LAMP Detection Assays for Boxwood Blight Pathogens: A Comparative Genomics Approach

Malapi‐Wight, Martha; Demers, Jill E.; Veltri, Daniel; Marra, Robert E.; Crouch, Jo Anne

doi:10.1038/srep26140

Cited by 29 publications

(19 citation statements)

References 44 publications

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“…The 55.0 Mb genome assembly of a North American isolate CBS 139707 (also known as cpsCT1) is assembled into just 27 scaffolds and is predicted to contain 16,304 genes (Crouch et al 2017 ). These genome sequences, along with assemblies for related fungi C. leucothoes , C. naviculata , and C. pseudoreteaudii , have already been employed for studies of mating-type, diagnostic marker development, and whole genome-scale sequence comparison of isolates from different hosts (Malapi-Wight et al 2014b , 2016a , b ; Ye et al 2017 ). Moving forward, these resources are likely to yield additional information about the genetic diversity of the boxwood blight pathogens.…”

Section: Genetic Variation and Reproduction Of The Fungi Causing Boxwmentioning

confidence: 99%

“…Multiple molecular diagnostic assays have been developed for the detection and quantification of the causal pathogens of boxwood blight. All of the currently available assays are based on some application of polymerase chain reaction (PCR) or isothermal amplification technology to identify the DNA of target pathogens (Gehesquière et al 2013 , 2016 ; Malapi-Wight et al 2016a ). However, the approaches to develop individual assays as well as their ease of use and effectiveness differ.…”

Section: Diagnostic Assays For Pathogen Detectionmentioning

confidence: 99%

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Boxwood blight: an ongoing threat to ornamental and native boxwood

LeBlanc

Salgado‐Salazar

Crouch

2018

Appl Microbiol Biotechnol

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Boxwood blight is an emerging disease of ornamental and native boxwood plants in the family Buxaceae. First documented in the 1990s at a single location in England, the disease is now reported throughout Europe, Asia, New Zealand, and North America. To address the growing concern over boxwood blight, ongoing research focuses on multiple biological and genetic aspects of the causal pathogens and susceptible host plants. Characterization of genetic variation among the Calonectria fungi that cause boxwood blight shows that two unique sister species with different geographic distributions incite the disease. Studies of the pathogen life cycle show the formation of long-lived survival structures and that host infection is dependent on inoculum density, temperature, and humidity. Host range investigations detail high levels of susceptibility among boxwood as well as the potential for asymptomatic boxwood infection and for other plants in the family Buxaceae to serve as additional hosts. Multiple DNA-based diagnostic assays are available, ranging from probe-based quantitative PCR assays to the use of comparative genomics to develop robust diagnostic markers or provide whole genome-scale identifications. Though many questions remain, the research that continues to address boxwood blight demonstrates the importance of applying a multidisciplinary approach to understand and control emerging plant diseases.

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Section: Genetic Variation and Reproduction Of The Fungi Causing Boxwmentioning

confidence: 99%

Section: Diagnostic Assays For Pathogen Detectionmentioning

confidence: 99%

Boxwood blight: an ongoing threat to ornamental and native boxwood

LeBlanc

Salgado‐Salazar

Crouch

2018

Appl Microbiol Biotechnol

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“…These characteristics provide results in a fast, efficient, and cost-effective method that theoretically should amplify target sequences with higher specificity than PCR. Since its introduction, LAMP has seen wide interdisciplinary application, for example in detecting microorganisms (e.g., bacteria23, fungi4, and viruses56) diagnosing genetic diseases7, and even early sex determination8.…”

