WO1995014084A1 - Biological control agent comprising an isolated polyhedrosis virus - Google Patents
Biological control agent comprising an isolated polyhedrosis virus Download PDFInfo
- Publication number
- WO1995014084A1 WO1995014084A1 PCT/GB1994/002438 GB9402438W WO9514084A1 WO 1995014084 A1 WO1995014084 A1 WO 1995014084A1 GB 9402438 W GB9402438 W GB 9402438W WO 9514084 A1 WO9514084 A1 WO 9514084A1
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- WO
- WIPO (PCT)
- Prior art keywords
- virus
- cells
- biological control
- control agent
- hasnpv
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
- C12N2710/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
Definitions
- This invention relates to a previously unisolated and unidentified strain of an entomopathogenic virus and to biological control agents comprising it and to its replication and use in the control of insect pests.
- the nuclear polyhedrosis viruses are insect-specific DNA viruses of the family Baculoviridae, in which enveloped rod-shaped nucleocapsids, containing a circular double-stranded DNA genome ranging from 50 to 100 M daltons in size, are occluded within a protein matrix known as a polyhedron.
- NPVs may be singly-embedded (SNPV) or multiply-embedded (MNPV) , according to the number of nucleocapsids per envelope. Because these viruses are pathogenic to several of the major lepidopteran pests of agricultural crops and forestry, they have been intensively studied as biological pest control agents. Although they have been sold commercially for this purpose, the widespread use of such products has been limited by their poor persistence, slow speed of kill, and high cost per unit area treated.
- a new strain of singly-embedded Heliothis zea nuclear polyhedrosis virus is described, deposited in the European Collection of Animal Cell Cultures under the accession number V93071301. It is characterised by high virulence, which provides an advantage over existing strains.
- HzSNPV singly-embedded Heliothis zea nuclear polyhedrosis virus
- the virus may be used for control of pest species such as species of the genera Heliothis and Helicoverpa. for example when formulated as an t agricultural spray.
- a biological control agent comprising the virus > of the present invention, and a method of combating an insect pest at a locus which comprises treating the insect or locus with the biological control agent of the present invention.
- the present ivention also provides a method for the j_n vitro replication of the virus of the present invention comprising infecting a culture of insect cells in a medium with the virus, culturing the cells for a period until polyhedral inclusion bodies are detected within the nuclei of the cells, and harvesting the virus from cells and/or the medium.
- the virus was originally propagated from three dead larvae of Hel coverpa zea. discovered in Pikeville, North Carolina, U.S.A.
- the virus was propagated by transferring cadavers into plastic cups containing freshly-prepared insect diet, crushing the cadaver on the surface of the diet, and introducing a larva of Heliothis virescens or Helicoverpa zea which was allowed to feed on the contaminated diet. Infection was normally carried out at 20-25°C. Propagation was carried out at intervals varying between two weeks and one year. Cadavers were stored in plastic diet cups at ambient temperature. Purification
- the virus was propagated in Heliothis virescens as described above. Symptoms were consistent with those of virus infection. Infected larvae became flaccid, blackened, and tended to rupture and liquefy. Inoculum of the pathogen was produced by further transfers to diet, by streaking the surface of the diet with a sterile plastic loop containing tissue from diseased insects. Cadavers were stored at -20°C. Fifty infected larvae were obtained in this way.
- the cadavers were collected, triturated in sterile distilled water, and filtered through two layers of muslin. The filtrate was repeatedly centrifuged at 1000 rpm for five minutes to remove insect debris. The supernatant was then centrifuged at 4,100 rpm for 20 minutes, and the pellet retained after washing. The pellet was resuspended in water and examined under the microscope. Particles typical of a nuclear polyhedrosis virus (NPV) were observed. Approximately 5 X 10 polyhedral inclusion bodies (PIB) were obtained per larva.
- NPV nuclear polyhedrosis virus
- the virus was bioassayed together with a characterised strain of Heliothis armiqera nuclear polyhedrosis virus, HaSNPV A44EB, which had previously been shown to infect Heliothis virescens.
- Helicoverpa armiqera and Helicoverpa punctiqera This strain had been cloned from an isolate collected by Dr. Robert Teakle from infected Helicoverpa armiqera on cotton in Queensland, Australia in 1974.
- Plugs of pinto bean insect diet were placed in wells of icrotitre dishes. Polyhedra, at varying concentrations, were then applied to the surface of each plug in a volume of l ⁇ l water. After 24 hours, all larvae which had consumed the diet plugs were transferred to plastic pots containing diet. The insects were examined after seven days.
- HzNPV V93071301 is more virulent by at least an order of magnitude than HaSNPV A44EB against at least _ virescens and ⁇ . zea.
