KR20180079240A - Novel Beauveria bassiana M130 and biological control method of greenhouse whitefly and bemisia tabaci using the same - Google Patents

Abstract

The present invention relates to a method for biological control of greenhouse whitefly and tobacco bemisia tabaci using an insecticidal insect pathogenic fungus Beauveria bassiana M130 (KCTC12104BP) strain and its base sequence According to the present invention, an insect pathogenic fungus Buergeriviana M130 strain having an insecticidal activity against powdery mildew is separated from a carcass of a melon-packed aphid, and this strain is suitable for maximization of insecticidal activity by mass spore production using rice bran extract The present invention provides a biologic method for preparing greenhouse and tobacco by directly spraying the greenhouse / tobacco with Louisiana by diluting the cultured spore culture medium, thereby providing an excellent microorganism insecticide against the greenhouse powder, It is effective.

Description

{Novel Beauveria bassiana M130 and biological control method of greenhouse whitefly and bemisia tabaci using the same}

Unlike trees, the present invention is Beauveria bassiana that exhibits insecticidal power against greenhouse whitefly and bemisia tabaci derived from domestic melon packaging, which are vegetables. bassiana ) M130 (KCTC12104BP) strain and a method for producing spores of the strain using the gene and rice bran extract, and an effective method for controlling the pests using the spores thereof.

About 1,200 types of powdery mildew are recorded to be distributed around the world, and 7 species are recorded to be distributed in Korea, and the representative problem is greenhouse and tobacco powder. Life history is developed through the stage of 2-4 instars, which are fixed and developed without eggs, mobile 1 instar, crawler, and legs, and 6 stages of adult insects. Damage to crops caused by greenhouse and tobacco powder is caused by insects and adult insects ingesting juice from leaves and secreting nectar. In particular, when the density is high, the absorption of juice affects the physiological metabolism of plants and inhibits the growth of shoots. In severe cases, it can lead to fading, forgery, fallen leaves, and death. In addition, such leaf damage affects the growth of fruits and causes yield reduction. In addition, the fruit becomes sticky by indirectly secreted nectar and is not good because it is shiny in appearance, and soot mold is generated and the fruit becomes dirty, resulting in poor marketability. In addition, it reduces photosynthesis and respiration of crops and mediates viruses.

Currently, as a biological control means for controlling greenhouse and tobacco dust, natural enemy insects, greenhouse and tobacco dust, are mainly used, and the control success rate is reported to be very high. However, in all cases, greenhouse and tobacco dust bees are not the most effective means. In the case of greenhouse/tobacco powdery crops with high density or with a lot of hair on leaves such as cucumbers, the behavior of greenhouse/tobacco powdery bees is hindered, resulting in poor control efficiency. In addition, even in low temperature and low light conditions, the activity of the greenhouse and tobacco powdery bees is inferior. The development of microbial pesticides is required as an alternative to such disadvantages of mothballs and various problems with chemical pesticides.

Greenhouse/tobacco powder is absorbed by stabbing goji into the leaf meat tissue of a crop. Therefore, it is impossible to control greenhouse powder by spraying microorganisms on the leaf surface and killing pests by feeding them. For reasons like this, Beauveria bassiana , Verticillium Development of microbial insecticides for controlling suckling insects using insect pathogenic fungi such as lecanii is ongoing worldwide.

An object of the present invention is to provide a microbial pesticide as a biological control agent suitable for greenhouses and tobacco powder.

Another object of the present invention is a newly isolated insect pathogenic fungus Beauveria bassiana derived from aphids of melon packaging of vegetables, unlike those known in trees such as peaches, apricots, plums, sakura, tangerine trees, etc. bassiana ) to provide the M130 strain (KCTC12104BP).

Another object of the present invention is to provide a mass spore production method using a rice bran extract using the above new strain. In addition, the present invention is to provide a biological control method capable of controlling greenhouse and tobacco powder using its spores through solid culture of the strain, Beauveria bassiana, M130 strain (KCTC12104BP).

The above object of the present invention is Beauveria bassiana derived from melon packaging, which is a fruit or vegetable that exhibits insecticidal action against greenhouse and tobacco powder. bassiana ) Through the steps of separating M130 strain (KCTC12104BP) from aphids, optimizing mass spore production using rice bran extract, and identifying biological control methods for greenhouse and tobacco powder contained as active ingredients of spores. Achieved.

