Fixation of Dormant Tilletia Teliospores for thin Sectioning

Dormant Tilletia caries teliospores in fixative solution or distilled water were frozen onto specimen chucks of an FTS Sorvall- Christensen frozen thin sectioner and cut or fractured at various temperatures (-20 C to -75 C) and thickness settings (10, 15, 20, and 25 μm). Cytoplasm of dormant spores was well preserved and organelles were found to differ from those of germinated spores in morphology. This procedure makes it possible to fix adequately dormant spores and thus compare the ultrastructurc and histochemistry of dormant spores with those of germinated spores.     

Fixation of Dormant Tilletia Teliospores for thin Sectioning

Dormant Tilletia caries teliospores in fixative solution or distilled water were frozen onto specimen chucks of an FTS Sorvall- Christensen frozen thin sectioner and cut or fractured at various temperatures (-20 C to -75 C) and thickness settings (10, 15, 20, and 25 μm). Cytoplasm of dormant spores was well preserved and organelles were found to differ from those of germinated spores in morphology. This procedure makes it possible to fix adequately dormant spores and thus compare the ultrastructurc and histochemistry of dormant spores with those of germinated spores.     

小麦网腥黑粉菌冬孢子总DNA提取方法比较

目的：研究提取不同时期小麦网腥黑粉菌冬孢子总DNA的最佳方法。方法：应用改良过的四种方法（CTAB法、SDS-CTAB法、SDS法和尿素法）对小麦网腥黑粉菌冬孢子总DNA进行提取。结果：提取当年小麦网腥黑粉菌冬孢子DNA,纯度（OD260/OD280）：CTAB法〉SDS-CTAB法〉SDS法〉尿素法=1.876〉1.7815〉1.7789〉1.6095;产率（μg/g）：SDS-CTAB法〉SDS法〉尿素法〉CTAB法=796.25〉664〉306〉291.5。提取15年的小麦网腥黑粉菌冬孢子DNA,纯度（OD260/OD280）：CTAB法〉SDS-CTAB法〉SDS法〉尿素法=1.91795〉1.8876〉1.65985〉1.55925;产率（μg/g）：尿素法〉CTAB法〉SDS法〉SDS-CTAB法=1529.25〉799〉687.25〉372.5。结论：SDS-CTAB法是提取当年小麦网腥黑粉菌冬孢子DNA的最佳方法。CTAB法是提取15年的小麦网腥黑粉菌冬孢子DNA的最佳方法。     

A Comparison of Polypeptides from Teliospores of Tilletia controversa (Kuhn) and Tilletia tritici (Bjerk) Wint.

Teliospores from 12 races of Tilletia tritici (Bjerk.) Wint. and twelve isolates of Tilletia controversa (Kuhn) were sampled from field-inoculated wheat (Triticum aestivum L.) differential cultivars. Proteins were extracted from the teliospores and analysed by one dimensional electrophoresis. An abundant 116 kD polypeptide detected in extracts from teliospores of all isolates of T. controversa was not detected in T. tritici teliospore extracts. Spores which were mechanically disrupted yielded greater quantities of the protein compared to intact teliospores, and this suggested the polypeptide was derived from within the teliospore. The presence of the polypeptide was correlated with dwarf bunt-causing Tilletia- Isolates of dwarf bunt-causing Tilletia that were intermediate between T. tritici and T. controversa in either morphology or germination characteristics contained the polypeptide while a common bunt-causing race of T. tritici (T18) with intermediate characteristics lacked the protein. The 116 kD polypeptide present in all T. controversa isolates may provide a stable biochemical marker for identification of these teliospores in wheat shipments.     

小麦印度腥黑穗病菌和黑麦草腥黑粉菌检测标准分子构建

以小麦印度腥黑穗病菌和黑麦草腥黑粉菌为研究对象,采用分子克隆技术,分别构建了该两种真菌分子检测的标准分子。前者包括线粒体2297 bp的DNA序列以及rDNA710 bp的ITS序列,后者包括线粒体2.3kb的DNA序列以及rDNA710 bp的ITS序列。分别对该两个标准分子进行了性能评估试验,测试结果显示所构建的标准分子具有良好的特异性、均匀性和稳定性,能够满足小麦印度腥黑穗病菌和黑麦草腥黑粉菌分子检测需求。     

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.     

