KR20010089868A - Microorganism having predacious ability for soil nematoda parasitic on plant from soil, and method for incubating the same - Google Patents

Abstract

PURPOSE: Provided are plant-parastic soil nematophagous microorganism, Arthrobotrys sp. microorganism(KCTC 8992P), devouring soil nematodes which damage the crops and being applied to insecticides. And its culture method is also provided. CONSTITUTION: The plant-parastic soil nematophagous microorganism, Arthrobotrys sp. microorganism(KCTC 8992P) is obtained by the steps of: sieving soil with a 1.0 mm sieve; suspending sieved soil with sterilized distilled water; centrifuging suspended solution and separating supernatant and precipitates; inoculating the precipitates on a corn meal 2 agar medium and culturing at 25 deg.C for 24 hours; adding 200-300 plant parastic nematophagous microorganisms thereto then culturing them at 25 deg.C for 7 days with aseptic culture technique to form spores of the plant parastic soil nematophagous microorganism; transplanting the spores to another corn meal 2 agar medium followed by culturing them; and re-culturing the culture with pure-culture technique, wherein a medium therefor is prepared by mixing 1.0-3.0 wt.% of peptone; 1.0-3.0 wt.% of beef-extract; 0.5-1.5 wt.% of yeast extract; 4.0-7.0 wt.% of glucose; 0.02-0.05 wt.% of KH2PO4; 0.003-0.008 wt.% of MgSO4; 0.003-0.008 wt.5 of KCl; and a balance of distilled water, and its pH is adjusted to pH 5-7 by using NH4OH.

Description

Microorganism having predacious ability for soil nematoda parasitic on plant from soil, and method for incubating the same

The present invention relates to a microorganism having a phagocytosis for plant parasitic soil nematode isolated from the soil, and to a method of culturing the same, and more specifically, to a high density of the soil, in particular, soil such as plant cultivation to perform the series, Control of soil nematodes by having a predatory ability to capture soil nematodes by forming special catching organs against plant parasitic soil nematodes, which invade the roots of crops, slowing crop growth, reducing harvests, and eventually killing crops. The present invention relates to a plant parasitic soil nematode nematode and its culture method.

Soil nematodes are small organisms belonging to all nematodes in all soils. Most phytosanitary soil nematodes are so small that they are difficult to identify with the naked eye and can be observed under a microscope. Plant parasitic soil nematodes are generally slender or fusiform, bilaterally symmetrical, and their length is usually 0.25-0.45 mm, very small, with no nodes, but with wrinkles on the body surface. The body wall is largely composed of epidermis, dermis, and muscle, and the nervous system is composed of the central nervous system and the nerve rings around the esophagus. It is reproduced by female adults that lay eggs, sometimes through hermaphrodite or sympathetic reproduction. Soil nematodes, hatched from eggs, hatch into adult stages after 4 stages of larvae and survive for about 1 month, and the digestive and reproductive organs are characteristically developed. Females lay about 50 to 600 eggs during their survival. . These plant-producing soil nematodes have a cough around the mouth and use them to puncture the roots of plants, absorb nutrients from plant tissues or invade the root tissues.

Plant Parasitic Soil nematodes are parasitic in all parts of plants, including seeds, leaves, stems, and roots. Depending on the species, external parasitics affecting plants, and internal parasitics and heads invading into plant tissues. It has a variety of lifestyles, such as semi-innervation, in which only parts are inserted into plants to consume nutrients. In addition, internal parasitic soil nematodes are those that are mobile and impaired, and do not migrate once they are added.

Root-knot nematodes mainly found in Korea are carrot root-knot nematodes, sweet potato root-knot nematodes, java root-knot nematodes, peanut root-knot nematodes, etc., and are mainly grown in melon, cucumber, tomato, watermelon, strawberry, red pepper, ginseng, etc. Doing damage.

The damage caused by phytosanitary soil nematodes is mild when the density of nematodes in the soil is low. However, as the growing period increases as the crop grows, host plants become fatally damaged or die. Plants infected with parasitic soil nematodes have reduced resistance to pathogens to encourage or aggravate the onset of pathogen invasion, and some soil nematodes may mediate phytopathogenic viruses.

Various methods have been tried to mitigate the damage caused by these plant parasitic nematodes and to control the soil nematodes.

