Causal Agent Of Karnal Bunt Research Articles

Pathogenic variability among Tilletia indica isolates and distribution of heterothallic alleles in the Northwestern Plains Zone of India

Tilletia indica Mitra, the causal agent of Karnal bunt of wheat (KB), is a quarantined disease of international importance whose pathogen shows high variability due to its heterothallic nature. Pathogenic variation was studied under artificially inoculated conditions on 13 wheat genotypes including bread and dicoccum wheat. This study used 34 T. indica isolates collected from 6 major wheat growing states in India (Rajasthan, Haryana, Punjab, Himachal Pradesh, Uttarakhand, and Uttar Pradesh). Mean coefficient of infection of the isolates ranged from 2.11% (KBR4) to 14.60% (KBH1). Among the 13 genotypes screened, only 1 genotype was found to be highly susceptible to infection. KBR1 and KBHP3 were the most virulent and least aggressive isolates and had coefficients of infection of 79.42% and 6.22%, respectively, on the susceptible variety HD 2009. Pathogenic variability test revealed the existence of 3 different aggressive groups among the 34 selected isolates on a set of host differentials. A total of 64 monosporidial (Ms) lines were developed from the isolates, which originated from 6 states, representing 19 locations in the North Western plains zone of India. Self-paired Ms lines revealed 2 mating alleles. A total of 15 mating alleles were postulated using 46 Ms lines. This study confirmed the heterothallic nature of T. indica and the existence of multiple alleles controlling pathogenicity.

In vitro evaluation of fungitoxicants and Phyto-extracts against Neovossia indica (Mitra) Mund. the causal agent of Karnal bunt of wheat

An effective management practice was developed; thirteen fungitoxicants and six phyto-extracts were evaluated under laboratory condition. N. indica was isolated from infected wheat grains. Among the systemic fungicides, Tilt 25 EC at 200 ppm was adjudged best to check mycelia growth and teliospore germination of fungus. In case of non-systemic fungicides, Dithane M- 45 was found highly effective at 2000 ppm. Among the phyto-extracts tested in laboratory, Lantana camara at 75 per cent concentration was found best for growth and teliospore germination inhibition of N. indica.

Pathogenic and genetic variability in Tilletia indica monosporidial culture lines using universal rice primer-PCR

Tilletia indica Mitra is the causal agent of Karnal bunt of wheat, an important disease prevalent in several countries. The disease is internationally quarantined and the pathogen due to its heterothallic nature shows high variability. In the present study, we compared the pathogenic behaviour of various isolates of T. indica collected from different geographical locations of India and genetically characterized monosporidial (Ms) culture lines raised from these isolates of the pathogen. Pathogenic variability revealed existence of three pathotypes based on aggressiveness on a set of differential host genotypes. Monosporidial culture lines viz., 5 each from KB1, KB2, KB4 and KB5 and three lines of KB3 were established and analyzed genetically using 12 Universal Rice Primers (URPs). Amplification showed 98.44% polymorphism and primer URP 13R produced 100% polymorphic bands. Maximum similarity (83%) was between KB1MsB and KB1MsD as calculated by Jaccard’s similarity coefficient, whereas, minimum similarity was between KB1MsC and KB4MsB; KB1MsE and KB3MsA (46%). Three groups were formed among all Ms culture lines. One major group consisted of 13 lines with approximately 70% similarity, the second group consisted of 7 culture lines showing 55% similarity and the third group consisted of 3 Ms lines. URPs were able to differentiate the Ms culture lines raised from different T. indica isolates and the results indicated heterogeneity in the pathogen population.

Determination of Genetic Divergence Based on DNA Markers Amongst Monosporidial Strains Derived from Fungal Isolates of Karnal Bunt of Wheat

Genetic variation among the base isolates and monosporidial strains derived from these isolates of Tilletia indica- the causal agent of Karnal bunt (KB) in wheat, was analyzed by morphological, growth behaviors and RAPD-ISSR based molecular polymorphism. Genetic make up of fungal cultures vary among each other. The magnitude of variation in KBPN group is less (narrow genetic base) when compared to the other groups KB3, KB9 and JK (broad genetic base) reflecting that variability is a genetically governed process. The generation of new variation with different growth characteristics is not a generalized feature and is totally dependant on the original genetic make-up of the base isolate generating new monosporidial strains. Thus, it can be concluded that monosporidial strains derived from mono-teliosporic isolate, consists of genetically heterogeneous population. The morphological and genetic variability further suggests that the variation in T. indica strains is predominantly derived through the genetic rearrangements through para sexual means.