Variability of Indian Isolates of Tilletia indica Assessed by Pathogenicity and Molecular Markers

Twenty isolates of Tilletia indica collected from sites in North and North‐western India showed pathogenic variation on 18 host differentials. Sixteen aggressive pathotypes were identified on the basis of percent coefficient of infection (PCI). Two major clusters were apparent in the dendrogram; cluster 1 comprised 13 isolates and cluster two consisted of seven isolates. One of the isolate Kashipur had a high PCI on most of the host differentials compared to other isolates. Polymerase chain reaction‐based random amplified polymorphic DNA (PCR – RAPD) analysis also divided isolates into two major clusters, one comprising of 5 isolates collected from hill and foot‐hill sites and another group comprising of 15 isolates collected from plain sites. Thus, the clusters identified based on PCI did not match closely with those identified by molecular analysis based on RAPD. Although diversity among the isolates of T. indica was absent in the rDNA‐ITS region, our study based on pathogenicity and molecular markers confirms the existence of great diversity in the pathogen, also shifting of ‘hot spot’ areas from one place to another within Karnal bunt prevailing areas.     

Immuno-pathotyping of Karnal bunt (Tilletia indica) isolates of wheat using anti-mycelial antibodies

Two types of polyclonal antibodies raised against whole lyophilized (LMA) and fractionated mycelial antigen (FMA) of most virulent, Pantnagar isolate of T. indica were used for the development of immunoassay systems, viz. dot immuno-binding assay (DIBA) and indirect enzyme linked immuno-sorbent assay (ELISA) procedures. The immuno-assays were developed by performing antigen concentration kinetics and antibody dilution curves analyses. These assays were employed for immuno-analysis of diversity amongst KB pathogen based on antibodies reactivity pattern and subsequently categorization into distinct sero-groups. The reactivity of two polyclonal antibodies was tested with 15 (P1-P15) isolates of T. indica. When anti-LMA antibodies were tested, four serologically distinct groups were formed based on percent reactivity (>75%, highly reactive; 60-75%; moderately reactive, <50-25%; low reactive and <25%, non-reactive). However, when anti-FMA antibodies were used, two distinct sero-groups were formed based on reactivity patterns (group I, highly reactive P1, P3, P4, P11 and P13, group II, less reactive P2, P4, P5, P6, P7, P8, P9, P10, P12, P14 and P15).     

Germination of Teliospores of Tilletia bardayana, the Causal Agent of Kernel Smut of Rice, in Relation to Some Physical Factors

Teliospores of Tilletia harclayana germinated equally well on the surface of 2 % water agar, soil extract agar and in cavities made in water agar. Maximum germination occurred at 29 °C under 12 h photoperiod with artificial daylight (cool fluorescent light). There was marked increase in germination when the teliospores in bunted grains were pre-treated for 30 min at 60 °C. Seventeen-week-old tehospores did not germinate while 73 week-old-tehospores showed retention of good germinability. Teliospores buried at 2 mm depth showed no germination whereas those at the moist surface germinated to produce sporidia. Implications of these results in the disease development are discussed.     

Development of Seed Immunoblot Binding Assay for Detection of Karnal bunt (Tilletia indica) of Wheat

Polyclonal antisera were produced in albino white rabbits against intact teliospores of Karnal bunt (Tilletia indica). The Immunoprobe generated was used for the development of Immunoblot binding assay for detecting Karnal bunt (KB) infections in wheat seed samples. The antiserum reacted strongly with intact teliospores of T. indica, Pantnagar isolate in agglutination reaction. Wheat grains with different grades of infection could be readily detected by Seed Immunoblot Binding Assay (SIBA). Karnal bunt infected wheat seeds when kept for vigour testing on nitrocellulose paper, formed a coloured imprint after the paper was immunoprocessed. The SIBA would not only be a better Indication of teliospores load on seed but also quality of seed in terms of vigour. This method is expected to be useful in routine monitoring of wheat lots for the presence of KB teliospores.     

Triacylglycerol Profiles of Tilletia controversa and Tilletia tritici

The triacylglycerol (TG) profiles of teliospores of Tilletia controversa and Tilletia tritici were examined by high-performance liquid chromatography (HPLC) and gas-liquid chromatography. Boiling isopropanol was used to ensure enzyme inactivation during homogenization. The largest lipid component as determined by thin-layer chromatography was TGs. On the basis of thin-layer chromatography of crude lipid extracts, T. controversa and T. tritici do not contain a large amount of free fatty acids. TG profiles of T. controversa and T. tritici were very similar, with 18 species of TGs resolved by HPLC and gas-liquid chromatography. In both organisms, PLL (palmitic, linoleic, linoleic) was the major component, followed by LLL (trilinolein) and PLO (palmitic, linoleic, oleic). The ratio of PLO to PLL was 1:6 and 1:4 in T. tritici and T. controversa, respectively. The TGs of both organisms contain long-chain (>22 carbons) mono- and dienoic acids. Linoleic acid was the major fatty acid found in TGs from both organisms. The differences of TGs were not considered significant for differentiation purposes.     

