Title of Invention

A BIO-PESTICIDE BASED ON NUCLEOPOLYHEDROVIRUS FOR THE MANAGEMENT OF TEAK PEST HYBLAEA PUERA

Abstract

This invention relates to the production of a wettable powder biopesticide fonnulation containing a naturally occurring microorganism, the nucleopolyhedrovirus of the teak defoliator, Hyblaea puera as the active ingredient and talc as carrier, activated carbon as protectant against deterioration by ultra violet rays in sunlight and polyvinyl alcohol as dispersing agent, all mixed in a specific proportion, which is effective in controlling the teak defoliator, Hyblaea puera, which can withstand the damaging effect of ultraviolet rays in sunlight during field application and which has a storage life with retention of over fifty percent infectivity upto eighteen months when stored, in dark at room temperature of about 28°C +- 4°C.

Full Text

Field of invention This invention in general relates to plant protection through biological means and in particular relates to development of a biopesticide for managing the teak defoliator pest Hyhlaeapuera. The invention more particularly relates to a biopesticide formulation for controlling the teak defoliator, Hyblaea puera containing the major ingredient-a naturally occurring nucleopolyhedrovirus of the virus group baculovirus causing disease on the teak defoliator which is storable with retention of its original infectivity for a long period of time and which can withstand the deleterious effect of ultra violet radiations on exposure to sunlight during field application. Background of the invention and prior art Teak, (Tectonagrandis L.f.) is one of the most valued timbers of India. Hyblaea puera is an insect belonging to the insect order Lepidoptera, a pest of the teak tree which is commonly called as teak defoliator. The caterpillars of this insect feed on the foliage of teak which results in volume increment loss of high economic significance (Nair et al, 1985). Attempt has been made to develop an ecologically acceptable and environmentally safe control strategy to contain this pest. Biological control involving natural enemies of the pest such as parasitic and predatory insects, bacteria, fungus and virus has always been advocated for managing insect pests which is safer when compared to chemical pesticides. Among the bio-control agents, the microbial pauiugcu, nucleopolyhedrovirus coming under the virus family, Baculoviruses have been successfully used for managing insect pests in various parts of the world and they are known to be environmentally safe and non-hazardous to other living beings. (Hunter Fugita et al.l998). A naturally occurring nucleopolyhedrovirus has been discovered to cause disease in the teak defoliator under field conditions. (Sudheendrakumar, et al, 1988).The virus particles (Polyhedral Occlusion Bodies- POBs) on consumption by the insect caterpillars results in development of disease leading to their death. The disadvantage of the nucleopolyhedrovirus is that it easily get degraded resulting in loss of virulence on exposure to ultra violet rays in sunlight and such exposure cannot be avoided when the virus is used in field conditions as a biopesticide. Another disadvantage is that when the virus is stored as a suspension in aqua media, it gets contaminated with bacterial growth. The crude virus stored and kept under room temperature loses its virulence within a few days. The present invention relates to a biopesticide formulation containing a particular strain of a nucleopolyhedrovirus, a pathogen of the teak defoliator Hyblaea puera designated and hereinafter referred to as HpNPV which can be stored for a long time by retaining a high percentage of its infectivity for over a period of one year, and which can withstand the degradation by ultra violet rays in sunlight and which is effective in controlling the teak defoliator. The present invention relates to a naturally occurring microbial pathogen active against Hybiaea puera, a storable biopesdcide composition containing this agent or any strains of this agent. This invention is based on the discovery that HpNPV when ingested by the Hyb/aea puera caterpillars, causes considerable mortality among these caterpillars thereby preventing loss of teak leaves and subsequent loss of volume increment of teak tree. Object of the invention Accordingly, the primary object