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Title:
FORMULATION OF ENTOMOPATHOGENIC FUNGUS FOR USE AS A BIOPESTICIDE
Document Type and Number:
WIPO Patent Application WO/2009/093261
Kind Code:
A2
Abstract:
The present invention relates to a biopesticide based on or more entomopathogenic fungi which is formulated as a novel highly compressed tablet and combinations thereof. The formulation exhibits longer shelf life and lesser contamination by saprophytic fungi. It also relates to a process of preparing the biopesticide formulation and modes of application of the same.

Inventors:
SATYASAYEE, Divi ("Mayuresh" Niwas, P.R. More & SonsPodi No. 1, Sector-15A,Near Panvel Railway Station,New Panvel 6, Dist. Raigad, Maharashtra, 410 20, IN)
Application Number:
IN2008/000620
Publication Date:
July 30, 2009
Filing Date:
September 26, 2008
Export Citation:
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Assignee:
SATYASAYEE, Divi ("Mayuresh" Niwas, P.R. More & SonsPodi No. 1, Sector-15A,Near Panvel Railway Station,New Panvel 6, Dist. Raigad, Maharashtra, 410 20, IN)
Attorney, Agent or Firm:
KAUSALYA, Santhanam (203 Quiescent Heights, Mindspace Malad ,Mumbai 4, Maharashtra, 400 06, IN)
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Claims:

Claims

What is claimed is:

1. A biopesticide formulation comprising conidia of one or more entomopathogenic fungi and other excipients in the form of a compressed tablet.

2. A biopesticide formulation comprising conidia of one or more entomopathogenic fungi, UV protectant, anti-saprophytic agent, dessicant, lubricant, binding agent, disintegrant and diluent.

3. The biopesticide formulation of claim 1 , wherein the said entomopathogenic fungi is selected from a group consisting of genera Beauveria, Metarhizium, Paecilomyces, Verticillium and Nomuraea.

4. The biopesticide formulation of claim 2, wherein the said UV protectant is selected from a group consisting of Tinopal, Blankophor BBH and Leucophor AP.

5. The biopesticide formulation of claim 2, wherein the said anti-saprophytic agent is chitin.

6. The biopesticide formulation of claim 2, wherein the said dessicant is dicalcium phosphate.

7. The biopesticide formulation of claim 2, wherein the said lubricant is selected from a group consisting of talc, magnesium stearate, calcium stearate, zinc stearate, boric acid, polyethylene glycol and sodium stearyl fumarate.

8. The biopesticide formulation of claim 2, wherein the said binding agent is selected from a group consisting of microcrystalline cellulose, cellulose acetate, carrageenan, dextrin, glucose, ethyl cellulose and polyvinylpyrrolidone.

9. The biopesticide formulation of claim 2, wherein the said disintegrant is selected from a group consisting of primogel, sodium starch glycolate, crospovidone and croscarmellose sodium.

10. The biopesticide formulation of claim 2, wherein the said diluent is water.

11. A biopesticide formulation comprising by weight of about 20% of conidia of Beauveria bassiana, about 0.5 to 2% Tinopal LPW UNPA GX, about 2% to 10% of chitin, about 4% to 6% Primogel, about 50% to 60% dicalcium phosphate, in

the range of 2% to 5% magnesium sterate, about 10% to 16% micro crystalline cellulose and 3% to 8% residual water content.

12. The biopesticide formulation of claim 1, wherein the said compressed tablet is packaged in moisture resistant strips or bottles.

13. A biopesticide formulation of claim 1 , wherein the said tablet is applied in combination with one or more pesticides selected from a group consisting of microbial, chemical and botanical insecticides.

14. The biopesticide formulation of claim 1, wherein the shelf life of the said formulation is at least one year at a temperature ranging from 0 0 C to 38°C.

15. The biopesticide formulation of claim 1, wherein the shelf life of the said formulation is at least one year at a temperature of about 25 ± 2°C.

16. A method of killing an insect comprising administering the biopesticide formulation of claim 1 dispersed in water and sprayed on plants or dispensed directly in irrigation water or stagnant water.

17. A process of preparing the formulation of claim 1 comprising

(a) mass culturing one or more entomopathogenic fungi through biphasic solid state fermentation to obtain a dry powder of conidia containing at least 10 12 CFU of conidia per gram;

(b) geometrical mixing of the conidia with binders, lubricants, diluents, disintegrating agents, UV protectants, dessicants and anti saprophytic agents;

(c) reducing the water content of the mixture up to <4% by slow drying to obtain a dried composition; and

(d) compressing the mixture in tablet press to form a compressed tablet.

