KR100485604B1 - Microbial nematocide containing a novel nematocidal microorganism , process for producing and using the same - Google Patents

Abstract

The present invention relates to a microbial nematode, a manufacturing method and a method for treating the plant by feeding the parasitic nematodes of the plant to protect the plant from damage such as root growth or root rot caused by plant growth. The microbial nematicide of the present invention is Arthrobotrys sp. A2003 (KCTC 10095 BP), Arthrobotrys sp. A2008 (KCTC 10096 BP), Arthrobotrys sp. A2010 (KCTC 10097 BP) and Monacrosporium sp. M3015 (KCTC 10116 BP) contains at least one novel nematode microbial strain, and contains a microbial delivery medium that can maintain the viability and activity of these strains, root root nematodes and root rot The nematode activity of plant parasitic nematodes, such as nematodes, is excellent and can be treated in various ways, including seeds, plants, soil and bedding.

Description

Microbial nematicides comprising a novel strain having nematicidal activity, a method for preparing the same and a method for treating the same {Microbial nematocide containing a novel nematocidal microorganism, process for producing and using the same}

The present invention relates to novel strains having nematicidal activity and microbial nematicides comprising them.

Plant parasitic nematodes can damage plant roots or damage the roots of plants as they inhabit the soil. Root-knot nematode (Meloidogyne sp.), Root rot nematode (Pratylenchus sp.), Spiral nematodes (Helicotylenchus sp.), Ssiseu root nematodes (Heterodera sp., Globodera sp. ) Damage caused by plant root parasitic nematodes due to the most It occurs in the roots of this soil, and the damage progresses slowly, so other symptoms on the ground are not obvious and often make difficult diagnosis. Therefore, the damage is difficult to identify properly before the roots of the plant are not properly controlled, and the damage is likely to proceed very seriously. Plant parasitic nematodes are parasitic to roots, stems and leaves of crops, blocking the passage of nutrients and depriving them of nutrients. Among the plant parasitic nematodes, Aphelenchoides fragariae , a nematode that damages the top of the plant, occurs on strawberry leaves and causes enormous damage to strawberry crops. In particular, when the roots of plants are damaged by nematodes, the roots cannot absorb nutrients or water effectively, resulting in poor growth of plants, poor fruit size and fidelity, resulting in reduced production, and economic loss such as killing crops. This is caused.

Root- knot nematodes ( Meloidogyne sp.) Were first discovered in cucumber root knots grown in greenhouses in England in the 1850s, and dozens of species have now been reported worldwide (Jepson, 1987). In Korea, carrot root gall nematodes ( M. hapla ), peanut root gall nematodes ( M. arenaria ), and sweet potato root gall nematodes ( M. incognita ) mainly damage crops. Peanut root-knot nematodes and sweet potato root-knot nematodes are damaging plant cultivated crops. These nematodes include pumpkin, melon, tomato, watermelon, cucumber, eggplant, strawberry, pepper, tobacco, carrot, peanut, radish, lettuce, spinach, pear, burdock, onion, sesame, ginseng, soybean, grape, peach, rose Briar) and potatoes are causing widespread damage. In Korea, as economic cultivation of economical crops is activated, the damage of root-knot nematodes caused by the series is very high. In Seongju, Gyeongbuk, for example, the damage of root-knot nematodes is very serious each year due to melon series.

As a method for controlling root-knot nematodes, various methods such as freshwater, fallow, rotation, heat treatment, drug control, and development of resistant crop varieties are known. Root-knot nematode control using non-host crops has the advantages of high effectiveness and low cost, but it is unrealistic considering the fact that root-knot nematodes have a wide host range and economical crops are repeatedly produced. Pharmaceutical nematicides include fumigants, contact agents, and penetrant nematodes. Since around 1950, ethylene bromide, a fumigant widely used for the control of plant parasitic nematodes, has been banned due to its high toxicity. Chemical nematicides are treated in soil before planting, and their use is severely restricted due to weakness due to non-selective toxicity, soil contamination, ecosystem destruction, and residual toxicity.

Examples of the use of environmentally friendly nematode microorganisms compared to toxic chemical pesticide nematicides include Arthrobotrys conoides (US Pat. No. 5,811,092) and Athrobotrys irregularis (US Pat. 4,666,714), Arthrobotrys amerospora (US Pat. No. 4,421,544), Arthrobotrys oligospora (EU Pat. No. 0,623,284, SU 1,688,818, RU 2,106,092). ), Arthrobotrys srobusta , Arthrobotrys cladodes , Trichothecium flagrans ( British patent 885,161), Monacrosporium salinum Control of parasitic nematodes using microorganisms such as) (French Patent No. 2,747,016). For the charging effect it was unclear.

It is an object of the present invention to provide a novel nematicidal microorganism strain with excellent activity for killing parasitic nematodes such as nematodes, in particular root knot nematodes and root rot nematodes.

It is also an object of the present invention to provide a microbial nematicide comprising the above strain, suitable for seed, plant root seedling or soil treatment, and suitable for maintaining nematicidal microbial strains in a viable state.

Another object of the present invention is to provide a method for producing the microbial nematicide.

In addition, an object of the present invention is to provide a method for simply using the microbial nematicide.

Strain of the present invention ahsseuro boat-less strain having the nematocide activity A2003 (Arthrobotrys sp. A2003, KCTC 10095 BP), ahsseuro boat-less strain A2008 (Arthrobotrys sp. A2008, KCTC 10096 BP), ahsseuro boat-less strain A2010 (Arthrobotrys sp A2010, KCTC 10097 BP) or Monacrosporium strain M3015 ( Monacrosporium sp. M3015, KCTC 10116 BP). These strains can enhance the healthy growth of plants by feeding on plant root parasitic nematodes, such as root nematodes and root rot nematodes, as novel strains.

The microbial nematicide of the present invention is characterized by comprising at least one of the above strains. The microbial nematicide, preferably microbial delivery medium. Plants to which this nematicide may be applied include, for example, pumpkins, melons, tomatoes, watermelons, cucumbers, eggplants, strawberries, peppers, tobacco, carrots, peanuts, radishes, lettuce, spinach, pears, Includes but is not limited to burdock, onion, sesame, ginseng, soybeans, grapes, peaches, or potatoes.

The delivery medium refers to a medium capable of maintaining and regenerating nematicidal activity of the strain, and may be classified into granular, powder and liquid phases according to its properties.

The dual granular delivery medium is in the form of granules, and any one may be included as long as it can induce the activity and regeneration of the strain. For example, a mixture of 1 part of sand (20 mesh) and 1 part of loam or loess; Chopped potatoes (1-5 mm); And PDB (Potato Dextrose Broth, Difco) (24 g / L) in a ratio of 4: 0.2 to 1.5: 0.2 to 0.9 (w / w / w), including wheat, barley or rice, sand (20) Mesh) and loam or a mixture comprising loess and chitin, corn flour or 100 parts by weight of corn flour and 0.1 to 25 parts by weight of chitin in a ratio of 0.8 to 0.9: 0.8 to 0.9: 0.2 to 0.4 (v / v / v); Chopped potatoes (1-5 mm); And PDB (24 g / L) in a ratio of 4: 0.2 to 1.5: 0.2 to 0.9 (w / w / w), respectively, a mixture of vermiculite, bran or vermiculite and 0.1 to 25 parts by weight of bran; Ground potatoes; PDB (24 g / L); And brown rice green tea at a ratio of 4: 0.2 to 1.5: 0.2 to 0.9: 0.001 to 0.01 (w / w / w / w), respectively.

In addition, the powder delivery medium has a powder form. As long as it can induce the activity and regeneration of the strain, any one may be included. For example, it is preferable to include a mixture of 100 parts by weight of wheat bran, starch, chitosan, sawdust, chitin, corn flour, soy flour, kaolin, diatomaceous earth, vermiculite or corn flour and 0.1 to 25 parts by weight of chitin.

