Development of isothermal detection methodology for Plasmopara viticola and Phytophthora infestans
Author(s)
Si Ammour, Melissa
Date Issued
March 24, 2017
Type
Doctoral Thesis
Abstract
This research concerns the development and application of isothermal amplification detection techniques to two important plant pathogenic Oomycete: Plasmopara viticola causing grapevine downy mildew and Phytophthora infestans, the causal agent of potato late blight. Real-time Loop-Mediated Isothermal Amplification (LAMP) assay was applied for the early detection of P. viticola from infected grapevine plants. A rapid crude plant extract (CPE) preparation method from infected leaves was developed for on-site testing. The LAMP assay targeting the large ribosomal subunit gene (LSU) was specific to P. viticola and sensitive to 10 sporangia/ml. In the inoculation experiments of greenhouse plants and leaf discs samples, LSU primers detected downy mildew infections at a concentration of 103 sporangia/ml after 24h of inoculation. In addition, Real-time LAMP and Recombinase Polymerase Amplification (RPA) assays were developed targeting the ITS2 region of the ribosomal DNA of P. infestans. Both LAMP and RPA assays showed specificity to P. infestans and the closely related species P. andina, P. mirabilis, P. phaseoli and P. ipomoeae. No cross-reaction occurred with the potato pathogens tested. LAMP and RPA assays detected DNA at 50 fg/ul and showed to be insensitive to CPE inhibition. The isothermal assays were validated with inoculated potato plants using a portable Smart-DART device. The LAMP and RPA assays effectively detected P. infestans DNA in symptomless leaf tissue 24 h and 72 h post-inoculation, respectively. Finally, the suitability of the latter LAMP assay was tested for a rapid detection and quantification of airborne inoculum of P. infestans. Standard curves of P. infestans sporangia and ITS2 copy were constructed. The quantitative LAMP (qLAMP) assay was validated in the laboratory with silicon-coated rods containing a known number of sporangia. The analysis was performed using a regression procedure. A linear relationship between the number of sporangia deposited onto the rods estimated with microscopy and the number of sporangia estimated with the qLAMP assay was obtained. A rapid and accurate on-site detection of P. infestans and P. viticola in plant material and spore samplers will contribute to improved disease diagnosis, early detection of first infections and facilitate prompt management decisions.
Additional information
Dottorato di ricerca in Scienze delle produzioni vegetali e animali
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