Real-time PCR assay for the early detection and relative quantification of the Rubus idaeus pathogen Aculeastrum americanum

Abstract

Late leaf rust of raspberry (Rubus idaeus), caused by Aculeastrum americanum (Farl.) M. Scholler & U. Braun (syn. Pucciniastrum americanum (Farlow) Arthur and syn. Thekopsora americana (Farl.) Aime McTaggart), is a fungal disease that inflicts significant losses and poses a serious threat to production. The absence of specific tests to detect late leaf rust before symptom onset allows the disease to spread undetected, leading to significant crop losses and complicating management. Thus, our objective was to establish an accurate and easy-to-apply semi-quantitative real-time PCR method to detect plants infected by A. americanum prior to commercialization, and to evaluate resistant cultivars in the breeding program. For this, primers were designed to amplify 272 bp and 119 bp fragments in the partial ITS1 and 5.8S rDNA regions of A. americanum and tested in semi-quantitative real-time PCR assays to screen for the fungus in a panel of cultivars displaying different degrees of symptoms, sourced from the INIAV raspberry germplasm collection. The assays showed consistent linear amplification from genomic DNA with both primer pairs, starting at 24 pg/uL, across all technical replicates. No cross-reactivity was observed with plant DNA. In the samples collected from the field, it was possible to detect A. americanum DNA in asymptomatic leaves, indicating the existence of weak and latent infections.

The assays designed in the present study represent the first specific test designed for the rapid detection and quantification of A. americanum, serving as a valuable tool for implementation of preventive strategies that mitigate the spread of the disease in Rubus idaeus.