KR101230787B1 - Agent and method for control insect using entomopathogenic nematode Rhabditis blumi - Google Patents

Abstract

The present invention provides a pest control agent and a pest control method comprising the insect pathogenic nematode Raptidis blummi . Insect control agents including the insect pathogenic nematode lapditis blueme of the present invention has a broad control spectrum for pests, including poultry and scarabs, can achieve a greater control effect than conventional biological control agents.

Description

Agent and method for control insect using entomopathogenic nematode Rhabditis blumi}

The present invention relates to a pest control agent and a pest control method including the insect pathogenic nematode Rhabditis blumi .

The market for eco-friendly agricultural products has exploded due to the improvement of the quality of people's living and the social interest in food safety due to economic growth, and the production of eco-friendly agricultural products has been growing more than 70% every year for the past 5 years since the late 1990s. .

As a pest control method to replace the chemical insecticides currently used for the production of eco-friendly agricultural products, biological control of the natural concept using the entomological pathogenic nematode (entomopathogenic nematode) is attracting attention as an effective method.

Currently known insect pathogenic nematodes are Steinernema spp. (Steinernematidae) and Heterorhabditis spp. (Heterorhabditidae), each of which is the symbiotic bacteria Xenorhabdus spp. and Photorhabdus It specifically binds to the host through the pores of the insect's mouth, anus, or epidermis and secretes symbiotic bacteria. The released symbiotic bacteria have a pesticidal mechanism that causes sepsis to kill the host within 48 hours. (Marcek et al., 1988; Clarke and Dowds, 1995; Gerritsen et al., 1998; Grewal and Georgis, 1999). These insect pathogenic nematodes are evaluated as natural biocontrol agents that are reproduced through long-term persistence in the field, wide host range, safety for useful animals, ease of spreading, and secondary growth through infected host pests, and are useful for controlling soil-type pests. (Kaya and Gaugler, 1993).

Insect pathogenic nematodes have various technically highly available characteristics, but there is a difficulty in improving the insecticidal properties of existing nematodes due to the tight symbiotic relationship with the symbiotic bacteria which have a major influence on the pathogenic expression of nematodes in host pests. . Therefore, there is a need to develop a new insect pathogenic nematode that is ecologically safe and highly insecticidal in the Korean environment.

The present invention is to develop a new insect pathogenic nematode and to provide a pest control and pest control method using the same.

The present inventors conducted a study on the presence of insect pathogenic nematodes in various host pests for the development of new insect pathogenic nematodes. As a result, a nematode that exhibited the characteristics of an insect pathogenic nematode was collected from a Blitopertha orientalis larva and identified using an ITS sequence and morphological analysis. The nematode was identified as Rhabditis blumi, Nematoda: Rhabditidae).

Until now, the fact that Micah has a blue tooth rapdi entomopathogenic was never found, rapdi Micah Blue tooth germs Providencia beomi Cola (Providencia vermicola), Flavobacterium (Flavobacterium sp.) and Alcaligenes faecalis have not been identified. The present invention relates to the fact that the lobidis blumi coexists with Providencia vermicola, flavobacterium and alkaline genes facalis, and nematodes invade the host pests to secrete these symbiotic bacteria to kill host pests, and It is the first time that rice is nutrientd by these symbiotic bacteria to grow and grow.

Accordingly, the present invention provides a pest control agent comprising the insect pathogenic nematode Raptitis blubmi.

Insect pathogenic nematodes secrete symbiotic bacteria after invading host pests through the pores of the insect's mouth, anus, or epidermis, and the released symbiotic bacteria have a pesticidal mechanism that kills host pests by causing sepsis. Therefore, the present inventors investigated the symbiotic bacteria of the entomopathogenic nematode rabditis blummi, and as a result, it was confirmed that Providencia vermicola, flavobacterium, and alkali genes facalis are the symbiotic bacteria of lobidis blummi. It became.

Thus, in one embodiment of the present invention, the pest control agent may include one or more nematode symbiotic bacteria selected from the group consisting of Providencia vermicola, Flavobacterium, and Alkogenes facalis. Such nematode bacteria may be present in the intestine of the lobidis blush, but may also be secreted out of the lobidis blush in the formulation of a pest control agent.