of the present invention is to invent a biopesticide agent which is effective against Hyb/aea puera, the said biopesticide formulation containing nucleopolyhedrovirus (HpNPV) a microbial pathogen causing disease in Hyblaeapuera. Another object of this invention is to invent a biopesticide formulation containing HpNPV which can be stored for a long time i.e over a year, retaining a high percentage of its infectivity. . A preferred embodiment of the present invention is: This biopesticide composition is in the form of a wettable dry powder comprising the said microorganism, HpNPV and a powdery vehicle, and other additives, e.g. additives capable of improving protection against deterioration on exposure to ultra violet rays etc. The present invention pertains to a method of preparing the above defined biopesticide formulation, this method being characterized in that it comprises the steps which consist of: a) infecting Hyblaea puera caterpillars with HpNPV. b) Extracting HpNPV from the caterpillars which have died following such infection and multifold multiplication of HpNPV within the caterpillars. c) Formulating the HpNPV using the method of sublimation along with other ingredients. Advantageously, step (a) of this method consists in causing said caterpillars to ingest a nutritional medium containing HpNPV, this medium comprising preferably the following nutritional elements: teak leaf powder, agar, powdered kabuligram (Cicer arietnum), yeast extract powder, casein, sorbic acid, ascorbic acid, multivitamins and minerals, tocopheryl acetate, methyl-para-hydroxybenzoate and streptomycin sulphate. As for step (b) of this method, it preferably comprises homogenisation of the caterpillars dead due to virus infection and the filtration of the slurry resulting from such crushing, and mixing of this suspension containing desired number of POBs/ml with desired quantity of additives like a suitable carrier and ultra violet protectant and mixing and freeze-drying and storing in dark at room temperature 28±4" C. Statement of the invention This invention in general relates to insect pest management through biological means and in particular relates to development of a biopesticide based on the microorganism, nucleopolyhedrovirus for managing the teak defoliator, Hyblaea puera, a serious pest of Teak (Tectona grandis L.f.) one of the most valued forest timber species. Hyblaea puera is an insect belonging to the insect order Lepidoptera. The caterpillars of this insect feed on the leaves of teak and repeated defoliation of the trees results in volume increment loss of high economic concern. Attempt has been made to develop an ecologically acceptable and environmentally safe control strategy to contain this pest. Among the biocontrol agents, the microbial pathogen, nucleopolyhedroviruses coming under the virus group, Baculovirus have been successfully used for managing insect pests in various parts of the world. Nucleopolyhedrorviruses are highly host specific. A naturally occurring nucleopolyhedrovirus has been observed to cause disease in Hyblaea puera under field conditions. The caterpillars of this insect feeding on teak leaves contaminated with nucleopolyhedrovirus particles develop disease and subsequentiy die due to infection. Thus the virus is useful as a biocontrol agent against this pest. The disadvantage of the virus is that it easily get degraded resulting in loss of virulence when exposed to the ultra violet radiation in sunlight and such exposure cannot be avoided when the virus is applied on teak leaves under field conditions as such in crude form. Another disadvantage is that when the virus is stored in aqua medium gets contaminated with harmful bacterial growth. The present invention thus relates to development of a wettable powder formulation of the virus containing the nucleopolyhedrovirus of Hyblaea puera having the scientific name HpNPV which can be stored for a long time, without being contaminated by any other harmful microorganisms, without loss of infectivity when applied on teak leaf in the field and with a long storage life over one year period and is effective in containing the teak defoliator. Caterpillars of Hyblaea puera are artificially inoculated with a small dose of HpNPV which get multiplied in their body leading to their death. From the cadaver of the caterpillars the HpNPV is