KR20030063955A - Lactobacillus sp. Probio-23 strain for controlling parasitic nematode and method of treating nematode infections in plants - Google Patents

Abstract

PURPOSE: A Lactobacillus sp. Probio-23 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 Lactobacillus sp. Probio-23 strain (KCCM-10327) for controlling plant parasitic nematode, especially root-knot nematode is provided. A composition for the prevention or treatment of plant parasitic nematode contains the Lactobacillus sp. Probio-23 strain (KCCM-10327) and agrichemically acceptable resins, wherein the composition optionally contains Bacillus cereus Probio-32 (KCCM-10297), 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 treating nematode infection in plants comprises treating plants, soil or seeds with the Lactobacillus sp. Probio-23 strain (KCCM-10327) cultured medium or the composition containing Lactobacillus sp. Probio-23 strain (KCCM-10327).

Description

A new Lactobacillus genus bacterium Probio-23 with nematicidal activity and an effective method for controlling nematode using the same {Lactobacillus sp. Probio-23 strain for controlling parasitic nematode and method of treating nematode infections in plants}

The present invention relates to Lactobacillus bacteria Probio-23 and an effective control method of nematodes using the same, and more specifically, to control nematodes causing great damage to various horticultural crops, edible crops, etc. A new nematode bacterium that has the effect of treating and preventing nematode infections using -23, thereby reducing the use of chemical pesticides, thereby reducing problems of human toxicity, killing soil microorganisms and environmental pollution, and enabling the production of safe natural produce. The present invention relates to a composition for preventing or treating nematode infection, and a method for controlling nematode 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 the root-knot nematodes (Meloidogyne), such as carrot root-knot nematodes, sweet potato root-knot nematodes, java root-knot nematodes, peanut root-knot nematodes, 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, and the damage is gradually increased due to the indiscriminate sterilization of useful microorganisms in the soil, which is beneficial to the growth of crops through symbiotic relationship with crops. 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, so they are low in practicality. 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 are known to include bacteria of Pasteria, bacteria of Bacillus, and fungi of Pacillomyces. No studies have yet been reported on the microorganisms of the genus Lactobacillus that act antagonistically or have strong nematicidal activity against root-knot nematodes.

The present inventors searched for microorganisms killing nematodes to control phyto-parasitic soil nematodes, which inhabit high density in domestic farmland, especially plant cultivation-based plant cultivation plants, which cause enormous damage to crops. Various lactobacillus strains antagonistic to infection and especially nematode activity against root-knot nematodes were isolated, among which microorganisms showing high activity were named Lactobacillus bacteria Probio-23. Experiment was confirmed that the effect is very excellent to complete the present invention.

Accordingly, an object of the present invention is to provide a novel Lactobacillus genus microorganism, preferably Lactobacillus genus microorganism Probio-23 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.

1 is a diagram showing the 16S rRNA nucleotide sequence of the present invention Lactobacillus bacteria Probio-23.

Figure 2 shows the shape of M. incognita larvae on anatomical 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 melon root gall nematodes (8 weeks after the diet) according to the nematode complex microbial treatment.

The object of the present invention is a Lactobacillus genus microorganism, preferably Lactobacillus genus microorganism Probio-23, which exhibits nematicidal activity, a composition containing the same in an effective amount, and a plant disease control method by nematodes using the strain or composition Was achieved by providing.

The present invention relates to a microbial pesticide composition prepared by separating the microorganism of the genus Lactobacillus, which is antagonistic and killing force against the plant noxious root root nematode, from the soil, and formulating the same, and a method for preventing and treating the root noxious nematode using the same.

