Title of Invention

"COLD PROCESS TECHNOLOGY FOR THE PREPARATION OF UREA MOLASSES MINERAL BLOCK"

Abstract

5. Abstract of Invention The invention relates to a "Process for rapid production of Nomuraea rileyi on crushed barley and storable formulation in sunflower oil". The insect pathogenic fungus Nomuraea rileyi, is a potential biological agent for the control of major insect pests such as Spodoptera litura, Helicoverpa armigera and Plusia spp., on several crops. Current recommendations for pest management do not include field application of Nomuraea rileyi primarily due to non-availability of a rapid and cost-effective mass production methodology. Multiplication of N. rileyi is constrained by its slow growing nature and specific growth requirements. A novel process is claimed for the mass production of Nomuraea rileyi on barley aged for a minimum of 6 months after harvest and crushed and supplemented with yeast extract in glass trays kept inside autoclavable and reusable polythene bags with 0.02µ, filter. The process leads to rapid production of conidia with yields ranging 6.0-7.5 x 109 conidia/ g substrate. Harvested conidia were formulated in sunflower oil with Triton-X-100 as an emulsifier. The formulation is of a storable nature. The formulation resulted in significant mortality of S. litura populations on groundnut and castor crops at the effective dose of 2 x 1011 conidia/1. Material costs for producing conidia for 1 acre at a spray volume of 100 liters is lowest at Rs 126.00 with the process claimed while it is Rs 4415.00 with the standard SMAY medium.

