KR20160000659A - The composition of biocontroller against soil nematode using plant extracts and microorganism - Google Patents

Abstract

The present invention relates to a composition which formulates a plant-derived extract and microorganisms in order to control soil nematode. More specifically, a plant-derived extract having neem-oil and Shrubby sophora extract, as active ingredients, and microorganisms which produces a protease. An emulsifier, a dispersing agent, and an antioxidant are added when producing liquid formulation, and thus stability and efficacy of the formulation can be maintained. A nematode control experiment is carried out using the liquid formulation, thereby confirming a nematode control effect.

Description

TECHNICAL FIELD The present invention relates to a composition for controlling soil nematodes using plant extracts and microorganisms,

The present invention relates to a composition capable of controlling soil nematides in soil, more precisely, a composition comprising a natural plant extract having anti-nematic activity and a microorganism that secretes protease .

Because of the cultivation of crops, vegetables and edible crops, invisible parasitic nematodes spread to produce high quality safe agricultural products. When the nematode is damaged, the growth of the ground part does not seem to be visually significant, but the size and quality of the fruit and seed are reduced, resulting in a reduction in yield of 20 to 30%. Plant pathogens Parasitic nematode plants are infected with a weak defense system, which can accelerate the infestation of several plant pathogens, leading to the development of complex diseases and early destruction of crops. Globally, damage to crops due to plant parasitic nematodes is increasing and there is a numerical loss of 11% of food crops and 14% of economic crops (US Nematode Society, 1987).

Globally, the damage caused by the root-knot nematode ( Meloidogyne spp.) Among plant parasitic nematodes is the most serious and 78 species are distributed worldwide. The three most common root-knot nematodes in Korea are M. arenaria (peanut root nematode), M. hapla (carrot root nematode), and M. incognita (sweet potato root nematode). Damage caused by root-knot nematodes is distributed throughout the world, especially in tropical or subtropical regions where the temperature is higher than in one region and the number of nematode breeding is high. In recent years, the area of domestic plant growing area has been continuously increasing, and planting facilities have been growing vegetables throughout the year by using facility heating during the winter, so that the root - knot nematodes adapted to the subtropical regions are generated. In Korea, major crops such as melon, melon, cucumber, pepper, tomato, eggplant, strawberry, etc., are damaged in major crops such as melon, horticultural crops, and plants that are severely infested by root-knot nematodes. The damage is serious. In particular, the cultivated crops such as melon, red pepper, strawberry, etc., which produce the same crop every year in the same package for more than 10 years, followed by cucumber, By promoting environmental conditions, the density of root-knot nematodes is high and damage is increasing.

[Document 1] KR 10-0860726 (B1) [Document 2] KR 10-1089318 (B1) [Document 3] KR 10-2012-0111568 (A)

[Document 1] Sela S. et al. 1998. Purification and characterization of Bacillus cereus collagenolytic / proteolytic enzyme and its effect on Meloidogyne javanica cuticular proteins. Eur. J. Plant. Pathol. 104. pp. 59-67 [Literature 2] Li W. et al. 2002. Broad spectrum anti-biotic activity and disease suppression by the potential biocontrol agent Burkholderia ambifaria BC-F. Crop Protection. 21. pp. 129-135

In Korea, organic nematocides are mostly used for nematode control, which causes environmental pollution of water quality and soil. There are many uses of soil fumigants in nematodes control outside the country, but their use is restricted due to environmental problems. Therefore, it is necessary to develop a new environmentally friendly nematocide.

In Korea, physical control such as soil improvement, inundation, solar thermal disinfection, anti-segregation control based on cropping system, biological control using natural fungi and bacteria, chemical treatment using nematicide And a variety of methods are being used. Although the history of nematode research in Korea is short, recent studies on nematode biosynthesis using microorganisms and plant extracts have been carried out, but the efficiency of the control has been somewhat lower than that of chemical control.

The advantages of natural plant extracts are biodegradation, activity at low concentrations, and rapidity of interest. Natural compounds also have the advantage of providing a novel mechanism for the development of safer organic synthetic pesticides.

There are many kinds of nematode, mites, bacteria, predatory fungi, and fungi. Among them, predator is the predominant fungus, and predatory fungus is the common species found in almost all the world. The advantage of the nematode is that it has persistence of efficacy, but it has no short-lived effect and is greatly affected by the soil environment.

In the present invention, an effective formulation for nematode control is established by mixing the advantages of a natural plant extract having a nematode activity and a bacterium, and the nematode controlling activity thereof is confirmed.

For eco-friendly farming, the use of organic chemical pesticides should be minimized and biological and biochemical materials should be used to control pest infestation. In the present invention, the advantages of the plant-derived extract which does not depend on the quick-acting nature of the nematode control and the soil environmental conditions, and the advantages of the microorganisms which can produce a sustained effect while producing the metabolites such as proteolytic enzymes in the soil, And was selected as an active ingredient for the formulation of a controlling agent. In order to optimize the advantages of the present invention, an emulsifier, a dispersant, and an antioxidant may be additionally added to form a composition suitable for controlling nematodes.

