KR20110011239A - Entomopathogenic fungi beauveria bassiana maw1 having insecticidal activity and its use - Google Patents

Abstract

PURPOSE: An entomopathogenic fungi Beauveria bassiana MaW1 having insecticidal activity and a composition containing the same for preventing Monochamus alternates or Moechotypa diphysis is provided to develop an effective insecticidal agent. CONSTITUTION: An entomopathogenic fungi Beauveria bassiana MaW1(KACC93076P) effectively prevents Monochamus alternates or Moechotypa diphysis. A composition for preventing Monochamus alternates or Moechotypa diphysis contains the entomopathogenic fungi Beauveria bassiana MaW1(KACC93076P) or spore thereof as an active ingredient. The entomopathogenic fungi Beauveria bassiana MaW1(KACC93076P) is isolated from Monochamus alternates which is infected by entomopathogenic fungi.

Description

Insect pathogenic fungus Boberia bassiana MA 1 strain with insecticidal activity and its use {Entomopathogenic Fungi Beauveria bassiana MaW1 having Insecticidal Activity and Its Use}

Insect pathogenic fungus Boberia Bassiana MaW1 strain, Monochamus alternatus or Moechotypa diphysis control composition comprising the same as an active ingredient, and a method for controlling the pests using the composition It is about.

Bursaphelenchus xylophylus ) is a pathogen that causes wilting disease and causes the death of pine trees. In Korea, the disease is called pine tree nematode disease. Pine tree nematodes are indigenous nematodes of North America, and native pines of origin are resistant, but indigenous pine trees of Northeast Asia including Korea are mostly susceptible and are known to cause severe damage. The first pine ash nematode damage began in Japan. After the damage caused by this nematode began to appear in 1905, the damage gradually spread, and since 1941, the amount of damage was peaked at 2.43 million m3 in 1979, and around 1 million m3 per year. . In China, the first occurrence was confirmed in Nanjing in 1982, and pine, sea, and horse pine were severely damaged. Korea has been trying to prevent the invasion by designating it as a prohibited pest under the Plant Defense Act, but the damage of pine ash nematode disease, which is believed to have invaded in Japan, was first discovered in Geumjeongsan area in Dongnae-gu, Busan in October 1988, In spite of the efforts of the relevant agencies, the area of damage has increased extensively.

Since the pine reptiles do not have the ability to move to other trees on their own, they move on to the body of the larva and then transfer to the healthy tree through the scars that occur when the larvae eat the bark of a healthy tree branch and tear off the bark. Lose. Pine reptiles invading host plants multiply rapidly, and after 20 days of infection, the yield of leaves is reduced, and the old leaves start to sag and fall down. Infected trees will die about 80% of the year, and the rest will be killed until May of the following year.

Tree injection, the most common method of controlling pine tree nematodes in Korea, prevents pine tree nematode diseases by injecting nematicides into healthy trees in areas of concern. The hole is about 9cm deep and the medicine is injected using an injection bottle. In Japan, green guards (morantel tartrate), levamis, and mesulfenfoss are used as tree-injection drugs, and they are mainly used for protection water and landscaping water in certain areas. In Korea, inexpensive abamectin and emamectin benzoate have been selected and used in a limited area or protected water. While the tree injection method requires a lot of manpower and control costs for its application, its use is limited because the area to be controlled is mainly a large forest area. It is necessary to prepare.

Therefore, the control of pine reptiles has no ability to move and the focus is on controlling the parasites rather than killing the nematodes themselves. The known mediators of pine reptiles are Monochamus genus, which is the representative of the monochamus alternatus in Korea. Currently, the National Forest Service applies the method of fumigation with methamsodium solution after harvesting and collecting trees infected with pine ash nematodes as a main control method. Air is being sprayed. However, this method is also a forest with a large processing area, which requires high cost and high manpower, along with environmental pollution and ecosystem disturbance caused by excessive treatment of synthetic pesticides. As a way of supplementing and replacing these problems, biological control methods using natural enemies of antler salt (ant punishment, large rice bandit, and ant paste) have been proposed. However, this method requires a lot of time to search for and settle effective natural enemies, has low parasitic efficiency, lacks host specificity for saltwater beetles, and can damage natural enemies in the air defenses conducted between May and July. And there is a problem that can cause damage to humans if multiplied in large quantities.

