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Home , Metarhizium acridum, Metarhizium anisopliae

US 2013 O156740A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/015674.0 A1 Leland (43) Pub. Date: Jun. 20, 2013

(54) BIO-PESTCIDE METHODS AND (52) U.S. Cl. COMPOSITIONS CPC ...... A0IN 63/04 (2013.01); A0IN 63/02 (71) Applicant: Novozymes Biologicals Holdings A/S, (2013.01) Bagsvaerd (DK) USPC ...... 424/93.5 (72) Inventor: Jarrod E. Leland, Blacksburg, VA (US) (57) ABSTRACT

(73) Assignee: NOVOZYMES BIOLOGICALS - 0 HOLDINGS A/S, Bagsvaerd (DK) The present invention is directed to the combination of bio pesticide and at least one exogenous cuticle degrading (21) Appl. No.: 13/719,624 enzymes (e.g., a protease, chitinase, lipase and/or cutinase) (22) Filed: Dec. 19, 2012 for controlling (preventing or eliminating) pests. The use of an exogenous cuticle degrading enzyme increases the effi Related U.S. Application Data cacy of the by increasing the speed and/or effi (60) Provisional application No. 61/577,224, filed on Dec. ciency of infestation of the pest resulting in faster or more 19, 2011. effective killing or disabling of the pest by the biopesticide. The present invention accordingly provides methods for con Publication Classification trolling a pest comprising treating a pest habitat with a com (51) Int. Cl. bination of pesticidally effective amounts of at least one bio AOIN 63/04 (2006.01) pesticide and at least one exogenous cuticle degrading AOIN 63/02 (2006.01) enzyme. compositions are also described. Patent Application Publication Jun. 20, 2013 US 2013/015674.0 A1

Effect of Protease and on Mortality at Varying Concentrations

O 1. 2 3 A. 5 6 7 8 s Days s0% Savinase No Met x&x 0.1% Savinase No Met NSw1% Savinase No Met xxx 10% Savinase No Met &O% Savinase 3E6 Met O.1% Savinase 3E6 Met w x 1%, Savinase 3E6 Met ·.10% Savinase 3E6 Met

FIG. 1 US 2013/0156740 A1 Jun. 20, 2013

BO-PESTCIDE METHODS AND effective amounts of at least one entomopathogenic COMPOSITIONS and at least one exogenously applied cuticle degrading enzyme. The present invention is also directed to an insecti CROSS-REFERENCE TO RELATED cide composition comprising insecticidally effective APPLICATIONS amounts of at least one and at least one exogenous cuticle degrading enzyme. 0001. This application claims the benefit under 35 U.S.C. 0007. In another aspect, the present invention is directed to 119 of U.S. provisional application No. 61/577,224 filed Dec. a method of controlling an infestation comprising treat 19, 2011, the contents of which are fully incorporated herein ing a pest habitat with a combination of pesticidally effective by reference. amounts of at least one acaripathogenic fungus and at least BACKGROUND one exogenously applied cuticle degrading enzyme. The present invention is also directed to a pesticide composition 0002 Pests, such as , Acari (mites and ticks) and comprising pesticidally effective amounts of at least one nematodes, are a major problem for the agriculture industry, acaripathogenic fungus and at least one exogenous cuticle limiting productivity, often significantly. Although chemical degrading enzyme. pesticides are used to control pests, excessive use of chemical 0008. The present invention is also directed to a method of pesticides leaves residues in Soil, water and air and also has controlling a nematode infestation comprising treating a adverse effects on the non-target organisms and the ecologi nematode habitat with a combination of nematicidally effec cal balance. In addition, pests can develop resistance to tive amounts of at least one nematopathogenic fungus and at chemical pesticides, limiting their effectiveness and applica least one exogenously applied cuticle degrading enzyme. The tion. Public concern overpotential health hazards of chemical present invention is also directed to a nematicide composition pesticides and the increase in cost of chemical pesticides has comprising nematicidally effective amounts of at least one also led to the exploration of more eco-friendly pest manage nematopathogenic fungus and at least one exogenous cuticle ment tactics. degrading enzyme. 0003 have been developed for use as an 0009. An entomopathogenic fungus may also have nem alternative, or in some cases as a Supplement, to chemical atopathogenic properties, and vice versa. Alternatively, at pesticides. Biopesticides are living organisms (e.g., fungi and least one entomopathogenic fungus and at least one nemato bacteria) that intervene in the life cycle of pests (by killing or pathogenic fungus may be used in combination as ingredients disabling the pest). Examples of biopesticides include the of pest treatment composition. In an embodiment, the present entomopathogenic fungus , which invention provides a method of controlling a pest comprising has been registered as a bio- for the control of treating a pest habitat with a combination of pesticidally pests in the United States and many other countries. effective amounts of at least one entomopathogenic fungus, at Metarhizium anisopliae has been reported to infect many least one nematopathogenic fungus and at least one exog insect types including Subterranean (Reticulitermes enously applied cuticle degrading enzyme, wherein the at and Coptotermes spp.), corn rootworms (Diabrotica spp). least one entomopathogenic fungus and the at least one nem black vine weevils (Otiorhynchus sulcatus), citrus root wee atopathogenic fungus may be the same fungus or a different vils (Diaprepes abbreviatus), Japanese (Popifia fungus. japonica), and European chafers (Rhizotrogus maialis). 0004 As natural agents, biopesticides offer more eco BRIEF DESCRIPTION OF THE DRAWING friendly solutions for controlling pests and/or for for use in combination with chemical pesticide. However, one major 0010 FIG. 1. is a graph that illustrates the effect of pro drawback of the use of biopesticides is in their efficacy com tease and Metarhizium on mortality at varying concentra pared to chemical pesticides. There is a need in the art for tions. biopesticides having greater efficacy for the replacement or Supplementation of chemical pesticides. DETAILED DESCRIPTION OF THE INVENTION 0011. A fungal pesticide is employed in the present inven BRIEF SUMMARY OF THE INVENTION tion to protect a habitat from pests (such as, an insect, an 0005. The present invention is directed to the combination Acari, and/or a nematode infestation) so as to prevent, elimi of at least one biopesticide and at least one exogenously nate or reduce a pest infestation in a habitat. As used herein, applied cuticle degrading enzymes (e.g., a protease, chitinase, the term “fungal pesticide” means a fungal organism that is lipase and/or cutinase) for use in controlling pests. The use of pathogenic to a target pest, Such as, an insect, Acari or a an exogenously applied cuticle degrading enzyme increases nematode. the efficacy of the biopesticide. The present invention accord 0012. As used herein, a “habitat” may be any area or ingly provides methods and compositions for controlling a environment where a pest lives or is able to live, that is, any pest comprising treating a pest habitat with a combination of area or environment that is infested or susceptible to infesta pesticidally effective amounts of at least one biopesticide and tion by a pest. at least one exogenously applied cuticle degrading enzyme. 0013 The habitat may be a plant, soil, or water, as well as, Pests which may be treated according to the present invention commercial or residential structures, storage containers (e.g., include, for example, insects, Acari (Such as, mites and ticks) shipping containers), and commercial products (e.g., food and/or nematodes (and, accordingly, the biopesticide may be products) and product packaging. The habitat may be agri used as an insecticide, Acaricide, and/or nematicide). cultural fields, orchards, greenhouses, gardens, lawns, orna 0006. In one aspect, the present invention is directed to a mental plants, or trees. method of controlling an insect infestation comprising treat 0014. The at least one fungal pesticide and the least one ing an insect habitat with a combination of insecticidally cuticle degrading enzyme are applied to the habitat in the US 2013/0156740 A1 Jun. 20, 2013