The cultivation methods include fallow, non-cultivation, crop rotation, cropping adjustment, planting of resistant varieties, planting attracted plants, and so on. However, fallow cultivation is not realistic due to the nature of cultivation in Korea, and the immersion treatment method is also not widely accepted due to the hassle of using the field as a field and then using it as a field again.

While crop rotation has traditionally been used as a method of reducing damage caused by crops, the actual crop rotation may be very limited in the trend of growing crops to crops that guarantee high incomes and selecting crops suitable for regional characteristics. There is nothing else. In addition, it is not realistic to adjust the planting time as the fallow method because of the climate characteristic of Korea, where the four seasons are clear and the growing season is limited, and the same is true for planting manned plants. These cultivation methods are only to mitigate and mitigate the damage, rather than to control the underlying soil nematodes.

As a physical method, hot water disinfection method, electrothermal heating method, ultraviolet irradiation, and ultrasonic wave method have been tried, but in the case of hot water sterilization method, the temperature rises, and in the summer, when there is a strong sun, the crops are not cultivated. Inevitably, there is a lack of confidence in the effectiveness of hot water disinfection by considering the nematode's ability to escape. The electrothermal heating method is a method of controlling soil nematodes by installing heating wires in the ground and heating the arable land, but it can only be applied to the interim period when crops are not cultivated, and the disadvantage of excessive facility and maintenance costs is required. have. This disadvantage is the same in the ultraviolet irradiation method and the sound wave control method.

Chemical control using pesticides is currently the most attempted, but the agents used to control soil nematodes are organophosphate compounds that inhibit the activity of acetylcholinesterase, which is a kind of neurotransmitters in the nervous system. Insecticides work by increasing the amount of phosphorus acetylcholine. Most of these organophosphate insecticides have been defined as hazardous substances.

In addition, these insecticides exist not only in soil nematodes, but also in the soil, and microorganisms beneficial to crop growth through symbiotic relations with crops cause indiscriminate killing and salt accumulation. . In addition, because the pests cause pesticide residues on the harvest, the polluted crops are placed on the table, causing the health threats of general consumers, and the damages caused by failing to dispose of the harvest to farmers.

An alternative solution to this problem is the biological control method using natural microorganisms.

Biological control methods for controlling plant parasitic soil nematodes include methods using predatory nematodes, predatory insects, mites, protozoa, bacteria, fungi, and viruses, but the most effective method is soil nematode or nematode fungi. It is a way. The method of controlling plant parasitic soil nematodes using such a fungus is very easy to carry out, and the control effect is long lasting through proper soil management. In addition, compared with chemical control methods, there is no problem of pesticides, soil pollution and residual pesticides caused by spraying pesticides.

Fungi with these characteristics include the genus Paecilomyces , genus Dactylaria , genus Dactylella , genus Arthrobotrys , genus Nematoctonus, and catenaria Catenaria genus, etc., but they are not commercialized due to the low insecticidal or high concentration culture is difficult.

In order to overcome these drawbacks, recently, preparations using Bacillus sp . Microorganisms as insecticides for nematodes have been studied.

For example, Korean Patent Application No. 91-9696 describes Bacillus thuringiennes active against nematodes, and Korean Patent Application No. 1997-709010 describes a method for preparing insecticidal activity enhancers using Bacillus microorganisms. , Korean Patent Application No. 91-34872 describes a microbial preparation using Bacillus microorganisms having antagonism against plant diseases, but the Bacillus microorganisms cannot form a trapping organ, so the plant parasitic soil nematode by a direct method Could not be removed.

Accordingly, the present inventors lived in domestic farmland, especially plant cultivation centers with high density, and the microorganisms that form trap organs against soil nematodes as a method for controlling plant parasitic soil nematodes that cause enormous damage to crops from the natural world. Pure water was isolated and confirmed that the isolated microorganisms were capable of capturing and feeding the soil nematode. It was tested whether or not it was suitable for the change of temperature of cultivated season. As a result, it was confirmed that the physiological characteristics of the new microorganisms matched the general cultivation environment, so that the nematode could be effectively controlled by using the new microorganisms. The invention has been completed based on this.

Accordingly, an object of the present invention is to provide a microorganism having a phagocytosis against plant parasitic soil nematodes.

Another object of the present invention to provide a method for culturing the microorganism.

