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A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum.

Stulberg MJ, Huang Q - PLoS ONE (2015)

Bottom Line: RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control.Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium.Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.

View Article: PubMed Central - PubMed

Affiliation: Floral and Nursery Plants Research Unit, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland, United States of America; Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee, United States of America.

ABSTRACT
Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.

No MeSH data available.


Test of latently infected plants by phylotype II IIB-1 or non-IIB-1&2 strains of Ralstonia solanacearum by dilution plating (A) and qPCR (B).Every sample had a positive Cox1 signal and the mean Cq was 21.72 (±0.17, n = 33). Asymptomatic plants with 107−9, 105−6, and 103−4 CFU/ml/cm of Rs had a mean Cq of 19.91 (±0.68, n = 8), 23.97 (±1.27, n = 8), and 28.28 (±1.31, n = 5) for RsII and 20.93 (±0.67, n = 6), 25.21 (±1.23, n = 4), and 30.42 (±0.8, n = 3) for RsSA2, respectively. Asymptomatic plants with undetectable amounts of Rs (UD) had average Cq values of 32.97 (±0.93, n = 12) for RsII and 34.9 (±1.09, n = 5) for RsSA2. Error bars represent standard error.
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pone.0139637.g002: Test of latently infected plants by phylotype II IIB-1 or non-IIB-1&2 strains of Ralstonia solanacearum by dilution plating (A) and qPCR (B).Every sample had a positive Cox1 signal and the mean Cq was 21.72 (±0.17, n = 33). Asymptomatic plants with 107−9, 105−6, and 103−4 CFU/ml/cm of Rs had a mean Cq of 19.91 (±0.68, n = 8), 23.97 (±1.27, n = 8), and 28.28 (±1.31, n = 5) for RsII and 20.93 (±0.67, n = 6), 25.21 (±1.23, n = 4), and 30.42 (±0.8, n = 3) for RsSA2, respectively. Asymptomatic plants with undetectable amounts of Rs (UD) had average Cq values of 32.97 (±0.93, n = 12) for RsII and 34.9 (±1.09, n = 5) for RsSA2. Error bars represent standard error.

Mentions: We further tested our multiplex qPCR assay with Qiagen-purified DNA extracted from a total of 86 asymptomatic tomato (32) and geranium (54). Similar to the symptomatic plants, our Cox1 duplexed probe set detected all 86 asymptomatic plants with a mean Cq value of 22.37 and standard error (SE) of 0.18. Of these 86 asymptomatic plants, 33 (12 tomatoes and 21 geraniums) were identified as latently infected by Rs with our multiplex qPCR assay, and were further tested by dilution plating to determine if they contained detectable amounts of Rs cells (Fig 2A). Twenty-one of the 33 plants were found to contain 103 to 109 CFU/ml/cm stem, while no bacterial cells were isolated from 12 other inoculated plants (Fig 2A). The qPCR detection was observed in a bacterial concentration dependent manner when Cqs were averaged for each detected cell concentration range, with values ranging from 28.28 (SE = 1.31) (103−4 CFU/ml/cm stem) to 19.91 (SE = 0.68) (107−9 CFU/ml/cm stem) for the RsII set (Fig 2B). A similar concentration-dependent manner was observed for the RsSA2 set when detecting the 18 plants infected by the IIB-1 strains, with Cq values ranging from 30.42 (SE = 0.8) for 103−4 to 20.93 (SE = 0.67) for 10 7–9 CFU/ml/cm stem (Fig 2B). Even for the 12 plants with undetectable amounts of bacterial cells, our RsII set recognized those plants as infected with an average Cq value of 32.97 (SE = 0.93) (Fig 2B). Similarly, our RsSA2 set recognized the five of the twelve plants with undetectable amount of bacterial cells that had been inoculated with IIB-1 strains, with an average Cq value of 34.90 (SE = 1.09) (Fig 2B).


A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum.

