ICHELCZIK, Dana (14 Ruth Street, Ramat Gan, 52524, IL)
BARAZANI, Avner (31A Marganit Street, Omer, 84965, IL)
YISSUM RESEARCH DEVELOPMENT COMPANY OF THE HEBREW UNIVERSITY OF JERUSALEM, LTD (Hi-Tech Park' Edmond J. Safra Campus, Givat Ra, P.O.B 91390 Jerusalem, 39135, IL)
APPLEBAUM, Shalom, W. (8 Hankin Street, Rehovot, 76354, IL)
ICHELCZIK, Dana (14 Ruth Street, Ramat Gan, 52524, IL)
BARAZANI, Avner (31A Marganit Street, Omer, 84965, IL)
A pesticidal composition comprising a combination of a chitin synthesis inhibitor (CSI) and the fungus Nomuraea rileyi, wherein said composition is insecticidally effective, and contains an insecticidally effective amount of a chitin synthesis inhibitor and an insecticidally effective amount of the fungus Nomuraea rileyi.
The composition of claim 1, wherein said chitin synthesis inhibitor is selected from bistrifluron, buprofezin, chlorfluazuron, cyromazine, diflubenzuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, noviflumuron, penfluron, teflubenzuron and tribflumuron .
The composition of claim 2, wherein said chitin synthesis inhibitor is Novaluron.
The composition of claim 3, said composition containing from about lxlO12 N. rileyi conidia per 1 gram of novaluron to about lxlO14 N. rileyi conidia per 1 gram of novaluron.
The composition of any of claims 1-4, further containing a carrier.
A method of treating or preventing pest infestation in a plant or in an environment in which said plant is grown or stored, the method comprising contacting said plant or said environment with a combination of a chitin synthesis inhibitor and of Nomuraea rileyi, further wherein said pest infestation is an insect infestation.
The method of claim 6, wherein said insect belongs to the order Lepidoptera. 2011/067757
8. The method of claim 6, wherein said combination of said chitin synthesis inhibitor and said Nomuraea rileyi is the composition of any one of claims 1-5.
9. The method of claim 6, wherein said combination of ' chitin synthesis inhibitor and N. rileyi is either applied together in a single formulation, or wherein each of said chitin synthesis inhibitor and said N. rileyi is applied separately.
10. The method . of claim 6, wherein said Contacting is conducted in one step or in consecutive steps.
1 1 . The method of claim 6, wherein the chitin synthesis inhibitor is Novaluron.
12. The method of claim 11, wherein said contacting is effected on crops selected from citrus, pome fruits and stone fruits crops, at an application rate ranging from about 0.5 grams novaluron per treated hectare to about 175 grams novaluron per treated hectare.
13. The method of claim 12, wherein said application rate ranges from about 0.5 grams novaluron per treated hectare to about 4 grams novaluron per treated hectare.
14. The method of claim 11, wherein said contacting is effected on crops selected from vegetables, cotton,' potato, tomato and field crops at an application rate ranging from about 0.1 grams . novaluron per treated hectare to about 40 grams novaluron per treated hectare.
15. The method of claim 14, wherein said application rate ranges from about 0.1 grams novaluron . per treated hectare to about 1 gram novaluron per treated hectare.
16. The method of any of claims 11-15, wherein said combination contains from about IxlO12 N. rileyi conidia per 1 gram of novaluron to about lxlO14 N. rileyx conidia per 1 gram of novaluron.
1 . A method for reducing the amount of a chitin synthesis inhibitor (CSI) required for controlling insects in a plant or in the environment in which said plant is grown or stored, the method comprising co-administering to a plant or said environment treated with said CSI with an effective amount of N. rileyi, so that the amount of CSI required for said control is at least 2 times smaller than the amount required to achieve the same control in the absence of said co-administration.
18. The method of claim 17, wherein said amount of chitin synthesis inhibitor required for said control is at least 10 times smaller than the amount required to achieve the same control in the absence of said coadministration.
19. The method of claim 18, wherein said amount of chitin synthesis inhibitor required for said control is at least 50 times smaller than the amount required to achieve the same control in the absence of said coadministration.
20. The method of claim 17, wherein said chitin synthesis inhibitor is novaluron.
21. The method of claim 20, wherein said effective amount of N. rileyi ranges from about lxl01? N. rileyi conidia per 1 gram of novaluron to about ;lxl014 N. rileyi conidia per 1 gram of novaluron.
22. Use of an insecticidal composition comprising a combination of a chitin synthesis inhibitor (CSI) and the fungus Nomuraea rileyi in controlling insects in a 2011/067757
32 plant or in the environment in which said plant is grown or stored.
23. The use of claim 22, wherein, said chitin synthesis inhibitor- is novaluron.
24. The use of claim 22, wherein said insect belongs to the order Lepidoptera.
Insect growth regulators (IGR) are materials that interrupt and/or inhibit the life cycle of insect pests. Examples include juvenile hormone mimics, ecdysone agonists and chitin synthesis inhibitors (CSIs) . Because molting must take place for the insect to progress from one larval stage to another, to metamorphose and ultimately reach the adult stage, an insect affected by an IGR cannot reproduce and eventually dies. These products are considered to have little or no human toxicity.
