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Timely prediction of pathogen is important key factor to reduce the quality and yield losses. Wheat is major crop in northern part of India. In Punjab, wheat face challenge by different diseases so the study was conducted for two locations viz. Ludhiana and Bathinda. The information regarding the occurrence of Karnal bunt in 12 consecutive crop seasons (from 2009-10 to 2020-21) in Ludhiana district and in 9 crop seasons (from 2010-11 to 2018-19) in Bathinda district, was collected from the Wheat Section of the Department of Plant Breeding and Genetics at Punjab Agricultural University (PAU), located in Ludhiana. The study aims to investigate the adequacy of various methods of machine learning for prediction of Karnal bunt using meteorological data for different time period viz. February, March, 15 February to 15 March and overall period obtained from Department of Climate Change and Agricultural Meteorology, PAU, Ludhiana. The most intriguing outcome is that for each period, different disease prediction models performed well. The random forest regression (RF) for February month, support vector regression (SVR) for March month, SVR and BLASSO for 15 February to 15 March period and random forest for overall period surpassed the performance than other models. The Taylor diagram was created to assess the effectiveness of intricate models by comparing various metrics such as root mean square error (RMSE), root relative square error (RRSE), correlation coefficient (r), relative mean absolute error (MAE), modified D-index, and modified NSE. It allows for a comprehensive evaluation of these models' performance. [ABSTRACT FROM AUTHOR]

Abstract Tilletia indica Mitra causes Karnal bunt (KB) in wheat by pathogenic dikaryophase. The present study is the first to provide the draft genomes of the dikaryon (PSWKBGD-3) and its two monosporidial lines (PSWKBGH-1 and 2) using Illumina and PacBio reads, their annotation and the comparative analyses among the three genomes by extracting polymorphic SSR markers. The trancriptome from infected wheat grains of the susceptible wheat cultivar WL711 at 24 h, 48h, and 7d after inoculation of PSWKBGH-1, 2 and PSWKBGD-3 were also isolated. Further, two transcriptome analyses were performed utilizing T. indica transcriptome to extract dikaryon genes responsible for pathogenesis, and wheat transcriptome to extract wheat genes affected by dikaryon involved in plant-pathogen interaction during progression of KB in wheat. A total of 54, 529, and 87 genes at 24hai, 48hai, and 7dai, respectively were upregulated in dikaryon stage while 21, 35, and 134 genes of T. indica at 24hai, 48hai, and 7dai, respectively, were activated only in dikaryon stage. While, a total of 23, 17, and 52 wheat genes at 24hai, 48hai, and 7dai, respectively were upregulated due to the presence of dikaryon stage only. The results obtained during this study have been compiled in a web resource called TiGeR ( http://backlin.cabgrid.res.in/tiger/ ), which is the first genomic resource for T. indica cataloguing genes, genomic and polymorphic SSRs of the three T. indica lines, wheat and T. indica DEGs as well as wheat genes affected by T. indica dikaryon along with the pathogenecity related proteins of T. indica dikaryon during incidence of KB at different time points. The present study would be helpful to understand the role of dikaryon in plant-pathogen interaction during progression of KB, which would be helpful to manage KB in wheat, and to develop KB-resistant wheat varieties.

Tilletia indica Mitra causes Karnal bunt (KB) in wheat by pathogenic dikaryophase. The present study is the first to provide the draft genomes of the dikaryon (PSWKBGD-3) and its two monosporidial lines (PSWKBGH-1 and 2) using Illumina and PacBio reads, their annotation and the comparative analyses among the three genomes by extracting polymorphic SSR markers. The trancriptome from infected wheat grains of the susceptible wheat cultivar WL711 at 24 h, 48h, and 7d after inoculation of PSWKBGH-1, 2 and PSWKBGD-3 were also isolated. Further, two transcriptome analyses were performed utilizing T. indica transcriptome to extract dikaryon genes responsible for pathogenesis, and wheat transcriptome to extract wheat genes affected by dikaryon involved in plant-pathogen interaction during progression of KB in wheat. A total of 54, 529, and 87 genes at 24hai, 48hai, and 7dai, respectively were upregulated in dikaryon stage while 21, 35, and 134 genes of T. indica at 24hai, 48hai, and 7dai, respectively, were activated only in dikaryon stage. While, a total of 23, 17, and 52 wheat genes at 24hai, 48hai, and 7dai, respectively were upregulated due to the presence of dikaryon stage only. The results obtained during this study have been compiled in a web resource called TiGeR (http://backlin.cabgrid.res.in/tiger/), which is the first genomic resource for T. indica cataloguing genes, genomic and polymorphic SSRs of the three T. indica lines, wheat and T. indica DEGs as well as wheat genes affected by T. indica dikaryon along with the pathogenecity related proteins of T. indica dikaryon during incidence of KB at different time points. The present study would be helpful to understand the role of dikaryon in plant-pathogen interaction during progression of KB, which would be helpful to manage KB in wheat, and to develop KB-resistant wheat varieties. [ABSTRACT FROM AUTHOR]

