Title of Invention	"IMMUNOLOGICAL MARKER TO ASSESS THE PATHOGENICITY LEVELS OF T.INDICA FUNGAL ISOLATES"
Abstract	This invention relates to a method for grouping the fungal isolates of Tilletia indica in to distinct pathogenic groups under induced conditions in presence of host determinant(s) or plant extract, by using immunological approach, comprising the steps of: (a) Liquid culture of fungal isolates with host determinants (b) Harvesting of mycelia at specific time intervals (c) Detection of immunoreactivity using indirect ELISA with mycelial antigen and anti mycelial antibodies (d) Grouping of Tilletia indica isolates in to virulent, moderately virulent and least virulent with reference to the growth behavior and immuno-reactivity of Tilletia indica isolates of known virulence levels.

Title of Invention

"IMMUNOLOGICAL MARKER TO ASSESS THE PATHOGENICITY LEVELS OF T.INDICA FUNGAL ISOLATES"

Abstract

This invention relates to a method for grouping the fungal isolates of Tilletia indica in to distinct pathogenic groups under induced conditions in presence of host determinant(s) or plant extract, by using immunological approach, comprising the steps of: (a) Liquid culture of fungal isolates with host determinants (b) Harvesting of mycelia at specific time intervals (c) Detection of immunoreactivity using indirect ELISA with mycelial antigen and anti mycelial antibodies (d) Grouping of Tilletia indica isolates in to virulent, moderately virulent and least virulent with reference to the growth behavior and immuno-reactivity of Tilletia indica isolates of known virulence levels.