In addition, the liquid delivery medium is in the form of a liquid, as long as it can induce the activity and regeneration of the strain, it may include any. For example, medium or sucrose (20 g / l) consisting of sucrose (20 g / l) and soybean meal (5 g / l), yeast extract (2 g / l), NaNO ₃ (1.82 g / l) _{), KH 2 PO 4 (0.25g} / ℓ), MgSO 4 · 7H 2 O (0.05g / ℓ), a very small amount small circle _{(1.0㎖ / ℓ) [MnSO 4} .4H 2 O (0.005g / 100㎖), FeSO ₄ . _{7H 2 O (0.002g / 100㎖)} , ZnSO 4 · 4H 2 O (0.02g / ℓ), CuSO 4 .7H 2 O (0.001g / 100㎖), CoSO 4 .7H 2 O (0.001g / 100㎖ It is preferable that it is a liquid culture medium consisting of).

In the microbial nematicide of the present invention, the strain may vary depending on the type of nematode to be applied to the plant or plant part of the plant, the soil or the control, for example, tomato and melon rootworm nematode If the purpose of the control, the strain is preferably included as a colony forming unit per g or ml of the carrier medium is 1.0x10 ³ or more cell number.

The preparation method of the granular microbial nematicide of the present invention is characterized by comprising the following steps:

(a) inoculating a strain having nematicidal activity on a granular microorganism delivery medium and incubating at 25 to 30 ° C; And

(b) drying the obtained culture.

The granular microorganism delivery medium may include any one that can maintain, grow and regenerate the activity of the strain having the nematicidal activity of the present invention, for example, sand (20 mesh) 1 part skin and loam Or a mixture of 1 part of loess; Chopped potatoes (1-5 mm); And a mixture of 100 parts by volume of vermiculite, vermiculite, bran or vermiculite and 0.1-25 parts of bran, comprising PDB (24 g / L) in a ratio of 4: 0.2 to 1.5: 0.2 to 0.9 (w / w / w), respectively; Ground potatoes; PDB (24 g / L); And delivery medium comprising brown rice green tea at a ratio of 4: 0.2 to 1.5: 0.2 to 0.9: 0.001 to 0.01 (v / w / w / w), 0.8 to 0.9 parts of sand (20 mesh), loam or loess 0.8 to A mixture of 0.9 parts by weight, chitin, corn flour or 100 parts by weight of corn flour and 0.1-25 parts by weight of chitin; Chopped potatoes (1-5 mm); And a delivery medium comprising PDB (24 g / L) in a ratio of 4: 0.2 to 1.5: 0.2 to 0.9 (w / w / w), respectively, and a particulate microbial delivery medium selected from the group consisting of wheat, barley and rice. Is preferred.

Preferably, the cultivation is performed in a solid culture, while shaking the culture well at 25 ° C. to 30 ° C. for 2 days at a constant 2-5 days, and shaking culture for 1 to 5 days at 28 ° C. and 140 rpm for shaking culture. will be.

The drying step may use a conventional drying method. For example, it may be carried out by drying slowly or freeze drying in the shade. If necessary, the method may include crushing the dried nematicide.

In addition, the preparation method of the granular microbial nematicide of the present invention is characterized by comprising the following steps:

(a) culturing the strain having nematicidal activity of the present invention to obtain the cells;

(b) uniformly mixing the obtained cells with a powdery delivery medium selected from sterilized bran, starch, chitosan, sawdust, chitin, corn flour, soy flour, kaolin, diatomaceous earth or vermiculite; And

(c) gelatin (0.2 to 3.0 w / v), lignin sulfate (2 to 30 w / v), agar (0.2 to 2.0 w / v), xanthan gum (1 to 20% w / v), starch ( 0.5-30% w / v), glycerol (6-30% v / v), Tween 20 (0.5-99% v / v). Tween 80 (0.5-99% v / v), pectin (10-30% w / v), alginic acid (3-25% w / v). And coating the coated wheat, barley or rice with a surface treatment material selected from the group consisting of a mixture comprising at least two of them.

Preferably, the cultivation is performed in a solid culture, while shaking the culture well at 25 ° C. to 30 ° C. for 2 days at a constant 2-5 days, and shaking culture for 1 to 5 days at 28 ° C. and 140 rpm for shaking culture. will be. In addition, the mixing may use a conventional mixing means generally known.

The method for preparing powdery microbial nematicides of the present invention is characterized by comprising the following steps:

(a) having the nematicidal activity on a microbial delivery medium comprising a mixture of 100 parts by weight of wheat bran, starch, chitosan, sawdust, chitin, corn flour, soy flour, kaolin, diatomaceous earth, vermiculite or corn flour and 0.1 to 25 parts by weight of chitin. Mixing the strains; And

(b) drying the mixture.

The mixing may use a conventional mixing means generally known. In addition, the drying may be a common drying means known in general, preferably lyophilization. In addition, the strain may be grown or maintained in the culture, or may be freeze-dried cells.

Method for producing a liquid microbial nematicide of the present invention, a medium consisting of sucrose (20g / l) and soybean meal (5g / l); Or sucrose (20 g / l), yeast extract (2 g / l), NaNO ₃ (1.82 g / l), KH ₂ PO ₄ (0.25 g / l), MgSO _{4 ·} 7H ₂ O (0.05 g / l), Trace Element Liquid (1.0 mL / L) [MnSO ₄ H ₂ O (0.005 g / 100 mL), FeSO ₄ . _{7H 2 O (0.002g / 100㎖)} , ZnSO 4 · 4H 2 O (0.02g / ℓ), CuSO 4 .7H 2 O (0.001g / 100㎖), CoSO 4 .7H 2 O (0.001g / 100㎖ Inoculating the strain having a nematicidal activity in the liquid culture medium consisting of) characterized in that it comprises the step of culturing.

In the nematicidal preparation method of the present invention, the strain may vary depending on the type of nematode to be applied to the plant or plant part, the soil, or to the control, for example, tomato and melon root nodules. If nematode control is desired, it is preferable to incubate for a time such that a cell number of 1.0 × 10 ³ or more as colony forming unit per g or ml of the delivery medium.

In addition, the method of using the microbial nematicide of the present invention comprises the steps of treating the seed or root of the plant with a microbial nematicidate comprising the strain having the nematicidal activity of the present invention; Mixing with the mother bed formulation or admixing the soil; Or irrigation to the growing plant.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example

Example 1 Isolation and Preservation of Nematode Microorganisms

(1) Collection of Soil for Isolation of Nematode Microorganisms

Nematode-contaminated soils were collected from Icheon, Busan, Seongju, Gyeongsangbuk-do, Miryang and Samyangjin, Gyeongcheongbuk-do, Goesan and Boeun, etc. Nematodes were separated from the collected soil, and nematode predators were purely separated.

(2) isolation of nematodes

300 g of soil samples were placed in 5 liters of water, and the well-mixed muddy water was filtered with 60 mesh (250 µm), and the obtained muddy water was again filtered with 400 mesh (38 µm), and the mixture containing nematodes was collected in 400 mesh. The nematode containing the mesh mesh was collected with water, and the collected nematode-containing muddy water was poured into a funnel with a Kimwipe (175 μm), filled with an appropriate amount of water, and allowed to stand for 24 hours. The nematodes were collected by filtration with 500 mesh (26 μm).

(3) Isolation of Nematode Predatory Microorganisms

Nematode predatory microorganisms were separated by dividing soil samples into soil, 60 mesh and 400 mesh soil samples. 2% agar medium, 25% CMA-2% agar medium (25% corn meal agar (CMA) + 2% agar) were inoculated with nematodes prepared by the nematode separation method described in (2) above, and then at 1-2 ° C. Incubated daily. Here, inoculated with raw soil, 60 mesh and 400 mesh soil samples prepared in advance, the nematode was fed and grown under a microscope for 5 to 14 days while incubated at 25 ° C., and the grown microbial conidia were purely separated. It was transplanted into sterilized CMA medium and cultured at 25 ° C. for nematode predation microorganisms.

Pure nematode predatory microorganisms were randomly numbered according to the pure separation order to distinguish strains.

(4) Cultivation and preservation of purely isolated nematode predators

Purely isolated nematode predators were inoculated in CMA medium and incubated at 25 ° C. for 3-7 days to allow the microorganisms to grow well, and then stored at 25 ° C. at 6-8 week intervals. Long-term storage of purely isolated microorganisms was stored by freeze drying.

