Title of Invention


Abstract A process for preparation of ready to use solid biopesticidal formulations with improved shelf life based on the biocontrol agent(s) such as the antagonist mycoparasite(s) ex. Trichoderma species and the biopesticidal product obtained thereof are reported. The dried and powdered solid hyphae of the agent are blended directly with an inorganic or organic inert carrier with or without the addition of other formulant(s) to obtain a ready to use product complying the application requirements for use in pest control. It passed the accelerated storage test (54 ± 1°C, FAO 1999) prescribed for assessing the shelf life of two years of the chemical pesticides.
Full Text 4. Description
Field of invention
The invention discloses a process of preparation of solid formulation(s) of biocontrol agent(s) with improved shelf life employing directly the dried hyphae of the biocontrol agent along with an inert carrier, with or without the addition of other formulant(s) to obtain ready to use viable product of the biocontrol agent for application in pest control. It is in the field of biological pest control.
Background of invention
It is very well known that the useful and the harmful arthropods and microorganisms coexist in nature. With an increasing emphasis on development of environmentally benign technologies for the control of pests and diseases, a judicious utilization of the beneficial organisms has come to the fore. The formulation technology till date has focused on development of usable forms and products of chemical pesticides. Lack of such products and technologies for application and use of biocontrol agents has been a serious constraint in their wide scale adoption. Efforts are being made all over the world to develop such products and technologies.
One of the key constraints in formulation of microbials is their short shelf life. Being living organisms, these require the basic necessities essential for their survival. Since such needs are not met by the commonly used formulants, the shelf life of the organisms is generally short. Therefore, a search for ways and means to develop formulations which extend survival of these organisms is essential..
Two logical approaches to formulate microbials could be:
1. Use of organisms in a form or at a stage when their survival is affected the least by the environmental factors or nutritional needs.
2. Use formulants capable of promoting environmental or nutritional factors that support life.

Whereas the second approach has been adopted in several attempts to formulate microbials, information on the first approach is lacking. It is known that the fungi reproduce through formation of sexually or asexually produced spores, which germinate only under favourable conditions. Till such conditions are available, these remain dormant. It is possible to preserve them under this stress condition. When the favourable conditions are restored, the spores germinate and become viable. In the present invention, the dormant stage of the agent has been explored to develop formulation with enhanced shelf life. Additionally, a medium capable of supporting energy needs of the microbes has been employed.
Prior art
Numerous fungi possess potential for use as biocontrol agents due to their ability to selectively infect and kill a variety of plants and insect pests or their ability to antagonize or remove plant disease-causing organisms through parasitism or competitive exclusion. Several factors must be considered while selecting microbial biocontrol agents for commercial development. These include need for non-chemical control, pathogen host range, economic importance of the target pest, potential of biocontrol organism to produce an effective propagule, availability of cost-effective production and stabilization technology for the required fungal propagule etc. Lack of suitable methods for economically producing the stable and effective fungal propagules continues to impede the commercial use of these agents. Selecting fungi for use as biocontrol agent and for concomitant amenability to liquid culture production requires an understanding of how the organism carries out its biocontrol function and in what environment will it be used.
For use as a foliar spray or in post-harvest disease control applications, production of the fungal biocontrol agent as yeast-like propagules is advantageous. The potential to control soil-borne plant diseases with fungal biocontrol agents is greatly enhanced if the agent produces propagules such as chlamydospores or sclerotia that are capable of persisting in soil environment.
The success of a biocontrol agent depends largely on its ability to establish itself in a new environment and maintain a threshold population on the planting material or rhizosphere. Biological control agents are living materials. Commercial production and application at farm

