WO2012108600A1 - Nouveau photorhabdus temperata j6 et procédé pour la lutte contre les organismes nuisibles utilisant celui-ci - Google Patents

Nouveau photorhabdus temperata j6 et procédé pour la lutte contre les organismes nuisibles utilisant celui-ci Download PDF

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WO2012108600A1
WO2012108600A1 PCT/KR2011/006980 KR2011006980W WO2012108600A1 WO 2012108600 A1 WO2012108600 A1 WO 2012108600A1 KR 2011006980 W KR2011006980 W KR 2011006980W WO 2012108600 A1 WO2012108600 A1 WO 2012108600A1
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moth
strain
temperata
pests
composition
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PCT/KR2011/006980
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Korean (ko)
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신재호
장은경
홍성준
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경북대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to a novel Photorhabdus temperata J6 strain having excellent pesticidal activity and antibacterial activity, a composition for controlling pests comprising the same, and a method for biologically controlling pests using the strain. .
  • BHC which is made of chlorine or benzene
  • BHC has attracted worldwide attention by leading development and synthesis in the United States because of its abundant raw materials, low cost, and strong insecticide, and less harmful to human beings.
  • various countries such as Western countries and Australia are strictly restricting the use of the pesticides.
  • organic chemical synthetic insecticides have been widely used to control pests, but due to decades of abuse and abuse, the emergence of abnormal or resistant pests of pest groups, toxic expression of non-target insects including humans, and environmental systems It causes many side effects such as pollution.
  • an international agreement was reached to refrain from using highly toxic organic synthetic pesticides in order to protect human health.
  • the world reduced production by 50% of the chemical synthetic organophosphorus and chlorine pesticides used in the past 10 years in 2004, and again reduced production of organophosphorus and organochlorine insecticides by 50% by 2010. It is in the phase of implementation, agreeing to international agreements to be made.
  • microorganisms have been able to reduce or replace the use of chemical fertilizers or pesticides, microorganisms capable of biological control techniques that are less harmful to livestock, do not harm crops, and have less damage to the environment, such as soil ecosystems.
  • chemical fertilizers or pesticides microorganisms capable of biological control techniques that are less harmful to livestock, do not harm crops, and have less damage to the environment, such as soil ecosystems.
  • biological pesticides that can complement the chemical pesticides.
  • Lepidoptera includes the nabiryu and nabangryu, species belonging to the neck are through the egg, larva, pupa, adult stage 4 to complete transformation, of Lepidoptera larvae herbivorous then large economic damage with respect to a wide range of crops It is a representative pest that causes.
  • the larvae of the genus Lepidoptera are insect pests affecting the honeycomb, and the larvae hatched from eggs laid by the moth adult in the beehive grow on the beehive and honey, so that the beehives are tattered. The state, and eventually collapses, is causing enormous economic losses to honey farmers.
  • tobacco castor moths belonging to the Lepidoptera are photogenic and omnivorous pests with more than 100 species of host, and these larvae are inherently resistant to drugs and have low susceptibility. It is known to be a very difficult representative heating pest (Choi et al., Kor. J. Appl. Entomol., 1996, 35: 249-253; Bae et al., Kor. J. Appl. Entomol., 2003, 42: 225-231). Night moth leaf moths penetrate into chestnut larvae to cool the flesh of the chestnuts. Perch moths are the main pests of rice, and the rice leaves dry vertically and eat the leaves in them, so that the grains of grain do not grow well. It is damaging economic crops. Therefore, there is a need for the development of low-toxic, environmentally friendly microbial preparations with excellent insecticidal properties that can be biologically controlled against lepidopteran pests that cause economic losses to farmers as described above.
  • the present inventors have diligently searched for new microorganisms with the aim of developing new biological pesticides having a wide range and excellent insecticidal properties against pests that cause widespread damage to crops.
  • photolabdus tempera isolated from insect pathogenic nematodes It was confirmed that other J6 strains showed excellent insecticidal activity against lepidopteran pests, and also confirmed that the strains showed excellent antibacterial activity against harmful bacteria, and completed the present invention.
  • Another object of the present invention is to provide a pest control composition or an antimicrobial composition comprising the strain or its culture as an active ingredient.
  • Still another object of the present invention is to provide a method for controlling a lepidopteran pest, comprising spraying the pest control composition on a host plant of a lepidopteran pest.