Example 2. Nematode predatory organs of nematode microorganisms

Place agar fragments containing each nematode predatory microorganism grown in CMA medium in the center of sterile 1.5% agar medium, with 1 repetition of 200 corrosive nematodes, 2 repetitions and 3 repetition of 300 root root nematodes. After 7 days of cultivation at, the living nematodes were examined under a microscope, and the organs having nematode phagocytic activity of each nematode microorganism isolated were examined. Corrosive nematodes were isolated from soil collected from Boeun, Chungbuk. Root-knot nematodes carefully washed the melon roots collected from Icheon cultivated field in Gyeonggi-do, and cut the roots to 1cm long. Next, place in a container containing 300 ml of 1% NaOCl (sodium hypochloride) solution, shake well, remove the root fragments with 60 mesh of the mixed root and nematode eggs, and use the eggs collected at 500 mesh for nematode prevalence. It was. The results of the observation of the predatory organs of purely isolated nematode microorganisms are shown in Table 1.

As shown in Table 1, nematode microorganisms isolated from pure soil collection in different regions were able to produce different nematode predatory organs such as adhesive networks, rings, knobs, and columns. It formed and showed nematicidal activity. In particular, A2003, A2008, A2010, and M3015 killed 95-100% of nematode inoculated with nematode predatory organ formation and excellent nematode activity.

Example 3. Atsurobotris sp. A2003, Astrobotris A2008, Astrobotris sp. A2010 or monacrosporium sp. Identification of M3015 Strain

For identification of purely isolated root-knot nematode predators, each nematode predatory microorganism was inoculated in 2% agar medium and incubated at 25 ° C. for 3 days. Next, inoculated nematodes on each medium and incubated for 5 to 14 days at 25 ℃ morphological characteristics and microscopic observation of the predatory organs, conidia spores, conidia spores of the microorganisms growing while feeding the nematodes and ITS1-5.8 Root-knot nematode predatory microorganisms isolated from pure S-ITS region sequences compared to previously reported microbial strains were identified. Morphological characteristics of purely isolated A2003, A2008, A2010, M3015, etc. are shown in Tables 2a, b and c, and the base sequences are shown in the sequence listing.

(1) Morphological features

A2003, A2008, A2010, and M3015 were grown by forming white mycelium and conidia on the CMA medium and 2% agar medium. M3015 showed slower growth on CMA and 2% agar compared to A2003, A2008 and A2010. As shown in Tables 2a, b and c, A2003, A2008, and A2010 all produced sticky net predators. M3015 produced a sticky rod predator. A2003, A2008, and A2010 all produced nodes, and when A2008 and A2010 were cultured in 0.2% CMA-2% agar medium, condensed spore branch was sometimes found. As many as 15 conidia-generating nodes of A2003 and up to 6 conidia-generating nodes of A2010 were observed, forming a capitate. M3015 did not create branches or nodes. The conidia of tip of A2003 was swollen and swollen with this shape denticle. The constellation of the conidia of the A2008 in the cortex was coral, and the corrugation was observed in this form. In the A2010, the conidia of the tip of the conidia showed a lot of this shape protrusion and coral. The conidia tip shape of M3015 blunted as if the mycelium was cut. The height of the conidia of the A2003 was 349.7 (254 to 514) ± 52.13 μm, the first diaphragm of the A2003 was at 3.613 (1 to 16) ± 2.185 μm, and the conidia of the butterfly was 7.696 (6 to 10). ) ± 0.981 μm. The height of conidia of A2008 was 392.9 (296-536) ± 55.16㎛, and the first diaphragm of A2008 was present at 3.07 (0.5-18) ± 3.98㎛, and the conidia of butterfly was 7.37 (5 ~ 10.5). ) ± 0.96 µm. The height of the conidia of the A2010 was 372.6 (222 to 512) ± 70.73 μm. The first diaphragm of the A2010 was located at 3.24 (0.5 to 20) ± 2.938 μm. The conidia of the butterfly was 8.162 (6.5 to 6.5). 10) ± 0.902 µm. The height of the conidia of the M3015 was 309.6 (202-368) ± 33.97 μm, and the first diaphragm of the M3015 was located at 2.384 (0.25-7) ± 1.6012 μm, and the conidia of the butterfly was 4.456 (3 to 6.5). ) ± 0.7066 μm. There were 6 to 8 conidia diaphragm diaphragms in A2003 and A2010, and 6 to 7 conidia diaphragm diaphragms in A2008. The conidia of A2003 produced 7 to 15, with 8 to 10 dominant. Up to four conidia producing nodes of A2008 were observed. Conidia produced 4-8 in the form of cosmos petals, with 4-6 conidia mostly. The conidia of A2010 produced 8-13 spores. The conidia of M3015 produced one. A2003 mainly produced broad obovoids and some elongate obovoid conidia. A2008 produced long obvoids, pyriforms and broad obvoid conidia, mostly long obvoids, followed by pyriforms and some broad obvoids. A2008 is also rarely observed for spores that are longer than long obvoids and have long and narrow butterflies that differ in the shape of pyriforms. A2010 produced wide obvoids and long obvoid conidia, with more broad obvoids observed. A2003 produces conidia with one diaphragm at 34.79 (27.5-44.87) ± 3.48%, A2010 produces 38.36 (27.78-62.22) ± 4.21%, and A2008 produces conidia with 1-4 diaphragms. Most of the conidia with one or two diaphragms were formed, conidia with three diaphragms were observed, and spores with four diaphragms were rarely observed. M3015 consists of a round head carrot (round cone) and a round head long carrot (fusiform), opposite the conidia of the conidia of the tip of a typical fusiform conidia with four diaphragms. elongate carrot), a conical conidia of the opposite direction of the conidia of the conidia tip, which is the half of a typical fusiform conidia with four diaphragms. M3015 produced conidia with two to four diaphragms, mostly conidia with four diaphragms, and conidia with two or three diaphragms. Concentric conidia of M3015 with two diaphragms were often round-shaped carrots (round cones) in the opposite direction of the conidia-diameter tip, and round-shaped long carrots and spindles were observed. The three diaphragm of the M3015 had a lot of spindle shape, a long carrot and a conical spore in the shape of the conidia in the direction opposite to the tip of the conidia. The locations of the conidia diaphragms of A2008 and M3015 are detailed in Tables 2a, b and c.

±: standard deviation

As shown in Tables 2a, b, and c, the conidia butterfly of A2003 was 12.13 (10.50 to 14.50) ± 0.74 m, and the conidia length was 21.22 (18.25 to 27.25) ± 1.90 m. The conidia butterfly of A2010 is 13.25 (10.25-16) ± 0.859 μm and the conidia length is 23.78 (20 to 27.75) ± 1.646 μm. The conidia butterfly of A2008 is 13.42 (9.25 to 16.25) ± 1.12 μm (diaphragm 1), 14.5 (10.75 to 20) ± 1.53 μm (diaphragm 2), 15.23 (11.5 to 19) ± 1.66 μm (diaphragm 3) Spore length was 26.47 (20.5-37.5) ± 2.82 µm (diaphragm 1), 30.29 (24, 25-38.5) ± 3.50 µm (diaphragm 2), 35.05 (25.75-45.5) ± 4.37 µm (diaphragm 3). The conidia of M3015 are 11.9 (5.25 to 17) ± 2.68 μm (diaphragm 2), 8.357 (6 to 15) ± 2.169 μm (diaphragm 3), 13.1 (10 to 17) ± 1.62 μm (diaphragm 4) The lengths are 32.7 (20 to 47) ± 6.18 µm (diaphragm 2), 35.58 (27.5 to 44.5) ± 3.712 µm (diaphragm 3), and 43.32 (37 to 55.5) ± 4.524 µm (diaphragm 4). Three diaphragm conidia of A2008 were observed less than one or two diaphragms. Two to three diaphragm conidia of M3015 were observed less than four diaphragm.

As shown in Tables 2a, b and c, A2003, A2008, A2010, and M3015 are new species with distinctive morphological features when compared to previously reported species of the genus Astrobotris and Monacrosporiium. Was identified.

