The current study describes a new diagnostic method for the rapid and accurate detection of Tilletia indica, the pathogen accountable for causing Karnal bunt (KB) disease in wheat. This method uses quantitative real-time polymerase chain reaction (qPCR) and a primer set derived from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of T. indica to identify the presence of the pathogen. The qPCR assay using this primer set was found highly sensitive, with a limit of detection (LOD) value of 4 pg of T. indica DNA. This level of sensitivity allows for the detection of the pathogen even in cases of different growth stages of wheat, where no visible symptoms of infection on the wheat plants can be seen by naked eyes. The study also validated the qPCR assay on ten different wheat cultivars. Overall, this study presents a valuable molecular tool for rapid, specific and sensitive detection of KB fungus in wheat host. This method has practical applications in disease management, screening of wheat genotypes against KB and can aid in the development of strategies to mitigate the impact of Karnal bunt disease on wheat production.

A high-quality genome sequence from an Indian isolate of Blumeria graminis f. sp. tritici Wtn1, a persistent threat in wheat farming, was obtained using a hybrid method. The assembly of over 9.24 million DNA-sequence reads resulted in 93 contigs, totaling a 140.61 Mb genome size, potentially encoding 8480 genes. Notably, more than 73.80% of the genome, spanning approximately 102.14 Mb, comprises retro-elements, LTR elements, and P elements, influencing evolution and adaptation significantly. The phylogenomic analysis placed B. graminis f. sp. tritici Wtn1 in a distinct monocot-infecting clade. A total of 583 tRNA anticodon sequences were identified from the whole genome of the native virulent strain B. graminis f. sp. tritici, which comprises distinct genome features with high counts of tRNA anticodons for leucine (70), cysteine (61), alanine (58), and arginine (45), with only two stop codons (Opal and Ochre) present and the absence of the Amber stop codon. Comparative InterProScan analysis unveiled “shared and unique” proteins in B. graminis f. sp. tritici Wtn1. Identified were 7707 protein-encoding genes, annotated to different categories such as 805 effectors, 156 CAZymes, 6102 orthologous proteins, and 3180 distinct protein families (PFAMs). Among the effectors, genes like Avra10, Avrk1, Bcg-7, BEC1005, CSEP0105, CSEP0162, BEC1016, BEC1040, and HopI1 closely linked to pathogenesis and virulence were recognized. Transcriptome analysis highlighted abundant proteins associated with RNA processing and modification, post-translational modification, protein turnover, chaperones, and signal transduction. Examining the Environmental Information Processing Pathways in B. graminis f. sp. tritici Wtn1 revealed 393 genes across 33 signal transduction pathways. The key pathways included yeast MAPK signaling (53 genes), mTOR signaling (38 genes), PI3K-Akt signaling (23 genes), and AMPK signaling (21 genes). Additionally, pathways like FoxO, Phosphatidylinositol, the two-component system, and Ras signaling showed significant gene representation, each with 15–16 genes, key SNPs, and Indels in specific chromosomes highlighting their relevance to environmental responses and pathotype evolution. The SNP and InDel analysis resulted in about 3.56 million variants, including 3.45 million SNPs, 5050 insertions, and 5651 deletions within the whole genome of B. graminis f. sp. tritici Wtn1. These comprehensive genome and transcriptome datasets serve as crucial resources for understanding the pathogenicity, virulence effectors, retro-elements, and evolutionary origins of B. graminis f. sp. tritici Wtn1, aiding in developing robust strategies for the effective management of wheat powdery mildew.

Karnal bunt (KB; Tilletia indica) is the prime quarantine concern for quality wheat production throughout the world. The most effective approach to dealing with this biotic stress is to breed KB-resistant wheat varieties, which warrants a better understanding of T. indica genome architecture. In India, the North Western Plain Zone is the prime hot spot for KB disease, but only limited efforts have been made to decipher T. indica diversity at the genomic level. Microsatellites offer a powerful and robust typing system for the characterization and genetic diversity assessment of plant pathogens. At present, inadequate information is available with respect to the development of genome-derived markers for revealing genetic variability in T. indica populations. In current research, nine complete genome sequences of T. indica (PSWKBGH_1, PSWKBGH_2, PSWKBGD_1_3, RAKB_UP_1, TiK_1, Tik, DAOMC236408, DAOMC236414, and DAOMC236416) that exist in the public domain were explored to know the dynamic distribution of microsatellites. Comparative genome analysis revealed a high level of relative abundance and relative density of microsatellites in the PSWKBGH_1 genome in contrast to other genomes. No significant correlation between microsatellite distribution for GC content and genome size was established. All the genomes showed the dominance of tri-nucleotide motifs, followed by mono-, di-, tetra-, hexa-, and penta-nucleotide motifs. Out of 50 tested markers, 36 showed successful amplification in T. indica isolates and produced 52 different alleles. A PCR assay along with analysis of the polymorphic information content (PIC) revealed 10 markers as neutral and polymorphic loci (PIC 0.37). The identified polymorphic SSR loci grouped a geographically distinct T. indica population of 50 isolates representing seven Indian regions (Jammu, Himachal Pradesh, Punjab, Haryana, Uttarakhand, Uttar Pradesh, and Rajasthan) into four distinct clusters. The results of the analysis of molecular variance identified 94% genetic variation within the population and 6% among the population. Structure analysis also confirmed the existence of four genetically diverse groups containing admixtures of T. indica isolates across populations. In nutshell, the current study was successful in identifying novel, neutral and polymorphic microsatellite markers that will be valuable in offering deep insight into the evolutionary relationship and dynamics of the T. indica population for devising effective KB management strategies in wheat.