vicinity of the target pest, such as, e.g., in agriculture, on the The term "isolated” means the enzyme is in a form or envi Surface of the plants to be protected (e.g., as a foliar applica ronment which does not occur in nature, that is, the enzyme is tion), as a seed coating, and/or to the soil, using conventional at least partially removed from one or more or all of the techniques. As used herein, “vicinity’ means a location effec naturally occurring constituents with which it is associated in tive to result in treatment of the pest by immediate or eventual nature. Thus, although enzymes produced endogenously by contact with the pest. The application or treating process will the fungal pesticide will impact efficacy, an isolated enzyme vary depending on the habitat. does not encompass an enzyme endogenously produced by 0015 The term “entomopathogenic’ means that the fun the fungal pesticide during treatment of apest in the processes gal pesticide is pathogenic to at least one targetinsect. As used of the present invention. An isolated enzyme may be present herein, "entomopathogenic fungus' is a fungus that can act as in the form of a purified enzyme composition or a fermenta a parasite of an insect to kill or seriously disable the insect and tion broth sample that contains the enzyme. is thus able to be used in the control or prevention of insect (0023 The term “pesticidally effective amount” or “effec infestation by adversely affecting the viability or growth of tive amount” (e.g., as in “insecticidally effective amount’ or the target insect. “nematicidally effective amount”) is used herein to mean the 0016. The term “acaripathogenic’ means that the fungal amount of the at least one fungus and the least one cuticle pesticide is pathogenic to at least one target Acari, Such as, as degrading enzyme is Sufficient to protect a habitat (e.g., mite or tick. As used herein, “acaripathogenic fungus' is a plants, soil or water) from pests, such as insects, Acari and/or fungus that can act as a parasite of an Acari to kill or seriously nematodes. Such protection can comprise a complete killing disable the Acari and is thus able to be used in the control or action, eradication, arresting in growth, reduction in number, prevention of Acari infestation by adversely affecting the prevention of infestation or any combination of these actions, viability or growth of the target nematode. collectively referred to herein as “efficacy.” 0017. The term “nematopathogenic’ means that the fungal 0024. An “amount effective of the exogenously applied pesticide is pathogenic to at least one target nematode. As cuticle degrading enzyme is an amount of the enzyme effec used herein, “nematopathogenic fungus' is a fungus that can tive to increase the efficacy of the fungal pesticide. Theat least act as a parasite of a nematode to kill or seriously disable the one fungal pesticide and the at least one exogenously applied nematode and is thus able to be used in the control or preven cuticle degrading enzyme will have an efficacy better than tion of nematode infestation by adversely affecting the viabil that of a control, that is, better than the application of the ity or growth of the target nematode. fungal pesticide without the at least one cuticle exogenously 0018. The fungal pesticide may in preferred embodiments applied degrading enzyme when applied under the same or be an “entomopathogenic fungus, a 'acaripathogenic fun comparable treatment conditions. Accordingly, the at least gus', a “nematopathogenic fungus' or a fungus which has one exogenously applied cuticle degrading enzyme is in an one or more of these properties. amount effective to improve the efficacy of the fungal pesti 0019. The fungal pesticide will generally function by cide as compared to treatment with fungal pesticide alone. attaching to the external body Surface of the pest (e.g., insect, The cuticle degradation products released from the surface of Arcari or nematode). Such as, in the form of microscopic the pest can also serve as signal for the conidia to turn on (usually asexual, mitosporic spores also called infestation pathways and/or also serve as nutrients for the conidia). Under permissive conditions of temperature and early germinating spores. The exogenously applied enzyme (usually high) moisture, these spores germinate, grow as may release products from the surface of the pest to provide hyphae and colonize the pest’s cuticle; eventually they bore this signal. through it and reach the pest’s body cavity (hemocoel). The 0025. The amount effective can increase colonization and/ fungal cells then proliferate in the host body cavity, usually as or boring through of the fungal pesticide into the body cavity walled hyphae or in the form of wall-less protoplasts (depend of the target pest to increase the efficacy of the fungal pesti ing on the fungus involved). After Some time, the pest is killed cide. The amount effective can also help degrade the per or disabled. itrophic membrane that covers the target pest gut to thereby 0020. As used herein in, a “cuticle degrading enzyme” is enhance penetration of the fungal pesticide into the pest gut. an enzyme that is able to at least partially degrade a cuticle of The amount effective can degrade the pest cuticle to release a pest, such as, the epicuticle and/or the procuticle. The exog cuticle Surface compounds from the pest cuticle, which serve enously applied cuticle degrading enzyme can increase the as a signal for the conidia to turn on one or more infection efficacy of the fungal pesticide by increasing the ability of the pathway. fungal pesticide to colonize and/or or bore through the pests 0026. The effective amounts used for the at least one fun cuticle to reach the pest’s body cavity. gal pesticide and the at least one cuticle degrading enzyme 0021. As used herein, “exogenously applied' means that will vary depending on many factors, such as, e.g., the habitat the cuticle degrading enzyme is applied independently (that treated, whether the use is for controlling an existing pest is, as a separate ingredient) from the fungal pesticide and any infestation or preventing a pest infestation, the target insect, enzyme produced by fungal pesticide. Although cuticle the density of the target insect population, and the method and degrading enzymes are naturally produced by a fungal pesti site of application. The effective amounts of the at least one cide (referred to herein as "endogenous” cuticle degrading fungal pesticide and the least one cuticle degrading enzyme enzymes) and are involved in the pesticidal activity of the may be determined by routine testing as amounts effective to fungal pesticide, the present invention is directed to the either kill or disable the target insect in the habitat (whether in enhancement of any Such endogenous pesticidal activity of an existing infestation or in preventing an infestation). the fungal pesticide through the use of an exogenously 0027 Fungal pesticides are well known in the art and applied cuticle degrading enzyme. include, for example, of , Alternaria, 0022. The “exogenously applied cuticle degrading Beauveria, Lecanicillium, Metarhizium, Verticillium, Tricho enzyme is in the form of an "isolated enzyme composition. derma, Aspergillus, Nomuraea, Paecilomyces, Isaria, Hirsu US 2013/0156740 A1 Jun. 20, 2013