Description:

FORMULATION OF ENTOMOPATHOGENIC FUNGUS FOR USE AS A

BIOPESTICIDE

Field of Invention

The present invention relates to a biopesticide based on one or more entomopathogenic fungi, which is formulated as a novel dispersible tablet resulting in longer shelf life preventing contamination with saprophytic fungi and facilitating easy application. It also relates to the process of preparing the formulation and use of it in combination with other pesticides.

Background

The development of a good formulation is important for the successful utilization of commercial biopesticides. Factors affecting commercial formulations include shorter shelf life, biological and physical properties of the formulation. It is desirable that the strength of the formulation is maintained over the two main cropping seasons and ideally even longer and hence a formulation with a longer shelf life is valuable for the manufacturers and end users alike.

Currently available biopesticide formulations in the market are in the form of powder, granules and oil which have significant disadvantages namely high level contamination by saprophytic fungi and very low viability of the microbes leading to low efficiency. The powder form further attracts moisture leading to contamination and subsequent loss of viability of the active ingredient (conidia) of the biopesticide and the oil form suffers from poor spraying characteristics. Moreover, spraying of powder formulations leaves a lot of residual talc (the carrier used in these formulations) on the plant surfaces. Therefore, the available formulations though based on a very effective biocontrol agent have not gained popularity among farmers. These drawbacks should not be allowed to remain as an obstacle for the widespread use of these very promising biopesticides (microbes) in biological control.

The present invention aims to develop an improved biopesticidal formulation. The said formulation is an entomopathogenic fungal-based biopesticide formulation, which shows considerably longer shelf life, minimized contamination and absorbance of moisture during storage and handling. The tabletted nature of the biopesticide formulation which is safely packed (contamination resistant packing ex: strip, bottle etc) is first of its kind in fungal based biopesticides. The quick water dispersible nature of the formulation with no or negligible contamination, long shelf life and components that ensure its effectivity in field conditions, all make it a valuable biopesticide addition handy in integrative crop insect management programmes.

Summary

The present invention relates to a biopesticide formulation comprising conidia of one or more entomopathogenic fungi and other excipients in the form of a compressed tablet. It also relates to the process of preparing the formulation and use of it in combination with other pesticides.

In one aspect, the invention provides a biopesticide formulation comprising of conidia of one or more entomopathogenic fungi, UV protectant, anti saprophytic agent, dessicant, lubricant, binding agent, disintegrant and diluent.

In another aspect, the invention provides a biopesticide formulation comprising by weight of about 20% conidia oϊBeauveria bassiana, about 0.5 to 2% Tinopal LPW UNPA GX, about 2 to 10% of chitin, about 4% to 6% Primogel, about 50% to 60% dicalcium phosphate, about 2 to 5% magnesium sterate, about 10% to 16% micro crystalline cellulose and 3% to 8% residual water content.

The invention also provides application of the biopesticide formulation in combination with other pesticides selected from the group consisting of microbial, chemical and botanical insecticides.

In yet another aspect, the invention provides a highly compressed tablet formulation of biopesticide packaged in moisture resistant strips or bottles to minimize the chance of contamination. The invention also provides a formulation with a shelf life of at least one

year at a temperature ranging from O 0 C to 38 0 C, more preferably at a temperature of about 25 ± 2 0 C.

In yet another aspect, the invention provides the process of preparation of the biopesticide formulation. The processs comprises the steps of:

(a) mass culturing one or more entomopathogenic fungi through biphasic solid state fermentation to obtain a dry powder of conidia containing at least 10 12 CFU conidia per gram;

(b) geometrical mixing of the conidia with binders, lubricants, diluents, disintegrating agents, UV protectants, dessicants and anti saprophytic agents;

(c) reducing the water content of the mixture up to <4% by slow drying to obtain a dried composition; and

(d) compressing the mixture in tablet press to form a compressed tablet.

Detailed description of the invention

The present invention provides a biopesticide formulation, comprising of conidia of one or more entomopathogenic fungi as the pesticidally active ingredient, in the form of a highly compressed tablet. It also relates to the process of preparing the same. The invention also encompasses combinations of the said formulation with other pesticides.

The biopesticide formulation of the present invention comprises conidia of one or more entomopathogenic fungi, UV protectant, anti saprophytic agent, dessicant, lubricant, binding agent, disintegrant and diluent.