The control of insect pathogenic fungus Boberia bassiana, which is a control method of insect pathogenic microorganisms that has been proposed as another biological control method, is currently presenting various studies and control methods in Japan, China, and Portugal. A nonwoven strips method using the Boberia Bassiana F-263 strain is used as a commercial microbial insecticide to control the sea salt, which is a carrier of disease. However, to date, there is no report or use of isolation of this type of fungal strain having pathogenicity in sea salt.

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.

The present inventors tried to develop an insect pathogenic fungal insecticide for controlling the pineal nematode, Monochamus alternatus , which is a mediator of pine reptiles, which harms Korean pines, by an environmentally friendly method without using chemical insecticides. As a result, we isolated and identified the Beauveria bassiana MaW1 strains from the larvae of the beetle larvae that were killed by infectious insect pathogenic fungi, and the insecticidal effect on the sperm and moechotypa diphysis . The present invention was completed by experimentally confirming that is very excellent.

Accordingly, an object of the present invention is to provide an insect pathogenic fungus Boveria bassiana MaW1 strain.

 It is another object of the present invention to provide a composition for controlling a salt brush or a hair toad, which comprises the strain or its spores.

Still another object of the present invention is to provide a method for controlling the brackishness sky cow or hair toad sky cow using the strain.

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

According to one aspect of the present invention, the present invention provides an insect pathogenic fungus Beauveria bassiana MaW1 strain (KACC93076P).

According to another aspect of the present invention, the present invention is an insect pathogenic fungus Beauveria bassiana Maw1 strain (KACC93076P) or its spores ( Monochamus alternatus ) or mole toads ( Moechotypa diphysis) Provided is a composition for controlling.

According to another aspect of the present invention, the present invention provides a composition comprising the insect pathogenic fungus Beauveria bassiana MaW1 strain (KACC93076P) or its spores, Monochamus alternatus or Moechotypa diphysis It provides a method of controlling the brushed sky cow or toad sky cow, comprising the step of spraying).

As used herein, the term "insect pathogenic fungus" refers to chitinase, protease, lipase, esterase, and the like, when the spores of the fungus adhere to the shell of the host insect and germinate. It penetrates through the cuticle layer of insects, and then proliferates in the insect's body, producing small molecules of toxic substances or toxic proteins such as glycoproteins, which block the insect's immune activity and kill host insects. Mean fungal bacteria having insecticidal activity. Insect pathogenic fungi are attracting attention as a control agent for agricultural and forest pests and are currently reported as more than 700 species, and in developed countries dozens of fungi have been developed and used as microbial insecticides.

Beauveria of the present invention bassiana ) MaW1 strain "is a fungal activity of the present inventors with excellent insecticidal activity against monochamus alternatus and Moechotypa diphysis among fungi isolated from dead male rhizome larvae infected by insect pathogenic fungi. The strain was isolated and finally identified.

The present inventors have found the strain Bo Beria Bridge Ana was the final identified as strains belonging to (Beauveria bassiana) it in the 'Bo Beria Bridge Ana (Beauveria bassiana) MaW1' naming and genetically National Academy of Agricultural Science Agriculture as of July 20, 2009 Support Center It was deposited (Accession Number: KACC93076P).

Beauveria bassiana MaW1 strain of the present invention has the following morphological characteristics, the mycelium is white in flat solid media and the back of the mycelium is initially white, but gradually changes from pale yellow to yellow (Fig. 2). Reference).

The Beauveria bassiana MaW1 strain of the present invention has conidia, which is characteristic of a typical Boberia genus, zigzag in conidia, and has one ovoid spore in each protrusion (see FIGS. 3 and 4).

