This invention relates to in vivo insect culture methed of producing spilarctia obliqua nuctear polyhedrosis viral pesticide.
BACKGROUND OF THE INVENTION:
Viral pesticides in general, can be produced either by in vitro- insect cell culture methed or by in vivo methed. Under the in vivo technique, the larvae can be reared either with artificial diet or with foliage of the food plant. But in vivo methed with natural foliage as food has been used only for small scale rearing especially for preliminary bioassays.
In the context of SoNPV, the polyhedra of the virus has so far been produced only for the purpose of lap bioassay experiments using leaf disc methed or leaf dip methed with maximum of 30 or 40 individuals per culture (Battu and Ramakrishnan, 1987, Chaudhari, 1997). The larvae were reared using the crop foliage. But iarge scale in vivo production of SoNPV polyhedra was not attempted earlier.
As this pest is known to infest nearly 126 plant species of 25 different families, it becomes imperative to controf their incidence. Further, Oil seeds, pulses, grams and Jute plants are the main target hosts in the Eastern India, where this pest maintains appreciable density during May to September. Field trials conducted during the DBT project tenure showed that the abundance of S. obliqua could be kept under check by spraying SoNPV @ 108 polyhedra per ml. Therefore, attempts have been made here to produce this viral pesticide.
However, viral pesticide in general, has been produced either through artificial diet - surface contamination methed (Armes ef al, 1992; Cunningham, 1982; Groner, 1986) or by insect cell culture technique (Granados and Federici, 1986).
Esrlier metheds available now for the mass production of viral pesticide describe and discuss either through In vivo artificial diet methed (surface contamination of respective polyhedra over the diet) or by in vttro-tnsect ceH culture methed (Granados and Federicl, 1986). Both these metheds are cumbersome and costly especially fn the context of developing countries like India. Therefore, the above practical difficulty forced to look for simple, cost effective and farmer's friendly methed and that led to the genesis of the present technique.
OBJECTS OF THE INVENTION:
An object of this invention is to propose an in vivo insect culture methed of producing spflarctia obliqua nuclear polyhedrosis viral pesticide.
Another object of this invention is to propose an in vivo insect culture methed of producing spilarctia obliqua nuclear poryhedrosfs viral pesticide which is simple.
Yet another object of this invention is to propose an in vivo insect culture methed of producing spilarctia obfiqua nuclear polyhedrosis viral pesticide which is ecofriendty.
Further, object of this invention is to propose an in vivo insect culture methed of producing spilarctia obliqua nuclear polyhedrosis viral pesticide which is cost effective.
Still further object of this invention is to propose an in vivo insect culture methed of producing spilarctia obUqua nuclear polyhedrosis viral pesticide which has an implication of weed (Ipomoea cornea) control.
REIF DESCRIPTION OF THE INVENTION
According to this invention there is provided a in vtvo insect culture methed of
producing spBarctia obtlqua nuclear polyhedrosis viral pesticide comprising the
steps of:
treating the twig of the weed in a suspension of SoNPV inserting the said treated
twig in water In a conical flask ;
placing the said flask In a big bucket;
releasing the starved caterpillars on the said twig and covering the bucket with
muslin cloth;
removing trie excreta and dead larvae daily and observing the larval mortality
everyday;
storing the dead larvae in a bottle containing water;
filtering the cadaver to romove the debris;
washing the filtrate with SDS & water alternately to get white precipitate of
SoNPV polyhedra;
diluting the precipitate with water and storing the same in refrigerator for use.
DETAILED DESCRIPTION OF THE INVENTION.
8.4X1011 polyhedral occlusion bodies (POBs) of Spfiarctla obliqua nuclear polyhedrosi!} virus (SoNPV) can be produced from 150 larvae of S. obliqua within 12 days by inoculating IV instar caterpillars with 5x105 POBs of SoNPV/ml and rearing them together in a plastic bucket (16 Htre), feeding them with foliage of the weed Ipomoea cornea Jac as there in the accompanying Figure -1.
The pathegenic cell suspension of SoNPV was prepared initially @ 5x105POBs. Young twig consisting of about 10 or 12 fresh foliages of l.cornea was collected from the field. The leaves were dipped inside the beaker containing the pofyhdra
4- isuspension or the leaves could be cproyod with the abovo suspension. On ofr
drying, the cut end of the twig was Inserted into a conical flask wtlh water to
provont drying up of the leaves. Plugging the conical flock with Ettle cotton
Ground the twig wou!d prevent the ertry of larvae into Iho conlcel ftoek, booidos
keeping the twig straight. Thfo cu3ure setup was placed Inside a ebon
transparent plasttc bucket (co 10 t:lro capacity). Keeping o fiDor paper mctdo the
bucket would enabte to collect the excreta and absorb the moisture. Tha
transparent bucket would facl^ote to oee the culluro set up (noWo. 130
individual of IV meter torvae of S. ob!!qira which were etorved for oboul 4 hours
iwere released on the twigs of each bucket. The bucket was covered wflh a
tdoubie layered muslin cloth and put the rubber bond (width 1 cm) ttgrdfy to prevent the escape of larvae from the culture. The rubber band was found to be convenient because of its easiness to take out or put the muslin ctoth ctowly witheut removing the band every tfine from the bucket. On feeding thd vtrus {inoculated folJago, fresh leaves were provided dafly. The cu!!ure woo chocked
doGy and kept clean. Their food consumption reduced gradual^ after 4 or S dayo and they showed typical symptoms of viral infection like restbssnsss, loose ond ohlny cuticle, eventualfy hanging upoldo down. At this stage, fl would bo better to pake out th@ dead larvae from the cuUure and put tt fit a bottle cordaWng water. Further delay in picking them would rooull in wastage of polyhedra along wtth the
oozing haonnolymph. The cadaver that wcs stored to the water cou!d be processed for purification by standard methed to get pure potyhedra. The present in vivo methed of producing viral pesticide (SoNPV) is vory easy to practice with minimum Infrastructure faci^teo. No need to employ person to carry out the works, rather the farmer tonoetf can took in to the cufturo dai^f end produce polyhedra according to his needs. Culture check up require
epproKimatory 30 minutes per day per cutlure. Eoch culture needs a (transparent) plastic bucket, conical flask (250ml capacity) with water and little cotton, muslin cloth (2 m2), Ipomoea weed twig, 150 larvae of S. oblique (IV motor), end SO ml of polyhedra inoculum of SoKPV. Since, augmentation of potyhadra will be carried ut during ths period of pest occurronco pay to September), the larval colony
eteo bo evcilcbb under ftefd condilfonc for Indoor roaring, it moons the farmers need not maintain a separate cufture stock of ths ccterplHar. Further, studios tnvo abo shown that the potyhodro could be otorcd of room tomporcturo for more tfion 12 months wdhoul effecting Its vtebiL'ty. Therefore, potyhadra produced during the current yoor can be used for the newt cropping coocon.
