Using Next-Generation Sequencing to Develop Molecular Diagnostics for <i>Pseudoperonospora cubensis</i>, the Cucurbit Downy Mildew Pathogen

Withers, Simon; Góngora-Castillo, Elsa; Gent, David H.; Thomas, Anna; Ojiambo, P. S.; Quesada‐Ocampo, Lina M.

doi:10.1094/phyto-10-15-0260-fi

Cited by 48 publications

(18 citation statements)

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“…Next generation sequencing (NGS) technologies have a huge potential in the diagnostic space as they can identify multiple pathogens in a single analysis without any previous knowledge of their nature ( Massart et al, 2014 ; Withers et al, 2016 ; Rott et al, 2017 ). Nevertheless, they still need to overcome a number of limitations before they can be used for POC applications.…”

Section: Discussionmentioning

confidence: 99%

Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection

2017

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Diagnostic technologies for the detection of plant pathogens with point-of-care capability and high multiplexing ability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability. This article describes and discusses various DNA-based point-of care diagnostic methods for applications in plant disease detection. Polymerase chain reaction (PCR) is the most common DNA amplification technology used for detecting various plant and animal pathogens. However, subsequent to PCR based assays, several types of nucleic acid amplification technologies have been developed to achieve higher sensitivity, rapid detection as well as suitable for field applications such as loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, recombinase polymerase amplification, and molecular inversion probe. The principle behind these technologies has been thoroughly discussed in several review papers; herein we emphasize the application of these technologies to detect plant pathogens by outlining the advantages and disadvantages of each technology in detail.

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Section: Discussionmentioning

confidence: 99%

Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection

2017

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“…A pathotype specific marker has been developed from the genome of M. oryzae f.sp. triticum (Pieck et al 2016) and comparative genomics has detected diagnostic regions in two Calonectria species (Malapi-Wight et al 2016) and in Pseudoperonospora cubensis (Withers et al 2016). Continued application of fungal genomes to generate identification tools is bound to increase the efficiency of quarantine procedures (McTaggart et al 2016).…”

Section: Impact Of Genomes On Plant Pathologymentioning

confidence: 99%

A plant pathology perspective of fungal genome sequencing

et al. 2017

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The majority of plant pathogens are fungi and many of these adversely affect food security. This mini-review aims to provide an analysis of the plant pathogenic fungi for which genome sequences are publically available, to assess their general genome characteristics, and to consider how genomics has impacted plant pathology. A list of sequenced fungal species was assembled, the taxonomy of all species verified, and the potential reason for sequencing each of the species considered. The genomes of 1090 fungal species are currently (October 2016) in the public domain and this number is rapidly rising. Pathogenic species comprised the largest category (35.5 %) and, amongst these, plant pathogens are predominant. Of the 191 plant pathogenic fungal species with available genomes, 61.3 % cause diseases on food crops, more than half of which are staple crops. The genomes of plant pathogens are slightly larger than those of other fungal species sequenced to date and they contain fewer coding sequences in relation to their genome size. Both of these factors can be attributed to the expansion of repeat elements. Sequenced genomes of plant pathogens provide blueprints from which potential virulence factors were identified and from which genes associated with different pathogenic strategies could be predicted. Genome sequences have also made it possible to evaluate adaptability of pathogen genomes and genomic regions that experience selection pressures. Some genomic patterns, however, remain poorly understood and plant pathogen genomes alone are not sufficient to unravel complex pathogen-host interactions. Genomes, therefore, cannot replace experimental studies that can be complex and tedious. Ultimately, the most promising application lies in using fungal plant pathogen genomics to inform disease management and risk assessment strategies. This will ultimately minimize the risks of future disease outbreaks and assist in preparation for emerging pathogen outbreaks.

