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

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 435
Author(s):  
Andres F. Salcedo ◽  
Savithri Purayannur ◽  
Jeffrey R. Standish ◽  
Timothy Miles ◽  
Lindsey Thiessen ◽  
...  
Keyword(s):  
Downy Mildew ◽  
Pathogen Detection ◽  
Plant Pathogens ◽  
High Throughput Sequencing ◽  
Rapid Development ◽  
Target Selection ◽  
Nucleic Acid Amplification ◽  
Accurate Identification ◽  
Downy Mildews ◽  
Pathogen Variability

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.

scholarly journals Pathogen Detection and Microbiome Analysis of Infected Wheat Using a Portable DNA Sequencer

2018 ◽  
Author(s):  
Yiheng Hu ◽  
Gamran S. Green ◽  
Andrew W. Milgate ◽  
Eric A. Stone ◽  
John P. Rathjen ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Pathogen Detection ◽  
Plant Pathogens ◽  
Puccinia Striiformis ◽  
Fungal Diseases ◽  
Septoria Tritici Blotch ◽  
Septoria Tritici ◽  
Zymoseptoria Tritici ◽  
Accurate Identification ◽  
Pyrenophora Tritici Repentis

ABSTRACTFungal diseases of plants are responsible for major losses in agriculture, highlighting the need for rapid and accurate identification of plant pathogens. Disease outcomes are often defined not only by the main pathogen but are influenced by diverse microbial communities known as the microbiome at sites of infection. Here we present the first use of whole genome shot-gun sequencing with a portable DNA sequencing device as a method for the detection of fungal pathogens from wheat(Triticum aestivum)in a standard molecular biology laboratory. The data revealed that our method is robust and applicable to the diagnosis of fungal diseases including wheat stripe rust (caused byPuccinia striiformisf. sp.tritici),septoria tritici blotch (caused byZymoseptoria tritici)and yellow leaf spot (caused byPyrenophora tritici repentis).We also identified the bacterial genusPseudomonasco-present withPucciniaandZymoseptoriabut notPyrenophorainfections. One limitation of the method is the over-representation of redundant wheat genome sequences from samples. This could be addressed by long-range amplicon-based sequencing approaches in future studies, which specifically target non-host organisms. Our work outlines a new approach for detection of a broad range of plant pathogens and associated microbes using a portable sequencer in a standard laboratory, providing the basis for future development of an on-site disease monitoring system.

scholarly journals Chamomile Floricolous Downy Mildew Caused by Peronospora radii

2019 ◽  
Vol 109 (11) ◽  
pp. 1900-1907
Author(s):  
Bojan Duduk ◽  
Nataša Duduk ◽  
Ivana Vico ◽  
Jelena Stepanović ◽  
Tatjana Marković ◽  
...  
Keyword(s):  
Downy Mildew ◽  
Plant Pathogens ◽  
Systemic Infection ◽  
Downy Mildews ◽  
New Findings ◽  
High Production ◽  
The Impact ◽  
Chamomile Flowers ◽  
Production Losses

Floricolous downy mildews (Peronospora, oomycetes) are a small, monophyletic group of mostly inconspicuous plant pathogens that induce symptoms exclusively on flowers. Characterization of this group of pathogens, and information about their biology, is particularly sparse. The recurrent presence of a disease causing flower malformation which, in turn, leads to high production losses of the medicinal herb Matricaria chamomilla in Serbia has enabled continuous experiments focusing on the pathogen and its biology. Peronospora radii was identified as the causal agent of the disease, and morphologically and molecularly characterized. Diseased chamomile flowers showed severe malformations of the disc and ray florets, including phyllody and secondary inflorescence formation, followed by the onset of downy mildew. Phylogeny, based on internal transcribed spacer and cox2, indicates clustering of the Serbian P. radii with other P. radii from chamomile although, in cox2 analyses, they formed a separate subcluster. Evidence pointing to systemic infection was provided through histological and molecular analyses, with related experiments validating the impact of soilborne and blossom infections. This study provides new findings in the biology of P. radii on chamomile, thus enabling the reconstruction of this floricolous Peronospora species’ life cycle.

scholarly journals Pathogen Detection and Microbiome Analysis of Infected Wheat Using a Portable DNA Sequencer