Full Text	"IMMUNOLOGICAL MARKER TO ASSESS THE PATHOGENICITY LEVELS OF T.INDICA FUNGAL ISOLATES". FIELD OF INVENTION This invention relates to the immunological detection of the pathogenecity levels of T.indica isolates known to be the etiologic agents of Karnal bunt disease in wheat. BACKGROUND OF INVENTION The incitant of Karnal bunt disease was first described by Mitra in 1931 under the name Tilletia indica and also referred to as "New bunt" (Mitra, 1931, 1935) or "Partial bunt" (Bedi et at, 1949). However it was later transferred by Mundukur (1940) from T. indica to Neovossia indica Mundukur (1940) because of its unbranched rather long promycelia, each with a whorl of non fusing conidia 32 - 123 or more at the apex. However, the Karnal bunt pathogen has been retained as Tilletia indica (Syn. Neovossia indica) by Fisher and Holton (1957). T.indica is facultative and heterothallic and septate mycelia are hyaline in nature. Based on a detailed taxonomic study (Munjal, 1970; Krishna and Singh (1982) justified the placement of N. indica under Neovossia. However, western scientific literature prefers to designate causal agent of KB as T. indica. Kingdom: Fungi Division: Eumycota Subdivision: Basidiomycotina Class: Teliomycetes Order: Ustilaginales Family: Tilletiaceae With respect to the present invention, members of the family Tilletiaceae, and particularly the genus Tilletia, are of particular interest. T. indica, a causative agent of Karnal bunt disease causes significant reduction in yield and quality. However, due to its mode of dissemination and control difficulties, many countries have imposed strict quarantines on the importation of infected seed. In many cases, any seed produced from an area where the disease has been identified cannot be imported. Wheat show considerable variation in degree of susceptibility among cultivars and in turn T. indica also exhibits high level of genetic variability among the isolates (Mishra et at, 2001) exhibiting varying degree of virulence. Development of resistant wheat varieties with a wide genetic base claims thorough knowledge of molecular mechanism in the development of variability in T. indica. Grouping of T. indica strains according to their pathogenic levels was predominantly carried out by field testing through artificial inoculations. This methodology is time consuming and require quite higher amount of inputs. The methodology is highly dependent on seasonal factors and due to this reason some erroneous judgments also can take place. This necessitated the need of simple and full proof method for categorization of fungal isolates based on their virulence levels. To identify variability in Tilletia indica in 14 isolates collected from different agroclimatic areas of Punjab, pathogenicity tests on wheat, isoenzyme analysis based on starch-gel electrophoresis were carried out. The isolates were grouped into 5 distinct groups when compared using pathogenicity tests. Based on the analysis of esterase and acid phosphatase isoenzymes the isolates could be grouped into 2 categories (Sharma et. al. 1998). A library of N. indica isolate Ni7 was constructed in a AZAPII 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 Nil, Ni7 and Ni8, thus suggesting a positive correlation between virulence typing and cluster analysis based on molecular data (Datta et.al 1999). In order to maximize the efficiency of grouping according to the pathogenic levels as well as to detect the pathogenic infection various methods have been tried and the immunological approaches have given better resolutions in identification of the pathogen and determination of pathogenic levels of several fungal isolates. Identification of an immunological marker(s) related with onset of pathogenicity provides a means of assessing the degree of variation based on their virulence levels. Development of polyclonal antibodies against mycelia and detection of variation in antigenic profile on T. indica, after the exposure to plant factors will provide valuable information on fungal surfaces which cause changes in pathogenicity. Serological (antibody based) techniques have been used for identification and taxonomic classification of plant pathogens as well as for diagnosing infection levels. Immunoassays have been used very successfully for a number of years for the detection of viruses in diseased plants but development for fungal pathogen has been slow. The difficulties encountered in producing antisera to fungi that have the required specificity have been one of the main reasons that has retreated the development of fungal immunodiagnostic techniques. When antisera raised to mycelia fragments, extracted from lyophilized mycelia, surface washing of solid culture or filtrates were tested by enzyme linked immunosorbant assay (ELISA) or immunofluorescence (IMF), they generally cross react with both related and unrelated fungi and host tissues of these extracts. Thus, there is need to improve the specificity of fungal antisera for their use in plant pathology diagnostics. OBJECTS FO INVENTION The main object of this invention is to develop immunological marker to assess the pathogenicity levels of T. Indica fungal isolates. Other object is to develop simple, reliable, and environmental factors independent invitro technique to detect the pathogenic levels of T. indica isolates. Another object is to develop a technique which is applicable for grouping of the pathogen in to virulent, moderately virulent and least virulent based on differential immunoreactivity patterns. STATEMENT OF INVENTION This invention relates to a method for grouping the fungal isolates of Tilletia indica in to distinct pathogenic groups under induced conditions in presence of host determinant(s) or plant extract, by using immunological approach, comprising the steps of: (a) Liquid culture of fungal isolates with host determinants (b) Harvesting of mycelia at specific time intervals (c) Detection of immunoreactivity using indirect ELISA with mycelial antigen and anti mycelial antibodies (d) Grouping of Tilletia indica isolates in to virulent, moderately virulent and least virulent with reference to the growth behavior and immuno-reactivity of Tilletia indica isolates of known virulence levels. BRIEF DISCRIPTION OF THE ACCOMPANYING DRAWING Fig 1. Shows Effect on different T. indica isolates grown in solid medium at different time intervals grown in absence and presence of host determinants [(-) Without host determinant(s) (+) With host determinant(s)] Fig 2. Shows Determination of optimal antigen concentration and antibody dilution for development of indirect ELISA by performing antigen concentration kinetics and antibodies dilution curves (checker board analysis) for mycelial antigen and anti-mycelial antibodies. Fig 3. Shows Variation in immunoreactivity (OD values) of anti-mycelial antibodies with mycelial antigens of different fungal isolates grown in absence and presence of host determinants [(-) Without host determinant(s) (+) With host determinant(s)]. DETAILED DESCRIPTION OF THE INVENTION Highly effective method for detection of pathogenic levels of different T. indica isolates was developed by the comparison of immuno reactivity of fungal isolates under natural and induced conditions by host determinant(s). Host determinant(s) which extracted from wheat spikes play(s) an important role in induction of mycelination of T. indica which caused faster growth of pathogen under in vitro cultures. As the real mode of action of host determinant(s) and the nature of the of the host determinant(s) is not known so far, the present work throws light upon immunological changes with respect to the exposure of the host determinant(s) to KB isolates differing in their pathogenic levels. The present study was carried out predominantly to distinguish the antigenic variation of fungal isolates and also to correlate with the variations with pathogenic levels. Time course antigenic variation also monitored in order to detect the variation with the age of the culture. In-order to detect the natural and induced antigenic variation among the isolates, indirect ELISA was performed using polyclonal antibodies specific to mycelium at different time intervals (7, 14, 21 and 30 day after inoculation). According to the results obtained through checker board analysis it was concluded thatl:250 to 1:500 primary antibody dilution is appropriate for mycelial antigenic concentration determination. In antigen concentration kinetics, it was concluded that 75 to 200ng of mycelial antigen per well is the optimal concentration. The three fungal stains P9, P16 and P7 with known pathogenic levels, which are used in the study and they were highly virulent, moderately virulent and least virulent in pathogenicity respectively. KBPN (Pantnagar isolate), KB3 (Delhi isolate), KB9 (Punjab isolate) and JK (Jammu Kashmir isolate) fungal isolates whose pathogenic levels were not known and analyzed in the present study. All seven fungal strains were cultured with and without host determinant(s) and variation in immunoreactivity was detected at four time intervals (7th day, 14th day, 21st day and 30th day). Fast growing mycelia exhibits more immuno-reactivity