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“…With this method, we were able to design primers that are able to distinguish between M. mycetomatis and M. pseudomycetomatis. Using this approach, other studies also succeeded in designing specific primers for their organism of choice (25-27). In a study by Withers et al , a similar genome comparison method was performed on Pseudoperonospora cubensis and Pseudoperonospora humuli (27).…”

Section: Resultsmentioning

confidence: 99%

“…Using this approach, other studies also succeeded in designing specific primers for their organism of choice (25-27). In a study by Withers et al , a similar genome comparison method was performed on Pseudoperonospora cubensis and Pseudoperonospora humuli (27). The comparison was first performed in silico and subsequently in vitro.…”

Section: Resultsmentioning

confidence: 99%

“…The comparison was first performed in silico and subsequently in vitro. Using this approach, they were able to identify and determine a large number of specific markers for their organism of interest (27). However, we were not able to perform a similar in silico approach here because at the time of data analysis and the preparation of this manuscript, only one M. mycetomatis isolate has ever been sequenced and none of M. fahalii, M. tropicana and M. pseudomycetomatis has ever been sequenced.…”

Section: Resultsmentioning

confidence: 99%

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The development of a novel diagnostic PCR forMadurella mycetomatisusing a comparative genome approach

Lim

Eadie

Siddig

et al. 2020

Preprint

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14 15 16 17 18 19 Corresponding author 20 Wendy WJ van de Sande 21 w.vandesande@erasmusmc.nl 22 23 Keyword: Mycetoma, Madurella mycetomatis, Comparative genome, Neglected tropical disease 24 25 26 ABSTRACT: 27 28 Eumycetoma is a neglected tropical disease characterized by large tumorous lesions. It is most 29 commonly caused by the fungus Madurella mycetomatis which accounts for more than 70% of cases 30 in central Africa. Currently, identification of the causative agent can only be reliably performed by a 31 species-specific PCR. However, we recently demonstrated that our M. mycetomatis specific PCR can 32 cross-react with Madurella pseudomycetomatis. We therefore used a comparative genome approach 33 to develop a new M. mycetomatis specific PCR for species identification. For this we compared the 34 published M. mycetomatis genome to genomes of other organisms in BLASTCLUST to identify unique 35 M. mycetomatis predicted protein coding sequences. Based on 16 of these unique sequences, PCR36 primers were developed. The specificity of these primers was further evaluated in other 37 eumycetoma causing agents including the Madurella sibling species. Out of the 16 tested sequences, 38 only one was unique for M. mycetomatis and this should be used as a novel diagnostic marker for M.39 mycetomatis. 40 41 42 43 INTRODUCTION: 44 45The neglected tropical disease mycetoma presents itself as a subcutaneous chronic granulomatous 46 infectious and inflammatory disease and is characterized by tumorous lesions (1, 2). This disease can 47 be caused by more than 70 different micro-organisms and is categorized into actinomycetoma 48 (caused by bacteria) and eumycetoma (caused by fungi). Most cases occur in the mycetoma belt 49 between the latitudes 15° South and 30° North. Diagnosis of eumycetoma is often only made 50 clinically in endemic areas due to the scarcity of facilities, expertise and financial capacity. 51Identification of the causative agent is time consuming and often limited to culture and histology 52 which can leads to misidentifications (1, 3, 4). The only way to properly identify eumycetoma 53 causative agents to the species level is through molecular identification, commonly PCR. PCR 54 identification's reliability, high turnover and sensitivity has made it widely used in diagnosing fungal 55 disease, and in most cases, it has already replaced culturing of the microorganism as the primary 56 diagnostic method (5-11). 58Madurella mycetomatis is a fungus only recognised as a pathogen in mycetoma and is responsible for 59 more than 70% of all mycetoma infections in endemic areas (1, 12, 13). In 1999, specific PCR primers 60 based on the internal transcribed spacer (ITS) region were developed for M. mycetomatis by our 61 group (14), however, recently, this M. mycetomatis specific PCR primer pair was discovered to cross-62 react with Madurella pseudomycetomatis (15). Back then, M. pseudomycetomatis was not yet 63 discovered (16). Since new fungi causing eumycetoma are still being discovered, there is clearly a 64 need...

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Using Next-Generation Sequencing to Develop Molecular Diagnostics for Pseudoperonospora cubensis, the Cucurbit Downy Mildew Pathogen

Cited by 48 publications

References 57 publications

Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection

Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection

A plant pathology perspective of fungal genome sequencing

The development of a novel diagnostic PCR forMadurella mycetomatisusing a comparative genome approach

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