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.     

Development of blunt Tilletia caries (D.C.) Tul. pathogen on embryogenic wheat callus

The development of Tilletia caries on embryogenic and morphogenic wheat calluses from germination of spores up to sorus formation have been investigated. Pathogene growth was similar in variants with wheat calluses of susceptible species Triticum aestivum and resistant species T. timopheevii, but intensity of its development of embryogenic calluses of both species was lower, than on morphogenic. Necrosis of parenchyma-like cells was more distinct in the embryogenic than in morphogenic calluses. It was combined with low accumulation rate of infected cells in embryogenic calluses. Necrosis reduced the healthy cell reproduction in meristem-like cells of morphogenic calluses and thereby additionally decreased resistance of morphogenic calluses to pathogene.     

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.     

Die Steuerung der Brandsporenkeimung und Sporidienbildung bei Tilletia caries (DC) Tul. durch Licht

Zusammenfassung Durch Licht wird bei Tilletia caries (DC) Tul. nicht nur die Keimungsgeschwindigkeit, sondern auch der Endwert der Keimung erhöht.Es konnte gezeigt werden, daß sowohl die Promycel- als auch die Sporidienbildung durch Licht gesteuert wird.Während es sich bei der Beeinflussung der Promycelbildung um eine induktive Lichtwirkung handelt, muß für eine maximale Förderung der Sporidienbildung während des entsprechenden Entwicklungsabschnittes Dauerlicht hoher Intensität geboten werden.Licht ist erst 3 Tage nach Sporenaussaat wirksam. Die Beeinflussung der Promycelbildung ist am stärksten, wenn das Licht zwischen dem 3. und 6. Tag geboten wird, während der Prozentsatz der Sporidienbildung um so größer ist, je später der Zeitpunkt der Belichtung liegt.Wie bei nahezu allen Photomorphosen bei Pilzen besitzt Blaulicht die stärkste Wirksamkeit. Grün ist zwar ebenfalls noch eindeutig wirksam, während zwischen dem Rotwert und der Dunkelkontrolle kein signifikanter Unterschied besteht.

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The role of light in spore germination and sporidial production of the wheat smut fungi Tilletia caries (DC.) Tul.

Summary In Tilletia caries, light hastens the beginning of spore germination and increases the maximum percentage of spores which germinate.It could be shown, that light is required for two different steps—the formation of germ tubes and the sporidial development.The stimulation of the spore germination is a photoinductive process; but the best sporidial development occurs in continuous light of high intensity.There is a sensitive period in the response of spores to light. High germination is induced when the light exposure occurs between the 3rd and 6th day after the spores were sowed on the medium. Best sporidia production results when the spores were irradiated at the 5th day or later.As in nearly all photoresponses in fungi, blue light is more effective than green. No significant difference exists between the red light and dark experiments.

     

Die synergistische Wirkung von Licht und Ionen auf den Keimungsverlauf beim Weizensteinbrand (Tilletia caries (DC) Tul.)

Summary In the wheat bunt fungi (Tilletia caries), the rate of teliospore germination is not only increased by light, but also by Ca(NO₃)₂ and KNO₃ when present in the medium (Fig. 1). CaCl₂ and KCl have no stimulative effect (Fig. 2). Therefore we assume, that NO ₃ ^- is the effective ion. The indifference of NH₄NO₃ in the medium may be explained by an antagonism of NH ₄ ⁺ and NO ₃ ^- .Under saturated light conditions, there is no increase in the germination rate after addition of Ca(NO₃)₂ or KNO₃ (Fig. 3). This indicates, that the effectiveness of the two external factors light and NO ₃ ^- is not independent.The growth of germ tubes is also stimulated with Ca(NO₃)₂ and KNO₃, but there is no influence on the sporidial development (Fig. 4).     

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.     

The detection of nonallelic to known genes of resistance to Tilletia caries (DC) Tul. in wheat strains from interspecific hybridization (Triticum aestivum x Aegilops cylindrica)

It was established by hybridological analysis that winter bread wheat lines 1/74-91, 3/36-91, 5/55-91 possess single dominant gene of resistance to bunt (Tilletia caries (DC) Tul.), but lines 8/2-91, 5/43-91, 4/11-91 and 8/16-91 have two independent dominant genes for this character. These genes originated from Aegilops cylindrica are not identical to Bt1-Bt17 genes and are unknown to date. The lines were obtained from crosses between winter bread wheat variety Odeskaya polukarlikovaya and Aegilops cylindrica.     

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.     