The microorganism of the present invention for achieving the above object of the present invention is an Athrobotrys sp. Microorganism deposited as KCTC 8992P in the Biotechnology Research Institute separated from the soil, and has a predatory ability against plant parasitic soil nematodes Have

Cultivation method of microorganisms having a phagocytic ability against the plant parasitic soil nematode of the present invention for achieving the above another object is peptone, beef extract, yeast extract, glucose from the genus Astrobotris sp. , KHPO, MgSO and KCl mixed solution as a medium, and consists of incubation at a temperature of pH 5-7 and 20-30 ℃.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a microorganism having a phagocytosis against plant parasitic soil nematodes inhabiting soil, the microorganism of the present invention shows a very fast growth ability and capture ability among the microorganisms having a phagocytic ability against a known soil nematode .

The microorganisms of the present invention are isolated from soil and identified based on the lookup table of the Saccardo system (Barnet & Hunter, 1972) and Coke & Godfrey (1964) and Hard (Haard, 1968). The isolated soil nematode trapping bacteria were identified as Arthrobotrys sp. The isolated soil nematode trapping bacteria were deposited with the Biotechnology Research Institute on February 11, 2000 (Accession No. KCTC 8992P). ). The bacterium of the present invention has better growth rate and trapping ability than the known asrobotic bacteria.

When the soil nematode is added to the culture medium in which the soil nematode predatory microorganism of the present invention is cultured, it forms a ring-shaped capture organ having a cell diameter of about 6 to 8 μm. When the soil nematode is caught in the ring-shaped trapping organ, it secretes an enzyme that breaks down the body wall of the nematode in the ring contacted with the nematode to digest the cuticle layer, thereby developing infiltration hyphae into the nematode. The soil nematode eventually dies.

On the other hand, looking at the cultivation method, first, the soil of the plant cultivation to perform the grinding is filtered to 1.0mm sieve, the soil is suspended in sterile distilled water. The suspension is centrifuged to separate the supernatant from the precipitate, and the precipitate is sterilely inoculated to Corn meal 2 agar medium and aseptically cultured at about 25 ° C. for about 24 hours. Here, 200 to 300 plant parasitic soil nematodes separated by a beer but funneling method are added and cultured at about 25 ° C. for about 7 days to form spores of soil nematode trapping bacteria on the medium. It is transplanted again to a wheat mill medium and then purely cultured, and then purely cultured as follows.

As a microbial nutrient, 1.0 to 3.0 weight of peptone, 1.0 to 3.0 weight of beef extract, 0.5 to 1.5 weight of yeast extract, 4.0 to 7.0 weight of glucose, 0.02 to 0.05 weight of KH ₂ PO ₄ , 0.003 to 0.008 weight of MgSO After mixing ₄ and 0.003-0.008 weight of KCl in distilled water (residual amount), the pH is adjusted to 5-7 with NH ₄ OH, and a medium used after sterilization is used. At this time, when the culture pH is less than 5 or more than 7, the growth rate of the microorganisms is significantly slowed down. Thereafter, the medium is inoculated with a culture solution incubated for 48 hours at 20-30 ° C in advance and incubated at 20-30 ° C. Here, when the incubation temperature is less than 20 ℃ or more than 30 ℃, the problem that the growth rate of the microorganisms can be significantly slowed.

The microbial cells obtained by centrifugation of the culture cultured by the culture method may be mixed with an excipient and dried to prepare a microorganism of the present invention.

The microbial preparation is a dry weight of 1 to 100 parts by weight of a microbial cell having the above characteristics with respect to 1000 parts by weight of an excipient mixed with skim steel, silkworm powder and zeolite in a ratio of 4 to 6: 1: 8 to 12 It is prepared by mixing. At this time, when the amount of microbial cells used for the excipient is less than 1 part by weight, the number of microorganisms required to form the nematode trapping organ to capture the soil nematodes is insufficient, so that the control effect as expected cannot be obtained, and 100 parts by weight. If exceeded, the balance of microbial flora in existing soils can be broken.

On the other hand, the degreasing steel is used as a nutrient, especially a carbon source for the growth of microorganisms, silkworm powder has a function of supplying a nitrogen source and crude ash, zeolite is not only a carrier function of the microorganism but also increase the moisture retention and drainage of the soil and soil It has a function of correcting the acidity of, and if the mixing ratio is out of the above range, when formulated, it is difficult to ensure the viability of the microorganisms in the formulation, it is difficult to form a capture organ quickly even when put into the soil.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples.