CSIs are defined as compounds that are capable of inhibiting chitin synthesis during the development of insects. Examples of CSIs are disclosed in Ishaaya, I. & Horowitz, A.R. 1998. Insecticides with novel modes of action: an overview. In: Ishaaya, I. and Degheele, D. (Eds.) Insecticides with Novel Modes of Action, Mechanism and Application, Chapter 1, pp. 1-24, Springer-Verlag, New- York". The water solubility of these compounds is typically extremely low, as is their mammalian toxicity. Insects exposed to CSIs are unable to form normal cuticle because the ability to synthesize chitin is inhibited. In the absence of chitin, the cuticle becomes thin and fragile, and is unable to support the insect or to withstand the rigors of molting.
One exemplary chitin synthesis inhibitor is novaluron, a benzoylurea having . the chemical formula of (+)-l-[3- chloro-4- (1,1, 2-trifluoro-2-trifluoromethoxyethoxy) phenyl] - 3- ( 2 , 6-difluorobenzoyl ) urea, depicted in Formula I below:
Novaluron acts by either ingestion or contact. At the level of the whole organism, novaluron disrupts post- apolytic cuticle formation, resulting in thinning of the pharate, subsequent stage cuticle. This disruption of new cuticle formation leads to failed ecdysis during the molting process. The preparation of Novaluron and insecticidal properties thereof are disclosed in U.S. Patent Nos . 4, 607, 044, 4, 833, 151, 4, 980, 376 and 5, 142, 064 and specifically in European patent No. 271,923.
The use of synthetic pesticides in agriculture is often limited by insufficient selectivity of . their toxicity, since synthetic pesticides may also affect humans, animals and useful plants. To some small extent, this is also true for IGRs and CSIs. Furthermore, many synthetic pesticides are characterized by relatively slow degradation and dissipation. Finally, many pest species develop resistance to the chemical pesticide over time.
Entomopathogenic microorganisms are an alternative group of bio-insecticides which act as biological control agents. The main advantages of entomopathogenic microorganisms are their relatively high selectivity for insects (and therefore their harmless effect on humans), as well as their low environmental impact. Amongst entomopathogenic microorganisms, fungal pathogens are unique in that they need not be ingested in order to invade their host. In most cases the fungal infection process is initiated by the landing of spores on the insect host cuticle. After docking and germinating, the fungal germ tube penetrates the host insect's cuticle, and if it manages to overcome the initial line of defense of the host., it commences to proliferate unimpeded, until it has exploited and exhausted most of the host reserves. Once the quality of the milieu in the host has degraded, the fungus produces a mycelial hyphal mass in the hemolymph and tissues. Although this results in a strong immune response of the insect larval host, it is often too late for the host to overcome the pathogen, which has by then depleted reserves and in many cases produced endogenous insecticidal metabolites. Finally, after the host dies, the fungal hyphae exit through the cuticle and under the right conditions produce spores externally.
. Combinations of two CSIs, novaluron and buprofezin (both being benzoylureas ) , with two species of entomogenous fungi: M. anisopliae and P. furnosoroseus, have been investigated by Skrobek A. (PhD. Thesis: "Investigations on the effect of entomopathogenic fungi on whiteflies", Bonn University, 2001), showing that they were compatible with each other.
However, CSIs have also been investigated as possible antifungal agents, since chitin is also present, to some extent, in fungal cell walls. Therefore, the compatibility and the insecticidal effect of combinations of CSIs and fungi are largely unpredictable, a priory described.
Nomuraea rileyi (N. rileyi, first disclosed by Farlow and Samson) is a fungus belonging to the deuteromycotina family, which is successfully infective to caterpillars of different lepidopteran species, being unharmful to other insect species, and may therefore be considered as a selective entomopathogenic biocontroi agent (e.g., Samson, R. A., 1974, Mycology 6: 80-85.; Ignoffo et al . , 1976, Journal of Invertebrate Pathology 27: 191-198; Vimala-Devi, P. S., 1994, Journal of Invertebrate Pathology 63: 145- 150) .
The inventors of the present invention have now found that combinations of Ή. rileyi with . CSIs, such as novaluron, form highly efficient yet inexpensive pesticide compositions, and may further have an unexpected synergistic effect on the target pests.
Thus, according to one aspect of the invention, there is provided a pesticidal composition comprising a combination of a chitin synthesis inhibitor (CSI) and the fungus Nomuraea rileyi.
As can be seen in the Examples section which follows, compositions combining N. rileyi . and a CSI proved to increase the mortality, lower the time to mortality of 50% of the treated larvae (LT 50 ) or of 90% of the treated larvae (LT 90 ) , lower the lethal concentration (LC) and further lower the percentage of insects pupating and/or emerging, normally, in the larvae of a model insect, Helicoverpa armigera.
Thus, according to a preferred embodiment of the present invention, the pesticidal composition described herein is in fact an insecticidal composition, effective for insects .
In the compositions described herein, the CSI and the fungus N. rileyi are used in insecticidal effective concentrations. As used herein, the terms "insecticidal effective amount" or "insecticidal effective concentration" respectively describe an amount or concentration of an active ingredient in a composition, or in a formulation, that will provide the insecticidal effect for which the composition or formulation is indicated, herein, controlling insects .
For the purpose of the present invention, the terms "composition" and "formulation" are used interchangeably and refer to the combination of the CSI and N. rileyi fungus, regardless of whether a carrier is included.
However, according to a preferred embodiment of the present invention, the composition described herein further contains at least one carrier.
