DNA-based detection and reference genome assembly of Aphanomyces cochlioides

Abstract

Aphanomyces root rot (ARR) and Aphanomyces damping-off, caused by the soil-borne oomycete A. cochlioides, are common diseases of sugar beet in major production regions. Management techniques are implemented to mitigate losses, but ultimately A. cochlioides is intractable and significantly reduces the sucrose content of the sugar beet taproot throughout the growing season, especially during periods with above average precipitation. Currently, a genome sequence for A. cochlioides is not available, and existing diagnostic assays are time consuming and have limitations. The first objective was to assemble and annotate a reference genome for A. cochlioides. We conducted a de novo genome assembly and annotation of A. cochlioides using 232x coverage of Nanopore long-reads, and error corrected with 77x coverage of Illumina short-reads. The assembled genome was 76.3 Mb, consisted of 97 contigs, and had a contig N50 of 2.6 Mb. The assembly contained 93.2% of complete benchmarking universal single-copy orthologs, a repeat content of 32.1%, and 20,274 gene models. This is the first report of a reference genome for A. cochlioides, which could serve as a platform for future investigations into virulence mechanisms, comparative genomics, and the development of diagnostic assays. The second objective was to develop a rapid, sensitive, and accurate DNA-based detection assay to quantify A. cochlioides inoculum in infested soil and infected sugar beet tissue. We developed a TaqMan qPCR assay that was specific to A. cochlioides. The qPCR assay was validated with 12 naturally infested soil samples, which had Ct (cycle threshold) values of 26.72 to 34.64 and ARR disease severity index (DSI) values of 48 to 100. The qPCR assay was further validated on infected adult sugar beets and seedlings. For 60 adult sugar beet roots, A. cochlioides DNA was detected in 63% of the samples, while a culture-based assay identified A. cochlioides in 15% of the samples. Furthermore, A. cochlioides DNA was detected in infected seedlings as early as 5 days after planting in a naturally infested soil. Finally, when oospore infested potting soil was tested with the qPCR assay and ARR bioassay, a strong correlation was observed between oospore density and Ct value (R2 = 0.96), as well as oospore density and DSI value (R2 = 0.968). The limit of detection (LOD) was 5 oospores per g soil (dry wt.), which had a mean Ct value of 34.58, and a mean DSI value of 23.33. Our DNA-based detection assay could provide growers with the A. cochlioides infestation level of field soils to help them make informed management decisions prior to planting.