scholarly journals Real-time PCR Quantification and Mycotoxin Production of Fusarium graminearum in Wheat Inoculated with Isolates Collected from Potato, Sugar Beet, and Wheat

2007 ◽  
Vol 97 (7) ◽  
pp. 835-841 ◽  
Author(s):  
Rishi R. Burlakoti ◽  
Rolando Estrada ◽  
Viviana V. Rivera ◽  
Anuradha Boddeda ◽  
Gary A. Secor ◽  
...  
Keyword(s):  
Sugar Beet ◽  
Real Time ◽  
Fusarium Graminearum ◽  
Real Time Pcr ◽  
The United States ◽  
Taqman Probe ◽  
Head Blight ◽  
Dry Rot ◽  
Trichodiene Synthase ◽  
Intergenic Sequences

Fusarium graminearum causes Fusarium head blight (FHB) in small grains worldwide. Although primarily a pathogen of cereals, it also can infect noncereal crops such as potato and sugar beet in the United States. We used a real-time polymerase chain reaction (PCR) method based on intergenic sequences specific to the trichodiene synthase gene (Tri5) from F. graminearum. TaqMan probe and primers were designed and used to estimate DNA content of the pathogen (FgDNA) in the susceptible wheat cv. Grandin after inoculation with the 21 isolates of F. graminearum collected from potato, sugar beet, and wheat. The presence of nine mycotoxins was analyzed in the inoculated wheat heads by gas chromatography and mass spectrometry. All isolates contained the Tri5 gene and were virulent to cv. Grandin. Isolates of F. graminearum differed significantly in virulence (expressed as disease severity), FgDNA content, and mycotoxin accumulation. Potato isolates showed greater variability in producing different mycotoxins than sugar beet and wheat isolates. Correlation analysis showed a significant (P < 0.001) positive relationship between FgDNA content and FHB severity or deoxynivalenol (DON) production. Moreover, a significant (P < 0.001) positive correlation between FHB severity and DON content was observed. Our findings revealed that F. graminearum causing potato dry rot and sugar beet decay could be potential sources of inoculum for FHB epidemics in wheat. Real-time PCR assay provides sensitive and accurate quantification of F. graminearum in wheat and can be useful for monitoring the colonization of wheat grains by F. graminearum in controlled environments, and evaluating wheat germplasms for resistance to FHB.

Quantification of Fusarium graminearum in infected wheat by species specific real-time PCR applying a TaqMan Probe

2004 ◽  
Vol 59 (1) ◽  
pp. 141-146 ◽  
Author(s):  
G.H. Reischer ◽  
M. Lemmens ◽  
A. Farnleitner ◽  
A. Adler ◽  
R.L. Mach
Keyword(s):  
Real Time ◽  
Fusarium Graminearum ◽  
Real Time Pcr ◽  
Taqman Probe ◽  
Species Specific

A Diagnostic TaqMan Real-Time PCR Assay for in planta Detection and Quantification of Colletotrichum theobromicola, Causal Agent of Boxwood Dieback

Plant Disease ◽  
2021 ◽  
Author(s):  
Harleen Kaur ◽  
Raghuwinder Singh ◽  
Vinson P. Doyle ◽  
Rodrigo Valverde
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Species Complex ◽  
The United States ◽  
Taqman Probe ◽  
In Planta ◽  
Pcr Assay ◽  
Phytophthora Root Rot ◽  
Specificity And Sensitivity ◽  
Detection And Quantification

Boxwood dieback, caused by Colletotrichum theobromicola, is spreading at an alarming rate in the boxwood industry in the United States. Although C. theobromicola has been accepted as a distinct species within the C. gloeosporioides species complex, it is difficult to distinguish it from other closely related species based on morphology. Moreover, molecular identification of C. theobromicola requires amplification and sequencing of multiple loci, which can be expensive and time consuming. Therefore, a diagnostic TaqMan real-time PCR assay was developed for early and accurate detection and quantification of C. theobromicola in boxwood. The study involved the design of species-specific primers and a TaqMan probe to differentiate C. theobromicola from other closely related Colletotrichum species. The primers and probe discriminate between C. theobromicola and other species in the C. gloeosporioides species complex and can detect C. theobromicola at very low concentrations, illustrating the high specificity and sensitivity of the assay. This TaqMan real-time PCR assay accurately and rapidly distinguishes boxwood dieback from other diseases with similar symptomatology including, Macrophoma blight, Phytophthora root rot, and Volutella blight, as well as some disorders produced by abiotic agents.

scholarly journals Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran

2021 ◽  
Vol 11 ◽  
Author(s):  
Reza Fotouhi-Ardakani ◽  
Seyedeh Maryam Ghafari ◽  
Paul Donald Ready ◽  
Parviz Parvizi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Taqman Probe ◽  
Amino Acid Permease ◽  
Specific Primers ◽  
Accurate Identification ◽  
Parasitic Load ◽  
Species Specific

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

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.

Evaluation of droplet digital PCR for the detection of black canker disease in tomato

Plant Disease ◽  
2021 ◽  
Author(s):  
Li Wang ◽  
Tian Qian ◽  
Pei Zhou ◽  
Wenjun Zhao ◽  
Xianchao Sun
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Plant Protection ◽  
Detection Threshold ◽  
Digital Pcr ◽  
Taqman Probe ◽  
Tomato Seed ◽  
Canker Disease ◽  
Probe Set

Clavibacter michiganensis subsp. michiganensis (Cmm), the cause of bacterial canker disease, is one of the most destructive pathogens in greenhouse and field tomato. The pathogen is now present in all main production areas of tomato and is quite widely distributed in the EPPO（European and Mediterranean Plant Protection Organization）region. The inspection and quarantine of the plant pathogens relies heavily on accurate detection tools. Primers and probes reported in previous studies do not distinguish the Cmm pathogen from other closely related subspecies of C. michiganensis, especially the non-pathogenic subspecies that were identified from tomato seeds recently. Here, we have developed a droplet digital polymerase chain reaction (ddPCR) method for the identification of this specific bacterium with primers/TaqMan probe set designed based on the pat-1 gene of Cmm. This new primers/probe set has been evaluated by qPCRthe real time PCR（qPCR） and ddPCR. The detection results suggest that the ddPCR method established in this study was highly specific for the target strains. The result showed the positive amplification for all 5 Cmm strains，and no amplification was observed for the other 43 tested bacteria, including the closely related C. michiganensis strains. The detection threshold of ddPCR was 10.8 CFU/mL for both pure Cmm cell suspensions and infected tomato seed, which was 100 times-fold more sensitive than that of the real-time PCR (qPCR ) performed using the same primers and probe. The data obtained suggest that our established ddPCR could detect Cmm even with low bacteria load, which could facilitate both Cmm inspection for pathogen quarantine and the routine pathogen detection for disease control of black canker in tomato.

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