Biases in the metabarcoding of plant pathogens using rust fungi as a model system

Plant pathogens such as rust fungi (Pucciniales) are of global economic and ecological importance. This means there is a critical need to reliably and cost-effectively detect, identify, and monitor these fungi at large scales. We investigated and analyzed the causes of differences between next-generation sequencing (NGS) metabarcoding approaches and traditional DNA cloning in the detection and quantification of recognized species of rust fungi from environmental samples. We found significant differences between observed and expected numbers of shared rust fungal operational taxonomic units (OTUs) among different methods. However, there was no significant difference in relative abundance of OTUs that all methods were capable of detecting. Differences among the methods were mainly driven by the method's ability to detect specific OTUs, likely caused by mismatches with the NGS metabarcoding primers to some Puccinia species. Furthermore, detection ability did not seem to be influenced by differences in sequence lengths among methods, the most appropriate bioinformatic pipeline used for each method, or the ability to detect rare species. Our findings are important to future metabarcoding studies, because they highlight the main sources of difference among methods, and rule out several mechanisms that could drive these differences. Furthermore, strong congruity among three fundamentally different and independent methods demonstrates the promising potential of NGS metabarcoding for tracking important taxa such as rust fungi from within larger NGS metabarcoding communities. Our results support the use of NGS metabarcoding for the large-scale detection and quantification of rust fungi, but not for confirming the absence of species.