levels indicating the presence of more antigens on the mycelia. In general, the immuno-reactivity level is high at initial growth in culture (up to 14 days of growth) and the same get decreased after 14th days of growth in old cultures. This phenomenon is more pronounced in fast growing strains than slow growing strains. This observation can be used to group a fungal population in to their pathogenic levels as the growth of the fungi positively correlate with the pathogenicity of the respective fungi. Host determinant(s) treatment tend to increase the immuno-reactivity of T. indica strains and the increment is very high in fast growing (Highly virulent) at the 7th days in culture. A significant reduction in immuno reactivity was observed after 14 days in cultures. This highly significant increase and decrease in immuno-reactivity is not that apparent in slow growing strains (less virulent). At the seventh day, all the fungal isolates have given significantly higher levels of reactivity in presence of host determinant(s) i.e. induced compared to the natural (control) conditions. This increase in immunoreactivity was very high in fast growing, highly virulent fungal strains (P9). After 14th days the immuno-reactivity was reduced in fast growing strains cultured with host extracts. In slow growing strains the immunoreactivity was reduced only after 14th days in control as well as in treated cultures. This unique behavior in exhibiting varied immuno-reactivity can be used in grouping fungal strains according to their pathogenicity. KBPN, KB3, KB9 and JK fungal strain has no known record in the pathogenic levels but KBPN, KB9 and JK acts some what similar to the P9 fungal strain which is highly virulent while KB3 acts some what similar to P7 fungal strain which is least virulent. Hence with this observations it is possible to conclude that KBPN, KB9 and JK strain also possess highly virulent pathogenic levels and KB3 strain possess least virulent pathogenic level as it was further revealed by pathogenicity tests. The findings of virulence behaviors of unknown isolates were verified after inoculation of these isolates in to different host differentials under field conditions. This finding can be used as a bio assay to discriminate an unknown population of T. indica into their respective pathogenic levels with respect to exposure to host determinant(s) derived from susceptible variety with out field testing. FLOW CHART FOR DETECTION OF PATHOGENICITY OF FUNGAL STRAINS LIQIUD CULTURE OF FUNGAL STRAINS WITH AND WITHOUT HOST DETERMINANTS HARVESTING OF MYCELIA IN SPECIFIC TIME INTERVALS (7TH, 14TH 21ST AND 30TH DAYS IN CULTURE) LYOPHILIZATION OF FUNGAL STRAINS FOR COMPLTE DRYNESS PRODUCTION OF MYCELIAL ANTIGENS DETECTION OF IMMUNOREACTIVITY USING INDIRECT ELISA (USING MYCELIAL ANTIGENS (75 to 200 ng PER WELL) AND ANTI MYCELIAL ANTIBODODIES (1:250 to 1:500 DILUTION)) GROUPING OF FUNGI POPULATION ACCORDING TO THE PATHOGENIC LEVELS STANDARDISED PROTOCOLS FOR DETECTION OF PATHOGENICITY TILLETIA JNDICA FUNGAL ISOLATES T. indica fungal isolates showing different virulence levels were generously provided by Dr. Indu Sharma, Principal Scientist, Department of Genetics and Plant Breeding, Punjab Agriculture University, Ludhiana (Punjab). P9 - Virulent PI6 - Moderately virulent P7 - Less virulent Three mono-teliosporic cultures T. indica isolates KB 3, KB 9 and JK prepared as described earlier (Geeta et ah, 2000), collected from Delhi, Punjab, Jammu & Kashmir provinces were received from Indian Agricultural Research Institute, New Delhi, India and one mono-teliosporic culture of KBPN isolate was collected from Uttarakhand province and obtained from Wheat Pathology Lab, Department of Plant Pathology, College of Agriculture, G. B. Pant University of Agriculture and Technology, Pantnagar, India. Fungal isolates and strains were cultured at Molecular Marker Laboratory, Department of Molecular Biology and Genetics Engineering, G. B. Pant University of Agriculture & Technology, Pantnagar, India on modified potato dextrose agar (PDA) and potato dextrose broth (PDB) medium in Petri plates and flasks respectively with and without host determinant. The cultures were incubated in BOD incubator at 22 ± 1°C under light and dark conditions. PREPARATION OF HOST DETERMINANTS Host determinant(s) was prepared from spike tissues collected from susceptible (WH-542) wheat variety in boot emergence stage (S2). 50g of plant parts were ground in liquid nitrogen to a fine powder using pestle and mortar. Finely ground plant tissues were suspended in cold acetone at the concentration of lg in 10 ml of acetone. The above suspension was agitated in cold condition for 5 hours and filtered through muslin cloth to remove larger debris and stored at 4°C in tightly capped bottles. Before starting the experiments acetone was evaporated at room temperature using flash evaporator or blowing hot air over the solution. Dried material obtained was re-suspended in l/l0th of the volume of sterilized distilled water and filtered through 0.22µ filter before incorporation to the culture mediums. In order to detect the induced growth with host determinants, culturing was done with plant extracts at 1: 10 host determinants and medium ratio. Harvesting of mycelium The growing liquid cultures of T. indica were harvested at desired time intervals. The media containing the mycelial mat of T. indica was filtered through a folded muslin cloth and washed several times in PBS (0.05 M, pH 7.2) and it was followed by washing with sterilized distilled water. The wet weight of the mycelial mass was taken and the wet masses were lyophilized for 5 hours to obtain the dry weight. Dried mycelial masses were stored in -20°C until use. PREPARATION OF IMMUNO PROBES Preparation of anti-mycelial antibodies against mycelial fragments. Dried mycelial masses were ground to a fine powder in liquid nitrogen and used as antigens to raise antibodies. Raised antibodies were used for the detection of changes in antigenic levels in fungal cultures treated with plant extracts. Antisera production New Zealand white female rabbits were used for production of anti mycelial antibody. Adjuvant used for immunization CFA- Complete Freund's adjuvant FIA - Freund's incomplete adjuvant Buffer used for immunization PBS- Phosphate buffer saline (0.15 M, pH 7.4) was used as buffer to prepare the antigen emulsions. Mode of Immunization The injections were given by following modes using 2 ml disposable syringe. (i) Subcutaneous (SC) (ii) Inter-peritoneal (IP) (iii) Foot pad (FP) Preparation of antigen emulsion Adjuvant, antigen dissolved in PBS in proper amount was mixed and emulsified with the help of 5ml syringe and 16 gauge needle. Immunization schedule Immunization schedule was carried out as per the table below. Table: 1 Immunization schedule using mycelial antigens (Table Removed) CFA = Complete Freund's adjuvant IFA = Incomplete Freund's adjuvant Bleeding of rabbits One week after the 4th booster dose, 5 ml of blood was withdrawn from the ear vain of rabbits immunized against mycelia. Preparation of antisera The blood samples were first clotted at room temperature by resting them in glass tubes for 2hr and kept overnight at 4°C. The clear sera were decanted into Eppendorf tubes, were spun at 4000 rpm for 10 min at 4°C to get rid off any remaining red blood cells. The straw colored clear supernatant was transferred into separately capped cryovials and stored at -20°C. ELISA FOR DETECTION OF CHANGES IN ANTIGENIC DETERMINANTS The ELISA was developed with minor modifications as described initially by Engvall and Perlman (1971) and later by Clark and Adams (1977). Principle Enzyme linked immuno-sorbent assay (ELISA) principle involves competitive binding of enzyme-linked antigen or unlabelled antigen to a high quality unlabelled antibody or enzyme linked secondary antibodies so an enzyme conjugated with an antibody react with a colorless substrate to generate a colored reaction product which can measured. Reagents prepared: 1. Phosphate buffer saline (PBS) [0.15 M] NaCl = 8.0g KC1 = 0.2g KH2 PO4 = 0.2g Na2HPO42H2O = 1.44g Distilled water = to make up to 1 L pH = 7.4 * Stored at R.T. 2. Coating buffer [0.05 M] Na2 CO3 = 1.59g Na2 H CO3 = 2.93g pH = 9.4 * Stored at R.T. 3. Blocking solution 2% BSA was prepared in PBS * This was prepared freshly 4. Antibody dilution buffer 0.25% BSA in PBS pH =7 * Stored at 4°C 5. Substrate buffer Diethanolamine = 9.7 ml MgCl2.6H2O = 0.lg pH = 9.8 * Volume was adjusted to 100ml 6. Substrate solution (1mg/ml) 0.01g of Na -salt of p-nitrophenyl phosphate dissolved in 10 ml of substrate buffer. Stored at 4°C in dark vials 7. Stop solution 6% NaOH in double distilled water Stored at R.T. Procedure: • Lyophilized mycelium were taken and dissolved in PBS at the rate of 20 mg/ml. This was homogenized in motorized hard homogenizer. Resultant solution was used as antigen. • Polystyrene micro plates were coated with mycelia fragments obtained from control and plant extract treated mycelium in two time intervals (14th day and 21st day). • Antigen fractions were dissolved in coating buffer in to different concentrations. 