KR20030063956A - Bacillus cereus Probio-32 strain for controlling parasitic nematode and method of treating nematode infections in plants - Google Patents

Abstract

PURPOSE: A Bacillus cereus Probio-32 strain for controlling parasitic nematode and a method for treating nematode infection in plants are provided, thereby biologically treating nematode infection in plants. CONSTITUTION: A Bacillus cereus Probio-32 strain (KCCM-10297) for controlling plant parasitic nematode is provided. A composition for the prevention or treatment of plant parasitic nematode contains the Bacillus cereus Probio-32 strain (KCCM-10297) and agrichemically acceptable resins, wherein the composition optionally contains Lactobacillus sp. Probio-23 (KCCM-10327), Bacillus sp. Probio-33 (KCCM-10298), Bacillus sp. Probio-35 (KCCM-10300), Enterococcus sp. Probio-36 (KCCM-10302) and a mixture thereof. A method for the treating nematode infection in plants comprises treating plants, soil or seeds with the Bacillus cereus Probio-32 strain (KCCM-10297) cultured medium or the composition containing Bacillus cereus Probio-32 strain (KCCM-10297), wherein the nematode is root-knot nematode.

Description

Bacillus cereus Probio-32 strain for controlling parasitic nematode and method of treating nematode infections in plants}

The present invention relates to a bacterium Probio-32 of the genus Bacillus and an effective method for controlling nematodes using the same, and more particularly, to control nematodes that cause great damage to various horticultural crops and edible crops. A new nematode bacterium that has the effect of reducing the use of chemical pesticides by treating and preventing nematode infections, thereby reducing problems of human toxicity, killing microorganisms of the soil, and environmental pollution and enabling the production of safe natural agricultural products. The present invention relates to a composition for treating and preventing nematode infections, and a method for controlling nematodes using the same.

Nematodes are small organisms belonging to the nematode, and most of them are so small that they are difficult to see with the naked eye and can be observed under a microscope. Common nematode forms are thin or fusiform, symmetrical in shape, and their length is usually 0.25-0.45 mm, very small, with no nodes, but with wrinkles on the body surface.

Hundreds of these nematodes are known to cause various diseases worldwide by feeding on live plants with coughing. Most nematodes killing plants spend part of their life history in the soil, most of them roaming freely in the soil, invading the roots and stems of the ground, or nutrients from sessile parasites, eggs and larvae. Spend all or part of life history in soil. By classifying pathogens, pathogens can be divided into external parasites, which take nutrients from cells outside the roots, and internal parasites, which enter and nourish host plants, without invading the root tissue. It can be divided into immigration, which roams freely within the plant and takes nutrients from the plant, or moves within the plant, and fixation that does not roam in the roots. External parasitic nematodes include sticky ring nematodes, migratory needles and live nematodes, while internal parasitic nematodes include sticky rootworm nematodes, cyst nematodes, citrus nematodes and migratory root nematode nematodes, citrus root nematodes, Leaf nematodes, atrophy nematodes and helix nematodes.

In this way, the nematodes are mostly present in the soil and parasitic roots of the host plants in the cultivated soil to ingest the nutrients of the host plants, thereby reducing the crop yield. Nematodes also cause diseases of plants on their own, but in many cases nematodes are closely related to these pathogens because they act surrounded by fungi and bacteria that can cause plant disease in the soil. Will cause. For example, when the second larvae of nematodes penetrate the roots of host plants, the invasion of by-product nematodes in the soil and secondary infections by phytopathogens cause much greater damage than each pathogen causes disease. It inhibits the growth of plants.

The annual loss of nematodes to edible crops such as cereals, legumes, bananas, cassava, coconuts, potatoes, sugar beets, sugar cane, and sweet potatoes is around 11% worldwide, and other major economic crops (vegetables, fruits, rain). The loss of edible crops is about 14%, resulting in more than $ 80 billion in economic losses each year.