level demands certain pre-requisites, such as, viability for long period (longer shelf life); tolerance to variable weather conditions and physiological stresses associated with transportation, storage and application, and cost-effective formulations that are easy to handle and have no adverse effect on seed germination or plant growth. Lack of effective and stable formulation has been a major obstacle in commercialization of biocontrol products. Granule, spray, dust, wettable powder, foam and other formulations are being developed to overcome these obstacles and attract commercial interest in biological control solutions to agricultural pest problems. Alginate based fungal antagonists have also been formulated by fluid-bed granulation using dextrin as a binder. Description of prior art
Biocontrol agents such as Trichoderma virens (formerly known as Gliocladium virens) have been recognized as mycoparasite and antibiotic-producing antagonists of plant pathogens, and have been used as effective biocontrol agents against several soil borne root or seedling diseases [Aluko and Hering, 1970; Beagle-Ristaino and Papavizas, 1985; Howell, 1982; Howell and Stipanovic, 1983;Weindling and Fawcett, 1936 and Wright, 1956 ]. T. virens produces gliotoxin, gliovirin and viridin, which are effective antifungal antibiotics, and heptelidic acid, which is antibacterial.
Neither the use of biological agents for control of plant diseases nor the controlled release of such agents from a matrix, are new ideas. For example, Lewis et al., (1985) described the use of alginate pellets containing propagules of potential fungal and bacterial biocontrol isolates to reduce the incidence of damping-off diseases in cotton, sugar beet and radish seedlings. Similarly, Fravel et al., (1985) disclosed a method for encapsulation in alginate pellets of microorganisms that have potential to control plant diseases . Backman et al., (1975) described a diatomaceous earth granule impregnated with molasses, as suitable for growth and delivery of Trichoderma harzianum. Paulitz et al., (1986) described addition of T. harzianum to mixes of Canadian sphagnum peat and vermiculite for increased growth of radish, free of plant pathogens. European Patent Application No. 86309438.9 discloses a fungal culture product in which a medium of ground vermiculite, a nutrient and water is prepared and inoculated with microbial culture.
EP 0124388 (A1) (1984) reports a new strain of Trichoderma harzianum isolated by a cloning technique for use as a biocontrol agent.

EP 0466133 (1992) reports an agronomically acceptable carrier which includes a food base for the antagonist T. harzianum Rifai T-39 (1-952) present in its culture in the form of conidia, chlamydospores or hyphal fragments or mixtures of these types of cells for control of gray mold caused by B. cinerea and S. sclerotiorum.
US Patent 4713342 reports conidia or chlamydospores or mixtures of T. harzianum, resistant to chemical pesticides and soil sterilants, in a carrier which includes a food base for the mycoparasite contained in culture, for controlling fungi.
US Patent 4,828,600 (1989) reports a biological inoculant comprising spores of T. humatum and T. harzianum for facilitating and fostering the growth of edible corn plants. It has been used with or without coating of a carrier.
The above approaches have used either the biocontrol agent per se in spore or hyphal or combination of these forms or combined with a carrier to apply the organism or provide nutrition to it. The present invention on the other hand focuses on increasing its shelf life which is one of the primary constraints in large scale adoption and use of biocontrol agents.
No information on improving the shelf life of the biocontrol agent by use on the use of dried and powdered mycelia biomass of fungi such as T. virens, T. harzianum into its solid or liquid formulation is available in literature. This invention discloses a novel approach of formulating the dry mycelium of the biocontrol agent into its solid powder formulation with considerable advantage in terms of enhanced shelf life and efficacy. Object of invention
An object of the invention is to propose a process for preparation of mycelium based solid biopesticidal formulations with improved shelf life based on the biocontrol agents such as T. virens. Another object is to report biopesticidal products with improved shelf life and efficacy obtained as a result of this process. The invention
The invention disclosed herein describes process for preparation of ready to use solid biopesticidal formulations with improved shelf life based on the biocontrol agent(s) such as the antagonist mycoparasite(s) ex. Trichoderma species and the biopesticidal products obtained thereof characterized by blending the dried and powdered solid hyphae of the agent directly with an inorganic or organic inert carrier, with or without the addition of other formulants to obtain a ready to use product complying with the shelf life and use requirements for application in pest