Background: Karnal bunt of wheat is an important quarantine disease, incited by Tilletia indica. It limits India's trade in wheat export. The teliospores are major source of inoculum to initiate and spread the Karnal bunt disease. The study aimed to identify the germination-related genes in the teliospores of T. indica. Methods and results: The candidate genes in the teliospores germination were identified through the differential gene expression analysis with suitable bioinformatics analysis. Keeping in soil-borne nature of fungi, the teliospores of T. indica (2015 and 2018) were subjected to the qPCR analysis. 20 candidate genes were identified having role in germination of teliospores of T. indica. Twenty genes, viz. Ti9297 (9.31, 7.87-fold), Ti8696 (5.13, 6.54-fold), Ti7699 (8.9, 7.7-fold), Ti7858 (10.33, 6.21-fold), Ti7954 (7.46, 5.54-fold), Ti7739 (5.46, 6.46-fold), Ti9665 (10.74, 7.64-fold), Ti9335 (6.75, 4.36-fold), Ti8396 (9.35, 7.72-fold), Ti8126 (8.87, 11.31-fold), Ti7326 (6.04, 7.7-fold), Ti10208 (13.83, 5.81-fold), Ti12356 (7.83, 8.02-fold), Ti14271 (9.98, 6.32-fold), Ti9234 (11.2, 8.72-fold), Ti 8876 (6.47, 3.55-fold), Ti 10,606 (4.97, 2.35-fold), Ti7758 (10.33, 8.78-fold), Ti4692 (6.89, 9.88-fold), and Ti3932 (5.77, 4.5-fold) were found highly expressed in the germinating teliospores of 2015 and 2018, respectively. Eight genes (Ti508, Ti4152, Ti5346, Ti2375, Ti3739, Ti1134, Ti4399, and Ti4422) were downregulated in the germinating teliospores but these eight genes were showed higher expression in the dormant teliospores. Conclusions: Twenty candidate genes were upregulated in the germinating teliospores are supposed to be involved in the process of germination. Eight genes were downregulated which were related to the process of the dormancy of teliospores. The study will be helpful to devise the newer management strategies for Karnal bunt disease of wheat. [ABSTRACT FROM AUTHOR]

Karnal bunt (KB) is a seed and airborne wheat disease caused by the fungus Tilletia indica Mitra. The floret-infecting fungus damages grains and adversely affects wheat quality through the production of trimethylamine. Deployment of host-plant resistance is the best strategy to combat this disease. The present study explored two sources of genetic resistance to KB by developing recombinant inbred lines (RILs) with a common susceptible parent. Munal × WL711 and SAUAL/KIRITTI/SAUL × WL711 produced 275 RILs each which were evaluated for KB disease resistance under four environments. The minimum value for disease incidence was 0% while the maximum values were 8.14% and 10.38% during 2018–2019 and 2019–2020, respectively. This study identified several RILs with durable and heritable resistance against KB, while SAUAL/KIRITTI/SAUL was identified as the highest-performance resistant parent. Based on the best linear unbiased estimates across environments, two bulk populations (resistant and susceptible) were developed from each set of progeny and were subjected to genome-wide single nucleotide polymorphism (SNP) genotyping using a 50K Affymetrix SNP array. Bulked segregation analysis showed that the maximum SNP index was identified on chr2A, chr6A, and chr7A in Munal × WL-711 while it was on chr4B and chr7B in the SAUAL/KIRITTI/SAUL × WL711 population. Taken together, the identified RILs might serve as breeding material for the development of KB-resistant wheat cultivars in the future. [ABSTRACT FROM AUTHOR]

Karnal bunt of wheat caused by Tilletia indica Mitra (Nevossia indica Mundkur), was first reported in Karnal, India, in 1931 hence named as 'Karnal bunt'. The disease mainly occurs in wheat growing regions with low winter temperatures and high humidity and affects mainly common wheat, durum wheat, triticale and other related species. This pathogen poses a serious quarantine problem and thus restricts the international trade of wheat. Therefore, quarantine restriction is applied by approximately 70 countries on wheat trade where Karnal bunt is known to occur. To access the status of Karnal bunt in major wheat growing regions of Meerut Division Western Uttar Pradesh, apost-harvest survey was conducted during the year 2021-22 and 2022-23. In the survey six Districtsof Meerut Division viz., Meerut, Bulandshahar, Hapur, Gautambuddhnagar, Gaziabadand Baghpat comprises 45 blocks were covered and 1125 samples from different wheat varietieswere collected from all blocks of six Districts. During the survey of both year the percent disease incidence of karnal bunt was not exceeding the permissible limit (0.25%) of Indian Minimum Seed Certification Standards for quality seed production except Kanaksona (0.288%) from Bulandshahar District and 1085 (0.42%) from Meerut District which is more than the permissible limit of standards. In both year (2021-22 and 2022-23) of survey the maximum incidence (0.42%) of karnal bunt was recorded in 1085 variety of Meerut District and 0.141% in HD 3226 of Baghpat District followed by 0.288% in Kanaksona of Bulandshahar District and 0.110% in UP 272 of Hapur District, whereas the minimum incidence (0.007%) was recorded in DBW 226 of Bulandshahar District and 0.001% in UP 303 of Meerut District respectively. The incidence percent of karnal bunt was recorded nil (0.00%) in both the year of all varieties of Gautambuddh Nagar District. Confirmation of karnal bunt pathogen was determined by species specific primers in PCR and RT-PCR on the basis of Diagnostic Protocol (DP4) of International Standard for Phytosanitary Measures (ISPM 27). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

