Activity of trypsin inhibitors in wheat seedlings exposed to pathogenic fungus Tilletia caries and phytohormones

We compared the effect of common bunt (caused by fungus Tilletia caries Tul.) and treatment with phytohormones IAA, ABA, and cytokinins (CK) on the activity of trypsin inhibitors (TI) in wheat seedlings. The experiments were conducted with pathogen-susceptible species of wheat Triticum aestivum L. (cv. Zhnitsa) and resistant species T. timopheevii Zhuk. (accession k-58666 from the collection of All-Russian Institute of Plant Industry). In the resistant wheat, the fungus elevated the activity of TI and the content of CK, whereas in the susceptible wheat, it induced accumulation of IAA. In the seedlings of wheat T. aestivum, TI activity increased under the effect of CK, same as upon the action of pathogen. ABA briefly increased the activity of TI, whereas IAA did not considerably affect it. It was concluded that among the investigated hormones, CK play a leading role in the regulation of defense responses of wheat plants involving TI.     

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.     

Sources of Resistance to Common Bunt (Tilletia foetida and T. caries) in Durum Wheat

Screening of durum wheat germplasm for resistance to common bunt (Tilletia foetida and T. caries) resulted in the identification of 26 resistant genotypes. The screening was made using nine common bunt isolates from the West Asia and North Africa (WANA) region. In one isolate the two pathogens were represented in ratio of 1:1, whereas eight isolates contained only T. foetida. The correlation, principal components and clustering analyses grouped the genotypes into three clusters., Cluster one comparised genotypes close to Senatore (S.) Cappelli and Haurani, the latter is a landrace from Syria. Cluster three comprised advanced genotypes containing resistance genes from Mindum, a Turkish landrace. Results indicated that donor sources of resistance appear to be related to the three major sources mentioned. Cultivar S. Cappelli is considered resistant since it has been grown by farmers on a large scale for many years and remained resistant to common bunt throughout 7 years of testing. This resistance is assumed to be of a durable type. The isolates were also grouped into three clusters representing different ecological areas and the wheat types from which the isolates originated. We infer that the different clusters reflect the presence of three pathotype groups of the pathogens.     

Markers to a common bunt resistance gene derived from ‘Blizzard’ wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) and mapped to chromosome arm 1BS

Common bunt, caused by Tilletia caries (DC.) Tul. &; C. Tul. and T. laevis J.G Kuhn, is an economically important disease of wheat (Triticum aestivum L.) worldwide. The resistance in the winter wheat cultivar ‘Blizzard’ is effective against known races of common bunt in western Canada. The incorporation of resistance from Blizzard into field-ready cultivars may be accelerated through the use of molecular markers. Using the maize pollen method, a doubled haploid population of 147 lines was developed from the F₁ of the second backcross of Blizzard (resistant) by breeding line ‘8405-JC3C’ (susceptible). Doubled haploid lines were inoculated at seeding with race T19 or T19 and L16 and disease reaction was examined under controlled conditions in 1999 and natural conditions in 2002, and 2003. Resistant:susceptible-doubled haploid lines segregated in a 1:1 ratio for bunt reaction, indicating single major gene segregation. Microsatellite primers polymorphic on the parents were screened on the population. Initial qualitative segregation analysis indicated that the wheat microsatellite markers Xgwm374, Xbarc128 and Xgwm264, located on wheat chromosome 1BS, were significantly linked to the resistance locus. Qualitative results were confirmed with quantitative trait locus analysis. The genetic distance, calculated with JoinMap^®, between the bunt resistance locus and overlapping markers Xgwm374, Xgwm264 and Xbarc128 was 3.9 cM. The three markers were validated on doubled haploid populations BW337/P9502&;DAF1BB and Blizzard/P9514-AR17A3E evaluated for common bunt reaction in the growth chamber in 2007. These markers will be useful in selecting for the common bunt resistance from Blizzard and assist in identifying the resistance among potential new sources of resistance.     

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.     

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.     

Changes in the Levels of IAA,ABA, and Cytokinins in Wheat Seedlings Infected with Tilletia caries

The seedling growth and the content of endogenous phytohormones in wheat seedlings were estimated 3, 6, and 9 days after infection with the bunt pathogen (Tilletia caries) (DC.)TUL. The infection of a pathogen-susceptible species Triticum aestivum L. and a resistant species T. timopheevii Zhuk. resulted, respectively, in an increase and a decrease in the seedling growth and the IAA content as compared to the control. The cytokinin content increased in both species, and the increase in T. timopheevii was more rapid. The pathogen-induced increase in auxin content is suggested to enhance fungal invasion of plants. In the susceptible species, a high ABA concentration was retained for a longer period of time and could act as a factor of virulence. At the same time, in the resistant species, an increase in ABA content was transient and seems to trigger plant defense mechanisms.     