tella, Fusarium, Cordyceps, Entomophthora, Zoophthora, (1998): 171-77, Clarkson, J. M. “Molecular biology of fungi Pandora, Entomophaga, Entomophthorales and Zygomy for the control of insects.” (1996): 123-35, Cole, S.C.J. A. K. cota. Examples of species of fungal pesticides include Tri Charnley, and R. M. Cooper. “Purification and partial char choderma hamatum, Trichoderma hazarium, Alternaria cas acterization of a novel trypsin-like cysteine protease from siae, Fusarium lateritum, Fusarium Solani, Lecanicillium metarhizium-aniisopliae.” FEMS Microbiology Letters 113.2 lecanii, Aspergillus parasiticus, Metarhizium anisopliae, and (1993): 189-96, Da Silva, M.V., et al. “Cuticle-induced endo/ . Any of the above organisms may be used exoacting chitinase CHIT30 from Metarhizium anisopliae is in the invention, including any combination thereof. In spe encoded by an ortholog of the chi3 gene.” Research in Micro cific embodiments, the fungal pesticide is a species from the genus Metarhizium. In more particular embodiments, the fun biology 156.3 (2005): 382-92, Dhar & Kaur, “Production of gal pesticide is Metarhizium anisopliae. Particular strains of cuticle-degrading proteases by Beauveria bassiana and their Metarhizium anisopliae include Metarhizium aniisopliae induction in different media, African Journal of Biochemis strain F52. The name of the species Metarhizium anisopliae try Research, Vol. 4(3), 65-72 (2010), Fang, W. G., et al. of the strain Metarhizium anisopliae F52 has recently been "Expressing a fusion protein with protease and chitinase changed to , and thus, may be referred activities increases the virulence of the insect pathogen Beau to in the art under both names. veria bassiana.” Journal of Invertebrate Pathology 102.2 (2009): 155-59, Freimoser, F. M., et al. “Expressed sequence 0028 Cuticle degrading enzymes are well known in the tag (EST) analysis of two subspecies of Metarhizium art, and include both naturally occurring (wild-type) enzymes anisopliae reveals a plethora of secreted proteins with poten and variant (modified by humans) enzymes. Examples of tial activity in insect hosts.” Microbiology-Sgm 149 (2003): cuticle degrading enzymes include proteases, peptidases, 239-47, Gimenez-Pecci, MdTP, et al. “Characterization of chitinases, chitosanase, cutinases, and lipases. In an embodi mycoviruses and analyses of chitinase secretion in the bio ment, the at least cuticle degrading enzymes is selected from control fungus Metarhizium anisopliae. Current Microbiol the group consisting of protease, peptidase, chitinase, chito ogy 45.5 (2002): 334-39, Hu, G. and R. J. S. Leger. “A sanase, lipase, cutinase and any combination thereof. In an phylogenomic approach to reconstructing the diversification embodiment the at least one cuticle degrading enzyme is a of serine proteases in fungi.” Journal of Evolutionary Biology protease. In an embodiment the at least one cuticle degrading 17.6 (2004): 1204-14, Hutwimmer, S., et al. “Algorithm enzyme is a chitinase. In an embodiment the at least one based design of synthetic growth media stimulating virulence cuticle degrading enzyme is a lipase. In an embodiment the at properties of Metarhizium anisopliae conidia.” Journal of least one cuticle degrading enzyme is a cutinase. Applied Microbiology 105.6 (2008): 2026-34, Joshi, L., R. S. 0029. In a particular embodiment, the at least one cuticle S. Leger, and D. W. Roberts. “Isolation of a cDNA encoding degrading enzymes is a combination of at least two cuticle a novel subtilisin-like protease (Pr1B) from the entomopatho degrading enzymes, such as, two cuticle degrading enzyme, genic fungus, Metarhizium aniisopliae using differential dis three cuticle degrading enzymes, four cuticle degrading play-RT-PCR. Gene (Amsterdam) 197.1-2 (1997): 1-8, enzymes, five cuticle degrading enzymes, etc. An example of Kim, H.K., et al. “Gene structure and expression of the gene this embodiment includes a protease and chitinase. In yet from Beauveria bassiana encoding bassiasin I, an insect another embodiment, the at least two cuticle degrading cuticle-degrading serine protease. Biotechnology Letters enzymes is a combination of at least two of the same type of 21.9 (1999): 777-83, Kim, J. S.“A novel biopesticide produc enzyme. Such as, at least two different proteases. In a particu tion: Attagel-mediated precipitation of chitinase from Beau lar embodiment, the at least one cuticle degrading enzymes is veria bassiana SFB-205 supernatant for thermotolerance.” a combination of at least three cuticle degrading enzymes. An Applied Microbiology and Biotechnology 87.5 (2010): 1639 example is a protease, a chitinase and a lipase. 48, “Relation of aphicidal activity with cuticular degradation 0030 The enzyme may possess one or more cuticle by beauveria bassiana SFB-205 Supernatant incorporated degrading activities. The cuticle degrading enzyme may be with polyoxyethylene-(3)-isotridecyl ether” Journal of obtained from any suitable source. In embodiments, the Microbiology and Biotechnology 20.3 (2010): 506-09, Kim, cuticle degrading enzyme may be obtained from a microor J. S., et al. “Influence of two FPLC fractions from Beauveria ganism (such as, bacterial or fungal sources). In another bassiana SFB-205 supernatant on the insecticidal activity embodiment, the cuticle degrading enzyme is the protease against cotton .’ Biocontrol Science and Technology described in WO 89/06279. Commercial proteases may also 20.1 (2010): 77-81, Kim, J. S., et al. “Correlation of the be used, such as, e.g. the product SAVINASE (available from aphicidal activity of Beauveria bassiana SFB-205 Superna Novozymes NS). tant with enzymes.” Fungal Biology 114.1 (2010): 120-28, 0031. The cuticle degrading enzyme may also be isolated Ko, H. J., et al. “Optimal production of protease from ento from an entomopathogenic, an acaripathogenic fungus or a mopathogenic fungus Beauveria bassiana. Agricultural nematopathogenic fungus and used as an exogenous enzyme, Chemistry and Biotechnology 39.6 (1996): 449-54, Ko, H.J., i.e., exogenously applied. Examples of cuticle degrading etal. “Purification and characterization of protease from ento enzymes are described in Bagga, S., etal. "Reconstructing the mopathogenic fungus Beauveria bassiana. Agricultural diversification of Subtilisins in the pathogenic fungus Metar Chemistry and Biotechnology 40.5 (1997): 388-94, Leal, S. hizium anisopliae. Gene 324 (2004): 159-69, Bidochka, M. C. M., et al. Amplification and restriction endonuclease J. and M.J. Melzer. “Genetic polymorphisms in three subtili digestion of the Pr1 gene for the detection and characteriza sin-like protease isoforms (Pr1A, Pr1B, and Pr1C) from tion of Metarhizium strains.” Mycological Research 101.3 Metarhizium strains. Canadian Journal of Microbiology (1997): 257-65, Liang et al., “The crystal structures of two 46.12 (2000): 1138-44, Braga, G.U.L., R. Vencovsky, and C. cuticle-degrading proteases from nematophagous fungi and L. Messias. “Estimates of genetic parameters related to chiti their contribution to infection against nematodes. The nase production by the entomopathogenic fungus Metarhi FASEB Journal, Vol. 24, 1391-1400, May 2010, Manalil, N. zium anisopliae. Genetics and Molecular Biology 21.2 S., et al. “Comparative analysis of the Metarhizium US 2013/0156740 A1 Jun. 20, 2013

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Applied and anisopliae host-pathogen interaction: differential immuno Environmental Microbiology 64.2 (1998): 709-13, St Leger, proteomics reveals proteins involved in the infection process R. J. R. C. Staples, and D. W. Roberts. “Entomopathogenic ofarthropods.” Fungal Biology 114.4 (2010): 312-19, Sasaki, isolates of metarhizium-aniisopliae, beauveria-bassiana, and S. D., et al. “BmSI-7, a novel subtilisin inhibitor from Boo aspergillus-flavus produce multiple extracellular chitinase philus microplus, with activity toward Pr1 proteases from the isozymes.” Journal of Invertebrate Pathology 61.1 (1993): fungus Metarhizium anisopliae. Experimental Parasitology 81-84, St. Leger et al., “Production of Cuticle-degrading 118.2 (2008): 214-20, Screen, S. E. G. Hu, and R. J. Leger. 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Synthesis in Culture cally and functionally related.” FEMS Microbiology Letters on Cuticle, Journal of Invertebrate Pathology, 48, 85-95 126.3 (1995): 227-31, Shah, F.A., C. S. Wang, and T. M. Butt. (1986), Todorova, S.I., et al. “Heterogeneity of two Beau “Nutrition influences growth and virulence of the insect veria bassiana strains revealed by biochemical tests, protein pathogenic fungus Metarhizium anisopliae.” FEMS Micro profiles and bio-assays on Leptinotarsa decemlineata (Col.: US 2013/0156740 A1 Jun. 20, 2013

