Karnal bunt: A minor disease that is now a threat to wheat

Karnal bunt of wheat, caused byNeovossia indica (Mitra) Mundkur, was first reported in 1931 from Northwestern India in experimental plantings. For many years it was a minor disease found only in Northwestern India. During the 1969–70 crop season it was unusually widespread in Northwest India and since 1974–75 Karnal bunt has been distributed throughout Northern India from West Bengal to the western border. Seed lots with more than 50% of the kernels infected have been collected from farm threshing sites. The disease is now established in Afghanistan, Iraq, Pakistan and Mexico and has been intercepted in India in wheat seed that was shipped from Lebanon and Syria. The increased geographic distribution of Karnal bunt may be due to the development and wide distribution of wheat cultivars that were more susceptible than the older wheats that were grown in India prior to 1969–70. Resistance to Karnal bunt has been reported in several Indian wheats but most cultivars are susceptible. Hexachlorobenzene and cyano (methylmercuric) guanidine each prevented germination of teliospores ofN. indica in tests in Mexico.     

Intraspecific genetic variability analysis of Neovossia indica causing Karnal bunt of wheat using repetitive elements

Neovossia indica (Tilletia indica), causing Karnal bunt of wheat, affects major wheat growing regions all over the world. Karnal bunt ranks as one of the major diseases of wheat causing quality losses and monetary losses due to international quarantine regulations. The present work is the first report of a genetic diversity analysis of Indian isolates of N. indica. A library of N. indica isolate Ni7 was constructed in a λZAPII system, and three repetitive elements were identified for molecular analysis. These repetitive elements generated complex hybridization profiles producing fingerprint patterns of all seven isolates. Copy-number estimation of these three elements, pNiR9, pNiR12 and pNiR16, indicated the presence of 32, 61 and 64 copies, respectively. Cluster analysis based on hybridization patterns grouped together moderately virulent isolates Ni1, Ni7 and Ni8, thus suggesting a positive correlation between virulence typing and cluster analysis based on molecular data. Variability analysis of N. indica isolates will aid in checking new resistant sources in host germplasm. Received: 20 April 1999 / Accepted: 29 July 1999     

Isolation and in-silico characterization of Peroxidase isoenzymes from Wheat (Triticum aestivum) against Karnal Bunt (Tilletia indica)

To investigate the role of Peroxidase and its physiological significance under Karnal Bunt (KB) were determined in resistant (HD-29) and susceptible genotype (WH-542) of wheat during different developmental stages. The enzymes were expressed constitutively in both the susceptible and resistant genotype. In gel assay and differential expression analysis of POD was significantly higher (p >0.05) in Sv and S2, than the S1 and S3 stages. in silico analysis of Peroxidase for eg. physico-chemical properties, secondary structural features and phylogenetic classification for comparative analysis. Motif and Domain analysis of Peroxidase by MEME, to be important for the biological functions, and studies of evolution. Our results clearly indicate that the enhanced expression of POD at the WS2 stage, which reinforces its role in stage dependent immunity against Karnal bunt and role of POD metabolism provides genotype and stage dependant structural barrier resistance in wheat against KB.     

Determination of genetic and pathological variance among Tilletia indica isolates and monosporidial lines using PCR based markers and host differentials

Karnal bunt of wheat caused by Tilletia indica is an important international quarantine disease in many countries. In this investigation, genetic and pathological variation among the 10 isolates and 15 monosporidial (Ms) lines belonged to different locations of North-West India was studied. Depending upon the pathogenic potential, most virulent and least aggressive isolate was found from Chaksu (Rajasthan) and Tarau (HP), which scored coefficients of infection 70.98 and 6.22, respectively, on susceptible host genotype HD 2009 under artificially inoculated conditions. Fifteen Ms lines were inoculated in 20 combinations. Most virulent compatible combination was found KB2MsD?×?KB6MsA, which scored co-efficient of infection 74.91%. Out of 32 Inter Simple Sequence Repeats based molecular markers, 28 were polymorphic generating 192 reproducible bands for all the T. indica isolates and Ms lines in this study. A grouping analysis using the unrooted neighbour – joining method was consistent with DARwin software and winboot analysis and combination approach suggested that self-paired Ms lines exhibit narrow genetic diversity. This result will be useful for developing integrated strategies for disease management and breeding programmes for improvement of the varieties.     

