of Phytophthora kernoviae and comparison of this method with a conventional culturing technique

Hughes, Kelvin; Tomlinson, J. A.; Giltrap, P. M.; Barton, V. C.; Hobden, E.; Boonham, Neil; Lane, Charles R.

doi:10.1007/s10658-011-9843-x

Cited by 15 publications

(10 citation statements)

References 22 publications

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“…The presence of G. smithogilvyi was revealed even in asymptomatic tissues, negative at culturing, with a DNA concentration value (ranged between 6 × 10 4 to 7.5 × 10 4 pg DNA per µg of total DNA) tenfold lower than in symptomatic tissues (ranged between 1.7 × 10 5 to 4.9 × 10 5 pg DNA per µg of total DNA extracted). This finding is in agreement with previous studies on different pathogens where latent infections were detected in asymptomatic host tissues (Migliorini et al 2015 ; Hughes et al 2011 ; Vettraino et al 2010 ).…”

Section: Discussionsupporting

confidence: 93%

Rapid diagnostics for Gnomoniopsis smithogilvyi (syn. Gnomoniopsis castaneae) in chestnut nuts: new challenges by using LAMP and real-time PCR methods

Vettraino

Luchi

Rizzo³

et al. 2021

AMB Expr

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Nuts of the sweet chestnut (Castanea sativa) are a widely appreciated traditional food in Europe. In recent years producers and consumers reported a drop of nut quality due to the presence of rot diseases caused by Gnomoniopsis smithogilvyi. Early detection of this pathogen is fundamental to the economic viability of the chestnut industry. In the present study, we developed three molecular methods based on real-time portable LAMP, visual LAMP and qPCR assays for G. smithogilvyi. The molecular assays were specific for G. smithogilvyi and did not amplify the other 11 Gnomoniopsis species and 11 other fungal species commonly associated with chestnuts. The detection limit of both the qPCR and real-time portable LAMP (P-LAMP) assays was 0.128 pg/µL, while the visual LAMP (V-LAMP) assay enabled the detection up to 0.64 pg/µL. By using these newly developed molecular tools, the pathogen was detected in symptomatic and asymptomatic nuts, but not in leaves. The reliability of these molecular methods, including the P-LAMP assay, was particularly useful in detecting G. smithogilvyi of harvested nuts in field, even in the absence of rot symptoms.

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Section: Discussionsupporting

confidence: 93%

Rapid diagnostics for Gnomoniopsis smithogilvyi (syn. Gnomoniopsis castaneae) in chestnut nuts: new challenges by using LAMP and real-time PCR methods

Vettraino

Luchi

Rizzo³

et al. 2021

AMB Expr

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“…Molecular diagnoses provide faster and more sensitive detection of Phytophthora spp. [16,45,[65][66][67][68][69][70][71][72].…”

Section: Discussionmentioning

confidence: 99%

The Use of qPCR Reveals a High Frequency of Phytophthora quercina in Two Spanish Holm Oak Areas

Mora-Sala

Berbegal

Abad-Campos

2018

Forests

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The struggling Spanish holm oak woodland situation associated with Phytophthora root rot has been studied for a long time. Phytophthora cinnamomi is considered the main, but not the only species responsible for the decline scenario. This study verifies the presence and/or detection of Phytophthora species in two holm oak areas of Spain (southwestern "dehesas" and northeastern woodland) using different isolation and detection approaches. Direct isolation and baiting methods in declining and non-declining holm oak trees revealed Phytophthora cambivora, Phytophthora cinnamomi, Phytophthora gonapodyides, Phytophthora megasperma, and Phytophthora pseudocryptogea in the dehesas, while in the northeastern woodland, no Phytophthora spp. were recovered. Statistical analyses indicated that there was not a significant relationship between the Phytophthora spp. isolation frequency and the disease expression of the holm oak stands in the dehesas. Phytophthora quercina and P. cinnamomi TaqMan real-time PCR probes showed that both P. cinnamomi and P. quercina are involved in the holm oak decline in Spain, but P. quercina was detected in a higher frequency than P. cinnamomi in both studied areas. Thus, this study demonstrates that molecular approaches complement direct isolation techniques in natural and seminatural ecosystem surveys to determine the presence and distribution of Phytophthora spp. This is the first report of P. pseudocryptogea in Europe and its role in the holm oak decline should be further studied.

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“…Molecular detection and quantification using real-time PCR approaches have been applied to detect oomycetes in soil and plant tissues [ 14 , 15 ]. Real-time PCR improves the speed, sensitivity and accuracy of DNA amplification in comparison with standard PCR [ 14 , 15 , 16 ], and has been used to identify and quantify Pythium and Phytophthora species including Phytophthora ramorum and P. kernoviae [ 17 , 18 , 19 , 20 ], which cause diseases of natural vegetation and horticultural crops. Different loci have been used to develop real-time PCR assays using TaqMan chemistry [ 21 ], the ITS region being most commonly selected for the design of primers and probes [ 14 , 17 ]; other loci have also been used, however, including mitochondrial loci such as atp9-nad9 and trnM-trnP-trnM [ 21 ].…”

Section: Introductionmentioning

confidence: 99%

Application of Real-Time PCR for the Detection and Quantification of Oomycetes in Ornamental Nursery Stock

Puértolas

Bonants²,

Boa

et al. 2021

JoF

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Numerous Phytophthora and Pythium disease outbreaks have occurred in Europe following inadvertent introduction of contaminated ornamental plants. Detection and identification of pathogens are crucial to reduce risks and improve plant biosecurity in Europe and globally. Oomycete diversity present in roots and compost was determined in 99 hardy woody plants bought from nurseries, retailers and internet sellers, using both isolations and molecular analyses. Oomycete DNA was quantified using real-time PCR of environmental DNA from the plants using three loci: ITS, trnM-trnP-trnM and atp9-nad9. At least one oomycete species was isolated from 89.9% of plants using classical techniques. In total, 10 Phytophthora spp., 17 Pythium spp. and 5 Phytopythium spp. were isolated. Oomycetes were isolated from 86% of asymptomatic plants, but real-time PCR demonstrated that oomycetes were associated with all plants tested. More oomycete DNA occurred in composts in comparison with roots and filters from baiting water (a mean of 7.91 ng g−1, 6.55 × 10−1 ng g−1 and 5.62 × 10−1 ng g−1 of oomycete DNA detected in compost with ITS, trnM and atp9 probes, respectively); the ITS probe detected the highest quantities of oomycete DNA. No significant differences were found in quantities of oomycete DNA detected using real-time PCR in plants purchased online or from traditional retailers.

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Development of a real-time PCR assay for detection of Phytophthora kernoviae and comparison of this method with a conventional culturing technique

Cited by 15 publications

References 22 publications

Rapid diagnostics for Gnomoniopsis smithogilvyi (syn. Gnomoniopsis castaneae) in chestnut nuts: new challenges by using LAMP and real-time PCR methods

Rapid diagnostics for Gnomoniopsis smithogilvyi (syn. Gnomoniopsis castaneae) in chestnut nuts: new challenges by using LAMP and real-time PCR methods

The Use of qPCR Reveals a High Frequency of Phytophthora quercina in Two Spanish Holm Oak Areas

Application of Real-Time PCR for the Detection and Quantification of Oomycetes in Ornamental Nursery Stock

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