COMPOSITION COMPRISING MULTIPLE BACULOVIRUSES TO TARGET DIFFICULT INSECT SPECIES

Abstract

The present invention relates to an agricultural composition comprising at least three insect pathogenic viruses, selected from Autographica californica (Alfalfa Looper) multiple nucleopolyhedrovirus (AcMNPV), Helicoverpa armigera nucleopolyhedrosis virus (HaNPV), Plutella xylostella granulovirus (PxGV), Spodoptera litura (oriental leafworm moth) nucleopolyhedrovirus (SpltNPV) and Spodoptera exigua (beet armyworm) nucleopolyhedrovirus (SeNPV).

Claims

1. An agricultural composition comprising at least three insect pathogenic viruses, selected from Autographica californica (Alfalfa Looper) multiple nucleopolyhedrovirus (AcMNPV), Helicoverpa armigera nucleopolyhedrosis virus (HaNPV), Plutella xylostella granulovirus (PxGV), Spodoptera litura (oriental leafworm moth) nucleopolyhedrovirus (SpltNPV) and Spodoptera exigua (beet armyworm) nucleopolyhedrovirus (SeNPV).

2. The agricultural composition according to claim 1 wherein said at least three insect pathogenic viruses are AcMNPV, HaNPV and PxGV.

3. The agricultural composition according to claim 1 wherein said at least three insect pathogenic viruses are SeNPV, HaNPV and PxGV.

4. The agricultural composition according to claim 1, comprising the insect pathogenic viruses in a ratio of between 5:1:1 and 1:1:5.

5. The agricultural composition according to claim 2, wherein the ratio of the insect pathogenic viruses is 1:1:1.

6. The agricultural composition according to claim 1, comprising at least one further insect pathogenic virus.

7. The agricultural composition according to claim 2, comprising at least one further insect pathogenic virus selected from the group consisting of Spodoptera litura (oriental leafworm moth) nucleopolyhedrovirus (SpltNPV) and Spodoptera exigua (beet armyworm) nucleopolyhedrovirus (SeNPV).

8. The agricultural composition according to claim 1, comprising AcMNPV, HaNPV, PxGV and SpltNPV.

9. The agricultural composition according to claim 1, comprising AcMNPV, HaNPV, PxGV and SeNPV.

10. The agricultural composition according to claim 1 comprising AcMNPV, HaNPV, PxGV, SpltNPV and SeNPV.

11. The agricultural composition according to claim 1 wherein each insect pathogenic virus is present in an amount of between 1×10.sup.8 and 1×10.sup.12 occlusion bodies per mL or per gram.

12. The agricultural composition according to claim 1, wherein said insect pathogenic viruses are selected from AcMNPV isolates comprised in VPN-ULTRA from Agricola El Sol, LOOPEX from Andermatt Biocontrol, LEPIGEN from AgBiTech and isolate C6, HaNPV isolates comprises in VIVUS® MAX and ARMIGEN from AgBiTech, HELICOVEX from Andermatt Biocontrol and Keyun HaNPV, PxGV isolates comprised in PLUTELLAVEX® from Keyun, and isolate K1, SpltNPV isolate K1 and SeNPV isolates comprised in KEYUN SeNPV.

13. The agricultural composition according to claim 1, wherein at least one insect pathogenic virus is a recombinant virus.

14. The agricultural composition according to claim 1, which is effective against at least one insect selected from Tuta absoluta, Spodoptera frugiperda, Spodoptera exigua and Helicoverpa armigera.

15. The agricultural composition according to claim 1, further comprising at least one auxiliary selected from carriers, ultraviolet protectants, frost protectants, diluents, coating polymers, surfactants and pH regulators.

16. A method for protecting a plant from insect pests comprising applying to such insect pest or its habitat or plant an insecticidally effective amount of the agricultural composition according to claim 1.

17. A method for reducing feeding damage on plants caused by insect pests comprising applying to such insects or insect habitat or plant an insecticidally effective amount of the agricultural composition according to claim 1.

18. The method of claim 16, wherein said insect pest is selected from Tuta absoluta, Spodoptera frugiperda, Spodoptera exigua, Plutella xylostella and Helicoverpa armigera.

19-22. (canceled)

23. The method for producing an agricultural composition according to claim 1, comprising mixing said insect pathogenic viruses and optionally at least one auxiliary.