There are many methods for nematode control, but in practice there are only a few limited uses depending on factors such as costs and crop type factors. Controls generally include seedling methods such as crop rotation, fallow, etc., resistant varieties, biological methods using antagonists or molds, physical methods using immersion, and various kinds of nematicides (eg, 1,3-dichloropropene; 2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbomate; ethyl 3-methyl-4- (methylthio) phenyl- (1-methylethyl) phosphoramidate; and methyl N, N'-dimethyl-N-[(methylcarbamoyl) oxy] -1-thiooxamidate), and the like. Since the 1950s, however, nematicides have been mainly used to effectively control nematodes in high value-added crops such as flowers, vegetables, strawberries, tobacco and seedling crops. In particular, damage to crops by root-knot nematodes has been known for a long time in Korea, and the current situation is severe in the melon farmers in Seongju region, but most of them are controlled by the input of chemicals. Nematodes mainly found in Korea are melon root nematodes (Meloidogyne), for example, carrot root hump nematode, sweet potato root hump nematode, java root hump nematode, peanut root hump nematode, etc. , Cucumbers, tomatoes, watermelons, strawberries, peppers and other fruits and ginseng, such as damage is the most damaging and difficult to control pests. 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. However, in order to alleviate the nematode damage, when the chemicals are used, a cost of about 500,000 won per ha is required, and the drug efficacy is not continuous. In addition, the control of root-knot nematodes by the introduction of chemicals is present not only in environmental pollution but also in the soil. In addition to being a depressive cause, in some cases the use / frequency of these nematode killers may be limited by the regulatory authority. On the other hand, when resistant varieties are used as a control method other than using chemicals, they are limited to only a few crops, and thus practicality is low, and methods such as soil freshwater, solar disinfection, land and soil are difficult to apply depending on the region and crops. There is a costly disadvantage.

Thus, a great deal of research has been conducted to identify effective means of suppressing nematode damage while avoiding or reducing the use of chemical pesticides, and one solution is, instead of chemical pesticides, specific modes of action and relatively safer toxicological profiles. The control of root-knot nematodes by microorganisms having been studied in recent years, and microorganisms killing the root-knot nematodes have been known so far as bacteria in Pasteria, bacteria in Bacillus, and fungi in Pacillomyces.

The present inventors searched for microorganisms that kill nematodes to control phyto-parasitic soil nematodes, which inhabit high density in domestic farmland, especially plant-based facility cultivation medium, which cause enormous damage to crops, resulting in a new Bacillus cereus Probio-32. Isolation and nematode control experiments with the strain confirmed that the effect is very excellent to complete the present invention.

It is therefore an object of the present invention to provide a novel Bacillus cereus Probio-32 exhibiting nematicidal activity.

Another object of the present invention is to provide a plant parasitic nematode infection prevention or treatment composition comprising the microorganism having an nematicidal activity and an agrochemically acceptable carrier.

Still another object of the present invention is to provide a method for preventing or treating an infection of phytoparasitic nematodes by treating the microbial cells or their culture solution or a composition containing an effective amount thereof with plant parts, soil, or seeds. .

Hereinafter, the configuration of the present invention will be described in detail. Other objects and advantages of the invention will be more clearly understood by the following examples.

Figure 1 shows the 16S rRNA sequence of Bacillus cereus Probio-32 of the present invention.

Figure 2 shows the shape of the larvae of M. incognita on a dissecting microscope (100X); At this time, A is a live larvae in tap water, B represents the form of larvae that died after 24 hours after treatment with the microbial culture.

Figure 3 shows the effect of controlling the melon root gall nematode (8 weeks after the diet) according to the nematicide complex microbial treatment.

The above objects of the present invention have been achieved by providing Bacillus cereus Probio-32 exhibiting nematicidal activity, a composition containing the same in an effective amount, and a plant disease control method by nematodes using the strain or composition.

The present invention isolates Bacillus cereus Probio-32, an antagonistic and killing bacterium against plant pests, Roots nematodes, and a microbial pesticide composition prepared therein and a method for preventing and treating diseases of nematodes using the same. It is about.

The nematicidal strain Probio-32 of the present invention isolated from nature is a strain belonging to the genus Bacillus based on 16S rDNA nucleotide sequence, and 100% and 99.7% of 16S rDNA nucleotide sequence (SEQ ID NO: 1), respectively. It was identified as the strain showing the highest molecular relationship between the standard strain of Bacillus cereus and the standard strain of Bacillus thuringiensis .