The obtained composition which includes the carrier may be provided in a form of a solid, a powder, a solution, dispersion, a suspension, a paste, an aerosol or a spray, wherein the active ingredients of the present invention (namely, the CSI and the N. Rileyi fungus) are formulated in a manner which suits the specific application. Non- limiting examples of suitable, formulations are; emulsion concentrates (EC), suspension concentrates (SC), water dispersible granules (WDG) and wettable powders (WP) .
Regarding compositions formulated as liquids, the active ingredients may be combined with a liquid carrier, which may be an aqueous carrier, an oily carrier, an organic carrier or any combination thereof.
For example, fungus species are usually stored and kept active in a carrier, such as an oily carrier, and therefore the synergistic combination of the present invention may contain the N. rileyi spores in an oily suspension. Thus, according to one preferred embodiment, the carrier is an oily carrier.
Examples of oily carriers include a variety of vegetable oils (such as, soybean oil, safflower oil, linseed oil, corn oil, sunflower- oil, olive oil, canola oil, sesame oil, cottonseed oil, palm oil, rapeseed oil, tung oil) , fish oil, or a blend of any of these oils .
Alternatively the N. rileyi spores may be added "as is" to the CSI solution, to be mixed within the carrier (or mixture of carriers) of the CSI, which may include aqueous carriers .
Non-limiting examples of aqueous carriers include water, saline solutions, buffered saline solutions or glycerol solutions .
Organic carriers may include N-methyl pyrolidolne (NMP) , dimethyl sulfoxide (DMSO), sulfolane, acetone, ethanol, dimethylformamide (DMF) , acetophenone, methanol, butyrolactone, cyclohexanone, dimethyl acetamide (DMA), methyl, ethyl, iso-propyl and butyl-lactate esters.
For example, two common formulations of novaluron, an exemplary CSI, are "Rimon lOEC" and "Rimon supra". In the "Rimon supra" formulation, the carriers are DMSO and soft water. In the lOEC formulation, the carrier is NMP. These formulations are commercially available from Makhteshim Chemical Works Ltd. under the names of "Rimon lOEC" and "Rimon supra". For example, "Rimon lOEC" provides novaluron at a concentration of about 100 grams/liter in N- methyl pyrrolidone.
It should be stressed that the choice of carrier is done such that the carrier will be unharmful to the activity of either fungus or CSI. In addition to the carrier, the composition described herein may further comprise at least one agent selected from the group consisting of a protective colloid, an acidifying agent, an adhesive, a thickening agent, a penetrating agent, a stabilizing agent, a sequestering agent, a fertilizer, an anti-freeze agent, a repellent, a color additive, a corrosion inhibitor, a water-repelling agent, a siccative, a UV-stabilizer , a pigment, a dye, an emulsifier, a detergent and a polymer. Examples of such agents are well known in the art.
The formulations may further contain at least one additional pesticide, for example a nematicide, a herbicide, an insecticide etc., which is added to enhance the pesticidal and/or herbicidal properties of the formulation and/or other entomopathogens , in order to simultaneously treat other families of pests.
The compositions of the present invention can be easily prepared by mixing the CSI and the N. rileyi, whereas each is prepared according to methods known in the art, and which are also discussed in the examples section which follows. N. rileyi may . be obtained from a collection of Entomopathogenic Fungi, and can then be continuously grown under laboratory or industrial conditions.
Thus, according to another aspect of the invention, there is provided a process of preparing the insecticide compositions or formulations described herein, the process comprising mixing a CSI and the fungus N. rileyi.
Optionally, the mixing is effected in the presence of at least one carrier, suitable for either the CSI and/or for N. rileyi, thereby forming the insecticidal formulations of the present invention. The additional agents or ingredients described hereinabove may be# added during any stage of the preparation of the final compositions/formulations, either in one step or in several consecutive or non-consecutive steps.
CSIs suitable for the purpose of the present invention include, but are not limited to, bistrifluron, buprofezin, chlorfluazuron, cyromazine, diflubenzuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, noviflumuron, penfluron, teflubenzuron and tribflumuron .
Preferably, the CSI is novaluron. It has now been found by the present inventors that various combinations of N. rileyi and novaluron may serve as efficient yet inexpensive pesticides, in particular as insecticide. In fact, the combinations of the present invention were able to decrease the amount of the synthetic pesticide (novaluron) , while retaining and even increasing the overall insecticidal effectiveness.
As can be seen in the experimental section which follows, in almost every case, combinations of the CSI and the N. rileyi improved the effectiveness of the treatment, as compared to each component when used at the same concentration on its own.
For example, from Table 1 it appears that 0.001 grams/1 novaluron alone obtained only 9.8% mortality, which increased to about 81% with the addition of the fungus (at 2x1ο 11 conidia/1) . Similarly, 0.005 grams/1 novaluron alone obtained only about 32% mortality, which increased to 66- 88% with the addition of the fungus; 0.008 grams/1 novaluron alone obtained only 52% mortality, which increased to 94-98% with the addition of the fungus; and 0.01 grams/1 novaluron alone obtained only 53% mortality, which increased to 86-97% with the addition of the fungus . . Referring now to the fungus improved activity as part of the combinations, the same effect can be seen (Table 1) . For example, N. riley alone at 2xl0 7 conidia/1 elicited no (0%) mortality, which increased to 65% with the addition of the novaluron (0.005 grams/1); N. riley alone at 2xl0 8 conidia/1 obtained only 7% mortality, which increased to 86% with the addition of the novaluron (0.005 grams /l ) ; N. riley alone at 2xl0 9 conidia/1 obtained only 19% mortality, which increased to 88% with the addition of the novaluron (0.01 grams/1) ; and N. riley alone at 2xl0 10 conidia/1 obtained only 50% mortality, which increased to 94% with the addition of the novaluron (0.008 grams/1) .
