Special Issue "Detection and Diagnostics of Fungal and Oomycete Plant Pathogens"

A special issue of Plants (ISSN 2223-7747). This special issue belongs to the section "Plant Protection and Biotic Interactions".

Deadline for manuscript submissions: 30 November 2020.

Special Issue Editor

Dr. Guillaume Bilodeau
Website
Guest Editor
Canadian Food Inspection Agency, Ottawa Plant Laboratory, Ottawa, ON, K2J 4S1, Canada
Interests: technologies for detection and identification of plant pests (fungi-oomycetes) of regulatory significance; fungal detection and genotyping; phytophthora; Verticillium; real-time PCR; genomic; metagenomic

Special Issue Information

Dear Colleagues,

In agriculture and forestry the development and validation of molecular detection tools for different organisms is important for phytosanitary export certification, which relies on having methods to identify plant pathogens associated with plants, grains and seeds. To develop detection–identification assays, DNA sequence information and genomic resources are important. Over the last decade, several genomes of plant pathogens have been made available, largely due to the development of high-throughput sequencing (HTS) technologies that have enabled lower costs for sequencing genomes and the acquisition of data conducive to running metagenomic analyses. Knowledge of the genome and its structure can help us better understand these organisms and is a valuable resource for the development of detection and genotyping tools. Often disease may be present in plants despite no symptoms being visible. This underscores the importance of having molecular methods for the detection of pathogens for improved sensitivity and to allow high-throughput sample processing while decreasing the dependency on time-consuming culturing methods. The development of technologies for the detection and identification of pathogenic fungi and oomycetes is continuously evolving as new and innovative tools for sequencing and analyzing genomes become available. Advances in techniques such as qPCR, isothermal amplification, AmpliSeq technologies and portable tools that provide faster and easier detection capabilities will be presented in this Special Issue.

Dr. Guillaume Bilodeau
Guest Editor

Manuscript Submission Information

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Keywords

  • detection and identification of plant pests
  • fungi-oomycetes
  • molecular detection
  • molecular tools
  • HTS detection
  • genomics

Published Papers (8 papers)

