Identification of powdery mildew disease on Cleome rutidosperma caused by Podosphaera xanthii in Odisha, India

Nayak, Ashish Kumar; Mund, Nitesh Kumar; Bandamaravuri, Kishore Babu

doi:10.1007/s42360-018-0059-2

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“…The rDNA gene cluster, including ITS1, 5.8S and ITS2 regions of selected mildew pathogens listed in Table 1 , were subjected to PCR amplification and direct sequencing using Big Dye™ Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, USA), as described earlier (Nayak et al 2018 ). The PCR products of both powdery and downy mildew pathogens such as P. xanthii CsKP07 and P. cubensis CsKD11 were then ligated into pGEM-T (Promega, USA) cloning vector according to manufacturer instructions.…”

Section: Methodsmentioning

confidence: 99%

Simultaneous detection of downy mildew and powdery mildew pathogens on Cucumis sativus and other cucurbits using duplex-qPCR and HRM analysis

et al. 2020

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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 speciesspecific 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.

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Section: Methodsmentioning

confidence: 99%