The Beauveria bassiana MaW1 strain of the present invention is a Boberia strain, Boberia bassiana strains Bobergia by a molecular biological identification method using a PCR (Polymerase Chain Reaction) primer specific (Hegedus and Khachatourians , 1996) Identified as Beauveria bassiana (see FIG. 5).

In addition, the Beauveria bassiana MaW1 strain of the present invention has a high sequencing sequence of the PCR product obtained by using the specific PCR primer of the genus Boberia compared with those of other strains already identified as Boberia bassiana . Homology is shown (see FIG. 6).

Insects herein "Beechtail ( Monochamus) alternatus ) "is a well-known pest known as a mediator of pine reptiles in Korea , and" Moechotypa diphysis "has an ecological characteristic similar to that of sea salted beetle, and is an important pest against mushroom shiitake and forest oak. Invention of " Boberia Bassiana ( Beauveria bassiana) MaW1 strain "is the sole beard sawyer (Monochamus alternatus ) "and" Moechotypa diphysis ) "has a very good insecticidal effect.

The Beauveria bassiana MaW1 strain of the present invention is very resistant to the echidonia ( Monochamus alternatus ) and the toaded larvae ( Moechotypa diphysis ) among fungi isolated from adult insects of dead pathogenic rhizome . It is an insect pathogenic fungi strain that has been identified and finally identified as having excellent pesticidal activity. The Beauveria bassiana MaW1 strain of the present invention is highly likely to be developed as an effective biological microbial insecticide against pine wood nematode mediators, pine bark and shiitake mushrooms and forest oak pests. By using these microbial pesticides can reduce the amount of chemical pesticides used to contribute to environmentally friendly control.

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 .

Example

Example 1: Sea salt Monochamus alternatus From the insecticide Boberia Bassiana ( Beauveria bassiana ) MaW1  Isolation of Strains

The inventors of the present invention collected an adult dead beetle larvae infected with an insect pathogenic fungus in the open air, thereby separating the Boberia bassiana MaW1 strain of the present invention.

The conidia of the dead canine infected with insect pathogenic fungi were removed and diluted in plated solid medium of Potato dextrose agar with penicillin-Streptomycin solution antibiotic. This was incubated for 5 days at 25 ℃ dark conditions, and then separated by screening Fresh potato dextrose agar was inoculated into a flat solid medium and re-incubated for 2 weeks under the same conditions and stored at 4 ° C. until use. To isolate the highly pathogenic entomopathogenic fungi from the isolated fungi, the conidia of each fungus were concentrated in a concentration of 1 × 10 ⁸ conidia / ml in 10 ml of sterile water containing 0.02% Tween 80. It was suspended using a hemocytometer measurement. Subsequently, the toad sky cow was immersed in each conidia spore suspension for 10-15 seconds, and then put into an insect cage to examine mortality at 25 ° C. and a relative humidity of 90% or more. As a result, only one fungal strain showed high insecticidal activity in the toad sky cow, and after separating it was identified through the following process.

Figure 1 is a photograph showing a beetle sky larva collected by infection with a fungus isolated from the Boberia Bassiana MaW1 strain, it can be seen that the adult is infected with white mold. Figure 2 is a photograph showing that the Boberia bassiana MaW1 isolated from the brine raspberry grows in a flat solid medium, it can be seen that the mycelium is the same as the white from the raspberry beetle.

Example 2: Boberia Bassiana Beauveria bassiana Morphological Identification of MaW1 Strains

In order to observe the isolated fungal strain with a phase contrast microscope, the mycelium cultured on a slide glass was carefully removed and the shape of the conidia attached to the conidia disease was observed using a Nikon microscope. In addition, scanning electron microscopy was performed for more detailed observation of the isolated fungal strains. First, the cultured fungus mycelium was carefully peeled off and immediately coated with gold and observed by scanning electron microscope (Carl Zeiss LEO-1530 SEM).