A molecular protocol using quenched FRET probes for the quarantine surveillance of Tilletia indica, the causal agent of Karnal bunt of wheat

The current surveillance protocol for Karnal bunt of wheat in most countries, including the USA, European Union (EU), and Australia, involves the tentative identification of the spores based on morphology followed by a molecular analysis. Germination of spores is required for confirmation which incurs a delay of about two weeks, which is highly unsatisfactory in a quarantine situation. A two-step PCR protocol using FRET probes for the direct detection and identification of Tilletia indica from a very few number of spores (≤10) is presented. The protocol involves amplification of the ITS1 DNA segment in the highly repeated rDNA unit from any Tilletia species, followed by FRET analysis to detect and unequivocably distinguish T. indica and the closely related T. walkeri. This rapid, highly sensitive, fluorescent molecular tool is species-specific, and could supersede the conventional microscopic diagnosis used in a quarantine surveillance protocol for Karnal bunt which is often confounded by overlapping morphological characters of closely related species.     

Mapping of a resistance gene effective against Karnal bunt pathogen of wheat

A set of 130 wheat recombinant inbred lines (RILs) developed from a cross between parents susceptible (WL711) and resistant (HD29) to Karnal bunt (caused by Tilletia indica), were screened for 3 years with the pathogen populations prevalent in northern India. When 90 simple sequence repeats (SSRs) and 81 amplified fragment length polymorphism (AFLP) loci were mapped on the RILs, markers on chromosomes 2A, 4B and 7B accounted collectively for about one-third of the variation in the disease reaction. The genomic region of largest effect, identified on the long arm of chromosome 4B, reduced Karnal bunt disease by half in three different experiments and accounted for up to 25% of the phenotypic variation for KB reaction. A closely linked SSR marker, GWM538, may be useful in marker-assisted selection for Karnal bunt resistance in wheat.     

Towards Molecular Tagging of Karnal Bunt Resistance Gene(s) in Wheat

We describe preliminary results of cosegregation of a DNA probe with Karnal bunt resistance genets) in wheat. The tagging strategy utilized 4 uniformly resistant and 13 uniformly susceptible F³ families from a cross of Karnal bunt resistant (HD29) and susceptible (WL711) lines of Triticum aestivum. Bulked DNA of 8–10 plants in each family were tested by gel blot DNA hybridization. Thirty three per cent of the probes (11/33) detected restriction fragment length polymorphism (RFLP) between Karnal bunt resistant and susceptible lines. Probe Cxp1 from chromosome group 6 detected the resistant parent (HD29) polymorphic band in all the resistant family bulks but was missing in 6 uniformly susceptible family bulks. A similar pattern was observed with probe XKsu G34 from 1DL. The intensity of the resistant parent band in the susceptible family bulks was less as compared to the resistant family bulks. Probe Xksu H8 which mapped on chromosomes 2, 3, 5 and 7D on the T. tauschii map did not differentiate resistant family bulks from susceptible family bulks. Our results indicated that one or more resistance genets) are located on chromosome groups 1 and 6 of wheat.     

Determination of variability in monosporidial lines of Tilletia indica by RAPD analysis

Genetic variability in 23 monosporidial lines developed from five isolates of Tilletia indica causing Karnal bunt of wheat isolated from four wheat growing states of India was determined by using 19 rapid amplified polymorphic DNA (RAPD) markers. Amplification profile generated with all the 19 primers produced 3–16 numbers of bands of 1.5–5 kb size. High level of polymorphism (95.2%) suggested wide range of variability. Maximum Jaccard's similarity coefficient (80%) was observed between KB2MsB and KB2MsC followed by KB5MsC and KB5MsE with 75% similarity, whereas it was minimum between KB3MsA and Kb4MsB (47%). The dendrogram derived from the fingerprint analysis with 19 RAPD primers by using UPGMA showed different levels of genetic similarity among monosporidial lines. At 35% genetic similarity, the monosporidial lines were grouped in two clusters. Some primers, viz., OPN-1, OPN-6, OPN-9, OPN-12, OPN-13, OPN-18, OPM-2, OPM-8, OPM-10, OPB-8, OPB-17 and OPB-20 showed 100% polymorphism. The RAPD fingerprint generated by OPN-1 and OPM-3 were analysed and showed high range of variation in genetic make-up of monosporidial lines.     

