Title of Invention

A METHOD OF STABILIZATION OF SOFT-WALLED CONIDIAL SPORES OF A NEMATODE TRAPPING FUNGUS

Abstract

1. A process of stabilization of nematode trapping fungal conidiopores suitable for application in the field comprising of the following steps : a. Culture the nematode trapping fungus and produce the conidial spores by known methods b. Count the number of conidial spores c. Suspend the conidial spores in 15% glycerol inn deionized water at a concentration of 3 to 4 x 104 per ml d Store the suspension at -20°C e. Evaluate the stabilization of the conidial spores by testing for viability, mycelial predaciousness and for larvicidal effect on nematodes such as Haemonchus contortus Dated 23rd August 2004 Signature of the agent (HM JAGANNATH) To, The Controller of patents, The Patent Office, Mumbai

Full Text

FORM 2 THE PATENTS ACT, 1970 (39 of 1970) COMPLETE SPECIFICATION (see Section 10) A method of stabilization of soft-walled conidial spores of a nematode trapping fungus We, NATIONAL DAIRY DEVELOPMENT BOARD, a body corporate constituted under the National Dairy Development Act 1987 (37 of 1987), Anand, State of Gujarat, India and Sardar Patel University, Vallabh Vidyanagar, Gujarat, India. ORIGINAL The following specification particularly describes the nature of this invention and the manner in which it is to be performed. 1324/MUM/2003 GRANTED 15/12/2004 Field of invention : This invention pertains to the field of stabilization of nematode trapping fungal conidiospores with potential for use as systemic application in animal agriculture Prior art: Usage of anthelmintic drugs for control of animal nematodes came under scrutiny in recent times with the increasing predominance of undesirable effects, drug resistance of the worms being one of them (Sanyal 2000). Biological control, a key component under the larger umbrella of Integrated Pest Management or the IPM had offered until date, the most promising and economically sustainable approach to an eco-friendly method of worm control. Fungi, the group of organism that are evolutionarily closest to present day plants and animals than others, yet distinct from them, and one which represent the ultimate branches of the 'eukarya' domain has taken the lead in this aspect with several members of the sub-group (class) Deuteromycota demonstrating potential for nematode predatory activity. Since this important finding, there has been a systematic approach to study of these organisms, whereby several sub-disciplines had opened up, modes and means of application of the organism into the field for effective decrease in pasture larval burden of animal nematodes being one of them. Mycostasis or fungistatis is a microbial-induced phenomenon. It is often observed in form of spore dormancy on the topsoil particularly when microbial population is significantly high. Among several factors that are potential causes for the phenomenon including certain volatile inhibitors, nutrient depletion by microorganisms happened to be the strongest body of evidence. However, preservation of viability in spores for an extended period of time happen to be the single important aspect of mycostasis that has potential for use in application related studies in these economically important microorganisms. Spore phenotype assist in dividing the nematophagous fungi into two loose groups, one being those producing tough-walled chlamydospores while the other, fragile and comparatively thin-walled conidia. Ability to survive gut passage provides an obvious advantage to chlamydospores-forming nematophagous fungi such as Duddingtonia flagrans (Hertzberg et al., 2002.) as it becomes amenable to a natural, economical and grossly uniform method of spore dispersion in the pastureland. Nematophagous fungi producing tough-walled chlamydospores remain a favorite for delivery device application studies among the biological control community at large. There are fungi that have documented nematophagous ability which do not produce chlamydospores for their propagation. Example of such fungi are Arthmbotrys oligospora, Arthrobotrys oviforrnis, Geniculifera eudermata (Waller et al., 1994) Because of this reason, they are not included in delivery device formulations, which are heavily dependent on gut passage. From the point of view of evolution, fungi producing conidia are less adaptive to nature compared to their counterparts that produce chlamydospores. This is primarily because chlamydospores are tough walled and is capable of maintaining