scholarly journals Detection and Quantification of Pythium tracheiphilum in Soil by Multiplex Real-Time qPCR

Plant Disease ◽  
2019 ◽  
Vol 103 (3) ◽  
pp. 475-483 ◽  
Author(s):  
Hervé Van der Heyden ◽  
Thérèse Wallon ◽  
C. André Lévesque ◽  
Odile Carisse
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Disease Incidence ◽  
Quantitative Polymerase Chain Reaction ◽  
Pcr Primers ◽  
Qpcr Assay ◽  
Inoculum Concentration ◽  
Soil P ◽  
Dry Soil ◽  
Detection And Quantification

In Canada, head lettuce (Lactuca sativa capitata) is extensively produced in the muck soils of southwestern Québec. However, yields are increasingly affected by various soilborne pathogens, including Pythium spp., which cause wilt and damping off. In a survey conducted in Québec muck soils in 2010 and 2011, Pythium tracheiphilum Matta was identified as the predominant Pythium sp. in the root of head lettuce showing Pythium stunt symptoms. Therefore, to improve risk assessment and help further understanding of disease epidemiology, a specific and sensitive real-time quantitative polymerase chain reaction (qPCR) assay based on TaqMan-minor groove binder (MGB) technology was developed for P. tracheiphilum. The PCR primers along with a TaqMan-MGB probe were designed from the ribosomal internal transcribed spacer 2 region. A 100-bp product was amplified by PCR from all P. tracheiphilum isolates tested while no PCR product was obtained from 38 other Pythium spp. or from a selection of additional lettuce pathogens tested. In addition to P. tracheiphilum, the assay was multiplexed with an internal control allowing for the individual validation of each PCR. In artificially infested soils, the sensitivity of the qPCR assay was established as 10 oospores/g of dry soil. P. tracheiphilum was not detected in soils in which lettuce has never been grown; however, inoculum ranged from 0 to more than 200,000 oospores/g of dry soil in commercial lettuce fields. Also, disease incidence was positively correlated with inoculum concentration (r = 0.764). The results suggest that inoculum concentration should be considered when making Pythium stunt management decisions. The developed qPCR assay will facilitate reliable detection and quantification of P. tracheiphilum from field soil.

scholarly journals Detection and Quantification of Airborne Claviceps purpurea sensu lato Ascospores from Hirst-Type Spore Traps using Real-Time Quantitative PCR

Plant Disease ◽  
2018 ◽  
Vol 102 (12) ◽  
pp. 2487-2493 ◽  
Author(s):  
Jeremiah K.S. Dung ◽  
Jeness C. Scott ◽  
Qunkang Cheng ◽  
Stephen C. Alderman ◽  
Navneet Kaur ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Management Approach ◽  
Conidial Suspension ◽  
Claviceps Purpurea ◽  
Spore Trap ◽  
Grass Seed ◽  
Wide Range ◽  
Detection And Quantification

The U.S. Pacific Northwest states of Oregon and Washington are major producers of cool-season grass seed. Ergot, caused by fungi in the Claviceps purpurea sensu lato group, is an important seed replacement disease of grass worldwide. Microscopic methods that are currently used to quantify airborne Claviceps ascospores captured by spore traps are not currently rapid enough to allow for detecting and reporting of spore numbers in a timely manner, hindering growers from using this information to help manage ergot. We developed a SYBR Green real-time quantitative polymerase chain reaction (qPCR)-based assay for fast and efficient detection and quantification of C. purpurea sensu lato ascospores from Hirst-type spore traps. Species-specificity of the qPCR assay was confirmed against 41 C. purpurea sensu lato isolates collected from six hosts and six other Claviceps spp. Significant relationships were observed between cycle threshold (Ct) values and standard curves of serial dilutions of DNA ranging from 1 pg to 10 ng (R2 = –0.99; P = 0.0002) and DNA extracted from a conidial suspension representing 8 to 80,000 conidia (R2 = –0.99; P = 0.0004). Ct values from qPCR were significantly correlated with results from microscopic examination of spore trap samples from the field (r = –0.68; P < 0.0001) and the procedure was able to detect a single ascospore from spore trap tape samples. The qPCR procedure developed in this study provided a means for quantifying airborne Claviceps ascospores that was highly specific and useful over a wide range of spore densities, and could be performed in a matter of hours instead of days. The qPCR assay developed in this study could be part of an integrated pest management approach to help grass seed growers make risk-based fungicide application decisions for ergot management in grass grown for seed.

scholarly journals Rapid and Quantitative Detection ofLeifsonia xylisubsp.xyliin Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan) PCR Assay