Full Text

Details of Invention This invention relates to a process for rapid and cost-effective multiplication of an insect pathogenic (entomopathogenic) fungus Nomuraea rileyi and its storable formulation. The fungus has potential for the control of several economically important insect pests of global importance viz., Helicoverpa armigera, Spodoptera litura, Plusia sp. etc. attacking crops such as groundnut, soybean, cotton, castor and tomato. The fungus has the ability to cause epizootics under congenial conditions. The fungus is reportedly safe to human beings and other non-target organisms including insect parasites and predators. Field use of fungal agents for crop protection is possible only when a rapid, cost-effective and efficient mass production technique is available. We hereby report such a rapid process for mass production and method for formulation of N. rileyi. Prior Art: The standard semi-synthetic medium used world over for multiplication and maintenance of Nomuraea rileyi is the Saboraud's maltose agar medium (SMAY) (Bell, 1975) where sporulation is initiated at 10 days at 24 + 10°C and spores harvested after 21 days. Ignoffo et al, 1976 reported average time to initial sporulation as 8.8 + 0.7 days at 25°C. The cost of ingredients in the existing medium is prohibitive. Consequently, the production of the fungus using this medium although reliable is not cost-effective for commercialisation. SMAY composition Agar-15 g Maltose - 40 g Peptone-10 g Yeast extract - 5 g Water- 1000ml The fungus multiplication is reported only on two solid substrates viz., polished rice grains (Silva and Loch, 1987) and crushed sorghum yielding 1.4 x 109 conidia per g of the substrate (Vimala Devi, 1994). However, the processes have not been used for scale-up production/ commercialization. Use of the solid substrates for mass production of fungi proves highly cost-effective. Although labour intensive, they require less capital investment and are suitable for mass production in developing countries where labour is cheap. Due to the recent technological developments, they can be mass produced by automation also and therefore suitable for even the developed countries. Identification of amenable virulent isolates and their registration, a cost-effective protocol for multiplication are the pre-requisites for ensuring a scale-up production. Some fungi associated with soybean pests, such as Nomuraea, Entomophthora and Neozygites, are difficult to produce in either the conidial or the mycelial form, which limits their use as microbial pesticides (F. Moscardi & D.R. Sosa-Gomez. 1996). N. rileyi requires aerobic conditions for optimal growth and sporulation. Formulating pathogens in oils increases their effect (Moore and Prior, 1993). Oil based ULV formulations of the entomopathogenic fungus Metarhizium flavoviridae have been developed to enhance the infectivity to desert locusts (Bateman et al., 1993). Vegetable oils were found to be compatible when combined with N. rileyi just at the time of spraying (Vimala Devi and Prasad, 1996). However, no storable formulations of N. rileyi with vegetable oils have been developed. These formulations serve to improve the shelf life and field persistence and can be readily suspended in water just before spraying. Keeping in view the importance of the pathogen with respect to the International market and also its effectiveness against the economically important pests Helicoverpa armigera, Spodoptera litura and Plusia sp., studies were carried out to develop cost-effective and reliable protocol for its multiplication using a solid substrate and development of a storable formulation of the spores in oil. Importance of the fungal pathogen for crop pest control (Background information) The fungus has potential for the control of economically important insect pests of global importance viz., Helicoverpa armigera, Spodoptera litura, Plusia sp. attacking crops such as groundnut, soybean, cotton and tomato. The details of yield losses due to these pests on the various crops and the current management practices are furnished below: Groundnut. The gram pod borer Helicoverpa armigera and tobacco caterpillar Spodoptera litura are important on rabi groundnut causing 20 - 25% yield losses. During the seedling stage, infestation by H. armigera could result in 100% yield loss. Both the pests have developed resistance to insecticides (Reddy and Prasad, 1991). Normal recommended pest management is through IPM i.e., cultural practices, use of resistant cultivars, use of pheromone traps, spraying of NPV, bio-pesticides of neem origin and need based pesticide application using endosulfan. The environmental conditions during the rabi groundnut favour the use of Nomuraea rileyi for the management of both the pests i.e., tobacco caterpillar and gram pod borer. The fungus can be used against these pests in the coastal belts and humid conditions. Soybean Soybean is a luxuriant crop with lush green soft, succulent and nutritive dense foliage attracting 273 pests of which a dozen insects are of significant importance. The important noctuid pests damaging foliage and pods with susceptiblity to the entomopathogenic fungus N. rileyi are as follows: (Table Removed)The control measures A, B, C and D indicated below do not include application of N. rileyi formulation. A. Use of light traps for collecting and destroying nocturnal flying adults. B. Deep ploughing after harvesting to expose hibernating larvae/ pupae to sunlight and birds. C. Sprays of 0.1% Endosulfan 35 EC / 0.04% Quinalphos 25 EC / 0.005% Cypermethrin 25 EC / 0.1% Fenvalerate 20 EC / 0.05% Methyl parathion 50 EC D. Dusting Endosulfan 4% / Quinalphos 1 .5% / Parathion 2%. The close canopy of soybean (congenial microclimate) and the favourable environmental conditions during the pest activity period are found to favour the use of the fungus Nomuraea for effective control of the above mentioned pests which are highly susceptible to it. Cotton As a commodity crop, cotton has huge national significance and much of the export earnings (> 30%) are from finished and value-added goods of cotton fibre. In India, cotton is cultivated in about 5% of the cropped area and almost 50% of the insecticides formulated in India are used on this crop which adversely influence the balance of nature. Growth of cotton plant is indeterminate and this feature supports a wide range of pests and diseases. Early season sucking pests followed by bollworms and foliage feeders and late season stainers among insects are the important yield limiting factors in cotton production. Yield loss due to cotton pests measure up to 40-60% and the current effort to control them through pesticides has led to a serious economic, ecological and social consequences. The pest has developed resistance to many insecticides (Mc Caffery et al., 1989). Among the key pests, Helicoverpa armigera is wide spread in occurrence and devastates the crop. Mortality of Helicoverpa armigera and Spodoptera litura due to Nomuraea rileyi was high on cotton inspite of heavy usage of insecticides. (Table Removed)The current recommended control measures of these pests do not include application of Nomuraea. The period during which the pest occurs is conducive for fungus development and Nomuraea can be effectively used for its management in cotton where indiscriminate use of pesticides is continuously causing serious environment problems and development of pesticide resistance in major pests. Tomato: Heavy infestation of tomato fruit borer Helicoverpa armigera has been reported during July -November. Yield losses to the extent of 65% have been reported due to this pest in Punjab (Singh and Singh, 1975). Current pest control in tomato is predominantly dependent on use of pesticides. The fungal pathogen has the potential for control of