Characterisation of potential antigen(s) of Tilletia indica teliospore walls to develop a specific immunoassay for Karnal bunt detection

Characterisation of potential antigen(s) in the teliospore wall of Tilletia indica, the causal agent of Karnal bunt (KB) of wheat (Triticum aestivum) is necessary to generate specific immunoprobes for the development of reliable immunodiagnostic procedures. The intact teliospores of KB were used to generate anti-teliospore antibodies for the development of microtitre ELISA. The anti-teliospore antibodies cross-reacted with Tilletia foetida, Puccinia recondita and Puccinia striiformis suggesting that these species share one or more antigenic components. These antibodies recognised two unique immunoreactive bands of molecular weight ~34 kDa and ~15 kDa present in the spore wall fraction of T. indica after western blotting. The periodate treatment alone and sequential treatment of periodate followed by trypsin lost the immunoreactivity partially and completely, respectively. This suggests that cross-reactivity among bunt and rust of wheat fungal pathogens is most likely due to peptidal/oligosaccharide epitopes overlapping the spore wall's glycoprotein.

Survival of Tilletia indica teliospores under European soil conditions

As part of developing a European Pest Risk Analysis (PRA) for Tilletia indica, the causal agent of Karnal bunt of wheat, teliospore survival studies were done outside under quarantine containment at three European sites (Norway, UK, Italy). At each site, experiments were set up in three consecutive years (Experiments 1, 2 & 3) to determine teliospore survival over time (1–3 years) at 5, 10 and 20 cm depths. Experiments were sampled annually and survival assessed in relation to teliospore recovery and to germination at recovery (T0) and 3 months after recovery in case of burial‐induced dormancy (T3). Teliospores survived at all three sites at all depths over all the time periods studied. At each site, there was no evidence of a marked decline in teliospore recovery between sampling years, except in one set of years in one Norwegian experiment. There was no consistent effect of depth on recovery. In general there was little evidence for a marked decline in teliospore germination between sampling years. There was some evidence of a decrease in germination with increasing depth in the UK, and for some time‐depth interactions. After 3 years’ incubation (Experiment 1), mean teliospore recovery and mean germination were: UK: 61% recovery and 31% (33%) germination for T0 (and T3); Italy: 30% recovery and 36% (29%) germination; and Norway: 12% recovery and 19% (49%) germination. Germination for laboratory controls ranged from 20–59% (UK), 18–41% (Italy) and 28–59% (Norway). There was no evidence for burial‐induced dormancy except in Norway. Teliospores of T. indica can survive for at least three years in European soils. This prolonged period of survival could support establishment of the pathogen if it were introduced into areas of European cereal production.

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.

Use of the fluorchrome vital dye acridine orange to determine viability and germination of Tilletia indica teliospores in soil

A vital staining method using the fluorochrome acridine orange (AO) was developed for staining teliospores and primary sporidia (basidiospores) of Tilletia indica the causal agent of Karnal bunt of wheat. This procedure allows determination of the viability of teliospores in soil. Also the method makes it possible to monitor germination of teliospores in different soil types under different soil moisture levels. The primary sporidia produced from germinated teliospores could be easily detected in small soil samples (10 g). This is the first report on evaluation of germination of viable teliospores directly in different soil types. This method makes it possible qualitatively to determine the germination of teliospores in nonsterile soil taken directly from the field. The AO procedure, as a supplement to the quarantine inspection of seed lots, could be used for routine testing of the viability of teliospores of T. indica extracted from seed lots.

Improved Detection of Tilletia indica Teliospores in Seed or Soil by Elimination of Contaminating Microorganisms with Acidic Electrolyzed Water.