- HzNPV V93071301 was established in cell culture by extracting hemolymph from infected insects into a static culture of Helicoverpa zea cells. The virus successfully infected the cells, forming polyhedra within the nucleus, and could be propagated in cell culture. Plaques were formed in a confluent layer of H. zea cells when exposed to the non-occluded form
- Infected insects were triturated in an aqueous solution of 1% sodium dodecyl sulphate (SDS).
- SDS sodium dodecyl sulphate
- the suspension of polyhedra was adjusted to q approximately 5X10 PIB per sample, filtered, centrifuged, and washed as described previously, removing the SDS.
- Polyhedra were then dissolved in a solution of Na 2 C0 3 buffer at pHlO. The suspension was centrifuged at 5000 rpm for five minutes to remove debris, and the supernatant centrifuged at
- the digest was incubated for 3-4 hours at 65°C with gentle agitation, mixed with 400 ⁇ l phenol/CHC isoamyl alcohol (50:48:2) and centrifuged for five minutes at 13000 rpm. This extraction was performed twice. Excess salts, detergents, phenol and chloroform were removed by dialysis for approximately 12 hours. The DNA preparations were then digested for 5 to 6 hours with each of the restriction endonucleases EcoRI,
- the DNA preparations were analysed by agarose gel electrophoresis (0.7 - 0.8% agarose).
- HzNPV V93071301 As a singly-embedded Heliothis zea nuclear polyhedrosis virus (HzSNPV), strain V93011301 is clearly distinct, according to DNA profile, from either of the other strains which were tested.
- Figure 1 shows an electron micrograph of a section of a polyhedron from the virus of the present invention.
- a sample was fixed in 3% gluteraldehyde in 0.05M phosphate buffer for 16 hours at 4°C, postfixed in 1% osmium texroxide in 0.05M phosphate buffer, set in agarose gel, dehydrated and infiltrated. The sample was then set in Spurr's resin. Ultrathin sections (about 70-90nm) were stained with 0.2% lead citrate in 0.1N sodium hydroxide for 5 minutes. The sections were examined using a Phillips PW 6002 Transmission Electron Microscope.
- the novel virus strain of the invention is suitable for use as a biological control agent for the control of insect pests, particularly larval lepidopterous pests of agriculturally useful crops, such as cotton, vegetables, friut, soya, cereals and the like.
- insect pests particularly larval lepidopterous pests of agriculturally useful crops, such as cotton, vegetables, friut, soya, cereals and the like.
- insects which may be combated and controlled by the use of the virus are Heliothis and Helicoverpa spp. such as H. zea. H. virescens. H. armi ⁇ era. H., punctioera and the like.
- the virus is applied to the pests or the locus of the pests (e.g. a growing crop) in the form of a composition in which the virus is admixed with suitable diluents or carriers which may be solid or liquid, optionally in the presence of additional ingredients such as surfactants, wetting and dispersing agents, and ingredients intended to protect the viability of the virus such as U.V. absorbents, antioxidants and the like.
- suitable diluents or carriers which may be solid or liquid, optionally in the presence of additional ingredients such as surfactants, wetting and dispersing agents, and ingredients intended to protect the viability of the virus such as U.V. absorbents, antioxidants and the like.
- Preferred formulations include wettable powders, oil suspensions, and microcapsulated suspensions of the virus (e.g. wherein the capsule coat is obtained by a coascervation technique or by in-situ cross-linking to form a polyurea or polyamide shell).
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Environmental Sciences (AREA)
- Dentistry (AREA)
- Medicinal Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Biomedical Technology (AREA)
- Plant Pathology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Pest Control & Pesticides (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BR9408057A BR9408057A (en) | 1993-11-15 | 1994-11-07 | Isolated strain in nuclear polyhedrosis virus, biological control agent and processes to combat an insect pest in one place and for the in vitro replication of this virus |
AU81108/94A AU691843B2 (en) | 1993-11-15 | 1994-11-07 | Biological control agent comprising an isolated polyhedrosis virus |
EP95900205A EP0729508A1 (en) | 1993-11-15 | 1994-11-07 | Biological control agent comprising an isolated polyhedrosis virus |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9323495.3 | 1993-11-15 | ||
GB939323495A GB9323495D0 (en) | 1993-11-15 | 1993-11-15 | Biological control agent |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1995014084A1 true WO1995014084A1 (en) | 1995-05-26 |