Chrysomelidae) and Coleomegilla maculata lengi (Col. 0037. The fermentation may be conducted using conven Coccinellidae) larvae. Entomophaga 39.2 (1994): 159-69, tional fermentation processes, such as, aerobic liquid-culture Valadares, M. C. C. and J. L. Azevedo. “Production of amy techniques, shake flask cultivation, and Small-scale or large lases and proteases by wild-type and mutant strains of Metar scale fermentation (including continuous, batch, fed-batch, hizium anisopliae var. aniisopliae.” Revista de Microbiologia or solid state fermentation) in laboratory or industrial fermen 27.4 (1996): 237-41, Valadares-Inglis, M. C. and J. L. Aze tors, and Such processes are well known in the art. The fungal Vedo. 'Amylase and protease secretion in recombinant strains organism may be used as a pesticide directly from the culture of Metarhizium anisopliae var. aniisopliae following para medium or Subject to purification and/or further processing sexual crosses.” Brazilian Journal of Genetics 20.2 (1997): steps, such as, a drying process. In one embodiment, follow 171-75, Valadares-Inglis, M. C. and J. F. Peberdy. “Location ing fermentation, the fungal organism may be recovered of chitinolytic enzymes in protoplasts and whole cells of the using conventional techniques, such as by filtration or cen entomopathogenic fungus Metarhizium anisopliae.’ Myco trifugation. The fungal organism may alternatively be dried, logical Research 101.11 (1997): 1393-96, Wang, C. S., M.A. Such as by air-drying, freeze drying or spray drying, to a low Typas, and T. M. Butt. "Detection and characterisation of pr1 moisture level, and stored at a Suitable temperature (e.g., virulent gene deficiencies in the insect pathogenic fungus room temperature). Metarhizium anisopliae.” FEMS Microbiology Letters 213.2 0038. The pesticidal compositions are prepared according (2002): 251-55, Wei, Z. Y. Q. Cao, andY.X. Xia. “Cloning of to procedures and compositions which are conventional in the the subtilisin Pr1A gene from a strain of specific fun relevant art, e.g., agricultural chemical art when applied in gus, Metarhizium anisopliae, and functional expression of agricultural applications. The pesticidal composition prefer the protein in Pichia pastoris.’ World Journal of Microbiol ably comprises the at least one fungal pesticide and a suitable ogy and Biotechnology 24.11 (2008): 2481-88, U.S. Pat. No. carrier and/or at least one cuticle degrading enzyme and a 5,962,765, WO/2008/063011. An exogenous cuticle degrad suitable carrier. The carrier for the at least one fungal pesti ing enzyme may be obtained from any one of the sources cide and the at least one cuticle degrading enzyme may be the listed above. same or different. Thus, the at least one fungal pesticide may 0032. The at least one fungal pesticide and the at least one be applied with the at least one cuticle degrading enzyme in cuticle degrading enzyme may be applied separately (sequen the same composition or as ingredients of separate composi tially) or simultaneously. If applied simultaneously, the at tions. least fungal pesticide and the at least one cuticle degrading 0039 Examples of carriers include aqueous carriers, enzyme may be applied as ingredients of the same composi nutritional carriers, and inert carriers, such as, a phytologi tion or different compositions. cally-acceptable inert carrier. Examples of carriers also 0033. The treatment compositions (formulations) of the include solid inert carriers or diluents such as diatomaceous present invention will vary depending on the habitat treated earth, talc, clay, Vermiculite, calcium carbonate, alginate gels, and the intended application. The composition may be a dry starch matrices or synthetic polymers. In an embodiment, the or liquid composition, Such as concentrated Solid or liquid at least fungal pesticide and the at least one cuticle degrading formulations. The pesticidial composition may be in the form enzyme are applied to an agricultural habitat, such as, a crop, of wettable powders, dusts, granules, baits, solutions, emul field, plant or soil. In this aspect, the carrier is an agronomical sifiable concentrates, emulsions, Suspensions, concentrates, acceptable carrier, which carriers are known in the art. sprays (aerosols and fumigants aerosols), microparticles or 0040. The compositions may beformulated if desired with microcapsules, topical treatment, gels, seed coatings, baits, conventional additives, such as, polymers, Sticking agents or eartags, boluses, foggers, and many others. The composition adherents, adjuvants, emulsifying agents, Surfactants, foams, may be dispersed in water for application, or are dust or humectants, or wetting agents, antioxidants, colorants, UV granular formulations, which are applied without dispersion protectants, thickners, fillers, antifreeze agents, solvents, in water. nutritive additives, fertilizers, chemical pesticides and biope 0034. An important factor for any composition is provid sticides ( (including other bioinsecticides), fun ing a stable fungal pesticide and stable enzyme so that these gicides and/or herbicides), which exhibit low toxicity to the ingredients retain a sufficient effective amount of activity Subject fungal pesticide compositions of the present inven when used. Methods for producing stabilized fungal organ tion. isms are known in the art. In embodiment, the fungal pesticide 0041 Exemplary polymers include polyvinyl acetate, organism is present in the composition in the form of a stable polyvinyl alcohols with different degrees of hydrolysis, poly . vinylpyrrolidones, polyacrylates, acrylate-, polyol- or poly 0035 Methods for stabilizing enzymes are also known in ester-based paint system binders which are soluble or dispers the art in both solid and liquid formulations. In an embodi ible in water, moreovercopolymers of two or more monomers ment, the at least one enzyme is in powder form. In another Such as acrylic acid, methacrylic acid, itaconic acid, maleic embodiment, the at least one enzyme is in granule form. acid, fumaric acid, maleic anhydride, vinylpyrrolidone, eth 0036 Production of the fungal pesticide may be done in a ylenically unsaturated monomers such as ethylene, butadi liquid culture media or a solid culture media fermentation ene, isoprene, chloroprene, styrene, divinylbenzene, ot-me process. Generally, the media have high carbon and nitrogen thylstyrene or p-methylstyrene, further vinyl halides such as concentrations, which are are necessary for high yields. Suit vinyl chloride and vinylidene chloride, additionally vinyl able nitrogen sources include, but are not limited to hydro esters such as vinyl acetate, vinyl propionate or vinyl Stearate, lyzed casein, yeast extract, hydrolyzed soy protein, hydro moreover vinyl methyl ketone or esters of acrylic acid or lyzed cottonseed protein, and hydrolyzed corn gluten protein. methacrylic acid with monohydric alcohols or polyols such as Suitable carbon sources include, but are not limited to carbo methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl hydrates, including glucose, fructose, and Sucrose, and glyc ene methacrylate, lauryl acrylate, lauryl methacrylate, decyl erol. acrylate, N,N-dimethylamino-ethyl methacrylate, 2-hy US 2013/0156740 A1 Jun. 20, 2013

droxyethyl methacrylate, 2-hydroxypropyl methacrylate or thiocarboxime, thiodicarb and ; phenyl methylcar glycidyl methacrylate, furthermore diethyl esters or bamate insecticides Such as allyxycarb, , bufen monoesters of unsaturated dicarboxylic acids, furthermore carb, butacarb, carbanolate, cloethocarb, dicresyl, dioxacarb, (meth)acrylamido-N-methylol methyl ether, amides or EMPC, ethiofencarb, fenethacarb, , isoprocarb, nitriles such as acrylamide, methacrylamide, N-methylol , , mexacarbate, promacyl, promecarb, (meth)acrylamide, acrylonitrile, methacrylonitrile, and also , trimethacarb. XMC and xylylcarb; dinitrophenol N-substituted maleiraides and ethers such as vinylbutyl ether, insecticides such as dinex, dinoprop, dinosam and DNOC: vinyl isobutyl ether or vinyl phenyl ether. fluorine insecticides such as barium hexafluorosilicate, cryo 0.042 Examples of Surfactants are non-ionic and anionic emulsifiers, such as, polyoxyethylene fatty acid esters, poly lite, sodium fluoride, sodium hexafluorosilicate and sulflura oxyethylene fatty alcohol ethers, alkylaryl polyglycol ethers, mid; formamidine insecticides Such as amitraz, chlordime fatty amine ethoxylates, alkylsulphonates, alkyl Sulphates, form, and ; fumigant insecticides such as acrylonitrile, carbon disulfide, carbon tetrachloride, alkylarylsulphonates, aryl Sulphates and silicone surfactants. chloroform, chloropicrin, para-dichlorobenzene, 1,2-dichlo 0043. Examples of colorants are soluble or sparingly ropropane, ethyl formate, ethylene dibromide, ethylene soluble color pigments such as, for example, titanium diox dichloride, ethylene oxide, hydrogen cyanide, iodomethane, ide, color black or zinc oxide. methyl bromide, methylchloroform, methylene chloride, 0044 Examples of antioxidants are sterically hindered