The pest control agent may include a general carrier such as water for formulation in addition to the insect pathogenic nematode. In addition, it may further include components generally included in pest control agents including entomopathogenic nematodes. For example, it may include a component for the growth of and its symbiotic bacteria. Such ingredients may include yeast extracts, cholesterol, vegetable oils, and the like.

As a result of investigating the pathogenicity of the lobidis blummi, it was confirmed that the rabidis blummi has a pathogenicity against an insect pest or a scarab pest.

Pests belonging to the species are not limited thereto, but are not limited to Agrotis segetum (Lepidoptera: Noctuidae), Cabbage butterfly (Artogeia rapae) (Lepidoptera: Pieridae), and black-leaved moth (Autographa nigrisigna) (Lepidoptera: Noctuidae) ), Dichocrocis punctiferalis (Lepidoptera: Pyralidae), American White Moth (Hyphanria cunea) (Lepidoptera: Arctiidae), White Belted Moth (Lepidoptera: Pyralidae), Thief Moth (Mamestra brassicae) (Lepidoptera: Noctuidae, Palpita indica (Lepidoptera: Pieridae), Chinese cabbage moth (Plutella xylostella) (Lepidoptera: Yponomeutidae), Spodoptera exigua (Lepidoptera: Noctuidae), Spodoptera litura (Lepidoptera: Noctuidae) and Honeybee moth (Galleria mellonella) (Lepidoptera: Pyralidae).

The pests of the scarabs include, but are not limited to, Blitopertha orientalis (Coleoptera: Scarabaeidae) or Curkio sikkimensis (Coleoptera: Curculionidae).

The present invention also provides a method for controlling pests, comprising treating a plant with a pest control agent comprising an insect pathogenic nematode lapidis blush. As described above, the pest control agent may include at least one nematode symbiosis bacteria selected from the group consisting of Providencia vermicola, Flavobacterium, and alkali genes facalis. Such pest control agents can be treated on plants at a concentration of 1 × 10 ⁷ to 1 × 10 ¹² IJs / ha. Considering the cost and control effect, the pest control agent is preferably treated to plants at a concentration of 3 x 10 ¹⁰ to 1 x 10 ¹⁰ IJs / ha.

On the other hand, the plant that can prevent damage caused by pests using the pest control agent is not particularly limited. Any of the above mentioned plant or pest beetles may be a target for control. Such plants include fruit, leafy vegetables, flowers and the like. Preferably the plant may be a cruciferous plant. Cruciferous plants include major crops such as cabbage, radish, radish, cabbage, broccoli, fresh, mustard, and wasabi.

Insect control agents including the insect pathogenic nematode lapditis blueme of the present invention has a broad control spectrum for pests, including poultry and scarabs, can achieve a greater control effect than conventional biological control agents.

Figure 1A shows that the insect pathogenic nematode lapditis blubmi invade through the gate of the host pests, Figure 1B shows that the host pests after the invasion of the nematode turned black and lethal due to bacterial infection.
Figure 2 shows the results observed by fluorescence microscopy after staining the sympathetic bacteria of the insect pathogenic nematode lapditis blue rice staining.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

[ Example ]

Preparation of pests

For the pathogenicity test of Rabditis blummi nematodes, Geogeunbang in organic farms in Gongju and Suwon A grot is segetum (Lepidoptera: Noctuidae), Cabbage Butterfly Artogeia rapae (Lepidoptera: Pieridae) , Black Silver Moth Autographa nigrisigna (Lepidoptera: Noctuidae), Peach Moth Dichocrocis punctiferalis (Lepidoptera: Pyralidae), American White Bull Moth Hyphanria cunea (Lepidoptera: Arctiidae), White-Banded Moth Hymenia recurvalis (Lepidoptera: Pyralidae), Thief Moth Mamestra brassicae (Lepidoptera: Noctuidae), Cotton Monarch Palpita indica (Lepidoptera: Pieridae), Chinese cabbage moth Plutella xylostella (Lepidoptera: Yponomeutidae), Pabamoth Moth Spodoptera exigua (Lepidoptera: Noctuidae), Tobacco Giant Spodoptera litura 3 and 4 larvae of 11 species of insect pests including (Lepidoptera: Noctuidae) were collected and 3 and 4 bee-beeped moth Galleria from the Horticultural Research Institute mellonella (Lepidoptera: Pyralidae) was supplied to prepare a total of 12 species of species.

