Variation in the Internal Transcribed Spacer Region of Phakopsora pachyrhizi and Implications for Molecular Diagnostic Assays

Abstract

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.

The life cycle and field epidemiology of Uromycladium acaciae (Pucciniales) on Acacia mearnsii in South Africa

“…Whether U. acaciae is a complex of species remains a knowledge gap. However, intraspecific variability of rDNA is known in other species of Uromycladium (Doungsa‐ard et al, 2018) and other rust fungi (Rush et al, 2019). Resolution of this taxonomic uncertainty and the possible existence of a complex of species will hinge on wide sampling of this rust in its native range and application of molecular markers other than rDNA.…”

“…We tested for within‐genome variation in rDNA with high‐throughput sequencing from a telial isolate of U. acaciae , specifically to examine whether polymorphisms from uredinia were present in copies of rDNA from telia. We expected to observe diversity within copies of the ITS region from one isolate as teliospores have two independent nuclei (dikaryotic), both of which have multiple repeats of rDNA (Feau et al, 2011; McTaggart & Aime, 2018; Persoons et al, 2014; Rush et al, 2019; Virtudazo et al, 2001). Parts of rDNA are used by the mycological community as barcode loci and intraspecific diversity is expected to be less than interspecific diversity.…”

Morphological Formation, Fatty Acid Profile, and Molecular Identification of Some Landraces of Ethiopian Brassica as a Promising Crop to Support Breeding Programs

“…Sequences from a gene bank of different species were a dominant method of classification [ 47 ]. ITS sequences are widely applied in different field crop plants to study the phylogenetic relationship between plant genera [ 17 , 18 , 48 ]. In the present findings, using the ITS sequence, the GC content was about 49% in all accessions.…”

“…ITS sequences have been developed to be distinct for growing species and can be used as a barcode to distinguish between plant species [ 15 , 16 ]. A large amount of variability has been identified in ITS, including the presence of polymorphic copies of ITS [ 17 , 18 ]. Morphological, biochemical, and posterior molecular classification can be used to analyze and identify diversity.…”

Season-long infection of diverse hosts by the entomopathogenic fungus Batkoa major

“…for Entomophthora muscae (39) and Zoophthora species (40). Also, genomes in some fungi contain multiple ITS copies (41).…”