ARS | Publication request: Polymerase chain reaction protocol for detection of Pyrenophora teres in natural field soils

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: May 18, 2010
Publication Date: N/A

Technical Abstract: Net blotch of barley, a widely distributed foliar disease occurs extensively in the upper Midwest of the US. The causal agent, Pyrenophora teres Drechs. survives in infested straw in field soils which produces conidia and ascospores, probably the most important sources of primary inoculum. We developed and present a PCR technique for direct and fast detection of P. teres in field soils. Total DNA was purified from P. teres amended and barley planted field soils using PowerSoil DNA Kit (MO BIO Lab. Carlsbad, CA) as per manufacture’s instructions. The purified DNA was subjected to PCR reaction in Extract-N-Amp PCR mix (Sigma Aldrich, St Louis MO) with PTACTIN, a P. teres actin primer. The amplicons were resolved by electrophoresis in 1% agarose gels. The amplified fragment sizes of P. teres from amended and the natural field soils correlated with the expected size of from amplified control P. teres pure culture. All amplicons were excised from the gel, purified with QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA), and sequenced. Sequences alignment confirmed the detection of P. teres from soils. This protocol will enable rapid pre- and post-planting soil screening for potential problem of P. teres and determine the effect of soil applied chemicals, biocontrol agents or other management techniques on P. teres and inoculum in field soils.