Example 1

Cultivation of Soil Nematodes

Soil nematodes in the soil were separated by a Bearmann funnel method, transplanted into Nigon medium, and cultured in an incubator maintained at about 25 ° C. A part of the soil nematode cultured medium (25 mm2) was removed and transplanted into a new nigon medium, and the nematode was extracted from the soil nematode culture medium by the empty but funneling method after 3-4 days when the nematode density was highest, and then suspended in sterile distilled water. .

Example 2

Isolation and Cultivation of Nematode Capturing Microorganisms

After collecting the soil collected in various regions with a sieve of 1.0 mm in diameter, 50 g was suspended in 100 ml of sterile distilled water and centrifuged to precipitate the precipitate formed in a cornmeal (acorn meal 2) agar medium aseptically for 24 hours in a 25 ° C incubator. Incubated. In addition, the precipitate formed by centrifugation of the separated supernatant was also sterilely inoculated in Cornmeal (Corn meal 2) agar medium and incubated in a 25 ° C. incubator for 24 hours. A predetermined amount of soil nematode isolated by the cultivation method of beer butt funnel was added to the cultured wheat flour medium and cultured again at 25 ° C. for 7 days. After 7 days, the soil nematode medium with soil nematode was observed under a dissecting microscope, and the spores of soil nematode trapping bacteria formed at the point where the soil nematode was captured by the microorganism trapping organ were transplanted into a new cone wheat cloth. .

Four different strains (ME-9701, 9702, 9703, 9704) forming the isolated nematode trapping organs were inoculated into Saabrod agar medium to compare the size of colonies formed during stationary culture for 7 days. The flask was inoculated with the adjusted GY (glucose 4, yeast extract 2) 200ml liquid medium, and cultured for 5 days while maintaining the culture temperature at 25 ℃ to compare the growth. In addition, 200 to 300 soil nematodes were added to the cornmeal cloth medium, and each inoculated strain was inoculated, and then cultured for 7 days in an incubator at 25 ° C., using a dissecting microscope, The density and initial time point were compared and observed.

As a result, as shown in Table 1 below, strain ME-9702 formed the largest colonies on conical agar medium, showed the strongest growth in GY liquid medium, and had the highest frequency of capturing organs. The initial time of forming was also formed faster than other isolates.

Comparison of Growth and Capture Organ Formation of Isolated Soil Nematode division ME-9701 ME-9702 ME-9703 ME-9704 Colony size (mm) 54 76 65 62 Dry weight (g / L) 5.5 6.8 5.7 6.3 Frequency of catching organ formation +++ ++++ ++ +++ At the time of capture agency formation 14 hours 9 hours 13 hours 12 hours

Example 3

Type of isolate strain

While cultivating the four types of isolated soil nematode bacteria in a condensed cloth medium, the form of colonies formed on the medium was visually observed. In addition, after removing some colonies of soil nematode trapping bacteria cultured for 7 days, they were spread on a slide glass in which Lactophenol picrinic acid solution was dropped, covering the cover glass, and conidia and considio The size and shape of the conidiophore and hypae were observed.

Colonies formed by pure incubation in corn cloth were white or light yellow, and mycelia were colorless and transparent.

The results of the microscope showed that the condydia had a conical shape of 19-42 X 8-15 μm, divided into two cells by a slightly contracted diaphragm, and the ratio of the length of the terminal cell to the host cell was increased. 1.5: 1 and terminal cells were longer and swelled than host cells.

The head of conidia was swollen in the form of a hump, no branches, no nodes formed, and 5 to 30 congenia were formed along the nodes.

The length of the conidia was about 100-500 μm, and the diameter was measured at 4-8 μm at the base and 2-3 μm at the distal end.

Chlamidospores are yellow in color, 30-50 x 13-15 μm in size, and the organs trapping the soil nematodes form a three-dimensional network.

Morphological characteristics of the novel soil nematode trapping bacteria identified through microscopic observation are shown in Table 2 below.