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Research

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Open AccessArticle
Early Detection of Airborne Inoculum of Nothopassalora personata in Spore Trap Samples from Peanut Fields Using Quantitative PCR
Plants 2020, 9(10), 1327; https://doi.org/10.3390/plants9101327 - 09 Oct 2020
Abstract
A quantitative PCR (qPCR)-assay was developed to detect airborne inoculum of Nothopassalora personata, causal agent of late leaf spot (LLS) on peanut, collected with a modified impaction spore trap. The qPCR assay was able to consistently detect as few as 10 spores [...] Read more.
A quantitative PCR (qPCR)-assay was developed to detect airborne inoculum of Nothopassalora personata, causal agent of late leaf spot (LLS) on peanut, collected with a modified impaction spore trap. The qPCR assay was able to consistently detect as few as 10 spores with purified DNA and 25 spores based on crude DNA extraction from rods. In 2019, two spore traps were placed in two peanut fields with a history of LLS. Sampling units were replaced every 2 to 4 days and tested with the developed qPCR assay, while plots were monitored for symptom development. The system detected inoculum 35 to 56 days before visual symptoms developed in the field, with detection related to environmental parameters affecting pathogen life-cycle and disease development. This study develops the framework of the qPCR spore trap system and represents the initial steps towards validation of the performance of the system for use as a decision support tool to complement integrated management of LLS. Full article
(This article belongs to the Special Issue Detection and Diagnostics of Fungal and Oomycete Plant Pathogens)
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Open AccessArticle
Comparative Microbiome Study of Mummified Peach Fruits by Metagenomics and Metatranscriptomics
Plants 2020, 9(8), 1052; https://doi.org/10.3390/plants9081052 - 18 Aug 2020
Abstract
The dried peach fruits clinging to peach trees or lying on the ground nearby are known as mummified peach fruits. Here, we examined the microbiome communities of three different mummified peach fruits from the nectarine cultivar “Hahong” by DNA- and RNA-sequencing. We found [...] Read more.
The dried peach fruits clinging to peach trees or lying on the ground nearby are known as mummified peach fruits. Here, we examined the microbiome communities of three different mummified peach fruits from the nectarine cultivar “Hahong” by DNA- and RNA-sequencing. We found the dominance of Monilinia fructigena followed by Sclerotinia borealis, S. sclerotiorum, and Botrytis cinerea in the mummified peach fruits. Moreover, we found a high number of Proteobacteria, including Frateuria aurantia, Neoasaia chiangmaiensis, Robbsia andropogonis, and Ewingella Americana. Furthermore, we identified several viruses and viroids. Bacteriophages were identified by DNA- and RNA-sequencing, while viruses and viroids with RNA genomes were identified by only RNA-sequencing. Moreover, we identified a novel mycovirus referred to as Monilinia umbra-like virus 1 (MULV1) from M. fructigena. Our results revealed the co-inhabitance of fungi and bacteria in the mummified peach fruits, although dominant microorganisms were present. RNA-sequencing revealed that several fungal and bacterial genes were actively transcribed. Comparative analyses suggested that RNA-sequencing provides more detailed information on microbial communities; however, combining DNA- and RNA-sequencing results increased the diversity of microorganisms, suggesting the importance of databases and analysis tools for microbiome studies. Taken together, our study provides a comprehensive overview of microbial communities in mummified peach fruits by DNA shotgun sequencing and RNA-sequencing. Full article
(This article belongs to the Special Issue Detection and Diagnostics of Fungal and Oomycete Plant Pathogens)
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Open AccessArticle
Occurrence of Sweetpotato (Ipomoea batatas) Wilt and Surface Rot Disease and Determining Resistance of Selected Varieties to the Pathogen in Korea
Plants 2020, 9(4), 497; https://doi.org/10.3390/plants9040497 - 13 Apr 2020
Abstract
Fusarium wilt and Fusarium surface rot caused by Fusarium oxysporum Schltdl are the major diseases of sweetpotato (Ipomoea batatas) and was surveyed in different locations (Cheongju, Heanam, Iksan, Icheon, Kimje, Nonsan, Yeoungam, and Yeoju) in Korea from 2015 to 2017 in [...] Read more.
Fusarium wilt and Fusarium surface rot caused by Fusarium oxysporum Schltdl are the major diseases of sweetpotato (Ipomoea batatas) and was surveyed in different locations (Cheongju, Heanam, Iksan, Icheon, Kimje, Nonsan, Yeoungam, and Yeoju) in Korea from 2015 to 2017 in the field, after harvesting and in storehouse. The wilt incidence in the early stage represented 17.9%, 5.9%, and 8.3% in 2015, 2016, and 2017, respectively. Samples were collected, and the causal organism was isolated on potato dextrose agar (PDA). Ten pure cultures were stored at the Sweetpotato Research Laboratory, Bioenergy Crop Research Institute, Muan, Korea. Morphological analysis, along with molecular phylogeny of the sequences of internal-transcribed spacer (ITS) and elongation factor 1-α (EF-1α) genes and their combined phylogenetic analysis, confirmed the isolates as the Fusarium oxysporum Schltdl. Pathogenicity tests were conducted on sweetpotato stems, and storage roots by artificially inoculation methods, and the most virulent isolate was selected as SPL18019. A rapid screening method on 21 selected varieties for resistant variety selection was applied on stems. The Pungwanmi was found resistant to Fusarium wilt, whereas Annobeni was the most susceptible. On the other hand, six varieties were used to test surface rot resistance, and Yulmi and Yesumi were resistant and susceptible, respectively, to Fusarium surface rot. Full article
(This article belongs to the Special Issue Detection and Diagnostics of Fungal and Oomycete Plant Pathogens)
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Open AccessArticle
Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for Phytophthora Species
Plants 2020, 9(4), 466; https://doi.org/10.3390/plants9040466 - 07 Apr 2020
Cited by 1
Abstract
Recombinase polymerase amplification (RPA) assays are valuable molecular diagnostic tools that can detect and identify plant pathogens in the field without time-consuming DNA extractions. Historically, RPA assay reagents were commercially available as a lyophilized pellet in microfuge strip tubes, but have become available [...] Read more.