2019 ◽  
Vol 3 (2) ◽  
pp. 92-101 ◽  
Author(s):  
Yiheng Hu ◽  
Gamran S. Green ◽  
Andrew W. Milgate ◽  
Eric A. Stone ◽  
John P. Rathjen ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Pathogen Detection ◽  
Plant Pathogens ◽  
Puccinia Striiformis ◽  
Fungal Diseases ◽  
Septoria Tritici Blotch ◽  
Septoria Tritici ◽  
Zymoseptoria Tritici ◽  
Accurate Identification ◽  
Pyrenophora Tritici Repentis

Fungal diseases of plants are responsible for major losses in agriculture, highlighting the need for rapid and accurate identification of plant pathogens. Disease outcomes are often defined not only by the main pathogen but are influenced by diverse microbial communities known as the microbiome at sites of infection. Here we present the first use of whole genome shot-gun sequencing with a portable DNA sequencing device as a method for the detection of fungal pathogens from wheat (Triticum aestivum) in a standard molecular biology laboratory. The data revealed that our method is robust and applicable to the diagnosis of fungal diseases including wheat stripe rust (caused by Puccinia striiformis f. sp. tritici), Septoria tritici blotch (caused by Zymoseptoria tritici), and yellow leaf spot (caused by Pyrenophora tritici repentis). We also identified the bacterial genus Pseudomonas co-present with Puccinia and Zymoseptoria but not Pyrenophora infections. One limitation of the method is the over-representation of redundant wheat genome sequences from samples. This could be addressed by long-range amplicon-based sequencing approaches in future studies, which specifically target nonhost organisms. Our work outlines a new approach for detection of a broad range of plant pathogens and associated microbes using a portable sequencer in a standard laboratory, providing the basis for future development of an on-site disease monitoring system. [Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

scholarly journals Advances in Diagnostics of Downy Mildews: Lessons Learned from Other Oomycetes and Future Challenges

Plant Disease ◽  
2018 ◽  
Vol 102 (2) ◽  
pp. 265-275 ◽  
Author(s):  
Sharifa G. Crandall ◽  
Alamgir Rahman ◽  
Lina M. Quesada-Ocampo ◽  
Frank N. Martin ◽  
Guillaume J. Bilodeau ◽  
...  
Keyword(s):  
United States ◽  
Downy Mildew ◽  
Plant Pathogens ◽  
The United States ◽  
The Philippines ◽  
Economic Losses ◽  
Diagnostic Tools ◽  
Detection Techniques ◽  
Downy Mildews ◽  
Critical Control Points

Downy mildews are plant pathogens that damage crop quality and yield worldwide. Among the most severe and notorious crop epidemics of downy mildew occurred on grapes in the mid-1880s, which almost destroyed the wine industry in France. Since then, there have been multiple outbreaks on sorghum and millet in Africa, tobacco in Europe, and recent widespread epidemics on lettuce, basil, cucurbits, and spinach throughout North America. In the mid-1970s, loss of corn to downy mildew in the Philippines was estimated at US$23 million. Today, crops that are susceptible to downy mildews are worth at least $7.5 billion of the United States’ economy. Although downy mildews cause devastating economic losses in the United States and globally, this pathogen group remains understudied because they are difficult to culture and accurately identify. Early detection of downy mildews in the environment is critical to establish pathogen presence and identity, determine fungicide resistance, and understand how pathogen populations disperse. Knowing when and where pathogens emerge is also important for identifying critical control points to restrict movement and to contain populations. Reducing the spread of pathogens also decreases the likelihood of sexual recombination events and discourages the emergence of novel virulent strains. A major challenge in detecting downy mildews is that they are obligate pathogens and thus cannot be cultured in artificial media to identify and maintain specimens. However, advances in molecular detection techniques hold promise for rapid and in some cases, relatively inexpensive diagnosis. In this article, we discuss recent advances in diagnostic tools that can be used to detect downy mildews. First, we briefly describe downy mildew taxonomy and genetic loci used for detection. Next, we review issues encountered when identifying loci and compare various traditional and novel platforms for diagnostics. We discuss diagnosis of downy mildew traits and issues to consider when detecting this group of organisms in different environments. We conclude with challenges and future directions for successful downy mildew detection.