Larvae that were not killed by the treatment can theoretically undergo pupation (either normally or not), and if they pupate normally, may either emerge normally as a mature insect or emerge abnormally, or not emerge at all.
For the farmer, it is essential that if the larvae are not killed by the initial treatment, they will at least not be able to form a second generation of the pest, namely that the percent of "normal emergence" will be minimal. Another way to describe the desired effect is to aim at high percentages of "cycle termination", whereas this term includes: a) cases of larval mortality (the larvae died and therefore did not create a second generation) , b) cases of no emergence (signifying pupal mortality, whether the pupa was apparently flawed or not) , and c) cases of incomplete emergence (for both incomplete or no emergence, the larvae did not die, did pupate but did not eventually create a second generation) .
Looking at the cycle termination values presented, in
Table 2, the improvement in the combined treatments can be seen with regard to each of the two components: For example, 0.001 grams/1 novaluron alone obtained only 25% cycle termination, which increased to 93% with the addition of the fungus (at 2x1ο 11 conidia/1) . Similarly, 0.004 grams/1 novaluron alone obtained only 55% cycle termination, which increased to 99% with the addition of the fungus (at 2x1ο 11 conidia/1); 0.005 grams/1 novaluron alone obtained only 52% cycle termination, which increased to 72-96% with the addition of the fungus; and 0.008 grams/1 novaluron alone obtained only 64% cycle termination, which increased to 97-98% with the addition of the fungus .
Again referring to the fungus improved activity as. part of the combinations, the same effect can be seen. For example, N. riley alone at 2xl0 7 conidia/1 obtained only 13.4% cycle termination, which increased to 72% with the addition of the novaluron (0.005 grams/1); N. riley alone at 2xl0 8 conidia/1 obtained only 23% cycle termination, which increased to 47-91% with the addition of the novaluron; N. riley alone at 2xl0 9' conidia/1 obtained only 43% cycle termination, which increased to 92% with the- addition of the novaluron; and N. riley alone at 2xl0 10 conidia/1 obtained only 69% . cycle termination, which increased to 96-97% with the addition of the novaluron.
Furthermore, the superiority of the described compositions can further be manifested by calculating the decrease in the amount of Novaluron needed to achieve a certain effect, by the addition of the fungus. As can be seen from Tables 1 and 2 below, the combined treatments enabled lowering the effective novaluron concentration by considerable amounts: For example, using 0.05 grams/1 novaluron alone resulted in approximately 93% pest mortality (Table 1) . The same mortality value was reached by using 0.004 grams/1 novaluron combined with N. rileyi (for example, at 2x1ο 11 spores/1), thus enabling to use 12.5 times less novaluron. Looking at cycle termination cases for the same pest (Table 2), it can again be seen that by combining novaluron (for example at 0.001 grams/1) with N. rileyi (for example, at 2x1ο 11 spores/1) it was possible to reach the same level of % cycle termination as that obtained when using 0.05 grams/1 novaluron alone, thus enabling to use 50 times less novaluron!
Furthermore, The comparison of LCgo values (Fig. IB) between the treatments indicates that in the combined treatment with the fungus at 10 8 conidia/ml, mortality was attained with 1/25 of the concentration of novaluron used to elicit the same mortality without N. rileyi (LCgo of 0.05 grams/liter for novaluron alone (line 3), vs. LCgo of 0.002 grams/liter for novaluron and the fungi at 10 8 conidia/ml, line 1) . Even at lower fungi concentrations (10 5 conidia/ml, line 2), the combined composition was more than 4 times more effective (LCgo of 0.012 grams/liter) compared to novaluron alone.
LC 50 results (Fig. 1Ά) suggest that novaluron combined with the fungus at 10 8 conidia/ml is about 100-times more effective than was novaluron alone (LC50 of 7.7xl0 ~3 grams/liter for novaluron alone, line 3, vs. 8.4xl0 ~5 for novaluron and the fungi at 10 8 conidia/ml, line 1) . Even at lower fungi concentrations (10 s conidia/ml, line 2), the combined composition was more than 3 times more effective (LC50 of 2.3xl0 ~3 ) compared to novaluron alone.
Thus, according to a preferred embodiment of the present invention, the concentration of the novaluron can be less than 0.1 grams/1, reaching as low as about 0.001 grams/1 while maintaining the overall insecticidal effect of the composition.
According to another preferred embodiment of the present invention, this was achieved when the compositions contained at least lxlO 9 N. rileyi conidia per 1 gram of novaluron. Even more preferably, the composition contains from about lxlO 12 N. rileyi conidia per 1 gram of novaluron to about lxlO 14 N. rileyi conidia per 1 gram of novaluron.
These concentrations are equivalent to using fungus solutions containing less than lxlO 13 conidia/1 (or less than lxlO 10 conidia/ml) , preferably having a concentration ranging from about lxlO 8 conidia/1 to about lxlO 12 conidia/1.
Looking at Tables 1 and 2, it can be seen that in most cases, the combination of the CSI and the N. rileyi fungus showed a synergistic effect.
The terms "synergistic effect" or "synergy" refer to an effect which is greater than the predictive additive effect of the two individual components of the combination. In the present invention, ' the effect is a pesticidal effect, in particular an insecticidal effect.
