KR101033270B1 - Composition for anti-pine wood nematodes and method of controlling pine wood nematodes thereby - Google Patents

Abstract

PURPOSE: A composition containing fungi-infected pine wood nematode is provided to safely treat pine wilt disease. CONSTITUTION: A composition for preventing pine wood nematode contains E. vermicola-infected pine wood nematode. The E. vermicola is cultured in PDA(potato dextrose agar) medium. The infected pine wood nematode is B. mucronatus. A composition prevents the pine wood nematode by injecting the composition.

Description

Composition for controlling pine re-nematode and control method using same {{Composition for anti-pine wood nematodes and method of controlling pine wood nematodes}

The present invention relates to a composition for the prevention and treatment of pine re nematode disease using a nematode fungus against pine re nematodes and a method for controlling pine re nematode disease thereby.

The pine re-nematode is a plant parasitic nematode propagated by the mediator, Beethoven, and now there is no suitable control agent and there is no natural enemies of the insects. Once infected, it kills 99.9% of pine trees after one year of infection. The pine re-nematode disease was first discovered in Japan in 1905, but it was not until 1972 that the causative pest proved to be a pine re-nematode, and now it is known that most pine forests in Japan died. Since then, international trade has been spreading throughout Asia and the world. In China, it was gradually spread after it was confirmed in Nanjing in 1982, and in Taiwan in 1985, it was reported that the indigenous Yugu was almost wiped out. In Korea, it was first reported in Geumjeongsan, Busan in 1988 and spread to Gyeongbuk in 10 years. The Forest Service puts enormous budgets and manpower every year in order to eradicate and prevent pine nematode diseases, but the number of affected areas and dead trees is increasing every year.

Pine re-nematodes are taxonomy belonging to leaf nematodes and pine re-nematodes, and 49 species (Proc. Helminthol. Soc. Wahs. 55 (1), pp 1-11, 1988) have been reported worldwide. Only two species of Bursaphelenchus xylophilus and Bursaphelenchus mucronatus are known in Korea. Among them, B. mucronatus is parasitic in pine but does not die, so only B. xylophilus is the cause of pine nematode disease.

Re-nematodes have a size of 1 mm or less, and the days required for the first generation at 25 ° C are only 4-5 days. One pair multiplies to 200,000 animals in 20 days, blocks the catheter of host plants, blocks the increase in water, and releases toxins of cellulase (Cellulase) to destroy tissues and kill host plants. Re-nematodes in the pine are transferred through the chrysalis through the larvae of the beetle, and into the body of the beetle. Thus, the beetle sky cow has a special symbiotic relationship that transfers the nematode, which does not have the ability to move, to the host plant, and the nematode causes the pine to die and provide the spawning place of the beetle sky cow.

For the past 30 years, many researches have been carried out mainly on the saltwater beetle, which is a mediator for the control of pineal nematode disease, but the development of direct inhibitors on pineal nematode using biological approach is insufficient.

Currently, as a method of controlling pine worm nematodes, there are methods for controlling by logging and fumigation, methods for controlling by logging and crushing, and methods for controlling by logging and incineration. Among them, the method of controlling by felling and fumigation is a method of controlling by using a fumigation agent such as methamsodium after logging and accumulating the damage caused by pine ash nematode disease. However, metasodium, a nematode insecticide, is expensive and may be harmful to humans and livestock. The control method by felling and crushing is a method of cutting the trees killed by pine ash nematode disease and crushing them to a thickness of 6 mm or less so that the larval beetle larvae cannot live. The control method by felling and incineration kills by pine ash nematode. Stack a tree and burn it. Since the damage or dead wood where the larvae of reptiles and larvae are infiltrated are mainly located in the mountains, to control by the above method, the felled trees must be transported to the place where the crushing machine or the incinerator is installed. In the process there is a risk that the larvae and re-nematodes of the nymphs will spread to other trees. In addition, in case of incineration, the control period is extremely limited due to forest fires, and when burned in forests, thermal trees are generated and there is a problem of killing surrounding pine trees. Above all, the control methods are post-treatment methods aimed at preventing the further spread of pine recharged nematodes by fumigation, crushing, or incineration of the damage caused by pine recharged nematodes. The underlying problem is that it is not a way to prevent re-nematode disease or to cure and revive infected pine trees.