Helicoverpa on tomato under congenial conditions of temperature and humidity (September-December) with no residual effect. Invention Particulars: While the effectiveness of the fungal pathogen N. rileyi to control lepidopteran pests was known, this could not become practical due to the absence of a cost-effective and efficient method to produce N. rileyi spores and its formulation. The pathogen is hence not available commercially. Therefore studies were conducted to invent such a process. These studies deployed a solid substrate medium for the rapid mass production of the entomofungal pathogen N. rileyi in re-usable polythene bags with air filter and develop a storable formulation with a view to commercialise the production and use of the fungal pathogen for the control of the major lepidopteran pests. Other details of the methods and formulations are as follows A to E. A. Media composition, Preparation & Cultivation of the fungal pathogen: On Solid substrate using Barley: Solid substrate medium comprised of crushed barley of six months and above age - 30 g, Yeast extract Type I (Hi-media) - 0.75 g and single distilled water - 275 ml. The medium is prepared as follows: Take 30 g of crushed barley and 0.75 g yeast extract in a glass tray and add 275 ml of single distilled water. Place the tray in an autoclavable and reusable polythene bag (Sigma make 44 x 20.5 cm) with 24 mm 0.02µ filter disc. The filter provided on the bag ensures a continuous supply of sterile air in a passive way thereby providing ample aeration for growth and sporulation of the fungus. The bag can be autoclaved several times and can be used repeatedly. Seal the open end of the cover using a bag-sealer and autoclave at 15 psi for 20 minutes. For cultivation of the pathogen, the autoclaved bag is allowed to cool and one end of the bag then cut near the flame in a laminar flow and dry powder of conidia with a microspatula is inoculated. Seal the cut end of the bag near the flame itself to avoid any contamination. Incubate the tray in darkness at 25°C. Mycelial growth starts on the 3rd day and continues till 5 days. Sporulation starts on 5th/6th day and continues till 9 days. Then transfer the bagged trays to a refrigerator and store. Sporulation continues even in the refrigerator for a week. It is found that crushing of the grains to allow efficient utilization of the maltose present in the seed is essential and ageing of barley grains plays a very important role in terms of sporulation of the fungus. Barley which is less than 6 months old supports only mycelial growth and not sporulation. Barley which is six months old or more (post-harvest) supports good sporulation. Barley infested with storage pests also supports good sporulation and therefore can be used cost-effectively. This information is the first of its kind for production of N. rileyi on aged and crushed barley grains. In addition, for the first time, bags with 0.02u filter are identified for optimal sporulation. B. Extraction of spore and formulation i) Spore extraction - Seven days after transfer to the refrigerator, the trays are removed from the polythene bags and kept for overnight drying in the laminar flow. The spores are harvested from the surface of the medium with a spatula and used for formulation. One tray yields 1.5 g dry spore approximately. One gram of dry powder yields 1.8 - 2.0 x 1011 conidia based on the isolate. ii) Oil Emulsion -To every 1.0 g of dry spore, add 350 ul Triton X-100 followed by 350 ul distilled water. Mix well and then add 5.3 ml of refined sunflower oil. Stir well the mixture to avoid any clumps and store at 25°C. For spraying, suspend 6 ml of the formulated product in one litre water. Formulation of N. rileyi with sunflower oil, of a storable nature is for the first time. C. Spore Yield Spore production is high in crushed barley (solid medium) particularly six month old or more old and infested by storage in comparison to the standard SMAY medium. The yield details are presented in the table below() D. Bioassay with conidia: Bioassays were conducted with six month old oil formulations of conidia of several isolates of N. rileyi multiplied on the above solid medium against two insect pests i.e., early 3rd instar larvae of S. litura and H. armigera at the effective dose of 2 x 108 conidia/ml (Vimala Devi, 1994). The conidia caused significant mortality of the larvae in the laboratory.Bioassay of Spodoptera litura with different isolates of Nomuraea rileyi at the effective dose of 2 x 108 conidia/ml(Table Removed) Bioassay of Helicoverpa armigera with different isolates of Nomuraea rileyi at the effective dose of 2x 108 conidia/ml (Table Removed)D. Field efficacy of the oil formulation The formulations (six month after storage) have been field tested at a concentration of 2 x 1011 conidia/1 S. litura on castor and groundnut under three different conditions: *" During high rainfall, the formulation caused total mortality of S. litura on castor. *• Under conditions of temperature ranging 20-30°C but only with one shower within a week after spraying, fungal mortality occurred to the extent of 70-75% with 20-25% parasitisation by Apanteles sp. indicative of its safety to the parasitoid. In kharif '97, when the maximum temperatures during the 10 day period after spraying varied from 28-33.5°C, fungal mortality to the tune of 45-50% was recorded on groundnut. The pathogen should be sprayed in the field where the congenial environmental conditions for its effectiveness and establishment exist i.e., Temperatures ranging 20-30°C and Relative Humidity above 70%. E. Cost involved for producing inoculum for One Acre: The spore required for one acre is 2 x 1013 conidia applied in a spray volume of 100 - 200 litres based on the crop canopy. The material costs involved for production of this spore from the solid medium and standard SMAY medium at current prices worked out is presented in table below: (Table Removed) 4. I/We claim 1. A novel process for rapid production of Nomuraea rileyi, an entomofungal pathogen, and preparing storable formulation in oil of the same, using crushed barley grains of above six months post-harvest age as solid substrate, yeast extract and single distilled water as ingredients in glass trays kept inside autoclavable, re-usable polythene bags incubated in darkness at 25°C for 9 days resulting in early initiation of sporulation as well as high spore yield and formulation of harvested spore in oil in a specific manner 2. The method of claim 1 of preparing solid substrate comprising six months aged crushed barley grains even if the grains are storage pest-infested for rapid production of Nomuraea rileyi 3. The method of claim 1 of production of Nomuraea rileyi in autoclavable and re-usable polythene bags with an air filter (pore size 0.02(j.) for ensuring continuous passive supply of sterile air resulting in early mycelial growth (3rd day) and early initiation of sporulation in 5 to 6 days time continuing till 16 days after inoculation to give a high spore yield 4. The method of claim 3 ensuring high spore yield averaging 6.0-7.5 x 109 spores per gram of substrate, by transferring the bagged trays 9 days after inoculation to low temperature in a refrigerator and incubating for 7 days for allowing continuation of sporulation 5. The method of claim 1 wherein the storable formulation in the oil is done by mixing 350µl of emulsifier Triton-X-100,350µ1 single distilled water and 5.3 ml of refined sunflower oil in a sequential manner to each gram of dried spore.

Documents:

944-del-2002-abstract.pdf

944-del-2002-claims.pdf

944-del-2002-correspondence-others.pdf

944-del-2002-correspondence-po.pdf

944-del-2002-description (complete).pdf

944-del-2002-drawings.pdf

944-del-2002-form-1.pdf

944-del-2002-form-19.pdf

944-del-2002-form-2.pdf

944-del-2002-form-3.pdf