Compositions and methods for controlling insect pests

Abstract

Disclosed herein are methods for controlling invertebrate pest infestations, particularly in plants; compositions and insecticidal polynucleotides useful in such methods; and plants having improved resistance to the invertebrate pests. More specifically, insecticidal polynucleotides and methods of use thereof for modifying the expression of genes in an insect pest, particularly through RNA interference are disclosed.

What is claimed is: 
     
         1 . A method of causing mortality or stunting in an insect, comprising providing in the diet of an insect at least one insecticidal polynucleotide comprising at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of said insect, wherein said target gene sequence is selected from the group consisting of SEQ ID NOs:1-859, and wherein ingestion of said insecticidal polynucleotide by said insect results in mortality or stunting in said insect. 
     
     
         2 . The method of  claim 1 , wherein said silencing element comprises:
 (a) at least one segment of 18 or more contiguous nucleotides comprising a sequence of 100% complementarity with a fragment of said target gene of said insect, wherein said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-859;   (b) at least one segment of 18 or more contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860-1718 and 1722-1730 and 1771-1975;   (c) a sequence selected from the group consisting of SEQ ID NOs:860-1718 and 1722-1730 and 1771-1975.   
     
     
         3 . The method of  claim 1 , wherein said insecticidal polynucleotide is provided in a microbial or plant cell that expresses said insecticidal polynucleotide, or in a microbial fermentation product, or is chemically synthesized. 
     
     
         4 . The method of  claim 1 , wherein said at least one insecticidal polynucleotide is provided in a composition comprising said at least one insecticidal polynucleotide, wherein said composition is applied to a surface of said insect or to a surface of a seed or plant in need of protection from infestation by said insect. 
     
     
         5 . The method of  claim 4 , wherein said composition:
 (a) comprises a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, or seed treatment;   (b) further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, a fertilizer, a micronutrient, an insect attractant, and an insect growth regulator;   (c) further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a  Bacillus thuringiensis  insecticidal protein, a  Xenorhabdus  insecticidal protein, a  Photorhabdus  insecticidal protein, a  Bacillus laterosporous  insecticidal protein, a  Bacillus sphaericus  insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species,  Lysinibacillus sphaericus  ( Bacillus sphaericus ),  Brevibacillus laterosporus  ( Bacillus laterosporus ),  Chromobacterium  species,  Chromobacterium subtsugae, Paenibacillus  species,  Paenibacillus lentimorbus , and  Paenibacillus popilliae ; or   (d) is ingested by said insect.   
     
     
         6 . A method for controlling an insect infestation of a plant comprising contacting, with a dsRNA, an insect that infests a plant, wherein said dsRNA comprises at least one segment of 18 or more contiguous nucleotides of a sequence of about 95% to about 100% complementarity with a fragment of a target gene of said insect, and wherein said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-859. 
     
     
         7 . The method of  claim 6 , wherein said contacting comprises:
 (a) oral delivery to said insect, non-oral delivery to said insect, or a combination of oral and non-oral delivery to said insect;   (b) application of a composition comprising said dsRNA to a surface of said insect or to a surface of said plant infested by said insect;   (c) providing said dsRNA in a composition that further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, a fertilizer, a micronutrient, an insect attractant, and an insect growth regulator;   (d) providing said dsRNA in a composition that further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a  Bacillus thuringiensis  insecticidal protein, a  Xenorhabdus  insecticidal protein, a  Photorhabdus  insecticidal protein, a  Bacillus laterosporous  insecticidal protein, a  Bacillus sphaericus  insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species,  Lysinibacillus sphaericus  ( Bacillus sphaericus ),  Brevibacillus laterosporus  ( Bacillus laterosporus ),  Chromobacterium  species,  Chromobacterium subtsugae, Paenibacillus  species,  Paenibacillus lentimorbus , and  Paenibacillus popilliae ; or   (e) providing said dsRNA in a composition that is ingested by said insect.   
     
     
         8 . An insecticidal composition comprising an insecticidally effective amount of a insecticidal polynucleotide, wherein said insecticidal polynucleotide comprises at least one segment of 18 or more contiguous nucleotides comprising a sequence of about 95% to about 100% complementarity with a fragment of a target gene of an insect that infests a plant, and wherein said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-859. 
     