Morphological features of isolated soil nematode trapping bacteria Haifa diameter 2-7㎛ A vein woolliness Condiodiopor shape Non-Side Chain Sintering diameter 100 to 500㎛ diameter Base 4 ~ 8㎛ Tip 2-3㎛ Prominence Hump shape Conydia shape Extended Oval size 19 to 42 x 8 to 15 µm Diaphragm One Number 5-30 A predatory organ Three dimensional structure

As a result of identifying the observation result of Table 2 based on the search table of the Saccardo system (Barnet & Hunter, 1972), Cook and Guardfrey (Cooke & Godfrey, 1964) and Hard (Haard, 1968), All of the isolated soil nematode trapping bacteria were identified as Arthrobotrys sp. However, the ME-9702 strain was relatively superior in growth rate and capture power. It was deposited with the Biotechnology Research Institute on the 11th day (Accession No. KCTC 8992P).

Example 4

Formation of Soil Nematode Capturing Organs from Isolated Strains

Soil nematode trapping bacteria (ME-9702) inoculated on a condensed mill cloth and incubated for 7 days in an incubator at 25 ℃, the addition of the soil nematode cultured in Example 1 above the culture of the soil nematode capture organs The formation process was observed. As a result, capture organs were first formed within 9 hours of the addition culture, and after 24 hours, most capture organs were observed. At this time, the diameter of the cells constituting the trapping organ formed was about 6 to 8 µm.

The trapping organ is a complex ring-shaped complex, and at the beginning of the formation phase, the organs of the trapping organ begin to protrude in the trophic mycelia, stretch and curve, and when the cell is stretched to some extent, the second or third cell continues to grow. It was confirmed that they formed and eventually formed a ring-shaped capture organ composed of 3 to 4 cells. As time goes by, a ring-shaped trapping engine having a three-dimensional solid structure is formed, and each of the ring-shaped trapping organs can identify 3 to 4 diaphragms.

Soil nematodes that move in the soil are trapped in the ring-shaped trapping organ thus formed to secrete an enzyme that breaks down the nematode body wall inside the ring in contact with the nematode, digesting the cuticle layer, and penetrating into the nematode. Develop, and the soil nematode eventually dies.

Example 5

Cell Fatty Acid Composition of Isolated Soil Nematode Capture Bacteria

For the analysis of cell fatty acid of isolated soil nematode trapping bacteria, isolated strain (ME-9702) was inoculated into 50 ml of sabrod-dextrose liquid medium and shaken at 25 ° C for 48 hours. The cultured cells were collected, washed three times with sterile distilled water, and lyophilized. After taking 40 mg of dry cells, the fatty acid was isolated, extracted with hexane (hexane) to induce a methyl reaction with BF ₃ , the fatty acid content was analyzed using gas chromatography, the results are shown in Table 3.

Cell Fatty Acid Composition of Isolated Soil Nematode Capture Bacteria fatty acid content C 16: 0 12.7-15.6 C 18: 0 1.7 to 2.1 C 18: 2 70.9-74.4

As a result of analyzing the mycelial fatty acid composition of the isolated soil nematode trapping bacteria (ME-9702), it was confirmed that the same as the mycelial fatty acid composition of the genus Astrobotris genus known as shown in Table 3. The major components of the cell fatty acid contained C 18: 2 having cis-type double bonds in the 9th and 16th carbons, and a small amount of C 16: 0 and C 18: 0.

Example 6

G + C content and quinone analysis of isolated soil nematode capture bacteria

Analysis of G + C content (molar) of the isolated soil nematode trapping bacteria gene was performed according to the method of Tamaoka & Komagata (1984), and G + C of the gene under the conditions of Table 4 using HPLC. The content was measured.

HPLC conditions for measuring G + C content of genes machine Shimazu column Cosmosil filled column 5C-184.6 × 150mm, Nakarai detection UV absorbance at 260nm Eluate NH ₄ H ₂ PO ₄ : Acetate (20: 1, v / v) Flow rate 1ml / min Temperature Room temperature

Isoprenoid quinone analysis for the identification of isolated soil nematode trapping bacteria was performed with a slight modification of the method of Yamada et al. (Yamada et al, 1973), and was measured under the conditions of Table 5 using HPLC.

HPLC conditions for measuring G + C content of genes machine Shimazu column Cosmosil filled column 5C-184.6 × 150mm, Nakarai detection UV absorbance at 270nm Eluate Methanol: Isopropanol (4: 1, v / v) Flow rate 1ml / min Temperature Room temperature

The G + C content of the isolated soil nematode trapping bacteria and the analysis results of the isoprenoid quinone system are shown in Table 6 below.

