Material And Methods Used By Powdery Mildew Essay

720 Words Jul 24th, 2016 3 Pages
Materials and Methods
Powdery mildew samples
Powdery mildew isolates included in this study were obtained from the symptomatic sweet cherry leaves collected from the orchards in cherry growing regions of Washington States. The fungus that causes powdery mildew on sweet cherry fit the description of Podosphaera prunicola U. Braun (Braun and Cook, 2012). Powdery mildew conidia and mycelium were harvested from leaf surfaces by scraping using a disposable scalpel and placed on 2 ml Eppendorf tube and stored at – 80 ºC until further procedure. For subsequent usages, P. Prunicola was also grown in young cherry plants in the greenhouse. Mycelium and conidia were also harvested from the greenhouse plants and stored for double – stranded RNA (dsRNA) extraction.

Double – stranded RNA (dsRNA) extraction and next generation sequencing
To isolate double – stranded RNA (dsRNA), at first fungal mass was grounded in liquid nitrogen to a fine powder and then double- RNA viral extraction mini kit (iNtRON Biotechnology, Inc.) was used as directed by the manufacturers. To visualize dsRNA each extraction was run on 1% agarose gel containing TAE buffer using the standard electrophoresis technique.
Almost 1.5 μg of dsRNA per-sample was submitted for sequencing library construction using Illumina True Seq Stranded Total RNA preparation kit with Plant Ribo-Zero and 50 –bp single – end read high throughput sequencing on the Illumina Hi Seq 2000 platform with multiplexing (Microarray and…

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