The present invention is effective in providing a composition for nematode control by formulating a plant extract and a microorganism. The present invention relates to plant-derived extracts such as nymph oil and gossam extract, and protease-producing microorganisms such as Bacillus sp. K4 and Brevibacillus sp. Mix K9. An emulsifier, a dispersant and an antioxidant are additionally added in the preparation of the mixed formulation to form a liquid formulation. This formulation was found to be effective in controlling nematodes. Therefore, it is expected that the composition of the present invention can be a new nematide controlling agent capable of controlling nematodes.

Figure 1 shows a nematode isolated from soil
Fig. 2 shows the nematode growth curve in the NGM medium
Fig. 3 shows the effect of nematode inhibition
Fig. 4 shows the effect of inhibiting nematodes
FIG. 5 is a graph showing the nematode inhibitory effect of a culture concentrate of a proteolytic microorganism
Figure 6 shows the effect of the nematocide inhibitory effect

1. Isolation of soil nematode

The nematodes used in the experiment were separated from the rhizosphere soil of the cultivated cucumber. 5 ~ 10 Cm of the surface of the nematode was removed and cucumber roots were collected and it was confirmed whether or not the root nematode was infected. Then, about 5 kg of soil was collected using a shovel. The soil was crushed and water was added to make a soil suspension, which was filtered with a 2 mm sieve. The soil suspension was passed through a standard netting (60, 325, 400 mesh) and the soil suspension, which had finally passed through 400 mesh, was separated by funnel method. The nematodes collected under the funnel were observed under a microscope, as shown in Fig.

2. Cultivation and proliferation in soil nematode

The nematodes separated by the funnel method were washed in sterile distilled water containing antibiotics for about 30 minutes and then for 30 minutes in sterile distilled water containing no antibiotics. This washing was repeated 2-3 times. The washed nematode was treated with NGM agar plate (Agar 20g / L, Peptone 4g / L, NaCl 1.5g / L, Cholesterol 0.0008g / L, Potassium phosphate monobasic 3g / L, Potassium phosphate dibasic 0.5g / Lt; / RTI > About 100 ~ 150 per plate were inoculated and cultured at 25 ℃ for 5 days under dark condition. The cultured plates were cut into approximately 1 cm length with a sterilized knife and placed in a 60 mm plate. 2 mL of NGM broth (Peptone 4 g / L, NaCl 1.5 g / L, Cholesterol 0.0008 g / L, Potassium phosphate monobasic 3 g / dibasic 0.5 g / L, pH 5.5 ~ 6.5) was added and incubated at 25 ℃ for 3 days under dark conditions. Nematodes grown in the agar block migrated to the liquid medium and grew. Nematodes grown in liquid medium were subcultured in NGM borth at 25 ℃ for 7 days under dark conditions. In order to confirm the growth rate of nematodes, 50 nematodes were separated into 3 plates for 10 days. This experiment was repeated three times, and the result is shown in FIG. On the first day, the average number was 62 and on the second day, the average was 1052. The average number of 1313 on the 4th day, 1225 on the 6th day, 1139 on the 8th day and 1066 on the 10th day. It was confirmed that the number of surviving nematodes decreased from day 6.

1. Review of nematode plant extracts

Nematode, ginseng extract (cold water extract) and wood vinegar were purchased to examine plant extracts for nematode control. The control and treatment groups were each inoculated with 2 mL of NGM broth at a rate of 1/100, 1/500, and 1/1000, respectively, and diluted in the treatment group inoculated with nematode at 25 ° C. under dark conditions Lt; / RTI > On the 1st and 4th day after plant extract treatment, nematode mortality was examined under a microscope. This experiment was repeated three times, and the results are shown in FIG. 3 and FIG. FIG. 3 shows the nematode mortality rate of nematocysts. In the control group, nematode proliferation was observed. In the nim oil 1/100 treatment group, the mortality rate was 65% in the first day and 61% in the fourth day treatment group. In the nim oil treatment group, the mortality rate was 54% in the first day and 47% in the fourth day , The nematode treated group showed a mortality rate of 17% on day 1, but the nematode was proliferated on day 4. FIG. 4 is a graph showing the mortality rate of nematodes against the extract of Gosam. In the control group, proliferation of nematodes was observed. In the 1/100 treatment group, the mortality rate was 46% in the 1st day and 43% in the 4th day treatment. In the 1/500 treatment group, the mortality rate was 27% in the first day and 25% in the fourth day , And 1/1000 treatment group, respectively, but it was confirmed that the nematodes were proliferated on the 4th day. Finally, it was confirmed that the wood vinegar had no nematode inhibitory effect. As a result, nematicide was found to be superior to goose extract in nematode control.

