New multiplex conventional PCR and quadruplex real-time PCR assays for one-tube detection of Phyllosticta citricarpa, Elsinoë fawcettii, Elsinoë australis, and Pseudocercospora angolensis in Citrus: development and validation

Ahmed, Yosra; Hussein, Ahmed S.; Hubert, Jacqueline; Fourrier‐Jeandel, Céline; Aguayo, Jaime; Ioos, Renaud

doi:10.1007/s00253-020-10880-w

Cited by 3 publications

(4 citation statements)

References 40 publications

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“…This clearly shows how the Real-Time PCR systems that enabled inference of curves with lower intercept values guaranteed the detection of the same target DNA quantity—thus also of the LoD—at a lower C t than the other systems. The capacity of the Rotor-Gene TM 6000 for detection at a lower C t than other platforms was also previously observed in a comparison with Lightcycler 480 [ 31 , 32 ]. This would also imply that Rotor-Gene TM 6000 has the potential to lower the LoD of our method, which had been established using the CFX96 TM [ 18 ].…”

Section: Discussionsupporting

confidence: 64%

Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystis platani, the Agent of Canker Stain of Platanus spp.

Brunetti¹,

Heungens²,

Hubert

et al. 2022

JoF

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Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diagnostic assays are ineffective, a Real-Time PCR detection method based on EvaGreen, SYBR Green, and Taqman assays was previously developed, validated in-house, and included in the official EPPO standard PM7/14 (2). Here, we describe the results of a test performance study performed by nine European laboratories for the purpose of an interlaboratory validation. Verification of the DNA extracted from biological samples guaranteed the high quality of preparations, and the stability and the homogeneity of the aliquots intended for the laboratories. All of the laboratories reproduced nearly identical standard curves with efficiencies close to 100%. Testing of blind-coded DNA extracted from wood samples revealed that all performance parameters—diagnostic sensitivity, diagnostic specificity, accuracy and reproducibility—were best fit in most cases both at the laboratory and at the assay level. The previously established limit of detection, 3 fg per PCR reaction, was also validated with similar excellent results. The high interlaboratory performance of this Real-Time PCR method confirms its value as a primary tool to safeguard C. platani-free countries by way of an accurate monitoring, and to investigate the resistance level of potentially canker stain-resistant Platanus genotypes.

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Section: Discussionsupporting

confidence: 64%

Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystis platani, the Agent of Canker Stain of Platanus spp.

Brunetti¹,

Heungens²,

Hubert

et al. 2022

JoF

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“…False positive results can therefore be obtained when testing DNA of the non-target species P. paracitricarpa ( EPPO, 2020 ). Such cross-reactions with P. paracitricarpa DNA were also observed with the assay developed by Ahmed et al (2020) targeting phylogenetic marker MCM7. The method of Zajc et al (2022) is based on the tef1 gene, which should differ between P. citricarpa and P. paracitricarpa by five nucleotide changes ( Guarnaccia et al, 2017 ).…”

Section: Introductionmentioning

confidence: 55%

“…The current diagnostic protocols for detecting and identifying P. citricarpa include several real-time PCR (qPCR) methods ( Ahmed et al, 2020 ; Schirmacher et al, 2019 ; van Gent-Pelzer et al, 2007 ; Zajc et al, 2022 ) and conventional PCR (cPCR) methods ( Baayen et al, 2002 ; Peres et al, 2007 ). Most of these assays are based on the amplification of a specific region of the internal transcribed spacer (ITS) region of rDNA ( Bonants et al, 2003 ; Peres et al, 2007 ; Schirmacher et al, 2019 ; van Gent-Pelzer et al, 2007 ).…”

Section: Introductionmentioning

confidence: 99%

Harnessing the power of comparative genomics to support the distinction of sister species within Phyllosticta and development of highly specific detection of Phyllosticta citricarpa causing citrus black spot by real-time PCR

Ioos,

Puertolas,

Renault

et al. 2023

PeerJ

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Citrus crops are affected by many fungal diseases. Among them, Citrus Black Spot caused by the ascomycete Phyllosticta citricarpa is particularly economically damaging wherever it occurs. Many other species of Phyllosticta are described on Citrus, but only P. citricarpa is considered a quarantine pest on the European continent. In order to prevent the introduction of this species into Europe, it is essential to have a detection test which can reliably identify it, and not confuse it with other species present on citrus, notably P. paracitricarpa. The latter taxon has recently been described as very close to P. citricarpa, and most detection tests do not allow to distinguish the two species. In this work, we exploited the genomic data of 37 isolates of Phyllosticta spp. from citrus, firstly to assess their phylogenetic relationships, and secondly to search for genomic regions that allowed the definition of species-specific markers of P. citricarpa. Analysis of 51 concatenated genes separated P. citricarpa and P. paracitricarpa in two phylogenetic clades. A locus was selected to define a hydrolysis probe and primers combination that could be used in real-time PCR for the specific detection of the quarantine species, to the exclusion of all others present on Citrus. This test was then thoroughly validated on a set of strains covering a wide geographical diversity, and on numerous biological samples to demonstrate its reliability for regulatory control. The validation data highlighted the need to check the reliability of the test in advance, when a change of reagents was being considered.

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“…For instance, both P. citricarpa and P. paracitricarpa can cause CBS symptoms on citrus fruits and the two species differ from each other just for some nucleotides related to tef1 and LSU genes (Guarnaccia et al 2017). A range of molecular tests, including multiplex methods for the detection of different citrus pathogens simultaneously, have been developed to detect P. citricarpa (Ahmed et al 2020;EPPO 2020). Nevertheless, these tests cannot distinguish P. citricarpa from the closely related species P. paracitricarpa and P. citriasiana.…”

Section: Septoria Spot Of Citrusmentioning

confidence: 99%

Green solutions and new technologies for sustainable management of fungus and oomycete diseases in the citrus fruit supply chain

Rovetto,

La Spada,

Aloi

et al. 2024

J Plant Pathol

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This review deals with major diseases caused by fungi and oomycetes in the citrus supply chain, including post-harvest fruit diseases, and summarizes the strategies and techniques that may be adopted to prevent the damages and losses they cause. Its scope is to highlight the contribute that smart technologies provide towards new solutions for sustainable and safe management strategies of these diseases. Particular attention is given to the application of biopesticides, natural substances, resistance inducers and biostimulants to prevent fruit rots. The review focuses also on mycotoxins and mycotoxigenic fungi that contaminate fresh fruit and food products derived from citrus fruit, an aspect that has been little investigated and regulated so far. An additional relevant aspect addressed by the review is the early detection and routine diagnosis of fungal and oomycete pathogens that threat the international trade and long-distance shipment of citrus fruit, with a particular emphasis on quarantine pathogens. In this respect, the opportunities offered by new practical, rapid, sensitive and robust molecular diagnostic methods are briefly discussed.

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New multiplex conventional PCR and quadruplex real-time PCR assays for one-tube detection of Phyllosticta citricarpa, Elsinoë fawcettii, Elsinoë australis, and Pseudocercospora angolensis in Citrus: development and validation

Cited by 3 publications

References 40 publications

Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystis platani, the Agent of Canker Stain of Platanus spp.

Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystis platani, the Agent of Canker Stain of Platanus spp.

Harnessing the power of comparative genomics to support the distinction of sister species within Phyllosticta and development of highly specific detection of Phyllosticta citricarpa causing citrus black spot by real-time PCR

Green solutions and new technologies for sustainable management of fungus and oomycete diseases in the citrus fruit supply chain

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