longer viability under adverse conditions and thus more suited for survival. Conidia by morphology are fragile spores primarily intended for mitotic propagation of fungi that fail to demonstrate sexual reproduction and clear formation of gametes. It is therefore necessary to develop methods whereby the inherent fragility of conidia is addressed rendering them to be useful in biological control of nematodes of veterinary importance The present study was undertaken to investigate into the mode and means of compensating this disadvantage in conidia-producing counterparts, employing an Indian isolate of nematode-trapping Artobotrys oviforrnis as the model system, documented for profuse production of oval-shaped conidia (Nagee et al., 2001). Summary of invention : This specification describes a process of stabilization of nematode trapping fungal conidiospores suitable for potential systemic use in animal agriculture using Arthrobotrys oviforrnis as an example comprising of the following steps: a. Culture the nematode trapping fungus and produce the conidial spores by known methods b. Count the number of conidial spores c. Suspend the conidial spores in 15% glycerol inn deionized water at a concentration of 3 to 4 x 104 per ml d. Store the suspension at -20°C e. Evaluate the stabilization of the conidial spores by testing for viability, mycelial predaciousness and for larvicidal effect on nematodes such as Haemonchus contortus Worm management in intensive farming practice involves stationing large number of animals in comparatively small area. As dispersion of fungal material for biological control in such location is less of a challenge, artificial administration of the preserved spores on suitable growth substrate in such intensive farming may prove convenient, effective and economical. While the efficacy of the procedure of stabilization of the conidial population preserving their viability is demonstrated in the present study, the future remains open for further improvement of the storage medium that may possibly be dispersible in natural for pressurised application in the farm. Further, amending the spore dispersion medium with other antibacterial or sanitation-related chemicals and biological material that do not interfere with the spore viability would be further directions for improvement. Detailed description of invention : Example 1: Fungal isolate ' Fungal culture of Arthrobotrys oviformis was maintained in corn meal agar plate (Hi Media, India) at 28°C, amended with tetracycline (Amersham Pharmacia, USA) at a final concentration of 35 μg/ml to prevent bacterial growth. For growth in liquid medium, a 10 day old plate was washed with 1 ml of sterile distilled water and used to inoculate 50 ml of liquid Czapex Dox medium (Sigma Aldrich, USA or Himedia India) in a 250 ml flask, that was then maintained under shaking condition (250 rpm at 28°C) for 7 days prior to mycelial extraction. The strain of Arthrobotrys oviformis has been deposited at Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Institute of the Royal Netherlands Academy of Arts and Sciences, PO Box 85167, 3608 AD Utrecht and the deposit number is : "CBS 110209" . Example 2.0 : Production of conidial spores Conidial spores were produced on large scale using dried corn seeds as substrate in accordance with Sanyal, 2000. Briefly the procedure is as follows : Artobotrys Oviformis was grown on 200 grams of com grains presoaked overnight in 200ml water in a one liter conical flask. A piece of medium (about 25mm2) containing fungal growth obtained as in example 1 was put into the flask and incubated at 27°C for 4 weeks. The flask is shaken daily for a week to get uniform growth. Harvesting of the spores : Method I: The conidial spores were harvested from the grains by vigorous washing with).6M sterile Potassium chloride and sieving in a 0.18mm nylon sieve Method II : Alternatively, the medium was aseptically subjected to vacuum-assisted pressurized filtration (Sigma Aldrich chemical company, USA) through sterile laboratory-grade filter paper in order to assist selective blocking of the newly formed mycelia and forced passage of un-germinated spores if any, that were further concentrated through centrifugation at 7500 RPM for 10 minutes at 8°C. The number of spores were counted using a haemocytometer Example 3.0: Identification of the appropriate storage medium and temperature conditions. Storage buffer Three different storage medium, viz., 15% glycerol in sterile de-ionized water, 