Modified ITS7 primer enhances oomycete richness detection and the amplification of economically important taxa.

Metagenomic analysis of oomycetes through deep amplicon sequencing has been conducted primarily using a ITS6/ ITS7 primer set that targets the ITS1 region. However, the ITS7 primer set contains three mismatches to the corresponding site in Aphanomyces euteiches, which is an important root rot pathogen of pea and other legume crops. This mismatch may explain why previous studies had little success in detecting this pathogen in soil, even though based on symptoms, A. euteiches is a relatively abundant component of the root rot disease complex in infested fields in western Canada. End-point PCR analysis with A. euteiches-specific primers showed that A. euteiches was present in 25 40% of fields tested in 2014 and 2015. In an attempt to overcome the impact of the reverse primer mismatches on A. euteiches detection, mismatched nucleotides were replaced by corresponding ambiguous nucleotides in a manner that contained the original ITS7 primer as well as nucleotides suitable for A. euteiches. Oomycete communities from 35 soil samples were analyzed simultaneously using both ITS6/ ITS7 and ITS6 /ITS7a.e (modified version of ITS7) primer sets on one Illumina Miseq run. The number of high quality reads obtained by ITS6/ ITS7a.e was more than twice that of ITS6/ ITS7. The total number of OTUs resulting from clustering of high quality reads was always higher in ITS7a.e, regardless of clustering criteria. The relative abundance of Pythium spp. was reduced, and Aphanomyces spp. and Phytophthora spp. increased, with the average percent reads assigned to A. euteiches increasing approximately 15 times when ITS7a.e was used. These results indicate that using ITS7a.e in oomycete metagenomic studies provides a more comprehensive picture of oomycete communities than ITS7, and enhances the detection power for important taxa such as Aphanomyces.