Genus-Specific Primers for Study of Fusarium Communities in Field Samples

Abstract

Fusarium is a large and diverse genus of fungi of great agricultural and economic importance, containing many plant pathogens and mycotoxin producers. To date, high-throughput sequencing of Fusarium communities has been limited by the lack of genusspecific primers targeting regions with high discriminatory power at the species level. In the present study, we evaluated two Fusarium-specific primer pairs targeting translation elongation factor 1 (TEF1). We also present the new primer pair Fa؉7/Ra؉6. Mock Fusarium communities reflecting phylogenetic diversity were used to evaluate the accuracy of the primers in reflecting the relative abundance of the species. TEF1 amplicons were subjected to 454 high-throughput sequencing to characterize Fusarium communities. Field samples from soil and wheat kernels were included to test the method on more-complex material. For kernel samples, a single PCR was sufficient, while for soil samples, nested PCR was necessary. The newly developed primer pairs Fa؉7/Ra؉6 and Fa/Ra accurately reflected Fusarium species composition in mock DNA communities. In field samples, 47 Fusarium operational taxonomic units were identified, with the highest Fusarium diversity in soil. The Fusarium community in soil was dominated by members of the Fusarium incarnatum-Fusarium equiseti species complex, contradicting findings in previous studies. The method was successfully applied to analyze Fusarium communities in soil and plant material and can facilitate further studies of Fusarium ecology.

Agricultural factors affecting Fusarium communities in wheat kernels

“…The TEF1 is single-copy in Fusarium (Geiser et al, 2004), which allows for quantitative comparisons of both total and relative abundances between species. In our previous study, the relative abundance of Fusarium species in mock communities varied ±8% between F. graminearum, F. avenaceum, F. langsethiae and F. tricinctum, indicating comparable amplification efficiencies (Karlsson et al, 2016). We conclude that the combination of these two methods provided a detailed description of the Fusarium community present on winter wheat kernels.…”

“…Size and concentration of amplicons was evaluated on 1% agarose gels. The Fa primer was tagged with a 9 bp sample identifying tag (Karlsson et al, 2016, supplemental file 1). In order to limit the risk of errors associated with many PCR cycles, the number of cycles was adapted for each sample to give weak to moderately strong bands on the gel.…”

“…Raw sequence data were deposited in the Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra) under accession number SRP061762. 2.3 Bioinformatics and species identification Raw 454 sequence data were quality filtered and clustered using the SCATA pipeline (Brandström Durling et al, [http://scata.mykopat.slu.se]) as described previously (Karlsson et al, 2016). Briefly, sequences were screened for matches to the primer sequences (allowing 1 mismatch) and the sample identifying tags.…”

“…Sequences passing the quality control step were clustered into operational taxonomic units (OTUs) at 1.5% dissimilarity cut-off in SCATA. Mock communities of 10 Fusarium species were included as positive controls as described in a previous publication (Karlsson et al, 2016). The mock communities were used to determine the cut-off which best reflected Fusarium species and to set a limit for low-abundance OTUs as these may be the result of sequencing and PCR errors (Tedersoo et al, 2010).…”

“…However, the internal transcribed spacer (ITS), which is commonly used as a barcode for fungi (Schoch et al, 2012), lack species-level resolution for Fusarium (O'Donnell and Cigelnik, 1997). Recently, new Fusarium-specific primers targeting the translation elongation factor 1-a (TEF1) gene has been developed and employed to characterise Fusarium communities using HTS (Edel-Hermann et al, 2015;Karlsson et al, 2016). This new method enables the study of Fusarium in cereals as a community.…”