The isolated strain was named Bacillus cereus Probio-32 by identifying microorganisms according to physiological, morphological and biochemical results, and the Korean Federation of Culture Colleftions (Hongje-dong, Seodaemun-gu, Seoul) The Korean Culture Center of Microorganisms (hereinafter referred to as "KCCM", Korean Culture Center of Microorganisms, 2nd floor of Yurim Building, 221, Accession No. KCCM-10297 (identification No. 18, 2001) reference: deposited as Bacillus sp.

It will be fully understood by those skilled in the art that the strains of the present invention can be improved or improved to have equivalent activity and excellent field stability or to exhibit better anti nematode activity by conventional physicochemical mutation methods and the like.

Since the present invention nematode microorganisms exhibit nematicidal activity against nematodes, it can be used as a microbial pesticide for the treatment and prevention of plant diseases caused by nematode infection. Here, the "biocontrol agent" is a pesticide used to control insects, mites, nematodes, and other plant pathogens or weeds that harm crops by directly using microorganisms or by using agents containing microorganisms. Compared with organic synthetic pesticides, these biopesticides have the characteristics of low toxicity, little impact on the ecosystem, and no drug resistance or resistance. Biological control using the bacterium of the present invention has the advantage of being safer, more environmentally friendly, more efficient and convenient as an alternative to synthetic chemicals. The strain of the present invention may be different depending on the habitat characteristics of the nematode according to the climate, soil and plant type, the root nematode nematode ( Meloidogyne spp.) Generally showing plant pathogenicity; Cyst nematodes, such as Heterodera spp., Globoderra spp., And the like; Root nematode (Pratylenchus.); Citrus nematodes (Tylenchulus semipenetrans); Citrus root nematodes (Radopholus); Stem bulb nematodes (Ditylenchus); Wheat nematodes (Anguina); It can be used for the inhibition of growth of plant nematodes (Aphelenchoides) and the control of plant diseases. Especially preferably, it is widely distributed all over the world, and it stops the growth of root growth point, stops growth and forms excessive roots on plants. It is very effective in controlling plant diseases caused by damaging root-knot nematodes.

When a plant is infected with root-knot nematodes, the roots are always small and swell up, resulting in the formation of typical root nodules and several stages of necrosis. In one preferred embodiment of the present invention, Bacillus cereus Probio-32 was confirmed to have excellent nematicidal activity killing both root-knot nematode larvae in a single day of inoculation on 96 well plates. In addition, the present invention Bacillus cereus ( Bacillus cereus ) Probio-32 strains in order to find out the effect on the packaging directly in the plastic house that has been infected with root-knot nematodes in Seongju area divided into treated strains and untreated plants in the root of melon As a result of examining the weight, the number of the nodules and the number of the egg sacs, the root weight was much increased and the number of the nodules and the number of the egg sacs was reduced. Moreover, the nematicidal microorganism preparation treatment of the present invention showed an increase in fruit as well as an increase in dry matter, and thus, it was found that the nematicidal microorganism fertilizer was effective as well as the nematicidal effect.

In one aspect of the present invention, the present invention provides a composition for preventing and treating nematode infection, comprising an effective amount of Bacillus cereus Probio-32 of the present invention and comprising an agrochemically acceptable carrier. The composition of the present invention is formulated as a powder, pellet or granules, microcapsules, etc. according to a microbial pesticide preparation method of a method known in the art, such as to include culture of cultured cells, lyophilized cells and starch, crude protein and rock powder, etc. It can be used as it is. The composition is a surface activator, inert carrier, preservative, wetting agent, feed promoter, attractant, encapsulating agent, binder, emulsifier, dye, U.V. It can be obtained by adding other ingredients that are easy to apply depending on the desired nematodes and crops such as protecting agents and buffers. The composition of the present invention may be in a form or concentrate or primary composition suitable for direct application by dilution with an appropriate amount of water or other diluent prior to application. It will be apparent to those skilled in the art that the nematicidal concentration may vary, particularly with concentration or direct use, depending on the nature of the particular formulation, the type of plant applied, the soil and climate of the growing region, and the like.

In addition, in order to double the synergistic effects of nematode eradication, other additional biological nematicides, such as the novel Lactobacillus microorganism Probio-23 (KCCM-10327), Bacillus cereus ( Bacillus cereus ) Microorganisms that antagonize nematode activity or nematodes such as Probio-33 (KCCM-10298), Bacillus microorganism Probio-35 (KCCM-10300), Enterococcus microorganism Probio-36 (KCCM-10302) Alternatively, the nematode may be effectively removed by applying a chemical nematicide (eg chitosan, etc.) which is not toxic to the mixture or nematode microorganisms thereof and which has selective toxicity to nematodes. Effective removal means reducing plant damage with a nematode elimination treatment that is cost effective and sufficient to avoid significant economic losses without further nematode inhibition.