Description

EXAMPLES

[0073] The examples illustrate the present invention in a non-limiting fashion.

Example 1: Material and Methods

Insects and Viruses:

[0074] The laboratory colonies of Spodoptera frugiperda, Spodoptera exigua and Helicoverpa armigera were reared on standard noctuid artificial diet, Tuta absoluta were reared on tomato plants under controlled conditions 25±1° C. and a 55±5% relative humidity. The origin of the populations is described in Table 1.

[0075] Other insects used in the bioassay were purchased from Benzon.

[0076] Five different baculovirus formulations comprising Autographica californica multiple nucleopolyhedrovirus (AcMNPV), Helicoverpa armigera nucleopolyhedrosis virus (HaNPV), Plutella xylostella granulovirus (PxGV), Spodoptera litura nucleopolyhedrovirus (SpltNPV) and Spodoptera exigua nucleopolyhedrovirus (SeNPV) were tested against the above-mentioned lepidopteran species. The application rate was: 6.67×106/mL for AcMNPV, 1.20×107/mL for HaNPV, 6.00×106/mL for SeNPV, 1.33×107 for SpltNPV and 1.00×108 for PxGV in 450 L water/ha (see Table 2). The mix of viruses contained all five viruses with mentioned concentrations in 450 L water/ha (6.67×106 AcMNPV+1.20×107 HaNPV+6.00×106 SeNPV+1.33×107 SpltNPV+1.00×108 PxGV in 450 L water/ha). The final application rate per ha is described in Table 2. Commercial product based on Bt-toxins (4 mL/L) served as a positive control.

[0077] Experiments were conducted at RT using maize (Zea mays subsp. mays) for Spodoptera frugiperda and Spodoptera exigua, cotton (Gossypium herbaceum) for Helicoverpa armigera and tomato (Solanumlycopersicum) for Tuta absoluta.

Insect Bioassays:

[0078] Twelve second- to third-instar larvae of S. frugiperda, S. exigua, T. ni; P. xylostella; H. zea; H. virescens; or H. armigera were prepared for each treatment. Leaf discs of cotton, corn and cabbage as well as S. frugiperda, S. exigua and H. armigera larvae were dipped for two seconds in the prepared solutions and placed in 12-well plates. To avoid leaf disc desiccation either wet filter papers or 1% agar were placed under the leaf disc in the plates. In Examples 2 to 4, larval survival and feeding damage (% severity of damage) was observed on day four and day seven post treatment. Larvae were considered as dead when completely immobile. Affected larvae were considered as alive. In examples 5 to 7, the leaf damage was recorded after seven days post treatment. Abbott was calculated as below formula:

[00001]$\mathrm{Abbott}\mathrm{of}\mathrm{leaf}\mathrm{consumption}\%=\left(1-\frac{n\mathrm{in}T\mathrm{after}\mathrm{Treatment}}{n\mathrm{in}\mathrm{Co}\mathrm{after}\mathrm{Treatment}}\right)*100$

[0079] n: % of leaf consumption; T: treatment, Co: Control without treatment

[0080] For Tuta absoluta five tomato leaves infected with first- to second-instar larvae were used in the bioassays. For T. absoluta whole tomato leaves infected with the insects were dipped in the different virus solutions and incubated in petri dishes.

TABLE-US-00001 TABLE 1a The origin of lepidopteran species used in the bioassay (Examples 2 to 4). Species Country City Crop Season Spodoptera Brazil São Paulo 2005 frugiperda Spodoptera exigua England ICI 1989 Helicoverpa armigera Germany Darmstadt 2000 Tuta absoluta Brazil Paulinia 2008

TABLE-US-00002 TABLE 1b The origin of lepidopteran species used in the bioassay (Examples 5 to 7). Species Source Country Spodoptera frugiperda Brazil Spodoptera exigua England Helivoverpa amigera Germany Tuta absoluta Brazil Trichoplusia ni United States Plutella xylostella United States Heliothis virescens United States Helicoverpa zea United States Spodoptera exigua United States

Examples 2 to 4

[0081]