15% mineral oil and 100 mMolar Tris-CI (pH-7.0) were tested in the study. For identification of the appropriate storage medium and temperature conditions, 105 conidial spores were inoculated in 3 sets of 6 ml test storage buffers each, in a transparent Petri dish of 40 mm diameter and incubated at -20°C, 4°C and 27°C respectively. Growth was monitored with a Nikon-make low power binocular microscope for the next 5 days and results subsequently recorded in terms of presence or absence of spore germination. Initial experiments suggested that spores failed to remain under imposed dormancy in mineral oil as well as Tris-CI buffer. This was evident from the fact that significant spore germination was recorded within the initial 5 days of incubation at 27°C as well as 4°C. Inorganic mineral oil constituents are unlikely to support growth of fungi as the taxon lack chemolithotrophic mode of nutrition. However, impurities and additives in the mineral stock as well as presence of the Trisma base with chloride as the ionic partner as a stable yet feebly degradable chemical component may have provided a minimal substrate to the spore structures of the organism, the group of which is well known for its ability to exhibit extreme oligotrophy. Although glycerol buffer exhibited trace spore germination (1-3/3.6 x 104) on the 15th day of storage at 4°C as well as 28°C, it was chosen for further experimentation, partly because of historical reason, it being the preferred medium for cryopreservation of many microorganisms and its chemical status as poor carbon source, along with the associated anti-freezing properties at low temperature. Further, glycerol medium imparted satisfactory apparent dormancy to the spores at -20°C temperature and to a significant level at 4°C and 28°C. Therefore, 15% glycerol in sterile de-ionized water was chosen for further evaluation. Example 3.0: Storage conditions The spore concentration of the master stock collected from fungal culture on com seeds suspended in 50 ml of 15% glycerol in sterile de-ionized water, was 3.6 x 104 per ml as measured through haemocytometer counting. (a) Identifying storage temperature for glycerol buffer Prior to long term storage, in a small-scale pilot experiment, five 40 mm transparent Petri dishes containing 6 ml of conidia-suspended glycerol buffer were stored at -20°C, 4°C and 28°C respectively. After 5 days interval, each of the plates was monitored for spore transformation to mycelial state under a binocular low power microscope, up to maximum of 25 days (5 observations, each with 5 day increment). (b) Viability assay For viability experiment, ten grams of worm-egg free cattle faecal matter was placed in an open Petri dish of 90 mm diameter and placed on to a larger Petri dish containing 10 ml of sterile de ionized water, covered to prevent evaporation and incubated at 28°C for different number of days prior to recording of the data. A single set of experiment consisted of six such Petri dish setups and 3.6 x 104 fungal spores were inoculated on the faecal matter in each plate at the rate of one per day for the next 5 days and a single one was left un-inoculated to serve as control. On the fifth day, all the Petri plates were uniformly mixed with ~ 500 infective larvae per ml of Haemonchus contorfus larval suspension in water obtained by known methods and incubated for another five days prior to larval counting, in the 10 ml water barrier. All experiments were done in triplicate and the mean value determined for analysis and conclusions. (c.) Normalization of larval counts Following 5 days of incubation after larval application, the total number of larvae that escaped in control setup was calculated and assumed as normal. There after, larval escape (LE) value of all other setups was expressed as a fraction assuming the control as unity. (d) Qualitative assay of mycelial predaciousness The predatory property of the fungal mycelia was assayed through the thin-film technique. Ten ml of Czapex Dox liquid medium, in 90 mm transparent Petri plate was inoculated with 3.6 x 104 spores (1 ml of preserved spore suspension) and co-incubated with 400 larvae at 28°C. Trapping was monitored in situ after 36 hours employing a Leitz-make high power microscope and documented using a WILD MPS11-make microscope photographic system attached to a semiautomatic photometer. (e) Isolate confirmation employing DNA markers For confirming the mycelial identity generated from conidia, molecular signature obtained from