Durum wheat (Triticum turgidum L.) is the only tetraploid species of wheat that has been used commercially and consumed as a food product in human diets. It is used in various ways for making several food products such as macaroni, pasta, spaghetti, semolina, couscous, and unleavened bread. Durum wheat is generally used for pasta making due to the sufficient amount of yellow pigment, protein content, and its gluten characteristic. The low molecular weight gluten subunit provides its extensibility. Durum wheat also has a high source of pro-vitamin A, antioxidants, carbohydrates, and non-starch polysaccharides. Due to anthocyanin and its antioxidant properties, it has been recognized as a healthy cereal crop and is recommended in the diet for those suffering from allergies, diabetes, and high blood cholesterol. The Glu-D1 locus is essential for the bread-making quality and in durum wheat it is absent. Efforts have been made by transferring the Glu-D1 locus to improve bread-making quality. Nutritional and end-use qualities are the two key determinants of durum products. [ABSTRACT FROM AUTHOR]

Karnal bunt of wheat is an important quarantine disease that interrupts India's wheat trade in the international market. The whole transcriptome of germinating and dormant teliospores of Tilletia indica was performed using the RNA Seq approach to identify germination-related genes. Approximately 63 million reads were generated using the RNA sequencing by the Illumina NextSeq500 platform. The high-quality reads were deposited in NCBI SRA database (accession: PRJNA522347). The unigenes from the pooled teliospores were 16,575 having unigenes length of 28,998,753 bases. The high-quality reads of germinating teliospores mapped on to 21,505 predicted CDSs. 9,680 CDSs were common between dormant and germinating teliospores of T. indica. 11,825 CDSs were found to be in germinating teliospores while only 91 were unique in dormant spores of pathogen. The pathway analysis showed the highest number of pathways was found in germinating spores than dormant spores. The highest numbers of CDSs were found to be associated with translation (431 in number), transport and catabolism (340), signal transduction (326), and carbohydrate metabolism (283). The differential expression analysis (DESeq) of germinating and dormant teliospores showed that 686 CDS were up-regulated and 114 CDS were down-regulated in the germinating teliospores. Significant germination-related genes in the spores were validated using qPCR analysis. Ten genes viz. Ti3931, Ti6828, Ti7098, Ti7462, Ti7522, Ti 9289, Ti 8670, Ti 7959, Ti 7809, and Ti10095 were highly up-regulated in germinated teliospores which may have role in germination of spores. Further, these differentially expressed genes provide insights into the molecular events. This first study of transcriptome will be helpful to devise better management strategies to manage Karnal bunt disease. [ABSTRACT FROM AUTHOR]

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.

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Karnal bunt (Tilletia indica Mitra) is an internationally quarantined disease of wheat. Until now, very little information has been available on the molecular basis of resistance and pathogenicity of T. indica. To investigate the molecular basis of host–pathogen interaction, the transcriptome of T. indica inoculated resistant (HD29) and susceptible (WH542) genotypes of wheat were analyzed. Approximately 58 million reads were generated using RNA sequencing by the Illumina NextSeq500 platform. These sequence reads were aligned to a reference genome of wheat to compare the expression level of genes in resistant and susceptible genotypes. The high-quality reads were deposited in the NCBI SRA database (SRP159223). More than 80,000 genes were expressed in both the resistant and susceptible wheat genotypes. Of these, 76,088 were commonly expressed genes, including 3184 significantly upregulated and 1778 downregulated genes. Four thousand one hundred thirteen and 5604 genes were exclusively expressed in susceptible and resistant genotypes, respectively. Based on the significance, 503 genes were upregulated and 387 genes were downregulated. Using gene ontology, the majority of coding sequences were associated with response to stimuli, stress, carbohydrate metabolism, developmental process, and catalytic activity. Highly differentially expressed genes (integral component of membrane, exonuclease activity, nucleic acid binding, DNA binding, metal ion binding) were validated in resistant and susceptible genotypes using qPCR analysis and similar expression levels were found in RNA-Seq. Apart from the wheat, the mapping of T. indica was 7.07% and 7.63% of resistant and susceptible hosts, respectively, upon infection, which revealed significant pathogenesis-related genes. This first study provided in-depth information and new insights into wheat–T. indica interaction for managing Karnal bunt disease of wheat. [ABSTRACT FROM AUTHOR]

