The application of real‐time PCR to the identification, detection and quantification of Pyrenophora species in barley seed

Abstract

Summary A real‐time quantitative PCR technique has been used to develop a rapid and sensitive seed health test for Pyrenophora spp. on barley seed. Using the fluorescent reporter dye SYBR Green I for real‐time detection of PCR amplification, pathogen DNA extracted from infected seed can be quantified to the picogram level. The amount of Pyrenophora DNA extracted from seed samples of an artificial infection level gradient, constructed by mixing infected and uninfected seed, correlated with good agreement (r = 0.931) to percentage infection levels of the same samples measured by agar plate testing. In addition, a correlation of r = 0.883 was obtained between the two testing methods for naturally infected seed, ranging from 0% to 89% infection. Samples could be quantified to below the 2% voluntary threshold required for deciding on seed treatment. The proposed test was performed in three parts: (i) quantification of Pyrenophora spp. infection using Pyrenophora‐specific PCR primers; (ii) test of any negative samples from (i) with barley‐specific PCR primers to check the DNA extraction process; (iii) test of positive samples from (i) for the presence of Pyrenophora graminea using P. graminea‐specific PCR primers. All PCRs were performed in the LightCycler™ instrument allowing each PCR run and analysis to be completed within 30 min. With the current daily receipt of samples (batches up to 16) the test can be completed in 8 h, compared to 7 days for the traditional agar plate test.

Detection and Quantification ofPlectosphaerella cucumerina, a Potential Biological Control Agent of Potato Cyst Nematodes, by Using Conventional PCR, Real-Time PCR, Selective Media, and Baiting

“…Real-time PCR relies on the combination of a primer set and an additional dual-labeled fluorogenic probe to allow continuous monitoring of amplicon synthesis during thermocycling and requires no post-PCR handling for target quantification (21). It has been used to successfully determine the population levels of a number of fungal species (5,6,8,26). In this paper, the development of molecular tools for the detection and quantification of P. cucumerina is described, and they are compared to more conventional methods, such as soil baiting and serial dilution on a selective medium.…”

Pathogenic variation in populations of Drechslera teres f. teres and D. teres f. maculata and differences in host cultivar responses

“…Estimations of fungal biomass in the host tissue either by ergosterol levels or quantitative PCR could clarify the relationship between visible symptoms and damage to the host. Bates et al (2001) developed a real-time PCR-based assay for the quantification of Pyrenophora species including P. teres in infected seeds. This could be adapted to examine the fungal biomass in leaf tissue in resistance studies.…”

Quantification of airborne spores of Monilinia fructicola in stone fruit orchards of California using real-time PCR

“…A more efficient and quantitative method is therefore needed. In addition to high sensitivity in detection of target DNA, the real-time PCR method has successfully quantified airborne, seedborne and soilborne inoculum of other fungi (Bates et al 2001;Cullen et al 2001Cullen et al 2005Van de Graaf et al 2003).…”