(2) Astrobotris sp. A2003, Astrobotris sp. A2008, Astrobotris sp. A2010 or monacrosporium sp. Nucleotide Sequences of the ITS1-5.8S-ITS2 Region of M3015

To identify the nucleotide sequences of the ITS1-5.8S-ITS2 region of A2003, A2008, A2010 and M3015, PCR using ITS5 primers starting from the end of the nuclear small rDNA and nuclear large rDNA (nuclear) Base sequences were read bidirectionally using ITS4 primers starting at the beginning of large rDNA), and the nucleotide sequences of the ITS1-5.8S-ITS2 region were shown in SEQ ID NOs: 1 to 4, respectively. As shown in SEQ ID NOs: 1 and 3, the sequence of the ITS1-5.8S-ITS2 region of A2003 and A2010 is significantly different due to the large number of insertions of previously unreported bases, compared to the previously reported base sequences of the genus Astrobotris. Was identified as a new species. The ITS1-5.8S-ITS2 region sequence of A2008 was also identified as a new species with significant differences compared to the previously reported sequence of the genus Astrobotris. In addition, the ITS1-5.8S-ITS2 region sequence of M3015 was classified as a novel species with a significant difference when compared with the previously reported sequence of the genus Monacrossporium.

According to the identification method of nematode predatory microorganisms, the morphological characteristics (Tables 2a, b and c) and the nucleotide sequences of the ITS1-5.8S-ITS2 region of SEQ ID NOS: 1 to 4 were analyzed. Genus M3015 was identified as Monacrosporium. A2003, A2008 and A2010 are the most important considerations in the identification of predator organs, conidia speculum shape, conidia tip shape, conidia diameter height, conidia generation node number, conidia number, conidia shape, conidia diaphragm location, A2003, A2008, and A2010 are new species when comparing the number of conidia diaphragm, conidia butterfly, conidia spore length, and nucleotide sequence of ITS1-5.8S-ITS2 region with those of known genus Astrobotris. Arthrobotrys genospora A2003, Arthrobotrys innospora A2008, and Arthrobotrys novospora A2010. In addition, M3015 was identified as Monacrosporium monosporum M3015 as a novel species when compared to a known species of the genus Monacrosporium . On the other hand, Atsurobotris sp. A2003, Astrobotris sp. A2008, Astrobotris sp. A2010 and Monacrossporium sp. On October 29, 2001 and November 13, 2001, M3015 was deposited with the Gene Bank of the Institute of Biotechnology, Daejeon Metropolitan City, an international depository under the Treaty of Budapest, with the deposit numbers KCTC 10095 BP, KCTC 10096 BP, KCTC 10097 BP and KCTC 10116. Deposited as BP.

Example 4. Preparation of Liquid Microbial Nematodes

(1) Determination of Cell Number of Nematode Microorganisms

The growth of nematicidal microorganisms was expressed as the number of cells / ml through microscopic observation or cfu / ml by continuous dilution and smearing.

The growth of nematode microbial strains of microbial nematicides produced by solid culture was investigated by incubating for 1-5 days at 25 ° C. using CMA medium by continuous dilution and smearing method using sterile water, and the number of cells per 1g colonies. It is expressed as a colony forming unit (cfu).

(2) spawn culture

Seed of Arthrobotrys in nematocides microorganism CMA (17g / ℓ) was used for each strain grown in the medium, Monacrosporium in live inoculum of nematodes microorganism CMA (17g / ℓ), glucose (6g / ℓ), yeast extract (0.5 g / l), asparagine (0.5 g / l), KH ₂ PO ₄ (0.1 g / l), _and respective strains grown in MgSO _{4 ·} 7H ₂ O (0.05 g / l) medium were used. As the spawn culture medium, a sterile medium composed of sucrose (20 g / l) and soybean meal (5 g / l) was used. Nematode predatory microorganisms of Arthrobotrys were inoculated into a 250 ml Erlenmeyer flask containing 50 ml of sterilized liquid medium and incubated at 140 rpm at 28 ° C. for 1 to 5 days to be used as a seed for liquid shaking culture or large fermentation.

(3) Production of liquid microbial nematicides

20 ml of each spawn culture medium prepared in (2) was inoculated into a 1,000 ml Erlenmeyer flask containing 200 ml of sterilized liquid medium and incubated at 28 ° C. at 140 rpm for 1 to 5 days. Liquid medium is sucrose (20g / l), yeast extract (2g / l), NaNO ₃ (1.82g / l), KH ₂ PO ₄ (0.25g / l), MgSO _{4 ·} 7H ₂ O (0.05g / l ), A sterile medium consisting of a trace element liquid (1.0 mL / L) was used. Trace elements solution _{MnSO 4 .4H 2 O (0.005g /} 100㎖), FeSO 4 .7H 2 O (0.002g / 100㎖), ZnSO 4 · 4H 2 O (0.02g / ℓ), CuSO 4 .7H 2 O (0.001g / 100㎖), CoSO were composed of _{4 .7H 2 O (0.001g / 100㎖} ). Liquid microbial nematicides were produced by aseptically harvesting nematode microorganisms, and the number of cells (cfu / ml) was recorded as an average value and is shown in Table 3.

As shown in Table 3, the above liquid medium was used to effectively produce a microorganism nematicide of 10 ³ cfu / ml or more having nematicidal activity.

Example 4. Production of powdery microbial nematicides

Nematode microorganisms produced by liquid culture were mixed with chitin or corn flour sterilized at 121 ° C., or a delivery medium consisting of chitin and corn flour. Nematode microorganism-delivery medium was frozen in a -70 ° C. cryogenic freezer and dried using a freeze dryer, so that the nematic microorganism content was 1.0 x 10 ^3-4 cfu / g or higher. Nematodes were produced. As the delivery medium, chitin or corn flour was used alone, or 100 parts by weight of corn flour and 0.1-25 parts by weight of the mixed delivery medium were sterilized at 121 ° C. for 30 minutes.

Example 5. Production of Granular Microbial Nematodes

Inoculate each nematode microorganism grown on CMA medium to a sterile delivery medium at 121 ° C., and granulate microorganism nematode by incubating for 5 to 14 days while shaking the culture well at 25 ° C. to 30 ° C. for 2 days. Produced.

In addition, potato, PDB (24 g / L), and brown rice green tea, which are finely pulverized in a mixture of 100 parts by weight of vermiculite, bran or vermiculite and 0.1 to 25 parts by weight of bran, respectively, from 4: 0.2 to 1.5: 0.2 to 0.9: 0.001 to A microbial nematicide was produced by constructing a ratio of 0.01 (v / w / w / w).

As an example of the delivery medium, the mixture was sterilized at 121 ° C. for 30 minutes in vermiculite (1 liter), liver potatoes (135 g), PDB (3.24 g) and brown rice green tea (1.5 g), followed by A2003, A2008, A2010, and M3015. Inoculation and incubated at 28 ℃. Further, a delivery medium composed of ground potato (135 g), PDB (3.24 g), and brown rice green tea (1.5 g) was added to a mixture of 100 parts by weight of bran (1 liter) or vermiculite and 0.1 to 25 parts by weight of bran (1 liter). After sterilization at 30 ° C. for 30 minutes, A2003, A2008, A2010, and M3015 were inoculated, and cultured at 28 ° C. for 5-14 days to produce microbial nematode. The activity of the granular microorganism nematode produced was shown in Table 4 by examining colonies in which each nematode microorganism grew in CMA medium.

As shown in Table 4, A2003, A2008, A2010, and M3015 granular microbial nematicides were effectively produced using the above-described delivery medium.

division Growth of granular microbial nematode A2003 A2008 A2010 A3015 Vermiculite (1ℓ) + Potato (135g) + PDB (3.24g) + Brown Rice Green Tea (1.5g) + + + + Bran (1ℓ) + Potato (135g) + PDB (3.24g) + Brown Brown Tea (1.5g) + + + + Vermiculite (833 ml) + Bran (167 ml) + Potato (135 g) + PDB (3.24 g) + Brown rice green tea (1.5 g) + + + +

+: Cell growth

In the production of another granular microbial nematode, the delivery medium used wheat, barley or rice. Wheat, barley or rice was put 250g each in a 1,000ml Erlenmeyer flask and sterilized for 30 minutes at 121 ℃ and then inoculated each nematode predatory microorganisms and incubated at 25 ℃ for 5-14 days. The cell activity of the granular microbial nematode was confirmed by cell growth from the granules on CMA medium, and produced an effective microbial nematode having the growth of nematicidal microorganisms of each of the granular microbial nematicides.

In another method, a nematic microorganism was coated on wheat, barley or rice to produce a granular microbial nematicide. That is, each of A2003, A2008, A2010 or M3015 is produced as described in Examples 4 (2) and 4 (3), and then bran, starch, chitosan, sawdust, chitin, corn flour, soy flour, kaolin, diatomaceous earth. Alternatively, the microorganisms were mixed with each other and then freeze-dried to prepare a highly concentrated powdery microbial nematicide (Table 5a).