extracted and purified and used as the major ingredients of the said biopesticide formulation. The other ingredients include talc as carrier, activated carbon as UV (ultra violet rays) protectant, polyvinyl alcohol as dispersing agent. All the ingredients are mixed in a specific proportion in aqueous medium and the mixture is freeze dried and a wettable powder of the formulation is obtained. The formulated HpNPV is equal to or more effective in causing disease in Hyblaea puera compared to the crude virus. Compared to the crude HpNPV, the formulation HpNPV can retain its original infectivity for a 1 period not less than 24 hours when exposed to ultraviolet rays in the sunlight during use under field conditions. Compared to the crude HpNPV, the formulated HpNPV can be stored for a longer period upto 18 months under room temperature (28±4°C) in dark by retaining over fifty percentage of its original infectivity. Thus in sum, this invention relates to the production of a wettable powder biopesticide formulation containing a naturally occurring microorganism, the nucleopolyhedrovirus of the teak defoliator, Hyblaea puera as the active ingredient and talc as carrier, activated carbon as protectant against deterioration by ultra violet rays in sunlight and polyvinyl alcohol as dispersing agent, all mixed in a specific proportion, which is effective in controlling the teak defoliator, Hyblaea puera, which can withstand the damaging effect of ultraviolet rays in sunlight during field application and which has a storage Ufe with retention of over fifty percent infectivity upto eighteen months when stored, in dark at room temperature of about 28"C±4"C. Details of invention The following non-limitative examples are given by way of illustration only and will not limit the scope of the present invention. EXAMPLE I: Production of HpNPV Hyblaea puera caterpillars are reared in an artificial nutritional medium described in Table 1. Each caterpillar is separately maintained in a rearing tube containing the nutritional medium. Caterpillar of the fifth instar growth stage is then transferred to fresh rearing tube containing the artificial nutrition medium as described in Table 1 but excluding formaldehyde, contaminated with HpNPV at a dosage of 1 x 10^ polyhedral oclussion bodies (POBs). The caterpillars feeding on the said virus contaminated food die within 96 hours. The dead larvae are stored at-20"C until processed further. The frozen larvae are macerated in a homogenizer with sterile 0.1 per cent sodium dodecyl sulphate (SDS) to prevent the clumping of polyhedral occlusion bodies (POBs). The homogenate is filtered using a muslin cloth to remove coarse insect debris. The filtrate is again filtered, this time using muslin cloth in three layers. The virus suspension is centrifuged at 1000 rpm for 10 minutes for removing the large contaminant particles and the supernatant is collected and again centrifuged at 7000 rpm for 25 minutes in a REMI R-24 centrifuge (with angle rotor) to produce a pellet, comprising mainly of virus POBs. For removing the SDS the pellet is re-suspended in distilled water, once the POBs settie to the bottom, the supernatant is removed. This process is repeated thrice and the retrieved POBs are enumerated using Improved Neubauer"s hemocytometer, and stored at 4 C. The infectivity of the preparations obtained as described above is determined by a series of counts using a neubauer"s hemocytometer which makes it possible to count an average number of POBs, then by biological tests measuring the infectivity on the caterpillar. Table 1 showing the ingredients for 1 litre of the nutritional medium mentioned in example 1. Ingredients Quantity Binders Agar agar 20g Distilled Water 1000 ml Nutritional elements Teak leaf powder 20g Kabuligram flour (Cicer arietnum) lOOg Yeast extract powder lOg Casein (purified) 30g Multivitamin and mineral mixture 400mg Tocopheryl acetate 400 mg Sorbic acid Ig Ascorbic acid 3.5g Antimicrobial agents Streptomycin sulphate 120 mg Tetracycline hydrochloride 125mg Potassium methylparabenzote 2g Methyl-para- hydroxybenzoate l-5g Formaldehyde solution (10%) 2ml EXAMPLE 2: Process of Formulating HpNPV To formulate the HpNPV virus it is necessary to have a suspension of the virus in distilled water having a minimum of 3.1 x 10^ POBs/ml. To 100 ml of the above virus suspension is added