Acidic electrolyzed water (AEW) is a germicidal product of electrolysis of a dilute solution (e.g., 0.4% vol/vol) of sodium chloride. This solution can be used to disinfest wheat seed or soil samples being tested for teliospores of Tilletia indica, causal agent of Karnal bunt, without risk of damaging the teliospores. The AEW used in this study had a pH of 2.5 to 2.8 and oxidation-reduction potential of approximately 1,130 mV. In simulations of routine extractions of wheat seed to detect teliospores of T. indica, the effectiveness of a 30-min AEW treatment was compared with a 2-min 0.4% sodium hypochlorite (NaOCl) treatment to eradicate bacteria and nonsmut fungi. Each treatment reduced bacterial and fungal populations in wheat seed extracts by 6 to 7 log10 units when determined on 2% water agar with antibiotics. Reductions of 5 log10 units or more were observed on other media. NaOCl and AEW also were very effective at eliminating bacteria and fungi from soil extracts. In studies to detect and quantitate T. indica teliospores in soil, AEW was nearly 100% effective at eliminating all nonsmut organisms. Free chlorine levels in AEW were very low, suggesting that compounds other than those with chlorine play a significant role in sanitation by AEW. The low pH of AEW was shown to contribute substantially to the effectiveness of AEW to reduce microorganisms. A standardized protocol is described for a 30-min AEW treatment of wheat seed washes or soil extracts to eliminate contaminating microorganisms. A significant advantage of the use of AEW over NaOCl is that, with AEW, teliospore germination is not reduced and usually is stimulated, whereas teliospore germination declines after contact with NaOCl. The protocol facilitates detection and enumeration of viable teliospores of T. indica in wheat seed or soil and the isolation of pure cultures for identification by polymerase chain reaction. The germicidal effects of AEW, as demonstrated in this study, illustrate the potential of AEW as an alternative to presently used seed disinfestants.

Reactions of western Canadian spring wheat and triticale varieties to Tilletia indica, the causal agent of Karnal bunt

The reactions of 47 Canadian wheat and triticale cultivars to Tilletia indica Mitra, the causal agent of Karnal Bunt (KB), were evaluated in the field at Centro de Investigaciones Agricolas del Noroeste (CIANO), Yaqui Valley, Mexico, during 1998 and 1999. The majority of lines and cultivars flowered during January and February, which coincided with the onset of cooler temperatures and high relative humidity required for optimum infection by the fungus. Canada Western Amber Durum (CWAD) and triticale were resistant, ranging from 0 to 6% infection. Canada Western Red Spring (CWRS) wheats were also generally resistant, but the cultivars Roblin, BW90 and Laura, and the experimental line Roblin*2/BW 553, were susceptible, ranging from 11 to 28% in treatments with the highest infection levels. Canada Western Extra Strong (CWES) wheats varied in reaction from moderately resistant to susceptible, ranging from 1 to 15% infection. Canada Prairie Spring (CPS) wheats were generally susceptible, exhibiting infection levels ranging from 5 to 31% infection in the most severely infected treatments. Canada Western Soft White Spring (CWSWS) wheats were uniformly susceptible; the highest recorded infection level among Canadian wheats was observed on AC Reed (38%) during 1999. The susceptibility of CPS and CWSWS cultivars may represent an increased risk to the establishment of KB if the fungus were to become introduced into western Canada. Key words: Karnal bunt, wheat, disease resistance, disease susceptibility

Development of Immunochemical Techniques for Detecting Karnal Bunt in Wheat

The protein profile of Tilletia indica, the causal agent of Karnal bunt, was analyzed by SDS-PAGE and compared to protein profiles of T. barclayana, T. tritici, T. controversa and T. laevis. Based on these results, antibodies were developed against a 64 kDa T. indica protein and used to construct both a microwell sandwich ELISA and a dipstick immunoassay. Both of these immunoassays can detect 1.25 ng/well of purified T. indica protein or 40 ng/well of crude spore extract, distinguishing Karnal bunt from all wheat smuts and, to some degree, the rice smut, T. barclayana. To enhance specificity, an innovative approach to species-specific antibody production was utilized to develop antibodies that discern Karnal bunt from all other fungal species. Using these new antibodies, a Karnal bunt specific immunoassay was developed that has all the attributes of the original Karnal bunt immunoassays and detects Karnal bunt in the presence of wheat. This is the first report of immunochemical diagnostic tools that will aid in controlling the spread of this wheat pathogen by accurately and efficiently detecting Karnal bunt teliospores in wheat.

Comparison of Effects of Acidic Electrolyzed Water and NaOCl on Tilletia indica Teliospore Germination.

Definitive identification of free teliospores of Tilletia indica, causal agent of Karnal bunt of wheat, requires polymerase chain reaction (PCR)-based diagnostic tests. Since direct PCR amplification from teliospores has not been reliable, teliospores first must be germinated in order to obtain adequate DNA. We have routinely surface-sterilized teliospores for 2 min with 0.4% (vol/vol) sodium hypochlorite (NaOCl) to stimulate germination and produce axenic cultures. However, we observed that some spores were killed even with a 2-min NaOCl treatment, the shortest feasible duration. Decreasing the NaOCl concentration in our study from 0.4% to 0.3 and 0.2%, respectively, increased teliospore germination, but treatment times longer than 2 min still progressively reduced the germination percentages. In testing alternative methods, we found "acidic electrolyzed water" (AEW), generated by electrolysis of a weak solution of sodium chloride, also surface-sterilized and increased the rate of T. indica teliospore germination. In a representative experiment comparing the two methods, NaOCl (0.4%) for 2 min and AEW for 30 min increased germination from 19% (control) to 41 and 54%, respectively, by 7 days after treatment. Because teliospores can be treated with AEW for up to 2 h with little, if any, loss of viability, compared with 1 to 2 min for NaOCl, treatment with AEW has certain advantages over NaOCl for surface sterilizing and increasing germination of teliospores of suspect T. indica.