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Hua-Ying Fu ◽  
Sheng-Ren Sun ◽  
Jin-Da Wang ◽  
Kashif Ahmad ◽  
Heng-Bo Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Disease Incidence ◽  
Quantitative Polymerase Chain Reaction ◽  
Field Trials ◽  
Diagnostic Methods ◽  
Qpcr Assay ◽  
Taqman Probe ◽  
Quantitative Detection ◽  
Conventional Pcr ◽  
Sugarcane Stalk

Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agentLeifsonia xylisubsp.xyli(Lxx). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification ofLxxdetection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting thePart1gene ofLxxwere used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg ofLxxgenomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods forLxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

Developing a real-time PCR assay for direct detection and quantification of Pratylenchus scribneri in field soil

Nematology ◽  
2020 ◽  
Vol 22 (7) ◽  
pp. 733-744
Author(s):  
Deepika Arora ◽  
Guiping Yan ◽  
Richard Baidoo
Keyword(s):  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Parasitic Nematodes ◽  
Field Soil ◽  
Accurate Identification ◽  
Soil Dna ◽  
Field Soils ◽  
Pratylenchus Scribneri ◽  
Detection And Quantification

Summary The endomigratory root-lesion nematode, Pratylenchus scribneri, is one of the major plant-parasitic nematodes infecting potato. Accurate identification and quantification of this nematode are essential to develop management strategies but microscopic observations are particularly challenging and time consuming. In this study, a SYBR Green I-based real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. scribneri from field soil DNA extracts. A primer set was designed from the internal transcribed spacer (ITS) region of the P. scribneri rDNA gene. Primer specificity to the target nematode was evaluated by both in silico analysis and qPCR and no detection or non-specific amplification was observed for other non-target nematode species/communities tested in this study. Standard curves were generated using DNA extracts from autoclaved soil infested with varying nematode numbers for calibration. The curves were supported by a high correlation between the P. scribneri numbers artificially added to soil or estimated from naturally infested field soils by traditional methods, and the numbers quantified using the qPCR assay. The assay was able to detect 1 out of 128 (0.0078) equivalents of the DNA of a single nematode in 0.5 g of soil. The qPCR assay developed in this study provides a specific and sensitive detection and quantification of P. scribneri from field soils and a rapid alternative to time-consuming traditional nematode identification and enumeration.

scholarly journals Detection and Quantification of Rhizoctonia solani AG-1 IA, the Rice Sheath Blight Pathogen, in Rice Using Real-Time PCR

Plant Disease ◽  
2007 ◽  
Vol 91 (12) ◽  
pp. 1663-1668 ◽  
Author(s):  
Ronald J. Sayler ◽  
Yinong Yang
Keyword(s):  
Rhizoctonia Solani ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Sheath Blight ◽  
Qpcr Assay ◽  
Rice Sheath Blight ◽  
Internal Transcribed Spacer Region ◽  
Causal Organism ◽  
Rice Disease ◽  
Detection And Quantification

Rhizoctonia solani Kühn is the causal organism of sheath blight, a major rice disease worldwide that severely impairs yield and quality. It is difficult to identify the pathogen in the early phase of the infection and to accurately quantify the fungal development based on visual inspection. Therefore, a rapid and reliable method is advantageous for the detection and quantification of the pathogen causing this important rice disease. In this study, a real-time, quantitative polymerase chain reaction (QPCR) assay was developed to detect and quantify R. solani AG-1 IA DNA from infected rice plants. A specific primer pair was designed based on the internal transcribed spacer region of the fungal ribosomal DNA. The specific detection of R. solani DNA was successful with quantities as low as 1 pg. The QPCR assay could be used for detecting the rice sheath blight pathogen, quantifying fungal aggressiveness, and evaluating the resistance level of rice cultivars.

Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains

2006 ◽  
Vol 52 (5) ◽  
pp. 451-461 ◽  
Author(s):  
S S Hynes ◽  
O Chaudhry ◽  
M A Providenti ◽  
M L Smith
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Environmental Persistence ◽  
Fungal Strains ◽  
Target Dna ◽  
Polymerase Chain

The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (~14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.Key words: Canada domestic substances list (DSL), Canadian Environmental Protection Act (CEPA), genetically modified organisms (GMO), quantitative polymerase chain reaction (qPCR).