naphthalene, phosphine, sulfuryl fluoride and tetrachloroet phenols and alkyl-substituted hydroxyanisoles and hydroxy hane; inorganic insecticides Such as borax, calcium polysul toluenes. fide, copper oleate, mercurous chloride, potassium thiocyan 0045 Examples of thickeners are organic polymers such ate and Sodium thiocyanate; chitin synthesis inhibitors such as partially or fully neutralized polyacrylic acids, polyvi as bistrifluoron, , chlorfluaZuron, cyromazine, nylpyrrolidone homo- or copolymers, polyethylene glycols, , flucycloxuron, , hexaflumuron, ethylene oxide?propylene oxide copolymers, polyvinyl alco lufenuron, novaluron, noviflumuron, penfluoron, teflubenzu hols and non-ionically or ionically modified celluloses, thixo ron and triflumuron; juvenile hormone mimics such as tropic Xanthan-based thickeners, and moreover inorganic dis epolfenonane, , , kinoprene, methop perse thickeners such as precipitated or pyrogenic silicas, rene, and triprene; juvenile hormones Such as kaolins, bentonites, aluminum/silicon mixed oxides, and sili juvenile hormone I, juvenile hormone II and juvenile hor Cates. mone III; moulting hormone agonists such as chroma 0046 Examples of antifreeze agents are urea, glycerol or fenozide, halofenozide, methoxyfenozide and : propylene glycol. moulting hormones such as alpha.-ecdysone and ecdyster 0047. Examples of fillers are ground minerals, calcium one; moulting inhibitors such as diofenolan; precocenes Such carbonate, ground quartz and aluminum/silicon mixed oxides as precocene I, precocene II and precocene III; unclassified or mixed hydroxides. insect growth regulators such as dicyclanil; ana 0.048 One or more other insecticides, acaricides, and/or logue insecticides such as benSultap, cartap, thiocyclam and nematicides, may be applied, either simultaneously or thiosultap; nicotinoid insecticides such as flonicamid; applied sequentially, with the biopesticide compositions of nitroguanidine insecticides such as , , the present invention. In addition, it may be beneficial in some and , nitromethylene insecticides embodiments to apply one or more fungicides and/or herbi Such as and ; pyridylmethylamine cides, either simultaneously or applied sequentially, with the insecticides such as , imidacloprid, nitenpyram biopesticide compositions of the present invention. and ; organochlorine insecticides Such as bromo 0049. Examples of additional insecticides that can be DDT, camphechlor, DDT, pp'-DDT, ethyl-DDD, HCH, employed beneficially include: antibiotic insecticides such as gamma-HCH, , , pentachlorophenol allosamidin and thuringiensin; macrocyclic lactone insecti and TDE; cyclodiene insecticides such as , bromocy cides such as spinosad, spinetoram, and other spinosyns clen, chlorbicyclen, , , , dilor, including the 21-butenyl spinosyns and their derivatives; , , HEOD, , HHDN, , avermectin insecticides such as abamectin, doramectin, ema isodrin, kelevan and , insecticides mectin, eprinomectin, ivermectin and Selamectin; milbemy Such as bromfenVinfos, , crotoxyphos, cin insecticides such as lepimectin, milbemectin, milbemycin , , dimethylvinphos, fospirate, hep Oxime and moxidectin; arsenical insecticides such as calcium tenophos, methocrotophos, , , arsenate, copper acetoarsenite, copper arsenate, lead arsen , naftalofos, , propaphos, TEPP and tet ate, potassium arsenite and sodium arsenite; other biological rachlorvinphos; organothiophosphate insecticides such as insecticides, plant incorporated protectant insecticides Such dioxabenzofos, foSmethilan and ; aliphatic organ as Cry1Ab, Cry1Ac, Cry1F, Cry1A.105, Cry2Ab2, Cry3A, othiophosphate insecticides such as acethion, amiton, mir Cry3A, Cry3Bb1, Cry34, Cry35, and VIP3A; botanical cadusafos, , chlormephos, , deme insecticides Such as , azadirachtin, d-limonene, phion-O, demephion-S, demeton, demeton-O, demeton-S, nicotine, , cinerins, cinerin I, cinerin II, jasmolin I. demeton-methyl, demeton-O-methyl, demeton-S-methyl, jasmolin II, I, pyrethrin II, quassia, rotenone, ryania demeton-S-methylsulphon, , , ethoprophos, and Sabadilla; insecticides Such as and IPSP, isothioate, , methacrifos, oxydemeton-me ; benzofuranyl methylcarbamate insecticides such as thyl, oxydeprofos, oxydisulfoton, , Sulfotep, benfuracarb, , , decarbofuran and and thiometon; aliphatic amide organothiophosphate insecti furathiocarb; dimethylcarbamate insecticides dimitan, dime cides such as amidithion, cyanthoate, , ethoate tilan, hyduincarb and ; oxime carbamate insecti methyl, , mecarbam, , , cides Such as alanycarb, , aldoxycarb, , sophamide and vamidothion; oXime organothiophosphate butoxycarboxim, , nitrilacarb, , tazimcarb, insecticides such as chlorphoxim, and phoxim-me US 2013/0156740 A1 Jun. 20, 2013 thyl; heterocyclic organothiophosphate insecticides Such as diamide, HGW86, , IKI-2002, isoprothi , , coumithoate, , endot olane, malonoben, metaflumizone, metoxadiaZone, niflurid hion, menazon, morphothion, , pyraclofos, ide, NNI-9850, NNI-0101, pymetrozine, pyridaben, pyridaphenthion and quinothion; benzothiopyran organ pyridalyl pyrifluquinazon, Qcide, rafoxanide, RynaxypyrTM. othiophosphate insecticides such as dithicrofos and thicrofos; SYJ-159, triarathene and triazamate and any combinations benzotriazine organothiophosphate insecticides such as azin thereof. phos-ethyl and azinphos-methyl, isoindole organothiophos 0050 Examples of fungicides that can be employed phate insecticides Such as dialifos and , isoxazole include: 2-(thiocyanatomethylthio)-benzothiazole, 2-phe organothiophosphate insecticides such as and nylphenol, 8-hydroxyquinoline Sulfate, Ampelomyces, Zolaprofos; pyrazolopyrimidine organothiophosphate insec quisqualis, azaconazole, azoxystrobin, Bacillus subtilis, ticides such as chlorpraZophos and pyrazophos; pyridine benalaxyl, benomyl, benthiavalicarb-isopropyl, benzylami organothiophosphate insecticides such as and nobenzene-sulfonate (BABS) salt, bicarbonates, biphenyl, chlorpyrifos-methyl, pyrimidine organothiophosphate insec bismerthiazol, bitertanol, blasticidin-S, borax, Bordeaux ticides Such as butathiofos, , etrimfos, lirimfos, pir mixture, boScalid, bromuconazole, bupirimate, calcium imiphos-ethyl, pirimiphos-methyl, primidophos, pyrimitate polysulfide, captafol, captan, carbendazim, carboxin, carpro and ; quinoxaline organothiophosphate insecti pamid, carvone, chloroneb, chlorothalonil, chloZolinate, cides such as and quinallphos-methyl; thiadiazole Coniothyrium minitans, copper hydroxide, copper octanoate, organothiophosphate insecticides such as athidathion, lythi copper oxychloride, copper Sulfate, copper Sulfate (tribasic), dathion, and prothidathion; triazole organ cuprous oxide, cyaZofamid, cyflufenamid, cymoxanil, cypro othiophosphate insecticides Such as isazofos and triaZophos: conazole, cyprodinil, dazomet, debacarb, diammonium eth phenyl organothiophosphate insecticides such as azothoate, ylenebis-(dithiocarbamate), dichlofluanid, dichlorophen, bromophos, bromophos-ethyl, , chlorthio diclocymet, diclomeZine, dichloran, diethofencarb, difeno phos, , cythioate, dicapthon, dichlofenthion, eta conazole, difenZoquat ion, diflumetorim, dimethomorph, phos, famphur, fenchlorphos, fensulfothion, dimoxystrobin, diniconazole, diniconazole-M, dinobuton, , fenthion-ethyl, heterophos, jodfenphos, mesulfen dinocap, diphenylamine, dithianon, dodemorph, dodemorph fos, , parathion-methyl, phenkapton, phosnichlor, acetate, dodine, dodine free base, edifenphos, epoxiconazole, , prothiofos, Sulprofos, temephos, trichlormeta ethaboxam, ethoxyquin, etridiazole, famoxadone, fenami phos-3 and trifenofos; phosphonate insecticides Such as buto done, fenarimol, fenbuconazole, fenfuram, fenhexamid, nate and trichlorfon; phosphonothioate insecticides such as fenoxanil, fenpiclonil, fenpropidin, fenpropimorph, fentin, mecarphon; phenyl ethylphosphonothioate insecticides Such fentin acetate, fentin hydroxide, ferbam, ferimzone, fluazi as and trichloronat, phenyl phenylphosphonothioate nam, fludioxonil, flumorph, fluopicolide, fluoroimide, fluox insecticides Such as cyanofenphos, EPN and ; phos astrobin, fluguinconazole, flusilaZole, flusulfamide, flutola phoramidate insecticides such as crufomate, , fos nil, flutriafol, folpet, formaldehyde, fosetyl, fosetyl thietan, imicyafos, mephosfolan, and pirimeta aluminium, fuberidazole, furalaxyl, furametpyr, guazatline, phos; phosphoramidothioate