Antigenic glycoproteins in the teliospore wall of Tilletia indica

We describe the partial characterization of immunoreactive glycoproteins in the teliospore wall of Tilletia indica, the causal agent of Karnal bunt of wheat (Triticum aestivum). Proteins from phenol-extracted and precipitated spore wall preparations were used as inject antigens to immunize New Zealand rabbits. Immunoblots of crude preparations of spore wall proteins probed with anti-T. indica spore wall IgG showed that the antibodies recognized a 37–43 kDa band present in the spore wall fraction of T. indica. A weakly immunoreactive band of the same mobility was detected in the spore wall of T. barclayana, a closely related fungal pathogen and causal agent of kernel smut of rice. The 37–43 kDa polypeptide could not be detected in the teliospore walls of the related fungal wheat pathogens T. controversa and T. tritici. Differential centrifugation of pulverized T. indica teliospores confirmed that the immunoreactive T. indica polypeptide was primarily localized in a 1500 g spore wall-enriched fraction and not in cytosolic or membrane fractions of the teliospores. The lectin concanavalin A bound to a 36–41 kDa polypeptide of T. indica but not T. barclayana. Treatment of spore wall proteins with glycosidase enzymes to remove oligosaccharide residues revealed that the 37–43 kDa polypeptide of T. indica was composed of two distinct immunoreactive polypeptides of lower molecular weight than those detected in undigested spore wall protein immunoblots. The immunoreactivity of the 37–43 kDa T. barclayana protein was lost upon enzymatic deglycosylation. This suggests the cross reactivity between the 37–43 kDa antigen of T. barclayana and T. indica is most likely due to oligosaccharide epitopes common to the glycoproteins. The differential reactivity of the deglycosylated proteins comprising the 37–43 kDa antigens provides a potential target for development of rapid immunological diagnostic assays for teliospores of different smut pathogens.

Antigenic Glycoproteins in the Teliospore Wall of Tilletia indica

We describe the partial characterization of immunoreactive glycoproteins in the teliospore wall of Tilletia indica, the causal agent of Karnal bunt of wheat (Triticum aestivum). Proteins from phenol-extracted and precipitated spore wall preparations were used as inject antigens to immunize New Zealand rabbits. Immunoblots of crude preparations of spore wall proteins probed with anti-T. indica spore wall IgG showed that the antibodies recognized a 37–43 kDa band present in the spore wall fraction of T. indica. A weakly immunoreactive band of the same mobility was detected in the spore wall of T. barclayana, a closely related fungal pathogen and causal agent of kernel smut of rice. The 37–43 kDa polypeptide could not be detected in the teliospore walls of the related fungal wheat pathogens T. controversa and T. tritici. Differential centrifugation of pulverized T. indica teliospores confirmed that the immunoreactive T. indica polypeptide was primarily localized in a 1500 g spore wall-enriched fraction and not in cytosolic or membrane fractions of the teliospores. The lectin concanavalin A bound to a 36–41 kDa polypeptide of T. indica but not T. barclayana. Treatment of spore wall proteins with glycosidase enzymes to remove oligosaccharide residues revealed that the 37–43 kDa polypeptide of T. indica was composed of two distinct immunoreactive polypeptides of lower molecular weight than those detected in undigested spore wall protein immunoblots. The immunoreactivity of the 37–43 kDa T. barclayana protein was lost upon enzymatic deglycosylation. This suggests the cross reactivity between the 37–43 kDa antigen of T. barclayana and T. indica is most likely due to oligosaccharide epitopes common to the glycoproteins. The differential reactivity of the deglycosylated proteins comprising the 37–43 kDa antigens provides a potential target for development of rapid immunological diagnostic assays for teliospores of different smut pathogens.

Risk Assessment for Karnal Bunt Occurrence in the Pacific Northwest.