In the field of agriculture, it is often understood that the term "synergy" is as defined by Colby S. R. in an article entitled "Calculation of the synergistic and antagonistic responses of herbicide combinations" published in the journal Weeds, 1967, 15, p. 20-22. The latter article, uses the formula:
in which E represents the expected percentage of inhibition of the disease for the combination of the two insecticides at defined doses (for example equal to x and y respectively) , X is the percentage of. inhibition observed for the disease by the compound (I) at a defined dose (equal to x) , Y is the percentage of inhibition observed for the disease by the compound (II) at a defined dose (equal to y) . When the percentage of inhibition observed for the combination is greater than E, there is a synergistic effect.
The term "synergistic effect" is sometimes also calculated by the application of the Tammes method ("Isoboles, a graphic representation of synergism in pesticides" Netherlands Journal of Plant Pathology, 70 (1964) , p. 73-80) .
For example, the pesticidal composition of the present invention acts synergistically for the combination of Novaluron and Nomuraea rileyi, for both the percentage of mortality (Table 1) and for the percentage of cycle termination (Table 2), as reflected by the Colby Synergy effect in these tables, being almost 1 or much higher.
In these cases, it can be seen that synergy is obtained when novaluron concentration was as low as 0.003 grams/1 and until 0.05 grams/1,, possibly even at higher concentrations, and wherein the fungus/novaluron ratio was in the range of 10 9 to 10 14 conidia/gram novaluron.
Preferably, since it is usually desired that the insecticidal effect will have at least 80%, more preferably 90% success (be it mortality or cycle termination) , the acceptable range may be further limited to include only the novaluron values and N. rileyi values that, when combined, achieved such high success rates. Thus, the desired pesticidal effect is yet another determining factor in designing the ratio of CSI and fungus in the compositions of the invention. Accordingly, to achieve at least about 90% success in mortality, the novaluron concentration ranges from 0.004 grams/1 to 0.05 grams/1, possibly even at higher concentrations, as long as the composition contained at least 10 12 conidia/gram novaluron (for example 10 12 , 10 13 and ΙΌ 14 conidia/gram novaluron) .
In all of these concentrations, the observed mortality was at least 85%, and in most cases over 90%, reaching about 98% mortality for a combination of 0.01 grams/liter novaluron and 2xl0 9 fungi.
Based on the results presented herein, it is obvious that combinations of CSIs and the entomopathogenic fungus N. rileyi are most suitable for the controlling of pests in a plant or in the environment in which such a plant is grown or stored, such as soil, storage containers etc.
The term "controlling" includes both treating and preventing a pest infestation.
Thus, according to another aspect of the present invention, there is provided a method for treating or preventing pest infestation in a plant or in an environment in which said plant is grown or stored, the method comprising contacting said plant or said environment with a combination of a CSI and of Nomuraea rileyi.
According to a preferred embodiment of the present invention, this method is particularly suitable for controlling insects and comprises contacting the plant or the environment with a combination of a CSI and N. rileyi, wherein this combination is insecticidally effective.
When the combination is administered directly to the plant, it may be applied to any part of said plant, e.g. foliar application or seed treatment. As used herein, the term "plant" or "plant material" includes any or all of the physical parts of a plant, including seeds, seedlings, saplings, roots, tubers, stems, stalks, foliage, and fruits.
In particular, the formulations of the present invention were successful in treating a representative agricultural insect species, Helicoverpa armigera (Htibner) , which belongs to the Lepidoptera order (Noctuidae family) . This species is widely distributed and common in Africa, Asia, Australia, New Zealand and the Pacific Islands, as well as the Mediterranean region and the Middle East and was used as a model insect since it is both widely distributed and has developed resistance to many of the common pesticides, such as DDT, carbamates, organophosphates , pyrethroids and endosulfan. Together with comparable species in the American continent (e.g., Heliothis zea) , larvae of the Helicoverpa/Heliothis complex impose extensive economic damage worldwide. This emphasizes the need for their integrated control.
Thus, according to a preferred embodiment of the present invention, the methods, compositions and formulations of the present invention are used to control insects belonging to the Lepidoptera order, more preferably to the Noctuidae family and, at this stage, even more preferably to the Heliothinae subfamily.
The presently claimed methods, compositions and formulations are specifically suitable for controlling plant-destructive feeding lepidopteran insects in ornamentals and crops of useful plants, especially in cotton (e.g. against Spodoptera littoralis and Heliothis virescens) , flowers, fruits and vegetables. Examples of fruits and vegetables infested by Noctuidae include, among others, cotton, maize, sorghum and related cereals, legumes (soybeans, peas, chickpeas, clover) solanaceous crops (tomatoes, potatoes, tobacco) , cucurbits, crucifers (cabbage and related crops)., sweet potatoes arid onions.
Moreover, it is an advantage of the present invention that due to the selectivity of the N. rileyi fungus, the present methods, compositions and formulations are particularly useful for controlling insects of the order Lepidoptera.
Thus, the method described herein is advantageously safe for other desirable insects, other than those belonging to the order Lepidoptera, for example this method is safe and will not harm honey bees and pollinating bees, natural enemies of various insect orders (such as Coleoptera, Homoptera and Hymenoptera) , and predatory mites .
According to a preferred embodiment of the present invention, the application of the present invention is conducted at all larval instars of the insect.