scholarly journals Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Rachelle Bester ◽  
Glynnis Cook ◽  
Johannes H. J. Breytenbach ◽  
Chanel Steyn ◽  
Rochelle De Bruyn ◽  
...  
Keyword(s):  
High Throughput ◽  
Extraction Method ◽  
Pathogen Detection ◽  
High Throughput Sequencing ◽  
Expression Profiles ◽  
Rna Extraction ◽  
Healthy Plant ◽  
Ion Torrent ◽  
Extraction Protocol ◽  
Zymo Research

Abstract Background High-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount. Methods Plant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis. Results The study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols. Conclusions This study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.

scholarly journals High-Throughput Sequencing of Phage Display Libraries Reveals Parasitic Enrichment of Indel Mutants Caused by Amplification Bias

2021 ◽  
Vol 22 (11) ◽  
pp. 5513
Author(s):  
Sander Plessers ◽  
Vincent Van Deuren ◽  
Rob Lavigne ◽  
Johan Robben
Keyword(s):  
Amino Acid ◽  
Phage Display ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Target Selection ◽  
Acid Modification ◽  
Phage Display Technology ◽  
Amplification Bias ◽  
Large Deletions ◽  
Depth Analysis

The combination of phage display technology with high-throughput sequencing enables in-depth analysis of library diversity and selection-driven dynamics. We applied short-read sequencing of the mutagenized region on focused display libraries of two homologous nucleic acid modification eraser proteins—AlkB and FTO—biopanned against methylated DNA. This revealed enriched genotypes with small indels and concomitant doubtful amino acid motifs within the FTO library. Nanopore sequencing of the entire display vector showed additional enrichment of large deletions overlooked by region-specific sequencing, and further impacted the interpretation of the obtained amino acid motifs. We could attribute enrichment of these corrupted clones to amplification bias due to arduous FTO display slowing down host cell growth as well as phage production. This amplification bias appeared to be stronger than affinity-based target selection. Recommendations are provided for proper sequence analysis of phage display data, which can improve motive discovery in libraries of proteins that are difficult to display.

scholarly journals A High-Quality Grapevine Downy Mildew Genome Assembly Reveals Rapidly Evolving and Lineage-Specific Putative Host Adaptation Genes

2019 ◽  
Vol 11 (3) ◽  
pp. 954-969 ◽  
Author(s):  
Yann Dussert ◽  
Isabelle D Mazet ◽  
Carole Couture ◽  
Jérôme Gouzy ◽  
Marie-Christine Piron ◽  
...  
Keyword(s):  
Downy Mildew ◽  
Genome Assembly ◽  
Population Genomics ◽  
Plasmopara Viticola ◽  
Plant Diseases ◽  
Host Specialization ◽  
Grapevine Downy Mildew ◽  
Downy Mildews ◽  
Genes Encoding ◽  
High Level

Abstract Downy mildews are obligate biotrophic oomycete pathogens that cause devastating plant diseases on economically important crops. Plasmopara viticola is the causal agent of grapevine downy mildew, a major disease in vineyards worldwide. We sequenced the genome of Pl. viticola with PacBio long reads and obtained a new 92.94 Mb assembly with high contiguity (359 scaffolds for a N50 of 706.5 kb) due to a better resolution of repeat regions. This assembly presented a high level of gene completeness, recovering 1,592 genes encoding secreted proteins involved in plant–pathogen interactions. Plasmopara viticola had a two-speed genome architecture, with secreted protein-encoding genes preferentially located in gene-sparse, repeat-rich regions and evolving rapidly, as indicated by pairwise dN/dS values. We also used short reads to assemble the genome of Plasmopara muralis, a closely related species infecting grape ivy (Parthenocissus tricuspidata). The lineage-specific proteins identified by comparative genomics analysis included a large proportion of RxLR cytoplasmic effectors and, more generally, genes with high dN/dS values. We identified 270 candidate genes under positive selection, including several genes encoding transporters and components of the RNA machinery potentially involved in host specialization. Finally, the Pl. viticola genome assembly generated here will allow the development of robust population genomics approaches for investigating the mechanisms involved in adaptation to biotic and abiotic selective pressures in this species.

scholarly journals Nucleic acid amplification-based pathogen detection in the blood of severe sepsis patients