G + C Content and Quinones of Isolated Soil Nematode Capture Bacteria Genes Isolate G + C content (mol) Master Quinone Genus Astrobotris 42 to 45 Q10 (Hn)

The G + C content of the chromosomal DNA of the capture bacterium ranges from 42 to 45, which is the same as the G + C content of the genus Astrobotris strain, and the analysis of the isoprenoid quinone system shows that quinone is the standard It was detected later than used Q-10 and earlier than Q-11, so it was identified as Q-10 (Hn) and contained a very small amount of Q-10 and Q-7.

Example 7

Optimum Culture Temperature of Isolated Strains

The isolated strain (ME-9702) was inoculated into a flask containing potato-dextrose liquid medium, and growth characteristics were investigated while shaking culture for 7 days at different incubation temperatures up to 10-40 ° C. Samples were taken every 24 hours, washed three times with sterile distilled water using a centrifugal separator, and the growth rate was measured by vacuum freeze drying the recovered cells and measuring the dry cell weight.

The measurement results, as shown in Table 7 below, there was a big difference in the overall growth rate according to the culture temperature, it was shown that the growth rate of the isolated cells was the largest when incubated at 25 ℃. In addition, the cultured cells showed maximum growth at 5 days of culture.

In the case of 10 ℃, the growth of the cells was very weak, but when the temperature was raised to 25 ℃ at the end of the culture, the rapid growth was seen as showing that the viability was maintained, whereas incubation at 40 ℃ In addition, the growth of the cells was not seen, of course, even after 7 days of temperature down to 25 ℃ culture did not grow.

When incubated at 20 ° C., the growth of the cells was slightly weaker than the culture at 25 ° C., but very vigorous, and similar results were obtained when cultured at 30 ° C.

Growth Characteristics of Separated Strains with Temperature Change (Unit: g / l) division 10 ℃ 20 ℃ 25 ℃ 30 ℃ 40 ℃ 0 hours 0.17 0.14 0.15 0.17 0.15 24 hours 0.19 0.94 1.27 0.84 0.13 48 hours 0.34 2.95 4.33 2.45 0.15 72 hours 0.91 4.18 6.45 3.76 0.17 96 hours 0.98 7.67 9.11 6.58 0.21 120 hours 1.31 9.15 10.28 8.66 0.14 148 hours 1.46 9.33 10.17 7.41 0.17 172 hours 1.49 8.95 10.23 7.56 0.15

As shown in Table 7, the isolated strain Arthrobotrys sp. (KCTC 8992P) having a soil nematode capturing ability has a viability at 10 ° C, proliferates slowly, and has an optimal growth temperature range at 20-30 ° C. It can be confirmed that it is suitable for general cultivation temperature.

Example 8

Growth Characteristics of Separated Strains with pH Change

The isolated strains were inoculated into flasks containing each medium-dextrose liquid medium adjusted to an initial pH of 3 to 9, and cultured at 25 ° C. for 5 days with culture. Samples were taken every 24 hours, washed three times with sterile distilled water using a centrifugal separator, and the growth rate was measured by vacuum freeze drying the recovered cells and measuring the dry cell weight.

As a result of the measurement, as shown in Table 8, there was a big difference in the overall growth rate according to the initial pH of the medium, showed the growth of the most viable cells under the condition of adjusting the initial pH to 6.0, the initial pH 5 ~ Expected growth between 7 However, no growth occurred when the initial pH was adjusted to 3 and 9, and the growth of the cells was weak even when the initial pH was adjusted to 4 and 8.

Growth Characteristics of Separated Strains According to pH Change (Unit: g / l) division pH 3 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 0 hours 0.13 0.17 0.16 0.15 0.13 0.14 0.17 24 hours 0.15 0.15 0.44 1.25 1.33 0.18 0.19 48 hours 0.19 0.18 1.29 4.36 4.28 0.37 0.15 72 hours 0.18 0.24 1.97 6.55 5.96 1.45 0.14 96 hours 0.16 0.27 3.25 9.24 8.87 1.54 0.17 120 hours 0.17 0.25 3.47 10.11 9.74 1.78 0.19

As shown in Table 8, the isolated strain Arthrobotrys sp. (KCTC 8992P) having a soil nematode capturing ability shows a relatively strong growth between pH 5-7, and particularly has an optimal growth pH at pH 6, It can be confirmed that the pH of the cropland soil is met.

