US 20140154223A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0154223 A1 KANG et al. (43) Pub. Date: Jun. 5, 2014 (54) METHODS OF PEST CONTROL Publication Classification (71) Applicants: Beijing Dabeinong Technology Group (51) Int. Cl. Co., Ltd., Beijing (CN); Beijing Green AOIN 63/02 (2006.01) Agrosino Plant Protection Technology CI2N 5/82 (2006.01) Co., Ltd., Beijing (CN); Beijing (52) U.S. Cl. Dabeinong Technology Group Co., CPC ............ A0IN 63/02 (2013.01); C12N 15/8286 Ltd. Biotech Center, Beijing (CN) (2013.01) USPC .......................... 424/93.21: 514/4.5: 800/302 (72) Inventors: Yuejing KANG, Beijing (CN); Jie PANG, Beijing (CN); Aihong ZHANG, Beijing (CN); Peng CHENG, Beijing (57) ABSTRACT (CN); Xu YANG, Beijing (CN); Lihong NIU, Beijing (CN); Zhiwei JIA, Beijing Certain embodiments of the present invention provide a (CN); Luoxu AN. Beijing (CN); Kangle method for controlling Athetis lepigone, which comprises TIAN, Beijing (CN); Ziqin JIANG, contacting Athetis lepigone with Cry1A protein. Aspects of Beijing (CN) the present invention can achieve control of Athetis lepigone by enabling plants to produce Cry1A protein in vivo, which (21) Appl. No.: 14/093,684 G be EE E. ch R other instances, the (22) Filed: Dec. 2, 2013 method can control Athetis lepigone throughout the growth period of the plants and provide the plants with a full protec (30) Foreign Application Priority Data tion. Additionally, the method, in certain embodiments, can be one or more of stable, complete, simple, convenient, eco Dec. 3, 2012 (CN) ......................... 2012.105098,172 nomical, pollution-free or residue-free. p(Et- \si. s 8 -: \ - \ * - ^ * Y -k x Crick 883. Patent Application Publication Jun. 5, 2014 Sheet 1 of 3 US 2014/O154223 A1 FIG. 1 p:Ext-I , tici rat 888. Patent Application Publication Jun. 5, 2014 Sheet 2 of 3 US 2014/O154223 A1 FG, 2. 888. ES3883: Patent Application Publication Jun. 5, 2014 Sheet 3 of 3 US 2014/O154223 A1 F.G. 3 Cry1Ab-01 Cry1Ab-02 FIG. 4 CK Cry1Ab-01 Cry1Ab-02 CK US 2014/O 154223 A1 Jun. 5, 2014 METHODS OF PEST CONTROL the safety of local human and livestock; and as Athetis lepi gone prefers a moist and dark micro-habitat, it generally hides CROSS-REFERENCE TO RELATED under coverings such as wheat Straws or below the topsoil. APPLICATION making the direct contact between chemicals and Athetis lepigone difficult, which can render the chemical control 0001. This application claims priority under 35 U.S.C. ineffective. S119(a)-(d) of Chinese Patent Application No. 0007 To overcome one or more of the limitations of the 2012 105098.17.2 filed Dec. 3, 2012, entitled “Method of Pest agricultural control method and/or of the chemical control Control” which is herein incorporated by reference in its method, researchers have found that, in Some instances, entirety. inserting genes coding for pesticidal proteins into plant genome can produce pest-resistant plants. Pesticidal protein BACKGROUND Cry1A, among a large group of pesticidal proteins, is a 0002 Some embodiments of the present invention relate parasporal crystalliferous protein produced by a subspecies to methods for pest control. Such as methods for preventing of Bacillus thuringiensis (Bacillus thuringiensis Subsp. Athetis lepigone from damaging plants by expressing Cry1A kurstaki, B.t.k). protein therein. 0008 Cry1A protein, ifingested by pests, can be dissolved 0003 Athetis lepigone belongs to the order of Lepidoptera in the alkaline environment of the pests midgut and releases and the family of Noctuidae. As an omnivorous pest, it some protoxin, a precursor to a toxin. Further, alkaline protease times feeds on maize. It can inhabit, in the Summer, maize digests the protoxin at its N- and C-terminus and can produce agricultural district of Huang-Huai-Hai region in China, and an active fragment, which can Subsequently bind to a mem has also been found in other areas such as in Japan, Korea, brane receptor of epithelial cells of the pests midgut and can Russia and Europe. It can damage aerial roots of maize in insert itself into the intestinal membrane, resulting in delete topsoil, can eat out maize brace roots and stems, can distort or rious effects to the pest, such as one or more of cell membrane even kill maize plants. The damaged maize field can show perforation, disequilibrating the pH homeostasis and/or large empty areas or even become sterile if under severe osmotic pressure across the cell membrane; this can disturb attacks. the digestion of the pest, and sometimes eventually lead to the 0004 Maize is a major food crop in China. On Jul.9 2011, death of the pest. the CCTV's “News Broadcast” reported outbreaks of Athetis 0009. There are no reports on controlling Athetis lepigone lepigone in China. From the autumn of 2011 until May 31 by generating transgenic plants producing a Cry1A protein. 