100 µl of respective antigen was delivered to a well. Antigen coated plates were incubated for 2hr at room temperature and followed with overnight incubation at 4°C. • The plates were washed with PBS+ Tween-20 (0.01%) • The plates were filled with PBS containing 5% skimmed milk for 2hr. at room temperature. Binding was done to prevent adventitious binding. • Washing was done with PBS + Tween-20 (0.01%) + 0.5% skimmed milk • l00µl of first antibody (Raised against respective antigens) were added and incubated for 2hrs at R.T. the antibody dilution (1:1000) was done with PBS + 0.5% skimmed milk. • Washing was done thrice with PBS + Tween-20 (0.01%) + 0.25% BSA • 100 µl of 1:1000 diluted alkaline phosphatase conjugated secondary antibody was incubated for 2hr at R.T. • The plate was washed with PBS containing 0.25% BSA + Tween-20 (0.01%) thrice • Alkaline phosphatase activity was assayed with p-nitrophenyl phosphate sodium salt dissolved in substrate buffer (lmg/ml). • The plates were incubated for 30 min in dark and the reaction was stopped with 100µl of 1.5 M NaOH solution • Reading was taken at 405nm in ELISA reader. Result: Three T. indica isolates (P7, P9 and PI6) with known virulence and four isolates (KBPN, KB3 JK and KB9) of unknown virulence levels were cultured in modified Potato Dextrose Agar media in-order to detect the behaviors of fungal isolates. Statistically Significant variation in radial diameter was observed in all three main isolates studied at all three time intervals. Host determinants have given a boost to the increment compare to the control. Fast growing isolate P9 and KBPN has shown higher radial diameter and the increment in radial diameter was always higher compare to least and moderately virulent isolates (P7 and P16 respectively). The growth behaviors of JK and KB9 were almost similar as shown by P16 isolates and KB3 growth was similar to P7 isolates as indicated in the Figure 1. At the sixth day, the variation in radial diameter with respect to the treatments was clearly depicted and the variation in the same at 9th day is not that apparent as the isolates have already covered the whole Petri plate indicating a rapid growth in them with host determinants treatment. Radial diameter variation with respect to the host factors was statistically analyzed individually with the sixth day data and it was revealed that the variation among the treatments (Control and WH 542) was significant (p >0.05) in all seven isolates compared. Host determinants treatment on fungal strains tends to increase their mycelination and it can be concluded that increase in mycelination of fungi leads to impose more virulence levels in the host and prolific multiplication of pathogen inside host lead to more damage to developing grains. In order to determine the virulence level of Tilletia indica isolates/strains, the causative agent of Karnal bunt disease of wheat and differentiating it from similar, Tilletia indica isolate, mycelial fragment antigen from all isolates were subjected to indirect ELISA determination at 7 day to 30 days incubation. In order to determine the optimum antibodies dilution and optimum antigen concentration for the development of indirect ELISA, different dilution of antibodies were used to determine immunoreactivity with 100 ng of mycelia antigen. From the immunoreactivity results of indirect ELISA, it was concluded that dilution in the range of 1:250 to 1:500 primary anti-mycelial antibody is appropriate for determine the virulence level of T. indica. Similarly results of optimum antigen concentration using different concentrations of mycelia antigens and 1: 250 dilution of anti-mycelial antibodies, it was concluded that 75 to 200 ng of mycelial antigen per well is the appropriate concentration (Figure 2). All fungal isolates P9, P16, P7, KBPN, KB3, KB9 and JK were cultured on modified Potato Dextrose Broth media with and without host determinants (Acetone extracts from S2 stage of WH-542 wheat spike). Acetone extracts were incorporated at 1: 10 media to extract ratio and culturing was carried out without replenishing of host determinants until the harvesting at desired time intervals. A time series estimation of antigenic variation was carried out on 7th, 14th, 21st and 30th days after the inoculation. Indirect ELISA was performed on mycelial antigen in order to detect any variation in immune-reactivity after the induction with host factors. Variation in immunoreactivity in terms of OD405 on mycelial antigen was detected using mycelial antigen concentration 125ng per well with 1:500 dilution of anti-mycelial antibody developed against mycelial antigens (Figure 3). It was interesting to see that the isolates with known virulence level, virulent (P9), moderately virulent (P16) and least virulent (P7) exhibited very clear increase in OD (201.35 %, 141.17 % and 136.36% respectively) at 7th day in the presence of host determinants. At the same time interval, the isolates (KBPN, KB3, KB9 and JK) with unknown virulence level also exhibited an increase in OD405 (212.69%, 134.54%, 172.37% and 162.50% respectively) when cultured in presence of host determinants. At the 14th 21st and 30th days of growth in culture, a drastic reduction in OD405 was observed with all the isolates P9, P16, P7, KBPN, KB3, KB9 and JK (121.52%, 122.85%, 103.89%, 119.72%, 114.72%, 106.41% and 112.34% at 14th days), (123.88%, 104.76%, 120.32%, 105.88%, 104.47%, 104.92%, and 129.16% at 21st days), and (118.75, 100.00%, 108.95%, 107.82%, 113.12%, 115.68% and 115.22% at 30th days) treated with host determinants. It is quite evident that all the isolates have exhibited increase in OD at the 7th day with the presence of host determinants with considerable variations respectively to their inherent growth behavior. As a thumb rule all the isolates have exhibited increase in OD405 at the 7th day with the presence of host determinants with considerable variations respectively to their inherent growth behavior. In general fast growing strains tend to show high reactivity levels. As the virulence levels of P9, P16 and P7 already known this finding can be matched with virulence levels of the pathogens and also can be used as a bio marker for detection of pathogenicity of a pathogen with an unknown history. The pathogenic level of KBPN is unknown but with the present observations, it can be considered as a highly pathogenic strain. This bio marker for detection of pathogenicity can be used in-order to group fungal strains in to the respective virulent groups more economically without doing any field testing. Contradictory to the conclusion made above (fast growing strains show low reactivity without host determinants) the fungal strains treated with host determinants exhibited fast growth as well high levels of immuno-reactivity. This can happen due to the expression of more antigenic epitopes on the mycelial surfaces such as some signal receptors. The signaling molecules present in the host determinants can enhance the expression of those genes leading to the more immuno reactivity as observed in the above experiment. With all these observations it is possible to conclude that the host determinants are acting as growth factors (Nutritional) as well as hormones which enhance expression of specific genes involved in the alteration of surface properties. This finding can be used as a bio assay to discriminate a unknown population of T. indica into their respective pathogenic levels with respect to a known host determinants derived from susceptible variety without field testing. WE Claim 1. A method for grouping the fungal isolates of Tilletia indica in to distinct pathogenic groups under induced conditions in presence of host determinant(s) or plant extract, by using immunological approach, comprising the steps of: (a) Liquid culture of fungal isolates with host determinants (b) Harvesting of mycelia at specific time intervals (c) Detection of immunoreactivity using indirect ELISA with mycelial antigen and anti mycelial antibodies (d) Grouping of Tilletia indica isolates in to virulent, moderately virulent and least virulent with reference to the growth behavior and immuno-reactivity of Tilletia indica isolates of known virulence levels. 2. The method of claim 1 where in mycelial fragments of Tilletia indica are used as antigen. 3. The method as claimed in claim 1, wherein mycelial antigen concentration is used 75-200 ng in the indirect ELISA 4. The method of claim 1 where in the using anti-mycelial polyclonal antibodies of Tilletia indica used in the range of 1:250 to 1:500. 5. The method of claim 1 where in the host determinant(s) or plant extract prepared from S2 -stage of developing wheat spike induced the growth of T. indica. (Z score). 6. The method as claimed in claim 1, wherein plant extract and fungal growth medium is used in 1:10 ratio. 7. The method as claimed in claim 1, wherein in the growth of fungal isolates, mycelial mass harvested at 7th day to 30 days for isolation of mycelial fragments and determination of differential immune reactivity under induced conditions. 8. The method for developing immunological marker to assess the pathogenicity levels of T. Indica fungal isolates as described and illustrated herein.