Such formulated microbial pesticide compositions can be treated on the soil in which the plant is growing, and also on the surface of the growing plant or on the surface of the seed in storage. When the composition of the present invention is treated on various plant cultivation or cultivated soils of horticultural crops, edible crops, and the like, cultivated soils, there is an excellent effect of suppressing plant diseases caused by root-knot nematodes and other nematode diseases.

The present invention also relates to a method of protecting and treating a plant and its environment by treating the present invention with an effective amount of a root-knot nematode. The nematicidal microorganism of the present invention or the composition containing the same, as a preventive protective measure before the nematode starts or appears on the plant or cultivated soil, grains (for example, wheat, barley, rye, oats, rice, sugar cane) And related crops), sugar beets (eg sugar beet and feed beets), cucumber plants (eg oil, zucchini, melon), vegetables (eg spinach, lettuce, asparagus, cabbage and brassica, carrots, onions, tomatoes, potatoes ), Citrus fruit (e.g. orange, lemon, grapefruit, Chinese tangerine), fruit (e.g. apple, pear, plum, peach, almond, cherry, strawberry, raspberry and blackberry), legume (e.g. alfalfa, Green beans, lentils, peas, soybeans, oily plants (e.g. rape seeds, mustard seeds, poppies, olives, sunflower seeds, coconuts, castor oil plants, cocoa seeds, peanuts), fiber plants (e.g. cotton, flax, hemp , Jute), laurase (e.g., Large, avocado, cinnamon, camphor), deciduous trees and conifers (e.g. yew, chestnut, alder, poplar, birch, fir, larch, pine) or plants (e.g. corn, grass, tobacco, nuts, Coffee, sugar cane, tea trees, vines, hops, bananas and natural gum trees), or the like, or delivered to or sprayed on the soil or seeds of these plants to inhibit or reduce the damage caused by nematodes. Can be. On the other hand, a mixture having the nematicidal activity of the present invention and / or other anti nematode activity in a plant area, soil, seed, etc., where it is necessary to prevent or treat nematode infection or where soil purification is necessary through pre-cultivation nematode removal. Microorganisms and chemical pesticides may be administered at the same time to prevent damage from nematodes.

The anti-nematode microorganisms of the present invention and the composition for preventing or treating nematodes, including the same, live in a high density in soil such as a plant cultivation plant that performs serialization, invade the roots of crops, slow down the growth of crops and reduce the harvest, and eventually As it can be used for plant parasitic nematode control, which causes crops to die, the interest in pollution-free, safe agricultural products is increasing, demand is increasing, and the awareness of residual toxicity and environmental pollution caused by the use of pesticides is increasing. It can contribute to the production of pollution-free safe agricultural products.

Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to Examples. However, the following examples are only for the purpose of illustrating the present invention and the scope of the present invention is not limited to the following examples.

Example 1 Identification of Strains

Strain Probio-32 was incubated at 30 ° C. in NA medium (Difco).

In order to identify strain Probio-32, sequencing and analysis of the nucleotide sequence of the 16S rDNA gene was performed. Base sequence determination and analysis of 16S rDNA gene was performed using the method of Yoon et al., Int. J. Syst. Bacteriol ., 47, 933, 1997. The base sequences of the 16SrRNA genes of Probio-32 are each set forth in SEQ ID NO: 1.

Strain Probio-32 is a strain belonging to the genus Bacillus based on 16S rDNA sequencing analysis. It is 100% and 99.7% homologous to the 16S rDNA sequencing, respectively, and the standard strain of Bacillus cereus It was identified as the strain showing the highest molecular relationship in the standard strain of Bacillus thuringiensis . Therefore, based on the above results, Probio-32 was identified as a Bacillus cereus strain, and the strain of the present invention was deposited on July 18, 2001 to the Korea Microorganism Conservation Center (Accession Number: KCCM-10297).