The only control method that directly targets pine re-nematodes is by injecting prophylactic agents into live pines. However, in the case of crown injection, there is no drug developed in Korea and it is known that abamectin and other drugs are being tested. In Japan, three types of Levamis, Mesulfenfos, and Morantel tartrate are available on the market. Therefore, it is urgent to develop a new control method that can prevent and treat the infection of pine re-nematode.

Korean Patent Laid-Open Publication No. 2000-56182 reports that the Etella vermicola, a nematode fungus, was isolated while restoring pine re-nematodes from dead black pine. Since then, it has been reported that the Esteja vermicola has been isolated from olive trees, European oak bark beetles and soil. These fungi have been shown to kill pine re-nematodes by showing high infectivity against pine re-nematodes, but only the possibility of principle is mentioned for the method of controlling pine re-nematodes.

Republic of Korea Patent Publication No. 2000-56182

 Proc. Helminthol. Soc. Wahs. 55 (1), pp 1-11, 1988

An object of the present invention is to provide a composition for pine re-nematode control effective in the prevention and treatment as well as the post-mortem control of pine re-nematode disease.

In another aspect, the present invention is to provide a pine pine nematode control composition is safe because there is no fear of soil pollution and toxicity to uninfected pines.

The present invention for achieving the above object relates to a pine re-nematode control composition comprising a pine re-nematode infected with fungi having a nematode efficacy against pine re-nematode.

The fungus is irrelevant as long as it is infected with pine re-nematode and acts as a nematode, and is preferably Esteya vermicola ( E. vermicola ). E. vermicola is known only four strains of CBS 100821, CBS 115803 and CNU 120806, including ACTC 74485 described in the Republic of Korea Patent Publication No. 2000-56182, but is not limited to all fungi and known fungal characteristics characteristic of E. vermicola It is natural to include all variants derived from strains already known by the method.

E. vermicola has two types of conidia , lunate and columnar. Among them, only the crescent-shaped conidia show adhesiveness and are adsorbed by the pine nematode to induce death. According to the preliminary experiments, the ratio of two conidia spores differed according to the type of medium, and the growth rate was fast in PDA (Potato Dextrose Agar) medium, and the rate of the formation of crescent conidia spores was high. Therefore, the E. vermicola is preferably E. vermicola cultured in PDA medium.

The pine re-nematode infected with the fungus may be any of the reported pine re- nematodes , but more preferably, B. mucronatus . B. mucronatus is a pine re-nematode that parasitic to pine but does not die. Therefore, when the fungus is used by infecting B. mucronatus , a more safe composition can be provided. However, when B. xylophilus infected with E. vermicola was used as confirmed in the following examples, the infection of pine re-nematodes due to the composition itself did not occur, thereby providing a safe composition.

The composition may be used in a form mixed with a pharmaceutically acceptable carrier, excipient (forming agent), diluent and the like.

The present invention also relates to a pine re-nematode control method using the pine re-nematode control composition. The composition may be sprayed on a tree, or sprayed on the soil around the tree, but it is more preferable to inject by canal injection, considering that pine re-nematodes are parasitic in the crown. The canal injection can be made by a conventional method, and thus a detailed description thereof is omitted.

The dosage of the active ingredient of the composition of the present invention is appropriately selected in consideration of the age, size, environment, and the extent of infection for treatment, but the spores 10 ⁸ ~ 10 ¹⁰ or spore infection nematodes based on pine trees 15 cm in diameter It is preferable to inject 5,000-10,000. At this time, spore-infected nematodes are more preferably at least 75% of all nematodes infected with spores. Naturally, more medicine should be given for larger diameter pine trees or severely infected pine trees.

As can be seen in the examples below in the control test of pine re-nematode, the composition of the present invention showed a higher pine re-nematode control effect compared to the composition consisting simply of E. vermicola spore suspension. This is because nematodes are free to move along the canal compared to spores without motility, which can infect fungi more nematodes when infected nematodes are used.