Design and testing of real-time PCR primers for the quantification ofMethanoculleus,Methanosarcina,Methanothermobacter, and a group of uncultured methanogens

2009 ◽  
Vol 55 (5) ◽  
pp. 611-616 ◽  
Author(s):  
Ingrid H. Franke-Whittle ◽  
Marta Goberna ◽  
Heribert Insam
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Biogas Production ◽  
Pcr Primers ◽  
Sybr Green ◽  
Rrna Gene ◽  
Pcr Assays ◽  
Design And Testing ◽  
Uncultured Archaea ◽  
Detection And Quantification

In this study, 16S rRNA gene primers were designed to complement the suite of already available PCR primers for the detection of different methanogens involved in biogas production through anaerobic digestion by SYBR Green real-time PCR. Primers designed for use in TaqMan real-time PCR for the organisms Methanosaeta , Methanosarcina , and Methanoculleus have been described previously; however, we found that (i) the Methanoculleus primers were not specific to members of the genus and that (ii) the Methanosarcina primers did not work specifically with SYBR Green real-time PCR. Thus, we designed new primers for these and other methanogens, and we optimized SYBR Green real-time PCR assays. Primers were tested by end-point and real-time PCR, and they were found to work specifically and sensitively. Application of these primers will allow the detection and quantification of Methanoculleus, Methanosarcina, Methanothermobacter , and a group of yet uncultured archaea from anaerobic habitats.

scholarly journals Real-Time PCR Assays for the Quantification of Phialophora gregata f. sp. sojae IGS Genotypes A and B

2009 ◽  
Vol 99 (9) ◽  
pp. 1008-1014 ◽  
Author(s):  
T. J. Hughes ◽  
Z. K. Atallah ◽  
C. R. Grau
Keyword(s):  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Intergenic Spacer ◽  
Qpcr Assay ◽  
Soil Dna ◽  
Intergenic Spacer Region ◽  
Brown Stem Rot ◽  
Phialophora Gregata ◽  
Probe Set ◽  
Genotype A

Populations of Phialophora gregata f. sp. sojae, the causal agent of brown stem rot (BSR) of soybean, consist of two genotypes, designated A and B. These genotypes are differentiated by an insertion or deletion in the intergenic spacer region (IGS) of ribosomal DNA. The two genotypes differ in the type and severity of symptoms they cause and have displayed preferential host colonization. Methods to quantify populations of P. gregata f. sp. sojae and to distinguish between the two genotypes are essential to understanding this host–pathogen interaction and to improving control of BSR. A real-time, quantitative polymerase chain reaction (qPCR) assay was developed for the specific detection and quantification of P. gregata f. sp. sojae genotype A. This assay is specific to P. gregata f. sp. sojae genotype A, sensitive to 50 fg of DNA, and unaffected by the presence of soybean or soil DNA. When the P. gregata f. sp. sojae genotype A-specific primer/probe set is used in a multiplex qPCR assay with a previously developed primer/probe set which indiscriminately amplifies both genotypes, the quantity of P. gregata f. sp. sojae genotype B can be indirectly determined. This multiplex assay provides a rapid and robust method for studying both the population size and genetic structure of P. gregata f. sp. sojae in its soybean host and in the soil.

scholarly journals A Newly Developed Real-Time PCR Assay for Detection and Quantification of Fusarium oxysporum and Its Use in Compatible and Incompatible Interactions with Grafted Melon Genotypes

2013 ◽  
Vol 103 (8) ◽  
pp. 802-810 ◽  
Author(s):  
Anita Haegi ◽  
Valentina Catalano ◽  
Laura Luongo ◽  
Salvatore Vitale ◽  
Michele Scotton ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Elongation Factor ◽  
Incompatible Interaction ◽  
Fungal Development ◽  
Race 1 ◽  
Pathogen Colonization ◽  
Species Specific ◽  
Detection And Quantification

A reliable and species-specific real-time quantitative polymerase chain reaction (qPCR) assay was developed for detection of the complex soilborne anamorphic fungus Fusarium oxysporum. The new primer pair, designed on the translation elongation factor 1-α gene with an amplicon of 142 bp, was highly specific to F. oxysporum without cross reactions with other Fusarium spp. The protocol was applied to grafted melon plants for the detection and quantification of F. oxysporum f. sp. melonis, a devastating pathogen of this cucurbit. Grafting technologies are widely used in melon to confer resistance against new virulent races of F. oxysporum f. sp. melonis, while maintaining the properties of valuable commercial varieties. However, the effects on the vascular pathogen colonization have not been fully investigated. Analyses were performed on ‘Charentais-T’ (susceptible) and ‘Nad-1’ (resistant) melon cultivars, both used either as rootstock and scion, and inoculated with F. oxysporum f. sp. melonis race 1 and race 1,2. Pathogen development was compared using qPCR and isolations from stem tissues. Early asymptomatic melon infections were detected with a quantification limit of 1 pg of fungal DNA. The qPCR protocol clearly showed that fungal development was highly affected by host–pathogen interaction (compatible or incompatible) and time (days postinoculation). The principal significant effect (P ≤ 0.01) on fungal development was due to the melon genotype used as rootstock, and this effect had a significant interaction with time and F. oxysporum f. sp. melonis race. In particular, the amount of race 1,2 DNA was significantly higher compared with that estimated for race 1 in the incompatible interaction at 18 days postinoculation. The two fungal races were always present in both the rootstock and scion of grafted plants in either the compatible or incompatible interaction.

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