An instar is the period or stage between molts, numbered to designate the various periods; e.g. the first instar is the stage between the egg and first molt; the second instar is the stage between the first molt and the second molt, and the third instar is the stage of the life cycle between the second and third molting cycles, etc. Molting is the process in which an immature insect casts its skin in order to grow a new skin (which develops under the old skin and is of larger size) .
For the use of the artisan in the . field, according to the methods described herein, the combinations of the present invention may be available as a kit or an article of manufacture. Thus, according to another aspect of the invention there is provided an article-of-manufacture comprising a packaging material and a composition comprising an effective amount of a chitin synthesis inhibitor (CSI) and an effective amount of the fungus Nomuraea rileyi, being packaged in a packaging material, this article-of-manufacture being identified in print, in or on said packaging material, for use in controlling insects, as described hereinabove. The CSI and the Nomuraea rileyi may be packaged separately or in combination. Preferably, at least one of the CSI and/or the Nomuraea rileyi is packaged in at least one carrier.
According to yet another aspect of the invention, there is also provided the use of any of the compositions described herein for controlling insect infestations as described hereinabove.
As can be understood by the skilled artisan, the ^methods described herein may be applied in various alternative ways, such that the combination of CSI and N. rileyi is applied either together ih a single formulation, or such that the CSI and the N. rileyi are applied separately.
Contacting between the insecticidal.. compositions or insecticidal formulations and the treated substance may be effected by several methods of application, such as : spraying, drenching, soaking, dipping, mixing, coating, dispersing, injecting, irrigating or impregnating.
Any of these methods of application may be employed, as the skilled artisan may appreciate. However, spraying is the preferred method of application. In other cases, for example in field crops, systemic application via an irrigation system can be combined with foliar application.
According to a particular embodiment of the present method, the insecticidal composition is used to prepare a tank mix which is then applied via spraying to the area, plant matter or crop needing treatment. The active ingredient concentration in the tank mix is adjusted to the particular application, depending on agricultural or non- agricultural application, the crop and the pest.
The contacting may be conducted either in one step or in several consecutive or non-consecutive steps. As described hereinabove, according to a preferred embodiment of the method of the present invention, the application of each active ingredient (a.i.), for ; example the CSI and the N. rileyi, may be conducted simultaneously, separately or sequentially, as is also true for : the application of any additional agent to the same crop.
Furthermore, the combined, optionally synergistic, effect exerted by the formulations described herein enables the use of relatively lower amounts of the active ingredients, particularly as compared with the amounts required to achieve the same effect with each of these components alone. This feature is particularly advantageous since (i) it renders the use of such a composition relatively cost-efficient; and (ii) any adverse side effects induced by each of the components are substantially reduced .
Thus, according to yet another . important aspect of the present invention, there is provided a method for reducing the amount of a chitin synthesis inhibitor (CSI) required for controlling insects in a plant or in the environment in which this plant is grown or stored, the method comprising co-administering to a plant or said environment treated with said CSI with an effective amount of N. rileyi, so that the amount of CSI required for said control is at least 2 times smaller and may be many fold more than the amount required to achieve the same control in the absence of said co-administration.
Thus, as is exemplified below, the amount of CSI required for said control can be at least 5 times smaller, 10 times smaller, 25 times smaller, 50 times smaller and even 100 times smaller than the amount required to achieve the same control in the absence of said co-administration.
Clearly, this reduction in the amount of synthetic CSIs is of particular importance in order to retard the development of any insect resistance thereto and to decrease any detrimental environmental affects.
In particular, and according to a preferred embodiment of the present invention, the CSI of the methods described herein is novaluron.
As can be seen from published application labels on novaluron products (Rimon Biological Data Sheet, published by Makhteshim-Agan Industries in January 2002) , novaluron is typically applied at between 10-75 grams /hectare for crops selected from vegetables, cotton, potato, tomato and other field crops, and at between 50-300 grams/hectare for crops selected from citrus, pome friits and stone fruits.
The term "field crops" refers to a range of grains (wheat, oats, barley, corn rye, etc.), as well as potatoes, peas, hay, and other fodder.
For purposes of this invention, the term "pome fruit" is used herein to designate fruits having a fleshy outer layer and a central core with seeds enclosed in a capsule. Examples of pome fruits include, but not limited to, all cultivars of apples, pears, quinces and the like.
The term "stone fruit" as used herein is intended to embrace, inter alia, fruit of the genus Prunus and covers, for example, cherries, plums (including greengages and damsons), apricots, peaches and nectarines.
The term "citrus" includes, but is not limited to, tangerines, oranges, limes, lemons, grapefruit, and related plants .
As exemplified below, it has now become possible to use much lowers amounts of novaluron, by a factor of 2, 5, 10, 25, 50 and even 100, when combined with the N. rileyi fungus .
Thus, according to a preferred embodiment of the present invention, the contacting is effected on crops selected from citrus, pome fruits and stone fruits, at an application rate ranging from about 0.5 grams novaluron per treated hectare to about 175 grams novaluron per treated hectare, more preferably such that the application rate ranges from about 0.5 grams novaluron per treated hectare to about 4 grams novaluron per treated hectare.
Similarly, 'according to another preferred embodiment of the present invention, the contacting is effected on crops selected from vegetables, cotton, potato, tomato and other field crops, at an application rate ranging from about 0.1 grams novaluron per treated hectare to about 40 grams novaluron per treated hectare, preferably ranging from about 0.1 grams novaluron per treated hectare to about 1 gram novaluron per treated hectare.