Full Text

"IMMUNOLOGICAL MARKER TO ASSESS THE PATHOGENICITY LEVELS OF T.INDICA FUNGAL ISOLATES".
FIELD OF INVENTION
This invention relates to the immunological detection of the pathogenecity levels of T.indica isolates known to be the etiologic agents of Karnal bunt disease in wheat.
BACKGROUND OF INVENTION
The incitant of Karnal bunt disease was first described by Mitra in 1931 under the name Tilletia indica and also referred to as "New bunt" (Mitra, 1931, 1935) or "Partial bunt" (Bedi et at, 1949). However it was later transferred by Mundukur (1940) from T. indica to Neovossia indica Mundukur (1940) because of its unbranched rather long promycelia, each with a whorl of non fusing conidia 32 - 123 or more at the apex. However, the Karnal bunt pathogen has been retained as Tilletia indica (Syn. Neovossia indica) by Fisher and Holton (1957). T.indica is facultative and heterothallic and septate mycelia are hyaline in nature. Based on a detailed taxonomic study (Munjal, 1970; Krishna and Singh (1982) justified the placement of N. indica under Neovossia. However, western scientific literature prefers to designate causal agent of KB as T. indica.
Kingdom: Fungi
Division: Eumycota
Subdivision: Basidiomycotina
Class: Teliomycetes
Order: Ustilaginales
Family: Tilletiaceae
With respect to the present invention, members of the family Tilletiaceae, and particularly the genus Tilletia, are of particular interest. T. indica, a causative agent of Karnal bunt disease causes significant reduction in yield and quality. However, due to its mode of dissemination and control difficulties, many countries have imposed strict quarantines on the importation of infected seed. In many cases, any seed produced from an area where the disease has been identified cannot be imported. Wheat show considerable variation in degree of susceptibility among cultivars and in turn T. indica also exhibits high level of genetic variability among the isolates (Mishra et at, 2001) exhibiting varying degree of virulence. Development of resistant wheat varieties with a wide genetic base claims thorough knowledge of molecular mechanism in the development of variability in T. indica. Grouping of T. indica strains according to their pathogenic levels was predominantly carried out by field testing through artificial inoculations. This methodology is time consuming and require quite higher amount of inputs. The methodology is highly dependent on seasonal factors and due to this reason some erroneous judgments also can take place. This necessitated the need of simple and full proof method for categorization of fungal isolates based on their virulence levels. To identify variability in Tilletia indica in 14 isolates collected from different agroclimatic areas of Punjab, pathogenicity tests on wheat, isoenzyme analysis based on starch-gel electrophoresis were carried out. The isolates were grouped into 5 distinct groups when compared using pathogenicity tests. Based on the analysis of esterase and acid phosphatase isoenzymes the isolates could be grouped into 2 categories (Sharma et. al. 1998). A library of N. indica isolate Ni7 was constructed in a AZAPII 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 Nil, Ni7 and Ni8, thus suggesting a positive correlation between virulence typing and cluster analysis based on molecular data (Datta et.al 1999). In order to maximize the efficiency of grouping according to the pathogenic levels as well as to detect the pathogenic infection various methods have been tried and the immunological approaches have given better resolutions in identification of the pathogen and determination of pathogenic levels of several fungal isolates. Identification of an immunological marker(s) related with onset of pathogenicity provides a means of assessing the degree of variation based on their virulence levels. Development of polyclonal antibodies against mycelia and detection of variation in antigenic profile on T. indica, after the exposure to plant factors will provide valuable information on fungal surfaces which cause changes in pathogenicity.
Serological (antibody based) techniques have been used for identification and taxonomic classification of plant pathogens as well as for diagnosing infection levels. Immunoassays have been used very successfully for a number of years for the detection of viruses in diseased plants but development for fungal pathogen has been slow. The difficulties encountered in producing antisera to fungi that have the required specificity have been one of the main reasons that has retreated the development of fungal immunodiagnostic techniques. When antisera raised to mycelia fragments, extracted from lyophilized mycelia, surface washing of solid culture or filtrates were tested by enzyme linked immunosorbant assay (ELISA) or immunofluorescence (IMF), they generally cross react with both related and unrelated fungi and host tissues of these extracts. Thus, there is need to improve the specificity of fungal antisera for their use in plant pathology diagnostics.
OBJECTS FO INVENTION
The main object of this invention is to develop immunological marker to assess the pathogenicity levels of T. Indica fungal isolates.
Other object is to develop simple, reliable, and environmental factors independent invitro technique to detect the pathogenic levels of T. indica isolates.
Another object is to develop a technique which is applicable for grouping of the pathogen in to virulent, moderately virulent and least virulent based on differential immunoreactivity patterns.
STATEMENT OF INVENTION
This invention relates to a method for grouping the fungal isolates of Tilletia indica in to distinct pathogenic groups under induced conditions in presence of host determinant(s) or plant extract, by using immunological approach, comprising the steps of: (a) Liquid culture of fungal isolates with host determinants (b) Harvesting of mycelia at specific time intervals (c) Detection of immunoreactivity using indirect ELISA with mycelial antigen and anti mycelial antibodies (d) Grouping of Tilletia indica isolates in to virulent, moderately virulent and least virulent with reference to the growth behavior and immuno-reactivity of Tilletia indica isolates of known virulence levels.
BRIEF DISCRIPTION OF THE ACCOMPANYING DRAWING
Fig 1. Shows Effect on different T. indica isolates grown in solid medium at different time intervals grown in absence and presence of host determinants [(-) Without host determinant(s) (+) With host determinant(s)]
Fig 2. Shows Determination of optimal antigen concentration and antibody dilution for development of indirect ELISA by performing antigen concentration kinetics and antibodies dilution curves (checker board analysis) for mycelial antigen and anti-mycelial antibodies.
Fig 3. Shows Variation in immunoreactivity (OD values) of anti-mycelial antibodies with mycelial antigens of different fungal isolates grown in absence and presence of host determinants [(-) Without host determinant(s) (+) With host determinant(s)].
DETAILED DESCRIPTION OF THE INVENTION
Highly effective method for detection of pathogenic levels of different T. indica isolates was developed by the comparison of immuno reactivity of fungal isolates under natural and induced conditions by host determinant(s).
Host determinant(s) which extracted from wheat spikes play(s) an important role in induction of mycelination of T. indica which caused faster growth of pathogen under in vitro cultures. As the real mode of action of host determinant(s) and the nature of the of the host determinant(s) is not known so far, the present work throws light upon immunological changes with respect to the exposure of the host determinant(s) to KB isolates differing in
their pathogenic levels. The present study was carried out predominantly to distinguish the antigenic variation of fungal isolates and also to correlate with the variations with pathogenic levels. Time course antigenic variation also monitored in order to detect the variation with the age of the culture. In-order to detect the natural and induced antigenic variation among the isolates, indirect ELISA was performed using polyclonal antibodies specific to mycelium at different time intervals (7, 14, 21 and 30 day after inoculation). According to the results obtained through checker board analysis it was concluded thatl:250 to 1:500 primary antibody dilution is appropriate for mycelial antigenic concentration determination. In antigen concentration kinetics, it was concluded that 75 to 200ng of mycelial antigen per well is the optimal concentration. The three fungal stains P9, P16 and P7 with known pathogenic levels, which are used in the study and they were highly virulent, moderately virulent and least virulent in pathogenicity respectively. KBPN (Pantnagar isolate), KB3 (Delhi isolate), KB9 (Punjab isolate) and JK (Jammu Kashmir isolate) fungal isolates whose pathogenic levels were not known and analyzed in the present study. All seven fungal strains were cultured with and without host determinant(s) and variation in immunoreactivity was detected at four time intervals (7th day, 14th day, 21st day and 30th day).
Fast growing mycelia exhibits more immuno-reactivity levels indicating the presence of more antigens on the mycelia. In general, the immuno-reactivity level is high at initial growth in culture (up to 14 days of growth) and the same get decreased after 14th days of growth in old cultures. This phenomenon is more pronounced in fast growing strains than slow growing strains. This observation can be used to group a fungal population in to their pathogenic levels as the growth of the fungi positively correlate with the pathogenicity of the respective fungi. Host determinant(s) treatment tend
to increase the immuno-reactivity of T. indica strains and the increment is very high in fast growing (Highly virulent) at the 7th days in culture. A significant reduction in immuno reactivity was observed after 14 days in cultures. This highly significant increase and decrease in immuno-reactivity is not that apparent in slow growing strains (less virulent).
At the seventh day, all the fungal isolates have given significantly higher levels of reactivity in presence of host determinant(s) i.e. induced compared to the natural (control) conditions. This increase in immunoreactivity was very high in fast growing, highly virulent fungal strains (P9). After 14th days the immuno-reactivity was reduced in fast growing strains cultured with host extracts. In slow growing strains the immunoreactivity was reduced only after 14th days in control as well as in treated cultures. This unique behavior in exhibiting varied immuno-reactivity can be used in grouping fungal strains according to their pathogenicity. KBPN, KB3, KB9 and JK fungal strain has no known record in the pathogenic levels but KBPN, KB9 and JK acts some what similar to the P9 fungal strain which is highly virulent while KB3 acts some what similar to P7 fungal strain which is least virulent. Hence with this observations it is possible to conclude that KBPN, KB9 and JK strain also possess highly virulent pathogenic levels and KB3 strain possess least virulent pathogenic level as it was further revealed by pathogenicity tests. The findings of virulence behaviors of unknown isolates were verified after inoculation of these isolates in to different host differentials under field conditions. This finding can be used as a bio assay to discriminate an unknown population of T. indica into their respective pathogenic levels with respect to exposure to host determinant(s) derived from susceptible variety with out field testing.
FLOW CHART FOR DETECTION OF PATHOGENICITY OF FUNGAL
STRAINS
LIQIUD CULTURE OF FUNGAL STRAINS WITH AND WITHOUT HOST
DETERMINANTS
HARVESTING OF MYCELIA IN SPECIFIC TIME INTERVALS (7TH, 14TH 21ST AND 30TH DAYS IN CULTURE)
LYOPHILIZATION OF FUNGAL STRAINS FOR COMPLTE DRYNESS
PRODUCTION OF MYCELIAL ANTIGENS
DETECTION OF IMMUNOREACTIVITY USING INDIRECT ELISA (USING
MYCELIAL ANTIGENS (75 to 200 ng PER WELL) AND ANTI MYCELIAL
ANTIBODODIES (1:250 to 1:500 DILUTION))
GROUPING OF FUNGI POPULATION ACCORDING TO THE PATHOGENIC
LEVELS
STANDARDISED PROTOCOLS FOR DETECTION OF PATHOGENICITY
TILLETIA JNDICA FUNGAL ISOLATES
T. indica fungal isolates showing different virulence levels were generously provided by Dr. Indu Sharma, Principal Scientist, Department of Genetics and Plant Breeding, Punjab Agriculture University, Ludhiana (Punjab).
P9 - Virulent
PI6 - Moderately virulent
P7 - Less virulent