Scarab beetle Blitopertha orientalis (Coleoptera: Scarabaeidae) and Bab wee Curculio , respectively, at the Seowon Golf Club in Paju Two species of scarab pests were prepared by collecting sikkimensis (Coleoptera: Curculionidae).

Example  1: against various pests Rabditis Blume's  Pathogenicity test ( Host Range Search )

In order to test the pathogenicity of Rapditis blummi for each pest at the laboratory level, concentrations of 100 infectious nematodes (IJs) / larva according to Petri dishes using 30 healthy 3 and 4 larvae per treatment. Inoculated with nematode nematode, and maintained the temperature at 25 ^o C in the incubator and confirmed the presence of nematodes in dead carcasses every 24 hours to calculate the mortality of host pests after 5 days and 10 days.

As a result of microscopic observation, it can be seen that the rabdis bleu nematode invades through the gate or anus of the host pest (FIG. 1A). After the nematode invasion, the host pests turned black due to bacterial infection and eventually died (FIG. 1B).

The pathogenicity of nematodes differed according to the type of pest (F = 40.39; df = 13,56; P <0.001). As shown in Table 1 below, on day 5 after treatment with nematodes, a mortality of 70-90% was observed in all pests except castration moth and peach moth. When the exposure time was extended from 5 days to 10 days, the mortality rate of macropods and peach moths increased from 0 to 81.1% and 34.4% to 85.6%, respectively, but the mortality of other pests did not increase. The dorsal beetle, which had been isolated from Rabditis blueme, had the highest mortality rate of 94.4%.

[Table 1]

Lobditis blummi used in this study was isolated from the scarab larvae and showed the highest pathogenicity in the scarab beetle larvae. However, it has a relatively high pathogenicity against moths and fly larvae. Judging.

Example  2: Cruciferous family  Against three pests that harm plants Rabditis Blume's  Pathogenicity test

The pathogenicity of the lobidis bumimi against three representative pests-cabbage butterfly, burglar moth, and cabbage moth on cruciferous plants was tested at four concentrations. Inoculate nematodes at concentrations of 10, 20, 40, and 80 IJs / larva according to Petri Dish using 90 healthy 3 and 4 larvae per treatment, and maintain the temperature at 25 ^o C in the incubator. Pest mortality was observed every 24 hours.

As shown in Table 2 below, as the nematode treatment concentration (F = 1064.4; df = 4,60; P <0.001) was increased, the mortality of each pest was observed, and the age of the pest (F = 37.3; df = 1). , 60; P <0.001), higher mortality was observed. In the case of cabbage butterfly, when the nematodes were treated at the concentration of 10 to 80 per pest, the mortality rate was 24.5% to 79.4% and 23.4% to 88.2% at 3 and 4 years, respectively. In case of thief moths, the mortality rate was 72.0% in 3rd age and 77.8% in 4th age, and the cabbage moths showed 87.5% and 93.5% in 3rd and 4th age, respectively. The highest mortality was observed when 80 insect infectious nematodes were treated to four insects of Chinese cabbage moth.

[Table 2]

The reason why susceptibility to nematodes was increased at 4 ages of each pest was that the larger the size of the pests, the larger the size of the gate, mouth and anus, which the nematodes can invade, and thus the invasion of nematodes was relatively favorable. Other Insect Pathogenic Nematodes, S. carpocapsae In the case of nematodes, nematodes easily invaded as the number of cabbage moths and cabbage butterfly increased. (Beair, G., Fournier, Y., Dauphinais, N. (2003) Efficacy of steinernematid nematodes against three insect pests of crucifers in Quebec. J. Nematol . 35, 259-265).

Using the data measured in Table 2 was measured the half-numerous concentration (LD ₅₀ ) and half-numbered mortality (LT ₅₀ ). As a result, as shown in Table 3, as the number of pests increased, the LD ₅₀ value decreased and the LT ₅₀ value increased. The LT ₅₀ and LT ₅₀ of the three pests were estimated to be approximately 25.7-48.2 IJs / larva and 53.2-88.0 h, respectively. Based on the semi-lethal concentration, the pathogenic expression of Rhabditis blummi was measured in the order of Chinese cabbage moth, Chinese cabbage butterfly, and thief moth.