      1. Protein-degrading Microorganisms Review

It has been reported that it can be controlled by secondary metabolites such as decomposition enzymes and antibiotics secreted by microorganisms during nematocarcinoma control. Bacillus There was a report that M. javanica epidermis was degraded by the protease produced by S. cereus , and the protease and chitinolytic enzyme in the culture medium of B. ambifaria inhibited egg hatching and larvae.

To isolate proteolytic microorganisms, soil samples were diluted to a concentration of 10 ^-3 to 10 ^-5 on nutrient skim milk agar (NSKA: nutrient broth 0.8%, agar 1.5%, skim milk 1%, Difco) For 24 hrs, and a transparent ring - forming bacterium was firstly selected around the colony. As a result of the morphological analysis of the selected microorganisms, two candidate microorganisms having Gram - positive, Bacillus, endogenous spore formation and motility were isolated. As a result of identification of these two species, Bacillus sp. And Brevibacillus sp., Which were identified as Bacillus sp. K4 and Brevibacillus sp. K9. These two strains were inoculated with nutrient broth containing 1% skim milk and cultured at 30 ℃ for 48 hours. After culturing, the culture supernatant was centrifuged and the culture supernatant was concentrated to about 1/10 with a 10 kDa membrane filter. This concentrated culture supernatant was added to the culture medium of the nematodes at a ratio of 1: 1 to examine the nematocidal activity. The number of nematodes per each sample was 100. After incubation at 25 ° C, the mortality of the nematodes was confirmed after 48 hours. This experiment was repeated three times, and the results are shown in FIG. The nematode lethality of the concentrated culture supernatant was examined. In the control group, nematode proliferation was observed. 81% in the K4 medium, 86% in the K9 medium, and 89% in the K4 and K9 medium. Since microorganisms have less rapid effect than biochemical components but are superior in terms of sustained effects, the effect of controlling nematodes at high concentrations was examined. The higher nematode mortality was observed in the K4 and K9 mixed cultures treated group than the single treated group, respectively.

1. Formulation

In order to formulate nematocides, nematode, ginseng extract and K4 / K9 culture medium were selected as effective ingredients. Antioxidants, emulsifiers and dispersants were studied for formulation. Since nem oil is a vegetable oil, stability with the use of antioxidants should be ensured in order to maintain physico-chemical stability and continuous insecticidal effect in the air. Ethoxyquin, which is widely used for foods and feeds, was found to have the best sleeping pattern after 20 days of 0.04% treatment. In order to emulsify the emulsifying component, it must be completely emulsified. The emulsifier was found to be excellent in physical properties and emulsification in 10% of polyoxyethylene (1,1,3,3-tetramethylbutyl) phenyl ether, polyethylene glycol, and polyoxyethylene nonylphenol. Polyethylene glycol was selected by judging final handling and unit cost. As a dispersing agent for improving efficacy, 10% of polyoxyethylene sorbitan monoleate showed good degree of aggregation and dispersion of active ingredients. The final configuration is shown in Table 1.

division Raw material name Input ratio (%) Active ingredient Nem oil 40 Gosam extract 25 Microbial culture 10 Antioxidant Ethoxyquin 0.04 Emulsifier Polyethylene glycol 10 Dispersant Polyoxyethylene sorbitan monoleate 10 extender Water 4.96 Sum 100

1. Nematode Control Test

In order to examine the nematocidal effect of the formulation formulated in Table 1, 100 nematodes of two days old were transferred to a 2 mL NGM borth / plate and the nematode was stabilized for 12 hours. And 1 / 1,000 ratio. Experimental conditions were observed for 4 days at 25 ℃ in dark condition, and the mortality of nematodes was examined. This experiment was repeated three times, and the results are shown in FIG. In the control group treated with the preparation, it was confirmed that the number of nematodes increased by about 1,000 on the 4th day of the nematode. In the 1/100 treatment group, the mortality rate was 97% on day 1, 98% on day 2, 98% on day 3, and 100% on day 4. In the 1/500 treatment group, the mortality rate was 88.3% on day 1, 91% on day 2, 97.3% on day 3, and 99% on day 4. In the 1/1000 treatment group, the mortality rate was 92% on day 1, 90.6% on day 2, 95.3% on day 3, and 97.6% on day 4.

As a result, it was confirmed that the formulation containing the nimule oil, the ginseng extract and the proteolytic enzyme - secreting microorganism was more than 90% in controlling the nematode.

Claims (3)

In nematode control, a composition consisting of nimoo oil, gossam extract, microorganism producing protease, emulsifier, dispersant and antioxidant

The method according to claim 1, wherein the microorganism producing the protease is Bacillus sp. K4, Brevibacillus sp. K9, a nematocidal composition comprising polyethylene glycol as the emulsifier, polyoxyethylene sorbitan monoleate as the dispersing agent, and Ethoxyquin as the antioxidant

Characterized in that it comprises 30 to 50%, 25 to 35%, 25 to 35%, and 5 to 15% of an emulsifier, 5 to 15%, and 0.1 to 0.01% of an antioxidant Of nematode

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