Three mono-teliosporic cultures T. indica isolates KB 3, KB 9 and JK prepared as described earlier (Geeta et ah, 2000), collected from Delhi, Punjab, Jammu & Kashmir provinces were received from Indian Agricultural Research Institute, New Delhi, India and one mono-teliosporic culture of KBPN isolate was collected from Uttarakhand province and obtained from Wheat Pathology Lab, Department of Plant Pathology, College of Agriculture, G. B. Pant University of Agriculture and Technology, Pantnagar, India. Fungal isolates and strains were cultured at Molecular Marker Laboratory, Department of Molecular Biology and Genetics Engineering, G. B. Pant University of Agriculture & Technology, Pantnagar, India on modified potato dextrose agar (PDA) and potato dextrose broth (PDB) medium in Petri plates and flasks respectively with and without host determinant. The cultures were incubated in BOD incubator at 22 ± 1°C under light and dark conditions.
PREPARATION OF HOST DETERMINANTS
Host determinant(s) was prepared from spike tissues collected from susceptible (WH-542) wheat variety in boot emergence stage (S2). 50g of plant parts were ground in liquid nitrogen to a fine powder using pestle and mortar. Finely ground plant tissues were suspended in cold acetone at the concentration of lg in 10 ml of acetone. The above suspension was agitated in cold condition for 5 hours and filtered through muslin cloth to remove larger debris and stored at 4°C in tightly capped bottles. Before starting the experiments acetone was evaporated at room temperature using flash evaporator or blowing hot air over the solution. Dried material obtained was re-suspended in l/l0th of the volume of sterilized distilled water and filtered through 0.22µ filter before incorporation to the culture mediums. In order to detect the induced growth with host determinants, culturing was done with plant extracts at 1: 10 host determinants and medium ratio. Harvesting of mycelium
The growing liquid cultures of T. indica were harvested at desired time intervals. The media containing the mycelial mat of T. indica was filtered through a folded muslin cloth and washed several times in PBS (0.05 M, pH 7.2) and it was followed by washing with sterilized distilled water. The wet weight of the mycelial mass was taken and the wet masses were lyophilized for 5 hours to obtain the dry weight. Dried mycelial masses were stored in -20°C until use. PREPARATION OF IMMUNO PROBES Preparation of anti-mycelial antibodies against mycelial fragments.
Dried mycelial masses were ground to a fine powder in liquid nitrogen and used as antigens to raise antibodies. Raised antibodies were
used for the detection of changes in antigenic levels in fungal cultures treated with plant extracts. Antisera production
New Zealand white female rabbits were used for production of anti mycelial antibody.
Adjuvant used for immunization
CFA- Complete Freund's adjuvant FIA - Freund's incomplete adjuvant
Buffer used for immunization
PBS- Phosphate buffer saline (0.15 M, pH 7.4) was used as buffer to prepare the antigen emulsions.
Mode of Immunization
The injections were given by following modes using 2 ml disposable syringe.
(i) Subcutaneous (SC)
(ii) Inter-peritoneal (IP)
(iii) Foot pad (FP)
Preparation of antigen emulsion
Adjuvant, antigen dissolved in PBS in proper amount was mixed and emulsified with the help of 5ml syringe and 16 gauge needle.
Immunization schedule
Immunization schedule was carried out as per the table below.
Table: 1 Immunization schedule using mycelial antigens
(Table Removed)
CFA = Complete Freund's adjuvant IFA = Incomplete Freund's
adjuvant
Bleeding of rabbits
One week after the 4th booster dose, 5 ml of blood was withdrawn from the ear vain of rabbits immunized against mycelia.
Preparation of antisera
The blood samples were first clotted at room temperature by resting them in glass tubes for 2hr and kept overnight at 4°C. The clear sera were decanted into Eppendorf tubes, were spun at 4000 rpm for 10 min at 4°C to get rid off any remaining red blood cells. The straw colored clear supernatant was transferred into separately capped cryovials and stored at -20°C.
ELISA FOR DETECTION OF CHANGES IN ANTIGENIC DETERMINANTS
The ELISA was developed with minor modifications as described initially by Engvall and Perlman (1971) and later by Clark and Adams (1977).
Principle
Enzyme linked immuno-sorbent assay (ELISA) principle involves competitive binding of enzyme-linked antigen or unlabelled antigen to a high quality unlabelled antibody or enzyme linked secondary antibodies so an enzyme conjugated with an antibody react with a colorless substrate to generate a colored reaction product which can measured.
Reagents prepared:
1. Phosphate buffer saline (PBS) [0.15 M]
NaCl = 8.0g
KC1 = 0.2g
KH2 PO4 = 0.2g
Na2HPO42H2O = 1.44g
Distilled water = to make up to 1 L
pH = 7.4
* Stored at R.T.
2. Coating buffer [0.05 M]
Na2 CO3 = 1.59g
Na2 H CO3 = 2.93g
pH = 9.4
* Stored at R.T.
3. Blocking solution
2% BSA was prepared in PBS
* This was prepared freshly
4. Antibody dilution buffer
0.25% BSA in PBS
pH =7
* Stored at 4°C
5. Substrate buffer
Diethanolamine = 9.7 ml
MgCl2.6H2O = 0.lg
pH = 9.8
* Volume was adjusted to 100ml
6. Substrate solution (1mg/ml)
0.01g of Na -salt of p-nitrophenyl phosphate dissolved in 10 ml of substrate buffer.
*Stored at 4°C in dark vials
7. Stop solution
6% NaOH in double distilled water * Stored at R.T.
Procedure:
• Lyophilized mycelium were taken and dissolved in PBS at the rate of 20 mg/ml. This was homogenized in motorized hard homogenizer. Resultant solution was used as antigen.
• Polystyrene micro plates were coated with mycelia fragments obtained from control and plant extract treated mycelium in two time intervals (14th day and 21st day).
• Antigen fractions were dissolved in coating buffer in to different concentrations. 100 µl of respective antigen was delivered to a well. Antigen coated plates were incubated for 2hr at room temperature and followed with overnight incubation at 4°C.
• The plates were washed with PBS+ Tween-20 (0.01%)
• The plates were filled with PBS containing 5% skimmed milk for 2hr. at room temperature. Binding was done to prevent adventitious binding.
• Washing was done with PBS + Tween-20 (0.01%) + 0.5% skimmed milk
• l00µl of first antibody (Raised against respective antigens) were added and incubated for 2hrs at R.T. the antibody dilution (1:1000) was done with PBS + 0.5% skimmed milk.
• Washing was done thrice with PBS + Tween-20 (0.01%) + 0.25% BSA
• 100 µl of 1:1000 diluted alkaline phosphatase conjugated secondary antibody was incubated for 2hr at R.T.
• The plate was washed with PBS containing 0.25% BSA + Tween-20 (0.01%) thrice
• Alkaline phosphatase activity was assayed with p-nitrophenyl phosphate sodium salt dissolved in substrate buffer (lmg/ml).
• The plates were incubated for 30 min in dark and the reaction was stopped with 100µl of 1.5 M NaOH solution
• Reading was taken at 405nm in ELISA reader.
Result:
Three T. indica isolates (P7, P9 and PI6) with known virulence and four isolates (KBPN, KB3 JK and KB9) of unknown virulence levels were cultured in modified Potato Dextrose Agar media in-order to detect the behaviors of fungal isolates. Statistically Significant variation in radial diameter was observed in all three main isolates studied at all three time intervals. Host determinants have given a boost to the increment compare to the control. Fast growing isolate P9 and KBPN has shown higher radial diameter and the increment in radial diameter was always higher compare to least and moderately virulent isolates (P7 and P16 respectively). The growth behaviors of JK and KB9 were almost similar as shown by P16 isolates and KB3 growth was similar to P7
isolates as indicated in the Figure 1. At the sixth day, the variation in radial diameter with respect to the treatments was clearly depicted and the variation in the same at 9th day is not that apparent as the isolates have already covered the whole Petri plate indicating a rapid growth in them with host determinants treatment.
Radial diameter variation with respect to the host factors was statistically analyzed individually with the sixth day data and it was revealed that the variation among the treatments (Control and WH 542) was significant (p >0.05) in all seven isolates compared. Host determinants treatment on fungal strains tends to increase their mycelination and it can be concluded that increase in mycelination of fungi leads to impose more virulence levels in the host and prolific multiplication of pathogen inside host lead to more damage to developing grains.
In order to determine the virulence level of Tilletia indica isolates/strains, the causative agent of Karnal bunt disease of wheat and differentiating it from similar, Tilletia indica isolate, mycelial fragment antigen from all isolates were subjected to indirect ELISA determination at 7 day to 30 days incubation. In order to determine the optimum antibodies dilution and optimum antigen concentration for the development of indirect ELISA, different dilution of antibodies were used to determine immunoreactivity with 100 ng of mycelia antigen. From the
immunoreactivity results of indirect ELISA, it was concluded that dilution in the range of 1:250 to 1:500 primary anti-mycelial antibody is appropriate for determine the virulence level of T. indica. Similarly results of optimum antigen concentration using different concentrations of mycelia antigens and 1: 250 dilution of anti-mycelial antibodies, it was concluded that 75 to 200 ng of mycelial antigen per well is the appropriate concentration (Figure 2).
All fungal isolates P9, P16, P7, KBPN, KB3, KB9 and JK were cultured on modified Potato Dextrose Broth media with and without host determinants (Acetone extracts from S2 stage of WH-542 wheat spike). Acetone extracts were incorporated at 1: 10 media to extract ratio and culturing was carried out without replenishing of host determinants until the harvesting at desired time intervals. A time series estimation of antigenic variation was carried out on 7th, 14th, 21st and 30th days after the inoculation. Indirect ELISA was performed on mycelial antigen in order to detect any variation in immune-reactivity after the induction with host factors. Variation in immunoreactivity in terms of OD405 on mycelial antigen was detected using mycelial antigen concentration 125ng per well with 1:500 dilution of anti-mycelial antibody developed against mycelial antigens (Figure 3). It was interesting to see that the isolates with known virulence level, virulent (P9), moderately virulent (P16) and least virulent (P7)
exhibited very clear increase in OD (201.35 %, 141.17 % and 136.36%
respectively) at 7th day in the presence of host determinants. At the same
time interval, the isolates (KBPN, KB3, KB9 and JK) with unknown
virulence level also exhibited an increase in OD405 (212.69%, 134.54%,
172.37% and 162.50% respectively) when cultured in presence of host
determinants. At the 14th 21st and 30th days of growth in culture, a drastic
reduction in OD405 was observed with all the isolates P9, P16, P7, KBPN,
KB3, KB9 and JK (121.52%, 122.85%, 103.89%, 119.72%, 114.72%,
106.41% and 112.34% at 14th days), (123.88%, 104.76%, 120.32%,
105.88%, 104.47%, 104.92%, and 129.16% at 21st days), and (118.75,
100.00%, 108.95%, 107.82%, 113.12%, 115.68% and 115.22% at 30th
days) treated with host determinants. It is quite evident that all the isolates
have exhibited increase in OD at the 7th day with the presence of host
determinants with considerable variations respectively to their inherent
growth behavior.
As a thumb rule all the isolates have exhibited increase in OD405 at the 7th day with the presence of host determinants with considerable variations respectively to their inherent growth behavior. In general fast growing strains tend to show high reactivity levels. As the virulence levels of P9, P16 and P7 already known this finding can be matched with virulence levels of the pathogens and also can be used as a bio marker for detection of pathogenicity of a pathogen with an unknown history. The pathogenic level of KBPN is unknown but with the present
observations, it can be considered as a highly pathogenic strain. This bio marker for detection of pathogenicity can be used in-order to group fungal strains in to the respective virulent groups more economically without doing any field testing.
Contradictory to the conclusion made above (fast growing strains show low reactivity without host determinants) the fungal strains treated with host determinants exhibited fast growth as well high levels of immuno-reactivity. This can happen due to the expression of more antigenic epitopes on the mycelial surfaces such as some signal receptors. The signaling molecules present in the host determinants can enhance the expression of those genes leading to the more immuno reactivity as observed in the above experiment. With all these observations it is possible to conclude that the host determinants are acting as growth factors (Nutritional) as well as hormones which enhance expression of specific genes involved in the alteration of surface properties.
This finding can be used as a bio assay to discriminate a unknown population of T. indica into their respective pathogenic levels with respect to a known host determinants derived from susceptible variety without field testing.