     
         9 . The insecticidal composition of  claim 8 , wherein said insecticidal composition:
 (a) further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, a fertilizer, a micronutrient, an insect attractant, and an insect growth regulator;   (b) further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a  Bacillus thuringiensis  insecticidal protein, a  Xenorhabdus  insecticidal protein, a  Photorhabdus  insecticidal protein, a  Bacillus laterosporous  insecticidal protein, a  Bacillus sphaericus  insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species,  Lysinibacillus sphaericus  ( Bacillus sphaericus ),  Brevibacillus laterosporus  ( Bacillus laterosporus ),  Chromobacterium  species,  Chromobacterium subtsugae, Paenibacillus  species,  Paenibacillus lentimorbus , and  Paenibacillus popilliae;      (c) is in a form selected from the group consisting of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, insect diet or insect bait, and seed treatment; or   (d) is provided in a microbial or plant cell that expresses said recombinant RNA, or in a microbial fermentation product.   
     
     
         10 . A plant treated with the insecticidal composition of  claim 8 , or a plant grown from seed treated with the insecticidal composition of  claim 8 , wherein said plant exhibits improved resistance to said insect. 
     
     
         11 . A method of providing a plant having improved resistance to an insect, comprising expressing in said plant a recombinant DNA construct, wherein said recombinant DNA construct comprises DNA encoding an RNA having a sequence essentially identical or essentially complementary to at least 18 or more contiguous nucleotides of said target gene, wherein said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-859, and wherein ingestion of said RNA by said insect results in mortality or stunting in said insect. 
     
     
         12 . The method of  claim 1  or  11 , or the insecticidal composition of  claim 8 , wherein said RNA:
 (a) comprises a strand comprising at least 21 contiguous nucleotides comprising a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860-1718 and 1722-1975; or 
 (b) comprises dsRNA. 
 
     
     
         13 . The method or insecticidal composition of  claim 12 , wherein:
 (a) said dsRNA is blunt-ended, or has an overhang at at least one terminus, or comprises at least one stem-loop;   (b) said dsRNA is chemically synthesized, produced by expression in a microorganism, produced by expression in a plant cell, or produced by microbial fermentation;   (c) said dsRNA is chemically modified; or   (d) said dsRNA comprises at least one RNA strand comprising a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860-1718 and 1722-1730 and 1731-1975.   
     
     
         14 . The method of  claim 6  or  12 , wherein said dsRNA comprises:
 (a) a sequence selected from the group consisting of SEQ ID NOs:860-1718 and 1722-1730 and 1731-1975, or the complement thereof, 
 (b) at least one segment of 18 or more contiguous nucleotides with a sequence of 100% complementarity with a fragment of said target gene of said insect, wherein said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-859; or 
 (c) at least one segment of 18 or more contiguous nucleotides of an RNA sequence selected from the group consisting of SEQ ID NOs:860-1718 and 1722-1730 and 1731-1975. 
 
     
     