As described above, the composition for controlling pine nematode nematode of the present invention is safe and effective for the treatment of pine nematode disease, and in particular, the prevention effect is excellent and can be used for efficient control of pine nematode.

1 is a micrograph of a pine re-nematode infected with E. vermicola by one embodiment of the present invention.
Figure 2 is a photograph showing the pine repellent prevention effect in the greenhouse.
3 is a pressure canal infusion vessel used for canal injection;
4 is a photograph showing the control effect in the test forest.

The present invention will be described in more detail with reference to the following Examples. However, such an embodiment is only an example for easily describing the content and scope of the technical idea of the present invention, whereby the technical scope of the present invention is not limited or changed. In addition, it will be apparent to those skilled in the art that various modifications and changes can be made within the scope of the present invention based on these examples.

< Example >

Example 1: E. vermicola Preparation of Infected Nematodes

One) Pine tree nematode  detach

B. xylophilus was isolated from dead wood samples sampled from pine reptile infected area by Baermann funneling.

More specifically, using a pruning shears to cut the wood samples to 1cm size all around, placed the sample on a funnel connected with a rubber tube filled with water. After 24 hours, the pine reptiles gathered under the rubber tube were checked, and the tube was removed by hanging forceps.

The separated suspension was observed under a microscope (Olympus BX41) to count the number of pine reptiles to determine the concentration was found to be about 20,000 / ml.

2) E. vermicola Incubation

CNU 120806 of E. vermicola was dispensed from the Department of Microbiological Engineering, College of Agriculture and Life Science, Chungnam National University, and incubated in 26 ° C PDA (Potato Dextrose Agar) medium for 7 days. After 7 days, a sterile 0.05% Tween 80 aqueous solution was added thereto, and the surface of the medium was scraped to prepare a spore suspension. As a result of measuring the spore concentration by a haemocytometer, the concentration of spores in the spore suspension was 10 ⁸ / ml.

3) E. vermicola Preparation of Infected Nematodes

1 ml (approximately 40,000 rats) of pine re-nematode ( B. xylophilus ) suspension prepared in 1) was added to 1 ml of 2. E. vermicola spore suspension and incubated at 28 ° C. for 10 hours. The cultured pine nematode was isolated from the medium by the baerman funnel and washed with sterile water to prepare a suspension. The number of spore-infected nematodes in the suspension was measured under a microscope and the concentration was converted. As a result, the concentration of the nematode was 10,000 / ml, of which about 80% of the nematodes were infected with spores, and the concentration of spore-infected nematodes was 8,000 / ml. It was.

Figure 1 is a photograph of the suspension observed under a microscope (Olympus BX41), it was confirmed that the spores of E. vermicola adsorbed on the surface of the pine re-nematode . Overall, about 80% of the pine re-nematode surface adsorbed spores of E. vermicola .

Example 3 Control Test of Pineal Nematode Disease

1) Spore Infection B. xylophilus Control test in greenhouse by

A 100-year-old tree of four-year-old red pine in the greenhouse was tested for prevention of pineal nematode disease caused by artificial infection.

More specifically, 200 μl of the E. vermicola spore infection nematode suspension prepared in Example 1, 3) by peeling the epidermis at the height of about 5 cm above the red pine and putting cotton wool in the endothelium (about 1,500 infected nematodes) using a pipette Absorbed by cotton wool. After that, the epidermis was wound with a sealing film to prevent evaporation. One month after the spore infection nematode injection, 200 μl (about 4,000 pine re nematode suspensions) prepared in Example 1) were injected into pine by the same method as the E. vermicola spore infection nematode suspension. After 6 months of infection, the number of dead and healthy trees was visually compared to compare the survival rates. The control group showed a survival rate of 10%, whereas the treatment group of spore-infected nematodes showed a survival rate of 80%.