Granular microbial nematicides were produced by coating the prepared powdery microbial nematicides on wheat, barley or rice delivery media, and the results are shown in Table 5b below. Wheat, barley or rice delivery media include gelatin (0.2 to 3.0 w / v), lignin sulfate (2 to 30 w / v), agar (0.2 to 2.0 w / v), xanthan gum (1 to 20% w / v), Starch (0.5-30% w / v), glycerol (6-30% v / v), tween 20 (0.5-99% v / v). Surface selected from the group consisting of tween 80 (0.5-99% v / v), pectin (10-30% w / v), alginic acid (3-25% w / v) and mixtures comprising at least two of these The coating was used as a treatment material and sterilized at 121 ° C. for 30 minutes.

As shown in Table 5b, 10 ⁴ to 10 ⁵ cfu / g of granular microbial nematicide was effectively prepared depending on the strain used.

microbe Cell count (cfu / g) Wheat ¹ Barley ² Rice ³ Atsu Robotris Strain A2003 ^1.5 x 10 ⁵ 1.7 x 10 ^{5 1.5} x 10 ⁵ Atsu Robotris Strain A2008 1.3 x 10 ⁵ 2.0 x 10 ⁵ 1.0 x 10 ⁵ Atsu Robotris Strain A2010 1.1 x 10 ⁵ 1.1 x 10 ⁵ 1.1 x 10 ⁵ Monacrossporium strain M3015 1.7 x 10 ⁴ 1.3 x 10 ⁴ 1.0 x 10 ⁴

¹ Chitin powdery microbial pesticide coating 2 Soy flour powdery microbial pesticide coating 3 Kaolin powdery microbial pesticide coating

Example 6. Plant Parasitic Nematode Control Activity of Microbial Nematodes

Meloidogyne sp., Pratylenchus sp., Helicotylenchus sp., Tylenchus sp., Nematodes ( Criconematidae sp.), The parasitic nematode control activity of Aphelenchus sp. Was investigated by cultivating pots (15 cm in diameter and 15 cm in height) in tomato plants. Root-knot nematodes ( M. arenaria, M. incognita , etc.) (195 cats / 100 g infected soil), Pratylenchus sp., Helicotylenchus to induce nematode damage in tomato ( Totorak yoko ) plants sp.), Tylenchus sp., C.nematomatidae sp. and Aphelenchus sp. . Tomato pot cultivation was used by mixing 2: 1 (v / v) of nematode infected soil and peat moss soil. The microbial nematicide treatment was treated by mixing 20 g of microbial nematicides in 800 g [(in the building) / port of nematode infected soil and peat moss topological mixture [2: 1 (v / v)]. The microbial nematicide was prepared by the method described in Example 5. Chemical pesticide treatment is based on the use, the cadusarfoss granules in 800 g [in the building] / 800 g of nematode infected soil and peat moss tops mixture [2: 1 (v / v)] 1 week before transplanting the tomato into the pot. 0.2 g of active ingredient cadusafos 3%) was mixed well and then used for tomato transplantation. Tomato seedlings used tomato seedlings (20㎝) grown on the bed for 30 days, planted in the pot, watered, and put the pot outdoors and managed. Tomato plant management was carried out in the same way as the general tomato cultivation, and irrigation treatment with water and four kinds of complex fertilizer as needed.

No treatment, microbial nematode treatment, chemical pesticide treatment, and the like were carried out in three replicates. After 45 days after transplanting the tomatoes, the tomatoes were extracted and root-knot nematodes, parasitic nematodes ( Pratylenchus sp.), And nematodes ( Tylenchus ) in the soil in the same manner as the separation of nematodes described in Example 1 (2). sp.), Criconematidae sp., Aphelenchus sp.], and free-living nematodes.

division Cell count (cfu / g) Average (mari / 800 g building / pot) Root-knot nematodes Parasitic nematodes a Free life nematodes No treatment 1,020  300 720 Chemicals b  180 120 600 A2008 8.3 x 10 ⁵   60  60 240 M3003 0.4 x 10 ⁴  180 290 710 M3014 0.7x10 ⁴  420 240 420

^{A number} of parasitic nematodes such as Pratylenchus sp., Helicotylenchus sp., Tylenchus sp., Nematodes ( Criconematidae sp.) and Aphelenchus sp. ^b 0.2 g of Kadusafos granules (3% active ingredient cadusafos) per pot before planting tomato seedlings.

As shown in Table 6, when the microbial nematode A2008, M3003, and M3014 were mixed with the pot formulation, and then transplanted into a tomato, compared to the control group without the microbial nematode, Meloidogyne sp. , Parasitic nematodes ( Pratylenchus sp., Helicotylenchus sp.), Nematodes ( Tylenchus sp.), Nematodes ( Criconematidae sp.), Nematodes ( Aphelenchus sp.) Life nematodes, etc. were greatly reduced. In particular, when treated with the microbial nematode A2008, root-knot nematodes, parasitic nematodes and free-living nematodes decreased by 94%, 80% and 67%, respectively, compared to the control group. , Parasitic nematodes and free-living nematodes decreased 82%, 60% and 17%, respectively. The microbial nematode A2008 was much more effective than chemical pesticides in the control of root-knot nematodes and parasitic nematodes. In other words, when the microorganism nematode A2008 was mixed with the pot formulation, the nematode control effect was reduced by 67% and 50%, respectively, compared with the chemical pesticide control agent. Excellent activity in control was shown.

Example 7. Tomato Root-knot Nematode Control Activity of the Microbial Nematode of the Present Invention

Root-knot nematode control activity of the nematicide containing the microorganism purely separated in Example 3 was investigated by cultivating tomato plant pot (15 cm in diameter, 15 cm in height). Root nocturnal disease induction of tomato ( Dotorak yoko ) was carried out using the melon plantation soil of Seongju area infected with M. arenaria, M. incognita , etc. ( 201/100/100 infected soil). Root-knot nematode infected soil and peat moss soil were mixed 2: 1 (v / v) in the pot to grow tomatoes. Chemical pesticide treatment is based on the use of Kadusapos granules in 800 g [in buildings] of 800 g [(building) / root] of root-knot nematode infected soils and peat moss tops mixture [2: 1 (v / v)] one week prior to transplanting tomatoes into pots. 0.2 g of active ingredient cadusafos 3%) was mixed well and then tomato was transplanted. Tomato seedlings were used after planting tomato seedlings (20 cm) grown in the bed for 30 days in a pot and placed in the open field. Tomato cultivation management was carried out in the same way as the daily tomato cultivation and irrigation treatment with water and four kinds of complex fertilizer as needed. The microbial nematode treatment was carried out after transplanting tomato seedlings into 800 g [in buildings] of 800 g [potatoes / pot] of root-knot nematode infected soils and peat moss soil mixtures. Separated from the pot and evenly sprayed 20 g of the granule microbial nematode containing each nematode microorganism around the pot compound and the stub roots growing roots, and then planted the tomato trees again and soaked water.

The microbial nematicide was prepared from Example 5. Treatment of the liquid microbial nematode A2008 produced by the seed culture medium of Example 4 (2) was 20ml (1.9x10 ⁴ cells / mL) of the liquid microbial nematode in the pot grown for 30 days after transplanting tomato seedlings into the pot. Was diluted 50 times in water and irrigated in a pot where tomatoes grow. No treatment, microbial nematode treatment, chemical pesticide treatment, and the like were carried out. Root-knot nematode control was performed after 60 days of treatment, and the roots and soil were carefully separated to examine the root-knots and egg sacs that occurred in the tomato roots. The ovum was immersed in 0.15% Phloxin B (sigma) solution and stained for 10 minutes, and the number of ovules stained red in a white container with the bottom was visually recorded. Root-knot nematodes were examined by the method described in Example 1 (2). That is, muddy water, which was well suspended in soil, was filtered through 60 mesh and muddy water was again filtered to 400 mesh to collect a mixture containing nematodes in 400 mesh. The nematode containing 400 mesh was collected in a beaker with water, poured into a funnel with a Kimwipe (175 μm), filled with an appropriate amount of water, and allowed to stand for 24 hours. The waste was collected by filtering. The number of collected nematodes was measured using a microscope, and the average values were recorded and shown in Table 7.