mentioning

confidence: 99%

Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay

Liu

Huang

Liu

et al. 2017

Sci Rep

102

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This study established a constant-temperature fluorescence quantitative detection method, combining loop-mediated isothermal amplification (LAMP) with molecular beacons. The advantages of LAMP are its convenience and efficiency, as it does not require a thermocycler and results are easily visualized by the naked eye. However, a major disadvantage of current LAMP techniques is the use of indirect evaluation methods (e.g., electrophoresis, SYBR Green I dye, precipitation, hydroxynaphthol blue dye, the turbidimetric method, calcein/Mn2+ dye, and the composite probe method), which cannot distinguish between the desired products and products of nonspecific amplification, thereby leading to false positives. Use of molecular beacons avoids this problem because molecular beacons produce fluorescence signals only when binding to target DNA, thus acting as a direct indicator of amplification products. Our analyses determined the optimal conditions for molecular beacons as an evaluation tool in LAMP: beacon length of 25–45 bp, beacon concentration of 0.6–1 pmol/μL, and reaction temperature of 60–65 °C. In conclusion, we validated a novel molecular beacon loop-mediated isothermal amplification method (MB-LAMP), realizing the direct detection of LAMP product.

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“…The genome of Cps isolate CB002 was assembled from 12 779 054 Illumina paired‐end reads passing quality threshold (average Phred score = 34.34 = 99.96 base accuracy) into 6439 scaffolds (≥500 bp) with an average depth of coverage of 81.0×. The estimated genome size was 49.4 Mb (N 50 = 17 153; average contig size = 6.5 kb), consistent with assemblies generated for other Cps isolates (Malapi‐Wight et al ., ,b; Crouch et al ., ). The scaffold accumulation curve reached a plateau at scaffold 4796.…”

Section: Resultsmentioning

confidence: 97%

“…Assessment of conserved single copy orthologues from the CB002 assembly identified 95.1% of the eukaryotic and 96.6% of the fungal lineage busco gene models. A total of 13 733 gene models were predicted from the assembly, similar to predictions made for the genomes of Cps CBS 139394 ( n = 13 103, Malapi‐Wight et al ., ,b). Together these data indicated a dataset that largely represented the complete Cps genome sequence.…”

Section: Resultsmentioning

confidence: 99%

Limited genetic diversity across pathogen populations responsible for the global emergence of boxwood blight identified using SSRs

LeBlanc

Gehesquière²,

Salgado‐Salazar

et al. 2019

Plant Pathology

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Boxwood blight is an emerging disease of ornamental as well as native boxwood. The disease became widely established in Europe at the beginning of the 21st century, prior to its more recent discovery in North America and Asia. Two sister‐species of fungi cause the disease, Calonectria pseudonaviculata (Cps) and C. henricotiae (Che). Prior efforts to quantify intraspecific genetic polymorphisms of Cps and Che have yielded little information, limiting the ability to understand the evolution and migration of these pathogens. This study describes the development and implementation of simple sequence repeat (SSR) markers to analyse genetic diversity from a global collection of Cps and Che isolates, representing major blight outbreaks since the disease was first identified in the UK in the late 1990s. Analysis of the Cps CB002 genome sequence identified 180 single copy SSR loci using stringent search criteria, 11 of which were polymorphic and used to screen a global sample of 306 isolates. Fourteen multilocus genotypes of Cps and two multilocus genotypes of Che were identified. Twelve of the 14 Cps genotypes differed from each other by a single allele. The most common Cps genotype was found on all continents where boxwood blight is confirmed. Based on measurement of linkage disequilibrium, Cps showed no evidence of sexual recombination. Further in silico analysis identified 1594 SSRs using relaxed SSR definition criteria. Comparison of these SSR‐containing loci with Cps and Che genome sequences representing three different genotypes demonstrated that single nucleotide polymorphisms might serve as informative genetic markers for future studies.

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LAMP Detection Assays for Boxwood Blight Pathogens: A Comparative Genomics Approach

Cited by 29 publications

References 44 publications

Boxwood blight: an ongoing threat to ornamental and native boxwood

Boxwood blight: an ongoing threat to ornamental and native boxwood

Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay

Limited genetic diversity across pathogen populations responsible for the global emergence of boxwood blight identified using SSRs

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