[Table 3]

Example  3: port experiment

Cabbage seedlings, 50 days after planting, were placed in plastic boxes (30 x 30 x 28.5 cm), 3 each. The larvae per cabbage seedlings and the number of larvae were separated and attached to the leaves of 10 each, and left for 2 hours to fully settle the larvae. After confirming the larvae settled, 30 ml of nematode solution containing 3,000, 9,000, and 12,000 nematodes in each box was sprayed using a portable sprayer. The untreated group was sprayed with 30 ml of sterilized water. The plastic crates were placed on growth at 25 ^° C. and 60 ± 5% relative humidity, and then observed for 7 days after treatment. The experiment was performed in three iterations.

As a result, as shown in the following Table 4, the pathogenicity of the lobitis bemi nematodes is a pest type (F = 30.6; df = 2,24; P <0.001) and the treatment concentration of nematodes (F = 279.42; df = 3). , 24; P <0.001) increased. White cabbage moths showed 36.7% to 76.7%, thief moths to 26.6% to 66.7%, and cabbage moths to 53.3% to 93.3%.

[Table 4]

Example  4: packaging experiment

In order to test the pathogenicity of the lobidis bluemi against cabbage white butterfly, thief moth, and Chinese cabbage moth in a facility cultivation field, packaging experiments were carried out in the plant house in Chungnam Yeongi-gun and the plant house in Suwon, Gyeonggi-do. . Each plant was planted at 30 cm intervals, and each plant was sprayed with 10 liters of nematode solution at concentrations of 1 x 10 ¹⁰ , 2 x 10 ¹⁰ , and 3 x 10 ¹⁰ IJs per ha, each with an area of 3.31 m ² . . The treated area was sprayed with 10 liters of water, and after 7 days of treatment, the pathogenicity was calculated using the following formula.

LR = [1- (N _t / N ₀ ) / (C _t / C ₀ )] x 100

N ₀ : pest density after nematode application in nematode, N _t : density of pest after 7 days in nematode, C ₀ : density of pest immediately after water spraying in untreated, C _t : density of pest after 7 days in untreated.

As shown in Table 5, the spreading effect of nematodes differed significantly depending on the types of vegetables and pests in the facility cultivation medium. In the case of vegetables, the decrease of pest density by nematode treatment was observed in the order of Ssamchu, red mustard, and kale, and the susceptibility to nematodes was increased in the order of Chinese cabbage moth, Chinese cabbage butterfly, and thief moth. In the case of Ssamchu, when the nematode concentration increased from 1 x 10 ¹⁰ to 3 x 10 ¹⁰ IJs per ha, the treatment effect was different according to the types of pests (F = 134.2; df = 2,54; P < 0.001). The density of Chinese cabbage moth, Chinese cabbage butterfly, and thief moth decreased from 59% to 88%, 8% to 29%, and 8% to 37%, respectively. Among the three pests, the most effective control against Chinese cabbage larvae was. In the case of Chinese cabbage moth, the packaging effect (F = 10.8; df = 2,18; P <0.001) was different according to the kinds of vegetables, and the treatment concentration of nematode was changed from 1 x 10 ¹⁰ to 3 x 10 ¹⁰ IJs per ha. When increased, the lowest control effect was observed in kale, from 47% to 72%.

The packaging effect in the facility cultivation was highly correlated with the results of nematode persistence according to the types of leafy vegetables in the pot test. It was judged that fine hairs on the foliar surface of vegetables favored the retention of nematode solution and consequently increased the packaging effect.

[Table 5]

Example  5: using symbiotic bacteria Insecticidal  black

Symbiotic bacteria cannot penetrate into the body blood cavity of host pests alone. Therefore, symbiotic bacteria were cultured in a liquid medium for 24 hours, and the lethality of the host pests was assayed by the blood injection method.

5 ul of symbiotic bacterium solution (2 x 10 ⁵ , 2 x 10 ⁶ , and 2 x 10 ⁷ cells / ml in Ringer's solution) of Providencia vermicola, Flavobacterium and Alkaliness facalis The pathogenicity of honeybee moth larvae was tested by direct administration to BLU's body cavity.

As can be seen in Table 6, the pathogenicity of each bacterium was significantly different (F = 283.8; df = 2,36; P <0.001).