The study of molecular interactions, especially the inter-species protein-protein interactions, is crucial for understanding the disease infection mechanism in plants. These interactions play an important role in disease infection and host immune responses against pathogen attack. Among various critical fungal diseases, the incidences of Karnal bunt (Tilletia indica) around the world have hindered the export of the crops such as wheat from infected regions, thus causing substantial economic losses. Due to sparse information on T. indica, limited insight is available with regard to gaining in-depth knowledge of the interaction mechanisms between the host and pathogen proteins during the disease infection process. Here, we report the development of a comprehensive database and webserver, TritiKBdb, that implements various tools to study the protein-protein interactions in the Triticum species-Tilletia indica pathosystem. The novel 'interactomics' tool allows the user to visualize/compare the networks of the predicted interactions in an enriched manner. TritiKBdb is a user-friendly database that provides functional annotations such as subcellular localization, available domains, KEGG pathways, and GO terms of the host and pathogen proteins. Additionally, the information about the host and pathogen proteins that serve as transcription factors and effectors, respectively, is also made available. We believe that TritiKBdb will serve as a beneficial resource for the research community, and aid the community in better understanding the infection mechanisms of Karnal bunt and its interactions with wheat. The database is freely available for public use at http://bioinfo.usu.edu/tritikbdb/. [ABSTRACT FROM AUTHOR]

Karnal bunt caused by Tilletia indica is a quarantine disease of wheat causing huge economic losses due to the ban on the import of bunted grains. This study was designed to characterize pathogenicity, aggressiveness and genetic diversity of 68 Tilletia indica isolates collected from different geographic regions of Pakistan. Forty-six isolates were tested for their pathogenicity on eight wheat varieties, out of which three were non-aggressive. The coefficient of infection (CI) ranged from 15.73% (PB-25) to 10% (PB-68, PB-60, and PB-43). The isolates collected from central Punjab showed higher infestation compared to other isolates. Among the wheat varieties used for the aggressiveness study, WL-711 showed susceptible reaction with 10.88% CI, while NIFA-Barsat, HD-29, Janbaz, Bakhtawar-92, Tatara, and AARI 2011 showed resistance to the highly resistant response. These isolates were amplified using 31 random amplified polymorphic DNA (RAPD) markers and 32 inter-simple sequence repeat (ISSR) markers for diversity analysis. The principal component analysis (PCA) and analysis of molecular variance (AMOVA) showed greater divergence among isolates collected from Punjab and Khyber Pakhtunkhwa (KPK), with a moderate level of admixture. The isolates from Faisalabad (Punjab) were more aggressive compared to isolates from KPK and were clearly separated based on PCA, indicating the significant genetic distance in the populations. Our findings will assist breeders and pathologists in better understanding the pathogenic variability in Tilletia indica and in subsequent disease management. [ABSTRACT FROM AUTHOR]

To quantify the effect of meteorological parameters on incidence of Karnal bunt in wheat crop, an investigation was done using 9 to 12 season's data of Bathinda and Ludhiana stations of Punjab. Maximum temperature during March in range of 25-31oC, minimum temperature of February (8.5-11.0oC), morning and evening relative humidity of March in range of 85-95 and 40-60 per cent respectively, rainfall more than 25 mm with sunshine hours 5.5-9.0 hrs/day during mid-February to mid-March favour Karnal bunt in wheat crop. Maximum temperature of March showed significant negative correlation with incidence of Karnal bunt whereas minimum temperature of February showed significant positive correlation with disease incidence at both locations. Morning and evening relative humidity showed significant positive correlation with disease incidence. Rain amount and rainy days during mid-February to mid-March significantly influenced disease incidence. Sunshine hours had negative correlation with disease incidence. Backward multiple linear regression (BMLR) analysis indicated maximum temperature, rainfall and sunshine hours play significant role in Karnal bunt incidence at Ludhiana. However, at Bathinda, maximum temperature, evening time relative humidity, rain amount and rainy days played significant role. [ABSTRACT FROM AUTHOR]