TABLE-US-00003 Final Final Original Original mL or g per Application Application Baculoviruses Formulation PIB/mL or g 450 L/ha Rate/mL Rate/ha AcMNPV liquid 7.50 × 10.sup.9  400 6.67 × 10.sup.6 3.00 × 10.sup.12 HaNPV WG 6.00 × 10.sup.10 90 1.20 × 10.sup.7 5.40 × 10.sup.12 PxGV liquid 3.00 × 10.sup.10 1500 1.00 × 10.sup.8 4.50 × 10.sup.13 SpltNPV WG 2.00 × 10.sup.10 300 1.33 × 10.sup.7 6.00 × 10.sup.12 SeNPV WG 3.00 × 10.sup.10 90 6.00 × 10.sup.6 2.70 × 10.sup.12 Combination 6.67 × 10.sup.6 3.00 × 10.sup.12 of all 5 1.20 × 10.sup.7 5.40 × 10.sup.12 viruses: 1.00 × 10.sup.8 4.50 × 10.sup.13 1:1:1:1 1.33 × 10.sup.7 6.00 × 10.sup.12 6.00 × 10.sup.6 2.70 × 10.sup.12

Examples 5 to 7

[0082]

TABLE-US-00004 Original Original Polyhedral Final Application Formulation Inclusion Bodies (PIB) Rate/mL AcMNPV WG 3.2 × 10.sup.10/g.sup.  1 × 10.sup.8 HaNPV WG 6 × 10.sup.10/g 1 × 10.sup.8 PxGV liquid  3 × 10.sup.10/ml 1 × 10.sup.8 SpltNPV WG 2 × 10.sup.10/g 1 × 10.sup.8 SeNPV WG 3 × 10.sup.10/g 1 × 10.sup.8 AcMNPV 1 × 10.sup.8 HaNPV 1 × 10.sup.8 PxGV 1 × 10.sup.8 SpltNPV 1 × 10.sup.8 SeNPV 1 × 10.sup.8

Example 8

[0083]

TABLE-US-00005 Baculoviruses Original Rate g Field Combination Original PIB/mL or Application of 5 viruses Formulation or g ml/ha Rate/ha AcMNPV liquid 7.50 × 10.sup.9  200 1.5 × 10.sup.12 HaNPV WG 6.00 × 10.sup.10 90 5.4 × 10.sup.12 PxGV liquid 3.00 × 10.sup.10 1500 4.5 × 10.sup.13 SpltNPV WG 2.00 × 10.sup.10 300 6.0 × 10.sup.12 SeNPV WG 3.00 × 10.sup.10 90 2.7 × 10.sup.12 HaNPV WG 6.00 × 10.sup.10 90 5.4 × 10.sup.12 PxGV liquid 3.00 × 10.sup.10 1500 4.5 × 10.sup.13 SeNPV WG 3.00 × 10.sup.10 90 2.7 × 10.sup.12

Example 2: A Combination of Five Baculoviruses Exert Excellent Activity Against Tuta absoluta

[0084] The experimental setup is described in Example 1. The five single viruses AcMNPV, HaNPV, PxGV, SpltNPV and SeNPV were tested as well as a mixture of all five viruses. 10 leaves per treatment were used. As can be seen in FIG. 1, the mixture of five viruses exert an unexpected efficacy against Tuta absoluta already 4 days after treatment which was not derivable from the activity of any of the single viruses.

Example 3: A Combination of Five Baculoviruses Exert Excellent Activity Against Spodoptera frugiperda

[0085] The experimental setup is described in Example 1. The five single viruses AcMNPV, HaNPV, PxGV, SpltNPV and SeNPV were tested as well as a mixture of all five viruses. 24 larvae per treatment were used. As can be seen in FIG. 2, the mixture of five viruses, at least 7 days after treatment, exert an unexpected efficacy against Spodoptera frugiperda which was not derivable from the activity of any of the single viruses.

Example 4: A Combination of Five Baculoviruses Exert Excellent Immediate Activity Against Helicoverpa armigera and Spodoptera exigua

[0086] The experimental setup is described in Example 1. The five single viruses AcMNPV, HaNPV, PxGV, SpltNPV and SeNPV were tested as well as a mixture of all five viruses. 24 larvae per treatment were used. As can be seen in FIGS. 3 and 4, the mixture of five viruses exert an unexpected efficacy against Helicoverpa armigera and Spodoptera exigua 4 days after treatment which was not derivable from the activity of any of the single viruses. Most notably, the damage effected to the plants is very low.

