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Pathogens
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Diagnostic Methods of Emerging Plant Pathogens (Viruses, Viroids and Phytoplasmas)
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Diagnostic Methods of Emerging Plant Pathogens (Viruses, Viroids and Phytoplasmas)

A special issue of Pathogens (ISSN 2076-0817). This special issue belongs to the section "Emerging Pathogens".

Deadline for manuscript submissions: closed (31 December 2021) | Viewed by 26005

Special Issue Editor

Dr. Nataša Mehle

E-Mail Website
Guest Editor
Department of Biotechnology and Systems Biology, National Institute of Biology, 1000 Ljubljana, Slovenia
Interests: plant virology; viroids; phytoplasma; molecular diagnosis; development and validation of new tests

Special Issue Information

Dear Colleagues,

Emerging pathogens of crops (e.g., viruses, viroids, and phytoplasmas) threaten food security and are increasingly problematic due to intensive agriculture and high volumes of trade and transport in plants and plant products. These plant pathogens can be managed most effectively when control measures are implemented at an early stage of infestation. In order to take action against these pathogens, they must be accurately identified. To enable safe trade, their diagnosis must further be completed quickly and to a high level of confidence. Diagnostics of plant viruses, viroids, and phytoplasmas are based on the use of a range of methods such as biological indexing, conventional PCR, real-time PCR, digital PCR, LAMP, etc. In addition, with the advent of high-throughput sequencing (HTS), new pathogens have been discovered, facilitating the association and identification of the etiological agents of unknown diseases.

For the forthcoming Special Issue of Pathogens, we invite you to submit research or review articles related to the latest advances in different aspects of the diagnosis of the emerging plant viruses, viroids, and phytoplasmas. Potential topics of interest to this Special Issue include, but are not limited to, new detection and identification tests for these pathogens. Contributions highlighting the usefulness of new techniques to improve the understanding and prediction of disease spread or disease control, and articles providing deep validation data or extensive comparison studies of existing tests will also be considered. We look forward to your contribution.

Adj. Prof. Dr. Nataša Mehle
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Pathogens is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2200 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • detection methods
  • HTS
  • validation of tests
  • plant viruses
  • viroids
  • phytoplasmas

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Published Papers (6 papers)