insecticides such as , guazatline acetates, GY-81, hexachlorobenzene, hexacona isocarbophos, isofenphos, and propetam Zole, hymexaZol, imazalil, imazalil Sulfate, imibenconazole, phos; phosphorodiamide insecticides Such as , mazi iminoctadine, iminoctadine triacetate, iminoctadine tris(al dox, and , oxadiazine insecticides such as besilate), ipconazole, iprobenfos, iprodione, iprovalicarb, indoxacarb; phthalimide insecticides such as dialifos, phos isoprothiolane, kasugamycin, kasugamycin hydrochloride met and ; pyrazole insecticides such as aceto hydrate, kresoxim-methyl, mancopper, mancoZeb, maneb, prole, ethiprole, , pyrafluprole, , tebufen mepanipyrim, mepronil, mercuric chloride, mercuric oxide, pyrad, tolfenpyrad and Vaniliprole; ester mercurous chloride, metalaxyl, mefenoxam, metalaxyl-M, insecticides such as , allethrin, , bar metam, metam-ammonium, metam-potassium, metam-So thrin, , bioethanomethrin, cyclethrin, cyclopro dium, metconazole, methasulfocarb, methyl iodide, methyl thrin, , beta-cyfluthrin, , gamma-cyha isothiocyanate, metiram, metominostrobin, metrafenone, lothrin, lambda-cyhalothrin, , alpha mildiomycin, myclobutanil, nabam, nitrothal-isopropyl, nua cypermethrin, beta-cypermethrin, theta-cypermethrin, Zeta rimol, octhillinone, ofurace, oleic acid (fatty acids), orysas cypermethrin, , , dimefluthrin, trobin, oxadixyl, oxine-copper, Oxpoconazole fumarate, oxy dimethrin, , fenfluthrin, fempirithrin, fenpropath carboxin, pefurazoate, penconazole, pencycuron, rin, , , flucythrinate, fluvalinate, tau pentachlorophenol, pentachlorophenyl laurate, penthiopy fluvalinate, furethrin, , , , rad, phenylmercury acetate, phosphonic acid, phthalide, biopermethrin, transpermethrin, , , picoxystrobin, polyoxin B, polyoxins, polyoxorim, potas profluthrin, pyresmethrin, , biopermethrin, cis sium bicarbonate, potassium hydroxyquinoline Sulfate, methrin, , terallethrin, tetramethrin, probenazole, prochloraz, procymidone, propamocarb, pro and ; pyrethroidether insecticides such as etofen pamocarb hydrochloride, propiconazole, propineb, pro prox, flufenprox, halfenprox, protrifenbute and ; quinazid, prothioconazole, pyraclostrobin, pyrazophos, pyrimidinamine insecticides such as flufenerim and pyrimid pyributicarb, pyrifenox, pyrimethanil, pyroquilon, quinoc ifen; pyrrole insecticides Such as chlorfenapyr, tetronic acid lamine, quinoxyfen, quintoZene, Reynoutria Sachalinensis insecticides such as spirodiclofen, Spiromesifen and spirotet extract, silthiofam, Simeconazole, Sodium 2-phenylphenox ramat; thiourea insecticides such as diafenthiuron; urea insec ide, Sodium bicarbonate, Sodium pentachlorophenoxide, ticides Such as flucofuron and Sulcofuron; and unclassified spiroxamine, sulfur, SYP-Z071, tar oils, tebuconazole, tecna insecticides such as AKD-3088, , closan Zene, tetraconazole, thiabendazole, thifluZamide, thiophan tel, crotamiton, cyflumetofen, E2Y45, EXD, fenazaflor, ate-methyl, thiram, tiadinil, tolclofos-methyl, tolylfluanid, fenazaquin, fenoxacrim, fenpyroximate, FKI-1033, fluben triadimefon, triadimenol, triaZoxide, tricyclazole, tridemo US 2013/0156740 A1 Jun. 20, 2013 rph, trifloxystrobin, triflumizole, triforine, triticonazole, val bispyribac and pyriminobac, pyrimidinylthiobenzoic acid idamycin, VincloZolin, Zineb, Ziram, Zoxamide, Candida herbicides such as pyrithiobac; phthalic acid herbicides such Oleophila, Fusarium oxysporum, Gliocladium spp., Phlebi as chlorthal; picolinic acid herbicides such as aminopyralid, opsis gigantean, Streptomyces griseoviridis, Trichoderma clopyralid and picloram; quinolinecarboxylic acid herbicides spp., (RS)-N-(3,5-dichlorophenyl)-2-(methoxymethyl)-suc Such as quinclorac and quinmerac, arsenical herbicides Such cinimide, 1,2-dichloropropane, 1,3-dichloro-1,1,3,3-tet as cacodylic acid, CMA, DSMA, hexaflurate, MAA, rafluoroacetone hydrate, 1-chloro-2,4-dinitronaphthalene, MAMA, MSMA, potassium arsenite and sodium arsenite: 1-chloro-2-nitropropane, 2-(2-heptadecyl-2-imidazolin-1- benzoylcyclohexanedione herbicides Such as mesotrione, yl)ethanol, 2,3-dihydro-5-phenyl-1,4-dithi-line 1,1,4,4-tet sulcotrione, tefuryltrione and tembotrione; benzofuranyl raoxide, 2-methoxyethylmercury acetate, 2-methoxyethylm ercury chloride, 2-methoxyethylmercury silicate, 3-(4- alkylsulfonate herbicides such as benfuresate and ethofume chlorophenyl)-5-methylrhodanine, 4-(2-nitroprop-1-enyl) sate; carbamate herbicides such as asulam, carboxazole chlo phenyl thiocyanateme: ampropylfos, anilazine, azithiram, rprocarb, dichlormate, fenasulam, karbutilate and terbucarb: barium polysulfide, Bayer 32394, benodanil, benquinox, carbanilate herbicides such as barban, BCPC, carbasulam, bentaluron, benzamacril; benzamacril-isobutyl, benzamorf, carbetamide, CEPC, chlorbufam, chlorpropham, CPPC, des binapacryl, bis(methylmercury) sulfate, bis(tributyltin) medipham, phenisopham, phenmedipham, phenmedipham oxide, buthiobate, cadmium calcium copper Zinc chromate ethyl, propham and Swep; cyclohexene oxime herbicides sulfate, carbamorph, CECA, chlobenthiazone, chlorani Such as alloxydim, butroxydim, clethodim, cloproxydim, formethan, chlorfenazole, chlorquinox, climbazole, copper cycloxydim, profoxydim, Sethoxydim, tepraloxydim and bis(3-phenylsalicylate), copper Zinc chromate, cufraneb, tralkoxydim; cyclopropylisoxazole herbicides such as isox cupric hydrazinium sulfate, cuprobam, cyclafuramid, achlortole and isoxaflutole; dicarboximide herbicides such as benzfendizone, cinidon-ethyl, flumezin, flumiclorac, flumi cypendazole, cyprofuram, decafentin, dichlone, dichloZo oxazin and flumipropyn, dinitroaniline herbicides such as line, diclobutraZol, dimethirimol, dinocton, dinosulfon, benfluralin, butralin, dinitramine, ethalfluralin, fluchloralin, dinoterbon, dipyrithione, ditalimfos, dodicin, draZOXolon, isopropalin, methalpropalin, nitralin, oryzalin, pendimetha EBP, ESBP. etaconazole, etem, ethirim, fenaminosulf, fena panil, fenitropan, fluotrimazole, furcarbanil, furconazole, flu lin, prodiamine, profluralin and trifluralin; dinitrophenolher conazole-cis, furmecyclox, furophanate, glyodine, griseoful bicides Such as dinofenate, dinoprop, dinosam, dinoseb, vin, halacrinate, Hercules 3944, hexylthiofos, ICIA0858, dinoterb, DNOC, etinofen and medinoterb; diphenyl ether isopamphos, isovaledione, mebenil, mecarbinzid, metazoxo herbicides such as ethoxyfen; nitrophenyl ether herbicides lon, methfuroxam, methylmercury dicyandiamide, metSul such as acifluorfen, aclonifen, bifenox, chlomethoxyfen, foVax, milineb, mucochloric anhydride, myclozolin, N-3,5- chlomitrofen, ethipromid, fluorodifen, fluoroglycofen, fluo dichlorophenyl-succinimide, N-3-nitrophenylitaconimide, ronitrofen, fomesafen, furyloxyfen, halosafen, lactofen, natamycin, N-ethylmercurio-4-toluenesulfonanilide, nickel nitrofen, nitrofluorfen and oxyfluorfen: dithiocarbamate her bis(dimethyldithiocarbamate), OCH, phenylmercury dim bicides such as dazomet and metam; halogenated aliphatic ethyldithiocarbamate, phenylmercury nitrate, phosdiphen, herbicides such as alorac, chloropon, dalapon, flupropanate, prothiocarb; prothiocarb hydrochloride, pyracarbolid, pyri hexachloroacetone, iodomethane, methyl bromide, dinitril, pyroxychlor, pyroxyfur, quinacetol; quinacetol Sul monochloroacetic acid, SMA and TCA; imidazolinone her bicides such as imazamethabenZ, imaZamox, imazapic, fate, quinaZamid, quinconazole, rabenzazole, salicylanilide, imazapyr, imaZaquin and imazethapyr; inorganic herbicides SSF-109, Sultropen, tecoram, thiadifluor, thicyofen, thiochlo Such as ammonium Sulfamate, borax, calcium chlorate, cop rfemphim, thiophanate, thioquinox, tioxymid, triamiphos, per Sulfate, ferrous Sulfate, potassium azide, potassium cyan triarimol, triazbutil, trichlamide, urbacid, XRD-563, and ate, Sodium azide, Sodium chlorate and Sulfuric acid; nitrile Zarilamid, and any combinations thereof. herbicides such as bromobonil, bromoxynil, chloroxynil, 0051 Examples of herbicides that can be employed dichlobenil, iodobonil, ioxynil and pyraclonil; organophos include: amide herbicides such as allidochlor, beflubutamid, phorus herbicides such as amiprofoS-methyl, anilofos, ben benzadox, benzipram, bromobutide, cafenstrole, CDEA, Sulide, bilanafos, butamifos, 2,4-DEP, DMPA, EBEP, fos chlorthiamid, cyprazole, dimethenamid, dimethenamid-P, amine, glufosinate, glyphosate and piperophos; phenoxy