IN WHEAT (Triticum aestivum L.), the production of disease resistant genotypes is crucial for managing diseases. Although, breeding programmes are challenging in disease-free areas owing to quarantine regulations but it is a necessity for upgrading the wheat genotypes that are adapted to current climate change situations. Karnal bunt caused by Tilletia indica has a significant influence on production and quality parameters of what as it adds a fishy odour and flavor to the wheat. A screening study was undertaken for Karnal bunt observation in two growing seasons in the high yielding area of South Punjab, i.e, Bahawalpur. Twenty one wheat commercial varieties developed in Regional Agricultural Research Institute Bahawalpur, Pakistan were screened for disease comparison between two wheat growing seasons (2019-2020 and 2020-2021). The area between the top of the spike and the surrounding boot sheath was inoculated with sporidial inoculum using a 3CC syringe with a 22-gauge needle. To assess the resistance/susceptibility of each line/cultivar of karnal bunt, a modified rating system was used. Disease incidence varied in cultivars during 2020-2021 growing season. In 2019-2020, Kohi Noor, Aas-09, Millat-11, and WL-711 had lower disease incidence than infected cultivars. While Shafaq-06, Johar-16, and Galaxy-13 have no disease incidence in the 2020-2021. Overall, the disease occurance, persistence and severity is relatively low in Bahawalpur, that indicates arid environment is a favorable indicator for developing karnal bunt resistant genotypes. However, it is important that regular disease screening of Karnal bunt through monitoring at larger scale should be promoted in other wheat-growing areas as well for ensuring the adaptation/selection of disease resistant wheat genotypes. [ABSTRACT FROM AUTHOR]

To quantify the effect of meteorological parameters on incidence of Karnal bunt in wheat crop, an investigation was done using 9 to 12 season’s data of Bathinda and Ludhiana stations of Punjab. Maximum temperature during March in range of 25-31oC, minimum temperature of February (8.5-11.0oC), morning and evening relative humidity of March in range of 85-95 and 40-60 per cent respectively, rainfall more than 25 mm with sunshine hours 5.5-9.0 hrs/day during mid February to mid March favour Karnal bunt in wheat crop. Maximum temperature of March showed significant negative correlation with incidence of Karnal bunt whereas minimum temperature of February showed significant positive correlation with disease incidence at both locations. Morning and evening relative humidity showed significant positive correlation with disease incidence. Rain amount and rainy days during mid February to mid March significantly influenced disease incidence. Sunshine hours had negative correlation with disease incidence. Backward multiple linear regression (BMLR) analysis indicated maximum temperature, rainfall and sunshine hours play significant role in Karnal bunt incidence at Ludhiana. However, at Bathinda, maximum temperature, evening time relative humidity, rain amount and rainy days played significant role.

Karnal bunt, a disease of wheat, durum, rye, and triticale, is subject to strict quarantine restrictions worldwide. The disease is considered a major threat to food security, due to its use as a non‐tariff trade barrier by several wheat‐importing countries. In this paper, we analysed seven years of phenotypic data to search for quantitative trait loci (QTLs) associated with resistance in common wheat, validated the QTLs using an independent population, and assessed the potential of genomic selection as a tool for pre‐emptive breeding. The QTL study used phenotypic data collected from artificially inoculated field experiments involving two historical Karnal bunt resistance populations: WH542 × HD29 and WH542 × W485. QTL analyses detected four significant (p < 0.001) QTLs on chromosomes 1A, 3A, 4B, and 6B, which explained between 13.7% and 15.7% of the phenotypic variation. A panel of 130 cultivars was used to validate QTL effects. These were genotyped with the same DArTseq protocol, and two of the four QTLs were significantly (p < 0.001) associated with Karnal bunt resistance in the validation panel. The potential of genomic selection was investigated by comparing accuracies of a model trained with all available markers and a model based solely on validated QTL information from the biparental populations. Genomic prediction accuracy, based on the two scenarios, averaged 0.43 and 0.33, respectively, suggesting that even in situations where phenotyping is difficult due to quarantine restrictions, the prospects for pre‐emptive breeding against Karnal bunt are encouraging, and resources are now available that will reduce the cost burden. [ABSTRACT FROM AUTHOR]

Abstract Utilization of modern breeding techniques for developing high yielding and uniform plant types ultimately narrowing the genetic makeup of most crops. Narrowed genetic makeup of these crops has made them vulnerable towards disease and insect epidemics. For sustainable crop production, genetic variability of these crops must be broadened against various biotic and abiotic stresses. One of the ways to widen genetic configuration of these crops is to identify novel additional sources of durable resistance. In this regard crops wild relatives are providing valuable sources of allelic diversity towards various biotic, abiotic stress tolerance and quality components. For incorporating novel variability from wild relative’s wide hybridization technique has become a promising breeding method. For this purpose, wheat-Th. bessarabicum amphiploid, addition and translocation lines have been screened in field and screen house conditions to get novel sources of yellow rust and Karnal bunt resistant. Stripe rust screening under field conditions has revealed addition lines 4JJ and 6JJ as resistant to moderately resistant while addition lines 3JJ, 5JJ, 7JJ and translocation lines Tr-3, Tr-6 as moderately resistant wheat-Thinopyrum-bessarabicum genetic stock. Karnal bunt screening depicted addition lines 5JJ and 4JJ as highly resistant genetic stock. These genetic stocks may be used to introgression novel stripe rust and Karnal bunt resistance from the tertiary gene pool into susceptible wheat backgrounds.