As described above, the present invention is a novel insect pathogenic fungus derived from aphid carcasses of melon packaging, Beauveria bassiana (Beauveria). bassiana ) It has the effect of providing the M130 strain, and not only provides microbial preparations with insecticidal activity on greenhouse whitefly and bemisia tabaci, but also can replace chemical pesticides through biological control using this. It has an excellent effect of providing a biological pesticide

FIG. 1 is a photographic view of the Beauveria bassiana M130 strain derived from aphid carcasses of melon-packed growing in the flat solid medium of the present invention.
Figure 2 is the Beauveria bassiana of the present invention (Beauveria bassiana ) is the result of 18S rRNA gene analysis of M130 strain.
Figure 3 is the Beauveria bassiana of the present invention (Beauveria bassiana ) is a systematic classification diagram using the results of the identification of the M130 strain.
Figure 4 is the Beauveria bassiana of the present invention (Beauveria bassiana ) These are the results of experiments on the activity of protease and chitinase of M130 strain.
5 is a result of measuring the spore concentration of the Beauveria bassiana M130 according to the concentration of the rice bran extract of the present invention.
Figure 6 is a measurement result of the cell mass of the Beauveria bassiana (Beauveria bassiana) M130 according to the concentration of the rice bran extract of the present invention.
7 is an experiment result of the active view Beria Bridge Ana (Beauveria bassiana) better kitty of the M130 (chitinase) of the rice bran extract, the concentration of the present invention.
8 is a result of the protease activity experiment of the Beauveria bassiana M130 according to the concentration of the rice bran extract of the present invention.
9 is the Beauveria bassiana of the present invention (Beauveria bassiana ) This is a photograph showing the results of spore production by culturing the inoculum of the strain in a solid culture medium.
10 is a photograph of the proliferation and bioverification of greenhouse powder for bioverification of the present invention.
11 is a result of bioverification after spraying the spores of the Beauberia bassiana strain cultured in the solid culture medium of the present invention on greenhouse powder.

The present invention is a step of separating the Beauveria bassiana M130 strain (KCTC12104BP) exhibiting insecticidal action against greenhouse and tobacco powder from aphids of domestic melon packaging, optimizing mass spore production using rice bran extract , It consists of confirming the biological control method for greenhouse·tobacco powder contained as an active ingredient of spores.

In the present invention, the microbial insecticide using a fungus acts as an insecticidal mechanism in which the sprayed spores contact, germinate, and invade the insect body. Therefore, when the spores contact the insect body, there are many environmental effects, and the spores have a long time to express the insecticidal effect. itgie is a problem that it takes time for new strains that can produce excellent insecticidal effect by mass spores were first separated from aphids dead domestic melon packing E. that the results of identification, views Beria Bridge Ana (Beauveria bassiana) M130 (KCTC12104BP) strain It was found that the microbial preparation using this has insecticidal activity in greenhouse and tobacco powder, and the present invention was completed by confirming the insecticidal activity through bioverification.

Hereinafter, the specific contents of the present invention will be described in detail according to examples, but the scope of the present invention is not limited only to these examples, and it goes without saying that the scope of the present invention is easily changed by those skilled in the art.

Example 1: Insect pathogenic fungi separation, selection and characterization

Aphid carcasses collected from domestic melon packaging were collected individually in sterilized vials, dried slightly to prevent secondary infection, and then stored with a piece of filter paper and transported to the laboratory. After disinfecting the aphid disease body with 70% alcohol, it was washed three times with sterile water, and then a part of the tissue inside the infected insect was removed with a sterilized dissecting knife. This was placed on a PDA medium containing an antibiotic (chloramphenicol) prepared in advance, and cultured in an incubator at 25° C. for 5 days to isolate insect pathogenic fungi (FIG. 1).

Thereafter, for taxonomic identification of insect pathogenic fungi, they were identified through 18S rRNA sequencing, which is a genetic analysis method (FIG. 2), and the genealogy and morphological characteristics thereof were investigated (FIG. 3).

The insect pathogenic fungus strain of the present invention is Beauveria bassiana (Beauveria bassiana ) M130 and deposited with the international depository as KCTC12104BP dated December 16, 2011.

Example 2: Invention Beauberia Ana Bridge (Beauveria bassiana ) Measurement of protease and chitinase activity using M130 strain

In order to confirm that the purely isolated strain according to Example 1 has insecticidal power against the greenhouse and tobacco powder, a spore culture solution cultivated in the PDB medium was made to finally select the strain having the best insecticidal activity against the greenhouse and tobacco powder. To do this, protease and chitinase activities were measured.

