scholarly journals Comparison of multiplex real-time PCR and ergosterol assays in quantifying Heterobasidion annosum in planta

2017 ◽  
Vol 38 (SI 2 - 6th Conf EFPP 2002) ◽  
pp. 406-407
Author(s):  
A.M. Hietala ◽  
M. Eikenes ◽  
M. Kvaalen ◽  
H. Solheim ◽  
C.G. Fossdal
Keyword(s):  
Real Time ◽  
Norway Spruce ◽  
Real Time Pcr ◽  
Comparative Method ◽  
Heterobasidion Annosum ◽  
Fungal Colonization ◽  
In Planta ◽  
Advantages And Disadvantages ◽  
Very High

A quantitative multiplex real-time PCR procedure was developed to monitor the dynamics in Norway spruce-Heterobasidion annosum pathosystem. The assay reliably detected down to 1 pg of H. annosum DNA and 1 ng of host DNA in multiplex conditions. As a comparative method for quantifying fungal colonization, we applied the ergosterol assay. There was a very high correlation between the results obtained with the two methods, this strengthening the credibility of both assays. The advantages and disadvantages of these assays are discussed.

scholarly journals Multiplex Real-Time PCR for Monitoring Heterobasidion annosum Colonization in Norway Spruce Clones That Differ in Disease Resistance

2003 ◽  
Vol 69 (8) ◽  
pp. 4413-4420 ◽  
Author(s):  
Ari M. Hietala ◽  
Morten Eikenes ◽  
Harald Kvaalen ◽  
Halvor Solheim ◽  
Carl G. Fossdal
Keyword(s):  
Tissue Culture ◽  
Real Time ◽  
High Resistance ◽  
Real Time Pcr ◽  
Heterobasidion Annosum ◽  
Fungal Colonization ◽  
Pcr Assay ◽  
Culture Experiment ◽  
The Real ◽  
Low Resistance

ABSTRACT A multiplex real-time PCR assay was developed to monitor the dynamics of the Picea abies-Heterobasidion annosum pathosystem. Tissue cultures and 32-year-old trees with low or high resistance to this pathogen were used as the host material. Probes and primers were based on a laccase gene for the pathogen and a polyubiquitin gene for the host. The real-time PCR procedure was compared to an ergosterol-based quantification method in a tissue culture experiment, and there was a strong correlation (product moment correlation coefficient, 0.908) between the data sets. The multiplex real-time PCR procedure had higher resolution and sensitivity during the early stages of colonization and also could be used to monitor the host. In the tissue culture experiment, host DNA was degraded more rapidly in the clone with low resistance than in the clone with high resistance. In the field experiment, the lesions elicited were not strictly proportional to the area colonized by the pathogen. Fungal colonization was more restricted and localized in the lesion in the clone with high resistance, whereas in the clone with low resistance, the fungus could be detected until the visible end of the lesion. Thus, the real-time PCR assay gives better resolution than does the traditionally used lesion length measurement when screening host clones for resistance.

scholarly journals A Set of Conventional and Multiplex Real-Time PCR Assays for Direct Detection of Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis in Citrus Fruits

Plant Disease ◽  
2019 ◽  
Vol 103 (2) ◽  
pp. 345-356 ◽  
Author(s):  
Yosra Ahmed ◽  
Jacqueline Hubert ◽  
Céline Fourrier-Jeandel ◽  
Megan M. Dewdney ◽  
Jaime Aguayo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Media ◽  
Direct Detection ◽  
Elongation Factor ◽  
Single Copy ◽  
The United States ◽  
Performance Criteria ◽  
In Planta ◽  
Conventional Pcr

Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis are causal agents of citrus scab and spot diseases. The three pathogens are listed as quarantine pests in many countries and are subject to phytosanitary measures to prevent their entry. Diagnosis of these diseases based on visual symptoms is problematic, as they could be confused with other citrus diseases. Isolation of E. fawcettii, E. australis, and P. angolensis from infected tissues is challenging because they grow slowly on culture media. This study developed rapid and specific detection tools for the in planta detection of these pathogens, using either conventional PCR or one-tube multiplex real-time PCR. Primers and hybridization probes were designed to target the single-copy protein-coding gene MS204 for E. fawcettii and E. australis and the translation elongation factor (Tef-1α) gene for P. angolensis. The specificity of the assays was evaluated by testing against DNA extracted from a large number of isolates (102) collected from different citrus-growing areas in the world and from other hosts. The newly described species E. citricola was not included in the specificity test due to its unavailability from the CBS collection. The detection limits of conventional PCR for the three pathogens were 100, 100, and 10 pg μl−1 gDNA per reaction for E. fawcettii, E. australis, and P. angolensis, respectively. The quadruplex qPCR was fully validated assessing the following performance criteria: sensitivity, specificity, repeatability, reproducibility, and robustness. The quadruplex real-time PCR proved to be highly sensitive, detecting as low as 243, 241, and 242 plasmidic copies (pc) μl−1 of E. fawcettii, E. australis, and P. angolensis, respectively. Sensitivity and specificity of this quadruplex assay were further confirmed using 176 naturally infected citrus samples collected from Ethiopia, Cameroon, the United States, and Australia. The quadruplex assay developed in this study is robust, cost-effective, and capable of high-throughput detection of the three targets directly from citrus samples. This new detection tool will substantially reduce the turnaround time for reliable species identification and allow rapid response and appropriate action.