2) spore infection B. mucronatus Control test in greenhouse by

Peel off the epidermis at about 5cm above the surface of 100-year-old red pine in the greenhouse, and put cotton wool on the endothelium. Pipette 200μl suspension of E. vermicola spore infection ( B. mucronatus ) (approximately 1,500 infected nematodes) Absorbed by cotton wool. After that, the epidermis was wound with a sealing film to prevent evaporation. B. mucronatus was obtained from the Korea Forest Research Institute. B. mucronatus was infected with E. vermicloa by the same method as B. xylophilus of Example 1 using a suspension of B. mucronatus .

One month after the spore infection nematode injection, 200 μl of B. xylophilus suspension prepared in Example 1) was injected into pine by the same method as E. vermicola spore infection nematode suspension. . After 6 months of infection, the number of dead and healthy trees was visually compared to compare the survival rates. The control group showed a survival rate of 10%, whereas the treatment group of spore-infected nematodes showed 87% survival rate.

3) Control test in test rim

On-site prevention and control tests were conducted on 40 uninfected pines of about 15cm in diameter in the test forest of the Southern Forest Research Institute, Jinju, Gyeongsangnam-do.

In the following experiments, pine re-nematodes, spore-infected nematodes or spores were injected in the following manner. First, the pine re-nematode is drilled at a height of about 50 cm from the ground of the red pine, using a drill, and injecting 1 ml of pine re- nematode suspension prepared in Example 1) (about 20,000 pine re- nematodes) into the perforated hole using a pipette. The hole was blocked using cotton wool. E. vermicola spore-infected nematodes were injected in the same manner as pine re-nematodes, except that 1 ml of the spore-infected nematode suspension prepared in Example 1 3) (about 8,000 infected nematodes) was injected instead of pine re-nematodes.

Infusion of E. vermicola spores for comparison was injected with 30 ml of spore suspension (concentration of ˜10 ⁸ / ml) to achieve a level comparable to the number of spores contained in 1 ml of the spore-infected nematode suspension. At this time, since the volume of the suspension to be injected is large, the pine is punched and then injected using the pressure main injection container of FIG. 3 instead of the pipette, and the additives described in Table 1 below to ensure the stability of the spores during the injection The composition was prepared by mixing the suspension to a concentration of 1 (v / v) and injecting it.

More specifically, the control group was divided into a group of injecting pine re-nematode and a group of injecting spore-infected nematodes into a total of 10 trees, each carried out 5 copies. The prevention group injected 20 spore-infected nematodes or 10 spores each, and then, after 90 days, the pine re-nematodes were infected. In the treatment group, 10 pine trees were first injected with pine re-nematodes, and 14 days later, spore-infected nematodes were injected. Five months later, the number of dead trees and healthy trees was visually confirmed, and the results are shown in Table 2 and FIG. 4.

As can be seen in Table 2, the trees infected with pine re- nematodes in the control group died , whereas the trees injected with E. vermicola -infected nematodes showed 100% survival rate, indicating that the composition of the present invention was safe.

Trees injected with E. vermicola infected nematodes showed a survival rate of 40% in the treatment group and 80% survival rate in the treatment group, indicating that they were effective in both the treatment and the preventive groups. Seemed. In addition, the preventive group injected with E. vermicola spore suspension showed a survival rate of 60%, indicating that the control by E. vermicola nematode was more effective.

Claims (7)

A pine re-nematode control composition comprising pine re- nematodes infected with Esteja vermicola having a nematode efficacy against pine re- nematodes.
delete The method of claim 1,
The Esteya vermicola ( E. vermicola ) is a pine pine nematode control composition, characterized in that cultured in PDA (Potato Dextrose Agar) medium.
The method of claim 1,
The pine re nematode infected with the Esteya vermicola ( E. vermicola ) is B. mucronatus ( B. mucronatus ) composition for controlling pine re-nematode.
A pine re-nematode control method, comprising using the composition for pine re- nematode control according to claim 1.
The method of claim 5, wherein
Method for controlling pine re-nematode, characterized in that the composition is injected by water injection.
The method of claim 5, wherein
Method for controlling pine re-nematode, characterized in that the composition is used so that the spore infection nematode injected per pine tree is 5,000 to 10,000.

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