WE Claim
1. A method for grouping the fungal isolates of Tilletia indica in to distinct pathogenic groups under induced conditions in presence of host determinant(s) or plant extract, by using immunological approach, comprising the steps of: (a) Liquid culture of fungal isolates with host determinants (b) Harvesting of mycelia at specific time intervals (c) Detection of immunoreactivity using indirect ELISA with mycelial antigen and anti mycelial antibodies (d) Grouping of Tilletia indica isolates in to virulent, moderately virulent and least virulent with reference to the growth behavior and immuno-reactivity of Tilletia indica isolates of known virulence levels.
2. The method of claim 1 where in mycelial fragments of Tilletia indica are used as antigen.
3. The method as claimed in claim 1, wherein mycelial antigen
concentration is used 75-200 ng in the indirect ELISA
4. The method of claim 1 where in the using anti-mycelial polyclonal antibodies of Tilletia indica used in the range of 1:250 to 1:500.
5. The method of claim 1 where in the host determinant(s) or plant extract prepared from S2 -stage of developing wheat spike induced the growth of T. indica. (Z score).
6. The method as claimed in claim 1, wherein plant extract and fungal
growth medium is used in 1:10 ratio.
7. The method as claimed in claim 1, wherein in the growth of fungal
isolates, mycelial mass harvested at 7th day to 30 days for isolation of
mycelial fragments and determination of differential immune
reactivity under induced conditions.
8. The method for developing immunological marker to assess the
pathogenicity levels of T. Indica fungal isolates as described and
illustrated herein.

