Simultaneous detection of downy mildew and powdery mildew pathogens on Cucumis sativus and other cucurbits using duplex-qPCR and HRM analysis
Autor: | Anu Sharma Bandamaravuri, Abdul Samad, Ashish Kumar Nayak, Kishore Babu Bandamaravuri |
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Jazyk: | angličtina |
Rok vydání: | 2020 |
Předmět: |
0106 biological sciences
lcsh:Biotechnology Biophysics lcsh:QR1-502 Oligonucleotide Primer 01 natural sciences Applied Microbiology and Biotechnology lcsh:Microbiology 03 medical and health sciences Powdery mildew lcsh:TP248.13-248.65 Internal transcribed spacer 030304 developmental biology Detection limit 0303 health sciences Mildew biology Cucumber biology.organism_classification Horticulture qPCR Downy mildew Pseudoperonospora cubensis Original Article SYBR green Cucumis 010606 plant biology & botany |
Zdroj: | AMB Express, Vol 10, Iss 1, Pp 1-11 (2020) AMB Express |
ISSN: | 2191-0855 |
DOI: | 10.1186/s13568-020-01071-x |
Popis: | Powdery mildew and downy mildew are two devastating diseases on cucumber and other cucurbit crops caused by Podosphaera xanthii and Pseudoperonospora cubensis, respectively. Identification and detection of these pathogens from field and plant material could be significant for the selection of resistant varieties and formulation of disease management strategies. In the present study, a duplex qPCR assay developed for simultaneous detection and quantification of both pathogens from different samples. Two sets of species-specific primers developed for the detection of P. xanthii and P. cubensis pathogens by targeting the internal transcribed spacer (ITS) region of the rDNA gene cluster. The specificity of designed primers was also evaluated against the different microbial, plant, soil, and environmental samples. Initially, the individual assays for P. cubensis and P. xanthii were validated using their corresponding species-specific primers, which amplified the prominent and distinctive products of ~ 705 bp and ~ 290 bp size, respectively. SYBR green-based duplex real-time PCR assay was developed to detect and quantify both mildew pathogens from different field samples. The species-specific oligonucleotide primer sets showed high specificity with melt curve peaks at 85.83 °C and 88.05 °C, for P. xanthii and P. cubensis, respectively. The relative quantification and lowest detection limit of qPCR assays using tenfold diluted plasmid (Csp1 and Csd1) DNA were estimated (0.1 pg/µl) through a standard curve. In this study, the species-specific PCR and qPCR assays in both simplex and duplex formats have been validated successfully. These assays could be useful for efficient detection and quantification of mildew pathogens from the cucumber and other cucurbit crops. |
Databáze: | OpenAIRE |
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