pure culture were compared with that obtained from regenerated mycelia following cryopreserved conidial inoculation in liquid Czapex Dox medium. Three different multiple arbitrary amplicon profiles (MAAP) were generated using the technique of Arbitrarily Primed PCR (Welsh and McClelland, 1990) employing three oligonucleotide primers unrelated to known fungal genome sequences. The primers were: A: 5'-ACAGACAGAAACTCAATGAAAGCA-3'; B: 5'-CGAATTCCAMTCTGTTAATTTGCT-3'; C: 5'-TCTCTCTAATGGAGTTGG1111IG-3'. For this study, DNA was extracted from fungal mycelia by the method of Raeder and Broda 1988 and thermal cycling performed in a 12.5 μl reaction volume that comprised of 50 ng of purified template DNA, 5 pmoles of each primer, 200 μl Molar dNTPs, 0.5 unit of Taq DNA polymerase and Taq DNA polymerase buffer at a final concentration of 1x, containing 1.5 molar MgCI2. Thirty-five cycles were performed, each with a denaturation step of 94°C - 30 seconds, an annealing step of 27°C -1 minute and an extension step of 72°C -1 minute. PCR amplicons were resolved in a 1.5% agarose gel, stained with Ethidium Bromide and documented using a BioRad-make CCD camera system under control of the 'Quantity One' software. Band profiles were analyzed employing Kodak Digital Science 1D Image analysis software. Lane scanning paths were manually marked and bands detected with moderate sensitivity covering 70% bandwidth. Scan images were subsequently generated as a function of the signal profile detected in the lanes. As these images were de-linked from the band sensitivity parameters of the software with provisions for manual setting, they reflected true signal complexity of a lane. Example 4.0: Evaluation of cryopreserved conidial spores The cryopreserved conidial spores of Aovifoimis were evaluated in two ways. Following storage for a period of 10 months at -20°C in 15% glycerol in deionized water, spore suspension was used to inoculate 10 ml of liquid Czapex Dox medium in 90 mm sterile, transparent Petri dish and co-incubated with live larval suspension. Monitoring under low power microscope provided important information. First, transformation of agglomerated spores to mycelial mass could be documented in situ (Figure 1A). Further, trapping of the live larvae could be clearly observed that substantiated preservation of this single crucial phenotype for the organism. Spores gradually formed mycelia after 24 hours of incubation at 28°C. This transition delayed with increase in temperature reflecting its growth temperature optima identified through earlier experiments. This report is also the first to document larval trapping in a liquid medium for this isolate. Careful observation showed that larvae in the liquid medium were in greater dynamic state and its contact with newly germinated mycelial mass spread across the medium was more frequent and from predatory point, more effective In a separate experiment, viability test as well predatory activity was assayed using worm-egg free bovine faecal matter as the target of inoculation. The faecal matter comprised of fungal as well as larval population that were allowed to co-incubate for varying period. Fungal predatory activity prevented live larva from escaping into the peripheral water source. On the contrary, the faecal matter un-inoculated with cryopreserved conidia and confirmed for nil indigenous fungal population through traditional nematophagous fungi isolation technique (Sanyal 2000), allowed 280 [mean deviation (MD): 6] larvae to escape, thus providing a mean LE value of 280 for the control set up. Microscopic examination of the larval suspension revealed a heterogeneous population of live (motile) and dead (non motile) larval population. In view of the fact that dead larva shall not be able to exhibit the typical hydrophilic behavior that generate LE value count, it would contribute to overestimation thereby adding an LE count even to the control that is hypothetically erroneous. Therefore, this value for control was assumed as unity (280=1) irrespective of the true count of larvae in the stock suspension. Absence of worm egg in the faeces confirmed inoculated larval suspension as the only source of nematodes. Spore inoculation at an interval of 1 day provided a varying degree of Fungal Maturity Index (FMI) that was indicative of the period of spore growth that supported larval trapping. FMI index further comprised of a fixed value (FMIfx) of 5 days that represented uniform incubation period with larvae prior to LE value