As shown in Table 7, microbial nematicides A2003 (2.5x10 ⁴ cfu / g), A2008 (8.3x10 ⁵ cfu / g), A2010 when the microbial nematode was treated after 20 days after transplanting tomatoes (2.0x10 ³ cfu / g) and M3015 (2.5x10 ³ cfu / g) were reduced by 88-91% and root-lump by 83-93% compared to the untreated control, whereas the chemical pesticide control Root-knot nematodes were reduced by 86% and root-knots by 84%, and the control of root-knot nematodes by microorganism nematodes A2003, A2008, A2010 and M3015 was more effective than chemical pesticides. In other words, the control effect of the root nocturnal nematode of the microorganism nematode A2003, A2008, A2010 was reduced by 11-33% and root nodules 8-52% of the root nocturnal nematode compared with the chemical pesticides, effectively controlling the root nocturnal nematode. In addition, M3015's root-knot nematode control effect was decreased by 28%, and root-knot nematode increased slightly (7%) compared with the chemical pesticide control. When the microorganism nematode M3015 was treated, the root nocturnal nematode was reduced by 95% compared with the control group, and 84% less than the chemical pesticide treatment, showing that it was effective in the control of the root nocturnal nematode. In addition, the treatment of the liquid microbial nematode A2008 (1.9x10 ⁴ cells / ml) decreased by 76% in the root nocturnal nematode, 88% in the root nocturnal nematode, and 88% in the root nocturnal nematode compared to the control group, whereas the chemical pesticide control drug was nodal root nematode. The root nocturnal nematode was reduced by 68%, root nocturnal nematode, 86% and root nocturnal 84%. That is, the microbial nematode A2003, A2008, A2010 and M3015 showed better nematode control effect than chemical pesticides.

^a 0.2 g / pot treatment with Kadusafos granules (3% active ingredient cadusafos) before planting tomato seedlings.

^b Treatment of 20 ml of liquid microbial nematode A2008 (1.9 x 10 ⁴ cells / ml) grown in liquid culture spawn medium [Sucrose (20 g / l) and Corn meal (2-10 g / l)].

As shown in the results of Table 7, microbial nematicides A2003 and A2008 effectively controlled root-knot nematodes at cell numbers of 10 ⁴ cfu / g and microbial nematicides A2010 and M3015 at 10 ³ cfu / g.

Example 8. Production of solid cultured granular microbial nematode

Cleanly washed wheat, barley or rice 1kg each into a 5 liter flask and sterilized for 30 minutes at 121 ℃ and incubated by inoculating the strain to be cultured. After inoculation, the cells were incubated at 25 to 30 ° C. for 5 to 14 days and dried to produce granular microbial nematicides (Table 8).

In addition, a 5 liter flask containing a delivery medium in which 200 to 500 ml of corn meal agar (CMA, corn meal agar, difco) was uniformly mixed with 1 kg of clean wheat, barley or rice was sterilized at 121 ° C. for 30 minutes. Spawn 100 ml each. Thereafter, the cells were left to incubate at 25 to 30 ° C. for 5 to 14 days and dried to produce granular microbial nematicides.

As shown in Table 8, A2003, A2008, A2010 and M3015 showed moderate and good cell growth, sporulation, and regeneration activity in solid culture delivery media consisting of wheat, barley or rice. It has been shown to be effective for solid culture production and stabilization.

microbe Cell growth Spore generation Play field active wheat barley rice wheat barley rice wheat barley rice Atsu Robotris Strain A2003 ++ ++ ++ ++ ++ ++ ++ ++ ++ Atsu Robotris Strain A2008 ++ ++ ++ ++ ++ ++ ++ ++ ++ Atsu Robotris Strain A2010 ++ ++ ++ ++ ++ ++ ++ ++ ++ Monacrossporium strain M3015 + + + + + + + + +

+: Normal, ++: good.

As shown in Table 9 below, the production and stabilization of effective microbial nematicides of 10 ⁶ ~ 10 ⁷ cfu / g through the solid culture using wheat, barley or rice as a delivery medium.

microbe Cell count (cfu / g) Wheat ^a Barley ^a Rice ^a Atsu Robotris Strain A2003 (KCTC 10095 BP) 1.6 x 10 ⁷ 2.1 x 10 ⁷ 1.3 x 10 ⁷ Atsu Robotris Strain A2008 (KCTC 10096 BP) 4.1 x 10 ⁷ 3.2 x 10 ⁷ 1.2 x 10 ⁷ Atsu Robotris Strain A2010 (KCTC 10097 BP) 5.2 x 10 ⁷ 7.6 x 10 ⁷ 3.9 x 10 ⁷ Monacrossporium strain M3015 (KCTC 10116 BP) 1.6 x 10 ⁶ 3.0 x 10 ⁶ 1.8 x 10 ⁶

^a granular microbial nematicide prepared by coating with CMA medium and then culturing after inoculation of microorganisms.

In order to investigate the stability of the granular microbial nematicide prepared by the method described above, the microbial strain viability and the regeneration field activity of the viable microorganism were measured and shown in Table 10a, b and c. In the case of the viability test, 1 g of each of the finally obtained samples was taken in 9 ml of sterilized distilled water, vortexed, and colony forming units (cfu) were measured by serial dilution and smearing.

microbe Initial cell count (cfu / g) Cell count after 3 months (cfu / g) Regeneration Field Activity of Surviving Cells Wheat ^a Wheat ^a Wheat ^a Atsu Robotris Strain A2003 (KCTC 10095 BP) 2.2 x 10 ⁷ 6.2 x 10 ⁶ +++ Atsu Robotris Strain A2008 (KCTC 10096 BP) 1.6 x 10 ⁷ 5.3 x 10 ⁶ +++ Atsu Robotris Strain A2010 (KCTC 10097 BP) 1.1 x 10 ⁷ 4.9 x 10 ⁶ +++ Monacrossporium strain M3015 (KCTC 10116 BP) 2.8 x 10 ⁶ 3.2 x 10 ⁵ +++

^a granular microbial nematicide prepared by coating with CMA medium and then culturing after inoculation of microorganisms.

+: Normal, ++: good, +++: very good

microbe Initial cell count (cfu / g) Cell count after 3 months (cfu / g) Regeneration Field Activity of Surviving Cells Barley ^a Barley ^a Barley ^a Atsu Robotris Strain A2003 (KCTC 10095 BP) 1.7 x 10 ⁷ 3.1 x 10 ⁶ +++ Atsu Robotris Strain A2008 (KCTC 10096 BP) 1.9 x 10 ⁷ 2.7 x 10 ⁶ +++ Atsu Robotris Strain A2010 (KCTC 10097 BP) 2.6 x 10 ⁷ 3.5 x 10 ⁶ +++ Monacrossporium strain M3015 (KCTC 10116 BP) 2.3 x 10 ⁶ 4.3 x 10 ⁵ +++

^a granular microbial nematicide prepared by coating with CMA medium and then culturing after inoculation of microorganisms.

+: Normal, ++: good, +++: very good

microbe Initial cell count (cfu / g) Cell count after 3 months (cfu / g) Regeneration Field Activity of Surviving Cells Rice ^a Rice ^a Rice ^a Atsu Robotris Strain A2003 (KCTC 10095 BP) 2.4 x 10 ⁷ 2.7 x 10 ⁶ +++ Atsu Robotris Strain A2008 (KCTC 10096 BP) 1.3 x 10 ⁷ 2.4 x 10 ⁶ +++ Atsu Robotris Strain A2010 (KCTC 10097 BP) 2.3 x 10 ⁷ 3.1 x 10 ⁶ +++ Monacrossporium strain M3015 (KCTC 10116 BP) 2.1 x 10 ⁶ 3.7 x 10 ⁵ +++

^a granular microbial nematicide prepared by coating with CMA medium and then culturing after inoculation of microorganisms.

+: Normal, ++: good, +++: very good

As shown in Table 10a, b and c, granular microbial nematicides survived stably after a certain period of time to maintain regeneration activity.