Characteristics of the Infection of Tilletia laevis Kühn (syn. Tilletia foetida (Wallr.) Liro.) in Compatible Wheat

Tilletia laevis Kühn (syn. Tilletia foetida (Wallr.) Liro.) causes wheat common bunt, which is one of the most devastating plant diseases in the world. Common bunt can result in a reduction of 80% or even a total loss of wheat production. In this study, the characteristics of T. laevis infection in compatible wheat plants were defined based on the combination of scanning electron microscopy, transmission electron microscopy and laser scanning confocal microscopy. We found T. laevis could lead to the abnormal growth of wheat tissues and cells, such as leakage of chloroplasts, deformities, disordered arrangements of mesophyll cells and also thickening of the cell wall of mesophyll cells in leaf tissue. What’s more, T. laevis teliospores were found in the roots, stems, flag leaves, and glumes of infected wheat plants instead of just in the ovaries, as previously reported. The abnormal characteristics caused by T. laevis may be used for early detection of this pathogen instead of molecular markers in addition to providing theoretical insights into T. laevis and wheat interactions for breeding of common bunt resistance.     

Identification of RAPD markers linked to the karnal bunt resistance genes in wheat

A set of 104 wheat recombinant inbred lines (RILs) obtained from a cross between parents resistant (HD 29) and susceptible (WH 542) to karnal bunt (KB) (caused by Neovossia indica) were screened and used to identify random amplified polymorphic DNA (RAPD) markers linked with resistance to karnal bunt as these would allow indirect marker assisted selection of KB resistant genotypes. The two parents were analysed with 92 RAPD primers. A total of 65 primers proved functional by giving scorable polymerase chain reaction (PCR) products. Of these, 21 (32 %) primers detected polymorphism between the two parental genotypes. Using these primers, bulked segregant analysis was carried out on two bulk DNAs, one obtained by pooling DNA from 10 KB resistant RILs and the other similarly derived by pooling 10 KB susceptible RILs. One marker, OPM-20 showed apparent association with resistance to KB. This was confirmed following selective genotyping of individual RILs included in the bulks.     

Secretome Analysis Identifies Potential Pathogenicity/Virulence Factors of Tilletia indica,a Quarantined Fungal Pathogen Inciting Karnal Bunt Disease in Wheat

Tilletia indica is a smut fungus that incites Karnal bunt in wheat. It has been considered as quarantine pest in more than 70 countries. Despite its quarantine significance, there is meager knowledge regarding the molecular mechanisms of disease pathogenesis. Moreover, various disease management strategies have proven futile. Development of effective disease management strategy requires identification of pathogenicity / virulence factors. With this aim, the present study was conducted to compare the secretomes of T. indica isolates, that is, highly (TiK) and low (TiP) virulent isolates. About 120 and 95 protein spots were detected reproducibly in TiK and TiP secretome gel images. Nineteen protein spots, which were consistently observed as upregulated/differential in the secretome of TiK isolate, were selected for their identification by MALDI‐TOF/TOF. Identified proteins exhibited homology with fungal proteins playing important role in fungal adhesion, penetration, invasion, protection against host‐derived reactive oxygen species, production of virulence factors, cellular signaling, and degradation of host cell wall proteins and antifungal proteins. These results were complemented with T. indica genome sequence leading to identification of candidate pathogenicity / virulence factors homologs that were further subjected to sequence‐ and structure‐based functional annotation. Thus, present study reports the first comparative secretome analysis of T. indica for identification of pathogenicity / virulence factors. This would provide insights into pathogenic mechanisms of T. indica and aid in devising effective disease management strategies.     

