Development of real-time PCR assay using TaqMan probe for detection and quantification of Rosellinia necatrix in plant and soil

Invasive alien tree pathogens can cause significant economic losses as well as large-scale damage to natural ecosystems. Early detection to prevent their establishment and spread is an important approach used by several national plant protection organizations (NPPOs). Molecular detection tools targeting 10 of the most unwanted alien forest pathogens in Canada were developed as part of the TAIGA project (http://taigaforesthealth.com/). Forest pathogens were selected following an independent prioritization. Specific TaqMan real-time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96- or 384-well plate format arrays for high-throughput testing of large numbers of samples against multiple targets. Assays were validated for 1) specificity, 2) sensitivity, 3) precision, and 4) robustness on environmental samples. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely-related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and ten target gene region copies. Precision or repeatability of each assay revealed a mean coefficient of variation of 3.4%. All assays successfully allowed detection of target pathogen on positive environmental samples, without any non-specific amplification. These molecular detection tools will allow for rapid and reliable detection of 10 of the most unwanted alien forest pathogens in Canada.

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“…ramorum [ 7 , 55 , 63 – 65 ], R . necatrix [ 66 – 68 ]), or 3) they did not target the specific race of interest ( G . abietina EU race [ 69 ]).…”

Section: Resultsmentioning

confidence: 99%

Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR

et al. 2015

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“…aNested polymerase chain reaction assays were conducted using the primer pairs of ITS1/ITS4 followed by R2 and R5 as described by Shishido et al (10). …”

Section: Tablementioning

confidence: 99%

“…Nested PCR was performed for every DNA sample as described previously by Shishido et al (10). Initial PCR amplified the fungal ITS-rDNA region using the primer pair ITS1 and ITS4 (12), with the following cycling conditions: initial denaturation at 95°C for 10 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and an extension at 72°C for 30 s; and a final extension at 72°C for 7 min, using GeneAmp PCR System 2700 (Applied Biosystems, Thermo Fisher Scientific, Tokyo, Japan).…”

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confidence: 99%

“…The reaction mixture consisted of BIO Taq (0.5 U), Ampdirect (10 μL, Shimadzu, Kyoto, Japan), 0.5 μM of the ITS1 and ITS4 primers, and 2 μL of extracted DNA as a template in a final volume of 20 μL. Since the weeds used in the present study were grown in volcanic ash soil (Andosol), which strongly inhibits DNA extraction and subsequent PCR (10), we applied nested PCR assays along with a gene amplification reagent (Ampdirect) to neutralize PCR inhibitors.…”

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confidence: 99%

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Weed Roots Facilitate the Spread of <i>Rosellinia necatrix</i>, the Causal Agent of White Root Rot

2019

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Rosellinia necatrix causes white root rot in various plants, including the Japanese pear. PCR assays using specific primers for R. necatrix detected the fungus on the roots of nine weed species from infested pear orchards. The soil inoculation experiment revealed that the spread of R. necatrix was similar between weed-mowed and non-weed-mowed treatments under field conditions. The spread of R. necatrix was also observed when rescue grass (Bromus catharticus) was grown in planter boxes under greenhouse conditions, but was limited without the grass, suggesting that some weeds facilitate the spread of R. necatrix in soil.

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“…It is possible to further improve the specificity of qPCR using a probe. For example, a qPCR assays using TaqMan probes to detect and quantify Rosellinia necatrix was developed and their detection limits was reported to be as low as 1 fg of template DNA (Shishido et al 2012). For DNA obtained from samples extracted from soil, PCR inhibition due to the influence of humic acid can be an important issue.…”

Section: Introductionmentioning

confidence: 99%

Developing a qPCR assay for the quantification of Calonectria ilicicola in soil of soybean field

Ochi

Kuroda

2020

Trop. plant pathol.

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Effective quantification of Calonectria ilicicola in naturally infested soils is critical for evaluating and validating control measures for managing red crown rot disease in soybean. Counting of microsclerotia has been used to quantify this pathogen, but the method is laborious and time-consuming to process large numbers of soil samples using wet sieving. To overcome these issues, we developed and tested a qPCR method to detect and quantify C. ilicicola in naturally infested soil of soybean fields. The primer pairs and probe, designed based on the intergenic spacer region of the ribosomal DNA, showed high specificity with a single melting curve peak in DNA samples from nine C. ilicicola isolates. No amplification product was detected with 2 isolates of other Calonectria species and 12 isolates of common soil-borne fungi. There was a significant positive correlation between the amount of C. ilicicola DNA and the number of C. ilicicola microsclerotia. The qPCR method is a promising tool to quantify C. ilicicola in soil.

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Development of real-time PCR assay using TaqMan probe for detection and quantification of Rosellinia necatrix in plant and soil

Cited by 9 publications

References 16 publications

Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR

Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR

Weed Roots Facilitate the Spread of <i>Rosellinia necatrix</i>, the Causal Agent of White Root Rot

Developing a qPCR assay for the quantification of Calonectria ilicicola in soil of soybean field

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