Honco, by ail moanc the proconl tcchntquo is bettor thsn trvo oitbllrtg methed. to ovorcome the problem of ovorcrowding the optimum rooring copcctiy woo selected to be ISO larvae per culture wflh about 5% canniboCsm. This methed of rearing the caterpC&r using the weed for the production of
SoNPV polyhedra is not known both at the National and Inlornetional tavol. Evon for other typoo of bocuSovfrujtoc, orrfy the artLTcial diet end in vttro coO cuDuro techniqua have been followed In majority of the cases especlafry for the targe scoto production of viral pcstfcfdo. Therefore, the idea of using weed oo o food source to rear virus Infected larvae and cu3uring nearly 150 larvae together In a
simple bucket is first of its kind for viral pecticido production In gonorol ond SoNPV in pEirticuJar. Rearing the tervae using artfficial dbt is dofinHoty costlier than that of the natural foliage. Further, to vUro eel) cuffuro is costlier than In v&o ntethed. Farmers always prefer simple and coct offocttvo mot ho do. More ovor, in
the present methed, onty wood foliage is used which Is abundantly and frooly ovaltebfa in our country. Therefore, this methed toe good uiffty voluo In thedeveloping countries.
EXAMPLE:
In the new methed, a small twig or two win 10 or 12 leaves of Ipomoea cornea were taken and dipped hi pathegenic suspension of SoNPV containing 5x105 POBs/mi (=tCso value of IV instar). After draining the water, the cut end of the twig was inserted in to a smatt conical flask with water. This set up was kept Inside the plastic bucket of 16 fire capacity. The caterpHars 0V Instar.), which
were starved for 4 hours, were released on the tpomoea twig Q 190 larvaeA)uckt1. To prevent their escape, the bucket was tightly covered wlh a double layered muslin cloth. On feeding the virus Inoculated leaf, fresh leaves of tpomoea (virus untreated) were provided. Care was taken to remove the excreta and dead larvae daily. Observation on the larval tnortaflty of individual culture
was noted every day. The dead larvae were stored to a bottle containing water and kept for purification under room temperature for 10 days, later, the cadaver was filtered through a double layered muslin cloth in order to remove the debris.
|Ns filtrate can be washed with 80S (0.5% final concentration) and water alternately by following the metheds of Sudhakar et at., 1997). Finally the peHeted white precipitate containing SoNPV polyhedra was diluted wMh little water and stored in a refrigerator for future use. (In case, purification by SOS
methed Is difficult, the fanner can dHute the above filtrate accordingly and apply on the crops concerned).

We Claim
1. A method of producing spilarctia obliqua nuclear polyhedrosis
viral pesticide comprising the steps of: -
i) treating the 1 or 2 twig of the weed in a suspension of
spilarctia obliqua nuclear polyhedrosis virus, containing
5xl05 POBs/ml (Poly hedral occlusion bodies) in a conical
flask ii) Placing the said flask in a plastic bucket of 16 litres
capacity, iii) releasing the caterpillars (IV instar) that are starved for 4
hours on the ipomoea twig @ 150 larvae/bucket and
covering the bucket with muslin cloth, iv) removing the excreta and dead larvae daily and observing
the larval mortality everyday v) Storing dead larvae in a bottle containing water and
keeping at room temperature for 10 days, vi) filtering the cadver through double layered muslin cloth to
remove the debris vii) washing the filtrate with 0.5% SDS and water alternately so
as to get white precipitate of SONPV polydedra. viii) diluting the precipitate with 5-7 ml water and storing in
refrigerator.
2. The method as claimed in claim 1, wherein the said weed twig
is of ipomoea cornea.

3. The method as claimed in claim 1, wherein the said suspension of SONPV contains 5xl05 polyhedral occlusion Bodies per ml of suspension, wherein it donotes the concentration of the virus, in the form of occlusion bodies visible under microscope and their number is used as concentration or dose.
4. The method as claimed in claim 1, wherein the dead larvae are stored in a bottle containing water at room temperature for 10 days for purification.
5. The method as claimed in claim 1, wherein the said SDS is used for washing the filtrate at 0.5% concentration.