Molecular mapping of QTLs for Karnal bunt resistance in two recombinant inbred populations of bread wheat

Karnal bunt (KB) of wheat, caused by the fungus Tilletia indica, is a challenge to the grain industry, owing not to direct yield loss but to quarantine regulations that may restrict international movement of affected grain. Several different sources of resistance to KB have been reported. Understanding the genetics of resistance will facilitate the introgression of resistance into new wheat cultivars. The objectives of this study were to identify quantitative trait loci (QTLs) associated with KB resistance and to identify DNA markers in two recombinant inbred line populations derived from crosses of the susceptible cultivar WH542 with resistant lines HD29 and W485. Populations were evaluated for resistance against the KB pathogen for 3 years at Punjab Agricultural University, Ludhiana, India. Two new QTLs (Qkb.ksu-5BL.1 and Qkb.ksu-6BS.1) with resistance alleles from HD29 were identified and mapped in the intervals Xgdm116–Xwmc235 on chromosome 5B (deletion bin 5BL9-0.76-0.79) and Xwmc105–Xgwm88 on chromosome 6B (C-6BS5-0.76). They explained up to 19 and 13% of phenotypic variance, respectively. Another QTL (Qkb.ksu-4BL.1) with a resistance allele from W485 mapped in the interval Xgwm6–Xwmc349 on chromosome 4B (4BL5-0.86-1.00) and explained up to 15% of phenotypic variance. Qkb.ksu-6BS.1 showed pairwise interactions with loci on chromosomes 3B and 6A. Markers suitable for marker-assisted selection are available for all three QTLs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Surface Rodlets of Tilletia indica Teliospores

Tilletia indica teliospores were studied by use of thin sections and freeze-etch replicas. Surfaces of these spores have rodlet patterns which differ from those previously reported for spores of other fungi. The rodlets on T. indica teliospores average 240 nm in length and are not grouped into fascicles.     

PCR based molecular technique for identification and discrimination of quarantined and non-quarantined Tilletia sps

Polymerase chain reaction (PCR) based RAPD profiles, in conjunction with six primers, of Karnal bunt of wheat and rice bunt exhibiting distinct polymorphic DNA. A total of 84 RAPD loci were observed on polyacrylamide gel for both Tilletia sps. Out of 84, 16 loci were found monomorphic, while other 68 loci were unique. Usefulness of random primers was also checked with other seed borne fungal pathogens of wheat and rice. None of primers gave amplification with Magnaporthe grisea, a causative agent of rice blast. However, distinct RAPD profiles were obtained with Alternaria triticina, Fusarium monaliforme, Helminthosporium sativum and Rhizoctonia solani. These six arbitrary primers could distinguish T. indica, a quarantine fungal pathogen from a non-quarantine fungal pathogen, T. barclayana. The two Tilletia sps. could be discriminated not only on the basis of distinct RAPD profiles, but also by presence of few unique gene fragments amplified using all six primers.     

Aerobiological aspects of quarantine risks in grain warehouses: a study on bunt (Tilletia spp.) dispersal

Several countries refuse the import of wheat if it is contaminated with Tilletia spp. Local quarantine regulations are in effect regarding Tilletia indica and Tilletia controversa as part of a strategy to first hinder the disease from establishing in the country or eradicate the disease and, more recently, to prohibit the spread of it. Because of the economic importance of these fungal pathogens in the warehouses, the aerobiology of Tilletia spp. should be known better. Air (with a portable Hirst-type volumetric impaction sampler) and wheat seeds were collected in two central grain warehouses in Budapest (Bp) and Jászapáti (Ja) and in 14 farm warehouses. Quarantine organisms (T. controversa and T. indica) were not detected in any of the samples. Tilletia caries dominated in the air and seed samples, while Tilletia laevis were present only in a low concentration in the samples collected in Bp, but this fungus was absent in both Ja and the farm warehouses. Teliospore levels of the two different sites of wheat storage rooms in Bp were compared, but no significant difference was found between them (p = 0.16). Similarly, the volume of stored wheat did not significantly affect the Tilletia spp. concentrations (p = 0.46) in the air. Teliospore levels in seed samples correlated with those in the air samples which were collected from above the wheat heaps (r = 0.596, p < 0.05). In Bp, teliospore dispersal takes place rapidly between storage rooms and floors via open-top walls and spouts, respectively. Consequently, there is a higher risk of contamination because of the high teliospore concentration in the air and the high probability of teliospore dispersal. It is proposed that warehouses could be tested for quarantine or economically important Tilletia spp. not only by seed sampling but by aerobiological methods as well. Our results claim attention on the quarantine risk of airborne contamination with Tilletia spp. teliospores of grain warehouses, and some modification in control strategy against warehouse contamination is suggested.     