control. The other formulants include surfactants, stabilizers, UV screens, binders and drying agents. A suitable amount of the dried hyphae is blended with the carrier , preferably fly ash and or/ other formulants to yield the formulation that passes the accelerated storage tests to meet the shelf life requirement as prescribed for chemical pesticides (FAO, 1999).
The biopesticidal formulation is prepared for delivery by: (1) growing the selected fungus and allowing it to sporulate under conditions favoring production of resistant spores or other survival structures, (2) harvesting and drying the biomass/mycelium and (3) mixing the biomass with carrier and other formulants. This dried preparation may be applied to soil or a growth medium adjacent to the plants or other targets to be protected, or as a seed dressing or coating. The spores germinate to produce vegetative cells, proliferate, and combat target plant pathogens or otherwise enhance the health of the plant.
The formulations described herein meet the application requirements. The carrier/biomass composition is versatile enough to be applied in agricultural, horticultural and other consumer settings in such a manner that the biocontrol agent permeates the infested soil and proliferates. The dried product may be applied on or as a mixture with the soil or soil less mix in which seeds or seedlings are to be planted. Upon wetting of the soil and biopesticidal formulations, the spores germinate and attack the target pathogens
Generally, the method comprises the following steps:
I: Production of fungal biomass in very high yield in fermenter(s) in the form of filamentous mycelium.
II: Harvesting the filamentous mycelium, drying and formulating with a solid carrier particularly fly ash / saw dust / attapulgite / China clay / talc and the likes.
Stage III: Subjecting the product so obtained to shelf life and bioefficacy evaluation
The invention is further illustrated by the following examples.
Preparation of mycelial biomass stock
Culture of the biocontrol agent T. virens was obtained from the repository of Indian Agricultural Research Institute, New Delhi. It was maintained on standard potato dextrose agar

medium (potatoes 300gL_1, dextrose 20g L-1 and agar 17g L-1). For production of spores in 1L capacity Erlenmeyer shake flasks, the culture was raised on 250 mL of the potato dextrose medium without agar, pre-autoclaved at 120°C, 15psi for 15 min. The medium was inoculated with spores at a concentration of 1 times 10. sup. 6 conidia mL-1 from one week old slant and incubated in a BOD at 28 + 1°C for 10-15 days. At the end of fermentation period, no vegetative cells were detected under microscope. The viability of spores as determined by colony forming units on standard potato dextrose agar (PDA) was of the order of 95 to 100%. The mycelium was separated from culture filtrate by filtration through a muslin cloth fitted on a buchner funnel. The solid mass collected on the cloth can be weighed and used wet or dry. The mass can also be washed with sterilized water and air dried in a laminar flow, weighed and ground to pass through a suitable mesh screen and stored for future use.
Preparation of 5 and 10% (biomass based) powder formulation of T. virens
Five or ten g of the dried biomass of T. virens was taken and mixed thoroughly with 95 or 90 g of fly ash. The mix was blended and sieved through a BS-200 mesh sieve to obtain a free flowing powder.
Stability and viability of spores in the formulation
The powder biocontrol formulation of example 2 was stored at ambient temperature (30 °C) for 30 days Viability of the spores was evaluated after 3, 5, 7, 10, 15, 30 days by inoculating a pinch of it on petri plates containing sterilized PDA. There was 100% germination of the spores of T. virens. No changes in the vegetative and reproductive growth of the fungus as compared to slant were observed, confirming its viability in the formulation. Lack of change of the strain or presence of other bacteria or fungi confirmed that there was no contamination of the hyphal mass stored per se or in the formulation.
Stability at accelerated storage

