Four In Silico Designed and Validated qPCR Assays to Detect and Discriminate Tilletia indica and T. walkeri, Individually or as a Complex

Simple Summary Plant pathogens represent a constant threat to human and animal food, as well as the economy. International trading is constantly expanding and has been known as a means of transportation and introduction for plant pests (e.g., bacteria, viruses, fungi, and insects) in new areas. They can damage or completely ruin a harvest and there are often strict regulations for the most unwanted plant pests in order to keep their incidence confined. The fungal plant pathogen Tilletia indica causes Karnal bunt, a wheat disease that breaks or hollows grains, grows in dark powdery masses, and emits a foul fishy odor, and is therefore highly regulated by a number of country authorities, many of which respond by imposing quarantine regulations. While there are many diagnostic methods developed (microscopy, molecular assays, etc.) to identify Karnal bunt, they have limitations. This study presents four highly sensitive quantitative PCR assays with molecular probes targeting unknown genomic regions for the detection and identification of T. indica and T. walkeri-its closest relative-and the species-complex including both species. Bioinformatics analyses of DNA sequences were used to design the toolkit presented. 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.