3 and 4 are photographs showing the conidia of Boberia bassiana MaW1 strain observed with a phase contrast microscope (400 times) and a scanning electron microscope (5000 times), respectively, the fungus of the present invention is a typical form of the genus Boberia The conidia, which are the characteristic features, emerged in a zigzag form from the conidia, and had one ovoid spore in each protrusion. Morphological analysis through these microscopic observations could strongly suggest that the isolated fungus is a strain belonging to the genus Boberia.

Example 3: Boberia Bassiana Beauveria bassiana Molecular Biology of MaW1 Strains

Molecular biological PCR techniques were used for identification at the species level, as well as more accurate identification of isolated fungal strains. Morphological observations confirmed that MaW1 was of the genus Boberia, and the genus Boberia specific PCR primer pairs (P1-P3 primer pairs) and the Boberia bassiana specific PCR primer pairs (P1-P5 primer pairs) (Hegedus DD, Khachatourians GG (1996) J Invert Path 67, 289-299.) Was prepared as follows.

primer  order

Primer Name Primer type Primer Sequence P1 forward primer 5'-AAGCTTCGACATGGTCTG-3 ' P3 backward primer 5'-GGAGGTGGTGAGGTTCTGTT-3 ' P5      backward primer 5'-AGGAGAGAGCTCGACGGTCA-3 '

Then, genomic DNA (gDNA) of MaW1 strain was extracted using I-genomic BYF DNA extraction mini kit (Intron, Seoul). MaW1 gDNA extracted using the prepared PCR primers was amplified using a PCR premix kit (Bionia, Daejeon). At this time, PCR conditions were denatured at 94 ° C. for 3 minutes, followed by a series of 35 reactions of annealing reaction at 53 ° C. for 30 seconds, and polymerization reaction at 72 ° C. for 45 seconds. PCR products were analyzed by 1.0% agarose gel electrophoresis.

Figure 5 shows the results of PCR amplification on gDNA of MaW1 strain using Boberia specific PCR primers (P1-P3 primer pair) and Boberia Basciana specific PCR primers (P1-P5 primer pair) It is a photograph. In case of PCR amplification with P1-P3 primer pair, the PCR product appeared near 500bp, and MaW1 strain was identified as Boberia. PCR amplification with P1-P5 primer pair showed PCR product near 300bp. Identified as Veria Bassiana.

Based on the above results, the fungus MaW1 strain isolated by the present inventors was identified as Beauveria bassiana , and was deposited on July 20, 2009 at the National Institute of Agricultural Science, Agricultural Genetic Resource Center (Accession Number: KACC93076P).

Example 4 Boberia Bassiana Beauveria bassiana ) Sequence Analysis of PCR Amplification Product of MaW1 Strain and Homology Comparison with Other Boberia Bassiana Strains

P1-P3 PCR amplification products analyzed by agarose gel electrophoresis were cloned into pCR2.1-TOPO (Invitrogen Co., USA) T-vector. The cloned T-vector was transformed according to the manufacturer's method, and white colonies were randomly selected and cultured. Cells containing the recombinant plasmids were lysed and then isolated using a plasmid separation kit (Cosmogenetech Co., Seoul). Plasmids containing each PCR amplification product insert were run on an automated DNA sequencer [Applied Biosystems, Inc. (Applied Biosystem, Inc.). The analyzed sequences were arranged through DNA man (Lynnon BioSoft, Quebec, Canada) and compared to homology with previously reported Boberia bassiana strains using the Internet program hierarchical clustering (Corpet, 1988). As a result of homology comparison, the isolated fungus Boberia Bassiana MaW1 strain showed 99% homology with Boberia Bassiana SFB-13 strain and 97% with Boberia Bassiana GK2016 strain.

Figure 6 shows the sequence of the PCR product amplified using a P1-P3 PCR primer pair for the Boberia Bassiana MaW1 strains (Boberia Bassiana SFB-13 strain, Boberia Bassiana GK2016 Figure showing the comparison with the nucleotide sequence of the strain). Boberia Bassiana MaW1 is highly homologous to other Boberia Bassiana strains.

