KR100379019B1 - Pasteuria penetrans 99-13 isolate parasiting on Meloidogyne hapla and its isolation method - Google Patents

Abstract

The present invention relates to Pasteuria penetrans 99-13 bacteria, which is an absolute parasitic in a carrot root nematode (Meloidogyne hapla), and a method of separating the same from soil, and increases the number of consecutive years of various fruits, vegetables, flowers, special crops, etc. Pasteur phenanthrace 99-13 bacteria, which are nematodes and nematodes, which are the basis of the biological control system of carrot root-knot nematodes, which are parasitic at the roots of plants and cause crop growth and premature death. It is a method of capturing carrot root gall nematodes and separating them from soil in the form of endospores.

Description

Pasteuria penetrans 99-13 isolate parasiting on Meloidogyne hapla and its isolation method

The present invention relates to Pasteuria penetrans 99-13 bacteria, which is an absolute parasitic in a carrot root nematode (Meloidogyne hapla) and a method for separating the same from soil, and more particularly, various fruits, flowers, crops, etc. As the number of years increases, Pasteria penetrance 99-13 bacteria, which are nemesis organisms of nematodes, which are the root of the biological control system of carrot root gall nematodes, which are parasitic on the roots of plants and cause crop failure and early death. It is a method of capturing this as a host nematode, carrot root-knot nematode, and separating it from soil in the form of endospores.

Carrot root-knot nematodes are pests that paralyze the roots of crops, deodorizing nutrients and water, thereby degrading the growth of crops and, if severe, prematurely killing crops or invading other pathogens. Recently, as the cultivation area of horticultural crops and specialty crops is increased, and the number of consecutive years in limited farmland increases, the damage caused by root pests, root pests, is spreading. Root-knot nematodes are parasitic to the roots of plants and complete life cycles in about four weeks, so that females lay about 400 eggs. Therefore, farmers use nematicides to disinfect soil every year in order to control the pests, but it is impossible to control 100% of the pests, and re-contamination of nematodes through soil, watering, etc. has been repeated, and damage continues in the field. In addition, the nematicidal nematode used for the control of root-knot nematode costs about 10 billion won per year, and the time and labor required for the treatment are enormous.

On the other hand, some areas are treated with nematode, soil reversal, and flooding for nematode control, but these are also very costly and are not effective in the long run. Therefore, it is urgent to develop new eco-friendly and effective control techniques.

The present invention has been made in order to overcome the above-mentioned conventional problems in the control of carrot root-knot nematodes, the object of the present invention is the development of biological control system of carrot root-knot nematode (Meloidogyne hapla) that damage is widening in various field crops It is to provide Pasteuria penetrans 99-13 bacteria, which is a nematode's natural bacterium.

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

1. Isolation and Deposit of Novel Microorganisms of the Invention

The bacterium of the present invention is a bacterium selectively parasitic only to carrot root-knot nematodes, and is separated from the soil into endospores by a capturing method using nematodes. The separated endospores are stored in a refrigerator diluted in distilled water.

The endogenous spores of the present invention isolated from the soil by nematode capture were named Pasteuria penetrans 99-13 and deposited on July 19, 2000 with the KFCC. , Accession number KFCC-11186.

2. Identification of New Microorganisms of the Invention

Morphology and structural characteristics of the isolated strains are as follows.

The strain of the present invention is Gram-positive bacteria and forms mycelia with septum. Endospores are formed at the end of mycelia. Endospores are discoids similar to UFOs, with a nucleus in the center and a membranous structure used to attach to host nematodes. These endospores are approximately 4.7 um (4 to 5.5 um) in diameter, 2 um (1.6 to 2.4 um) high, and 2.3 um (1.9 to 2.9 um) in diameter. Endogenous spores do not have their own motility.

3. Action of novel microorganism of the present invention

The action of the bacterium of the present invention is to kill nematodes by parasitic carrot root gall nematodes. Bacteria remain in the soil in the form of endogenous spores and adhere to the surface of the larvae of the carrot root-knot nematodes, which are active around them. When nematodes begin to suck plants and grow, the endogenous spores of bacteria penetrate into the nematodes and nourish as the nematodes grow. In general, the root-knot nematode completes the growth and prepares laying in about 4 weeks under the appropriate temperature. At this time, the bacteria complete the nutrition and enter the reproduction stage to form the spores. In this process, the nematode loses its nutrients and dies, so it cannot be spawned and the nematode is filled with endospores of bacteria.

When the plant is finished growing and the roots are broken down, bacteria leak into the soil along with nematodes. About 2 million endospores are formed per nematode. Mature endospores are viable for many years after they leak into the soil, and bacteria that form endogenous spores are generally resistant to environmental changes, ie changes in temperature and humidity, and physical and chemical changes in the soil.

4. Soil Separation Method-Capture Method Using Host Nematodes

In order to mass-proliferate the bacterium of the present invention, firstly, a leading technology is required to separate the bacteria from the soil to secure a certain amount of endospores. The bacterium presented in the present invention is difficult to separate from the soil because it is impossible to separate culture using artificial media. Therefore, by inoculating the roots of the plants after the capture of the endospores using the host nematode and incubating them, it is possible to secure a large amount of endospores that can be used for subsequent bacterial mass growth test.