TABLE 6

After 24 hours of 1 x 10 ⁵ treatment with Providencia vermicola and Flavobacterium, the pathogenicity was about 20%, whereas the treatment with Alkogenes faecalis and saline did not. After 48 hours, Providencia vermicola and Flavobacterium showed 100% pathogenicity at all concentrations, while Alkogenes facalis showed less than 30% pathogenicity. Therefore, among three bacteria, Providencia vermicola and Flavobacterium were judged to be the determinants of pathogenic expression of nematodes.

Example  6: Sterile nematode  Pathogenicity test

In order to determine the pathogenicity of nematodes with or without symbiotic bacteria, aseptic nematodes and infectious nematodes were prepared at a concentration of 2,000 in 1 ml of sterile saline, respectively, and 5 ul of nematode solution was directly administered to honey bee moth larvae. In addition, antibiotics (500 U / ml of penicillin and 500 ppm of streptomycin) were treated with nematode solution directly to the blood cavity of honey bee moths.

As a result, as can be seen in Table 7, aseptic nematodes without symbiotic bacteria in nematodes did not show pathogenicity against honeybee larvae until 120 hours.

[Table 7]

In nematode treatment, there was a significant difference in pathogenic expression according to the presence or absence of antibiotics ( t = 203.9, P <0.001). In the case of antibiotics, pathogenicity was not expressed until 48 hours, but showed 100% pathogenicity at 120 hours, whereas in the absence of antibiotics, pathogenicity was expressed from 48 hours to 90%, indicating 100% pathogenicity at 120 hours.

In general, nematodes that are pathogenic to invertebrates have shown pathogenic differences depending on their combination with the associated symbiotic bacteria (Wilson, MJ, Glen, DM, George, SK and Pearce, JD (1995a) Selection of a bacterium for the mass production of Phasmarhabditis hermaphrodita (Nematoda: Rhabditidae) as a biocontrol agent for slugs. Fundam . appl . Nematol . 18, 419-425). Nematodes have a pathogenic mechanism that acts as a carrier of symbiotic bacteria, invades host pests, and then releases and multiplies symbiotic bacteria present in the intestine of the nematodes, paralyzing the host's immune system and killing host pests (Marcek). , Z., Hanzal, R., Kodrik , D. (1988) Sites of penetration of juvenile steinernematids and heterorhabditids (Nematoda) into the larvae of Galleria mellonella (Lepidoptera) J. Invertebr Pathol 52, 477-478;... Clarke , DJ and Dowds, BCA (1995) Virulence mechanisms of Potorhabdus sp. strain K122 toward wax moth larvae. J. Invertebr. Pathol . 66, 149-155; Gerritsen, LJM, Wiegers, GL and Smits, PH (1998) Pathogenicity of new combinations of Heterorhabditis spp. and Photorhabdus liminescens against Galleria mellonella and Tipula oleracea . Biol . Control 13, 9-15; Grewal, PS and Georgis, R. (1999) Entomopathogenic nematodes, In Hall, FR and Menn, JJ (eds), Methods in biotechnology, vol . 5: biopesticides : use and delivery . Humana press Inc., Totowa, NJ. pp. 271-299). Insecticidal activity of symbiotic bacteria acts at this time plays a major role in the expression of pathogenicity to invertebrates. Rhabditis at 48 hours when antibiotics are present Blue reason for pathogenicity is reduced by the US nematode is feed as due to the growth of the symbiotic bacteria secreted from nematodes in the host insect was inhibited by antibiotics, it is determined that the symbiotic bacteria to perform a central role in the virulence expression of the nematode It provides useful evidence.

Example  7: Nematode  Bacterial Symbiosis Analysis

To confirm the symbiotic relationship between nematodes and bacteria, the bacteria were stained with fluorescence staining reagents and cultured with nematodes. In order to stain the Providencia vermicola bacteria with fluorescent staining reagent, the culture medium was purely cultured in nutrient broth for 24 hours and centrifuged to remove the media components. The reaction was performed at room temperature for 60 minutes by adding 100 ul DMSO dissolved in 10 ug Alexa Fluor 488 succinimidyl ester (Molecular Probes-Invitrogen, UK) to about 3.0 x 10 ¹⁰ cells / ml of Providencia vermicola. Unreacted fluorescent dyes were removed using sterile water, and the fluorescently stained bacteria were added at concentrations of 3.0 x 10 ⁹ cells / ml and incubated with nematodes. After the nematode fed the bacteria, the locating and morphological changes of the bacteria in the intestine of the nematodes were observed by fluorescence microscopy (x 200). As can be seen in Figure 2, basal bulbs, vesicles, and nematodes of the nematode body were observed (Fig. 2a), and the same nematodes were observed in fluorescence microscopy under the basal bulb vesicle stained blue. The cluster of Sia vermicola could be confirmed (FIG. 2b). This is a prerequisite for the pathogenic expression and lifestyle of entomopathogenic nematodes. The green Providencia vermicola, which has been widely spread in the intestine of the nematodes during proliferation, is considered to be the remnant of bacteria digested in the intestines for propagation of nematodes (FIGS. 2C and 2D).