Karnal bunt (Tilletia indica Mitra) is an internationally quarantined disease of wheat. Until now, very little information has been available on the molecular basis of resistance and pathogenicity of T. indica. To investigate the molecular basis of host–pathogen interaction, the transcriptome of T. indica inoculated resistant (HD29) and susceptible (WH542) genotypes of wheat were analyzed. Approximately 58 million reads were generated using RNA sequencing by the Illumina NextSeq500 platform. These sequence reads were aligned to a reference genome of wheat to compare the expression level of genes in resistant and susceptible genotypes. The high-quality reads were deposited in the NCBI SRA database (SRP159223). More than 80,000 genes were expressed in both the resistant and susceptible wheat genotypes. Of these, 76,088 were commonly expressed genes, including 3184 significantly upregulated and 1778 downregulated genes. Four thousand one hundred thirteen and 5604 genes were exclusively expressed in susceptible and resistant genotypes, respectively. Based on the significance, 503 genes were upregulated and 387 genes were downregulated. Using gene ontology, the majority of coding sequences were associated with response to stimuli, stress, carbohydrate metabolism, developmental process, and catalytic activity. Highly differentially expressed genes (integral component of membrane, exonuclease activity, nucleic acid binding, DNA binding, metal ion binding) were validated in resistant and susceptible genotypes using qPCR analysis and similar expression levels were found in RNA-Seq. Apart from the wheat, the mapping of T. indica was 7.07% and 7.63% of resistant and susceptible hosts, respectively, upon infection, which revealed significant pathogenesis-related genes. This first study provided in-depth information and new insights into wheat–T. indica interaction for managing Karnal bunt disease of wheat.

The study of molecular interactions, especially the inter-species protein-protein interactions, is crucial for understanding the disease infection mechanism in plants. These interactions play an important role in disease infection and host immune responses against pathogen attack. Among various critical fungal diseases, the incidences of Karnal bunt (Tilletia indica) around the world have hindered the export of the crops such as wheat from infected regions, thus causing substantial economic losses. Due to sparse information on T. indica, limited insight is available with regard to gaining in-depth knowledge of the interaction mechanisms between the host and pathogen proteins during the disease infection process. Here, we report the development of a comprehensive database and webserver, TritiKBdb, that implements various tools to study the protein-protein interactions in the Triticum species-Tilletia indica pathosystem. The novel ‘interactomics’ tool allows the user to visualize/compare the networks of the predicted interactions in an enriched manner. TritiKBdb is a user-friendly database that provides functional annotations such as subcellular localization, available domains, KEGG pathways, and GO terms of the host and pathogen proteins. Additionally, the information about the host and pathogen proteins that serve as transcription factors and effectors, respectively, is also made available. We believe that TritiKBdb will serve as a beneficial resource for the research community, and aid the community in better understanding the infection mechanisms of Karnal bunt and its interactions with wheat. The database is freely available for public use at http://bioinfo.usu.edu/tritikbdb/.

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. [ABSTRACT FROM AUTHOR]

Wheat (Triticum aestivum L.) crop health assumes unprecedented significance in being the second most important staple crop of the world. It is host to an array of fungal pathogens attacking the plant at different developmental stages and accrues various degrees of yield losses owing to these. Tilletia indica that causes Karnal bunt (KB) disease in wheat is one such fungal pathogen of high quarantine importance restricting the free global trade of wheat besides the loss of grain yield as well as quality. With global climate change, the disease appears to be shifting from its traditional areas of occurrence with reports of increased vulnerabilities of new areas across the continents. This KB vulnerability of new geographies is of serious concern because once established, the disease is extremely difficult to eradicate and no known instance of its complete eradication using any management strategy has been reported yet. The host resistance to KB is the most successful as well as preferred strategy for its mitigation and control. However, breeding of KB resistant wheat cultivars has proven to be not so easy, and the low success rate owes to the scarcity of resistance sources, extremely laborious and regulated field screening protocols delaying identification/validation of putative resistance sources, and complex quantitative nature of resistance with multiple genes conferring only partial resistance. Moreover, given a lack of comprehensive understanding of the KB disease epidemiology, host-pathogen interaction, and pathogen evolution. Here, in this review, we attempt to summarize the progress made and efforts underway toward a holistic understanding of the disease itself with a specific focus on the host-pathogen interaction between T. indica and wheat as key elements in the development of resistant germplasm. In this context, we emphasize the tools and techniques being utilized in development of KB resistant germplasm by illuminating upon the genetics concerning the host responses to the KB pathogen including a future course. As such, this article could act as a one stop information primer on this economically important and re-emerging old foe threatening to cause devastating impacts on food security and well-being of communities that rely on wheat.

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.