The pesticidally active agent of the formulation is the conidia of one or more of the entomopathogenic fungi. Entomopathogenic fungus acts against insects by colonizing the cuticles after which the invasive hyphae begin to enter the host's tissues and ram ify through the hemocoel. Hyphal bodies or segments of the hyphae distribute throughout the hemocoel, filling the dying insect with mycelium. Emerging hyphae grow out through the insect's integument and produce spores on the external surface of the host. These spores, or conidia, are dispersed and capable of infecting new host insects. These characteristics of the entomopathogenic fungi make it a popular choice for use as a

biopesticides. Use of entomopathogenic fungi in the preparation of biopesticide is well known in the art. WO 1995010597 discloses the use of oil-based formulation comprising conidia of entomopathogenic fungi. US5413784 claims the use of Beauveria bassiana, ATCC-74040 a type of entomopathogenic fungi in the preparation of a biopesticide. The entomopathogenic fungi of the present invention is selected from the group comprising of the genera Beauveria, Metarhizium, Paecilomyces, Verticillium and Nomuraea or combinations thereof. In a preferred aspect of the invention, conidia of Beauveria bassiana are used in the formulation. About 20% of the conidia of one or more entomopathogenic fungi are used in the invention.

In one embodiment of the invention, the biopesticide formulation is provided as a highly compressed tablet form. Currently biopesticides available in the form of powder, oil or granules exhibit unfavorable characteristics such as absorbance of moisture, contamination with opportunistic saprophytic fungi which lead to a shorter shelf life. Most wettable formulations tend to leave a residual layer of pesticide which negatively affects photosynthesis. In the instant invention, the water-dispersible tablet is wettable in water, disperses very quickly and forms a suspension with excellent suspendability without forming a residual layer on the plants. The highly compressed nature of the tablet formulation also leads to lesser absorbance of moisture and contamination by opportunistic saprophytic fungi thus affording a longer shelf life.

The term conidia, as used in the current disclosure, are well recognized in the art and are intended to include asexual spores of fungi.

In another embodiment, the biopesticide formulation contains a UV protectant as a component. The UV protectant serves as a substance that imparts moderate tolerance of conidia to UV protection and also acts as a surfactant when applied to the field's thereby increasing viability. Commonly known UV protectants in the art include Tinopal, Blankophor BBH, Leucophor AP which can be used in the instant invention. In a preferred embodiment, the UV protectant is Tinopal LPW UNPA GX (Sigma Fine Chemicals, India). Tinopal LPW, a water soluble, non toxic (as per EPA reports),

stilbene-derived fluorescent optical brightener is reported to confer UV tolerance to conidia much better than other fluorescent dyes such as Blankophor and Congo red (Inglis et al 1995, Biological Control 5: 581-590). Tinopal is reported to have synergistic effect with Beauveria bassiana (Reddy et al 2008, Pest Management Science (In press), the entomopathogenic fungi used in the instant invention. The amount of the said UV protectant used in the formulation is about 0.5% to 2% of the total weight of the formulation.

One of the major impediments to the use of biopesticides is contamination by opportunistic saprophytic fungi. The formulation of the present invention includes the use of one or more agents against opportunistic fungi, for example, chitosan. Preferably, the anti-saprophytic agent used in the present formulation is chitin. The preferred amount of the said anti saprophytic agent used in the formulation is in the range of 2% to 10% of the total weight of the formulation.

In one embodiment of the invention the dessicant used is dicalcium phosphate. The amount of the said dessicant used in the formulation is about 50% to 60% of the total weight of the formulation.

The lubricant, which facilitate in the tablet making process, is selected from a group consisting of Talc, magnesium stearate, calcium stearate, zinc stearate, boric acid, polyethylene glycol and sodium stearyl fumarate. The amount of lubricant used in the invention is in the range of about 2% to 5% of the total weight of the formulation.

Yet another component of the compressed form of the tablet is a binding agent. The binding agent of the present invention is selected from a list including but not limited to microcrystalline cellulose, cellulose acetate, carrageenan, dextrin, glucose, ethyl cellulose and polyvinylpyrrolidone. The preferred binding agent is microcrystalline cellulose which has excellent compressing properties. The amount of binding agent used in the invention is about 10% to 16% of the total weight of the formulation.

The disintegrant used in the present invention is selected from a group consisting of primogel, sodium starch glycolate, crospovidone and croscarmellose sodium. The preferred disintegrant is Primogel. The amount of disintegrant used in the invention is about 4% to 6% of the total weight of the formulation.