In all of these cases, the applied composition contained at least lxlO 9 N. rileyi conidia per 1 gram of novaluron, more preferably from about lxlO 12 N. rileyi conidia per 1 gram of novaluron to about lxlO 14 N. rileyi conidia per 1 gram of novaluron.
It is expected than even lower concentrations of N. rileyi may be effectively combined with a CSI, such as novaluron, while maintaining the insecticidally effective properties of the formulation.
The minimal concentrations of the novaluron CSI in the formulations applied to the plant and/or its environment have been shown to be lower than 0.1 grams/liter, and even lower than 0.05 grams/liter, in some cases being as low as 0.01 grams/liter, 0.004 grams/liter and 0.001 grams/liter, without lowering, and even increasing, the insecticidal effect of the formulation.
Thus, it has now been 1 established that the combinations of Novaluron and N. rileyi have a superior effect and act synergistically in controlling insects. This combination is advantageous in that it enables the use of lower amounts of each insecticide, lowers the environmental impact of the insecticide, retards the development of insect resistance to the Novaluron, and generally provides an efficient, selective, 'effective insecticidal composition.
Reference is now made to the following examples, which together with the above, descriptions, illustrate the invention in a non limiting fashion ..
MATERIALS AND ANALYTICAL METHODS
Materials : Larvae of 2 nd" 3 rd instar H. armigera were provided by Professor Ada Rafaeli of Volcani Institute, ARO, Bet Dagan, Israel.
The fungus Nomurae rileyi (N. rileyi, ARSEF 1972 isolate) was provided by Professor Richard Humber, USDA-ARS collection of Entomopathogenic Fungi [ARSEF] , Ithaca, New York, USA.
Novaluron was obtained from Makhteshim, Israel under the trade name Rimon as either a 99% technical concentrate, or as a lOEC (100 grams/liter) formulation.
Novaluron carrier solution, as used in the Examples below, is an emulsiable concentrate (EC) blank formulation, obtained from Makhteshim, which contains N-methyl-2- pyrolidolne (NMP) .
"Manduca Premix-Heliothis Premix" insect diet was purchased from Arthro Feeds, Stonefly Industries, Inc., USA. ;
Sabouradau' s Maltose-Agar-Yeast (SMAY) medium was prepared by mixing Maltose (4%), Peptone (1%), Yeast extract (1%) and Agar (2%) in distilled water, stirring for several minutes and autoclaving.
Maltose was obtained from Sigma, Peptone, Yeast extract and Agar were obtained from Difco.
Soybean oil was obtained from a local commercial food store .
Hemocytometry was performed using a light microscope (CH2, Olympus) .
Protocol of Insect culture Larvae of 2 nd~ 3 rd instar H. afmigera were transferred into individual cells (J2 cavities, Nutrend) , each containing nutrition (approximately 3 grams of "Manduca Premix-Heliothis Premix" diet) , ; and reared through metamorphosis. Rearing was conducted in culture rooms at 25 °C with a photoperiod of 14 hours under light followed by 10 hours in the dark (hereinafter noted as ' 14L:10D), which prevents pupal diapause. In the conditions used for culture of this pest, the larvae develop from egg, through 5 larval instars and the pupal stage, to adult, within approximately one month, with the larval stages lasting approximately two weeks .
Preparation of Fungal culture
A sporulated N. rileyi culturS .was prepared using 9- centimeter Petri dishes on Sabouradau' s altose-Agar-Yeast (SMAY) medium, according to known protocols (Bell, J. V. , 1975. Production and pathogenicity of the fungus Spicaria rileyi from solid and liquid media. Journal of Invertebrate Pathology 26: 129-135) . The medium was inoculated with conidia collected from larval cadavers. Cultures were prepared periodically, mostly every month. Culture preparation was made under sterile conditions, in a vertical laminar flow hood. Plates were kept in a dedicated culture room at 25 °C with a photoperiod of 14L:10D.
Preparation of fungal N. rileyi conidial suspension
N. rileyi conidia from the culture were transferred to a glass vial by the addition of 4-5 ml of soybean oil to the plate, scraping off the conidia, mixing gently with a Drigalski spatula and filtering through gauze, then counted by hemocytometry, and adjusted to provide the required suspension conidia concentration by addition of soybean oil .
Preparation of Novaluron aqueous suspension
Aqueous stock solutions of Novaluron were prepared by diluting concentrated (lOEC, or 100; grams/liter) Novaluron with additional EC carrier to obtain aqueous suspensions containing 0.001, 0.003, 0.004, 0.005, 0.008, 0.01 and 0.05 grams/liter Novaluron (in the final suspensions, double these concentrations before addition of equal volumes of fungus) .
Preparation of combined N. rileyi - Novaluron mixtures
Fungal suspensions were mixed 1:1 by volume with novaluron suspensions, such that the original concentration of conidia in the fungal suspension was double the concentration specified as used for ; experiments , as was the original concentration of novaluron compared to its final concentration in the combined compositions.
For example, in order to reach; 2xl0 8 conidia/ml in the final formulation, a 4xl0 8 conidia/ml suspension was used, and in order to obtain 0.01 grams/liter . Novaluron in the final formulation, a 0.02 grams/litter suspension was made, and. then these two were mixed in a 1:1 volume ratio. The mixtures were vortexed to provide stable emulsions.
