Materials and Methods
Inoculum source and preparation
Cherry (Prunus avium (L.) L. cv. Bing) leaves showing symptoms of powdery mildew were used as a source of primary inoculum. Powdery mildew colonies were identified as P. Prunicola based on microscopic observation (Broun and Cook 2012). To produce fresh inoculum foliage of young cherry (Prunus avium (L.) L. cv. Black Tartarian) plants grown in the greenhouse were inoculated. To harvest inoculum, infested leaves were soaked in sterile 0.005% Tween-20 solution and shaken vigorously for a few minutes to loosen conidia. The suspension was filtrated through cheesecloth and conidia was counted using a hemocytometer. The spore concentration of 100 spores ml-1, 500 spores ml-1, 1000 spores ml-1, 5000 spores ml-1, 10,000 spores ml-1 were prepared by serial dilution.
Orchard …show more content…
prunicola intergenic spacer (IGS) region was use in both PCR and qPCR of this study. This primer set was previously designed and positively tested in the WSU Grove lab. In brief, polymorphic nucleotides unique to P. prunicola were first identified by analysis of IGS sequence data, then primer was designed using primer 3 and confirmed by testing against powdery mildew fungi common in central Washington using conventional PCR method.
Using 1 l DNA extracted from Rotorod sampler as a template, 1X PCR standard buffer (New England Biolabs, Ipswich, MA), 200 M dNTPs (New England Biolabs), 400 nM each primer and 2.5 unit Taq polymerase (New England Biolabs) 20L PCR mix were prepared. For initial denaturation one cycle 94°C was applied. After that 35 cycles of , 94 °C for 45 seconds (DNA denaturation) 54 °C for 45 sec (oligonucleotide primer annealing), and 72 °C for 1 min (Taq polymerase extension) were applied. Lastly, 1 cycle of 72 °C for 10 min was applied for a final
Inoculum source and preparation
Cherry (Prunus avium (L.) L. cv. Bing) leaves showing symptoms of powdery mildew were used as a source of primary inoculum. Powdery mildew colonies were identified as P. Prunicola based on microscopic observation (Broun and Cook 2012). To produce fresh inoculum foliage of young cherry (Prunus avium (L.) L. cv. Black Tartarian) plants grown in the greenhouse were inoculated. To harvest inoculum, infested leaves were soaked in sterile 0.005% Tween-20 solution and shaken vigorously for a few minutes to loosen conidia. The suspension was filtrated through cheesecloth and conidia was counted using a hemocytometer. The spore concentration of 100 spores ml-1, 500 spores ml-1, 1000 spores ml-1, 5000 spores ml-1, 10,000 spores ml-1 were prepared by serial dilution.
Orchard …show more content…
prunicola intergenic spacer (IGS) region was use in both PCR and qPCR of this study. This primer set was previously designed and positively tested in the WSU Grove lab. In brief, polymorphic nucleotides unique to P. prunicola were first identified by analysis of IGS sequence data, then primer was designed using primer 3 and confirmed by testing against powdery mildew fungi common in central Washington using conventional PCR method.
Using 1 l DNA extracted from Rotorod sampler as a template, 1X PCR standard buffer (New England Biolabs, Ipswich, MA), 200 M dNTPs (New England Biolabs), 400 nM each primer and 2.5 unit Taq polymerase (New England Biolabs) 20L PCR mix were prepared. For initial denaturation one cycle 94°C was applied. After that 35 cycles of , 94 °C for 45 seconds (DNA denaturation) 54 °C for 45 sec (oligonucleotide primer annealing), and 72 °C for 1 min (Taq polymerase extension) were applied. Lastly, 1 cycle of 72 °C for 10 min was applied for a final