The diluent used in the instant formulation is water which forms the residual 3% to 8% of the total weight of the formulation.

Table 1 provides a representative formulation of the invention, their proportion and properties.

Table 1: List of components, their proportion (Wt in tng per 250mg tablet) in the formulation and their properties

The tableted formulation of the biopesticide described in the instant disclosure provides more mechanical stability, thermo tolerance, protection from moisture and resistance against other microbial (especially saprophytic fungi) contaminants. Thus, a long shelf life of at least one year at storage temperature ranging from O 0 C to 37°C is provided by the invention. The shelf life of the formulation has been tested at: a) a constant temperature of 25 ± 2 0 C and b) a fluctuating temperature of day temperatures ranging

between 29 0 C - 38 0 C and night between 17°C - 27°C in different seasons during one whole year in Visakhapatnam, India.

hi yet another embodiment of the invention, a process to prepare the said biopesticide formulation in a tablet form is provided. Firstly the process comprises of culturing the entomopathogenic fungi to obtain the dry conidia for the preparation of the formulation. One or more entomopathogenic fungi are mass cultured through biphasic solid state fermentation which is well known in the art. This step yields a dry powder of the conidia. The second step comprises of geometrically mixing the conidia with the other agents present in the formulation such as binders, lubricants, diluents, disintegrating agents, UV protectants, dessicants and antisaprophytic agents. Consequently the water content of the mixture is reduced to <4%.

The procedure for preparing tablets is also well known in the art. The tablet form of formulation is made as follows:

1) The geometrically mixed composition of the pesticide was compressed into tablets with the following parameters: tablet press: Korsch 106, instrumented, 24 punches, rotary press punches: 010 mm, normal concave, target tablet weight: 200-500 mg. According to the invention, tablets containing 10 5 , 10 6 , 10 9 or even 10 n or 10 12 Colony Forming Units (CFU) per gram is manufactured.

2) Tablets were produced in a standard tableting process, but at reduced pressure, the compression being 200 - 300 kP. Friability was 0.31 and hardness 2.75 kP.

In another embodiment of the invention is described the application of the aforementioned biopesticide formulation. The tablet rapidly disperses in water forming a suspension whose viscosity is such that it can be dispensed through the nozzle of standard spraying equipment. The tablets are also directly dispensed with irrigation water for treatment of soil borne insects or with stagnant water for insects breeding in the water.

B. bassiana, one of the entomopathogenic fungus used in the formulation has a very wide host range of more than 700 insect species. The insect species to which B. bassiana has

been reported to be pathogenic, span a very expansive scope of the class Insecta, one of the biggest (with reference to number of species) classes in the biological world. The taxonomic orders to which the insects reported susceptible to B. bassaina belong are:

(I) Blattariae, (2) Coleoptera, (3) Dermaptera, (4) Diptera, (5) Embioptera,

(6) Hemiptera, (7) Homoptera, (8) Hymenoptera, (9) Isoptera, (10) Lepidoptera,

(I I) Mantodea, (12) Neuroptera, (13) Orthoptera, (14) Siphonaptera, (15) Thysanoptera. Examples of economically important insect pests that are reported (in literature) susceptible to B. bassiana are listed below.

Agricultural importance Crops

Tobacco caterpillar (Spodoptera litura ), tobacco budworm, cotton bollworm (Helicoverpa armigera), fall armyworm (Spodoptera frugiperda), corn ear worm (Helicoverpa zea), european corn borer (Ostrinia nubilalis), asian corn borer (Ostrinia furnacalis Guenee) sorghum stem borers (Chilo partellus, Coniesta ignefusalis, Busseola fusca, Chilo spp.), yellow stem borer of rice (Scirpophaga incertulas), rice leaffolder (Cnaphalocrocis medinalisi), brown plant hopper of rice(Nilaprvata lugens), rice thrip (Stenchaetothrips biformis), leaf hopper (Hishimonus phycitis), grasshoppers (Melanoplus spp.), cactus weevil (Metamasius spinolaei), silverleaf whitefly (Bemisia argentifolii), beet armyworm (Spodoptera exigua), cucumber beetle (Diabrotica undecimpunctata), alfalfa looper (Autographa californica), cotton aphid (Aphis gossypii), termites (Odontetermes obesus, Odontotermes spp., Trinervitermes biformis), jassid (Emboasca kerri), thrips (Frankliniella schultzei, Scirtothrips dorsalis, Podothrips bicolor).