one by one the following additives: 4% of activated carbon, 4 per cent of talc and 4 per cent of polyvinyl alcohol. Each ingredient on addition to the virus suspension is thoroughly mixed using a homogenizer. The ensuing slurry is dried in a freeze drier at -40°C. It involves freezing of aqueous suspension of HpNPV under vacuum for complete removal of moisture by sublimation. The freeze dried material is powdered and stored at room temperature 28±4"C. The final produce weighs 8.5 gm containing about 2.4 x 10"" POBs/gm. EXAMPLE 3: With this method of treatment, ability of the formulated virus product on Hyblaea puera caterpillars under laboratory conditions can be observed. A test is carried out against laboratory reared, pure line, Hyblaea puera caterpillars by providing them individually, equal does of HpNPV at three doses of 2 X 10", 2xl0\ 2x 10"^" POBs in batches of thirty, with crude, and formulated HpNPV provided on teak leaf discs along with that of 25 untreated test larvae fed with teak leaf disc treated with distilled water. Thus there were seven types of treatments- one batch of untreated control, three batches of crude virus with three different doses of HpNPV and three batches of formulated HpNPV with the same three doses as the crude virus. Then the mortality of Hyh/aea puera larvae is recorded at 24 hour intervals up to 96 hours. The results obtained are as follows: Untreated control: 70 late instar larvae lead to 67 pupae 3% mortality Crude virus dose (2x10^): 73 late instar larvae lead to 49 pupae 32.9 ± 3.2% mortality Crude virus dose (2x10"*): 55 late instar larvae lead to 1 pupae 98.2 ±1.7% mortality Crude virus dose (2x 10""): 59 eggs lead to 59 moths 100% mortality Formulated virus dose (2 x 10"): 35 late instar larvae lead to 16 pupae 54.3 ± 2.5% mortality Formulated virus dose (2x10"*): 43 late instar larvae lead to 8 pupae 81.4 ±3.8% mortality Formulated virus dose 2 (2x 10^"): 69 late instar larvae lead to 0 moths 100% mortality Which can be averaged as Untreated control: 3% mortality Crude virus: 77.03% mortality Formulated virus: 78.56% mortality The above results undeniably demonstrate that the power of formulated virus is equal or better than the crude virus in killing Hyblaea puera caterpillars under laboratory conditions. EXAMPLE 4: ^ith this method of treatment^ ability of the formulated virus product to retain its original infectivity by withstanding deleterious effect of ultra violet radiation in sunlight in field conditions can be observed. In a teak plantation, teak plants of comparable height (in the range 100-150cm) are selected and marked. Those plants with sufficient tender leaves well exposed to sunlight are selected for the study. 10 ^1 of the HpNPV formulation containing 1x10*^ POBs are applied to the marked area on the upper surface of a tender teak leaf in a selected tree and left to ultra violet rays in sunlight. Similarly 10 >4.1 of the crude virus containing 1x10"^" POBs are applied to the marked area on the upper surface of a tender teak leaf in a selected tree and left to direct sunlight. Different plants are used for the formulation and the crude virus. Sufficient replicates are kept for both the formulation and the virus. The leaves are removed from the plants at intervals of 0, 3, 6 and 9 h after virus exposure and the marked leaf area containing the known dose of virus is cut out and fed to individual H.puera larvae. After 5 h, the larvae were transferred individually to tubes containing artificial diet. For each formulation, the bioassay is replicated twice with 30 larvae per replicate. Observations on the mortality of larvae are recorded daily from 48 h of ingestion until death or pupation of the test insect. Cumulative larval mortality at 96h is used to compare differences between the various formulations and sunlight exposure periods. The percentage original virus infectivity remaining is computed. The results are as follows: Test samples Percentage original infectivity remaining after exposure to ultra violet rays in sunlight for different periods (Time in hours) Oh 3h 6h 9h 6Am 9 Am 12noon 3pm Crude H^^NPV 100 89 56.5 37.8 Formulated HpNPV 100 96.5 94 88.5 The above results undeniably demonstrate that the formulated product effectively retained its infectivity than the crude virus under field conditions. EXAMPLE 5 With this method of