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.

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.