Abstract
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.
Highlights
Cucurbits are one of the most cultivated vegetable crops worldwide, and a large number of annual species are under cultivation in tropical and sub-tropical regions (Judd et al 2008)
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
Pseudoperonospora cubensis and P. xanthii pathogens are devastating in nature with a broad host range of cucurbits and challenging to identify by morphological, biochemical features, which are similar to many obligate pathogens (Lee et al 2016)
Summary
Cucurbits are one of the most cultivated vegetable crops worldwide, and a large number of annual species are under cultivation in tropical and sub-tropical regions (Judd et al 2008). In India, the cucurbits are cultivated mainly as food crops, which share about 5.6% of the total vegetable production. Apart from food and vegetable use, many cucurbits seeds and fruits are reported to have medicinal properties due to the presence of secondary metabolite cucurbitacin (Kaushik et al 2015). Out of all fungal diseases, mildews are widespread and destructive plant diseases caused by obligate biotrophic pathogens, which produce large quantities of short-lived, asexual spores on the surface of host plant leaves. These asexual spores cause secondary infections and disperse through the air to infect fresh plants
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
