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Detection and Diagnostics of Fungal and Oomycete Plant Pathogens

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

Deadline for manuscript submissions: closed (31 January 2021) | Viewed by 56090

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Dr. Guillaume Bilodeau

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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
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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

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Keywords

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

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Published Papers (10 papers)

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Research

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17 pages, 6124 KiB

Open AccessArticle

Detection and Quantification of Rhizoctonia solani and Rhizoctonia solani AG1-IB Causing the Bottom Rot of Lettuce in Tissues and Soils by Multiplex qPCR

by Thérèse Wallon, Andréanne Sauvageau and Hervé Van der Heyden

Plants 2021, 10(1), 57; https://doi.org/10.3390/plants10010057 - 29 Dec 2020

Cited by 22 | Viewed by 4770

Abstract

In the muck soil region of southwestern Quebec, vegetable growers are threatened by several soilborne diseases, particularly the bottom rot of lettuce caused by the fungus Rhizoctonia solani. The particularly warm temperature of the few last seasons was marked by an increase in disease severity, and the associated yield losses were significant for Quebec lettuce growers. In the absence of registered fungicides and resistant cultivars, the management of Rhizoctonia solani-induced diseases in lettuce is based on good agricultural practices, which require detailed knowledge of the pathogen. In this study, Rhizoctonia solani fungal strains were isolated from infected field-grown lettuce plants presenting bottom rot symptoms to determine the anastomotic groups (AGs) of these isolates by internal transcribed spacer region (ITS) sequencing. Rhizoctonia solani AG 1-IB was identified as the main anastomotic group causing bottom rot lettuce in field-grown lettuce in organic soils in the Montérégie region. Two specific and sensitive quantitative PCR assays were then developed for R. solani AG1-IB and R. solani. The AG 1-IB qPCR assay amplified all strains of R. solani AG 1-IB tested, and no PCR product was obtained for any non-target strains. The R. solani qPCR assay amplified all strains of R. solani and did not amplify non-target strains, except for two strains of binucleate Rhizoctonia AG-E. In artificially inoculated soils, the sensitivity of both qPCR assays was set to 1 μg of sclerotia g⁻¹ of dry soil. In the growth chamber experiment, a minimum concentration between 14 and 42 μg sclerotia g⁻¹ of dry soil was required to induce the development of symptoms on the lettuce. Indeed, the AG 1-IB qPCR assay was sensitive enough to detect the lowest soil concentration of AG1-IB capable of inducing symptoms in head lettuce. In addition, the qPCR assays successfully detected R. solani and R. solani AG 1-IB from infected plant tissue samples and soil samples from lettuce fields. The qPCR assays developed in this study will be useful tools in lettuce bottom rot management. Full article

(This article belongs to the Special Issue Detection and Diagnostics of Fungal and Oomycete Plant Pathogens)

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15 pages, 2961 KiB

Open AccessArticle

Early Detection of Airborne Inoculum of Nothopassalora personata in Spore Trap Samples from Peanut Fields Using Quantitative PCR

by Misbakhul Munir, Hehe Wang, Nicholas S. Dufault and Daniel J. Anco

Plants 2020, 9(10), 1327; https://doi.org/10.3390/plants9101327 - 9 Oct 2020

Cited by 11 | Viewed by 3177

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 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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18 pages, 3556 KiB

Open AccessArticle

Comparative Microbiome Study of Mummified Peach Fruits by Metagenomics and Metatranscriptomics

by Yeonhwa Jo, Chang-Gi Back, Hoseong Choi and Won Kyong Cho

Plants 2020, 9(8), 1052; https://doi.org/10.3390/plants9081052 - 18 Aug 2020

Cited by 15 | Viewed by 4529

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 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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14 pages, 1899 KiB

Open AccessArticle

Occurrence of Sweetpotato (Ipomoea batatas) Wilt and Surface Rot Disease and Determining Resistance of Selected Varieties to the Pathogen in Korea

by Narayan Chandra Paul, Won Park, Seungyong Lee, Mi Nam Chung, Hyeong-Un Lee and Jung-Wook Yang