Rhabditis bumi nematodes were considered to have pathogenic mechanisms similar to those of the insect pathogenic nematodes Steinernema spp. And Heterorhabditis spp., Which are specifically associated with Xenorhabdus spp. And Photorhabdus spp.

Example  8: by type of symbiotic bacteria Nematode  Growth Trend Analysis

Three symbiotic bacteria of the entomopathogenic nematode Lapditis blummi-Providencia vermicola, Flavobacterium and Alkaliness faecalis are isolated from nematodes, and each bacterium is purified to 25 ° C in tryptic soy broth (TSB, Difco). Incubated.

The propagation trends of nematodes were analyzed according to the concentrations of three symbiotic bacteria derived from Rabditis bleu nematodes. Remove the media by centrifugation at 8,000 rpm for 10 minutes and remove at four different concentrations (1.5 x 10 ¹⁰ cfu / ml, 3.0 x 10 ¹⁰ cfu / ml, 6.0 x 10 ¹⁰ cfu / ml, and no bacteria in Ringer's solution). Nematodes were added at a concentration of 10,000 IJs / ml and incubated at 25 ° C. for 120 hours. Nematode proliferation was observed according to the concentration of two bacteria. As a result, as shown in Table 8, when the nematode was inoculated with Providencia vermicola, the higher the initial bacterial concentration was, the higher the productivity of the nematode was observed. It was.

[Table 8]

The initial concentration of Providencia vermicola at 1.5 ± 0.1 x 10 ¹⁰ When increased to 6.0 ± 0.2 × 10 ¹⁰ cfu / ml, after 72 hours the concentration of nematodes increased proportionally from 2.4 × 10 ⁴ to 5.2 × 10 ⁴ IJs / ml. Nematode proliferation was observed when the concentration of Providencia vermicola was maintained at 1 × 10 ⁹ cfu / ml or higher. In the case of Flabobacterium and Alkella nes facalis, nematode proliferation was observed proportionally as the concentration of early bacterium increased, but showed lower productivity compared to Providencia vermicola. Among the three symbiotic bacteria, Providencia vermicola and Flavobacterium were considered to be the decisive bacteria for nematode growth.

Example  9: Aseptic nematodes  Preparation and Growth Trend Analysis

As part of the investigation of the role of symbiotic bacteria, a sterile nematode containing no symbiotic bacteria was developed and the growth and proliferation trends of nematodes containing symbiotic bacteria were analyzed. Adults bearing eggs were killed for 10 min in 0.4 N NaOH, neutralized by addition of 50% phosphate, and only nematode eggs were separated and surface sterilization was performed with 0.2% sodium hypochlorite for 10 min to develop aseptic nematodes.

The 6-well plate was used to observe the growth rate and final yield of nematodes according to the presence of bacteria in vitro. As can be seen in Table 9, in In the presence of symbiotic bacteria in vitro, nematodes increased at a non-proliferative rate of about 0.2 h ^-1 . However, when bacteria were not present, nematodes grew slowly over time, but proliferation was not observed. After 240 hours, the concentration of common nematodes increased approximately 110-fold to 110,000 IJs / ml, but the concentration of aseptic nematodes remained constant without increasing or decreasing.

TABLE 9

In general, symbiotic bacteria of entomopathogenic nematodes are known to not only provide pathogenicity to nematodes, but also to maintain high productivity by creating an environment favorable for nematode growth in artificial liquid culture. In the case of Rabditis blummi, it can be seen that symbiotic bacteria play an important role in nematode growth.