Example 9

Culture Characteristics of Isolated Strains

Based on the results of Examples 7 and 8, the isolated strain was inoculated into a 5 L fermentor containing GY (glucose 4, yeast extract 2) liquid medium adjusted to initial pH 6.0, and maintained at 1 vvm while maintaining a culture temperature of 25 ° C. Aeration, the culture characteristics were examined while incubating at 150rpm conditions. Samples were taken every 24 hours, washed three times with sterile distilled water using a centrifugal separator, and the recovered cells were vacuum-dried and the growth was measured by measuring the dry cell weight.

As a result, the isolates grew very active at 2.7 g / l in 24 hours, 8.5 g / l at 48 hours, 12.6 g / l at 72 hours, 17.5 g / l at 96 hours, and 21.4 g / l at 120 hours. Showed.

Example 10

High concentration culture and formulation of microorganisms

To cultivate a detached soil nematode predatory microbes ahsseuro in Boris (Arthrobotrys sp.) (KCTC 8992P ) in bulk, peptone 200g, Beef Extract 200g, yeast extract, 100g, glucose _{500g, KH 2 PO 4 30g,} MgSO 4 5g, KCl 5 g was added to 10 L of water, dissolved, and sterilized to use as a medium. At this time, the pH is adjusted to 6.0 using NH ₄ OH, the air injection amount is 5ℓ / min, it is injected through a sterile filter. After inoculating 1 liter of the isolated strain culture medium incubated at 30 ° C. for 48 hours, the culture medium was incubated at 25 ° C. for 150 hours at 150 rpm. Cells were obtained by centrifugation in the culture medium after the incubation.

The cells thus obtained were mixed with 1 Kg of excipients in which the degreasing steel, silkworm powder and zeolite were mixed at a ratio of 5: 1: 10 at a dry cell weight of 100 g, and dried to form a formulation. Degreasing steel was considered as a source of carbon and crude ash for the survival of cells, and silkworm powder was considered as a source of nitrogen and crude ash.

Example 11

Confirmation of Soil Nematode Prediction of Formulated Culture Strains

The reproducibility of soil nematode predation was confirmed under laboratory conditions using the formulations prepared in Example 10, and the soil nematode predation ability was confirmed by directly spraying the soil nematode into the soil. Injecting the soil nematode cultivated in the laboratory and the preparation prepared in Example 10 into a Petri dish, and then incubated in a 25 ℃ stationary incubator to form a soil nematode trapping organ and to capture the soil nematode using a microscope Was observed.

After 3 hours of mixing the soil nematode and the preparation and starting the cultivation, the strain contained in the preparation grew actively with the extension of the hyphae. After 9 hours, the nematode trapping organ of the three-dimensional network structure began to form. It was. After 12 hours, some of the capture nematodes began to be captured in some of these capture organs, and after 24 hours, the density of the capture organs was highest.

In addition, 5 Kg of cropland soil was collected and sterilized, and soil nematodes cultured in a laboratory were added at a density of 1000 / Kg and mixed well. After dividing this into control and test sections, add 25 g of the formulation prepared in Example 10 to the test section, and then 200g of samples were taken daily in a stationary incubator at 25 ° C. for 7 days. Soil nematode capture and phagocytosis of the strains contained in the formulations in the soil were determined by measuring the population of soil nematodes present in the soil.

As a result, there was no significant change in nematode density of 1000 / Kg for 7 days in control, and no significant change in population until 3 days in test, but the number of remaining soil nematodes decreased over time. After 8, 5 days, 20, 6 days, 45, 7 days after the reduction was confirmed to 80.

As described above, the microorganism of the genus Astrobotris of the present invention cultured by separating from the soil has a predatory ability to the plant parasitic soil nematode, so that the soil, especially soils that cause enormous damage to crops, such as plant cultivation to perform a series It is very useful as a nematode's natural enemy, and its growth conditions are similar to the cultivated soil conditions, and its growth rate is fast, so that mass production is possible.

Claims (3)

Athrobotrys sp. Microorganism deposited as KCTC 8992P with Biotechnology Research Institute. The microorganism according to claim 1, wherein the microorganism is separated from the soil, and has a phagocytic ability by forming a capture organ for a plant parasitic soil nematode. The soil nematode predatory microorganism according to claim 1 isolated from soil is cultured at a temperature of pH 5-7 and 20-30 ° C using peptone, beef extract, yeast extract, glucose, KHPO, MgSO and KCl as a medium. Way.