Recombinase polymerase amplification (RPA) assays are valuable molecular diagnostic tools that can detect and identify plant pathogens in the field without time-consuming DNA extractions. Historically, RPA assay reagents were commercially available as a lyophilized pellet in microfuge strip tubes, but have become available in liquid form more recently—both require the addition of primers and probes prior to use, which can be challenging to handle in a field setting. Lyophilization of primers and probes, along with RPA reagents, contained within a single tube limits the risk of contamination, eliminates the need for refrigeration, as the lyophilized reagents are stable at ambient temperatures, and simplifies field use of the assays. This study investigates the potential effect of preformulation on assay performance using a previously validated Phytophthora genus-specific RPA assay, lyophilized with primers and probes included with the RPA reagents. The preformulated lyophilized Phytophthora RPA assay was compared with a quantitative polymerase chain reaction (qPCR) assay and commercially available RPA kits using three qPCR platforms (BioRad CFX96, QuantStudio 6 and Applied Biosystems ViiA7) and one isothermal platform (Axxin T16-ISO RPA), with experiments run in four separate labs. The assay was tested for sensitivity (ranging from 500 to 0.33 pg of DNA) and specificity using purified oomycete DNA, as well as crude extracts of Phytophthora-infected and non-infected plants. The limit of detection (LOD) using purified DNA was 33 pg in the CFX96 and ViiA7 qPCR platforms using the preformulated kits, while the Axxin T16-ISO RPA chamber and the QuantStudio 6 platform could detect down to 3.3 pg with or without added plant extract. The LOD using a crude plant extract for the BioRad CFX96 was 330 pg, whereas the LOD for the ViiA7 system was 33 pg. These trials demonstrate the consistency and uniformity of pathogen detection with preformulated RPA kits for Phytophthora detection when conducted by different labs using different instruments for measuring results. Full article
(This article belongs to the Special Issue Detection and Diagnostics of Fungal and Oomycete Plant Pathogens)
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Open AccessArticle
Phylogenetic Analysis and Development of Molecular Tool for Detection of Diaporthe citri Causing Melanose Disease of Citrus
Plants 2020, 9(3), 329; https://doi.org/10.3390/plants9030329 - 04 Mar 2020
Cited by 1
Abstract
Melanose disease caused by Diaporthe citri is considered as one of the most important and destructive diseases of citrus worldwide. In this study, isolates from melanose samples were obtained and analyzed. Firstly, the internal transcribed spacer (ITS) sequences were used to measure Diaporthe [...] Read more.
Melanose disease caused by Diaporthe citri is considered as one of the most important and destructive diseases of citrus worldwide. In this study, isolates from melanose samples were obtained and analyzed. Firstly, the internal transcribed spacer (ITS) sequences were used to measure Diaporthe-like boundary species. Then, a subset of thirty-eight representatives were selected to perform the phylogenetic analysis with combined sequences of ITS, beta-tubulin gene (TUB), translation elongation factor 1-α gene (TEF), calmodulin gene (CAL), and histone-3 gene (HIS). As a result, these representative isolates were identified belonging to D. citri, D. citriasiana, D. discoidispora, D. eres, D. sojae, and D. unshiuensis. Among these species, the D. citri was the predominant species that could be isolated at highest rate from different melanose diseased tissues. The morphological characteristics of representative isolates of D. citri were investigated on different media. Finally, a molecular tool based on the novel species-specific primer pair TUBDcitri-F1/TUBD-R1, which was designed from TUB gene, was developed to detect D. citri efficiently. A polymerase chain reaction (PCR) amplicon of 217 bp could be specifically amplified with the developed molecular tool. The sensitivity of the novel species-specific detection was upon to 10 pg of D. citri genomic DNA in a reaction. Therefore, the D. citri could be unequivocally identified from closely related Diaporthe species by using this simple PCR approach. Full article
(This article belongs to the Special Issue Detection and Diagnostics of Fungal and Oomycete Plant Pathogens)
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Open AccessArticle
A PCR, qPCR, and LAMP Toolkit for the Detection of the Wheat Blast Pathogen in Seeds
Plants 2020, 9(2), 277; https://doi.org/10.3390/plants9020277 - 21 Feb 2020
Cited by 1
Abstract
Wheat blast is a devastating disease caused by the pathogenic fungus Pyricularia oryzae. Wheat blast first emerged in South America before more recently reaching Bangladesh. Even though the pathogen can spread locally by air-dispersed spores, long-distance spread is likely to occur via infected [...] Read more.
Wheat blast is a devastating disease caused by the pathogenic fungus Pyricularia oryzae. Wheat blast first emerged in South America before more recently reaching Bangladesh. Even though the pathogen can spread locally by air-dispersed spores, long-distance spread is likely to occur via infected wheat seed or grain. Wheat blast epidemics are caused by a genetic lineage of the fungus, called the Triticum lineage, only differing from the other P. oryzae lineages by less than 1% genetic divergence. In order to prevent further spread of this pathogen to other wheat-growing areas in the world, sensitive and specific detection tools are needed to test for contamination of traded seed lots by the P. oryzae Triticum lineage. In this study, we adopted a comparative genomics approach to identify new loci specific to the P. oryzae Triticum lineage and used them to design a set of new markers that can be used in conventional polymerase chain reaction (PCR), real-time PCR, or loop-mediated isothermal amplification (LAMP) for the detection of the pathogen, with improved inclusivity and specificity compared to currently available tests. A preliminary biological enrichment step of the seeds was shown to improve the sensitivity of the tests, which enabled the detection of the target at an infection rate as low as 0.25%. Combined with others, this new toolkit may be particularly beneficial in preventing the trade of contaminated seeds and in limiting the spread of the disease. Full article
(This article belongs to the Special Issue Detection and Diagnostics of Fungal and Oomycete Plant Pathogens)
Open AccessCommunication
Uncovering the Host Range for Maize Pathogen Magnaporthiopsis maydis
Plants 2019, 8(8), 259; https://doi.org/10.3390/plants8080259 - 30 Jul 2019
Cited by 4
Abstract
The fungus Magnaporthiopsis maydis is a soil-borne, seed-borne vascular wilt pathogen that causes severe damage to sensitive Zea mays L. (maize) hybrids throughout Egypt, Israel, India, Spain, and other countries. It can undergo virulence variations and survive as spores, sclerotia, or mycelia on [...] Read more.
The fungus Magnaporthiopsis maydis is a soil-borne, seed-borne vascular wilt pathogen that causes severe damage to sensitive Zea mays L. (maize) hybrids throughout Egypt, Israel, India, Spain, and other countries. It can undergo virulence variations and survive as spores, sclerotia, or mycelia on plant residues. Maize, Lupinus termis L. (lupine) and Gossypium hirsutum L. (cotton) are the only known hosts of M. maydis. Identification of new plant hosts that can assist in the survival of the pathogen is an essential step in restricting disease outbreak and spread. Here, by field survey and growth chamber pathogenicity test, accompanied by real-time PCR analysis, the presence of the fungal DNA inside the roots of cotton (Pima cv.) plants was confirmed in infested soil. Moreover, we identified M. maydis in Setaria viridis (green foxtail) and Citrullus lanatus (watermelon, Malali cv.). Infected watermelon sprouts had delayed emergence and development, were shorter, and had reduced root and shoot biomass. M. maydis infection also affected root biomass and phenological development of cotton plants but caused only mild symptoms in green foxtail. No M. maydis DNA was detected in Hordeum vulgare (barley, Noga cv.) and the plants showed no disease symptoms except for reduced shoot weight. These findings are an important step towards uncovering the host range and endophytic behavior of M. maydis, encouraging expanding this evaluation to other plant species. Full article
(This article belongs to the Special Issue Detection and Diagnostics of Fungal and Oomycete Plant Pathogens)
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Review