diphenamid, epronaz, etnipromid, fentraZamide, flupoxam, herbicides such as bromofenoxim, clomeprop, 2,4-DEB, 2,4- fomesafen, halosafen, isocarbamid, isoxaben, napropamide, DEP difenopenten, disul, erbon, etnipromid, fenteracol and naptalam, pethoxamid, propyZamide, quinonamid and teb trifopsime; phenoxyacetic herbicides such as 4-CPA, 2,4-D, utam; anilide herbicides such as chloranocryl, cisanilide, 3,4-DA, MCPA, MCPA-thioethyl and 2,4,5-T: phenoxybu clomeprop, cypromid, diflufenican, etobenzanid, fenasulam, tyric herbicides such as 4-CPB, 2,4-DB,3,4-DB, MCPB and flufenacet, flufenican, mefenacet, mefluidide, metamifop, 2,4,5-TB; phenoxypropionic herbicides such as cloprop, monalide, naproanilide, pentanochlor, picolinafen and propa 4-CPP, dichlorprop, dichlorprop-P, 3,4-DP, fenoprop, meco nil, arylalanine herbicides Such as benzoylprop, flampropand propand mecoprop-P; aryloxyphenoxypropionic herbicides flamprop-M; chloroacetanilide herbicides such as acetochlor, Such as chloraZifop, clodinafop, clofop, cyhalofop, diclofop, alachlor, butachlor, butenachlor, delachlor, diethatyl, dimeth fenoxaprop, fenoxaprop-P, fenthiaprop, fluazifop, fluazifop achlor, metaZachlor, metolachlor, S-metolachlor, preti P. haloxyflop, haloxyfop-P, isoxapyrifop, metamifop, pro lachlor, propachlor, propisochlor, prynachlor, terbuchlor, the paquizafop, quizalofop, quizalofop-P and trifop; phenylene nylchlor and xylachlor; sulfonanilide herbicides such as diamine herbicides such as dinitramine and prodiamine; benZofluor, perfluidone, pyrimisulfan and profluaZol; Sul pyrazolylherbicides such as benzofenap, pyrazolynate, pyra fonamide herbicides such as asulam, carbasulam, fenasulam Sulfotole, pyrazoxyfen, pyroxasulfone and toprameZone; and oryzalin; antibiotic herbicides such as bilanafos; benzoic pyrazolylphenyl herbicides such as fluazolate and pyraflufen; acid herbicides such as chloramben, dicamba,2,3,6-TBA and pyridazine herbicides such as credazine, pyridafol and pyri tricamba; pyrimidinyloxybenzoic acid herbicides such as date; pyridaZinone herbicides such as brompyrazon, chlo US 2013/0156740 A1 Jun. 20, 2013

ridazon, dimidazon, flufenpyr, metflurazon, norflurazon, 0.052 A variety of pests may be targeted, including oxapyrazon and pydanon; pyridine herbicides Such as ami insects, Acari or nematodes. Insects and otherpests which can nopyralid, cliodinate, clopyralid, dithiopyr, fluoroxypyr, be targeted include, but are not limited to: Lepidoptera— haloxydine, picloram, picolinafen, pyriclor, thiazopyr and Heliothis spp., Helicoverpa spp., Spodoptera spp., Mythinna triclopyr; pyrimidinediamine herbicides such as iprymidam unipuncta, Agrotis ipsilon, Earias spp., Euxoa auxiliaris, Tri and tioclorim; quaternary ammonium herbicides such as choplusia ni, Anticarsia gemmatalis, Rachiplusia nu, Plutella cyperquat, diethamquat, difenZoquat, diguat, morfamquat xylostella, Chilo spp., Scirpophaga incertulas, Sesamia infe and paraquat; thiocarbamate herbicides such as butylate, rens, Cnaphalocrocis medinalis, Ostrinia nubilalis, Cydia cycloate, di-allate, EPTC, esprocarb, ethiolate, isopolinate, pomonella, Carposina niponensis, Adoxophyes Orana, Archips argyrospilus, Pandemis heparana, Epinotia methiobencarb, molinate, orbencarb, pebulate, prosulfocarb, aporema, Eupoecilia ambiguella, Lobesia botrana, Poly pyributicarb, sulfallate, thiobencarb, tiocarbazil, tri-allate chrosis viteana, Pectinophora gossypiella, Pieris rapae, and Vemolate; thiocarbonate herbicides such as dimexano, Phyllonorycter spp., Leucoptera malifoliella, Phyllocnisitis EXD and proxan; thiourea herbicides such as methiuron; citrella Coleoptera—Diabrotica spp., Leptinotarsa decem triazine herbicides Such as dipropetryn, triaziflam and trihy lineata, Oulema Oryzae, Anthonomus grandis, Lissorhoptrus droxytriazine; chlorotriazine herbicides such as atrazine, Oryzophilus, Agriotes spp., Melanotus communis, Popillia chlorazine, cyanazine, cyprazine, eglinazine, ipazine, meso japonica, Cyclocephala spp., Tribolium spp. Homoptera— prazine, procyazine, proglinazine, propazine, Sebuthylazine, Aphis spp., Myzus persicae, Rhopalosiphum spp., Dysaphis simazine, terbuthylazine and trietazine; methoxytriazine her plantaginea, Toxoptera spp., Macrosiphum euphorbiae, bicides such as atraton, methometon, prometon, secbumeton, Aulacorthum Solani, Sitobion avenae, Metopolophium dirho simeton and terbumeton; methylthiotriazine herbicides such dum, Schizaphis graminum, Brachycolus noxius, Nephotettix as ametryn, aZiprotryne, cyanatryn, desmetryn, dimetham spp., Nilaparvata lugens, Sogatella furcifera, Laodelphax etryn, methoprotryne, prometryn, simetryn and terbutryn; tri striatellus, Bemisia tabaci, Trialeurodes vaporariorum, aZinone herbicides Such as ametridione, amibuzin, hexazi Aleurodes proletella, Aleurothrixus floccosus, Ouadraspidio none, isomethiozin, metamitron and metribuzin, triazole tus perniciosus, Unaspis yanonensis, Ceroplastes rubens, herbicides such as amitrole, cafenstrole, epronaz and Aonidiella aurantii Hemiptera—Lygus spp., Eurygaster flupoxam, triazolone herbicides such as amicarbazone, ben maura, Nezara viridula, Piezodorus guildingi, Leptocorisa carbazone, carfentraZone, flucarbazone, propoxycarbazone, varicornis, Cimex lectularius, Cimex hemipterus Thysan SulfentraZone and thiencarbazone-methyl, triazolopyrimi optera—Frankliniella spp., spp., Scirtothrips dorsalis dine herbicides such as cloransulam, diclosulam, florasulam, Isoptera—Reticulitermes flavipes, Coptotermes formosanus, flumetSulam, metoSulam, penoXSulam and pyroxSulam; Reticulitermes virginicus, Heterotermes aureus, Reticuliter uracil herbicides Such as butafenacil, bromacil, flupropacil, mes hesperus, Coptotermes frenchii, Shedorhinotermes spp., isocil, lenacil and terbacil; 3-phenyluracils; urea herbicides Reticulitermes Santonensis, Reticulitermes grassei, Reticuli Such as benzthiaZuron, cumyluron, cycluron, dichloralurea, termes banyulensis, Reticulitermes speratus, Reticulitermes diflufenZopyr, isonoruron, isouron, methabenzthiazuron, hageni, Reticulitermes tibialis, Zootermopsis spp., Incisiter monisouron and noruron; phenylurea herbicides such as ani mes spp., Marginitermes spp., Macrotermes spp., Microcero Suron, buturon, chlorbromuron, chloreturon, chlorotoluron, termes spp., Microtermes spp. Diptera—Liriomyza spp., chloroXuron, daimuron, difenoXuron, dimefuron, diuron, Musca domestica, Aedes spp., Culex spp., Anopheles spp., fenuron, fluometuron, fluothiuron, isoproturon, linuron, Fannia spp., Stomoxys spp., Hymenoptera—Iridomyrmex methiuron, methyldymron, metobenzuron, metobromuron, humilis, Solenopsis spp., Monomorium pharaonis, Atta spp., metoxuron, monolinuron, monuron, neburon, parafluoron, Pogonomyrmex spp., Camponotus spp., Monomorium spp., phenobenzuron, Siduron, tetrafluoron and thidiaZuron; pyri Tapinoma sessile, Tetramorium spp., Xvlocapa spp., Vespula midinylsulfonylurea herbicides such as amidosulfuron, azim spp., Polistes spp. Mallophaga (chewing lice) Anoplura Sulfuron, benSulfuron, chlorimuron, cyclosulfamuron, ethox (sucking lice)—Pthirus pubis, Pediculus spp. ysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, (, crickets)—Melanoplus spp., Locusta migra foramsulfuron, halosulfuron, imaZoSulfuron, mesosulfuron, toria, Schistocerca gregaria, Gryllotalpidae (mole crickets). nicosulfuron, orthosulfamuron, oxasulfuron, primisulfuron, Blattoidea (cockroaches)—Blatta Orientalis, Blattella ger pyrazosulfuron, rimsulfuron, Sulfometuron, Sulfosulfuron manica, Periplaneta americana, Supella longipalpa, and trifloxysulfuron; triazinylsulfonylurea herbicides such as Periplaneta australasiae, Periplaneta brunnea, Parcoblatta chlorSulfuron, cinosulfuron, ethametSulfuron, iodosulfuron, pennsylvanica, Periplaneta fuliginosa, Pycnoscelus surina metsulfuron, prosulfuron, thifensulfuron, triasulfuron, tribe mensis, Siphonaptera—Ctenophalides spp., Pulex irritans nuron, triflusulfuron and tritosulfuron; thiadiazolylurea her Acari Tetranychus spp., Panonychus spp., Eotetranychus bicides such as buthiuron, ethidimuron, tebuthiuron, thiaz afluoron and thidiaZuron; and unclassified herbicides such as carpini, Phylocoptruta oleivora, Aculus pelekassi, Brevipal acrolein, allyl alcohol, azafenidin, benazolin, bentaZone, ben pus phoenicis, Boophilus spp., Dermacentor variabilis, Rhi Zobicyclon, buthidazole, calcium cyanamide, cambendichlor, picephalus sanguineus, Amblyomma americanum, Ixodes chlorfenac, chlorfenprop, chlorflurazole, chlorflurenol, cin spp., Notoedres cati, Sarcoptes scabiei, Dermatophagoides methylin, clomazone, CPMF, cresol, ortho-dichlorobenzene, spp. Nematoda—Dirofilaria immitis, Meloidogyne spp., Het dimepiperate, endothal, fluoromidine, fluridone, fluorochlo erodera spp., Hoplolaimus columbus, Belonolaimus spp., ridone, flurtamone, fluthiacet, indanofan, methazole, methyl Pratylenchus spp., Rotylenchus reniformis, Criconemella isothiocyanate, nipyraclofen, OCH, oxadiargyl, oxadiazon, ornata, Dity lenchus spp., Aphelenchoides besseyi, Hir oxaziclomefone, pentachlorophenol, pentoxazone, phenylm schmanniella spp. ercury acetate, pinoxaden, prosulfalin, pyribenzoxim, pyrift 0053 Examples of target pests include soil-born insects alid, quinoclamine, rhodethanil, Sulglycapin, thidiazimin, tri and ground- and canopy-dwelling insects. Without being lim diphane, trimeturon, tripropindan and tritac. ited thereto, pests which may be targeted include Acari (mites US 2013/0156740 A1 Jun. 20, 2013