Example 9. Effects of Microbial Nematodes on the Melon

In order to verify the nematicidal activity against the root-knot nematodes of each of the granular microbial nematicides prepared by the method described in Example 8, a melon (gold leaf) packaging cultivation test was performed. In the field cultivation test, the test zones were placed in three repetition of the egg mass method, and 30 weeks of melon were grown per treatment zone. The drug treatment was mixed with soil 6kg per 300 pyeong two weeks before the melon meal. As a control, a test piece without any drug was placed. The nematode density of the package before the drug treatment was 300 to 400 per 300 g of soil, and after 40 days of treatment, the nematode rate of root-knot nematodes was examined and shown in Table 11 below.

microbe Wire density (treatment / week) Cell count (cfu / g rice) % Viability Cell count (cfu / g wheat) % Viability Cell count (cfu / g barley) % Viability Average Average Average Untreated control 359 77.7 ^a 77.7 ^a 77.7 ^a Atsukatris oligospora IBT 5010 372 1.7 x 10 ⁵ 26.9 ^b 2.1 x 10 ⁶ 22.6 ^b 1.2 x 10 ⁷ 18.8 ^b Atsu Robotris Strain A2003 400 2.9 x 10 ⁵ 25.0 ^b 3.2 x 10 ⁶ 21.3 ^b 2.5 x 10 ⁷ 17.3 ^b Atsu Robotris Strain A2008 384 3.4 x 10 ⁵ 22.4 ^b 3.9 x 10 ⁶ 19.8 ^b 2.4 x 10 ⁷ 15.4 ^b Atsu Robotris Strain A2010 391 5.1 x 10 ⁵ 24.0 ^b 4.1 x 10 ⁶ 22.3 ^b 3.5 x 10 ⁷ 19.9 ^b Monacrossporium strain M3015 370 4.6 x 10 ⁴ 21.4 ^b 3.4 x 10 ⁵ 19.5 ^b 2.8 x 10 ⁶ 18.9 ^b Monacrossporium Ellipsos Forum IBT 13015 350 1.9 x 10 ⁴ 26.3 ^b 2.5 x 10 ⁵ 22.3 ^b 1.5 x 10 ⁶ 19.7 ^b

^x When comparing the results of the untreated control group and the treated experimental group of the same column statistically different in the range of p = 0.05 are represented by different alphabets (a, b).

As shown in Table 11, when treated with the granular microorganism pesticide of the present invention (10 ⁴ ~ 10 ⁷ cfu / g), the viability rate of melon root nodules nematode (%) was 15.4 ~ 19.9%, compared to 77.7% of the control group 74 The mortality rate was greatly reduced to -80%.

This indicates that the nematicidal activity of the root-knot nematode of the present invention is very effective in the actual field test environment.

According to the microbial nematicide of the present invention, it contains a novel nematicidal microorganism, and is particularly excellent in the control and control activity of root-knot nematodes and root parasitic nematodes.

In addition, the microbial nematicide of the present invention can stably maintain a microbial strain having nematicidal activity.

In addition, the microorganism itself, liquid phase, granular or powdery form can be treated in various ways, such as seeds, plants, bedding, soil.

<110> SUH, Hyung Won <120> Microbial nematocide containing a novel nematocidal microorganism and process for producing and using the same <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 637 <212> DNA <213> Arthrobotrys genospora A2003 <400> 1 ccaatacaag ccggccggtt tgctgttgca gcttgttcga aagagcggtt gcgctgtctt 60 ccggttggta agccagcacc cgccttcccc gcaaggggca ggtttgggta cctggtaaac 120 cctttgtgaa ccaaaacaaa cctttcgctt cggcagctgg gccccgtctg ggacccgtca 180 gcctgccgct agcaccaaac aaaaaaactt gttgtcaaaa cattgtctga taaccaaaat 240 tttcgaatga aaatcaaaac tttcaacaac ggatctcttg gttcccgcat cgatgaagaa 300 cgcagcgaaa cgcgatagtt aatgtgaatt gcagaattca gtgaatcatc gagtctttga 360 acgcacattg cgcccattgg tattcctttg ggcatgtctg tttgagcgtc attacaaccc 420 tcagctaccc gctggttttg aacccgaacg gtgcccccta accggggaac cgagccggtt 480 ttaaagttgt aagctctgct ggccgctccg ccccaaccag aacatagtaa aatactactt 540 ttgttagggt caagcggaac ggtttttcgg cctgaacaaa acctaccctt tttcaaggtt 600 tgacctcaga tcagacaagg atacccgctg aacttaa 637 <210> 2 <211> 599 <212> DNA <213> Arthrobotrys innospora A2008 <400> 2 tccgtaggtg aacctgcgga aggatcatta ccaatgcagc gagaaatcgc tacaaacaac 60 ctgctggtgg ccccatccgg gctgccaacc ggtcaaccct ttgtgaacca aaaaaccttt 120 cgcttcggca gctgggctcc ctaacctggg cctgtcagcc tgccgctagc acccaaccaa 180 aaaacctgtt gtcaaacatt gtctgataac caaattttcg aatgaaaatc aaaactttca 240 acaacggatc tcttggttcc cgcatcgatg aagaacgcag cgaaacgcga tagttaatgt 300 gaattgcaga attcagtgaa tcatcgagtc tttgaacgca cattgcgccc attggtattc 360 ctttgggcat gtctgtttga gcgtcattac aacccctcag ctaccgctgg ttttgaattg 420 gaacgggtca cagcccgcgc cggttttaaa gttgtaagct ctgctggccg ctctgcccca 480 accagaacat agtaagcaac tacttgttag ggtgaagctg aacggtacgg cctgaacaaa 540 acctaccctt tctcaaggtt tgacctcaga tcagataagg atacccgctg aacttaagc 599 <210> 3 <211> 636 <212> DNA <213> Arthrobotrys novospora A2010 <400> 3 tccgtaggtg aacctgcgga aggatcatta ccaatacaag ccggccggtt tgctgttgca 60 gcttgttcga aagagcggtt gcgctgtctt ccggttggta agccagcacc cgccttcccc 120 gcaaggggca ggtttgggta cctggtaaac cctttgtgaa ccaaaacaaa cctttcgctt 180 cggcagctgg gccccgtctg ggacccgtca gcctgccgct agcaccaaac aaaaaaactt 240 gttgtcaaaa cattgtctga taaccaaaat tttcgaatga aaatcaaaac tttcaacaac 300 ggatctcttg gttcccgcat cgatgaagaa cgcagcgaaa cgcgatagtt aatgtgaatt 360 gcagaattca gtgaatcatc gagtctttga acgcacattg cgcccattgg tattcctttg 420 ggcatgtctg tttgagcgtc attacaaccc tcagctaccc gctggttttg aacccgaacg 480 gtgcccccta accggggaac cgagccggtt ttaaagttgt aagctctgct ggccgctccg 540 ccccaaccag aacatagtaa aatactactt ttgttagggt caagcggaac ggtttttcgg 600 cctgaacaaa acctaccctt tttcaaggtt tgacct 636 <210> 4 <211> 593 <212> DNA <213> Monacrosporium monosporum M3015 <400> 4 tccgtaggtg aacctgcgga aggatcatta ccaatcatgt ctcccagccg aaaggttggt 60 gcaggcgctt aaccctctgt gaaccaaaaa acctttcgct tcggcagcag ctcggttggc 120 aacagcctct gcgtcagcct gccggtagca ccaatcatca aaacttgcag ttaataacat 180 tgtccgatta ccaaattttc gaatgaaaat caaaactttc aacaacggat ctcttggttc 240 ccgcatcgat gaagaacgca gcgaaacgcg atagttaatg tgaattgcag aattcagtga 300 atcatcgagt ctttgaacgc acattgcgcc cattggtatt ccattgggca tgtctgtttg 360 agcgtcatta caaccctcgg tcaccaccgg ttttgagcaa gcgaggtctc cggacccggc 420 tggctttaaa gttgtaagct ctgctggctg ccaggcccaa ccagaacata gtaaaatcat 480 gcttgttcac ggttcgcggt cgaagcggta cggcctgaac aatacctacc acctctcagg 540 tttgacctca gatcagacaa ggatacccgc tgaacttaag catatcaata agc 593

Claims (34)