in view of the fact that the fungal spores have been directly formulated in the past to obtain various products, their shelf life has been investigated under ambient conditions at temperatures below 30 C. Normally, shelf life of a few months has been successfully reported. In this study the accelerated storage test as applicable to chemical pest control agents was applied.
The formulation (5 or 10%) obtained in example 2 was incubated at 54 + 1°C, 25 g cm-2 for a period of 14 days (FAO, 1999) in an incubator. Samples were withdrawn after 1, 3,5,7,10,14 days and examined for spore viability. The data are reported in Table 1.
Table 1. Viability of spores in powder formulation subjected to accelerated stage
(Table Removed)
* Values represent Log. sub. 10-colony forming units g" formulation
It is seen from Table 1 that on subjecting the formulation to accelerated storage at 54 + 1°C, 25 g cm-2 (equivalent to 2 years shelf life), viable spores to the extent of 3.46 (10 colony forming units per gm) were still present in the formulation. These would be enough to serve as inoculum for multiplication of spores under favourable conditions. This will be a distinct advantage as the biocontrol agents are reported in literature to forego virulence after a few months of storage.
Viability of the formulation under favourable biotic conditions
The effect of temperature and humidity on the viability of the formulated spores was studied in the absence of growth medium. The formulation (1 gm) was mixed with soil (5 gm) in laboratory and water added to completely wet the soil. It provided moist and humid environment at the ambient temperature of 34°C. The germination of spores is reported in Table 2. It is seen from Table 2 that up to 6 weeks of incubation under the given conditions, the viability of spores in the formulation remained unaffected.

Table 2. Viability of powder formulated spores in moist and humid soil at ~ 34°C
(Table Removed)
Evaluation of the formulation in pot culture study
The formulation prepared in example 2 was evaluated in a pot culture study for its ability to prevent damping off of tomato, caused by Sclerotium rolfsii. Mycelia mats of the pathogen were multiplied in potato dextrose broth at 27°C. After 7 days, these were separated from the medium and homogenized in a small blender for 1 min. Inoculum from 10 flasks was thoroughly mixed with 2.5kg of sterilized soil ( 2 mm autoclaved at 120°C at 15 psi for 15 min). The infested soil was incubated in laboratory at room temperature (approximately 25°C) for 5-7 days. Sterilized, autoclaved, uninfested soil was used as control.
Treatments. The treatments for disease control were:
i) Infested soil, as above, moistened with water
ii) Infested and moistened soil as per no. (i) above was treated with the biocontrol formulation (5 and 10% hyphal concentration) at the rate of 5.0 gm in 150cm3 of soil
iii) Uninfested moist soil.
The treated soil was placed in polyethylene bags, closed tightly and incubated for 7 days. After the incubation period, the soil from each treatment was taken in 6.6 cm diameter standard plastic pots, in triplicate, and one 4-6 week old seedling of tomato transplanted per pot. The pots were placed randomly in the green house. Disease characteristics (wilting and mortality) were assessed after 7, 14, 21, 28 and 35 days of transplanting, and thereafter at weekly intervals. The occurrence of symptoms in different treatments was recorded at each assay date and expressed in terms of the proportion of symptomless plants. The results are shown in Table 3. The treatment

of soil with 5% formulation of biocontrol agent revealed 80% healthy tomato plants 5 weeks after transplanting. The formulation prevented tomato damping off significantly. It allowed a stand comparable to the uninfested control.
Table 3. Effect of T. virens formulation (5%) in suppressing S. rolfsi on tomato in pots
(Table Removed)
Thus, the formulation is effective in checking tomato wilt up to 5 weeks observation as reported above. Its efficacy is thus established.
1. Aluko and Hering, 1970, Trans. Br. Mycol. Soc, 55:173-179
2. Beagle-Ristaino and Papavizas, 1985, Phytopathology, 75:560-564
3. Howell, 1982, Phytopathology, 72:496-498
4. Howell and Stipanovic, 1983, Can. J. Microbiol, 29:321-324
5. Weindling and Fawcett, 1936, Hilgardia, 10:1-16
6. Wright, 1956, Plant and Soil, 8:132-140
7. Lewis et al., 1985, "Formulation and Delivery Systems for Biocontrol Agents Effective Against Soilborne Plant Pathogenic Fungi," Proceed. Intern. Symp. Control. Rel. Bioact. Mater, 12 :341-3
8. Fravel et al., (1985)"Encapsulation of Potential Biocontrol Agents in an Alginate-Clay Matrix," Phytopathology, 75 :772-7.
9. Backman et al., (1975) "A System for the Growth and Delivery of Biological Control Agents to the Soil," Phytopathology, 65 :819-21.