Stulberg MJ, Huang Q - PLoS ONE (2015)

Test of latently infected plants by phylotype II IIB-1 or non-IIB-1&2 strains of Ralstonia solanacearum by dilution plating (A) and qPCR (B).Every sample had a positive Cox1 signal and the mean Cq was 21.72 (±0.17, n = 33). Asymptomatic plants with 107−9, 105−6, and 103−4 CFU/ml/cm of Rs had a mean Cq of 19.91 (±0.68, n = 8), 23.97 (±1.27, n = 8), and 28.28 (±1.31, n = 5) for RsII and 20.93 (±0.67, n = 6), 25.21 (±1.23, n = 4), and 30.42 (±0.8, n = 3) for RsSA2, respectively. Asymptomatic plants with undetectable amounts of Rs (UD) had average Cq values of 32.97 (±0.93, n = 12) for RsII and 34.9 (±1.09, n = 5) for RsSA2. Error bars represent standard error.
© Copyright Policy
Related In: Results  -  Collection

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pone.0139637.g002: Test of latently infected plants by phylotype II IIB-1 or non-IIB-1&2 strains of Ralstonia solanacearum by dilution plating (A) and qPCR (B).Every sample had a positive Cox1 signal and the mean Cq was 21.72 (±0.17, n = 33). Asymptomatic plants with 107−9, 105−6, and 103−4 CFU/ml/cm of Rs had a mean Cq of 19.91 (±0.68, n = 8), 23.97 (±1.27, n = 8), and 28.28 (±1.31, n = 5) for RsII and 20.93 (±0.67, n = 6), 25.21 (±1.23, n = 4), and 30.42 (±0.8, n = 3) for RsSA2, respectively. Asymptomatic plants with undetectable amounts of Rs (UD) had average Cq values of 32.97 (±0.93, n = 12) for RsII and 34.9 (±1.09, n = 5) for RsSA2. Error bars represent standard error.
Mentions: We further tested our multiplex qPCR assay with Qiagen-purified DNA extracted from a total of 86 asymptomatic tomato (32) and geranium (54). Similar to the symptomatic plants, our Cox1 duplexed probe set detected all 86 asymptomatic plants with a mean Cq value of 22.37 and standard error (SE) of 0.18. Of these 86 asymptomatic plants, 33 (12 tomatoes and 21 geraniums) were identified as latently infected by Rs with our multiplex qPCR assay, and were further tested by dilution plating to determine if they contained detectable amounts of Rs cells (Fig 2A). Twenty-one of the 33 plants were found to contain 103 to 109 CFU/ml/cm stem, while no bacterial cells were isolated from 12 other inoculated plants (Fig 2A). The qPCR detection was observed in a bacterial concentration dependent manner when Cqs were averaged for each detected cell concentration range, with values ranging from 28.28 (SE = 1.31) (103−4 CFU/ml/cm stem) to 19.91 (SE = 0.68) (107−9 CFU/ml/cm stem) for the RsII set (Fig 2B). A similar concentration-dependent manner was observed for the RsSA2 set when detecting the 18 plants infected by the IIB-1 strains, with Cq values ranging from 30.42 (SE = 0.8) for 103−4 to 20.93 (SE = 0.67) for 10 7–9 CFU/ml/cm stem (Fig 2B). Even for the 12 plants with undetectable amounts of bacterial cells, our RsII set recognized those plants as infected with an average Cq value of 32.97 (SE = 0.93) (Fig 2B). Similarly, our RsSA2 set recognized the five of the twelve plants with undetectable amount of bacterial cells that had been inoculated with IIB-1 strains, with an average Cq value of 34.90 (SE = 1.09) (Fig 2B).

Bottom Line: RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control.Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium.Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.

View Article: PubMed Central - PubMed

Affiliation: Floral and Nursery Plants Research Unit, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland, United States of America; Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee, United States of America.

ABSTRACT
Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.

No MeSH data available.