Tilletia indica, the causal agent of Karnal bunt of wheat, was first detected and reported in the United States in 1996. Karnal bunt occurred in the southwestern United States as early as 1992. Wheat contaminated with teliospores of T. indica is likely to have been transported from the Southwest to other regions, including the Pacific Northwest, before presence of the pathogen was discovered. Teliospore and sporidial germination and infection are highly dependent on climatic conditions. The potential for T. indica to infect wheat in the Pacific Northwest has not been reported. The objective of this study was to use published information on environmental factors favorable for infection and historical climate data for the Pacific Northwest to analyze the environmental risk for Karnal bunt to occur if wheat fields in the Pacific Northwest become contaminated by T. indica. Conditions during the past four decades appeared favorable for infection in nonirrigated wheat during 1 of every 3 years at two (Corvallis, OR, and Spokane, WA) of 13 Idaho, Oregon, and Washington locations examined, and every year at all locations where wheat is irrigated. If introduced to the area, it appears possible for T. indica to become established in selected regions of the Pacific Northwest.

Development of a PCR-Based Method for Identification of Tilletia indica, Causal Agent of Karnal Bunt of Wheat

The polymerase chain reaction (PCR) was used to identify Tilletia indica, the causal agent of Karnal bunt of wheat. The method uses two sets of oligonucleotide primers developed by sequence analysis of cloned Dra I fragments of mitochondrial DNA of T. indica. The primer pair TI17M1 (5'-TCCCCTTGGATCAGAACGTA-3') and TI17M2 (5'-AGAAGTCTAACTCCCCCCTCT-3'), derived from clone pTI-MD17, amplified a single 825-bp product from all isolates of T. indica and no products for other Tilletia species. In addition, the primer pair TI57M1 (5'-TTTTCCCTCTCTC-CTTTTTTCA-3') and TI57M2 (5'-AGCAAAGACAAAGTAGGCTTCC-3'), derived from clone pTI-MD57, produced a product of 118 bp which was unique to T. indica.

Comparison of the Virulence of Isolates ofTilletia indica,Causal Agent of Karnal Bunt of Wheat, from India, Pakistan, and Mexico

Four Tilletia indica teliospore field populations, two from Mexico and one each from India and Pakistan, were tested for virulence on five Karnal bunt-resistant cultivars, one moderately susceptible, and two Karnal bunt highly susceptible wheat cultivars. The five resistant cultivars represented the most Karnal bunt-resistant germ plasm in the breeding programs at the International Maize and Wheat Improvement Center (Centro Internacional de Mejoramiento de Maiz y Trigo [CIMMYT]), Mexico, and the Department of Plant Breeding, Punjab Agricultural University, Ludhiana, India. Plants at the boot stage were inoculated by injecting into the boot 1 ml of a water suspension containing 10,000 allantoid sporidia per ml, incubated in a mist chamber for 3 days, then maintained until maturity in a greenhouse. All inoculated and control wheat spikes were harvested individually, and percentages of T. indica-infected seeds were determined. In addition, infected seeds from 10 randomly selected infected spikes per treatment were examined to estimate the proportion of each infected seed converted to a sorus. On the most resistant wheat cultivar (HD-29), percentage of seeds infected varied from 10 to 30%, depending on pathogen aggressiveness. On the most susceptible cultivar (Bacanora), infection varied from 55 to 84%. Although there were differences in pathogen aggressiveness, there was no evidence of the existence of races among the field populations. Wheat cultivars resistant to the Mexican fungal populations also were resistant to those from Asia, and vice versa; there was a significant correlation (P ≤ 0.05) between percentage of seeds infected and extent of fungal colonization of infected seeds with all but one pathogen population when comparing resistant versus other wheat cultivars.

Teliospore Survival and Plant Quarantine Significance of Tilletia Indies (causal agent of Karnal bunt) Particularly in Relation to China

The influence of temperature, light and soil conditions on teliospore germination of Tilletia (Neovossia) Indica was investigated. The teliospores were germinated by incubation in an aqueous suspension in a concavity on a water agar surface and the germination percentage was scored directly under low magnification of the compound microscope. The optimal temperature range for germination was found to be 15 to 22°C. Germination was extremely low at 2°C, but total inhibition was only observed after prolonged exposure to 35®C (in darkness) or to ‐ 18°C under dry conditions. Light was shown to have a stimulating effect on germination (artificial daylight was found to be better that near ultraviolet light). No stimulation was achieved by prolonging the illumination from 12 to 24 h. Teliospores from 5‐year‐old wheat samples were shown to be viable. The weather conditions of the various wheat‐ growing areas of China were compared with the results obtained for the survival of teliospores of T. indica. It is concluded that a considerable risk exists for establishment and spread of the Karnal bunt disease if first accidentally introduced into China. Strict quarantine precautions, and policies can minimize or even eliminate this risk.