Wheat (Triticum aestivum L.) crop health assumes unprecedented significance in being the second most important staple crop of the world. It is host to an array of fungal pathogens attacking the plant at different developmental stages and accrues various degrees of yield losses owing to these. Tilletia indica that causes Karnal bunt (KB) disease in wheat is one such fungal pathogen of high quarantine importance restricting the free global trade of wheat besides the loss of grain yield as well as quality. With global climate change, the disease appears to be shifting from its traditional areas of occurrence with reports of increased vulnerabilities of new areas across the continents. This KB vulnerability of new geographies is of serious concern because once established, the disease is extremely difficult to eradicate and no known instance of its complete eradication using any management strategy has been reported yet. The host resistance to KB is the most successful as well as preferred strategy for its mitigation and control. However, breeding of KB resistant wheat cultivars has proven to be not so easy, and the low success rate owes to the scarcity of resistance sources, extremely laborious and regulated field screening protocols delaying identification/validation of putative resistance sources, and complex quantitative nature of resistance with multiple genes conferring only partial resistance. Moreover, given a lack of comprehensive understanding of the KB disease epidemiology, host-pathogen interaction, and pathogen evolution. Here, in this review, we attempt to summarize the progress made and efforts underway toward a holistic understanding of the disease itself with a specific focus on the host-pathogen interaction between T. indica and wheat as key elements in the development of resistant germplasm. In this context, we emphasize the tools and techniques being utilized in development of KB resistant germplasm by illuminating upon the genetics concerning the host responses to the KB pathogen including a future course. As such, this article could act as a one stop information primer on this economically important and re-emerging old foe threatening to cause devastating impacts on food security and well-being of communities that rely on wheat. [ABSTRACT FROM AUTHOR]

Fungal pathogen, Tilletia indica, the cause of Karnal bunt disease in wheat, is severely affecting the yield and grain quality, worldwide. Thus, strict quarantine regulations by most wheat growing countries have to be followed, leading to trade barriers for wheat export. The conventional methods being used for pathogen detection at symptomatic stage requires the germination of Tilletia spores for the processing of samples. Thus, it is time-consuming and expensive. This study proposes a simulated microcantilever-based piezoelectric biosensor for the early detection of T. indica. Four different materials, SiO2, SiC, Si3N4, and Poly Si, were used for the microcantilever design. Microcantilever was coated with siliceous frustules of diatom that provides high surface area and enhanced sensitivity for specific antibody against the antigen, T. indica. Ansys software was used for the simulation analysis. Simulation results showed that microcantilever beam of SiO2 length of 150 µm, width of 30 µm and thickness 1 µm enhanced the sensitivity by two times against the antibody in comparison to normal microcantilever beam. The results concluded that SiO2 with coated diatom is the best material for the microcantilever fabrication, thus, providing an excellent protocol for fabrication of microcantilever-based biosensor which is both cost- and time effective. [ABSTRACT FROM AUTHOR]

This study was initiated to identify genomic regions conferring resistance to Karnal Bunt (KB) disease in wheat through a genome-wide association study (GWAS) on a set of 179 pre-breeding lines (PBLs). A GWAS of 6,382 high-quality DArTseq SNPs revealed 15 significant SNPs (P-value <10−3) on chromosomes 2D, 3B, 4D and 7B that were associated with KB resistance in individual years. In particular, two SNPs (chromosome 4D) had the maximum R2 values: SNP 1114200 | F | 0–63:T > C at 1.571 cM and R2 of 12.49% and SNP 1103052 | F | 0–61:C > A at 1.574 cM and R2 of 9.02%. These two SNPs displayed strong linkage disequilibrium (LD). An in silico analysis of SNPs on chromosome 4D identified two candidate gene hits, TraesCS4D02G352200 (TaNox8; an NADPH oxidase) and TraesCS4D02G350300 (a rhomboid-like protein belonging to family S54), with SNPs 1103052 | F | 0–61:C > A and 1101835 | F | 0–5:C > A, respectively, both of which function in biotic stress tolerance. The epistatic interaction analysis revealed significant interactions among 4D and 7B loci. A pedigree analysis of confirmed resistant PBLs revealed that Aegilops species is one of the parents and contributed the D genome in these resistant PBLs. These identified lines can be crossed with any elite cultivar across the globe to incorporate novel KB resistance identified on 4B. [ABSTRACT FROM AUTHOR]

Parental genotypes H567.71 and WH542 and 107 recombinant inbred lines (RILs) were grown in field conditions during the rabi season 2010-11 and 2011-12. The numbers of infected grains were recorded and percentage of infected grains and coefficients of infection were worked out. A wide range of variation was observed among the RILs evaluated for Karnal bunt resistance. The distribution of RILs based on Karnal bunt disease was higher towards the resistant parent (H567.71). The population also characterized morphologically for various traits and analysis of variance for morphological characters showed significant variations among the RILs. Seventy SSRs markers were used for screening of the parental genotypes out of these 42 were found polymorphic. These 42 SSR markers were used to screen selected RILs and a total of 88 alleles were detected, number of alleles per locus ranged from 2-3 with an average of 2.09 alleles per locus. The NTSYS-pc UPGMA cluster analysis was used to determine similarity between parental genotypes and selected RILs based on the SSR scoring data. The dendrogram represented two major groups; 1st major group consist of H567.71 (resistant parental genotype) along with resistant RILs and the 2nd major group comprised of WH542 (susceptible parental genotype) and susceptible RILs at the similarity coefficient value of 0.39. Genetic relationship was also assessed by Principal component analysis (PCA) and similar results were obtained.