10. Paulitz et al., (1986) "Effect of Peat: Vermiculite Mixes Containing Trichoderma harzianum on Increased Growth Response of Radish," J. Amer. Soc. Hort. Sci., 11: . 810-16.
11. European Patent Application No. 86309438.9 (Agracetus),
12. European Patent 0124388 (A) (1984) New strain of Trichoderma harzianum process of isolation of this strain, process culture of this strain, new peptides produced by this strain and application of this strain and these new peptides or proceeds obtained by the process culture as a means of biocontrol in the form of phytosanitary products.
13. European Patent 0466133 (1992) Novel isolate of Trichoderma harzianum, T-39, fungicidal compositions containing said isolate and use against B. cinerea and S. sclerotiorum.
14. US Patent 4713342. Novel isolate of Trichoderma, fungicidal compositions containing said isolate and use thereof.
15. US Patent 4828600 (1989) Biological inoculants for corn.
16. FAO (1999) FAO specification for Plant Protection Products. AGP: CP/30-306.

5. Claims I / we claim
1. A process for the preparation of ready to use solid biopesticidal formulations with improved shelf life based on the biocontrol agent(s) such as the antagonist mycoparasite(s) ex. Trichoderma species and the biopesticidal product obtained thereof characterized by blending the dried and powdered solid hyphae of the agent directly with an inorganic or organic inert carrier, with or without the addition of formulant(s) such as surfactants, stabilizers, UV screens, binders, drying agents and the likes to obtain a ready to use product complying the use requirements for application in pest control.
2. A process as claimed in claim 1 wherein the biopesticidal antagonist mycoparasites ex. Trichoderma sp. are more specifically exampled by the dried hyphae of T. virens, T. harzianum and the likes.
3. A process as claimed in claims 1 and 2 wherein the dried mycelium powder is obtained by inoculating the growth medium with fungal spores, promoting spore multiplication, separating hyphae from culture medium, washing with sterilized water, drying under ambient conditions and grinding. The powder is stored below 10°C to retain viability for over two years.
4. A process as claimed in claim 1 wherein the solid inert carrier is an inorganic clay or clay mineral such as attapulgite, China clay, talc and the like or fly ash, more particularly the fly ash or an organic carrier such as sawdust, plant leaf powder, powdered flowers, flours, synthetic or natural polymers such as alginate, starch, cellulose, dextrin, gelatin or the likes or their derivates.
5. A process as claimed in claim 1 wherein the formulants are the commonly used materials in pesticide formulation as exemplified by surfactants - ionic, nonionic, blend of ionic or/ and nonionic such as alkyarylsulphonates, alkyl sulphates and the like; stabilizers, such as epichlorohydrin, benzophenone, propyl gallate, poly (oxyethylene), dodecylamino ether and the like; UV screens such as Tinopal, LPW (Calcofluor white), stilbene, salicylates, benzophenones, benzotriazoles and the like; binders such as glues, gums, starch, shellac, resin or other polymeric materials capable of binding the particles together; drying agents

- such as calcium chloride, blue silica gel, sugars, water absorbing polymers and the like, and others as per need.
6. A process as claimed in claim 1 wherein the ready to use formulations for pest control are exemplified by a powder such as dustable or wettable powder; granule such as water dispersible granule, ordinary granule, micro granule, floating granule; polymeric products such as those based on hydrophilic and hydrophobic polymers and the like.
7. A process as claimed in any of the above claims, substantially as described in the body of the application with examples and wherein the product so obtained provides the desired spore viability even after 14 days of accelerated storage (54 ± 1°C, FAO 1999) for an effective pest management.
8. A process as well as the product as claimed in any of the above claims for obtaining materials with shelf life comparable to chemical pesticides, for use in pest management, particularly the pest management in agriculture.








405-DEL-2006-Description (Complete)-(23-09-2008).pdf

405-del-2006-description (complete).pdf





Patent Number 234480
Indian Patent Application Number 405/DEL/2006
PG Journal Number 26/2009
Publication Date 26-Jun-2009
Grant Date 29-May-2009
Date of Filing 14-Feb-2006
# Inventor's Name Inventor's Address
PCT International Classification Number A01N 63/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA