Intra-specific classification of Puccinia coronata has been controversial, with previous approaches falling into three major categories: 1. A two-species system, namely P. coronifera and P. coronata; 2. The same two-species system subdivided into many formae speciales, in which the host range of each is restricted to species within one genus of Poaceae; 3. A one-species system, P. coronata, subdivided into a few varieties with host ranges that may overlap. To re-assess these concepts in the context of multigene analyses and comparative morphological assessments, data were generated for a comprehensive set of herbarium and recently collected specimens, representing a broad range of hosts and geographic origins. Phylogenetic analyses of a combined data set of DNA sequences for four loci (BT, COI, ITS, and RPB2) revealed a high degree of genetic variation. Morphological differences among phylogenetic lineages were overlapping but nine lineages were differentiated using calculated means for teliospore and urediniospore length/width as well as measurements for the teliospore hilum and digitation. The taxon infecting Avena also comprises collections from a wide range of other grass hosts while other lineages, such as those on Bromus and Agrostis, were restricted in host association. Type specimen DNA sequences included in the analyses resolved the placement of five previously described varieties. Based on evidence of host specificity, morphology and multigene analyses, we recognized seven species, one of which was further divided into two varieties. Expanded descriptions, illustrations and a synoptic key are provided. A new series, Puccinia Series Coronata, was erected to accommodate all the lineages comprising P. coronata sensu lato.

The rust species Puccinia graminis and P. striiformis sensu stricto are important cereal pathogens, most well-known for causing the diseases stem rust and stripe rust on wheat and generating significant yield losses. Early and accurate detection of the pathogens would facilitate effective control of the diseases. In the present study, we developed real-time PCR assays to detect the specific lineages that include the wheat pathogens for each species complex, as identified using multi-gene DNA sequence analyses. Four DNA loci, for a comprehensive set of target and closely related fungi collected from diverse hosts and geographic regions, were explored to search for suitable lineage-specific probes: β-tubulin (BT), cytochrome c oxidase subunit 1 (COI), rDNA internal transcribed spacer (ITS), and RNA polymerase II second largest subunit (RPB2). Four TaqMan^® real-time PCR assays were designed based on either the BT or RPB2 genes: one targeting Puccinia Series Striiformis (PSBT), one targeting P. striiformis sensu stricto (PSstrRPB2), and two targeting the P. graminis lineages on wheat (Pg2⁺BT and Pg2RPB2). Sensitivities of the assays were determined to be 0.65 pg µL⁻¹ (Pg2) and 5 pg µL⁻¹ (PS). Specificity of each assay was confirmed using a broad diversity of rusts and other selected wheat-associated fungi. The ITS and COI loci were found to be unsuitable for diagnostic assay development but contributed phylogenetic signal to the multi-gene analyses.

The dispersion of fungal inocula such as the airborne spores of rust fungi (Pucciniales) can be monitored through metabarcoding of the internal transcribed spacer 2 (ITS2) of the rRNA gene in environmental DNAs. This method is largely dependent on a high-quality reference database (refDB) and primers with proper taxonomic coverage and specificity. For this study, a curated ITS2 reference database (named CR-ITS2-refDB) comprising representatives of the major cereal rust fungi and phylogenetically related species was compiled. Interspecific and intraspecific variation analyses suggested that the ITS2 region had reasonable discriminating power for the majority of the Puccinia species or species complexes in the database. In silico evaluation of nine forward and seven reverse ITS2 primers, including three newly designed, revealed marked variation in DNA amplification efficiency for the rusts. We validated the theoretical assessment of rust-enhanced (Rust2inv/ITS4var_H) and universal fungal (ITS9F/ITS4) ITS2 primer pairs by profiling the airborne rust fungal communities from environmental samples via a metabarcoding approach. Species- or subspecies-level identification of the rusts was improved by use of CR-ITS2-refDB and the Automated Oligonucleotide Design Pipeline (AODP), which identified all mutations distinguishing highly conserved DNA markers between close relatives. A generic bioinformatics pipeline was developed, including all steps used in this study from in silico evaluation of primers to accurate identification of short metabarcodes at the level of interest for defining phytopathogens. The results highlight the importance of primer selection, refDBs that are resolved to reflect phylogenetic relationships, and the use of AODP for improving the reliability of metabarcoding in phytopathogen biosurveillance.

Several fungi classified in the genus Tilletia are well-known to infect grass species including wheat (Triticum). Tilletia indica is a highly unwanted wheat pathogen causing Karnal bunt, subject to quarantine regulations in many countries. Historically, suspected Karnal bunt infections were identified by morphology, a labour-intensive process to rule out other tuberculate-spored species that may be found as contaminants in grain shipments, and the closely-related pathogen T. walkeri on ryegrass (Lolium). Molecular biology advances have brought numerous detection tools to discriminate Tilletia congeners (PCR, qPCR, etc.). While those tests may help to identify T. indica more rapidly, they share weaknesses of targeting insufficiently variable markers or lacking sensitivity in a zero-tolerance context. A recent approach used comparative genomics to identify unique regions within target species, and qPCR assays were designed in silico. This study validated four qPCR tests based on single-copy genomic regions and with highly sensitive limits of detection (~200 fg), two to detect T. indica and T. walkeri separately, and two newly designed, targeting both species as a complex. The assays were challenged with reference DNA of the targets, their close relatives, other crop pathogens, the wheat host, and environmental specimens, ensuring a high level of specificity for accurate discrimination.

A second species of Vestigium is described and contrasted with V. felicis known from Oregon and B.C. on Thuja plicata. The new species occurs on needles of Abies balsamea in New Brunswick and is compared with Rhizothyrium abietis, the anamorph of Rhizocalyx abietis. Molecular studies of the new species show affinity with Chlorencoeliaceae, Dermateaceae, Hemiphacidiaceae or Hyaloscyphaceae.

Triticale (× Triticosecale), was initially produced by crossing wheat (Triticum) with rye (Secale). Although still a minor crop in Canada, triticale grain is used both as human food (in bread, pastry products, and the brewing industry) and as livestock feed (Larter 2015). In September 2023 typical leaf rust samples were observed and collected in winter Triticale at Lacombe, Alberta. Typical exposed rust colored uredinia were mainly on adaxial leaf surface. Urediniospores obovoid, ellipsoid or globoid, measured 24-33 × 19-26 µm; with yellow-brown echinulate wall 1-1.5 µm thick. These spores mostly had 8 to 9 scattered germ pores, approximately bizonate with moderate to strong internal ring. Telia not present. Due to the absence of the telial stage, the morphological features of uredinia and urediniospores align our species with Puccinia triticina and P. recondita. These species have overlapping uredinial characteristics and can potentially both cause disease on Triticale (Yekelo et al. 2019). For testing pathogenicity on Triticale, two-week-old winter Triticale seedlings were infected with single pustule isolate from the Albertan leaf rust sourced from Triticale (AB-1). For seedling inoculation, we used 20 mg of pure urediniospores suspended in 5-6 ml of NOVEC 7100™ Engineered fluid, applied with an airbrush compressor system equipped with a 0.3 mm nozzle. All inoculated plants were kept in a dew chamber at 13°C for 24 hours in the dark, and then transferred to a Reach-In growth chamber with a 16-hour photoperiod (21°C day/15°C night). After 10 days, all inoculated seedlings showed uredinia. Control plants that were only sprayed with NOVEC 7100 and kept under the same conditions remained healthy, showing no rust symptoms. To clarify the identity of leaf rust, we sequenced the nuclear ribosomal rRNA internal transcribed spacer region (ITS) and elongation factor 1 alpha gene (EF1-a) for both the field collection (DAOM 985259) and the single pustule inoculated collection (DAOM 985258) and the data were deposited in NCBI GenBank (accession # PQ317950 and PQ317949 for ITS and PQ338170 and PQ338169 for EF1-a, respectively). Obtained sequences of both field and the inoculated collections were identical and shared %100 identity with available sequences of P. triticina in GenBank (MT955180 for ITS and JX533504 for EF1-a) (Liu et al. 2013). To further investigate the pathogenicity of the Albertan leaf rust isolate (AB-1) on Triticale, we conducted additional rounds of inoculation tests. In those experiments, two-week-old seedlings of winter rye and Avocet wheat were inoculated under the above-mentioned conditions and methods. After 9 to 10 days, both rye and wheat plants showed uredinia on the infected leaves. This indicates that the Triticale isolate of leaf rust was capable of infecting rye and wheat in addition to Triticale. Since Triticale is primarily planted as a forage or cover crop, its susceptibility to wheat leaf rust could contribute to the build-up of inoculum and subsequent infection of commercial wheat crops (Yekelo et al. 2019). Our research showed, there was no record of leaf rust (P. triticina) on Triticale based on USDA Fungal Databases (https://fungi.ars.usda.gov/) and also no herbarium specimen of leaf rust on Triticale in Canada and United States based on MyCoPortal (https://www.mycoportal.org). To our knowledge this is the first morphologically and molecularly documented report of P. triticina on Triticale in Alberta and probably in Canada.