Development and Application of Real-Time and Conventional SSR-PCR Assays for Rapid and Sensitive Detection of Didymella pisi Associated with Ascochyta Blight of Dry Pea

Abstract

Didymella pisi is the primary causal pathogen of Ascochyta blight (AB) of dry pea in Montana. Diagnosis of AB is challenging because there are six different species that cause AB worldwide and that can co-occur. Additionally, agar plate identification of D. pisi is challenging due to its slow growth rate. Currently, there are no PCR-based assays developed for specific detection of D. pisi or any fungal pathogen in the AB complex of dry pea. In this study, we evaluated simple sequence repeat (SSR) primer pairs for their specificity and sensitivity in real-time and conventional SSR-PCR both in vitro and in planta. The specificity of the assay was determined by testing DNA of 10 dry pea varieties, fungal species in the AB complex, and fungal species associated with dry pea. To avoid false-negative results, plant and fungal DNA markers were included as controls in a conventional multiplex SSR-PCR, to amplify any plant or fungal DNA in the absence of the D. pisi SSR target. SYBR Green SSR-quantitative PCR (qPCR) detection was conducted using the same primer pairs but in a uniplex format. D. pisi was specifically amplified, whereas other fungi and host DNA were not. Also, sensitivity experiments showed that the detection limit was 0.01 ng of DNA of D. pisi for both assays and 100 conidia in SSR-qPCR. These assays are valuable diagnostic tools for the detection of D. pisi.

Facilitating the adoption of high‐throughput sequencing technologies as a plant pest diagnostic test in laboratories: A step‐by‐step description

“…The X. fastidiosa sequences were also used to identify subspecies and to allow tracing the origin of the pathogen in an incursion (Cella et al, 2018). In addition, sequencing the genomes of many isolates/specimens/strains of a pest provides a broader overview of its genetic diversity and consequently improves the inclusivity of diagnostic primers and targeted diagnostic protocols (Adams et al, 2018; An et al, 2015; Bonants et al, 2015; Catara et al, 2021; Katsiani et al, 2018; Kikuchi et al, 2011; Owati et al, 2019; Pritchard et al, 2016).…”

Ascochyta blight in North Dakota field pea: the pathogen complex and its fungicide sensitivity

“…& A. Bloxam) Petr., and Didymella pinodella (L.K. Jones) Qian Chen & L. Cai have been documented as infecting field pea in the northern Great Plains region of North America ( Owati et al., 2019 ). These three fungal pathogens have been associated with the disease in many field pea producing countries including the USA ( Peever et al., 2007 ), Australia ( Ali et al., 1978 ), Canada ( Beasse et al., 1999 ), China ( Liu et al., 2016 ), France ( Le May et al., 2018 ), and Spain ( Barilli et al., 2016 ).…”