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

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.

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

Plant Disease ◽

10.1094/pdis-03-19-0563-re ◽

2019 ◽

Vol 103 (12) ◽

pp. 3031-3040 ◽

Cited By ~ 1

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.

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

Phytopathology ◽

10.1094/phyto-98-5-0592 ◽

2008 ◽

Vol 98 (5) ◽

pp. 592-599 ◽

Cited By ~ 151

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.

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

Plant Physiology and Biochemistry ◽

10.1016/j.plaphy.2005.07.003 ◽

2005 ◽

Vol 43 (9) ◽

pp. 890-899 ◽

Cited By ~ 71

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

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A robust method for identification and in-planta detection of Verticillium dahliae in the infected olive trees, using real-time PCR and nested PCR

Physiological and Molecular Plant Pathology ◽

10.1016/j.pmpp.2020.101559 ◽

2020 ◽

Vol 112 ◽

pp. 101559

Author(s):

Seyed Ali Mousavi ◽

Mojtaba Keykhasaber ◽

Leila Fahmideh ◽

Mehdi Aran

Keyword(s):

Real Time ◽

Verticillium Dahliae ◽

Real Time Pcr ◽

Nested Pcr ◽

Robust Method ◽

In Planta ◽

Olive Trees

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RT real-time PCR-based quantification ofUromyces fabae in planta

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.2011.02343.x ◽

2011 ◽

Vol 322 (2) ◽

pp. 131-137 ◽

Cited By ~ 9

Author(s):

Ralf T. Voegele ◽

Annette Schmid

Keyword(s):

Real Time ◽

Real Time Pcr ◽

In Planta

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Development of a DNA-Based Real-Time PCR Assay To Quantify Allorhizobium vitis Over Time in Grapevine (Vitis vinifera L.) Plantlets

Plant Disease ◽

10.1094/pdis-04-20-0732-re ◽

2020 ◽

pp. PDIS-04-20-0732

Author(s):

Trong Nguyen-Huu ◽

Jeanne Doré ◽

Essaïd Aït Barka ◽

Céline Lavire ◽

Christophe Clément ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Classical Method ◽

Population Level ◽

Endophytic Bacterium ◽

Vitis Vinifera L ◽

In Planta ◽

Pcr Assay ◽

Allorhizobium Vitis ◽

Over Time

Allorhizobium vitis is the primary causal pathogen of grapevine crown gall disease. Because this endophytic bacterium can survive as a systemic latent (symptomless) infection in grapevine, detecting and monitoring its development in planta is of great importance. In plant bacteria studies, plate counting is routinely used as a simple and reliable method to evaluate the bacterial population level in planta. However, isolation techniques are time-consuming and present some disadvantages such as the risk of contamination and the need for fresh samples for research. In this study, we developed a DNA-based real-time PCR assay that can replace the classical method to monitor the development of Allorhizobium vitis in grapevine plantlets. Primers targeting Allorhizobium vitis chromosomic genes and the virulent tumor-inducing plasmid were validated. The proposed quantitative real-time PCR technique is highly reliable and reproducible to assess Allorhizobium vitis numeration at the earliest stage of infection until tumor development in grapevine plantlets. Moreover, this low-cost technique provides rapid and robust in planta quantification of the pathogen and is suitable for fundamental research to monitor bacterial development over time.

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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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A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth

Letters in Applied Microbiology ◽

10.1111/j.1472-765x.2010.02942.x ◽

2010 ◽

Vol 51 (6) ◽

pp. 603-610 ◽

Cited By ~ 28

Author(s):

B. Llorente ◽

F. Bravo-Almonacid ◽

C. Cvitanich ◽

E. Orlowska ◽

H.N. Torres ◽

...

Keyword(s):

Phytophthora Infestans ◽

Real Time ◽

Real Time Pcr ◽

Quantitative Real Time Pcr ◽

In Planta ◽

Pcr Method

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In Planta Quantification of Plasmodiophora brassicae Using Signature Fatty Acids and Real-Time PCR

Plant Disease ◽

10.1094/pdis-94-4-0432 ◽

2010 ◽

Vol 94 (4) ◽

pp. 432-438 ◽

Cited By ~ 20

Author(s):

Thomas Sundelin ◽

Camilla Beck Christensen ◽

John Larsen ◽

Kaare Møller ◽

Mette Lübeck ◽

...

Keyword(s):

Fatty Acid ◽

Real Time ◽

Real Time Pcr ◽

Plasmodiophora Brassicae ◽

Disease Incidence ◽

Infested Soil ◽

In Planta ◽

Internal Transcribed Spacer Region ◽

Severity Scores ◽

Basic Studies

Until now, molecular and biochemical methods have only been used to show whether or not Plasmodiophora brassicae is present in plant or soil samples but not to what extent. Here, in planta quantification of P. brassicae by whole-cell fatty acid (WCFA) measurements and real-time polymerase chain reaction (PCR) was evaluated. Arachidonic acid (ARA, 20:4) was the most abundant fatty acid in resting spores and was only found in infected roots, which indicates a potential of ARA as a biomarker for P. brassicae. A real-time PCR assay was developed using primers designed from the internal transcribed spacer region of the ribosomal DNA. Using these primers, it was possible to detect P. brassicae in infected roots 10 days after germination of plants sown in infested soil. A bioassay showed that the amounts of ARA found by WCFA analysis and the DNA found by real-time PCR in infected plants were well correlated. These measurements also correlated with the soil spore content and the assessed disease incidence and disease severity scores. Therefore, we conclude that WCFA analysis and real-time PCR are good tools for P. brassicae quantification that can be applied to basic studies of the pathogen and in resistance screens.

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