Detection of Golovinomyces orontii using species-specific primers and high-resolution melting analysis

Abstract

This study presents the development of species-specific PCR primers for detection of Golovinomyces orontii, the causal agent of powdery mildew disease on different cucurbit hosts. The species-specific primers CgF2 and CgR2 were designed based on partial ITS and 5.8S rDNA sequences using an isolates collection of powdery mildew and downy mildew pathogens from different sources. After optimization of the PCR conditions, a 233 bp fragment was amplified only from DNA of G. orontii. Limit of detection was determined to be 0.01 ng of pure conidial genomic DNA. Further association of PCR and high-resolution melting (HRM) analysis showed that the designed primer set can be used for differentiation of G. orontii from different powdery mildew fungi in infected cucurbits tissue collected in the field.

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

“…Both the species-specific PCR primer pairs (PcK F&R and PxK F&R) validated under individual PCR assays, as mentioned above, were subjected for validation under duplex PCR assay mode. The specificity of the duplex PCR assay was evaluated against different test microbial isolates as mentioned in Table 2 , including the test microbes used in the Nayak et al ( 2019 ) and genomic DNA obtained from dually infected cucumber ( P. cubensis CsKD11 and P. xanthii CsKP07) leaf samples (Table 4 ) was used as a positive control. The duplex PCR assay showed amplification only in positive samples with two distinctly separated specific bands of ~ 705 bp and ~ 290 bp (Figs.…”

“…The ITS region of the rDNA gene cluster has established as a suitable target for fungal species-specific primers due to their high copy number, sequence variability, and fidelity among pathogen species or subspecies. Several researchers reported that the identification of obligate fungi causing powdery mildew and downy mildew diseases by developing species-specific primers targeted on ITS region of rDNA (Wang et al 2008 ; Pirondi et al 2015 ; Bandamaravuri et al 2015 ; Lee et al 2016 ; Nayak et al 2019 ).…”

“…Both conidia and sporangia collected into separate bottles containing 50 ml sterile distilled water (DW) at four °C with the help of vacuum suction (150 ppm). The samples were purified and subjected to genomic DNA extraction as per the protocol described by Nayak et al ( 2019 ). In the case of test microbes used in this study (Table 2 ), genomic DNA extraction was carried out as described previously, and the DNA from the cultures reported in the previous study Nayak et al ( 2019 ) were used as test samples for validation of PCR and qPCR assays.…”

“…Since both the pathogens produce similar symptoms and modes of infection, identification and differentiation of these pathogens become difficult. Though both are different species, morphologically distinct, and even epidemiological variations were reported, their molecular characterization and detection could provide an accurate estimate of each pathogen's distribution and economic impact (Nayak et al 2019 ). In early stages, white circular powdery patches on either side of infected leaves and later whole leaf, petiole, stem, and branches of the plant were covered with white powdery spores.…”