  • the present invention provides photolabdus temperata J6 (Accession No .: KCTC 12004BP) having both insecticidal activity and antimicrobial activity, isolated from entomopathogenic nematodes.
  • the strain of the present invention was isolated and identified from the insect pathogenic nematode, and compared with the known strain 16s rDNA sequence of the strain showed a 99% homology with Photorhabdus temperata (Fig. 1) Reference). However, it was found that it is a novel strain belonging to the species because it was not completely identical. According to the analysis result, the strain of the present invention was named photolabdus temperata J6, and deposited on January 4, 2011, with the accession number KACC 91625P to the National Institute of Agricultural Science, National Agricultural Science Institute, and issued on August 23, 2011 The depository was deposited with accession number KCTC 12004BP.
  • the growth temperature is 28 °C to 30 °C growth is active and culture temperature As the growth was inhibited growth was hardly seen above 36 °C (see Figure 8).
  • Strains of the invention can exhibit excellent pesticidal activity against pests.
  • the pest that is controlled in the present invention may be a Lepidopter pest.
  • the pest is a beetle moth ( Galleria mellonella ), Chinese cabbage moth ( Plutella xylostella ), tobacco moth ( Spodoptera litura ), moth ( Alcis angulifera ), moth pattern leaf moth ( Adoxophyes orana ), persimmon tree Leafy moth ( Ptycholoma lecheana ), Peach nettle moth ( Grapholita molesta ), silver-patterned moth ( Lyonetia prunifoliella ), peach moth ( Carposina sasakii ), moth ( Spodoptera exigua ), moth monk ( Diaphania indica ) Cnaphalocrocis medinali s), Chilo suppressalis , and Helicoverpa armigera , but not limited to
  • insect activity refers to the ability of endotoxin proteins secreted from the microorganisms or microorganisms of the present invention to increase the mortality or inhibit the growth rate of pests on plants.
  • the supernatant of the culture supernatant of the photolapdus temperata J6 strain of the present invention according to the culture time for the insect larvae of at least 5 years of age, the culture supernatant after 3-5 days
  • the culture supernatant after 3-5 days As a result of the treatment showed a high insecticidal activity of 80-100%, it was confirmed that it has an excellent pesticidal activity even in the aging larvae (see Figure 9).
  • the photolabduus temperata J6 strain of the present invention may exhibit excellent antimicrobial activity against harmful bacteria to mammals.
  • the bacteria to may be a Salmonella typhimurium (Salmonella typhimurium), Salmonella cholera desu device (Salmonella choleraesuis), E. coli (Escherichia coli), Streptococcus Agar Rock tiae (Streptococcus agalactiae), or micro Caucus Lou Proteus (Micrococcus luteus) .
  • the harmful bacteria may be Salmonella typhimurium and Micrococcus luteus .
  • Salmonella typhimurium is a pathogenic gram negative bacterium found in the intestinal lumen, which is toxic by LPS in the outer membrane and is known to cause gastroenteritis or typhoid-like symptoms in humans and mammals.
  • the micrococcus luteus is also known as a typical microorganism involved in food spoilage.
  • the photolapus temperata J6 strain shows excellent antimicrobial activity against Salmonella typhimurium and micrococcus luteus. It could be confirmed (see FIG. 7).
  • the photolabdus temperata J6 strain of the present invention has not only insecticidal activity but also antimicrobial activity. Therefore, the strain of the present invention can be used as an active ingredient of a microbial agent or antimicrobial agent for pest control. If the control using the strain stops the planting of insects by pests can produce a cleaner and higher quality agricultural products, it can bring labor savings and economic benefits for the control.
  • the present invention provides a pest control composition
  • a pest control composition comprising a novel photoractus temperata J6 (Accession No .: KCTC12004BP) strain or culture medium thereof having excellent insecticidal activity against a wide range of lepidopteran pests as an active ingredient. to provide.
  • Plants to which the composition for controlling pests of the present invention can be applied are not particularly limited, and include cabbage, cabbage, eggplant, persimmon, peach, cucumber, cabbage lettuce (lamb lettuce), strawberries, tomatoes, green onions, tobacco, rice, chestnuts, and the like.
  • they may be treated on the surface of plants that may be hosts of lepidopteran pests, such as flowers or special crops, or on the soil in which they are grown, or on the surface of fruits or vegetables in cultivation or transportation or storage.