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Open AccessReview
Molecular Diagnostics and Detection of Oomycetes on Fiber Crops
Plants 2020, 9(6), 769; https://doi.org/10.3390/plants9060769 - 19 Jun 2020
Abstract
Fiber crops are an important group of economic plants. Traditionally cultivated for fiber, fiber crops have also become sources of other materials such as food, animal feed, cosmetics and medicine. Asia and America are the two main production areas of fiber crops in [...] Read more.
Fiber crops are an important group of economic plants. Traditionally cultivated for fiber, fiber crops have also become sources of other materials such as food, animal feed, cosmetics and medicine. Asia and America are the two main production areas of fiber crops in the world. However, oomycete diseases have become an important factor limiting their yield and quality, causing devastating consequences for the production of fiber crops in many regions. To effectively control oomycete pathogens and reduce their negative impacts on these crops, it is very important to have fast and accurate detection systems, especially in the early stages of infection. With the rapid development of molecular biology, the diagnosis of plant pathogens has progressed from relying on traditional morphological features to the increasing use of molecular methods. The objective of this paper was to review the current status of research on molecular diagnosis of oomycete pathogens on fiber crops. Our search of PubMed identified nearly 30 species or subspecies of oomycetes on fiber crops, among which the top three species were Phytophthora boehmeriae, Phytophthora nicotianae and Pythium ultimum. The gene regions that have been used for molecular identifications of these pathogens include the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene cluster, and genes coding for translation elongation factor 1α (EF-1α) and mitochondrial cytochrome c oxidase subunits I and II (Cox 1, Cox 2), etc. We summarize the molecular assays that have been used to identify these pathogens and discuss potential areas of future development for fast, specific, and accurate diagnosis of oomycetes on fiber crops. Full article
(This article belongs to the Special Issue Detection and Diagnostics of Fungal and Oomycete Plant Pathogens)
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Planned Papers