Example 5: A Combination of Five Baculoviruses Exert Excellent Immediate Activity Against Helicoverpa armigera and Spodoptera exigua

[0087] The experimental setup is described in Example 1. The five single viruses AcMNPV, HaNPV, PxGV, SpltNPV and SeNPV were tested as well as a mixture of all five viruses. 24 larvae per treatment were used. As can be seen in FIG. 5, the mixture of five viruses exert an unexpected efficacy against Helicoverpa zea and Trichoplusia ni 7 days after treatment which was not derivable from the activity of any of the single viruses.

Example 6: A Combination of Three Baculoviruses Exert Excellent Activity Against Spodoptera exigua

[0088] The experimental setup is described in Example 1. The three single viruses AcMNPV, HaNPV, PxGV, were tested as well as a mixture of all three viruses. 12 larvae per treatment were used. As can be seen in FIG. 6, the mixture of three viruses exert an unexpected efficacy against Spodoptera exigua 7 days after treatment which was not derivable from the activity of any of the single viruses. The 3 viruses combination showed very good dose response.

Example 7: A Combination of 4 Baculoviruses Shows Broad Spectrum Against Lepidoptera Species

[0089] The experimental setup is described in Example 1. The different combinations tested are listed below:

[0090] Mix 1234 (AcMNPV, HaNPV, PxGV and SpltNPV)

[0091] Mix 1245 (AcMNPV, HaNPV, SpltNPV and SeNPV

[0092] Mix 2345 (HaNPV, PxGV, SpltNPV and SeNPV)

[0093] Mix 5 (AcMNPV, HaNPV, PxGV, SltNPV and SeNPV)

[0094] The combinations comprising four baculoviruses were tested at 107 and 108 PIB and five combination at 105, 106 and 107 against Spodoptera frugiperda, Trichoplusia ni and Helicoverpa zea. 12 larvae per treatment were used. Based on FIG. 7, it can be seen that all combinations have better efficacy than the single viruses (see above).

Example 8: Materials and Methods for Field Testing Baculoviruses Combinations

[0095] Complete Randomized block experiments were set up at sites in Italy and Spain in 2020. The five virus combination of AcMNPV, HaNPV, PxGV, SpltNPV and SeNPV were tested at 3 rates 100%, 50% and 10% of Field application rate (Table above). In addition a three virus combination HaNPV, PxGV and SeNPV were tested at 100%, 50% and 10% of Field application rate (Table above) Spray applications were made at 400-1200 L/ha water volume and repeated at 7-10 day intervals (applications A, B, C, D). Pest control, % incidence and severity of crop damage caused by the target pest were assessed 3, 7, 10, 14 days after the last application (DAA, DAB, DAC or DAD).

Example 9: A Combination of Five Baculoviruses Exert Excellent Activity Against Tuta absoluta

[0096] As can be seen in FIGS. 8 to 11 the mixture of five viruses exert a high level of efficacy against Tuta absoluta from 3-15 days after application, similar to reference products

Example 10: A Combination of Three Baculoviruses Exert Activity Against Tuta absoluta

[0097] As can be seen in FIGS. 8 to 11 the mixture of five viruses exert a high level of efficacy against Tuta absoluta from 3-15 days after application, similar to reference products

Example 11: A Combination of Five Baculoviruses Exert Excellent Activity Against Spodoptera exigua

[0098] As can be seen in FIG. 12, the mixture of five viruses exert a high level of efficacy against Spodoptera exigua from 8-20 days after application, better than the 3 virus combination and similar to Bt reference product.

Example 12: A Combination of Five Baculoviruses Exert Excellent Activity Against Helicoverpa armigera

[0099] As can be seen in FIG. 13, the mixture of five viruses exert a high level of efficacy against Helicoverpa armigera from 3-15 days after application, better than the 3 virus combination and similar to Bt reference product.

Example 13: A Combination of Five Baculoviruses Exert Activity Against Plutella xylostella

[0100] As can be seen in FIG. 14, the mixture of five viruses exert a moderate level of efficacy against Plutella xylostella from 7-21 days after application, better than the 3 virus combination and similar to Bt reference product.