  • compositions for controlling pests of the present invention are surfactants, inorganic salts, adjuvants, binders and extenders in photolabdus temperata J6 strain, or 5 to 90% by weight of the culture or dry powder thereof. And the like.
  • the surfactant is an amphiphilic material having both hydrophilic and lipophilic molecular groups in the molecule, and has excellent characteristics in cleaning, dispersing, emulsifying, solubilizing, wetting, sterilizing, foaming and penetrating properties according to the present invention.
  • the photolabduus temperata J6 strain in the pest control composition may act to hydrate, suspend, and disperse to effectively express the drug.
  • the surfactant may be a sodium salt of a sulfonate such as alkylbenzenesulfonate, alkylnaphthalenesulfonate, dialkylsulfosuccinate, ligninsulfonate, alkylnaphthalenesulfonate formalin condensate, polyoxyalkylene alkylphenylsulfonate or Sodium salts of sulfates such as calcium salts, alkyl sulfates, polyoxyalkylene alkyl sulfates, polyoxyalkylene alkylphenyl sulfates or sodium salts of succinates such as calcium salts, naphthalenesulfosuccinates, polyoxyalkylene succinates or Anionic surfactants such as calcium salts, ethoxylated alkyl ethers, polyoxyalkylene alkylphenyl polymers, and nonionic surfactants such as multiple alcohols may be used alone or in combination
  • a substance acting to induce physiological changes in the body of the pest to increase the effect of the toxin those skilled in the art will be able to easily purchase and use.
  • the binder serves to bind the drug enhancer, the extender, and the like, including the dry powder of the active ingredient Photorapdus temperata J6 strain, water-soluble starch, dextrin, carboxymethylcellulose, sodium polyacrylate, polyvinyl alcohol , Gum arabic or xanthan gum may be used alone or in combination of two or more thereof.
  • the extender may be used together with the surfactant to adsorb and powder the surfactant, and together with the surfactant, the drug enhancer, Photorabdus temperata J6 strain, culture medium thereof, or dry powder to form a particulate surface of the composition.
  • Starch, soybean meal, bran, granular fiber, yuan, diatomaceous earth, zeolite, bentonite, talc, kaolin, pyrophyllite, white carbon and the like may be used alone or in combination of two or more thereof.
  • the composition can be formulated into environmentally friendly microbial pesticides.
  • microbial pesticide refers to the production of a microorganism, a compound secreted by a microorganism, or the like selected or modified in nature or controlled for use in order to control pests of plants, pathogenic microorganisms, and the like.
  • Photolabdus temperata J6, or its culture solution may be produced or commercialized by including the endocrine toxin protein produced by the strain of the present invention as an active ingredient.
  • the pest control composition may be formulated in the form of granules, powders, liquid hydrating agents, hydrating agents, etc., but may be used as insecticides, but is not limited thereto.
  • Formulated pesticides may be used diluted 500 to 2,000 times, preferably about 1,000 times, in water before use.
  • their application amount is usually 0.1 to 100 g per 10 acres.
  • the pest control composition of the present invention may be used together with other insecticides, nematicides, acaricides, fungicides, fungicides, herbicides, plant growth regulators, synergists, fertilizers, soil conditioners and / or animal feeds. They can be used simultaneously, or separately from them.
  • the present invention is a pest control composition
  • the host plant is not particularly limited, and in addition to economic crops such as cabbage, cabbage, eggplant, persimmon, peach, cucumber, cabbage lettuce (lamb lettuce), strawberries, tomatoes, green onions, tobacco, rice, chestnuts, etc.
  • the composition may be treated on these surfaces or on the soil in which these plants are growing, or on the surface of fruits or vegetables in cultivation and transportation or storage.
  • the pest control method of the present invention includes applying the pesticide of the present invention in consideration of various factors, such as the formulation of the pesticide, the type of pest to be pesticide, the object, the place and method of pesticide.
  • the present invention provides an antimicrobial composition
  • a photolabduus temperata J6 strain (Accession Number: KCTC12004BP) or a culture thereof as an active ingredient.
  • the antimicrobial composition of the present invention exhibits an antimicrobial activity against a bacterium that is harmful to a mammal, and may preferably exhibit excellent antimicrobial activity against Salmonella typhimurium or Micrococcus luteus .
  • the novel photolabdus temperata J6 strain of the present invention has excellent insecticidal and pesticidal antimicrobial activity against pests, so that the microorganism preparation using the strain or its culture solution as an active ingredient, It is effective to effectively control and prevent damage caused by pests in various crops.
  • Figure 1 shows the phylogenetic schematic diagram of the 16s rDNA nucleotide sequence (1493bp) of the Photorabdus temperata J6 strain of the present invention compared to the known strain.
  • Figure 2 is a 16s rDNA sequencing results of the photolabduus temperata J6 strain of the present invention.
  • Figure 3 shows the colony (colony) form in MacConkey agar of the Photorabdus temperata J6 strain of the present invention.
  • Figure 4 shows the catalase reaction results of the photolabduus temperata J6 strain of the present invention according to the addition of H 2 O 2 .
  • Figure 5 shows the results of confirming the protease enzyme activity (Fig. 5a) and lipase enzyme activity (Fig. 5b) of the Photorabdus temperata J6 strain of the present invention.
  • E. coli DH5 ⁇ was used as a negative control
  • B. subtilis 2232 was used as a positive control
  • the production of the transparent ring in FIG. 5A was a result of decomposition of skim milk.
  • Figure 6 shows the fatty acid content (fatty acids) of the Photorabdus temperata J6 strain of the present invention.
  • Figure 7 shows the growth inhibitory activity against Salmonella typhimurium KCTC1926 (Fig. 7a) and Micrococcus luteus KACC 10488 (Fig. 7b) as a result of the antibiosis plate assay of the Photorabdus temperata J6 strain of the present invention.
  • Bacillus tequilensis 23 was used as a positive growth inhibitory bacterium for KCTC1926 and KACC10488, and Escherichia coli DH5 ⁇ was used as a negative growth inhibitory bacterium.
  • Figure 8 shows the growth of the photolabdus temperata J6 strain according to the culture temperature, was measured after culturing for two days in 100ml culture Tryptic soy broth.
  • Figure 9 shows the insecticidal ability against the bee larva moth larva of the culture supernatant of the Photorabdus temperata J6 strain according to the culture time.
  • soil samples were collected from all over the country to isolate microorganisms having insecticidal or antimicrobial activity from insect pathogenic nematodes.
  • the nematodes isolated from soil samples were baiting to honey bee moth larvae.
  • was separated. Separation of symbiotic bacteria from isolated insect pathogenic nematodes ( Heterorhabditidae sp.) was performed using MacConkey agar and NBTA (nutrient agar bromothymol blue triphenyltetrazolium chloride) medium.
  • Genomic DNA was isolated and identified by 16s rDNA PCR and sequencing for identification of isolated symbiotic bacteria.
  • 16S rDNA sequence of the isolated strain cells were recovered from the TSB culture medium of the strain, and genomic DNA was extracted from the recovered cells (Thompson, 1980). 16S rDNA was amplified by PCR (polymerase chain reaction) using the isolated genomic DNA as a template, and the universal primers used were 27F (5'-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5'-GGTTACCTTGTTACGACTT-3' ) Were used separately (Lane 1991). The amplified PCR product was purified using a Gel Purification system (Solgent, Daejon, Korea).
  • Gram-negative bacillus-Strepaking on MacConkey agar and forming pink or red colonies was determined as Gram-negative bacillus, and confirmed by optical microscopy after gram staining.
  • Bacillus subtilis 2232 strain was used as a gram positive strain as a control.
  • Antibiosis assays are Salmonella typhimurium KCTC 1926 (bacterial diarrhea), Salmonella choleraesuis KCTC 2929 (epidemic diarrhea), Escherichia coli KCCM 11234 (daejanggyunjeung poultry), Streptococcus agalactiae ATCC 13813 (bovine mastitis) and was carried out on Micrococcus luteus KACC 10488. First, each of the five harmful bacteria was incubated in 5 ml of TSB, and then cross-streaked several times on TSB agar plate. Incubated for 1-2 days at °C. E.
  • coli DH5 ⁇ was used as a negative control, and positive control strains were Pseudomonas putida 93-16 (antagonists against KCCM 11234) and Pseudomonas putida 55-30 (ATCC13813). Antagonists ), Bacillus tequilensis 23 (antagonists against KCTC1926 and KCTC2929) strains were used separately. Inhibition of harmful microorganisms was confirmed by the size of the resulting transparent ring.
  • the culture temperature was 28 °C, 30 °C, 34 °C, 35 °C, 36 °C, respectively, the medium was used for TSB and cultured for 5 days It was.
  • the 16S rRNA gene sequence of the strain was registered in GenBank of NCBI (GenBank accession number: HQ 685757), and the strain was named Photorabdus temperata J6, and the National Agricultural Science Institute Agricultural Genetic Resource Center January 4, 2011 It was deposited with the accession number KACC 91625P and deposited with the accession number KCTC 12004BP as of August 23, 2011 to the International Depositary.
  • Fatty acid content of the photolapux temperata J6 strain was analyzed and shown in Table 1 and FIG. 6.
  • the fatty acid content was analyzed using the Sherlock MIDI system according to the manufacturer's manual.
  • coli DH5a without the antimicrobial activity as shown in Figure 7 is that the transparent ring for S. typhimurium and M. luteus is not produced, there is no growth inhibition, and also growth inhibitory material for these two bacteria
  • J6 strain and positive control to produce an antimicrobial substance it can be seen that a transparent ring is formed due to the inhibition of growth, and the antimicrobial activity of the strain is also known depending on the size of the transparent ring.
  • Photolab doth temperata J6 strain was active at the growth temperature of 28 °C and 30 °C but appeared to inhibit the growth as the culture temperature increases, the growth was hardly seen above 36 °C (see Figure 8).
  • Insecticidal activity against the beetle larvae ( Galleria mellonella ) larvae of the culture supernatant according to the incubation time of the photolapux temperata J6 strain is shown in FIG. As shown in FIG. 9, high insecticidal activity was observed in the culture supernatant of late logarithmic growth (culture 3 to 5 days), and insecticidal activity was 80-100%.
  • novel photolabduus temperata J6 strain of the present invention has excellent insecticidal and pesticidal properties against harmful bacteria, it can effectively control and prevent damage caused by pests in various crops.

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Abstract

La présente invention concerne des souches de Photorhabdus temperata J6 qui ont d'excellentes activité insecticide et activité antimicrobienne simultanée. Les souches de la présente invention présentent d'excellentes activité insecticide contre des insectes nuisibles et activité antimicrobienne contre des pathogènes, qui conduisent à la préparation de formulations microbiennes avec les souches ou milieux de culture de celles-ci en tant que substances actives.
PCT/KR2011/006980 2011-02-10 2011-09-21 Nouveau photorhabdus temperata j6 et procédé pour la lutte contre les organismes nuisibles utilisant celui-ci WO2012108600A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100522667B1 (ko) * 2003-06-04 2005-10-20 김용균 포토랍두스 템페라타 템페라타 ａｎｕ101

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100522667B1 (ko) * 2003-06-04 2005-10-20 김용균 포토랍두스 템페라타 템페라타 ａｎｕ101

Non-Patent Citations (4)

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Title
JANG EUN-KYUNG ET AL.: "Insecticidal Toxin and Research Trends of Photorhabdus, Entomopathogenic Bacteria.", KOR. J. MICROBIOL. BIOTECHNOL., vol. 38, no. 2, 2010, pages 117 - 123 *
JANG EUN-KYUNG: "Molecular biological characterization of the toxin complexes and their insecticidal activity of the symbiotic bacteria Photorhabdus temperata M1021 isolated from an entomopathogenic nematode", THESIS OF D.AGR., June 2011 (2011-06-01), KYUNGPOOK NATIONAL UNIVERSITY, pages 25,52 - 70 *
SAMYEOL SEO ET AL.: "Comparative Analysis of Immunosuppressive Metabolites Synthesized by an Entomopathogenic Bacterium, Photorhabdus temperata ssp. temperata, to Select Economic Bacterial Culture Media", HAN EUNG GON JI, vol. 49, no. 4, 2010, pages 409 - 416 *
SHRESTHA, YAM KUMAR ET AL.: "Oral toxicity of symbiotic bacteria Photorhabdus spp. against immature stages of insects", JOURNAL OF ASIA-PACIFIC ENTOMOLOGY, vol. 14, 29 October 2010 (2010-10-29), pages 127 - 130 *

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