The capture method using nematodes is a first step in which a large number of carrot root worms (Meloidogyne hapla) are cultured in tomato roots in a greenhouse and the eggs of the nematodes are isolated and hatched: endogenous hatching of the hatched larvae of Pasteria penetrance 99-13. Step 2 of inoculating spores into the soil and then inoculating them into the root zone of the tomato: After cultivating the mixed inoculated tomato, dissecting the female adult insect of the carrot root-knot nematode to endocrine spores of bacteria in the female body It consists of a third step of separating.

Hereinafter, the capture method using the host nematode in detail.

In the first step, the carrot root-knot nematode, the host nematode of the present invention, is cultured in the tomato roots of the greenhouse, and the eggs of the nematodes are separated by an analyte decanting method and incubated in the laboratory by a funneling method. Let's do it. The larvae of hatched nematodes are then counted under a microscope after 2-3 days. In the second step, 50 g of the endogenous spores are dispensed by 50 g, and then 5,000 larvae hatched in the first step are inoculated with 15 cc of distilled water. At this time, if the number of spores is too much or too small for the number of nematodes, the number of bacterial spores is too much or too little to be attached to each larva. If too few bacterial spores are attached, the propagation efficiency of bacteria decreases. As a result, the invasion efficiency to the host plant is lowered, so the proliferation efficiency may be lowered. Therefore, it is most preferable to inoculate with distilled water so that there are 5,000 larvae per 50 g of soil in which endospores exist. After inoculation, the soil is sealed and placed in a 25 ° C incubator. After 7 days, the soil is removed from the incubator and mixed with the root of tomato. In general, the number of adherence to the endoscopic spores at about 25 ℃ is the maximum, it takes about 6 to 8 days for the endospores to enter the nematode at this temperature, so the soil mixed with the larvae at 25 ℃ Most preferably, it is removed after about 7 days after being injured in an incubator. In the third step, after cultivating the mixed inoculated tomatoes at 30 ℃, after eight weeks to isolate the female adult of the carrot root-knot nematodes parasitic in the root of the tomato. Female adult insects of the carrot root-knot nematodes are triturated with a tissue grinder to separate endogenous spores of bacteria in the female body.

After the regrowth of the endogenous spores separated through the above process several times to secure a large amount of endogenous spores and processed in farm packaging contaminated with nematodes, or by grinding the roots of tomatoes after 8 weeks in the capture method After the preparation, it can be used to prevent pests by treating nematode-contaminated packages .

Hereinafter, the nematode parasitic range of the novel microorganism of the present invention will be described through experimental examples.

Experimental Example 1

Parasitic Range Testing of New Microorganisms

In order to find out the degree of attachment to nematode larvae of the novel bacterium Pasteuria penetrans 99-13 (KFCC-11186) of the present invention, a mixture of nematode and bacterial endospores was carried out for 24 hours, and then The results are shown in Table 1.

TABLE 1

Body surface attachment by nematode type Carrot Root Peanut rootworm Sweet Potato Root Nematode Java Root Beetle Nonparasitic nematodes Attached or not O X X X X

From Table 1, it can be seen that the novel bacterium Pasteria penetrance (Pasteuriapenetrans) 99-13 (KFCC-11186) of the present invention selectively attaches only to a carrot root nematode.

As discussed above, it is urgent to develop new eco-friendly and sustainable effective control techniques, namely biological control systems using natural enemies. Therefore, if the biological control system of the nematode using the carrot root-knot nematode (Meloidogyne hapla) parasitic Pasteuria penetrans (Pasteuria penetrans) 99-13 bacteria (KFCC-11186) is a root-knot nematode which is a problem in the culture medium Low cost and high efficiency can be achieved. In addition, since the bacteria are not parasitic except for control bacteria, there are no adverse effects on the environment and no toxicity to human beings. If this technology is put into practical use, it will be expected to contribute to low pesticide input and high quality agricultural production by reducing pesticide use in the long term, preventing soil and water pollution, and drastically reducing the problems caused by pesticide residue in fresh and vegetable production.

Claims (4)

Carrot Root Nematode (Meloigogyne hapla) for the control of natural bacteria Pasteria penetrans (Pasteuria penetrans) 99-13 bacteria (KFCC-11186) A method for separating the Pasteria penetrance 99-13 bacterium (KFCC-11186) according to claim 1 from the soil by a capturing method using the host nematode, Meloigogyne hapla, wherein the carrot root-knot nematodes are separated from the tomato roots of the greenhouse. The first step of incubating the eggs of the nematode by incubating the mass of the nematode into the soil containing the endospores of Pasteria penetrance 99-13 bacteria and then inoculating them into the root zone of the tomato The second step: cultivating the mixed inoculated tomato and then dissected to isolate the female adult of the carrot root-knot nematode, the third step of separating the endogenous spores of bacteria in the female body, Pasteria penetrance 99 -13 Isolation of bacteria (KFCC-11186) The pasteeria of claim 2, wherein the first step comprises separating the eggs of the nematode mass cultured in tomato roots by an analytical decanting method and incubating the funnels with a funnel. Isolation Method of Penetrant 99-13 Bacteria (KFCC-11186) The method of claim 2, wherein the second step is divided into 50 g of the soil containing the endospores of Pasteria penetrance 99-13 bacteria and inoculated with each of the 5,000 larva hatched in the first step with distilled water Separation method of Pasteria penetrance 99-13 bacteria (KFCC-11186), characterized by inoculation in a 25 ℃ incubator, taken out and mixed inoculation to closely adhere to the tomato root.