Critical Care ◽  
2008 ◽  
Vol 12 (Suppl 5) ◽  
pp. P43 ◽  
Author(s):  
Frank Bloos ◽  
Svea Sachse ◽  
Karl-Herrmann Schmidt ◽  
Mark Lehmann ◽  
Roland Schmitz ◽  
...  
Keyword(s):  
Severe Sepsis ◽  
Nucleic Acid ◽  
Pathogen Detection ◽  
Nucleic Acid Amplification

scholarly journals Nontoxic Nep1-Like Proteins of the Downy Mildew Pathogen Hyaloperonospora arabidopsidis: Repression of Necrosis-Inducing Activity by a Surface-Exposed Region

2012 ◽  
Vol 25 (5) ◽  
pp. 697-708 ◽  
Author(s):  
Adriana Cabral ◽  
Stan Oome ◽  
Nick Sander ◽  
Isabell Küfner ◽  
Thorsten Nürnberger ◽  
...  
Keyword(s):  
Amino Acids ◽  
Downy Mildew ◽  
Plant Pathogens ◽  
Exposed Region ◽  
Alternative Function ◽  
Early Expression ◽  
Specific Expansion ◽  
Species Specific ◽  
Hyaloperonospora Arabidopsidis ◽  
Specific Cluster

The genome of the downy mildew pathogen Hyaloperonospora arabidopsidis encodes necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP). Although NLP are widely distributed in eukaryotic and prokaryotic plant pathogens, it was surprising to find these proteins in the obligate biotrophic oomycete H. arabidopsidis. Therefore, we analyzed the H. arabidopsidis NLP (HaNLP) family and identified 12 HaNLP genes and 15 pseudogenes. Most of the 27 genes form an H. arabidopsidis–specific cluster when compared with other oomycete NLP genes, suggesting this class of effectors has recently expanded in H. arabidopsidis. HaNLP transcripts were mainly detected during early infection stages. Agrobacterium tumefaciens–mediated transient expression and infiltration of recombinant NLP into tobacco and Arabidopsis leaves revealed that all HaNLP tested are noncytotoxic proteins. Even HaNLP3, which is most similar to necrosis-inducing NLP proteins of other oomycetes and which contains all amino acids that are critical for necrosis-inducing activity, did not induce necrosis. Chimeras constructed between HaNLP3 and the necrosis-inducing PsojNIP protein demonstrated that most of the HaNLP3 protein is functionally equivalent to PsojNIP, except for an exposed domain that prevents necrosis induction. The early expression and species-specific expansion of the HaNLP genes is suggestive of an alternative function of noncytolytic NLP proteins during biotrophic infection of plants.

scholarly journals Transcriptome analysis of lateral buds from Phyllostachys edulis rhizome during germination and early shoot stages

2020 ◽  
Author(s):  
Yuting Shou ◽  
Yihua Zhu ◽  
Yulong Ding
Keyword(s):  
Signaling Pathways ◽  
High Throughput Sequencing ◽  
Stem Elongation ◽  
Rapid Development ◽  
Development Stage ◽  
Fast Growth ◽  
Moso Bamboo ◽  
Phyllostachys Edulis ◽  
Lateral Buds ◽  
Meristem Development

Abstract Background: The vegetative growth is an important stage for plants when they conduct photosynthesis, accumulate and collect all resources needed and prepare for reproduction stage. Bamboo is one of the fastest growing plant species. The rapid growth of Phyllostachys edulis results from the expansion of intercalary meristem at the basal part of nodes, which are differentiated from the apical meristem of rhizome lateral buds. However, little is known about the major signaling pathways and players involved during this rapid development stage of bamboo. To study this question, we adopted the high-throughput sequencing technology and compared the transcriptomes of Moso bamboo rhizome buds in germination stage and late development stage. Results: We found that the development of Moso bamboo rhizome lateral buds was coordinated by multiple pathways, including meristem development, sugar metabolism and phytohormone signaling. Phytohormones have fundamental impacts on the plant development. We found the evidence of several major hormones participating in the development of Moso bamboo rhizome lateral bud. Furthermore, we showed direct evidence that Gibberellic Acids (GA) signaling participated in the Moso bamboo stem elongation. Conclusion: Significant changes occur in various signaling pathways during the development of rhizome lateral buds. It is crucial to understand how these changes are translated to Phyllostachys edulis fast growth. These results expand our knowledge on the Moso bamboo internodes fast growth and provide research basis for further study.

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