The Effect of Salicylic Acid on Peroxidase Activity in Wheat Calli Cocultured with the Bunt Pathogen Tilletia caries

The effect of salicylic acid (SA) on peroxidase activity in wheat (Triticum aestivum L.) calli cocultured with the bunt pathogen Tilletia caries was studied. Fungal infection was shown to activate cytoplasmic peroxidase. SA suppressed total peroxidase activity but did not inhibit the peroxidase with pI 9.8. A novel chitin-specific peroxidase with pI 3.5 appeared after the SA treatment. The infection of SA-treated cells with Tilletia caries activated the isoenzymes with pI 3.5, 4.8, and 7.5 and stimulated their secretion into the culture medium. The ability of SA to control wheat peroxidase activity during pathogenesis is discussed. The important role of this control in plant defense responses to the bunt pathogen is emphasized.     

Salicylic acid increases the defense reaction against bunt and smut pathogens in wheat calli

The role of salicylic acid (SA) in regulating wheat calli growth and peroxidase activity in the co-culture with bunt (Tilletia caries (D.C.) Tul.) and smut (Ustilago tritici Pers.) pathogens was studied in this work. We found that the influence of SA increased the number of globular high-density sites with meristema-like cells and reduced the number of low-structured sites with parenchyma-like cells in wheat calli. SA inhibited fungi spores germination, mycelium growth, and formation of its new spores during the co-cultivation of wheat calli with bunt or smut agent. Interaction of fungi mycelium with wheat calli treated by SA led to formation of zones with hypersensitive reaction in calli high-structured sites with meristema-like cells. The obtained data about the influence of SA on activation of wheat calli growth and the peroxidases with pI ~3.5 and ~9.8, which can bind to the mycelium of T. caries or U. tritici are discussed in this paper.     

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.     

Detection of Tilletia caries, Causal Agent of Common Bunt of Wheat, by ELISA and PCR

The paper reports about the development and evaluation of two methods, a PCR‐based assay and an enzyme‐linked immunosorbent assay (ELISA), for the detection of the common bunt fungus Tilletia caries (syn. T. tritici) in young wheat plants. Using the published primer pair Tcar2A/Tcar2B for polymerase chain reaction (PCR) with DNA from axenic cultures of T. caries or from T. caries‐infected plants, we obtained a single band after electrophoresis of the amplification products. By PCR the bunt pathogen could be detected in shoots (EC 12) as well as in leaves (EC 13–14) of infected plants. Immunological detection was performed using a double antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) with biotinylated detection antibodies. The antibodies were obtained after injection of mycelial homogenates of axenic cultures of T. caries into rabbits. The detection limit was 16 pg DNA per 100 mg plant fresh weight for the PCR and 7 ng/ml fungal protein for the ELISA, respectively. Except for the closely related T. controversa, no cross‐reactions with other fungi were observed with both methods. While it was possible to detect teliospores of T. caries by PCR, the ELISA did not react with spore extracts. Analysis by ELISA of shoots of individual plants grown from inoculated seeds revealed that at EC 10 all plants were infected. There was, however, a large variability in the amount of T. caries present in the plants. This observation and reports in the literature indicate quantitative differences in the degree of colonization of the tissue between individual plants even in a given variety. Regarding the use of modern diagnostics to assist in the development of resistant varieties we therefore suggest that for the wheat –T. caries pathosystem the non‐quantitative PCR‐assay employed here is less suited than the ELISA that allows precise quantification of the amount of fungal antigen present in the plant. However, to routinely employ the ELISA in resistance breeding further development work is needed.     

Development and validation of microsatellite markers for Karnal bunt (Tilletia indica) and loose smut (Ustilago segetum tritici) of wheat from related fungal species

Simple sequence repeats (SSRs) are preferred molecular markers because of their abundance, robustness, high reproducibility, high efficiency in detecting variation and suitability for high‐throughput analysis. In this study, an attempt was made to mine and analyse the SSRs from the genomes of two seed‐borne fungal pathogens, viz Ustilago maydis, which causes common smut of maize, and Tilletia horrida, the cause of rice kernel smut. After elimination of redundant sequences, 2,703 SSR loci of U. maydis were identified. Of the remaining SSRS, 44.5% accounted for di‐nucleotide repeats followed by 29.8% and 2.7% tri‐ and tetranucleotide repeats, respectively. Similarly, 2,638 SSR loci were identified in T. horrida, of which 20.2% were di‐nucleotide, 50.4% tri‐ and 20.5% tetra‐nucleotide repeats. A set of 65 SSRs designed from each fungus were validated, which yielded 23 polymorphic SSRs from Ustilago and 21 from Tilletia. These polymorphic SSR loci were also successfully cross‐amplified with the Ustilago segetum tritici and Tilletia indica. Principal coordinate analysis of SSR data clustered isolates according to their respective species. These newly developed and validated microsatellite markers may have immediate applications for detection of genetic variability and in population studies of bunt and smut of wheat and other related host plants. Moreover, this is first comprehensive report on molecular markers suitable for variability studies in wheat seed‐borne pathogens.     