The below list represents only planned manuscripts. Some of these manuscripts have not been received by the Editorial Office yet. Papers submitted to MDPI journals are subject to peer-review.

Identification and Diferentiation of Pathogenic Fungi Genus Verticillium with PCR Markers and Sequencing of ITS Region

Taja Jeseničnik1, Anela Kaurin1, Jernej Jakše1, Sebastjan Radišek2, Branka Javornik1, Nataša Štajner1*

1 University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, 1000 Ljubljana, Slovenia

2 Slovenian Institute for Hop Research and Brewing, Plant Protection Department, Cesta Žalskega tabora 2, 3310 Žalec, Slovenia

* Corresponding author (email: [email protected])

Abstract: The genus Verticillium is a group of ascomycete fungi, including plant-pathogenic species capable of affecting vasculature of many agricultural crops and therefore causing big economic losses worldwide. In 2011 a new taxonomic classification of the genus was established which now referred to as Verticillium sensu stricto, comprises of ten species of plant-pathogenic fungi.

The main objective of the work is linked to the taxonomic classification and nomenclature of genus Verticillium sensu stricto as suggested by Inderbitzin et al. (2013). First, the isolates collected in Slovenian Institute of Hop Research and Brewing were analysed with simplex and multiplex PCR assays, designed by Inderbitzin et al (2013) to identify their origin in accordance to the new taxonomic classification. Second, the isolates’ ITS region was sequenced, and the resulted phylogeny analysis were compared to PCR analyses to determine the compatibility with the newly introduced PCR markers. Since the unambiguous identification using molecular markers is crucial for the analyses considering fungi, the third objective was to test whether some more routinely used marker system, e.g. highly polymorphic simple sequence repeats (SSR markers) are enough effective for accurate identification of isolates. In addition, we used set of 9 markers originating from lethal region of Verticillium nonalfalafe to show the variability of certain regions within Verticillium sensu stricto species.

With new PCR primers we were able to identify 88 isolates out of 105 isolates obtained from different geographic locations in Europe, North America and Japan and from different host plants including hop, potato, tomato, cotton, olive and alfalfa. Among all determined isolates, 6 were identified as V. albo-atrum, 5 as V. alfalfae, 28 as V. dahliae, 3 as V. isaacii, 1 as V. longisporum lineage A1/D1, 41 as V. nonalfalfae, 1 as V. nubilum and 2 as V. tricorpus. Identification and differentiation of V. longisporum linegaes was implemented by multiplex PCR assay. 

Based on comparison of ITS sequencing and PCR/marker analysis reported previously we obtained some additional information about subgroups within species V. albo-atrum and V. dahliae.

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