Astrobotris sp. A2003 (KCTC 10095 BP) strain. delete delete delete delete delete delete delete delete delete delete delete Astrobotris sp. A2008 (KCTC 10096 BP) strain. Astrobotris sp. A2010 (KCTC 10097 BP) strain. Monacrosporium sp. Having nematicidal activity. M3015 (KCTC 10116 BP) strain. A microbial nematicide comprising one or more strains selected from the group consisting of the strains of claims 1, 13 and 15. The method of claim 16, further comprising a microbial delivery medium, the delivery medium A mixture of sand (20 mesh) and loam or loess [1: 1 (v / v)]; Chopped potatoes (1-5 mm); And a delivery medium comprising PDB (potato dextrose broth: 24 g / L) in a ratio of 4: 0.2 to 1.5: 0.2 to 0.9 (w / w / w) Delivery media including wheat, barley or rice, Sand (20 mesh); Loam or loess; And a mixture of 100 parts by weight of chitin, corn flour or corn flour and 0.1-25 parts by weight of chitin [0.8-0.9: 0.8-0.9: 0.2-0.4 (v / v / v)]; Chopped potatoes (1-5 mm); And a delivery medium comprising PDB (24 g / L) in a ratio of 4: 0.2 to 1.5: 0.2 to 0.9 (w / w / w), respectively, or A mixture of vermiculite, bran or vermiculite 100 parts by volume and bran 0.1-25 parts by volume; Ground potatoes; PDB (24 g / L); And granular microorganism nematicides comprising brown rice green tea in a ratio of 4: 0.2 to 1.5: 0.2 to 0.9: 0.001 to 0.01 (v / v / v / v), respectively. The method of claim 16, further comprising a microbial delivery medium, the delivery medium is 100 parts by weight of wheat bran, starch, chitosan, sawdust, chitin, corn flour, kaolin, diatomaceous earth, vermiculite or corn flour and chitin 0.1-25 parts by weight Powdery microbial nematode, characterized in that the powdery delivery medium comprising a. 17. The method of claim 16, further comprising a microbial delivery medium, said delivery medium comprising sucrose (20 g / l) and soybean meal (5 g / l) or sucrose (20 g / l), yeast Extract (2 g / L), NaNO ₃ (1.82 g / L), KH ₂ PO ₄ (0.25 g / L), MgSO _{4 ·} 7H ₂ O (0.05 g / L), trace element liquid (1.0 mL / L) [ MnSO ₄ H ₄ O (0.005 g / 100 mL), FeSO ₄ . _{7H 2 O (0.002g / 100㎖)} , ZnSO 4 · 4H 2 O (0.02g / ℓ), CuSO 4 .7H 2 O (0.001g / 100㎖), CoSO 4 .7H 2 O (0.001g / 100㎖ Liquid microorganism nematicide, characterized in that the liquid culture medium consisting of). 18. The microorganism nematicide according to claim 17, wherein said strain has 1.0x10 ³ or more cell numbers as a colony forming unit per g or ml of said delivery medium. 19. The microorganism nematicide according to claim 18, wherein said strain has a cell number of 1.0x10 ³ or more as colony forming unit per g or ml of said delivery medium. 20. The microorganism nematicide according to claim 19, wherein said strain has a cell number of 1.0x10 ³ or more as colony forming unit per g or ml of said delivery medium. A mixture of 1 part of sand (20 mesh) and 1 part of loam or loess; Chopped potatoes (1-5 mm); And a delivery medium comprising PDB (24 g / L) in a ratio of 4: 0.2 to 1.5: 0.2 to 0.9 (w / w / w), respectively. A mixture of vermiculite, bran or vermiculite 100 parts by volume and bran 0.1-25 parts by volume; Ground potatoes; PDB (24 g / L); And a delivery medium comprising brown rice green tea at a ratio of 4: 0.2 to 1.5: 0.2 to 0.9: 0.001 to 0.01 (v / w / w / w), A mixture of 0.8 to 0.9 parts of sand (20 mesh), 0.8 to 0.9 parts of loess or loess, 100 to parts of chitin, cornmeal or corn flour and 0.1 to 25 parts of chitin, and 0.2 to 0.4 parts of volume; Chopped potatoes (1-5 mm); And a delivery medium comprising PDB (24 g / L) in a ratio of 4: 0.2 to 1.5: 0.2 to 0.9 (w / w / w), respectively, and Inoculating a granular microorganism delivery medium selected from the group consisting of wheat, barley or rice containing a strain selected from the group consisting of the strain of claim 1, claim 13 to 15 and incubated at 25 ~ 30 ℃ step; And Method for producing a granular nematicide comprising the step of drying the obtained culture. A method for preparing a granular microbial nematicide comprising the following steps: Claim 1, the step of culturing a strain selected from the group consisting of the strain of claim 13 to obtain a cell; Uniformly mixing the obtained cells with a powdery delivery medium selected from the group consisting of sterile bran, starch, chitosan, sawdust, chitin, corn flour, soy flour, kaolin, diatomaceous earth and vermiculite; And The mixture was gelatin (0.2 to 3.0 w / v), lignin sulfate (2 to 30 w / v), agar (0.2 to 2.0 w / v), xanthan gum (1 to 20% w / v), starch (0.5 to 30). % w / v), glycerol (6-30% v / v), tween 20 (0.5-99% v / v). Tween 80 (0.5-99% v / v), pectin (10-30% w / v), alginic acid (3-25% w / v). And coating wheat, barley or rice coated with a surface treatment material selected from the group consisting of a mixture comprising at least two of them. Claims 1 and 13 to a microbial delivery medium comprising bran, starch, chitosan, sawdust, chitin, corn flour, soy flour, kaolin, diatomaceous earth, vermiculite, or a mixture of 100 parts by weight of corn flour and 0.1 to 25 parts by weight of chitin. Mixing a strain selected from the group consisting of the strain of claim 15; And Method for producing a powdery microbial nematicide comprising the step of drying the mixture. Medium consisting of sucrose (20 g / l) and soybean meal (5 g / l); Or sucrose (20 g / l), yeast extract (2 g / l), NaNO ₃ (1.82 g / l), KH ₂ PO ₄ (0.25 g / l), MgSO _{4 ·} 7H ₂ O (0.05 g / l), Trace Element Liquid (1.0 mL / L) [MnSO ₄ H ₂ O (0.005 g / 100 mL), FeSO ₄ . _{7H 2 O (0.002g / 100㎖)} , ZnSO 4 · 4H 2 O (0.02g / ℓ), CuSO 4 .7H 2 O (0.001g / 100㎖), CoSO 4 .7H 2 O (0.001g / 100㎖ A method for producing a liquid microorganism nematicide comprising the step of inoculating and culturing a strain selected from the group consisting of the strain of claim 1, claim 13 to claim 15 in a liquid culture medium consisting of. 24. The granular microorganism nematicide of claim 23, wherein the microorganism strain having nematicidal activity is incubated for a time such that the microorganism strain has a number of 1.0x10 ³ cells or more as a colony forming unit per g or ml of the delivery medium. Manufacturing method. 25. The granular microorganism nematicide of claim 24, wherein the microorganism strain having nematicidal activity is cultured for a time such that the microbial strain has a cell number of 1.0x10 ³ or more as a colony forming unit per g or ml of the delivery medium. Manufacturing method. 27. The method of claim 25, wherein the microbial strain having nematicidal activity is cultured for a time such that the microbial strain has a cell number of 1.0x10 ³ or more as a colony forming unit per g or ml of the delivery medium. Manufacturing method. 27. The method of claim 26, wherein the microbial strain having nematicidal activity is cultured for a period of time such that the microbial strain has a cell number of 1.0x10 ³ or more as a colony forming unit per g or ml of the delivery medium. Manufacturing method. Treating the seed or root of the plant with the microbial nematicide of claim 16; Mixing with root bed formulations or sprinkling on soil; Or a method of using a microbial nematicide comprising the step of irrigation to the growing plant. Treating the seed or root of the plant with the microbial nematicide of claim 17; Mixing with root bed formulations or sprinkling on soil; Or a method of using a microbial nematicide comprising the step of irrigation to the growing plant. Treating the seed or root of the plant with the microbial nematicide of claim 18; Mixing with root bed formulations or sprinkling on soil; Or a method of using a microbial nematicide comprising the step of irrigation to the growing plant. Treating the seed or root of the plant with the microbial nematicide of claim 19; Mixing with root bed formulations or sprinkling on soil; Or a method of using a microbial nematicide comprising the step of irrigation to the growing plant.