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.

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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Real-Time Polymerase Chain Reaction for One-Hour On-Site Diagnosis of Pierce's Disease of Grape in Early Season Asymptomatic Vines

Phytopathology ◽

10.1094/phyto.2002.92.7.721 ◽

2002 ◽

Vol 92 (7) ◽

pp. 721-728 ◽

Cited By ~ 100

Author(s):

N. W. Schaad ◽

D. Opgenorth ◽

P. Gaush

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Xylella Fastidiosa ◽

Molecular Techniques ◽

Plant Diseases ◽

Pierce's Disease ◽

Chain Reaction ◽

Pierce’S Disease ◽

Polymerase Chain

Molecular-based techniques, such as polymerase chain reaction (PCR), can reduce the time needed for diagnosis of plant diseases when compared with classical isolation and pathogenicity tests. However, molecular techniques still require 2 to 3 days to complete. To the best of our knowledge, we describe for the first time a real-time PCR technique using a portable Smart Cycler for one-hour on-site diagnosis of an asymptomatic plant disease. Pierce's disease (PD) of grape, caused by the fastidious bacterium Xylella fastidiosa, causes serious losses in grapes in California and the southeastern United States. The disease has been difficult to diagnose because typical leaf scorching symptoms do not appear until late (June and after) in the season and the organism is very difficult to isolate early in the season. Sap and samples of macerated chips of secondary xylem from trunks of vines were used in a direct real-time PCR without extraction of DNA. Using two different sets of primers and probe, we diagnosed PD in 7 of 27 vines (26%) from four of six vineyards sampled 10 to 12 days after bud break in Kern, Tulare, and Napa counties of California. The diagnosis was confirmed by isolation of Xylella fastidiosa from two of the original PCR positive samples and later from symptomatic leaf petioles of four out of four vines from one vineyard that were originally PCR positive.

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

Plant Protection Science ◽

10.17221/10507-pps ◽

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.

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Deconvolution of the confounding variations for reverse transcription quantitative real-time polymerase chain reaction by separate analysis of biological replicate data

Analytical Biochemistry ◽

10.1016/j.ab.2012.04.029 ◽

2012 ◽

Vol 427 (1) ◽

pp. 21-25 ◽

Cited By ~ 4

Author(s):

Daijun Ling ◽

Christian J. Pike ◽

Paul M. Salvaterra

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Reverse Transcription ◽

Separate Analysis ◽

Chain Reaction ◽

Polymerase Chain

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Multiplex Real-Time PCR Assays for the Identification of the Potato Cyst and Tobacco Cyst Nematodes

Plant Disease ◽

10.1094/pdis-94-8-0959 ◽

2010 ◽

Vol 94 (8) ◽

pp. 959-965 ◽

Cited By ~ 22

Author(s):

Mark K. Nakhla ◽

Kristina J. Owens ◽

Wenbin Li ◽

Gang Wei ◽

Andrea M. Skantar ◽

...

Keyword(s):

Real Time ◽

Cyst Nematode ◽

Potato Cyst Nematode ◽

Globodera Pallida ◽

Cyst Nematodes ◽

Detection And Identification ◽

Polymerase Chain ◽

Probe Set ◽

Internal Transcribed Spacer Rdna ◽

Quantitative Pcr Assay

TaqMan primer-probe sets were developed for the detection and identification of potato cyst nematodes (PCNs) Globodera pallida and G. rostochiensis using two-tube, multiplex real-time polymerase chain reaction (PCR). One tube contained a primer-probe set specific for G. pallida (pale potato cyst nematode) multiplexed with another primer-probe set specific for G. rostochiensis (golden potato cyst nematode). A second tube consisted of the G. pallida-specific primer-probe set multiplexed with a primer-probe set specific for G. tabacum (the morphologically similar tobacco cyst nematode). This internal transcribed spacer rDNA-based system was specific for the Globodera spp. of interest and successfully identified several populations of PCN. This rapid, sensitive, and specific quantitative PCR assay presents a useful tool for PCN regulatory response and management programs.