count and a variable value (FMIvr) that represented varying period of conidial incubation prior to larvae application (5,4,3, 2 and 1 days) thus generating the equation of FMI = FMIfx + FMIvr. Experimental design provided a minimum of six (FMIfx: 5; FMIvr: 1) and a maximum of 10 (FMIfx:: 5; FMIvr: 5) FMI value. Results show that at a FMI of 10 and 9, the LE count was nil. However, with gradual decrease in FMI from 8 to 6, there was a corresponding rise in LE count. As the FMIfx was fixed at a value of five, the change in LE count could directly be related to the FMIvr (Figure 2). From the data it could thus be concluded that a conidial pre¬incubation of 4-5 days provided adequate maturity to trap mature larvae at cent percent value leading to nil LE count. However, conidial pre-incubation of 3, 2 and 1 day allowed 17.6, 28.5 and 57.1% of larval escape respectively suggesting that pre-larval application period of less than 4 days were insufficient in demonstrating total larval predation. It is interesting to note that 24 hours of conidial growth was capable of trapping 42.9% (MD: 2.7) of the larvae under laboratory conditions. This observation is of importance in view of the fact that in the event of delayed spore application on to a zone of interest, predation is likely to initiate at an early stage thus delivering results even if larval maturity in the area had progressed significantly. Further, aligning the general observation that nematode maturation period from egg to larva stage was 5 days, with the one that nil LE count was recorded at minimum FMIvr value of 4, it could be concluded that in the event of co incubation of nematode egg with fungal conidia, this organism would enjoy an extra day of total maturity (5-4) when the nematode enter the final L2 phase on the fifth day. Long arbitrary primers are known to simplify DNA fingerprint profile by destabilizing annealing through extended 5' terminal mismatched sequences (Caetano-Anolles. 1994). Therefore, these could conceivably increase detection of polymorphic DNA in complex genomes by scanning extended annealing sites for sequence changes. In this study, we have employed three unrelated oligonucleotide primers (A: 24, B: 25, C: 24-mer) to generate multiple banding profiles that were found to be diagnostic for the organism. The genetic identity of the organism following preservation and revival could be adequately confirmed employing this molecular tool. In view of the specificity associated with DNA markers, this appeared to a plausible method for confirming strain identity and purity. A scan profile rather than DNA banding pattern was presented as molecular signature in view its ability to focus on minute lane signals that may be of diagnostic significance. References Caetano-Anolles, C. (1994): MAAP: a versatile and universal tool for genome analysis. PI Mol Biol. 25: 1011-1026. Nagee, A., Mukhopadhyaya, P.N., Sanyal, P.K., and Kothari, I.L., (2001): Isolation of Nematode- Trapping fungi with potential for biocontrol of parasitic nematodes in animal agriculture, from ecological niches of Gujarat. Intas Polivet 2: 27-29. Sanyal, P.K. (2000) Nematophagous activity of Arthrobotrys oligospora against pre-parasitic stage of Strongyloides papillosus. J Vet Parasitol. 14: 63-65. Sanyal, P.K. (2000) Screening for Indian Isolates of predaceous fungi for use in biological control against nematode parasites of ruminants. Vet Res Commu. 24: 55-62. Welsh, J., and McClelland, M. (1990): Fingerprinting genomes using PCR with arbitrary primers. Nuc. Acids Res. 19: 861-866. Waller PJ, Larsen M, Faedo M and Hennessy DR (1994). Veterinary Parasitology 51; 289-299 Hertzberg H, Larsen M and Maurer V (2002). Bert Munch Tiererztl Wochenschr 116:278-285 We claim: 1. A process of stabilization of nematode trapping fungal conidiopores suitable for application in the field comprising of the following steps : a. Culture the nematode trapping fungus and produce the conidial spores by known methods b. Count the number of conidial spores c. Suspend the conidial spores in 15% glycerol inn deionized water at a concentration of 3 to 4 x 104 per ml d Store the suspension at -20°C e. Evaluate the stabilization of the conidial spores by testing for viability, mycelial predaciousness and for larvicidal effect on nematodes such as Haemonchus contortus Dated 23rd August 2004 Signature of the agent (HM JAGANNATH) To, The Controller of patents, The Patent Office, Mumbai

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