scholarly journals Pathogenicity and a TaqMan Real-Time PCR for Specific Detection of Pantoea allii, a Bacterial Pathogen of Onions

Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3031-3040 ◽  
Author(s):  
Shabnam Rahimi-Khameneh ◽  
Sanni Hsieh ◽  
Renlin Xu ◽  
Tyler J. Avis ◽  
Sean Li ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Phylogenetic Analyses ◽  
Similarity Index ◽  
Taqman Probe ◽  
Economic Losses ◽  
In Planta ◽  
Pcr Assay ◽  
Blind Test ◽  
Reliable Detection

Bacterial diseases of onion are reported to cause significant economic losses. Pantoea allii Brady, one of the pathogens causing the center rot on onions, has not yet been reported in Canada. We report the pathogenicity of P. allii on commercially available Canadian green onions (scallions). All P. allii-inoculated plants, irrespective of the inoculum concentration, exhibited typical leaf chlorotic discoloration on green onion leaves, which can reduce their marketability. Reisolation of P. allii from infected scallion tissues and reidentification by sequencing and phylogenetic analyses of the leuS gene suggest that the pathogen can survive in infected tissues 21 days after inoculation. This is the first report of P. allii as a potential pathogen of green onions. This study also reports the development and validation of a TaqMan real-time PCR assay targeting the leuS gene for reliable detection of P. allii in pure cultures and in planta. A 642-bp leuS gene fragment was targeted because it showed high nucleotide diversity and positively correlated with genome-based average nucleotide identity with respect to percent similarity index and identity of Pantoea species. The assay specificity was validated using 61 bacterial and fungal strains. Under optimal conditions, the selected primers and FAM-labeled TaqMan probe were specific for the detection of nine reference P. allii strains by real-time PCR. The 52 strains of other Pantoea spp. (n = 25), non-Pantoea spp. (n = 20), and fungi/oomycetes (n = 7) tested negative (no detectable fluorescence). Onion tissues spiked with P. allii, naturally infested onion bulbs, greenhouse infected green onion leaf samples, as well as an interlaboratory blind test were used to validate the assay specificity. The sensitivities of a 1-pg DNA concentration and 30 CFU are comparable to previously reported real-time PCR assays of other bacterial pathogens. The TaqMan real-time PCR assay developed in this study will facilitate reliable detection of P. allii and could be a useful tool for screening onion imports or exports for the presence of this pathogen.

scholarly journals In Planta Distribution of ‘Candidatus Liberibacter asiaticus’ as Revealed by Polymerase Chain Reaction (PCR) and Real-Time PCR

2008 ◽  
Vol 98 (5) ◽  
pp. 592-599 ◽  
Author(s):  
Satyanarayana Tatineni ◽  
Uma Shankar Sagaram ◽  
Siddarame Gowda ◽  
Cecile J. Robertson ◽  
William O. Dawson ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Citrus Tree ◽  
Candidatus Liberibacter Asiaticus ◽  
In Planta ◽  
Chain Reaction ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide, and is caused by a phloem-limited fastidious prokaryotic α-proteobacterium that is yet to be cultured. In this study, a combination of traditional polymerase chain reaction (PCR) and real-time PCR targeting the putative DNA polymerase and 16S rDNA sequence of ‘Candidatus Liberibacter asiaticus,’ respectively, were used to examine the distribution and movement of the HLB pathogen in the infected citrus tree. We found that ‘Ca. Liberibacter asiaticus’ was distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. Quantification analysis of the HLB bacterium indicated that it was distributed unevenly in planta and ranged from 14 to 137,031 cells/μg of total DNA in different tissues. A relatively high concentration of ‘Ca. Liberibacter asiaticus’ was observed in fruit peduncles. Our data from greenhouse-infected plants also indicated that ‘Ca. Liberibacter asiaticus’ was transmitted systemically from infection site to different parts of the plant. Understanding the distribution and movement of the HLB bacterium inside an individual citrus tree is critical for discerning its virulence mechanism and to develop management strategies for HLB.

