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

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 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.

Effects of Nutrient Solution Irrigation Quantity and Downy Mildew Infection on Growth and Physiological Traits of Greenhouse Cucumber

“…Cucumber, as an important economic crop, is one of the main vegetables widely planted in the world. It has a status second only to tomato in fruit vegetables [8,9]. Because of its rich taste and nutritional value, cucumber is deeply loved by consumers and plays an increasingly important role in agricultural structure adjustment and farmers' income increase.…”

“…Severely infected plants produce retarded/deformed fruits, which leads to a considerable loss of production. As the disease progress, the yellow spots became brown and then necrotic, which leads to leaf fall and death [8,25]. Infection by downy mildew pathogens results in many changes in the plant's metabolic processes, including changes in the physiological structure of the leaves, which may affect the stomata that regulate transpiration and water loss from plants [26].…”

A Rapid Detection Method for Tomato Gray Mold Spores in Greenhouse Based on Microfluidic Chip Enrichment and Lens-Less Diffraction Image Processing

“…Laboratory testing techniques mainly include electron microscopy testing technology, polymerase chain reaction (PCR), and biological testing technology, etc. These testing technologies can accurately determine the type of disease, but laboratory testing technology is destructive, time-consuming, and labor-intensive [ 15 , 16 , 17 ]. Spectral detection technology and image processing technology are used to detect known or specific diseases.…”

Separation-enrichment method for airborne disease spores based on microfluidic chip

“…At present, the detection methods of fungal spores mainly include image processing [6,7] , weighing method [8] , and polymerase chain reaction (PCR) [9,10] . However, due to the presence of a large amount of impurity, the above methods are limited in the detection of fungal spores.…”