Vegetable crops

Diamondback moth (Plutella xylostella), green peach aphid (Aphis gossypii), potato aphid (Macrosiphum euphorbiae) , thrips (Anephothrips dorsalis, Thrips palmi), mealy bug (Maconellicoccus hirsutus), grass hopper (Melanoplus spp.), whitefly (Bemisia tabaci), brinjal shoot borer (Leucinodes orbonalis), stem borers, beet armyworm, (Spodoptera exigua, Spodoptera spp.), cabbage looper (Trichoplusia ni, Trichoplusia

spp.), spiny bollworm (Earias insulana), spotted bollworm (Earias vitella), leaf roller (Sylepta derogata), mites (Leyranychus telari), okra jassid (Amrasca biguttula). Medical Importance

Mosquito (Anopheles gambiae, Culex quinquefasciatus), house fly (Musca domestica), cockroaches (Periplanata americana) and ticks (Ixodes dammini).

Veterinary Importance

Triatoma infestans, Rhodnius prolixus, ticks- soft tick (Argas persicargas persicus), bovine tick (Boophilus microplus), Babesia microti, blacklegged tick or deer tick (Ixodes scapularis), sheep scab mite (Psoroptes ovis), tropical fowl mite (Ornithonyssus bursa), fleas- Siphonaptera, Cat flea (Ctenocephalides felis) and cattle louse (Haemaptopinus eurysternus).

B. bassiana used in the composition described herein was found to be highly virulent to all the insects bioassayed in our lab. These include 15 insects from various taxonomic divisions: the lepidopterans - tobacco caterpillar {Spodoptera liturά), cotton bollworm {Helicoverpa armigera), spotted bollworm (Earias vitella), sorghum stem borer (Chilo partellus), cotton leaf roller (Sylepta derogate) and brinjal shoot and fruit borer (Leucinodes orbonalis); coleopterans - brinjal beetle (Epilachna vigintioctopunctata), blister beetle (Mylabris pustulata), cucurbit beetle (Diabrotica speciosa), homopteran - pea aphid (Aphis craccivora); orthopteran - locust (Prionolopha serrata), hemipteran - pink hibiscus mealybug (Maconellicoccus hirsutus), dictyopteran - American cockroach (Periplanata americana), hymenopteran - tree/nest ant (Oecophylla smaragdinai) and dipteran - house fly (Musca domestica) (Mohan et al.1999, Biocontrol Science and Technol. 9: 29-33, Uma Devi et al. 2001, World J Microbiol and Biotechnol. 17: 131-137, Mohan 2002, Phenotypic and molecular characterization of Beauveria bassiana (BaIs.) Vuill. isolates suitable for integrated pest management of cotton boll worm (Helicoverpa armigera Hub.) in semi-arid tropics (Doctoral thesis), Uma Maheswara Rao 2004, Genotyping of entomopathogenic fungi Nomuraea rileyi (Farlow) Samson and Beauveria bassiana (BaIs.) Vuillemin from an epizootic population and a world wide

population through pathotyping and DNA fingerprinting (Doctoral thesis), Uma Devi and Uma Maheswara Rao 2006, Mycopathologia, 161 : 385 - 394).

B. bassiana used in the formulation was also found effective in field tests conducted from our laboratory for management of cotton leaf folder (Ramesh et al. 1999, J Entomol Research. 23: 267-271). Incidentally, house flies also died in large numbers in the treated cotton field. The cadavers were found stuck to the leaf tips (Ramesh et al. 1999, J Entomol Research. 23: 267-271). Our field tests targeted on the leaf skeletonizing beetle (Epilachna vigintioctopunctatά) infesting brinjal (aborigine - Solarium melongenά) crop was also successful. In this field treatment, the white fly and jassid infestation in the crop was also simultaneously controlled.

B. bassiana, the active ingredient in the formulation has been reported to colonize plants and exist as an endophyte conferring protection against insects with stem boring activity (Gomez et al. 2006, Micron 37: 624-632). Experiments from our laboratory confirmed this ability in field grown sorghum and its protection from stem borer (Chilo partellus) (Reddy et al. 2008, Int J Pest Management (In press).

B. bassiana has also been reported to be antagonistic to plant pathogenic fungi. It was found to effectively control Rhizoctonia solani (Ownley et al. 2004, Emerging concepts in plant health management, Research Signpost, 255 - 269) causing seedling wilt of tomato.