Leaf structure and histochemistry of Ecballium elaterium (L.) A. Rich. (squirting cucumber)

Light, scanning and transmission electron microscopy as well as histochemical reactions were employed to study the leaf structure and secretory activity of Ecballium elaterium, a hairy pharmaceutical perennial common in the Mediterranean region. The amphistomatic leaf has a peculiar structure due to special cells supporting the conductive bundles, a remarkable shortage of mechanical tissue, and the existence of pectin strands between mesophyll cells. The secreting activity is limited mostly to secretary hairs. These attributes of the Ecballium leaf fine structures do not resemble the common structure of leaves from Mediterranean plants and point to a peculiar strategy of this species coping with stress conditions of its habitat.     

Identification of a specific scar marker for detection of Tilletia foetida (Wall) Liro pathogen of wheat

Common bunt is one of the most important destructive diseases of wheat worldwide and is a domestic quarantined disease in China. However, a rapid and efficient method to identify the corresponding pathogens is currently limited. The objective of the present study was to develop a diagnostic molecular marker specific towards Tilletia foetida (Wall) Liro, a causal agent of the bunt disease. One specific DNA fragment for T. foetida (286 bp in length) was amplified using an Amplified Fragment Length Polymorphism (AFLP) assay and, this fragment was cloned and sequenced. One pair of specific primers (SC(286-1)/SC(286-2)), which was designed according to the sequence, could specifically amplify the corresponding fragment in all of the T. foetida isolates employed from both the People's Republic of China and United States, whereas this fragment could not be amplified by the other fungal species tested. Therefore, a specific Sequence Characterized Amplified Region (SCAR) marker was developed. This SCAR marker could distinguish T. foetida from related pathogenic fungi efficiently and could be used for the early diagnosis of the common bunt of wheat in the field, and provide an efficient way for disease surveillance and disease forecasting in cereal crop.     

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results.     

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.     

The Dimension of Trichomonas vaginalis as Measured by Scanning Electron Microscopy

It is known that physicochemical conditions (e.g., pH, temperature, and ionic strength) affect the size of trichomonads. In this study, the sizes of 4 isolates of Trichomonas vaginalis cultured for more than a year (called "old T") and 3 isolates freshly isolated from vaginitis cases (called "fresh T") were compared by scanning electron microscopy. Although the fresh T had shorter body length, body width, and flagellar length than old T, total length (about 26 µm), including body length, flagella length, and axostyle length was almost the same in the 2 groups. A striking difference was observed between the axostyles of the 2 groups; the axostyle length of the fresh T (8.2 µm) was more than twice as long as that of the old T (4.0 µm). However, in several parasitology textbooks, the length of T. vaginalis is said to vary widely from 7 to 32 µm, and its undulating membrane is said to extend about half way (53.5%) to the posterior end of the body. On the other hand, in our study, the undulating membrane was observed to extend more than 3/4 of the body length (72.1%) in old T, whereas in fresh T it could not be measured. Taken together, we suggest that T. vaginalis averages 26 (21-32) µm in total length, with 9.5 (7.4-11.4) µm of body length and 6.8 (5.3-7.7) µm of width, and its undulating membrane extending 3/4 of its body length. Therefore, these findings may provide useful information for morphological characteristics of T. vaginalis.     

Changes in the Phytohormone Levels in Wheat Calli as Affected by Salicylic Acid and Infection with Tilletia caries,a Bunt Pathogenic Agent

The role of salicylic acid (SA) in growth regulation and the change in the levels of phytohormones (IAA, ABA, and cytokinins) were studied in the wheat calli co-cultured with bunt pathogen Tilletia caries. Calli infection with T. caries resulted in the hypertrophied callus growth and simultaneous increase in phytohormone level. The addition of SA to the nutrient media decreased the callus growth induced by the pathogen, whereas the level of investigated phytohormones was not affected. In the SA-treated infected calli, the formation of necrotic lesions was observed in the zones of contact of the fungal mycelium with callus cells that limited pathogen growth. The authors suggest that the stabilization of the hormonal balance of plant cells at pathogenesis is one of the possible mechanisms of the SA protective action in vitro and in vivo. Hence, co-culturing wheat calli and T. caries fungus appeared to be a convenient model for assessing SA protective action.