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Research

12 pages, 1421 KiB  
Article
Inter-Laboratory Comparison of RT-PCR-Based Methods for the Detection of Tomato Brown Rugose Fruit Virus on Tomato
by Marta Luigi, Ariana Manglli, Antonio Tiberini, Sabrina Bertin, Luca Ferretti, Anna Taglienti, Francesco Faggioli and Laura Tomassoli
Pathogens 2022, 11(2), 207; https://doi.org/10.3390/pathogens11020207 - 3 Feb 2022
Cited by 11 | Viewed by 3095
Abstract
In 2020, a test performance study (TPS) for the specific detection of tomato brown rugose fruit virus (ToBRFV) was organized in the frame of the H2020 Valitest project. Since no validated tests were available, all the protocols reported in the literature were at [...] Read more.
In 2020, a test performance study (TPS) for the specific detection of tomato brown rugose fruit virus (ToBRFV) was organized in the frame of the H2020 Valitest project. Since no validated tests were available, all the protocols reported in the literature were at first screened, performing preliminary studies in accordance with the EPPO standard PM 7/98 (4). Five molecular tests, two conventional RT-PCR and three real-time RT-PCR were found to be suitable and were included in the TPS. Thirty-four laboratories from 18 countries worldwide took part in TPS, receiving a panel of 22 blind samples. The panel consisted of sap belonging to symptomatic or asymptomatic leaves of Solanum lycopersicum and Capsicum annuum. The results returned by each laboratory were analyzed and diagnostic parameters were assessed for each test: reproducibility, repeatability, analytical sensitivity, diagnostic sensitivity and diagnostic specificity. All the evaluated tests resulted in being reliable in detecting ToBRFV and were included in an EPPO Standard PM 7/146—Diagnostics. Full article
(This article belongs to the Special Issue Diagnostic Methods of Emerging Plant Pathogens (Viruses, Viroids and Phytoplasmas))
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11 pages, 1255 KiB  
Article
Real-Time On-Site Diagnosis of Quarantine Pathogens in Plant Tissues by Nanopore-Based Sequencing
by Luca Marcolungo, Alessandro Passera, Simone Maestri, Elena Segala, Massimiliano Alfano, Francesca Gaffuri, Giovanni Marturano, Paola Casati, Piero Attilio Bianco and Massimo Delledonne
Pathogens 2022, 11(2), 199; https://doi.org/10.3390/pathogens11020199 - 2 Feb 2022
Cited by 23 | Viewed by 5308
Abstract
Rapid and sensitive assays for the identification of plant pathogens are necessary for the effective management of crop diseases. The main limitation of current diagnostic testing is the inability to combine broad and sensitive pathogen detection with the identification of key strains, pathovars, [...] Read more.
Rapid and sensitive assays for the identification of plant pathogens are necessary for the effective management of crop diseases. The main limitation of current diagnostic testing is the inability to combine broad and sensitive pathogen detection with the identification of key strains, pathovars, and subspecies. Such discrimination is necessary for quarantine pathogens, whose management is strictly dependent on genotype identification. To address these needs, we have established and evaluated a novel all-in-one diagnostic assay based on nanopore sequencing for the detection and simultaneous characterization of quarantine pathogens, using Xylella fastidiosa as a case study. The assay proved to be at least as sensitive as standard diagnostic tests and the quantitative results agreed closely with qPCR-based analysis. The same sequencing results also allowed discrimination between subspecies when present either individually or in combination. Pathogen detection and typing were achieved within 13 min of sequencing owing to the use of an internal control that allowed to stop sequencing when sufficient data had accumulated. These advantages, combined with the use of portable equipment, will facilitate the development of next-generation diagnostic assays for the efficient monitoring of other plant pathogens. Full article
(This article belongs to the Special Issue Diagnostic Methods of Emerging Plant Pathogens (Viruses, Viroids and Phytoplasmas))
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16 pages, 1203 KiB  
Article
Interlaboratory Comparison Study on Ribodepleted Total RNA High-Throughput Sequencing for Plant Virus Diagnostics and Bioinformatic Competence
by Yahya Z. A. Gaafar, Marcel Westenberg, Marleen Botermans, Krizbai László, Kris De Jonghe, Yoika Foucart, Luca Ferretti, Denis Kutnjak, Anja Pecman, Nataša Mehle, Jan Kreuze, Giovanna Muller, Nikolaos Vakirlis, Despoina Beris, Christina Varveri and Heiko Ziebell
Pathogens 2021, 10(9), 1174; https://doi.org/10.3390/pathogens10091174 - 12 Sep 2021
Cited by 14 | Viewed by 4339
Abstract
High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS workflows from sample preparation to data analysis and results interpretation for plant virus detection [...] Read more.
High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS workflows from sample preparation to data analysis and results interpretation for plant virus detection and identification must be evaluated (verified and validated) to approve this tool for diagnostics. Many different extraction methods, library preparation protocols, and sequence and bioinformatic pipelines are available for virus sequence detection. To assess the performance of plant virology diagnostic laboratories in using the HTS of ribosomal RNA depleted total RNA (ribodepleted totRNA) as a diagnostic tool, we carried out an interlaboratory comparison study in which eight participants were required to use the same samples, (RNA) extraction kit, ribosomal RNA depletion kit, and commercial sequencing provider, but also their own bioinformatics pipeline, for analysis. The accuracy of virus detection ranged from 65% to 100%. The false-positive detection rate was very low and was related to the misinterpretation of results as well as to possible cross-contaminations in the lab or sequencing provider. The bioinformatic pipeline used by each laboratory influenced the correct detection of the viruses of this study. The main difficulty was the detection of a novel virus as its sequence was not available in a publicly accessible database at the time. The raw data were reanalysed using Virtool to assess its ability for virus detection. All virus sequences were detected using Virtool in the different pools. This study revealed that the ribodepletion target enrichment for sample preparation is a reliable approach for the detection of plant viruses with different genomes. A significant level of virology expertise is needed to correctly interpret the results. It is also important to improve and complete the reference data. Full article