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Etiology and Real-Time Polymerase Chain Reaction-Based Detection of Gremmeniella- and Phomopsis-Associated Disease in Norway Spruce Seedlings

Phytopathology ◽

10.1094/phyto-96-1305 ◽

2006 ◽

Vol 96 (12) ◽

pp. 1305-1314 ◽

Cited By ~ 18

Author(s):

Isabella Børja ◽

Halvor Solheim ◽

Ari M. Hietala ◽

Carl Gunnar Fossdal

Keyword(s):

Polymerase Chain Reaction ◽

Sequence Analysis ◽

Real Time ◽

Norway Spruce ◽

Internal Transcribed Spacer ◽

Chain Reaction ◽

Large Tree ◽

Nursery Stock ◽

European Race ◽

Polymerase Chain

In spring 2002, an unusual disease outburst was recorded on Norway spruce seedlings in southeast Norway. Extensive damage was recorded on 1- and 2-year-old Norway spruce seedlings that either had wintered in nursery cold storage or had been planted out in autumn 2001. The damage was characterized by leader shoot dieback and stem necroses on the upper or lower part of the shoot from 2001. Gremmeniella abietina and a Phomopsis sp. frequently were isolated from the diseased seedlings. Internal transcribed spacer (ITS) ribosomal (r)DNA sequence analysis and random amplified microsatellites profiling indicated that the G. abietina strains associated with diseased nursery seedlings belonged to the large-tree type (LTT) ecotype of the European race of G. abietina var. abietina, and inoculation tests confirmed their pathogenicity on Norway spruce. Based on ITS rDNA sequence analysis, the Phomopsis strains associated with diseased seedlings did not represent any characterized Phomopsis spp. associated with conifers. The Phomopsis sp. was not pathogenic in inoculation tests, indicating that it may be a secondary colonizer. ITS-based real-time polymerase chain reaction assays were developed in order to detect and quantify G. abietina and Phomopsis in the nursery stock. We describe here the G. abietina-associated shoot dieback symptoms on Norway spruce seedlings and conclude that the unusual disease outburst likely was related to the G. abietina var. abietina epidemic caused by the LTT on large Scots pines in 2001.

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Detection and Quantification of Phytophthora ramorum from California Forests Using a Real-Time Polymerase Chain Reaction Assay

Phytopathology ◽

10.1094/phyto.2004.94.10.1075 ◽

2004 ◽

Vol 94 (10) ◽

pp. 1075-1083 ◽

Cited By ~ 101

Author(s):

Katherine J. Hayden ◽

David Rizzo ◽

Justin Tse ◽

Matteo Garbelotto

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Pathogen Detection ◽

Phytophthora Species ◽

Phytophthora Ramorum ◽

Plant Diseases ◽

Chain Reaction ◽

Nested Polymerase Chain Reaction ◽

New Host ◽

Polymerase Chain

The timely and accurate detection of pathogens is a critical aid in the study of the epidemiology and biology of plant diseases. In the case of regulated organisms, the availability of a sensitive and reliable assay is essential when trying to achieve early detection of the pathogen. We developed and tested a real-time, nested polymerase chain reaction (PCR) assay for the detection of Phytophthora ramorum, causal agent of sudden oak death. This technique then was implemented as part of a widespread environmental screen throughout California. The method here described is sensitive, detecting less than 12 fg of pathogen DNA, and is specific for P. ramorum when tested across 21 Phytophthora spp. Hundreds of symptomatic samples from 33 sites in 14 California counties were assayed, resulting in the discovery of 10 new host species and 23 infested areas, including 4 new counties. With the exception of a single host, PCR-based discovery of new hosts and infested areas always was confirmed by traditional pathogen isolations and inoculation studies. Nonetheless, molecular diagnostics were key in early pathogen detection, and steered the direction of further research on this newly discovered and generalist Phytophthora species.