Development of a SCAR marker for molecular detection and diagnosis of Tilletia controversa Kühn,the causal fungus of wheat dwarf bunt

Tilletia controversa Kühn (TCK) is an important quarantine pathogen that causes wheat dwarf bunt and results in devastating damage to wheat production. The fungus is difficult to be distinguished from T. caries and T. laevis, which cause wheat common bunt, based on morphological, physiological and symptomatological characteristics of the pathogens. The traditional detection of the fungus can be a long and tedious process with poor accuracy. The inter-simple sequence repeat (ISSR) technique has been used for identifying molecular markers for detection of TCK. Of 28 ISSR primers screened, ISSR-859 amplified a specific 678 bp DNA fragment from all TCK isolates but not from any isolates of the common bunt fungi or other pathogenic fungi tested. Based on the fragment sequence, a pair of sequence characterized amplified region (SCAR) primers was designed, which amplified a 372 bp DNA fragment specifically in TCK. The SCAR marker was detected using as low as 1 ng template DNA of TCK, and was also detected using broken teliospores and DNA from asymptomatic wheat samples. We developed the SYBR Green I and TaqMan Green I and TaqMan real-time polymorphism chain reaction methods to detect TCK with the detection limit of 0.1 fg with asymptomatic wheat samples. Further work is needed to develop a rapid test kit for this pathogenic fungus using the designed specific primers.     

Screening of wheat genotypes for the presence of common bunt resistance genes

Common bunt is known to cause grain yield and quality losses in wheat due to bunt ball formation and infestation of the grain. The aim of this study is to identify for sources of resistance to common bunt in wheat genotypes using phytopathological and molecular methods. In general, studied 60 Kazakh and foreign wheat genotypes were found 15 samples with the Bt9, Bt8 and Bt11 genes. Carriers of the Bt10 gene include the five varieties. The four resistance genes, Bt8, Bt10, Bt11, Bt9, and Bt10 were identified in the Karasai variety. Phytopathological and molecular screening of Kazakh and foreign wheat genotypes selected 18 with genes for resistance to the disease. According to evaluation on an artificial infection 19 varieties showed an immune type of reaction. These varieties will be used in breeding programs as donors to create resistant varieties against the common bunt. Thus, approaches can reduce the level of fungicides use and the most effective method to control the common bunt.     

Basal expression studies of cystatins during specific growth stages of wheat spikes for defining their possible role in differential and stage dependent immunity against Karnal bunt (<Emphasis Type="Italic">Tilletia indica</Emphasis>)

Two genotypes showing differential immunity against Karnal bunt (Tilletia indica) were used to investigate the role of three members of cystatin gene family in growth stage dependent immunity in wheat (Triticum aestivum L.). Three members of cystatin gene family (WC1, WC2, and WC4) were cloned and sequenced. Analysis of sequenced data showed that there was 76–99% nucleotide and protein sequence identity between different genes of the wheat cystatin. In silico amino acid sequence analysis revealed the presence of a conserved signature pattern of residues and also the functional domains were presumed to be actively involved in imparting cysteine protease inhibition capability. The semi-quantitative and quantitative levels of these members were measured by means of RT-PCR, northern blotting, western blotting, and by ELISA techniques. The members of cystatin gene family were expressed in both resistant (HD 29) and susceptible genotypes (WH 542); however, the expression level was significantly (P < 0.001) higher in resistant compared to susceptible genotype at all the stages of wheat spikes. The patterns of expression of WC2, WC4 were similar except in the levels in S₁ and S₂ stages as it remained constant (P > 0.05) in contrary to WC1 family whose expression gradually increased from S_v to S₂ stage. According to the intensity of the detected band in RT PCR, northern blot and western blot, WC1 family seems to be expressed more than the other gene families. The immunoassay results further showed that WC1 protein was abundantly expressed in resistant genotype and high expression was observed at the S2 stage as compared to susceptible genotype (P < 0.001) suggesting that low level of expression of WC1 in S2 stage is responsible for KB infection. The results of the present study clearly indicate the role of cystatin gene family in differential and stage dependent immunity against KB.