Variation in the internal transcribed spacer region of phakopsora pachyrhizi and implications for molecular diagnostic assays

Phakopsora pachyrhizi, the causal agent of soybean rust (SBR), is a global threat to soybean production. Since the discovery of SBR in the continental United States, quantitative polymerase chain reaction assays based on the internal transcribed spacer (ITS) ribosomal DNA locus were established for its rapid detection. However, insufficient data were initially available to test assays against factors that could give rise to misidentification. This study aimed to reevaluate current assays for (i) the potential for false-positive detection caused by nontarget Phakopsora species and (ii) the potential for false-negative detection caused by intraspecific variation within the ITS locus of P. pachyrhizi. A large amount of intraspecific and intragenomic variation in ITS was detected, including the presence of polymorphic ITS copies within single leaf samples and within single rust sori. The diagnostic assays were not affected by polymorphisms in the ITS region; however, current assays are at risk of false positives when screened against other species of Phakopsora. This study raises caveats to the use of multicopy genes (e.g., ITS) in single-gene detection assays and discusses the pitfalls of inferences concerning the aerobiological pathways of disease spread made in the absence of an evaluation of intragenomic ITS heterogeneity.