Thus, this biopesticide has a potential to manage a wide array of domestic and agricultural insect pests and some phytopathogenic fungi besides being useful as a defense agent against stem borer attacks. B. bassiana has also been reported to clear oil spills.

The present invention also provides the use of the biopesticide with other pesticides. The other pesticides that are used in combination with the biopesticide of the invention is microbial, chemical or botanical insecticides that have proven insecticidal properties in field conditions. Data on the compatibility of the entomopathogenic fungal strain used in the formulation with the commonly used insecticides - Monocrotophos

(organophosphate), Cypermethrin ( pyrethriod), Quinolphos, Margocide (neem) - an azadirachtin based botanical pesticide and fungicides - Bavistin (carbendazim), copper oxychloride (Blitox), and Mancozeb (dithane M45) is available from the laboratory assays conducted in our laboratory (Mohan 2002, Phenotypic and molecular characterization of Beauveria bassiana (BaIs.) Vuill. isolates suitable for integrated pest management of cotton boll worm (Helicoverpa armigera Hub.) in semi-arid tropics (Doctoral thesis), Butters et al. 2003, Mycological Research 107: 260-266, Uma Devi et al. 2004, Pest Management Science, 60: 408-412, Mohan et al. 2007, Biocontrol Science and Technology, 17: 1059-1069). Thus, the integration of this biopesticide in pest management programmes which are based to a large extent on chemical management can be well planned.

The B. bassiana based biopesticide described here was found to act synergistically with neem (Azadirachtin) based botanical pesticide which is widely used in insect pest management (Mohan et al. 2007, Biocontrol Science Technol.17: 1059-1069) Data is also available from assays conducted in our lab on the temperature and desiccation tolerance of B. bassiana used in the formulation (Uma Devi et al. 2005, J Invertebrate Pathol. 88:181-88). So timing of its application in the field can be planned to utilize the effective window.

The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only, and should not be used to limit the scope of the present invention in any manner.

Example 1

Preparation of conidia of entomopathogenic fungi

The conidia of Beauveria bassiana was mass multiplied in a standard two-stage solid- state fermentation. B. bassiana inoculum developed in liquid medium is inoculated on rice with 2% vegetable oil and 30% moisture autoclaved in a polypropylene bag with a specially designed ventilation window. Conidia were harvested and used for formulation of tablet with a viability of 98%.

Example 2

Effect of conidial suspension of Beauveria bassiana on the insect population

Methodology

The insects used for the assay were the second instar larvae of HeI icoverpa armigera, Spodoptera litura, and adults of Epilachna vigintioctopunctat.

The assays were set up in triplicate and repeated thrice. Each treatment batch consisted of 20 insects. The bioassay was conducted as per standard norms. Each insect was treated with 100 μl of conidial suspension (10 8 conidia mlT 1 ). The inoculum was applied with a micropipette over the entire surface of the insect. Post treatment, mortality of the insects was recorded daily until all the insects in the treatment batch either died or pupated. The dead insects were then transferred individually into petri dishes lined with moist filter paper to facilitate mycosis. The mortality and mycosis data were % Varcsine transformed after Abbott correction (Abbott 1925, J Economic Entomol. 18: 265-267).

Results

The results of the insect assay are represented below in Table 2.

Table 2: Effect of conidial suspension of Beauveria bassiana on the insect population

H. armigera S. litura E. vigintioctopunctata

Mortality Mycosis Mortality Mycosis Mortality Mycosis

B. bassiana 93.33±0 91.36±1.5 92.29±1.5 91.57±1.1 94.72±7.6 86.22±1.3

7 5

Example 3

Viability of the tablet formulation

Formulated tablets were stored in plastic screw cap bottles to check the shelf-life. These formulations were tested at a) a constant temperature of 25 ±2 0 C and b) at fluctuating day temperature ranging between 29-38°C and night temperature ranging between 17-27 0 C during the entire year spanning all the seasons. Viability was checked at weekly

intervals for up to a month after formulation and at monthly intervals thereupon. Viability was checked as described by Reddy et al 2008, Pest Management Science (In press).

The results show that the conidial viability in the formulation declined over time, at different rates depending on the storage temperature. Conidial viability was similar when tested at 25 0 C and at room temperature with fluctuating day 29-38°C and night 17-27 0 C. Conidia from tablet formulations tested at 25 0 C contained -90% viable conidia for one year. When stored at lower temperatures (4-8 0 C) conidia will remain viable for more than 18 months (550 days).