Modulation of antigenicity of mycelial antigens during developmental cycle of Karnal bunt (Tilletia indica) of wheat

Indirect enzyme linked immunosorbent assays (ELISA) were developed using polyclonal antibodies against soluble cytoplasmic (SCA) and insoluble cell wall antigens (ICWA) for monitoring modulation of mycelial antigens during growth cycle of T. indica. With SCA, continuous decrease in ELISA reactivity was observed in maturing fungus cultures, suggesting that SCA were expressed predominantly during early vegetative phase and their decreasing role was apparent as the fungus matures possibly towards sporogenous mycelium. In case of ICWA, the reaction profile showed an increase up to exponential phase of growth probably due to increase in the cell division and branching of mycelium. But later, ICWA antibody reactivity was decreased which may be due to conversion of mycelial phase to sporogenous phase, a quiescent stage of growth. Characterization of changes in antigenic configuration during developmental cycle of Tilletia indica by these antibodies could prove to be useful in identification of developmentally related and virulence marker(s).     

A comparison of inoculation techniques for assessment of germplasm susceptibility to Karnal bunt (Tilletia indica) disease of wheat

Three inoculation techniques for Karnal bunt (Tilletia indica) disease of wheat were compared: 1) boot inoculation - injection of inoculum with a hypodermic syringe into the boot; 2) spray inoculation - inoculum sprayed at growth stages between heading and anthesis, and 3) cotton wool inoculation - small pieces of cotton wool saturated in inoculum placed either inside the floret or between the spikelet and rachis. Each inoculation technique was assessed using susceptible cultivars to determine the optimum inoculum concentration, the ideal plant growth stage and the humidity requirements for successful infection.
Boot inoculation did not require high humidity and gave reliable infection with low secondary sporidia concentrations (1000–10 000/ml). The ideal plant growth stages for inoculation were early-boot and mid-boot. Spray inoculation required high secondary sporidia concentrations (50 000/ml) and 48 h of high humidity, but infection was initiated over a range of growth stages throughout heading and anthesis. Cotton wool inoculation gave low levels of infection at growth stages throughout heading and anthesis, even with high secondary sporidia concentrations (100 000/ml).     

Isolation of a species-specific mitochondrial DNA sequence for identification of Tilletia indica, the Karnal bunt of wheat fungus.

Mitochondrial DNA (mtDNA) from five isolates of Tilletia indica was isolated and digested with several restriction enzymes. A 2.3-kb EcoRI fragment was chosen, cloned, and shown to hybridize with total DNA restricted with EcoRI from T. indica and not from a morphologically similar smut fungus, Tilletia barclayana. The clone was partially sequenced, and primers were designed and tested under high-stringency conditions in PCR assays. The primer pair Ti1/Ti4 amplified a 2.3-kb fragment from total DNA of 17 T. indica isolates from India, Pakistan, and Mexico. DNA from 25 isolates of other smut fungi (T. barclayana, Tilletia foetida, Tilletia caries, Tilletia fusca, and Tilletia controversa) did not produce any bands, as detected by ethidium bromide-stained agarose gels and Southern hybridizations. The sensitivity of the assay was determined and increased by using a single nested primer in a second round of amplification, so that 1 pg of total mycelial DNA could be detected. The results indicated that the primers which originated from a cloned mtDNA sequence can be used to differentiate T. indica from other Tilletia species and have the potential to identify teliospores contaminating wheat seeds.     

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.     

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.     

Generation of anti-teliospores antibodies for immunolocalization and characterization of antigenic epitopes of teliospores of Karnal bunt (Tilletia indica) of wheat

Polyclonal antibodies raised against intact teliospores of T. indica in New Zealand albino rabbits were used for the development of indirect immunofluorescence tests. Specificity of anti-teliospore antibodies was evaluated by cross reactivity studies on other bunt, smut and related pathogens. The characteristic reactivity pattern indicated that the antibodies reacted with Tilletia species only. Chemical modifications, heat and enzyme treatments followed by indirect immunofluorescence tests were employed to delineate the molecular nature of the surface antigens. There was partial or no loss in immunoreactivity by methanol, periodate, heat or trypsin treatments. Extensive periodate treatment altered the fluorescence pattern due to changes in configuration of carbohydrate antigen present in episporium. Sequential treatment of periodate and trypsin showed diminished fluorescence due to access of proteolytic enzyme into inner site of episporium thereby cleaving peptide epitope(s) after reorientation of carbohydrate moietiesby periodate treatment. It indicated glycoprotein nature or peptide nature of epitopes on the teliospore surface.