Family
ID=10745175
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1994/002438 WO1995014084A1 (en) | 1993-11-15 | 1994-11-07 | Biological control agent comprising an isolated polyhedrosis virus |
Country Status (12)
Country | Link |
---|---|
US (1) | US5681734A (en) |
EP (1) | EP0729508A1 (en) |
CN (1) | CN1141648A (en) |
AP (1) | AP659A (en) |
AU (1) | AU691843B2 (en) |
BR (1) | BR9408057A (en) |
GB (1) | GB9323495D0 (en) |
ID (1) | ID16156A (en) |
IL (1) | IL111604A (en) |
PH (1) | PH31408A (en) |
WO (1) | WO1995014084A1 (en) |
ZA (1) | ZA948984B (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AP659A (en) * | 1993-11-15 | 1998-08-18 | Zeneca Ltd | Biological control agent comprising an isolated polyhedrosis virus. |
US5885569A (en) * | 1994-03-25 | 1999-03-23 | Zeneca Limited | Biological insect control agent |
EP0908099A1 (en) * | 1997-09-16 | 1999-04-14 | Societe Des Produits Nestle S.A. | Natural insecticide affecting the growth of Heliothis armigera |
WO2017174430A1 (en) * | 2016-04-06 | 2017-10-12 | Bayer Cropscience Aktiengesellschaft | Combination of nuclear polyhedrosis virus and diamides |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990010387A1 (en) * | 1989-03-07 | 1990-09-20 | The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce | A multiple embedded nuclear polyhedrosis virus from celery looper with activity against lepidoptera |
US5023182A (en) * | 1988-06-28 | 1991-06-11 | The United States Of America As Represented By The Secretary Of Agriculture | Novel virus composition to protect agricultural commodities from insects |
US5132220A (en) * | 1989-06-30 | 1992-07-21 | The United States Of America As Represented By The Secretary Of Agriculture | More virulent biotype isolated from wild-type virus |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5298418A (en) * | 1991-09-16 | 1994-03-29 | Boyce Thompson Institute For Plant Research, Inc. | Cell line isolated from larval midgut tissue of Trichoplusia ni |
GB9323495D0 (en) * | 1993-11-15 | 1994-01-05 | Zeneca Ltd | Biological control agent |
-
1993
- 1993-11-15 GB GB939323495A patent/GB9323495D0/en active Pending
-
1994
- 1994-11-07 AP APAP/P/1996/000807A patent/AP659A/en active
- 1994-11-07 EP EP95900205A patent/EP0729508A1/en not_active Withdrawn
- 1994-11-07 WO PCT/GB1994/002438 patent/WO1995014084A1/en not_active Application Discontinuation
- 1994-11-07 BR BR9408057A patent/BR9408057A/en not_active Application Discontinuation
- 1994-11-07 AU AU81108/94A patent/AU691843B2/en not_active Ceased
- 1994-11-07 CN CN94194809A patent/CN1141648A/en active Pending
- 1994-11-10 US US08/337,360 patent/US5681734A/en not_active Expired - Fee Related
- 1994-11-11 IL IL11160494A patent/IL111604A/en not_active IP Right Cessation
- 1994-11-11 ZA ZA948984A patent/ZA948984B/en unknown
- 1994-11-11 ID IDP941945A patent/ID16156A/en unknown
- 1994-11-14 PH PH49363A patent/PH31408A/en unknown
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5023182A (en) * | 1988-06-28 | 1991-06-11 | The United States Of America As Represented By The Secretary Of Agriculture | Novel virus composition to protect agricultural commodities from insects |
WO1990010387A1 (en) * | 1989-03-07 | 1990-09-20 | The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce | A multiple embedded nuclear polyhedrosis virus from celery looper with activity against lepidoptera |
US5132220A (en) * | 1989-06-30 | 1992-07-21 | The United States Of America As Represented By The Secretary Of Agriculture | More virulent biotype isolated from wild-type virus |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AP659A (en) * | 1993-11-15 | 1998-08-18 | Zeneca Ltd | Biological control agent comprising an isolated polyhedrosis virus. |
US5885569A (en) * | 1994-03-25 | 1999-03-23 | Zeneca Limited | Biological insect control agent |
EP0908099A1 (en) * | 1997-09-16 | 1999-04-14 | Societe Des Produits Nestle S.A. | Natural insecticide affecting the growth of Heliothis armigera |
WO2017174430A1 (en) * | 2016-04-06 | 2017-10-12 | Bayer Cropscience Aktiengesellschaft | Combination of nuclear polyhedrosis virus and diamides |
Also Published As
Publication number | Publication date |
---|---|
AP9600807A0 (en) | 1996-07-31 |
US5681734A (en) | 1997-10-28 |
EP0729508A1 (en) | 1996-09-04 |
IL111604A (en) | 1999-01-26 |
ZA948984B (en) | 1996-02-12 |
AU691843B2 (en) | 1998-05-28 |
BR9408057A (en) | 1996-12-24 |
IL111604A0 (en) | 1995-01-24 |
ID16156A (en) | 1997-09-11 |
AP659A (en) | 1998-08-18 |
GB9323495D0 (en) | 1994-01-05 |
CN1141648A (en) | 1997-01-29 |
PH31408A (en) | 1998-01-01 |
AU8110894A (en) | 1995-06-06 |
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