Karnal bunt of wheat is an internationally quarantined disease. One of the major constraints in exporting wheat is prevalence of Karnal bunt (KB). It is caused by Tilletia indica which is a heterothallic fungus and requires fusion of different mating types for causing infection. The growth efficiency and aggressiveness of plant pathogens are often associated with their carbohydrate active enzymes (CAZymes). The present study was undertaken to identify the CAZymes in whole genome sequence of Tilletia indica, and conduct sporulation analysis. Total 315 predicted secretory proteins were annotated with the CAZyme database (dbCAN). The secreted carbohydrates active enzymes were identified as 105 glycosylhydrolases (GH), 85 glycosyltransferases (GT), 83 carbohydrate esterases (CE), 8 carbohydrate binding modules (CBM), 30 auxiliary activities (AA) and 4 polysaccharide lyases (PL). Based on sporulation analysis of T. indica isolates, KB1 and KB2 isolates were most sporulative among all the isolates of T. indica. These carbohydrate active enzymes can help to understand the pathogenesis mechanism(s) of Karnal bunt using functional genomics approach.

Tilletia indica Mitra is a fungal pathogen causing Karnal bunt of wheat. Tilletia indica is a quarantine pest in many countries worldwide. In the European Union, imported wheat grain from countries where the fungus is present must be checked for the presence of T. indica teliospores. The inspection services at the borders need rapid, sensitive and reliable detection tests to identify T. indica spores on wheat grain. In this work, validation was carried out according to EPPO Standard PM 7/98 to evaluate the multiplex real‐time PCR test described in ISPM 27 Diagnostic Protocol for regulated pests (Annex 4 Tilletia indica Mitra) by means of a test performance study with nine participating laboratories, and the performance characteristics of the test were established. The original protocol was modified with regard to the extraction of DNA from the pellet obtained from the 'washing test' and the enrichment PCR step in order to increase the amount of template DNA for the real‐time PCR. The optimized test still has five teliospores as the limit of detection for the contaminated pellet but has an increased analytical sensitivity and had positive results with three teliospores in 93% of cases instead of 43% for the original test. The two closest Tilletia species, Tilletia horrida and Tilletia walkeri, were used to evaluate analytical specificity (exclusivity) and no cross‐reactions were obtained. Diagnostic sensitivity, diagnostic specificity, accordance and concordance were also evaluated. [ABSTRACT FROM AUTHOR]

Karnal bunt is a seedborne disease of wheat caused by the fungus Tilletia indica Mitra and is a major biosecurity threat for the Australian wheat industry. Host-plant resistance is an effective means of controlling this disease. This study has identified Australian wheat genotypes with durable resistance should the pathogen enter Australia and become established. These genotypes provide a basis for breeding adapted genotypes that can be recommended for growers. In the study, 196 genotypes comprising 177 common (bread) wheat (hexaploid, Triticum aestivum), eight durum (tetraploid, T. durum) and 11 triticale (× Triticosecale) genotypes were evaluated in the field for their reaction to infection by the fungus. Six experiments were carried out at CIMMYT's research station at Obregon, Mexico, during three consecutive cropping seasons (2014–15, 2015–16 and 2016–17) and at two planting dates. In each experiment, the genotypes were screened for resistance to Karnal bunt by injecting an inoculum suspension with a hypodermic syringe into the boot at awn emergence. Disease incidence averaged 14.7% infection in 2015, 21.7% in 2016 and 25.6% in 2017. Resistant triticale genotypes, Tuckerbox, Berkshire and Hawkeye, were identified, along with three resistant wheat genotypes, Batavia, Pelsart and RAC-655, and two moderately resistant durum genotypes, Hyperno and Saintly. Stability analysis showed that RAC-655, Hyperno, Tuckerbox and Berkshire were consistently resistant to infection in different seasonal conditions and unaffected by the environment. Karnal bunt is a disease of wheat caused by the fungus Tilletia indica Mitra and is a major biosecurity threat for the Australian wheat industry. This study has identified Australian wheat genotypes with durable resistance to infection. The genotypes provide a basis for breeding adapted varieties that can be recommended for growers, should the pathogen enter Australia and become established. [ABSTRACT FROM AUTHOR]

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.