Example  5: Boberia Bassiana MaW1  Strain Toad Sky Cow  Pathogenicity Assay for Adults

Boberia Bassiana MaW1 conidia were spun in 10 ml sterile water containing 0.02% Tween 80 in 1 × 10 ⁴ , 1 X 10 ⁶ , It was suspended using a hemocytometer measurement to 1 × 10 ⁸ conidia / ml. Thereafter, the toad sky cow was immersed in the conidia of each concentration for 10-15 seconds, and then put into an insect breeding tank to examine mortality at 25 ° C. and relative humidity of 90% or more. The pineal salt was examined only in 1 × 10 ⁸ conidia spores / ㎖ and the mortality under these conditions. As a result, Boberia bassiana MaW1 showed a high insecticidal effect according to the concentration and the insecticidal effect on the brackish goat.

Figure 7 is a graph showing the pesticidal effect of the spore suspension concentration on hair toad sky cow. As can be seen in Figure 7, it was found that the higher the concentration of the spore suspension, the higher the insecticidal effect.

FIG. 8 is a photograph showing the adult hair toad sky cow infected with Boberia bassiana MaW1, and as shown in FIG. 8, the symptom of infection was the same as that of the isolated beetle bovine insect.

9 is a photograph showing a male rhizome larvae infected with Boberia bassiana MaW1, as shown in FIG.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

FIG. 1 is a photograph showing a beetle larvae adult collected from a beetle infected with a fungus isolated from a beaveria bassiana MaW1 strain.

Figure 2 is a photograph showing the Boveria bassiana MaW1 strains growing in a flat solid medium.

Figure 3 is a photograph showing the observation of the conidia of Beauveria bassiana MaW1 strain by phase contrast microscope.

Figure 4 is a photograph showing the observation of the conidia of the Beauveria bassiana MaW1 strain by scanning electron microscopy.

FIG. 5 shows PCR primer pairs (P1-P3 primers) and Boberia bassiana identification PCR primer pairs (P1-P5 primer pairs) for gDNA of the Boveria bassiana MaW1 strain. An electrophoretic photograph of each amplified product was used.

Figure 6 shows the gene sequence of the product amplified gDNA of Beauveria bassiana MaW1 strain using a PCR primer pair (P1-P3 primer pair) of the genus Boberia homology with other Boberia Bassiana strains Is a comparison.

7 is a graph showing the results of the insecticidal effect of Boberia bassiana MaW1 strain measured by immersion method using Moechotypa diphysis .

FIG. 8 is a photograph showing an adult adult Moechotypa diphysis infected with Boberia bassiana MaW1 strain.

FIG. 9 is a photograph showing an adult adult Monochamus alternatus infected with Boberia bassiana MaW1 strain.

<110> Chungbuk National University Industry Academic Cooperation Foundation <120> Entomopathogenic Fungi Beauveria bassiana Maw1 having          Insecticidal Activity and Its Use <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 18 <212> DNA <213> Beauveria bassiana <400> 1 aagcttcgac atggtctg 18 <210> 2 <211> 20 <212> DNA <213> Beauveria bassiana <400> 2 ggaggtggtg aggttctgtt 20 <210> 3 <211> 20 <212> DNA <213> Beauveria bassiana <400> 3 aggagagagc tcgacggtca 20  

Claims (4)

Insect Pathogenic Fungus Boberia Bassiana bassiana ) MaW1 strain (KACC93076P). The strain of claim 1, wherein the insect is a monochamus alternatus or a moechotypa diphysis . Insect pathogenic fungus Beauveria bassiana ( Beauveria bassiana ) MaW1 strain (KACC93076P) or a composition for the control of the brackish or black toads, including spores thereof as an active ingredient. Insect Pathogenic Fungus Boberia Bassiana bassiana ) A method of controlling a quinoa clarine or a hair toad, comprising a step of spraying a composition comprising MaW1 strain (KACC93076P) or its spores as an active ingredient on a quinine or cow toad.

Images