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Configuración Dermatoglífica, ACTN3 y ECA: Un estudio transcultural en deportistas de diferentes Disciplinas (Dermatoglyphic Configuration, ACTN3 and ECA: A transcultural study in athletes of different Disciplines)

Retos ◽

10.47197/retos.v44i0.90561 ◽

2021 ◽

Vol 44 ◽

pp. 87-94

Author(s):

Claudio Hernández-Mosqueira ◽

Humberto Castillo-Quezada ◽

Sebastian Peña-Troncoso ◽

Andrea Joao ◽

Rodrigo Moraga Muñoz ◽

...

Keyword(s):

Real Time ◽

Genetic Variants ◽

Saliva Sample ◽

High Association ◽

Group A ◽

Polymerase Chain ◽

Los Grupos ◽

Aerobic And Anaerobic ◽

Very High ◽

Transcultural Study

El rendimiento físico se ha asociado con diferentes variantes genéticas. El objetivo de este estudio fue determinar la asociación entre características dermatoglíficas y los genotipos ACTN3 y ECA. La muestra la constituyen 82 seleccionados nacionales de diferentes modalidades deportivas de Brasil, Japón y Chile. Los marcadores ACTN3 y ECA se obtuvieron a través de una muestra de saliva y se analizaron mediante el empleo de cadena de la polimerasa en tiempo real a partir del iQ5ThermalCycler, BioRad, mientras que para la configuración dermatoglifica se utilizó un lector de huella digital Verifier® 320 LC 2.0. Estos deportistas fueron clasificados en grupos de acuerdo a la configuración de sus patrones dermatoglíficos (A, L, W, D10 y SQTL) en los siguientes grupos: aeróbicos (n= 27); anaeróbicos (n= 55). En cuanto a la frecuencia de aparición de polimorfismos, para ambos grupos predomina el genotipo RX 48,0% y 49,1% de ACTN3, y DI 68,0% y 41,3% para ECA, en los grupos aeróbico y anaeróbico respectivamente. En el grupo aeróbico se observa una asociación muy alta entre ACTN3 con presilla, verticilo y D10 (r=0,90), en el grupo anaeróbico solo observa asociación alta en presilla (r=0,77), para el gen ECA se observan asociaciones moderadas entre presilla, verticilo y D10 (r=0,45), en el grupo aeróbico. Conclusión: Las características dermatoglíficas pueden estar asociados con la variante alélica del gen ACTN3 (RR) y ECA (DI), para perfiles deportivos de carácter anaeróbico. Abstract. Physical performance has been associated with different genetic variants. The objective of this study was to determine the association between dermatoglyphic characteristics and the ACTN3 and ECA genotypes. The sample is established by 82 national teams from different sports modalities in Brazil, Japan and Chile. The ACTN3 and ECA markers were obtained through a saliva sample and was analyzed using the polymerase chain in real time from the iQ5ThermalCycler, BioRad, while a Verifier® 320 LC fingerprint reader was used for the dermatoglyphic configuration. These athletes were classified into groups according to the configuration of their dermatoglyphic patterns (A, L, W, D10 and SQTL) in the following groups: aerobics (n = 27); anaerobic (n = 55). Regarding the frequency in the appearance of polymorphisms, the RX genotype 48.0% and 49.1% of ACTN3 predominated for both groups and DI 68.0% and 41.3% for ACE, in the aerobic and anaerobic groups respectively. In the aerobic group a very high association is observed between ACTN3 with a clip, verticil and D10 (r = 0.90), in the anaerobic group only a high association was observed in the clip (r = 0.77), for the ACE gene they are observed moderate associations between clip, verticil and D10 (r = 0.45), in the aerobic group. Conclusion: Dermatoglyphic characteristics may be associated with the allelic variant of the ACTN3 (RR) and RCT (DI) gene, for sports profiles of an anaerobic nature.

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