         15 . The method of any one of  claims 1 ,  6 , or  11 , or the insecticidal composition of  claim 8 , wherein:
 (a) said insect is a flea beetle;   (b) said insect is a species of a genus selected from the group consisting of the genera  Altica, Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus, Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea, Crepidodera, Derocrepis, Dibolia, Disonycha, Epitrix, Hermipyxis, Hermaeophaga, Hespera, Hippuriphila, Horaia, Hyphasis, Lipromima, Liprus, Longitarsus, Luperomorpha, Lythraria, Manobia, Mantura, Meishania, Minota, Mniophila, Neicrepidodera, Nonarthra, Novofoudrasia, Ochrosis, Oedionychis, Oglobinia, Omeisphaera, Ophrida, Orestia, Paragopus, Pentamesa, Philopona, Phygasia, Phyllotreta, Podagrica, Podagricomela, Podontia, Pseudodera, Psylliodes, Sangariola, Sinaltica, Sphaeroderma, Systena, Trachyaphthona, Xuthea , and  Zipangia;      (c) said insect is a species selected from the group consisting of  Altica ambiens  (alder flea beetle),  Altica canadensis  (prairie flea beetle),  Altica chalybaea  (grape flea beetle),  Altica prasina  (poplar flea beetle),  Altica rosae  (rose flea beetle),  Altica sylvia  (blueberry flea beetle),  Altica ulmi  (elm flea beetle),  Chaetocnema pulicaria  (corn flea beele),  Chaetocnema conofinis  (sweet potato flea beetle),  Epitrix cucumeris  (potato flea beetle),  Systena blanda  (palestripped fleabeetle), and  Systena frontalis  (redheaded flea beetle);   (d) said insect is a species selected from the group consisting of  Phyllotreta armoraciae  (horseradish flea beetle),  Phyllotreta cruciferae  (canola flea beetle),  Phyllotreta pusilla  (western black flea beetle),  Phyllotreta nemorum  (striped turnip flea beetle),  Phyllotreta atra  (turnip flea beetle),  Phyllotreta robusta  (garden flea beetle),  Phyllotreta striolata  (striped flea beetle),  Phyllotreta undulata, Psylliodes chrysocephala , and  Psylliodes punctulata  (hop flea beetle);   (e) said insect is an insect pest of a  Brassica  species;   (f) said insect is a  Phyllotreta  species and said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-551;   (g) said insect is  Phyllotreta atra  (turnip flea beetle) and said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-296;   (b) said insect is  Phyllotreta cruciferae  (canola flea beetle) and said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:297-532;   (i) said insect is  Phyllotreta striolata  (striped flea beetle) and said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:533-551;   (j) said insect is a  Psylliodes  species and said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:552-859;   (k) said insect is  Psylliodes chrysocephala  and said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:552-859;   (l) said insect is a  Phyllotreta  species and said silencing element comprises an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:860-1410;   (m) said insect is  Phyllotreta atra  (turnip flea beetle), said recombinant RNA molecule comprises dsRNA, and said dsRNA comprises at least one RNA sequence selected from the group consisting of SEQ ID NOs:860-1155;   (n) said insect is  Phyllotreta cruciferae  (canola flea beetle), said recombinant RNA molecule comprises dsRNA, and said dsRNA comprises at least one RNA sequence selected from the group consisting of SEQ ID NOs:1156-1391, 1731-1972, and 1974;   (o) said insect is  Phyllotreta striolata  (striped flea beetle), said recombinant RNA molecule comprises dsRNA, and said dsRNA comprises at least one RNA sequence selected from the group consisting of SEQ ID NOs:1392-1410, 1973, and 1975;   (p) said insect is a  Psylliodes  species and said silencing element comprises an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:1411-1718; or   (q) said insect is  Psylliodes chrysocephala , said recombinant RNA molecule comprises dsRNA, and said dsRNA comprises at least one RNA sequence selected from the group consisting of SEQ ID NOs:1411-1718.   
     
     
         16 . The method of  claim 6  or  11 , or the insecticidal composition of  claim 8 , wherein:
 (a) said plant is selected from an ornamental plant or a crop plant; 
 (b) said plant is a plant in the family Brassicaceae; 
 (c) said plant is a  Brassica  species selected from the group consisting of  B. napus, B. juncea, B. carinata, B. rapa, B. oleracea, B. rupestris, B. septiceps, B. nigra, B. narinosa, B. perviridus, B. tournefortii , and  B. fructiculosa;    
 (d) said plant is a  Brassica  plant selected from the group consisting canola, rapeseed, turnips, and field mustard or turnip rape; 
 (e) said plant is selected from the group consisting of  Glycine max, Linum usitatissimum, Zea mays, Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum, Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocos nucifera, Coriandrum sativum, Gossypium  spp.,  Arachis hypogaea, Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgare , and  Triticum aestivum ; or 
 (f) said plant is a potato plant and said insect is  Epitrix cucumeris  (potato flea beetle). 
 
     
     
         17 . The method of  claim 11 , wherein:
 (a) said method further comprises expression in said plant of at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a  Bacillus thuringiensis  insecticidal protein, a  Xenorhabdus  insecticidal protein, a  Photorhabdus  insecticidal protein, a  Bacillus laterosporous  insecticidal protein, a  Bacillus sphaericus  insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species,  Lysinibacillus sphaericus  ( Bacillus sphaericus ),  Brevibacillus laterosporus  ( Bacillus laterosporus ),  Chromobacterium  species,  Chromobacterium subtsugae, Paenibacillus  species,  Paenibacillus lentimorbus , and  Paenibacillus popilliae ; or   (b) said method further comprises expression in said plant of at least one protein conferring tolerance to an herbicide.   
     
     
         18 . The plant having improved resistance to an insect, provided by the method of  claim 11 , or fruit, seed, or propagatable parts of said plant. 
     
     
         19 . A recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an RNA transcript comprising at least 21 contiguous nucleotides of a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860-1718 and 1722-1730. 
     
     
         20 . The recombinant DNA construct of  claim 19 , wherein said heterologous promoter is functional for expression of said RNA transcript in a bacterium or in a plant cell. 
     
     
         21 . A recombinant vector, plant chromosome or plastid, or transgenic plant cell comprising the recombinant DNA construct of  claim 19 .