Example  10: Nematode  According to the time of inoculation Final yield measurement

The nematode artificial medium was inoculated with symbiotic bacteria and incubated in advance for 24 hours, and then the nematode inoculation time was adjusted differently to compare the productivity of nematodes. The final yield of nematodes was determined by adjusting the concentration of the lobidis blummi nematodes to 1,000 IJs / ml and varying in culture at 24, 48 and 72 hours. Nematode culture was carried out in a 6-well plate at 25 ° C. for 216 hours. Nematode culture was carried out in a 0.15 l bubble column fermentor by adjusting the aeration rate to 3 vvm at 25 ° C. for 216 hours.

As can be seen in Table 10 below, the final yield of the nematode was 31,000 IJs / ml when the inoculated nematode was incubated for 24 hours after inoculation of the symbiotic bacteria. At inoculation of nematodes, DOT was measured at 3% and remained below 10% by 48 hours thereafter (data not shown). When inoculated nematodes after incubation of symbiotic bacteria for 48 hours and 72 hours, the final concentration of nematodes was 119,000 ± 14,500 IJs / ml and 112,000 ± 3,800 IJs / ml, respectively. The timing of nematode inoculation was considered to have a significant effect on the final yield.

[Table 10]

Example  11: In the medium  According Nematode Increased productivity  black

The final yield of nematodes was determined using the five media shown in Table 11 below. After culturing symbiotic bacteria in each medium for 24 hours, the concentration of infectious nematodes was adjusted to 1,000 IJs / ml and cultured at 25 ° C. for 216 hours.

TABLE 11

As a result, as shown in Table 12, after 216 hours incubation in a 6-well plate infectious nematode was media No. The highest yield was shown at 2 to 213,000 ± 35,000 IJs / ml.

[Table 12]

The higher the yeast extract ratio in the media, the higher the final yield of nematodes. Media No. without vegetable oil In case of 5, the final yield of nematodes was significantly reduced to 34,000 ± 6,000 IJs / ml. It is considered that the growth of symbiotic bacteria that could be used for propagation of nematodes was limited due to the relatively low concentration of yeast extract, which can be used as a carbon source or nitrogen source. As a nutrient medium for symbiotic bacteria, media No. Nematode productivity at 4 was relatively low, and it was judged that whole milk powder was not suitable as a medium for nematode culture. The yield of nematodes showed a big difference according to the type and concentration of vegetable oil in each medium, but no direct comparison was possible. When cholesterol was added directly to the composition of the medium with sufficient basic carbon or nitrogen source for growth of symbiotic bacteria (media No. 2), or when the medium component (egg yolk) containing cholesterol was added (media No. 3) The nematode productivity was relatively high.

Yoo et al. (Yoo, SK, Brown, I. , Gaugler, R. (2000) Liquid media development for Heterorhabditis bacteriophora:.... Lipid source and concentration Appl Microbiol Biotechnol 54, 759-763) is the concentration of the vegetable oil in 2.5% When increased to 8%, the concentration of commensal bacteria did not increase, but the productivity of H. bacteriophora was reported to increase significantly. Vegetable oil is not a nutrient that affects the growth of bacteria, but it has been used as a major nutrient for nematode cholesterol biosynthesis (Ehlers, RU (2001) Mass production of entomopathogenic nematodes for plant protection). . Appl. Microbiol. Biotechnol. 56 , 623-633). In the case of Rhabditis blum nematodes, the direct addition of vegetable oil or cholesterol was considered to be an important factor that greatly increases the productivity of nematodes.

Example  12: bacteria according to the combination ratio of bacteria Cell productivity  black

Nematode production by artificial culture method is important to maintain a high concentration of bacteria that act as a nutrient source of nematodes. Therefore, the propagation trend of each bacterium was analyzed by artificially adjusting the inoculation amount of the three symbiotic bacteria.

Each bacterium incubated in tryptic soy broth (TSB) for 24 hours was divided into two groups: alkalizenes faecalis and flabobacterium, alkalinegenes faecalis and providencia vermicola, or flabobacterium and providencia vermicola. 10: 0, 10: 1, 5: 5, 1:10, and 0:10 were combined to observe the effect of the type and combination ratio of bacteria on the growth of each bacteria.

