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

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.

Centenary of Soil and Air Borne Wheat Karnal Bunt Disease Research: A Review

Karnal bunt (KB) of wheat (Triticum aestivum L.), known as partial bunt has its origin in Karnal, India and is caused by Tilletia indica (Ti). Its incidence had grown drastically since late 1960s from northwestern India to northern India in early 1970s. It is a seed, air and soil borne pathogen mainly affecting common wheat, durum wheat, triticale and other related species. The seeds become inedible, inviable and infertile with the precedence of trimethylamine secreted by teliospores in the infected seeds. Initially the causal pathogen was named Tilletia indica but was later renamed Neovossia indica. The black powdered smelly spores remain viable for years in soil, wheat straw and farmyard manure as primary sources of inoculum. The losses reported were as high as 40% in India and also the cumulative reduction of national farm income in USA was USD 5.3 billion due to KB. The present review utilizes information from literature of the past 100 years, since 1909, to provide a comprehensive and updated understanding of KB, its causal pathogen, biology, epidemiology, pathogenesis, etc. Next generation sequencing (NGS) is gaining popularity in revolutionizing KB genomics for understanding and improving agronomic traits like yield, disease tolerance and disease resistance. Genetic resistance is the best way to manage KB, which may be achieved through detection of genes/quantitative trait loci (QTLs). The genome-wide association studies can be applied to reveal the association mapping panel for understanding and obtaining the KB resistance locus on the wheat genome, which can be crossed with elite wheat cultivars globally for a diverse wheat breeding program. The review discusses the current NGS-based genomic studies, assembly, annotations, resistant QTLs, GWAS, technology landscape of diagnostics and management of KB. The compiled exhaustive information can be beneficial to the wheat breeders for better understanding of incidence of disease in endeavor of quality production of the crop.

Reaction of advanced lines of triticale to karnal bunt (Tilletia indica)

Twenty advanced lines of triticale were evaluated for resistance to karnal bunt during the 2014-2015 crop season, at the Norman E. Borlaug Experimental Station. The sowing dates were November 19 and 29, 2014. One mL of an allantoid sporidial suspension (10,000/mL) was injected during the boot stage of ten spikes per line. A mist-irrigation system was used to provide high humidity in the experimental area. Harvesting and threshing were done manually, and the counting of infected and healthy grains was perfomed by visual inspection. The range of infection for the first sowing date was 0 - 2.4%, with an average of 0.3, and for the second 0 - 1.4%, with an average of 0.4. Five lines did not show any infected grains in both dates and the rest was in the 0.1-2.5% infection category. The average of infection of the susceptible check during the period of inoculation of this group of lines was 86.6%. The highest average percentage of infection of both dates were shown by lines: TURACO/CENT.SARDEV/7/LIRON_2/5/DISB5/3/SPHD/ PVN//YOGUI_6/4/KER_3/6/BULL_10/MANATI_1/8/LIRON_2/5/DISB5/3/SPHD/PVN// YOGUI_6/4/KER_3/6/BULL_10/MANATI_1/9/BICEN (CTSS08Y00035S-099Y-026M-19Y-099M-2Y-2BMX-4Y) with 1.6 and POPP1_2/CAAL//THELIN#2/5/PRESTO//2*TESMO_1/ MUSX603/4/ARDI_1/TOPO1419//ERIZO_9/3/SUSI_2/6/ARDI/GNU//2*FAHAD_1/4/BULL_ 10/MANATI_1/3/ELK54/ BUF_2//NIMIR_3 with 1.3%. These lines also showed the highest percentage of infection in the first date with 1.8 and 2.4%, respectively.

Mass Spectrometry based Identification and Characterization of 28 kDa protein from Teliospores of Karnal Bunt Pathogen Tilletia Indica

Background Karnal bunt (KB) of wheat incited by Tilletia indica is an economically important quarantined fungal disease that cause huge economical loss to agricultural productivity. MethodsIn the present investigation, we have characterized the unique immunoreactive determinant present on the teliospore’s wall of Tilletia indica employing proteomic and in-silico approaches. The 28 kDa protein from the teliospore’s walls was eluted from the preparative gels and further subjected to two-dimensional gel electrophoresis. Mass spectrometry was carried out in search of teliosporic protein(s).ResultsProteomic analysis identified and characterized proteins as small Heat shock protein (sHSP20) and Trehalose-6-phosphatase synthase (TPS) in T.indica. In-silico analysis confirmed the function of the identified protein as a sHSP and TPS belonging to stress response and carbohydrate metabolism. These results were further complemented to identify the homologs of proteins by sequence and structure based functional annotation using genome sequence of T. indica. The potential diagnostic protein present in immunoreactive 28 kDa of teliospores wall entities are sHSP and TPS. Conclusion This study is the first to perform finding indicated that proteins sHSP and TPS act as functional protein, involve in the pathogen protection under stress condition. Together these proteins increase our understanding of the transition from vegetative to sporulation process of T. indica by activation of sHSP-TPS, especially in terms of energy metabolism and stimulus responses. Identification of such protein(s) may also provide an opportunity to generate specific immunoprobes for development of rapid immuno-diagnostic assay.

Multilocus Sequence Typing and Single Nucleotide Polymorphism Analysis in Tilletia indica Isolates Inciting Karnal Bunt of Wheat

Karnal bunt of wheat is an internationally quarantined disease affecting trade, quality, and production of wheat. During 2015–2016, a severe outbreak of Karnal bunt disease occurred in north-western plain zone of India. The present study was undertaken to decipher genetic variations in Indian isolates of Tilletia indica collected from different locations. Seven multilocus sequence fragments were selected to differentiate and characterize these T. indica isolates. A phylogenetic tree constructed based on pooled sequences of actin-related protein 2 (ARP2), β-tubulin (TUB), eukaryotic translation initiation factor 3 subunit A (EIF3A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone 2B (H2B), phosphoglycerate kinase (PGK), and serine/threonine-protein kinase (STPK) showed that isolate KB-11 (Kaithal, Haryana) was highly conserved as it was located in cluster 1 and has the maximum sequence similarity with the reference strain. Other isolates in cluster 1 included KB-16 and KB-17, both from Uttar Pradesh, and KB-19 from Haryana. Isolates KB-07 (Jind, Haryana) and KB-18 (Mujaffar Nagar, Uttar Pradesh) were the most diverse and grouped in a subgroup of cluster 2. Maximum numbers of single nucleotide polymorphisms (SNPs) (675) were in the PGK gene across the T. indica isolates. The minimum numbers of SNPs (67) were in KB-11 (Kaithal, Haryana), while the maximum number of SNPs (165) was identified in KB-18, followed by 164 SNPs in KB-14. KB-18 isolate was found to be the most diverse amongst all T. indica isolates. This first study on multilocus sequence typing (MLST) revealed that the population of T. indica was highly diverse.