Conidia were mixed with either novaluron, or with an EC carrier, in a concentration■ equal to that of Novaluron in the Novaluron treatment. For example, if Novaluron 0.01 grams/liter was tested, then in the N. rileyi + carrier treatment, or in the carrier treatment, the carrier was in 0.01 grams/liter concentration. : Various combinations of Novaluron and fungus, were prepared, containing from 0.001 grams/liter to 0.05 grams/liter Novaluron, together with from 2xl0 7 conidia/liter to 2xl0 12 conidia/liter of the N. rileyi fungus . Reference samples contained either Novaluron or fungus at the same concentrations .
EXAMPLE 2 '
Inoculation of larvae by different combinations of
Novaluron and N. rileyi
H. ar igera larvae were individually treated with different combinations of Novaluron ' suspensions, and/or N. rileyi suspensions in oil and/or in the Novaluron EC carrier, by topical application of 20 μΐ of each suspension immediately after ecdysis (shedding of the cuticle) to the 5 th (last) larval instar, within 4 hours from start of head slippage.
20-25 larvae were subjected to each treatment, which was repeated 3 times at different times and with different larval batches and different fungal cultures.
Following treatments, larvae were monitored several times daily either for mortality, symptomatic to N. rileyi mycosis, or the, formation of "chimera" . Larvae that pupated were monitored for flaws in pupation, pupal mortality and emergence, either incomplete or normal. Percent mortality or emergence was calculated, as well as LT-50 values where 50% mortality was obtained. Additionally, LC-50 values were calculated for Novaluron in the individual treatment versus Novaluron-N. rileyi combination at two different fungal conidial concentrations, using "LdP Line" software for probit analysis. One-tailed Student's t-test was used to compare % mortality or emergence and LT-50 of combined treatments and the respective individual treatments .
The compiled and analyzed results are shown in Figures 1 and 2:
Figure 1 is an "LDP-Line" Probit calculation of Novaluron lethal concentrations (LC) values: fig. 1A extrapolates to find LC 50 values and fig. IB extrapolates to find LC 90 values, whereas line #1 represents Novaluron and N. rileyi at 10 s conidia/ml, Line #2 represents Novaluron and N. rileyi at 10 5 conidia/ml and Line #3 represents Novaluron alone.
Figure 2 presents the Percent of larval mortality as a result of N. rileyi mycosis and percent "chimera" as a result of Novaluron treatment: Fig. 2A at a concentration of 10 5 conidia/ml N. rileyi and fig, 2B at a concentration of 10 8 conidia/ml N. rileyi. Values presented are means (n=4-8), ± standard error (full error bars for "chimera" and fragmented bars for % mortality) . Percent was calculated from sample sizes of 20-25 larvae per repeat.
Table 1 below summarizes the impacts of separate and combined treatments at different concentrations of novaluron and N. rileyi. Furthermore, the expected effect E was calculated by the following formula: E=X+Y-XY/ 100 , whereas X is the percentage of the larvae affected by novaluron only ("chimera") at a given novaluron concentration, and Y is the percentage of the larvae affected by fungi only at a given fungal concentration.
The synergy was calculated by dividing the observed mortality effect (0) by the expected combined effect, according to Colby (E).. A value larger than 1 signifies synergism by Colby. Table 1
Table 2 presents the percentages of cycle termination, as it has been defined hereinabove, in a variety of treatments, based on sample sizes of 20-25 larvae per repeat. In practice, the cycle termination presented in Table 2 was calculated as the complementary value to 100% of normal emergence (adults capable of reproducing a second generation of pests) .
The Colby synergy values in Table 2 were calculated in a similar way as in Table 1, based on the expected cycle termination (E, as calculated based on the cycle termination of novaluron only (X) and fungus only (Y) ) and the observed cycle termination (0) . Table 2
W. Rileyi Expected Observed Colby termination termination
Novaluron N.Riley spores/ Cycle Cycle Synergy gr/1 conidia/1 (%) by
gram (%) by termination termination factor
Novaluron W.i ley
Novaluron (%, E) (%, O) (O/E)
0.001 2.E+07 2.E+10 25.39 13.43 ■ 35.41 6 0.17
0.001 2.E+ll 2.E+14 25.39 96.41 97.32 93.06 0.96
0.003 2. E.+08 7.E+10 38.02 23.64. 52.67 47.48 0.90
0.004 2.E+ll 5.E+13 55.27 96.41 98.39 98.64 1.00
0.005 2. E+07 4.E+09 52.11 13.43 58.54 72.2 1.23
0.005 2.E+08 .E+10 52.11 23.64 63.43 89 1.40
0.005 2.E+10 .E+12 52.11 69.2 ; 85.25 96.19. 1.13
0.008 2.E+10 3. E+12 64.23 69.2 88.98 97.28 1.09
0.008 2. E+ll 3.E+13 64.23 96.41. 98.72 98 0.99
0.01 2.E+08 2.E+10 64.43 23.64 72.84 91.17 1.25
0.01 2.E+09 ■ 2.E+ll 64.43 42.92 79.70 92 1.15
0.01 2. E+10 2.E+12 64.43 69.2 89.04 96 1.08
0.01 2.E+ll 2.E+13 64.43 96.41 98.72 98.33 1.00
0.01 2.E+12 2.E+14 64.43 96.14 98.63 97.92 ■ 0.99
0.05 2. E+ll 4.E+12 94.3 96.41 99.80 96.43 0.97