Plants 2020, 9(4), 497; https://doi.org/10.3390/plants9040497 - 13 Apr 2020

Cited by 9 | Viewed by 5564

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 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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17 pages, 2925 KiB

Open AccessArticle

Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for Phytophthora Species

by Austin G. McCoy, Timothy D. Miles, Guillaume J. Bilodeau, Patrick Woods, Cheryl Blomquist, Frank N. Martin and Martin I. Chilvers

Plants 2020, 9(4), 466; https://doi.org/10.3390/plants9040466 - 7 Apr 2020

Cited by 19 | Viewed by 4577

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 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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22 pages, 2607 KiB

Open AccessArticle

Phylogenetic Analysis and Development of Molecular Tool for Detection of Diaporthe citri Causing Melanose Disease of Citrus

by Chingchai Chaisiri, Xiang-Yu Liu, Yang Lin, Jiang-Bo Li, Bin Xiong and Chao-Xi Luo

Plants 2020, 9(3), 329; https://doi.org/10.3390/plants9030329 - 4 Mar 2020

Cited by 26 | Viewed by 7574

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-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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14 pages, 259 KiB

Open AccessArticle

A PCR, qPCR, and LAMP Toolkit for the Detection of the Wheat Blast Pathogen in Seeds

by Maud Thierry, Axel Chatet, Elisabeth Fournier, Didier Tharreau and Renaud Ioos

Plants 2020, 9(2), 277; https://doi.org/10.3390/plants9020277 - 21 Feb 2020

Cited by 18 | Viewed by 4400

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 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)

9 pages, 1125 KiB

Open AccessCommunication

Uncovering the Host Range for Maize Pathogen Magnaporthiopsis maydis

by Shlomit Dor and Ofir Degani

Plants 2019, 8(8), 259; https://doi.org/10.3390/plants8080259 - 30 Jul 2019

Cited by 23 | Viewed by 3713

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 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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24 pages, 3332 KiB

Open AccessReview

Fantastic Downy Mildew Pathogens and How to Find Them: Advances in Detection and Diagnostics

by Andres F. Salcedo, Savithri Purayannur, Jeffrey R. Standish, Timothy Miles, Lindsey Thiessen and Lina M. Quesada-Ocampo

Plants 2021, 10(3), 435; https://doi.org/10.3390/plants10030435 - 25 Feb 2021

Cited by 17 | Viewed by 10881

Abstract

Downy mildews affect important crops and cause severe losses in production worldwide. Accurate identification and monitoring of these plant pathogens, especially at early stages of the disease, is fundamental in achieving effective disease control. The rapid development of molecular methods for diagnosis has provided more specific, fast, reliable, sensitive, and portable alternatives for plant pathogen detection and quantification than traditional approaches. In this review, we provide information on the use of molecular markers, serological techniques, and nucleic acid amplification technologies for downy mildew diagnosis, highlighting the benefits and disadvantages of the technologies and target selection. We emphasize the importance of incorporating information on pathogen variability in virulence and fungicide resistance for disease management and how the development and application of diagnostic assays based on standard and promising technologies, including high-throughput sequencing and genomics, are revolutionizing the development of species-specific assays suitable for in-field diagnosis. Our review provides an overview of molecular detection technologies and a practical guide for selecting the best approaches for diagnosis. Full article

(This article belongs to the Special Issue Detection and Diagnostics of Fungal and Oomycete Plant Pathogens)

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22 pages, 742 KiB

Open AccessReview

Molecular Diagnostics and Detection of Oomycetes on Fiber Crops

by Tuhong Wang, Chunsheng Gao, Yi Cheng, Zhimin Li, Jia Chen, Litao Guo and Jianping Xu

Plants 2020, 9(6), 769; https://doi.org/10.3390/plants9060769 - 19 Jun 2020

Cited by 16 | Viewed by 5048

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 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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