When two kinds of bacteria were co-cultured, one bacterium affected the growth of another. As can be seen in Table 13, the bacteria production of the two bacteria is 6.4-12.6 g / l, showing a better cell production than the pure culture of only one bacteria (4.1-5.3 g / l), each bacteria It was judged to have a positive effect on the growth of other bacteria. The total bacterial load was observed in the order of Alkénes Fabalis and Providencia vermicola, Flavobacterium and Providencia vermicola, and Alkogenes Facalis and Flavobacterium in the highest order. When Callis and Providencia vermicola were combined at 1:10, the result was 12.6 g / l. At this time, the weight of the Providencia vermicola was 10.9 g / l, and the optimal condition was considered in consideration of the fact that Providencia vermicola plays the most effective role in nematode growth. Alkaline ness faecalis does not have a direct effect on nematode growth and pathogenic expression, but it has a positive effect on the growth of Providencia vermicola and consequently plays a positive role in providing abundant nutrients to the artificial culture of nematodes. It was judged.

[Table 13]

Example  13: Bubble tower  In fermenter Nematode production  Perform optimization

Nematodes were produced by controlling the air flow in the bubble column fermenter based on the fermentation factor identified on a small scale. By adjusting the ratio of symbiotic bacteria to 10: 1 (Providencia Vermicola: Flavobacterium), aeration volume of 5 vvm in 0.15 l bubble column fermenter (3 cm dia. X 22 cm, Taegyung Sci., Korea) Incubated for 48 hours. Thereafter, 10,000 IJs / ml nematode was inoculated, and the aeration rate was adjusted to 2, 3, 5, and 6 vvm, and cultured at 25 ° C. for 216 hours to determine the final yield of nematodes.

As shown in Table 14 below, when the nematode was inoculated differently to produce infectious nematodes for 144 hours at 25 ° C., the highest yield was 170,000 ± 15,600 IJs / ml at 6 vvm. At this time, the concentration of symbiotic bacteria was measured as 3 x 10 ⁷ cfu / ml. After 72 hours of incubation at 2 vvm, the bacterial concentration increased to 5.4 ± 0.89 x 10 ¹⁰ cfu / ml, while the nematode concentration decreased to 900 ± 200 IJs / ml. After 72 hours of incubation at 3 vvm, the nematode concentration did not increase significantly, but the nematode length increased, indicating that growth was progressing (data not shown). As the amount of aeration increased, the productivity of infectious nematodes tended to increase, and the concentration of bacteria decreased. The yield change of infectious nematodes was due to the increase of dissolved oxygen in the culture medium due to the increase of the aeration rate. At this time, the feeding of bacteria by the nematodes increased the concentration of bacteria in the medium and the growth of nematodes was accelerated.

[Table 14]

Claims (10)

A pest control agent comprising an insect pathogenic nematode Rhabditis blumi .
The method of claim 1,
The pest control agent Providencia vermicola , Flavobacterium sp.) and Alcaligenes faecalis pest control agent comprising at least one nematode symbiosis bacteria selected from the group consisting of.
The method of claim 1,
The pest is a pest control agent that is an insect pest or a scarab pest.
The method of claim 3, wherein
The insect pests of the species are Agrotis segetum , cabbage butterfly ( Artogeia rapae ) , black silver moth ( Autographa) nigrisigna ), Peach moth ( Dichocrocis) punctiferalis ), American White Moth ( Hyphanria) cunea), ttimyeong white moth (Hymenia recurvalis ), thief moth ( Mamestra brassicae ), cotton monarch moth ( Palpita) indica ), Chinese cabbage moth ( Plutella) xylostella , Pamoth Moth ( Spodoptera) exigua ), tobacco giant seminar ( Spodoptera) litura ) and beetle moth ( Galleria mellonella ) pest control agent selected from the group consisting of.
The method of claim 3, wherein
The scarab pest is a Blitopertha orientalis (Coleoptera: Scarabaeidae) or chestnut weevil ( Culculio sikkimensis ) (Poleoptera: Curculionidae).
A method of controlling pests comprising treating a plant with a pest control agent comprising an insect pathogenic nematode Rhabditis blumi .

The method according to claim 6,
The pest control agent Providencia vermicola , Flavobacterium sp.) and Alcaligenes faecalis , and at least one nematode symbiotic bacterium selected from the group consisting of.
The method according to claim 6,
The pest control agent is a pest control method that is treated to plants at a concentration of 1 x 10 ⁷ to 1 x 10 ¹² IJs / ha.
The method according to claim 6,
The pest control agent is a pest control method that is treated to plants selected from fruit, leafy vegetables and flowers.
The method according to claim 6,
The pest control agent is a pest control method that is treated to cruciferous plants.

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