The measurement of protease was measured using casein as a substrate. 200 ul of 0.6% casein and 200 ul of coenzyme were mixed, reacted at 30° C. for 10 minutes, and then 250 ul of 0.4M trichloroacetic acid solution was added to terminate the reaction. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 10 minutes, 0.4M Na2CO3 and 1M proline were added to 600 ul of the supernatant, reacted at 40° C. for 30 minutes, and then measured at 660 nm.

Chitinase was measured using colloidal chitin as a substrate. 100 ul of 1% colloidal chitin and 100 ul of coenzyme were mixed, reacted at 50° C. for 30 minutes, centrifuged at 10,000 rpm for 5 minutes, and then the supernatant was mixed with a DNS (dinitrosalicylic acid) reagent and measured according to the DNS measurement method (Fig. 4, 7, 8).

Example 3: Optimization of spore production strains of the invention by utilizing the agricultural by-product of rice bran, in a culture medium using a rice bran extract

For the mass cultivation of the strain of the present invention, rice bran, which is an agricultural by-product rather than PDB (potato dextrose broth) medium, is used in consideration of economic efficiency, and it shows high growth and spore production in the medium using rice bran extract. The extract was used as the basic medium of the culture medium of this strain, and the cell mass and protease and chitinase activities were measured by the above method. The cell mass was measured by the dry mass of the mycelium, and Beauveria bassiana cultured in the medium (Beauveria bassiana ) After separating a certain amount of the culture medium of the M130 strain into mycelium and culture filtrate with filter paper, the weight of the mycelium completely dried at 110° C. for 8 hours was measured. For the measurement of spore concentration, the mycelium was separated from the culture medium with glass wool, and then the spores were measured with a hemocytometa. The results are shown in FIGS. 5 and 6.

Example 4: Invention Beauberia Ana Bridge (Beauveria bassiana ) solid medium culture using M130 strain

After putting 1 kg of rice into a high-temperature film bag, autoclaving (121°C, 15 minutes), inoculating a liquid medium inoculum using rice bran extract, spreading evenly, and placing it in a solid culture chamber at 28°C for 7 days and cultured. After 7 days of incubation, water generated by respiration is removed. On the 20th day of inoculation, spores develop. When the culture is complete, put it in a dryer and dry the spores. The spore drying temperature is about 40℃ for about 48 hours to be sufficiently dried so that the particles become hard. The generated large amount of spores is recovered by spore recovery method using water. First, the spores shaken in a mixture of Tween-80 in water were filtered with filter paper, and then the spores collected on filter paper were dried and recovered. (Fig. 9).

Example 5: Invention Beauberia Ana Bridge (Beauveria bassiana ) Bioverification of microbial pesticides containing M130 strain and its spores

Greenhouse powder and tobacco powder were bred for bioverification, and tomato, angel trumpet, and pepper were used as host plants. The host plant was planted in the breeding box and then inoculated with tobacco powder and greenhouse powder to induce spawning on the leaves. After inoculation of powdery (cigarette, greenhouse), eggs and larvae were checked about 15 days, and the present invention Beauveria bassiana prepared according to Example 4 bassiana ) M130 strain spores were diluted to 1.0 ~ 1.8 × 10 ⁸ cfu/mL and spread evenly on the foliar surface. In order to test the control effect for each number of treatments, it was treated up to 3 times (Table 1).

In order to induce spore germination, a temperature of 25°C and a humidity of 85% were maintained, and in order to increase significance, abandon was changed and repeated 5 times to investigate. The survey results are shown in FIGS. 10 and 11.

division 1st processing day 2nd processing day 3rd processing day Processed flowerpot Number of treatment abandonment microbe
process PDA
badge microbe
process PDA
badge microbe
process PDA
badge Treatment once 10.25 10.31 2 10 2 treatment zone 10.28 11.4 2 10 Treatment 3 times 10.31 11,7 2 10 No treatment 2 10

The present invention has the effect of providing a novel insect pathogenic fungus strain derived from a domestic melon-packed aphid, which has not been known so far, and the nucleotide sequence of the gene, and provides a microbial preparation having insecticidal activity against greenhouse powder and tobacco powder. Rather, it is a very useful method for eco-friendly agriculture because it has an excellent effect of providing biological pesticides that can replace chemical pesticides.

Korea Research Institute of Bioscience and Biotechnology KCTC12104BP 20111216

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Claims (1)

The genomic sequence of strain Beauveria bassiana M130 (KCTC12104BP) comprising SEQ ID NO: 1 derived from aphid carcass of a melon pavement in which the greenhouse powder or tobacco has a fungicidal activity.

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