treatment, the storage life of the formulated virus product can be observed. The formulated virus product is stored at room temperature (28±4°C) and sample taken out at 0 hour, 6 months, 12 months and 18 months post storing and used for experiments to determine the effect of storage on the virulence of virus. The virus formulation is fed at a dose of 1x10"* POBs to the test caterpillars of Hjblaea puera and the mortality of the caterpillars recorded. The results obtained are as follows: 0 hour storage - 76.3% mortality of test larvae obtained with formulated product as against 92.5% mortality obtained with crude unformulated virus 6 month storage - 72.1% mortality of test larvae obtained with formulated product as against 68.1% mortality obtained with crude unformulated virus 12 month storage - 67.6% mortality of test larvae obtained with formulated product as against 65.6% mortality obtained with crude unformulated virus 18 month storage - 52.5% mortality of test larvae obtained with formulated product as against 23.6% mortality obtained with crude unformulated virus The above results undeniably demonstrate that the formulated product retains high virulence even after 18 months of storage while the crude virus has almost lost its virulence. EXAMPLE 6 With this treatment the HpNPV can be characterized and genetic fingerprint generated and identity established. DNA characterization of the HpNPV strain used for preparing the formulation is done by following the basic methods of Smith and Summers (1979). The viral DNA is digested with the Restriction Endonuclease Enzyme, Hind III and subjected to electrophoresis using 0.6% agarose gel at 35V overnight. Lambda DNA cut with Eco RI and a Ikb molecular weight marker were used as standards. DNA bands stained with ethidium bromide were photographed. The restriction enzyme profiles of HPNPV, X, DNA and the ladder are given in Figure 1. The Size of restriction fragments of HpNPV DNA identified from digests of HpNPV with Hind III, was estimated from restriction profile. The size of the various restriction fragments and the total size of HpNPV genome estimated by the sum of the restriction fragments are shown in the Table 2. Fig.l REN profiles of HpNPV DNA electrophoresed through a 0.6% agarose gel and digested with Hind III. An EcoRl digest of X DNA and 1 Kb ladder were used as markers. The size of the marker DNA bands and HpNPV DNA fragment designations are shown Restriction fragment Molecular Weight A 22.43 B 20.93 C 18.61 D 11.03 E 9.15 F 8.73 G 7.39 H 6.76 Sum 105.02 Table 2. Size of restriction fragments identified from digests of HpNPV DNA with Hind III wasestimated from restriction profiles as in Fiq 1. The size of the genome as estimated by the sums of the molecular weight of the restriction fragments is shown . References Smith, G.E. and Summers, M.D.I979. Restriction maps of 5 A.californica MNPV variants, Trichiplusia ni MNPV and Galleria mellonella MNPV DNAs with ndonuclease Sma I, Kpn I, Bam HI, Sac I, Xho I, and Eco Rl.J.Virology, 30: 828-838. Hunter-Fugita, F.R., Entwistle, P.F., Evans, H.F. and Crook, N.E. 1998. Insect viruses and pest management. John Wiley & Sons, New York, 620pp. Nair, K.S.S., Sudheendrakumar, V.V., Varma, R.V. and Chacko, K.C.I985. Studies on the seasonal incidence of defoliators and the effect of defoliation on volume increment of teak. KFRI Research Report No.30, Kerala Forest Research Institute, Peechi, Kerala, 78pp. Sudheendrakumar, V.V., Mohamed Ali, M.I., and Varma, R.V. 1988. Nuclear polyhedrosis virus of the teak defoliator Hyblaea puera. Journal of Invertebrate Pathology, 51 : 307-308. We claim: 1. A stomble biopesticide wettable formulation for biological control of the pest Hyblaea puera, the teak defoliator wherein the major ingredient is HpNPV of the virus family baculoviridae, of genome size 105.02 kbps containing talc as carrier, activated carbon as UV protectant, and polyvinyl alcohol as dispersing agent mixed in a specific proportions; which can be stored in a dark room upto a period of 18 months wherein the room temperature is maintained at around 28°C±4"C. 2. A storable biopesticide wettable formulation as claimed in Claim 1, containing specific proportions of HpNPV -14.8 % w/w; activated carbon -56.8 % w/w; talc14.2 % w/w and polyvinyl alcohol -14.2 % w[vv).

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