Biology subjects

Sarkes, A.

Publications and source records attributed to Sarkes, A..

4 recordsLinked to original sources

bioRxiv2023

Detection and differentiation of Xanthomonas translucens pv. translucens and pv. undulosa from wheat and barley by duplex quantitative PCR

Two probe-based quantitative PCR (qPCR) systems, namely P-Xtt and P-Xtu, were developed for diagnosis of cereal bacterial leaf streak pathogens Xanthomonas translucens pv. translucens and pv. undulosa, respectively. P-Xtt is specific to pv. translucens. P-Xtu is specific to pv. undulosa, pv. cerealis, pv. secalis and pv. pistaciae. P-Xtt and P-Xtu worked on all accessible strains of pv. translucens and pv. undulosa, respectively. Both systems could detect 100 copies of the target gBlock DNA. The two systems could be used in both singleplex qPCR and duplex qPCR with similar efficiencies. On genomic DNA from strains of various X. translucens pathovars, both singleplex qPCR and duplex qPCR could specifically detect and differentiate pv. translucens and pv. undulosa. The duplex qPCR could detect pv. translucens and pv. undulosa from genomic DNA of 1,000 bacterial cells. On infected barley and wheat grain samples, and on one infected wheat leaf sample, the duplex qPCR showed similar efficiency compared to a previously published qPCR system but with the additional capability of pathovar differentiation. The duplex qPCR system developed in this study will be useful in studies on bacterial leaf streak and detection/differentiation of the pathogens.

plant biology↗

bioRxiv2021

Detection of Xanthomonas translucens pv. undulosa from wheat by quantitative PCR

A probe-based quantitative PCR (qPCR) protocol was developed for detection and evaluation of the wheat bacterial leaf streak pathogen Xanthomonas translucens pv. undulosa (Xtu). The protocol can also detect X. translucens pv. translucens (Xtt), but cant differentiate the two pathovars. When tested on DNA from plant, non-target bacteria and culture of microorganisms from wheat seeds, the qPCR showed a high specificity. On purified Xtu DNA, the qPCR was more sensitive than a loop-mediated isothermal amplification (LAMP) assay. When DNA samples from a set of serial dilutions of Xtu cells were tested, the qPCR method could repeatedly generate quantification cycle (Cq) values from the dilutions containing [≥]1,000 cells. Since 2 {micro}L of the total of 50 {micro}L DNA was used in one reaction, one qPCR reaction could detect the presence of the bacteria in samples containing as few as 40 bacterial cells. The qPCR could detect the bacteria from both infected seed and leaf tissues. For seed testing, a protocol for template preparation was standardized, which allowed one qPCR reaction to test DNA from the surface of one seed. Thus, the qPCR system could theoretically detect Xtu and/or Xtt in samples where the bacteria had an average concentration [≥]40 cells per seed.

microbiology↗

bioRxiv2020

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection of Tomato brown rugose fruit virus (ToBRFV)

Tomato brown rugose fruit virus (ToBRFV) is a member of Tobamovirus infecting tomato and pepper. Within North America, both the United States and Mexico consider ToBRFV to be a regulated pest. In Canada, the presence of ToBRFV has been reported, but an efficient diagnostic system has not yet been established. Here, we describe the development and assessment of a loop-mediated isothermal amplification (LAMP)-based assay to detect ToBRFV. The LAMP test was efficient and robust, and results could be obtained within 35 min with an available RNA sample. Amplification was possible when either water bath or oven were used to maintain the temperature at isothermal conditions (65{degrees}C), and results could be read by visual observation of colour change. Detection limit of the LAMP was eight target RNA molecules. Under the experimental conditions tested, LAMP was as sensitive as qPCR and 100 times more sensitive than the currently used rt-PCR. We recommend this sensitive, efficient LAMP protocol to be used for routine lab testing of ToBRFV.

microbiology↗

bioRxiv2019

Plasmodiophora brassicae in its environment-effects of temperature and light on resting spore survival in soil

Clubroot caused by Plasmodiophora brassicae is an important disease on cruciferous crops worldwide. Management of clubroot has been challenging, due largely to the millions of resting spores produced within an infected root that can survive dormant in the soil for many years. This study was conducted to investigate some of the environmental conditions that may affect the survival of resting spores in the soil. Soil samples containing clubroot resting spores (1 x 107 spores g-1 soil) were stored at various temperatures for two years. Additionally, other samples were buried in soil, or kept on the soil surface in the field. The content of P. brassicae DNA and the numbers of viable spores in the samples were assessed by quantitative polymerase chain reaction (qPCR) and pathogenicity bioassays, respectively. The results indicated that 4{degrees}C, 20{degrees}C and being buried in the soil were better conditions for spore survival than were -20{degrees}C, 30{degrees}C and at the soil surface. Most of the spores kept on the soil surface were killed, suggesting the negative effect of light on spore viability. Additional experiments confirmed that ultraviolet (UV) light contributed a large negative effect on spore viability as lower pathogenicity and less DNA content were observed from the 2-and 3-hour UV light treated spores compared to the untreated control. Finally, this work demonstrated that DNA-based quantification methods such as qPCR can be poor predictors of P. brassicae disease potential due to the presence and persistence of DNA from dead spores.

microbiology↗