Example 2 Toxicity Test of Root-knot Nematodes of Bacillus cereus Probio-32

In order to investigate the activity of killing the root-knot nematode of Bacillus cereus Probio-32 identified in Example 1, put about 15-20 root-knot nematode larvae in 96-well plate, and then incubate the above strains One day, two days after the insertion was directly examined under a microscope and the mobility of the second larvae was considered dead nematode (Fig. 2).

As a result, Probio-32 showed effects in 1/10 fold dilution and 1/20 fold dilution (Table 1).

TABLE 1

Example 4: Anti-nematode activity field experiment of Bacillus cereus Probio-32 strain

To find out the effect of the above strain on the pavement, we selected a vinyl house that had been infected with root-knot nematodes in Seongju area, and then irrigated 10 times so that 50 ml per week of melon would be about 10 ⁶ cfu / ml. In order to confirm the difference in treatment method, the test was performed by dividing into irrigation and only irrigation.

As a result, Table 2 shows the melon root weight and number of humps and egg sacs per subject at 8 weeks after treatment with nematode microbial agent after treatment. Root weight of untreated (nematode) was 54.5g, soaking and irrigation showed 62.0g, which increased about 13.8% root weight, and irrigation of 59.6g increased 9.4% compared to untreated. The number of root lumps was 556.7 per treatment, and the number of immersion and irrigation treatments was 168, which decreased by 69.8% compared to the treatments. The irrigation treatment was 58.3, which decreased about 89.5% of root roots. In addition, the number of ovarian sacs decreased similarly to root roots, with immersion and irrigation decreased by 141.7, compared with 62.1% compared with untreated, and 55.7 with irrigation decreased by 85.1% (Fig. 3).

TABLE 2

Table 3 shows the growth and fruit production of melon according to the treatment of root-knot nematode antagonist microorganisms. The total weight was 52.6g in the untreated group, and 84.2g in soaking and irrigation treatment, 31.6g higher than the untreated group, and 70.2 in the irrigated group. g increased by 27.6 g. In addition, when comparing the weights of the treatment sections divided into stems and leaves, the untreated section was the lowest at 29.1g, and the irrigation was 29.2g and the dipping and irrigation 36.8g. Leaf weights were also 23.5g with no treatments, 41.0g of irrigation treatments and 47.4g of soaking and dipping treatments. On the other hand, when comparing the number of fruits per individual, the untreated plot showed 5.7 fruits per individual, and the immersion and irrigation treatment showed 6.0 and the irrigation treatment showed 7.0. It can be seen that it is effective as a nutritional microbial fertilizer as well as nematicide effect.

TABLE 3

On the other hand, the number of antagonistic strains according to the treatment of root-knot nematode antagonist microorganisms was 2.8 x 10 ⁷ (cfu / g), and there was no difference in immersion and irrigation treatments as 2.8 x 10 ⁷ , and in the irrigation treatments as 6.7 x 10 ⁷ Many antagonistic strains were shown. In addition, the number of nematodes was 395, and the control group showed the highest number of root-knot nematodes. The number of root-knot nematodes was slightly reduced to 276 soaking and irrigation treatments, and the number of root-knot nematodes was 98. It is thought to be the most effective at reducing nematode density (Table 4).

TABLE 4

As described above, the Bacillus cereus Probio-32 strain of the present invention kills larvae and eggs, which are parasitic on crop roots and cause severe damage, and thus have an antagonistic effect on nematode infection. Treatment in severely damaged plant cultivation areas has a very good effect of reducing environmental pollution and natural ecosystem destruction caused by pesticides and producing pollution-free natural agricultural products.

Claims (5)

Bacillus cereus Probio-32 strain (KCCM-10297) with nematicidal activity. Plant parasitic nematode infection prevention or treatment comprising a strain having the nematicidal activity of claim 1 and an agrochemically acceptable carrier. The method according to claim 1, wherein the composition is Lactobacillus microorganism Probio-23 (KCCM-10327), Bacillus microorganism Probio-33 (KCCM-10298), Bacillus microorganism Probio-35 (KCCM) having antagonistic activity against nematodes -10300), Enterococcus microorganism Probio-36 (KCCM-10302), and mixtures thereof. A method of preventing or treating an infection of phytoparasitic nematodes by treating the microbial cells of claim 1, the culture medium thereof, or the composition of claim 2 or 3 with a plant part, soil, or seed. The method of claim 4, wherein the nematode is a root-knot nematode.