2012, several field surveys conducted by the Pest Prevention and Control Laboratory of National Maize Industry found SUMMARY that 2012’s Athetis lepigone included a large number of large 0010 Some embodiments of the present invention include size wintering populations with a high density of larvae, providing a pest control method by using transgenic plants indicating that the outbreak of Athetis lepigone is likely to expressing Cry1A protein to, for example, control damage flare up again in Huang-Huai-Hai region. Two methods that caused by Athetis lepigone. In certain embodiments, the can be used to control Athetis lepigone are the agricultural method can overcome one or more limitations of the agricul control method and the chemical control method. tural control method and the chemical control method. 0005. The agricultural control method is an integrated and 0011. In other embodiments, the method controls (e.g., coordinated management of multiple factors for the entire limits growth or kills) Athetis lepigone, by, for example, ecosystem of farmland, which regulates crops, pests and envi contacting (e.g., eating) Athetis lepigone with the Cry1A ronmental factors and establishes a farmland ecosystem con protein. In certain instances, the Cry1A protein is Cry1Ab ducive to crop growth but unfavorable to Athetis lepigone. For protein. example, the prompt removals of Straw, weeds and other 0012. In certain aspects, the transgenic plant expresses coverings from the roots of maize seedlings to a bigger space Cry1A protein in one or more plant parts, including but not between maize lines far away from the plants so as to expose limited to reproductive material. Such as seeds, seedlings, and the ground, is commonly used, in order to make Sure the next the like. step of pesticide spray can directly contact Athetis lepigone. 0013 The Cry1Ab protein can be present in a plant cell However, since the agricultural control must obey the require expressing the protein, and it can be, in some instances, con ments for crop layout and increasing production, Such this tacted with Athetis lepigone by ingestion of the plant cell. method has limited applications and cannot be used as an 0014 Further, in certain embodiments, the Cry1Ab pro emergency measure when Athetis lepigone outbreaks. tein is present in a transgenic plant expressing the Cry1Ab 0006. The chemical control method, also known as the protein, and Athetis lepigone contacts with the Cry1Ab pro pesticide control method, kills pests by using pesticides. As a tein by ingestion of a tissue of the transgenic plant. means for the comprehensive management of controlling, it 0015. In some embodiments, Athetis lepigone is detrimen can be a fast, convenient, simple and highly cost-effective tally effected, such as, but not limited to the inhibition of method. Particularly, it can be used as an emergency practice growth of Athetis lepigone or death of Athetis lepigone, dam to reduce the density of Athetis lepigone before damage has age to the plant resulting from Athetis lepigone can, in some occurred. Currently, the Some measures of the chemical con instances, be controlled. trol include poisoned bait, poisoned soil, as well as pesticide 0016. In certain embodiments, the transgenic plant can be drenching and spraying. However, the chemical control has in any growth period. In other aspects, the tissue of the trans its limitations: its improper use can cause devastating conse genic plant can be roots, leaves, stems, tassels, ears, anthers or quences, such as poisoning crops, pest resistance, killing filaments. predators and polluting the environment so as to destroy 0017. The control of the damage of Athetis lepigone to the farmland ecosystems; pesticide residues can pose a threat to plant may or may not depend on planting location. US 2014/O 154223 A1 Jun. 5, 2014 0018. The control of the damage of Athetis lepigone to the larvae per individual seedling. When maize seedlings are at plant may or may not depend on planting time. 3-leaf to 5-leaf stage, larvae feed mainly on its stalk base and 0019. The plant can be any suitable plant, including but leave behind round or oval holes of 3-4 mm in size, resulting not limited to maize. in the disruption of nutrition transport to leaves and eventu 0020. In some instances, prior to the step of contacting ally the wilting and death of interior leaves above ground. Athetis lepigone, a transgenic seedling containing a poly When targeting maize seedlings of 8-leaf to 10-leaf stage, nucleotide encoding the Cry1Ab protein is planted. larvae mainly feed on roots, including aerial roots and main 0021. In some embodiments, the amino acid sequence of roots, resulting in lodging or even death of the plants. The the Cry1Ab protein comprises an amino acid sequence of damaged plants count for 1% to 5% generally and reach up to SEQ ID NO:1 or SEQID NO:2.