Degenerate ITS7 primer enhances oomycete community coverage and PCR sensitivity to Aphanomyces species, economically important plant pathogens
Esmaeili Taheri, A., Chatterton, S., Gossen, B.D., McLaren, D.L. (2017). Degenerate ITS7 primer enhances oomycete community coverage and PCR sensitivity to Aphanomyces species, economically important plant pathogens, 63(9), 769-779. http://dx.doi.org/10.1139/cjm-2017-0100
Plain language summary
Root rot of pea is caused by many pathogens, including several in the Oomycete group (similar to fungi, but not fungi). DNA sequencing of the ITS1 region has been used to identify the oomycetes species in the soil from around pea roots in western Canada. However, Aphanomyces euteiches was consistently under-represented in these analyses. This was important because A. euteiches is a widespread and damaging pathogen of pea. An improved protocol (Illumina MiSeq + specific primer) was developed that detected A. euteiches and other Oomycestes in soil much more frequently than the standard protocol did. Since A. euteiches is the most damaging pathogen associated with root rot in the region, development of accurate identification and detection methods are needed to identify fields with high levels of pathogen inoculum, as well as to provide an accurate description of microbiological communities.
Metagenomic analysis of oomycetes through deep amplicon sequencing has been conducted primarily using the ITS6-ITS7 primer set that targets the ITS1 region. While this primer set shows a perfect match to most oomycete taxa, ITS7 contains 3 mismatches to the corresponding binding site of plant pathogens within the genus Aphanomyces. Polymerase chain reaction (PCR) efficiency differs for taxa with uneven primer matching characteristics, which may explain why previous studies have detected this genus at low abundance. To overcome the impact of these mismatches on PCR sensitivity, the mismatched nucleotides were replaced with degenerate nucleotides. Oomycete communities from 35 soil samples collected from asymptomatic and root rot diseased sites in pea fields across Alberta were analyzed simultaneously using ITS6-ITS7 and ITS6-ITS7-a.e. (modified version of ITS7) primer sets on 1 Illumina MiSeq run. The number of high-quality reads obtained by ITS6-ITS7-a.e. was more than twice that of ITS6-ITS7. The relative abundance of Pythium spp. was reduced and Aphanomyces spp. increased. Aphanomyces cf. cladogamus and Aphanomyces euteiches were the second and third most abundant species, respectively, in the pea rhizosphere using the ITS7-a.e. primer, but were rare using the ITS7 primer. These results indicate that use of ITS7-a.e. provides a more accurate picture of oomycete communities than ITS7 by enhancing PCR sensitivity to Aphanomyces.