Development of real-time PCR (TaqMan®) assays for the detection and quantification of Botrytis cinerea in planta

2005 ◽  
Vol 43 (9) ◽  
pp. 890-899 ◽  
Author(s):  
M. Belen Suarez ◽  
Kathryn Walsh ◽  
Neil Boonham ◽  
Tim O’Neill ◽  
Simon Pearson ◽  
...  
Keyword(s):  
Real Time ◽  
Botrytis Cinerea ◽  
Real Time Pcr ◽  
In Planta ◽  
Detection And Quantification

scholarly journals Multiplex real-time PCR revealed very high prevalence of soil-transmitted helminth infections among aborigines in Peninsular Malaysia

2020 ◽  
Vol 13 (12) ◽  
pp. 550
Author(s):  
Rahmah Noordin ◽  
Nurulhasanah Othman ◽  
Noorizan Miswan ◽  
Weng-kin Wong ◽  
Boon-huat Lim
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Peninsular Malaysia ◽  
Helminth Infections ◽  
High Prevalence ◽  
Very High

scholarly journals Early determination of fetal sex in goat a comparison between real time PCR and ultrasonography

2022 ◽  
Vol 951 (1) ◽  
pp. 012059
Author(s):  
S A Hussein ◽  
K M Karam
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Maternal Blood ◽  
Fetal Sex ◽  
Blood Specimens ◽  
High Level ◽  
Pcr Techniques ◽  
Very High ◽  
Genital Tubercle

Abstract The point of the current study is to assess the productivity of the real time PCR and ultrasound techniques in early determination of fetal sex in Iraqi singleton pregnant goats. Our investigation has been led in Iraq, Al-Diwanya city from 10/8/2020 – 15/1/2021. The examination incorporates 45 singleton pregnant Iraqi goats, which initially inspected by ultrasound to affirm pregnancy and to decide the fetal sex depending on the restriction of the genital tubercle of the goat fetuses, after that, blood specimens had been gathered from the jugular vein of all examined does to detect fetal sex by discovery of AMLX and SRY genes in the circling cells free fetal DNA (ccffDNA) in these maternal blood specimens by utilizing real time PCR. Our outcomes showed an exceptionally high level of accuracy in real time PCR in contrast with the ultrasound strategy. The outcomes were affirmed by the true fetal sex after parturition in the inspected does. The complete symptomatic rate were 51.11% (23/45) and 97.78% (44/45) for ultrasound and PCR strategies separately. The exactness level of genuine analyzed female and male caprine kidding were 58.33% (7/12), 48.48% (16/33), and 100% (12/12), 96.97% (32/33) for ultrasound and real time PCR techniques separately. While the exactness rates of the two techniques utilized in this investigation for early caprine fetal sexing in respect to early pregnancies periods analyzed uncovered 100% (13/13), 96.3% (26/27), 100% (5/5), and 61.54% (8/13), 40.74% (11/27), 80% (4/5) in early pregnancy periods (58-62, 63-67, 68-73) days for real time PCR and ultrasound strategies individually. In conclusion our outcomes revealed a huge predominant exactness and productivity in fetal sexing in Iraqi singleton pregnant does in early development periods, with very high accuracy in real time PCR in compare to ultrasound techniques.

A rapid infection assay for Armillaria and real-time PCR quantitation of the fungal biomass in planta

2010 ◽  
Vol 114 (1) ◽  
pp. 107-119 ◽  
Author(s):  
Kendra Baumgartner ◽  
Ravi Bhat ◽  
Phillip Fujiyoshi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fungal Biomass ◽  
In Planta ◽  
Infection Assay

scholarly journals Real-Time PCR-Based Monitoring of DNA Pools in the Tri-Trophic Interaction Between Norway Spruce, the Rust Thekopsora areolata, and an Opportunistic Ascomycetous Phomopsis sp.

2008 ◽  
Vol 98 (1) ◽  
pp. 51-58 ◽  
Author(s):  
Ari M. Hietala ◽  
Halvor Solheim ◽  
Carl Gunnar Fossdal
Keyword(s):  
Real Time ◽  
Norway Spruce ◽  
Plant Diseases ◽  
Separate Analysis ◽  
Latent Stage ◽  
Phloem Necrosis ◽  
Biotrophic Fungi ◽  
Polymerase Chain ◽  
Internal Transcribed Spacer Rdna ◽  
Very High

The difficulty in subculturing biotrophic fungi complicates etiological studies related to the associated plant diseases. By employing internal transcribed spacer rDNA-targeted quantitative real-time polymerase chain reaction, we now show that the heteroecious rust Thekopsora areolata, commonly associated in natural conditions to sapling shoots and cones of Norway spruce and leaves of wild bird cherry, frequently infects nursery-grown seedlings of the conifer. A spatial sampling scheme was used to investigate seedlings and saplings of Norway spruce showing phloem necrosis: the highest concentration of DNA of T. areolata was recorded in the area with necrotic phloem. The separate analysis of bark and wood tissues suggested that the initial spread of the rust to healthy tissues neighboring the infection site takes place in the bark. A Phomopsis species found to coexist with T. areolata in several seedlings showed very high DNA levels in the upper part of the lesion, and even in the visually healthy proximal tissues above the lesions, which indicates that the ascomycete, most probably a secondary invader following primary infection by T. areolata, has a latent stage during early host colonization. We hypothesize that this hemibiotrophic mode of infection contributes to the successful coexistence of Phomopsis with a biotrophic rust.

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