A Quantitative Real-Time PCR Method to Detect the Quinoa Downy Mildew Pathogen, Peronospora variabilis.

Quinoa downy mildew, caused by Peronospora variabilis, is the most devastating disease of quinoa globally. Rapid, sensitive diagnostic methods are needed to detect and quantify this pathogen in seeds and plant tissue. A hydrolysis probe-based quantitative real-time PCR (qPCR) assay including a competitive internal control was developed for P. variabilis detection. This assay could detect as low as 20 ag of DNA or approximately 25 internal transcribed spacer (ITS) copies per reaction with efficiencies ranging from 93.9 to 98.2%. No non-target amplification was observed when tested against DNA from other downy mildew pathogens and related oomycetes. Peronospora variabilis strains from multiple countries were detected using this assay. The assay was successfully applied to quantify the pathogen in quinoa seeds from a field trial conducted in Washington State. Downy mildew disease was recorded on all 14 genotypes with the genotypes 104.88 and 106.49 recording the highest area under the disease progress values (3,236 ± 303 SE and 2,851 ± 198, respectively) while J6 and Dutchess recorded the lowest (441 ± 107 and 409 ± 129, respectively). Seed washes obtained from field samples were subjected to the qPCR assay, and the pathogen was detected in all samples. The highest pathogen ITS copy number recorded with 106.49 (194,934 ± 38,171 SE), while the lowest was observed in Pasto (5,971 ± 1,435) and Riobamba (9,954 ± 4,243). This qPCR assay could lead to improved detection and quantification of P. variabilis as well as increased understanding of quinoa-P. variabilis interactions and epidemiology.

A Quantitative PCR Method to Detect the Tomato Corky Root Rot Pathogens, Pseudopyrenochaeta lycopersici and P. terrestris.

Corky root rot is an important disease in tomato production systems and is caused by Pseudopyrenochaeta terrestris and P. lycopersici (formerly Pyrenochaeta lycopersici Types 1 and 2, respectively). The corky root rot pathogens are slow growing and difficult to isolate and quantify in soil and plant tissue. A multiplex hydrolysis probe-based qPCR assay was designed to allow for simultaneous detection and quantification of P. lycopersici and P. terrestris with a competitive internal control to indicate if qPCR inhibitors are present. Single species and multiplex assays for Pseudopyrenochaeta spp. detected DNA levels above 0.013 pg of DNA per reaction. These highly specific assays had no nontarget amplification of other fungal and oomycete pathogens or rhizosphere-associated fungi of tomatoes that were tested. This assay can be used to quantify Pseudopyrenochaeta populations in roots and soils in tomato production systems to better determine the impacts of disease management strategies on Pseudopyrenochaeta spp. and provides a tool to study the biology of Pseudopyrenochaeta spp.