Compositions and methods for controlling insect pests

Abstract

Disclosed herein are polynucleotides, compositions, and methods for controlling insect pests, especially flea beetles, such as Phyllotreta spp. and Psylliodes spp., particularly in plants. More specifically, polynucleotides such as double-stranded RNA triggers and methods of use thereof for modifying the expression of genes in flea beetles.

1 . A method of causing mortality or stunting in an insect, comprising contacting-an insect with at least one recombinant RNA 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 recombinant RNA by said insect results in mortality or stunting in said insect. 
     
     
         2 . The method of  claim 1 , wherein said at least one silencing element comprises an RNA strand having 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 1722-1724. 
     
     
         3 . The method of  claim 1 , wherein said insect is:
 (a) a flea beetle;   (b) 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) 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) 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); or   (e) an insect pest of a  Brassica  species.   
     
     
         4 . The method of  claim 1 , wherein:
 (a) 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;   (b) 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;   (c) 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;   (d) 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;   (e) 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; or   (f) said insect is  Psylliodes chrysocephala  and said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:552-859.   
     
     
         5 . The method of  claim 1 , wherein said silencing element comprises:
 (a) an RNA strand having 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;   (b) an RNA strand having 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 1722-1724;   (c) an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:860-1718 and 1722-1724.   
     
     
         6 . The method of  claim 1 , wherein:
 (a) 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;   (b) said insect is  Phyllotreta atra  (turnip flea beetle) and said silencing element comprises an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:860-1155;   (c) said insect is  Phyllotreta cruciferae  (canola flea beetle) said silencing element comprises an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:1156-1391;   (d) said insect is  Phyllotreta striolata  (striped flea beetle) and said silencing element comprises an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:1392-1410;   (e) 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   (f) said insect is  Psylliodes chrysocephala  and said silencing element comprises an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:1411-1718.   
     
     
         7 . The method of  claim 1 , wherein said recombinant RNA is provided in a microbial or plant cell that expresses said recombinant RNA, or in a microbial fermentation product, or is chemically synthesized. 
     
     
         8 . The method of  claim 1 , wherein said silencing element comprises dsRNA. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein said at least one recombinant RNA is provided in a composition comprising said at least one recombinant RNA, wherein said composition is applied to a surface of said insect or to a surface of a seed or a plant in need of protection from infestation by said insect. 
     
     
         11 .- 14 . (canceled) 
     
     
         15 . The method of  claim 10 , wherein said plant is:
 (a) selected from an ornamental plant or a crop plant;   (b) a plant in the family Brassicaceae;   (c) 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) a  Brassica  plant selected from the group consisting canola, rapeseed, turnips, and field mustard or turnip rape;   (e) 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) a potato plant and said insect is  Epitrix cucumeris  (potato flea beetle).   
     
     
         16 . The method of  claim 10 , 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 recombinant RNA to a surface of said insect or to a surface of said plant infested by said insect;   (c) providing said recombinant RNA 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 recombinant RNA 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 recombinant RNA in a composition that is ingested by said insect.   
     
     
         17 . An insecticidal composition comprising an insecticidally effective amount of a recombinant RNA molecule, wherein said recombinant RNA molecule comprises at least one segment of 18 or more contiguous nucleotides with 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. 
     
     
         18 . The insecticidal composition of  claim 17 , wherein said recombinant RNA molecule:
 (a) comprises at least one RNA strand having 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 1722-1724;   (b) comprises dsRNA;   (c) comprises dsRNA of at least 50 base pairs in length;   (d) comprises blunt-ended dsRNA;   (e) comprises dsRNA with an overhang at at least one terminus;   (f) comprises dsRNA comprising at least one stem-loop;   (g) comprises dsRNA that is chemically modified;   (h) is a dsRNA comprising an RNA strand having 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 1722-1724;   (i) is a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:860-1718 and 1722-1724; or   (j) is provided in a microbial or plant cell that expresses said recombinant RNA, or in a microbial fermentation product; or is chemically synthesized.   
     
     
         19 . The insecticidal composition of  claim 17 , 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;   (h) 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  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;   (m) 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;   (n) 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;   (o) 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;   (p) said plant is selected from an ornamental plant or a crop plant;   (q) said plant is a plant in the family Brassicaceae;   (r) 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;      (s) said plant is a  Brassica  plant selected from the group consisting canola, rapeseed, turnips, and field mustard or turnip rape;   (t) 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   (u) said plant is a potato plant and said insect is  Epitrix cucumeris  (potato flea beetle).   
     
     
         20 . The insecticidal composition of  claim 17 , 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 ; or   (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.   
     
     
         21 . A plant treated with the insecticidal composition of  claim 17 , or a plant grown from seed treated with the insecticidal composition of  claim 17 , wherein said plant exhibits improved resistance to said insect. 
     
     
         22 .- 25 . (canceled) 
     
     
         26 . A recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an RNA transcript 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 1722-1724. 
     
     
         27 . (canceled) 
     
     
         28 . A recombinant vector, plant chromosome or plastid, or transgenic plant cell comprising the recombinant DNA construct of  claim 26 . 
     
     
         29 . A method of providing a plant having improved resistance to an insect, comprising expressing in said plant the recombinant DNA construct of  claim 26 . 
     
     
         30 . A plant having improved resistance to an insect, provided by the method of  claim 29 , or fruit, seed, or propagatable parts of said plant.