(This article belongs to the Special Issue Diagnostic Methods of Emerging Plant Pathogens (Viruses, Viroids and Phytoplasmas))
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14 pages, 987 KiB  
Article
Identification of Divergent Isolates of Banana Mild Mosaic Virus and Development of a New Diagnostic Primer to Improve Detection
by Marwa Hanafi, Rachid Tahzima, Sofiene Ben Kaab, Lucie Tamisier, Nicolas Roux and Sébastien Massart
Pathogens 2020, 9(12), 1045; https://doi.org/10.3390/pathogens9121045 - 12 Dec 2020
Cited by 6 | Viewed by 3450
Abstract
Banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus belonging to the Betaflexiviridae family. It infects Musa spp. with a very wide geographic distribution. The genome variability of plant viruses, including the members of the Betaflexiviridae family, makes their [...] Read more.
Banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus belonging to the Betaflexiviridae family. It infects Musa spp. with a very wide geographic distribution. The genome variability of plant viruses, including the members of the Betaflexiviridae family, makes their molecular detection by specific primers particularly challenging. During routine indexing of the Musa germplasm accessions, a discrepancy was observed between electron microscopy and immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) test results for one asymptomatic accession. Filamentous viral particles were observed while molecular tests failed to amplify any fragment. The accession underwent high-throughput sequencing and two complete genomes of BanMMV with 75.3% of identity were assembled. Based on these sequences and on the 54 coat protein sequences available from GenBank, a new forward primer, named BanMMV CP9, compatible with Poty1, an oligodT reverse primer already used in diagnostics, was designed. A retrospective analysis of 110 different germplasm accessions from diverse origins was conducted, comparing BanMMCP2 and BanMMV CP9 primers. Of these 110 accessions, 16 tested positive with both BanMMCP2 and BanMMV CP9, 3 were positive with only BanMMCP2 and 2 tested positive with only BanMMV CP9. Otherwise, 89 were negative with the two primers and free of flexuous virions. Sanger sequencing was performed from purified PCR products in order to confirm the amplification of the BanMMV sequence for the five accessions with contrasting results. It is highly recommended to use the two primers successively to improve the inclusiveness of the protocol. Full article
(This article belongs to the Special Issue Diagnostic Methods of Emerging Plant Pathogens (Viruses, Viroids and Phytoplasmas))
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20 pages, 4178 KiB  
Article
Efficient Confirmation of Plant Viral Proteins and Identification of Specific Viral Strains by nanoLC-ESI-Q-TOF Using Single-Leaf-Tissue Samples
by Pavel Cejnar, Štěpánka Kučková, Jiří Šantrůček, Miroslav Glasa, Petr Komínek, Daniel Mihálik, Lucie Slavíková, Leona Leišová-Svobodová, Tatiana Smirnova, Radovan Hynek, Jiban Kumar Kundu and Pavel Ryšánek
Pathogens 2020, 9(11), 966; https://doi.org/10.3390/pathogens9110966 - 19 Nov 2020
Cited by 3 | Viewed by 3367
Abstract
Plant viruses are important pathogens that cause significant crop losses. A plant protein extraction protocol that combines crushing the tissue by a pestle in liquid nitrogen with subsequent crushing by a roller-ball crusher in urea solution, followed by RuBisCO depletion, reduction, alkylation, protein [...] Read more.
Plant viruses are important pathogens that cause significant crop losses. A plant protein extraction protocol that combines crushing the tissue by a pestle in liquid nitrogen with subsequent crushing by a roller-ball crusher in urea solution, followed by RuBisCO depletion, reduction, alkylation, protein digestion, and ZipTip purification allowed us to substantially simplify the sample preparation by removing any other precipitation steps and to detect viral proteins from samples, even with less than 0.2 g of leaf tissue, by a medium resolution nanoLC-ESI-Q-TOF. The presence of capsid proteins or polyproteins of fourteen important viruses from seven different families (Geminiviridae, Luteoviridae, Bromoviridae, Caulimoviridae, Virgaviridae, Potyviridae, and Secoviridae) isolated from ten different economically important plant hosts was confirmed through many identified pathogen-specific peptides from a protein database of host proteins and potential pathogen proteins assembled separately for each host and based on existing online plant virus pathogen databases. The presented extraction protocol, combined with a medium resolution LC-MS/MS, represents a cost-efficient virus protein confirmation method that proved to be effective at identifying virus strains (as demonstrated for PPV, WDV) and distinct disease species of BYDV, as well as putative new viral protein sequences from single-plant-leaf tissue samples. Data are available via ProteomeXchange with identifier PXD022456. Full article
(This article belongs to the Special Issue Diagnostic Methods of Emerging Plant Pathogens (Viruses, Viroids and Phytoplasmas))
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13 pages, 1992 KiB  
Article
Development and Validation of a New TaqMan Real-Time PCR for Detection of ‘Candidatus Phytoplasma pruni’
by Zala Kogej, Marina Dermastia and Nataša Mehle
Pathogens 2020, 9(8), 642; https://doi.org/10.3390/pathogens9080642 - 7 Aug 2020
Cited by 13 | Viewed by 4856
Abstract
Phytoplasmas of the 16SrIII group are wide spread, and have a broad plant host range. Among these, ‘Candidatus phytoplasma pruni’ (‘Ca. P. pruni’; phytoplasmas of 16SrIII subgroup A) can cause serious diseases in Prunus species and ‘Ca. P. pruni’-related strains [...] Read more.
Phytoplasmas of the 16SrIII group are wide spread, and have a broad plant host range. Among these, ‘Candidatus phytoplasma pruni’ (‘Ca. P. pruni’; phytoplasmas of 16SrIII subgroup A) can cause serious diseases in Prunus species and ‘Ca. P. pruni’-related strains can infect other plant species, including grapevines. In this study, a new real-time PCR detection system was developed for ‘Ca. P. pruni’ using TaqMan chemistry. This test was designed to detect ‘Ca. P. pruni’, by amplifying the species-specific secY gene. In addition, a test to amplify the group-specific 16S rRNA gene region was also developed. The performances of both tests were evaluated. The test that amplifies the secY gene provided reliable and quick detection of ‘Ca. P. pruni’. Using the newly developed and validated test, ‘Ca. P. pruni’ was not found in any of the 434 field samples collected from different plants species grown in different regions of Slovenia. Full article
(This article belongs to the Special Issue Diagnostic Methods of Emerging Plant Pathogens (Viruses, Viroids and Phytoplasmas))
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