An ISSR‐based Approach for the Molecular Detection and Diagnosis of Dwarf Bunt of Wheat,Caused by Tilletia controversa Kühn

Dwarf bunt of wheat, caused by Tilletia controversa Kühn, is an important international quarantine disease in many countries. The objective of this investigation was to develop a diagnostic molecular marker generated from intersimple sequence repeat (ISSR) for rapid identification of T. controversa. A total of 60 primers were tested by ISSR to detect DNA polymorphisms between T. controversa and related species. The primer ISSR818 generated a polymorphic pattern displaying a 952‐ bp DNA fragment specific for T. controversa. The marker was converted into a sequence characterized amplified region (SCAR), and specific primers (TCKSF2/TCKSR2) were designed for use in a PCR detection assay. Its detection limit was 1 ng of DNA, which could be yielded by 1.1 μg of teliospores in a 25‐ μl PCR. Conclusively, a method to distinguish T. controversa from similar pathogenic fungi has been successfully developed based on the use of a SCAR marker.     

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.     

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.     

Peroxidase Activity in Leaves of Wheat Cultivars Differing in Resistance to Erysiphe graminis DC.

The peroxidase activities in leaves from resistant and susceptible cultivars of wheat infected and non-infected by Erysiphe graminis DC were studied. In non-infected wheat, soluble and ionic bound peroxidase activity level was found to be higher in the resistant cultivar than that in the one susceptible to Erysiphe graminis DC. After infecting wheat leaves with Erysiphe graminis DC a remarkable increase in the activity of soluble and ionic bound peroxidases was detected 5 days after inoculation only in the resistant cultivar. In the susceptible cultivar a high increase in the activity of the soluble and ionic bound peroxidases occurred only 15 days after inoculation. Using ion exchange chromatography four peroxidase fractions were obtained from infected susceptible and resistant cultivars as from non-infected ones. The fraction II in non-inoculated resistant cultivars was much higher than that in the susceptible one. This fraction increased after inoculation in both cases reaching a higher level in resistant cultivars. Fraction I was higher in the susceptible cultivar. Electrofocusing profiles of peroxidase from the susceptible and resistant cultivar differed from one another. New peroxidase bands after inoculation appeared only in the resistant cultivar.     

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.     

茉莉酸对小麦幼苗UV-B抗性的生理学效应研究

以小麦品种‘西农88’(Triticum aestivum L.,cv.Xinong 88)为材料,研究了外源施加不同浓度茉莉酸(1、2.5、5、10 mmol/L)对UV-B辐射(1.5 kJ·m-2·d-1)下小麦幼苗光合色素、抗氧化酶、丙二醛、游离脯氨酸、紫外吸收物、花青素、根系活力等生理指标以及对其生长的影响,探讨了茉莉酸在UV-B辐射胁迫中的可能作用及其作用机制.研究结果表明,外源茉莉酸对小麦幼苗生理指标产生显著影响,并且表现出浓度效应,其中较低浓度的茉莉酸(1 mmol/L和2.5 mmol/L)能明显提高小麦幼苗的UV-B抗性.表现为低浓度茉莉酸显著提高UV-B辐射下小麦幼苗叶片中的总叶绿素含量、过氧化氢酶(CAT)、过氧化物酶(POD)和超氧化物歧化酶(SOD)活性.并且外源施加的茉莉酸还能够增加小麦幼苗的游离脯氨酸含量,降低脂质过氧化水平,提高花青素含量,增强根系活力.可见,茉莉酸通过提高小麦幼苗的抗氧化酶活性,增加渗透调节物含量以及保护性色素含量,从而缓解膜脂过氧化程度和提高防御物质含量,进而增强植物抵抗UV-B辐射胁迫的能力,保证小麦幼苗正常生长.     

肠道菌对苏云金芽胞杆菌杀虫活性的研究

苏云金芽胞杆菌(Bacillus thuringiensis,Bt)在生长发育过程中伴随芽胞的形成高效表达对昆虫具有特异毒性的杀虫晶体蛋白,从而被广泛应用于害虫防治上。有关Bt的杀虫机制,近年来有学者提出了肠道菌模型,认为肠道菌在Bt发挥杀虫活性中是必须的,也有人提出相反的观点。以棉铃虫作为供试昆虫,利用Cry1Ac10晶体蛋白研究了棉铃虫肠道菌在Bt杀虫过程中所发挥的功能。结果发现,在棉铃虫中肠道菌并非Bt杀虫所必需,并且在肠道菌存在的情况下,Bt杀虫活性反而明显降低,通过肠道菌回接试验发现5号肠道菌对棉铃虫的保护作用最为明显。