and ticks); Blatteria, Coleoptera, Diptera, Orthoptera, Thys 39. The method of claim 32, wherein the least one exog anoptera, Hemiptera, Homoptera, Isoptera, Phthiraptera, enously applied cuticle degrading enzyme is selected from Siphonaptera, Lepidoptera, Hymenoptera. Target pests also the group consisting of protease, peptidase, chitinase, chito include root weevils, thrips, whiteflies, mites, ticks, root sanase, lipase, cutinase and any combination thereof. worms, wireworms, fruit flies, soil grubs, root maggots, ter 40. The method of claim32, wherein the treating comprises mites, and ants, particularly corn rootworm (Diabrotica spp). treating the habitat with a composition comprising the pesti black vine weevil (Otiorhynchus sulcatus), citrus root weevil cidally effective amounts of the at least one fungal pesticide, (Diaprepes abbreviatus), Sweet potato weevil (Cylas formi at least one exogenously applied cuticle degrading enzyme carius), Sugarbeet root maggot (Tetanops myopaeformis), and a carrier for the at least one fungal pesticide and the at cabbage maggot ( radicum), onion maggot (Delia anti least one exogenously applied cuticle degrading enzyme. gua), turnip maggot (Delia floralis), seedcorn maggot (Delia 41. The method of claim32, wherein the treating comprises platura), carrotrust fly (Psila rosae), Japanese (Popil treating the habitat with a composition comprising the at least lia japonica), European chafer (Rhizotrogus maialis), Subter one fungal pesticide and a carrier for the fungal pesticide and ranean (Reticulitermes and Coptotermes spp., emerald a composition comprising the at least one exogenously ash borer (Agrilus planipennis), gypsy moth (Lymantria dis applied cuticle degrading enzyme and a carrier for the at least par), and the pecan weevil (Curculio Carvae). one exogenously applied cuticle degrading enzyme. 42. The method of claim32, wherein theat leastonecuticle EXAMPLES degrading enzyme is in an amount effective to increase the boring through of fungal pesticide through the cuticle of the Example 1 target pest. 0054 An experiment was done with two doses of Metar 43. The method of claim 32, wherein the habitat is a plant, hizium anisopliae strain F52 (0 and 3.3x1OE6 conidia/mL) soil, or water environment. each combined with four concentrations of a protease (the 44. A biopesticide comprising pesticidally effective commercial protease enzyme SAVINASE available from amounts of at least one fungal pesticide and at least one Novozymes NS) in amount of 0, 0.1%, 1%, and 10% w/w. Ten exogenous cuticle degrading enzyme. mealworms were exposed to each of the resulting eight treat 45. The biopesticide of claim 44, wherein the at least one ments and monitored for mortality over 6 days. fungal pesticide comprises is at least one entomopathogenic 0055. The resulting mortality is presented in FIG. 1. As fungus. illustrated in FIG. 1, the combination of conidia at 3.3x10E6 46. The biopesticide of claim 44, wherein the at least one conidia/mL with concentrations of Savinase at 0.1% or higher fungal pesticide comprises at least one nematopathogenic resulted in a shorter time to mortality in treated mealworms. fungus. Savinase alone at 1% did not result in increased mortality over 47. The biopesticide of claim 44, wherein the at least one untreated mealworms. Savinase alone at 10% resulted in up to fungal pesticide comprises at least one acaripathogenic fun 20% mortality above untreated mealworms, but still lower guS. than mortality in mealworms treated with conidia alone at 48. The biopesticide of claim 44, wherein the at least one 3.3x1OE6 conidia/mL. fungal pesticide has one or more of the following properties: entomopathogenic activity, acaripathogenic activity, and 1-31. (canceled) nematopathogenic activity. 32. A method for controlling a pest comprising treating a 49. The biopesticide of claim 44, wherein the at least one pest habitat with a biopesticide comprising a pesticidally fungal pesticide is selected from the group consisting of spe effective amount of at least one fungal pesticide and at least cies of Ascomycota, Alternaria, Beauveria, Lecanicillium, one exogenously applied cuticle degrading enzyme. Metarhizium, Verticillium, Trichoderma, Aspergillus, Nomu 33. The method of claim 32, wherein the at least one fungal raea, Paecilomyces, Isaria, Hirsutella, Fusarium, pesticide comprises is at least one entomopathogenic fungus. Cordyceps, Entomophthora, Zoophthora, Pandora, Ento 34. The method of claim 32, wherein the at least one fungal mophaga, Entomophthorales and Zygomycota. pesticide comprises at least one nematopathogenic fungus. 50. The biopesticide of claim 44, wherein the at least one 35. The method of claim 32, wherein the at least one fungal fungal pesticide comprises Alternaria cassiae, Fusarium lat pesticide comprises at least one acaripathogenic fungus. eritum, Fusarium Solani, Verticillium lecanii, Aspergillus 36. The method of claim 32, wherein the at least one fungal parasiticus, Metarhizium aniisopliae, Beauveria bassiana pesticide has one or more of the following properties: ento and any combination thereof, more preferably, and Metarhi mopathogenic activity, acaripathogenic activity, and nemato Zium anisopliae. pathogenic properties. 51. The biopesticide of claim 44, wherein the least one 37. The method of claim 32, wherein the at least one fungal exogenous cuticle degrading enzyme is selected from the pesticide is selected from the group consisting of species of group consisting of protease, peptidase, chitinase, chitosan Ascomycota, Alternaria, Beauveria, Lecanicillium, Metarhi ase, lipase and any combination thereof. Zium, Verticillium, Trichoderma, Aspergillus, Nomuraea, 52. The biopesticide of claim 44, wherein the biopesticide Paecilomyces, Isaria, Hirsutella, Fusarium, Cordyceps, comprises a composition comprising the pesticidally effec Entomophthora, Zoophthora, Pandora, Entomophaga, Ento tive amounts of the at least one fungal pesticide, at least one mophthorales and Zygomycota. exogenous cuticle degrading enzyme and a carrier for the at 38. The method of claim 32, wherein the at least one fungal least one fungal pesticide and the at least one exogenous pesticide comprises Alternaria Cassiae, Fusarium lateritum, cuticle degrading enzyme. Fusarium Solani, Verticillium lecanii, Aspergillus parasiti 53. The biopesticide of claim 44, wherein the biopesticide cus, Metarhizium anisopliae, Beauveria bassiana and any comprises a composition comprising the at least one fungal combination thereof, preferably Metarhizium anisopliae. pesticide and a carrier for the fungal pesticide and a compo US 2013/0156740 A1 Jun. 20, 2013 11 sition comprising the at least one exogenous cuticle degrad ing enzyme and a carrier for the cuticle degrading enzyme. 54. The biopesticide of claim 44, wherein the at least one cuticle degrading enzyme is in an amount effective to increase the penetration of the fungal pesticide through the cuticle of the target pest. 55. The biopesticide of claim 44, wherein the biopesticide is in the form of a wettable powder, dust, granule, bait, solu tion, emulsifiable concentrate, emulsion, Suspensions, con centrate, spray, microparticle, microcapsules, topical treat ment, gel, seed coating, systemic uptake, bait, eartag, bolus, or fogger.