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Patent Number

279419

Indian Patent Application Number

1761/DEL/2010

PG Journal Number

04/2017

Publication Date

27-Jan-2017

Grant Date

20-Jan-2017

Date of Filing

28-Jul-2010

Name of Patentee

G.B. PANT UNIVERSITY OF AGRICULTURE & TECHNOLOGY

Applicant Address

PANT NAGAR-2633145, UTTARAKHAND, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	ANIL KUMAR DE	DEPARTEMENT OF MIOLECULAR BIOLOGY AND GENETIC ENGINEERING, COLLEGE OF BASIC SCIENCE & HUMANITIES, G.B. PANT UNIVERSITY OF AGRICULTURE & TECHNOLOGY, PANTNAGAR UTTARAKHAND-2633145.
2	J.M SENEVIRATNE	DEPARTEMENT OF MIOLECULAR BIOLOGY AND GENETIC ENGINEERING, COLLEGE OF BASIC SCIENCE & HUMANITIES, G.B. PANT UNIVERSITY OF AGRICULTURE & TECHNOLOGY, PANTNAGAR UTTARAKHAND-2633145.
3	ATUL KUMAR GUPTA	DEPARTEMENT OF MIOLECULAR BIOLOGY AND GENETIC ENGINEERING, COLLEGE OF BASIC SCIENCE & HUMANITIES, G.B. PANT UNIVERSITY OF AGRICULTURE & TECHNOLOGY, PANTNAGAR UTTARAKHAND-2633145.

PCT International Classification Number

A23J

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA