KR101487699B1 - Insecticidal composition comprising Pseudomonas sp. EML-PB1 Strain and the method for controlling harmful insects using the same - Google Patents

Abstract

The present invention relates to: a Pseudomonas sp. EML-PB1 (KCTC 12486BP) strain having an insecticidal activity against harmful insects; an insecticidal composition including the same; and a method for biologically repelling harmful insects using the same. According to the present invention, EML-PB1, which is a novel Pseudomonas sp. bacterial species, has a very remarkable effect in repelling harmful insects so that marketability and productivity can be remarkably improved when EML-PB1 is used for plants. Moreover, the composition of the present invention is a biological insecticide using a microorganism strain. The composition is very stable for animals and plants so that harmful insects can be controlled in an environmentally-friendly way.

Description

Composition for controlling insects including Pseudomonas sp. EML-PB1 and insecticidal composition comprising Pseudomonas sp. EML-PB1 Strain and the method for controlling harmful insects using the same}

The present invention relates to a composition for controlling insects including Pseudomonas sp. EML-PB1 and a method of controlling the same.

Organic chemical synthetic insecticides which are conventionally used are excellent in terms of insecticidal effect but their use is restricted in each country due to problems of harmfulness and environment pollution. In particular, since the Rio Environmental Conference, which aims to replace 20% of the world's pesticides with bio-pesticides, there is a trend to reduce the use of chemical pesticides worldwide and international agreements to regulate the use of chemical pesticides are being strengthened. Therefore, interest in bio - pesticides that can replace chemically synthesized pesticides has been increasing, and studies have been actively conducted to develop and resource microorganisms with excellent control activity.

Some pathogenic fungi also secrete enzymes such as chitinase, protease, and lipase, which break down the cuticle layer of the outer wall of insects, and may exhibit an insecticidal effect. The fungi that enter the insects produce toxins that kill the insects and then grow continuously to fill the inside of the insects with hyphae to form mummies. When the conditions such as moisture and temperature grow, the insects grow out of the cuticle layer and grow out do. Insect pathogenic fungi include Paecilomycyces, Veticilium and Aschersonia .

According to the EPA pesticide database (2013), bacillus thuringiensis is known to be an insecticide using bacteria (Ishiwata, 1901), (Dean, 1984, Biotechnology & Genetic Engineering Reviews 2: 341-363) To kill pests.

However, these microbial preparations still need to be repeatedly administered without high insecticidal effect, and a new microbial formulation is required in that an appropriate environment must be maintained. Particularly, it is necessary to use insecticides which are free from environmental pollution and can act specifically on the target organisms, without direct influence on the host plants by using microorganisms which are environmentally friendly preparations.

Pseudomonas species (Pseudomonas sp.) Are known to have antimicrobial activity as the biological control due to a gene that causes competition in the root colonization, colonization, mycorrhiza books by environmental factors in Gram-negative bacteria that distribution lot in the soil or the water column (Weller 2007; Dowling and O (2007)). It is known that PGPR is a plant-growth promoting rhizobacteria (PGPR), which induces systemic resistance induction (ISR) or siderophore to obtain resistance to pathogens, Gara 1994). It is also a microorganism capable of decomposing many organic substances such as aliphatic hydrocarbons, phenols, terpenes and steroids. Therefore, it has been used for the decomposition of oil and the removal of nitrogen in wastewater as disclosed in Korean Patent Publication Nos. 2003-0025560 and 2005-0079114 by using organic decomposition properties. The insecticidal effect of Pseudomonas sp. Has not yet been specifically reported.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

 Kim, J. S., Roh, J. Y., Choi, J. Y., Wang, Y., Shim, H. J., Je, Y. H. (2010) Correlation of the aphicidal activity of Beauveria bassiana SFB-205 supernatant with enzymes. Fungal Biology 114, 120-128.  Insecticidal activity of rhamnolipid isolated from Pseudomonas sp. Kim, S.K., Kim, Y.C., Lee, S., Kim, J.C., Yun, M.Y., Kim, EP-3 against green peach aphid (Myzus persicae). J Agric Food Chem 59, 934-938.  (2013) Pseudomonas fluorescens strain (Pseudomonas fluorescens strain) was isolated from a strain of Pseudomonas fluorescens strains. CL145A - A biopesticide for the control of zebra and quagga mussels (Bivalvia: Dreissenidae). J Invertebr Pathol 113, 104-114.  Alin, W. G., Siomne, P., Raul, L. B., Eliane, F. N., Carlos, R. F., Tatsya, N., Bergmann, M. R. (2010) Insecticidal of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses. Virology Journal 7: 143.  Siddiqui, I.A. and Shaukat, S. S. (2003) Suppression of root-knot disease by Pseudomonas fluorescens CHAO in tomato: important of bacterial secondary metabolite, 2,4 diacetylpholoroglucinol. Soil Biology and Biochemistry 35, 1615-1623.

Accordingly, an object of the present invention is to provide a microbial strain having insecticidal activity against harmful insects.

Another object of the present invention is to provide a composition for controlling insects, which comprises a microbial strain having insecticidal activity against harmful insects, a culture solution thereof or a culture medium extract as an active ingredient.

It is still another object of the present invention to provide a method for controlling harmful insects, comprising the step of spraying or applying a composition comprising an effective amount of a microbial strain, a culture solution thereof, or a culture medium extract as an active ingredient to a host plant of an insect.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

In one aspect to achieve the above object, the present invention provides Pseudomonas sp. EML-PB1 (accession number: KCTC 12486BP) having insecticidal activity against harmful insects. As a result of efforts to develop a biological control agent capable of effectively controlling harmful insects, the present inventors have found that when EML-PB1, a novel strain of Pseudomonas sp., Is used as a biological control agent, it is very safe for animals and plants, And have excellent efficacy.

As a result of analyzing the base sequence of 16S rDNA of Pseudomonas sp. EML-PB1 according to the present invention, it was confirmed that it has a close vicinity to Pseudomonas. The above-mentioned novel strain was named Pseudomonas sp. EML-PB1, and the Pseudomonas sp. Strain EML-PB1 was deposited on September 5, 2013 to the microorganism resource center in the Korea Biotechnology Research Institute No. KCTC 12486BP.

The Pseudomonas sp. EML-PB1 of the present invention can be used as a microorganism itself or a culture or an extract obtained by culturing in various media. For example, LB medium (Luria-Bertani, Difco ^™ , USA), NB medium (Nutrient Broth, Difco ^™ , USA) and YMB medium (Yeast Malt Broth, 3 g yeast extract, 3 g malt extract, dextrose 10 g, distilled water 1000 ml) and the like can be used. The incubation time is preferably 24 to 48 hours, and the incubation temperature is preferably 25 to 30 占 폚.

Strain of the present invention can be an excellent insecticidal effect on various insect pests are not limited control target in the present invention may be an insect of the aphids with (Aphidoidea) or Lepidoptera (Lepidoptera).

In another aspect, the present invention provides a composition for insect control comprising Pseudomonas sp. EML-PB1 (accession number: KCTC 12486BP) having a pesticidal activity against harmful insects, a culture thereof or a culture solution extract thereof as an active ingredient . The composition is suitable for controlling various kinds of insects. The type of the insect may be but are not limited to, preferably by aphid pests and (Aphidoidea) or Lepidoptera (Lepidoptera) of the crop.

Aphidoidea is a species that is parasitic on the stem or leaf of a plant, and has a variety of parasitic on large stems and roots. It is widely distributed all over the world. In Korea, aphids such as rose aphids ( Macrosiphum rosae ) and Rhopalosiphum pseudobrassicae Many species are closely related to agriculture as pests of various crops, vegetables, fruit trees, cotton, rose, Most aphids are very active in breeding and are harmful to the vegetable. In case of neglecting the control in the early stage, it is often harmful. Preferably, rose aphid (Macrosiphum rosae) and aphids, cabbage aphid (Rhopalosiphum pseudobrassicae), apple aphid (Aphis pomi), cabbage powder aphid (Brevicoryne brassicae), barley green aphid (Toxoptera graminum), cotton aphid (Aphis gossypii), Macrosiphum pisi , and Myzus persicae . In particular, peach aphid ( Myzus persicae ) is known as a pest having a very wide host range that damages plants.

According to an embodiment of the present invention, an insecticidal effect against aphids ( Myzus persicae ), a kind of aphid which is well known to be harmful to various crops, was tested and showed a mortality rate of 60% after 48 hours 3).

Lepidoptera (Lepidoptera) includes the nabiryu and nabangryu, species belonging to the neck are the egg, larva, pupa, to complete transformation after the adult stage 4, of Lepidoptera larvae herbivorous then causing great economic damage with respect to a wide range of crops It is a typical pest. Preferably Lepidoptera (Lepidoptera), the grain moth (Nemapogon granellur), apple moth (Argyresthia conjugella), cabbage moth (Plutella xylostella), Bee debts person moth (Galleria mellonella), persimmon ipmalyi moth (Ptycholoma lecheana), night Such as Cydia Kurokoi , Grapholita molesta , Lyonetia prunifoliella , Carposina sasakii , Spodoptera exigua , Diaphania indica and Moth moth ( Cnaphalocrocis medinalis ). In particular, moths and moths are well known as cruciferous pests, and most of the damage is from their larvae. According to one embodiment of the present invention, an insecticidal effect of the moth larvae, a species of the moths and moths, was tested and showed a mortality rate of 90% or more (see Table 2).

The Pseudomonas sp. EML-PB1 strain of the present invention, the culture or the culture extract thereof, contains a substratease having an insecticidal effect. Preferably, the proteolytic enzyme is at least one selected from the group consisting of chitinase and protease. Mechanism is the physical action of the microorganism of the strain to germinate on the outer wall of the insect and pierce the cuticle layer. At this time, insecticidal activity is caused by the action of chitinase or protease enzymes. The degree of activity of these enzymes can be used as an indicator of the insecticidal ability of the strain in the laboratory. The strain of the present invention shows a high amount of production from the 12th hour of culture as shown in Table 1 showing the amount of protease produced, It can be applied as a strain.

When the composition of the present invention is used for the control of aphids, it is prevention, avoidance or relief. The aphid can be prevented from accessing the host plant to be controlled, the aphid can avoid the object to be controlled, or the aphid already attached to the object to be controlled can be effectively destroyed.

The composition for controlling insects may be formulated in the form of granules, powders, liquid wettable powders, wettable powders and encapsulated powders, and may be used as an insecticide, but is not limited thereto, and is preferably a wettable powder. The formulated pesticide may be diluted with water to 500 to 2,000 times, preferably about 1,000 times, before use. When the insecticides of the present invention are used as agricultural insecticides, their application amount is usually 0.1 to 100 g per 10 acres. For emulsifiable concentrates, wettable powders, animal and other similar formulations used after dilution with water, the application concentration is usually in the range of 1 to 100,000 ppm. Such application amount and concentration may vary depending on the form of the formulation, application time, place and method, type of pest, degree of damage, and other factors, so that it is not limited to the above range and can be increased or decreased.

The insect control composition of the present invention may also be used together with or in addition to other insecticides, fungicides, fungicides, fungicides, herbicides, plant growth regulators, synergists, fertilizers, soil conditioners and / or animal feeds Can be used simultaneously.

In another embodiment, the present invention relates to a method for producing an effective amount of a Pseudomonas sp. EML-PB1 (accession number: KCTC 12486BP), a step of spraying or applying a composition comprising the culture liquid or a culture solution extract thereof as an active ingredient to an insect host plant The present invention provides a method of controlling harmful insects. Host plants are not particularly limited, but include aphids such as flowers or special crops in addition to economic crops such as cabbage, cabbage, eggplant, persimmon, peach, cucumber, lettuce, strawberry, tomato, Or a plant that can become a host of a lepidoptera or larva. The composition can be treated on these surfaces or on the soil in which these plants are growing or can be treated on fruit or vegetable surfaces during transport or storage. Specifically, the controlling method of the present invention includes application of the insecticide of the present invention in consideration of various factors such as formulation of pesticide, kind of insect pest to be insect, object to be insect, place and method.

As described above, the present invention provides a composition for controlling insects, which comprises Pseudomonas sp. EML-PB1 having an excellent harmful insect control activity, a culture solution thereof or a culture medium extract thereof as an active ingredient, , It is possible to greatly increase the commerciality and productivity when used in crops. Further, the composition of the present invention is a biological pest control agent using a microorganism strain, and it is very safe for animals and plants, and environment-friendly control of pests is possible.

FIG. 1 shows the result of confirming the phylogenetic position of 16S rDNA of a novel Pseudomonas sp. EML-PB1 (accession number: KCTC 12486BP) according to the present invention.
2 is a photograph showing the enzyme activity of Pseudomonas sp. EML-PB1 according to the present invention (A: cellulase, B: chitinase, C & D: protease, C: Culture filtrate).
Figure 3 shows the molecular weight of the protease determined by SDS-PAGE of EML-PB1 according to the present invention (lane 1: protein marker 100 kDa, lane 2: negative control, lane 3: supernatant; arrow shows the molecular weight of EML-PB1 protease Of about 27 kDa).
FIG. 4 shows the activity of EML-PB1 according to the present invention on Chinese cabbage moth (A & B: control, C & D: EML-PB1 medium).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Isolation and Identification of Pseudomonas sp. EML-PB1

This strain was isolated from the forest soil samples around Chonnam National University by dilution plate culture method and sequenced for the final identification of isolated strains. (5'-AGA GTT TGA TCM GGC TCA G-3 ') and 518r (5'-ATT ACC GCG GCT GCT GG-3') for the sequencing of 16S rDNA after isolating the EML- (PCR) method using 2 oligonucleotides as a primer. This procedure was performed by denaturation at 95 ° C for 1 minute, annealing at 53 ° C for 40 seconds, annealing at 72 ° C for 1 minute The DNA was amplified by enzymatic method under the condition of minute extension.

The amplified DNA was purified and then sequenced. EzTaxon (Chun et al., 2007 ) a As can be seen from the nucleotide sequence comparison result of the analysis of the type strain, 1 Using was confirmed that have close relatives related to the genus Pseudomonas (Pseudomonas sp.), Pseudomonas species (Pseudomonas sp.) EML-PB1.

The Pseudomonas sp. EML-PB1 strain was deposited with the Microbiological Resource Center of the Korea Biotechnology Research Institute, which is a depository of the Korean patented strain, on September 5, 2013, and received the deposit number KCTC 12486BP.

Enzyme activity of Pseudomonas sp. EML-PB1

(SMK; 1% skim milk, 1.5% agar) and chitinase agar (CA; Leisuer et al., 2008) were used to assay the protease and chitinase activities of Pseudomonas sp. EML-PB1 Respectively) were used.

EML-PB1 was cultivated in LB (Luria Bertani broth) medium. After centrifugation, the supernatant and supernatant were filtered using a syringe filter, and the culture filtrate was dispensed onto paper discs in an amount of 50 ul each. I put it up. And cultured at 30 ^° C.

As shown in FIG. 2, clear zones were observed around paper discs in SMA and CA medium, indicating that EML-PB1 has protease and chitinase activity.

Protease production and molecular weight of Pseudomonas sp. EML-PB1

To investigate the amount of protease production of Pseudomonas sp. EML-PB1, it was performed according to the method of Izrael-Zivkovic et al. (2010). The supernatant obtained by the ^{EML-PB1 50ml, 30 o C} , 150rpm , respectively 24, 48 and 72, the culture medium was cultured for 96 hours in separate 15 minutes and centrifuged at 10,000rpm were used in the experiments.

Protease production was observed at 12 hours after the incubation, 38.3 U / ml at 24 hours, and decreased at 48 hours.

 The amount of protease produced by Pseudomonas sp. EML-PB1 over time Incubation time Protease production (Unit / ml) 0 0 24 38.3 48 35.5 72 34.4 96 30 120 28

To confirm the molecular weight of the protease produced by EML-PB1, SDS-PAGE was performed by Laemmli (1970) and 12% polyacrylamide gel was used. After SDS-PAGE, the cells were stained with coomassie brilliant blue R-250 and observed to have a molecular weight of about 27 kDa (see arrows in FIG. 3), as can be seen in FIG.

Of Pseudomonas sp. EML-PB1 Invitro  Insecticidal effect

To investigate the insecticidal effect of Pseudomonas sp. EML-PB1 on cabbage moths, LB medium (Luria-Bertani, Difco ^TM , USA) was inoculated and cultured at 30 ° C for 24 hours with shaking at 150 rpm. Respectively.

The insecticidal effect test was carried out using a spraying method which is similar to the packaging condition and highly reproducible. The Chinese cabbage leaves were cut into 10 ㎝ in diameter and placed in a Petri dish (10 x 6 ㎝ in diameter) and inoculated with 20 rats of each 3rd instar larva. Using a small spray, the amount of each strain was sprayed on the petri dish to about 3 ml. Experimental conditions were as follows: temperature: 25 ℃; humidity: 50-60%; light period: 16L: 8D. The longevity of the insects was observed every 12 hours and the insecticidal power for 72 hours was ultimately assayed (C and D). Water treatment (A) and non - treatment (B) were used as control. All experiments were carried out three times, and the life and death of the pupa was considered as a mushroom by using a brush and no movement when the pupae were lifted.

As shown in the results of Table 2, the insecticidal activity of the cabbage moth was 80% or higher after 36 hours of treatment with the culture medium.

In addition, as shown in FIG. 4, the larvae of the dead Chinese cabbage moth after the treatment of EML-PB1 were observed to slow down the movement of the larvae body, and the color became soft and dry.

Insecticidal effect of EML-PB1 on cabbage moth Time (hr) * Average insecticidal rate (%) ** Culture solution Control 12 50 0 24 78.3 0 36 85 0 48 86.7 0 60 86.7 0 72 86.7 0

* Elapsed time after culture inoculated with cabbage moth, ** Number of larvae / Number of whole larvae × 100.

Insecticidal effect of Pseudomonas sp. EML-PB1 on peach aphid

To investigate the insecticidal effect of Pseudomonas sp. EML-PB1 on peach aphid, the culture was prepared by inoculating LB medium and shaking culture at 30 ° C for 24 hours at 150 rpm. The culture broth was centrifuged and syringe filter using a ^{0.1um, Acrodisc ® Syringe filter, PALL} Life Sciences, USA) was used for filtering a filtrate.

The spraying method was used to investigate insecticidal activity against peach aphid. The host plants, Chinese cabbage leaves, were cut into a diameter of 10 ㎝, placed in a Petri dish (10 x 6 ㎝ in diameter) and inoculated with 20 rats of each 3rd instar larva. Using a small spray, the amount of each strain was sprayed on the petri dish to about 3 ml. Experimental conditions were as follows: temperature: 25 ℃; humidity: 50-60%; light period: 16L: 8D. The longevity of the insects was observed every 12 hours and the insecticidal power was ultimately tested for 72 hours. Sterilized water and non - treated water were used as control. All experiments were carried out three times, and the life and death of the pupa was considered as a mushroom by using a brush and no movement when the pupae were lifted.

As shown in Table 3, the culture of EML-PB1 against peach aphid showed more than 50% insecticidal activity after 48 hours.

Insecticidal effect of EML-PB1 on peach aphid Time (hr) * Average insecticidal rate (%) ** EML-PB1 Control Culture solution Culture filtrate *** 12 15.5 10.1 2.1 24 27.6 10.1 2.1 36 36.6 14.6 2.7 48 54.2 14.6 3.4 60 60.0 24.7 3.4 72 62.9 34.8 6.2

* The culture medium inoculated with the peach aphid time elapsed, * mortality number / the total number of larvae x 100, *** broth was filtered by ^{syringe filter (0.1um, Acrodisc ® Syringe} Filter, PALL Life Sciences, USA) strain of After removing the supernatant.

The above results show that the culture solution of the Pseudomonas sp. EML-PB1 strain exhibits a high insecticidal effect on the Chinese cabbage moth control agent than the aphid.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Korea Biotechnology Research Institute KCTC12486BP 20130905

Claims (11)

Pseudomonas sp. EML-PB1 (accession number: KCTC 12486BP) having insecticidal activity against peach lycopene ( Myzus persicae ) or Plutella xylostella . delete ( Pseudomonas sp.) EML-PB1 (accession number: KCTC 12486BP) having an insecticidal activity against Myzus persicae or Plutella xylostella , a culture or a culture solution extract thereof Composition for controlling insects. delete delete delete delete delete 4. The composition of claim 3, wherein the control is a prophylactic, preventative or remedy. Aphid ( Myzus persicae) comprising a step of spraying or applying a composition comprising Pseudomonas sp. EML-PB1 (accession number: KCTC 12486BP), a culture or a culture extract thereof as an active ingredient, ) Or the control method of the Chinese cabbage moth ( Plutella xylostella ). delete

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