To date, no bacterium of the genus Lactobacillus with nematicidal activity has been reported, and the present inventors have isolated several lactobacillus strains that have antagonistic activity against nematode's plant infection in nature and especially have nematicidal activity against root-knot nematodes. Among them, strains of the present invention showing particularly high activity were selected and subjected to molecular phylogenetic analysis based on 16S rDNA nucleotide sequences. As a strain belonging to the genus Lactobacillus , 98.2% of 16S rDNA nucleotide sequences (SEQ ID NO: 1) Homologously, it was identified as the strain showing the highest molecular relationship in the standard strain of Lactobacillus hilgardii . The isolated strains were identified as microorganisms according to physiological, morphological and biochemical results, and were named Lactobacillus bacteria Probio-23, and Korean Federation of Culture Colleftions (361-221 Hongje-dong, Seodaemun-gu, Seoul) On October 27, 2001, the Korean Culture Center of Microorganisms (KCCM), a deposit deposit and preservation institution of the 2nd floor of the building, assigned accession number KCCM-10327 (identification reference: Lactobacillus). sp . Pro-23) 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. In addition, the microorganisms of the genus Lactobacillus are antagonistic to nematodes and The fact that the nematicidal activity was confirmed by the inventors for the first time, on the basis of this fact through the well-known methods of identifying microorganisms isolated Lactobacillus, anti- nematode activity and field crop experiment, etc. It will be fully understood by those skilled in the art that it is possible to screen microorganisms of the genus Lactobacillus which act antagonistically on nematode infections and, in particular, have nematicidal activity against root-knot nematodes.

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 vary depending on the habitat characteristics of the nematode according to the climate, soil, and plant type, but root knot nematodes ( Meloidogyne spp .), Which generally exhibits 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, the microbial Probio-23 of the genus Lactobacillus was confirmed to have excellent nematicidal activity that kills both root-knot nematode larvae in a single day of inoculation on a 96 well plate. In addition, in order to find out what effect the microbial Probio-23 strain of the genus Lactobacillus of the present invention has in direct packaging, the root weight of cultivated melon, divided into treated and untreated areas, strains in the plastic house that has been infected with root-knot nematodes in Seongju area, As a result of examining the number of humps and the number of egg sacs per subject, it was found that the root weight was much increased and the number of humps and egg sacs per subject was also 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, which comprises an effective amount of the present invention Lactobacillus bacteria Probio-23 and comprises an agrochemically acceptable carrier. The composition of the present invention is formulated in powder, pellet or granules, microcapsules, etc., according to a microbial pesticide preparation method in a manner known in the art, for example, to include culture of cultured cells, lyophilized cells and starch, crude protein and rock powder, and the like. Can be used as 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, to further double the synergistic effect of nematode eradication, other additional biological nematicides, such as the novel Bacillus cereus Probio-32 (KCCM-10297), which the inventors have isolated from the soil, Microorganisms that antagonize nematode activity or nematodes such as Bacillus microorganisms Probio-33 (KCCM-10298), Bacillus microorganisms Probio-35 (KCCM-10300), Enterococcus microorganisms 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, poppy, olives, sunflower seeds, coconut, castor oil plants, cocoa seeds, peanuts), fiber plants (e.g. cotton, flax, hemp , Jute), laurase (example 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 to plant soil or seeds of these plants, to prevent 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 the prevention or treatment of nematode infection is necessary or where the soil purification through nematode removal is necessary before cultivation. 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 nematode prevention or treatment composition comprising the same live in high density in soil such as a plant cultivation plant, which performs serialization, invade the roots of crops, slow 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-23 was incubated at 37 ° C. in MRS medium (Difco). In order to identify strain Probio-23, sequencing and analysis of 16S rDNA gene was performed. The sequencing and analysis of the 16s rDNA gene was performed using the method of Yoon et al ., Int . J. Syst. Bacteriol. , 47, 933, 1997.

The base sequences of the 16S rRNA genes of Probio-23 are each set forth in SEQ ID NO: 1 (FIG. 1). Strain Probio-23 is a strain belonging to the genus Lactobacillus based on 16S rDNA sequencing analysis, and has the highest homology with the standard strain of Lactobacillus hilgardii with 98.2% 16S rDNA sequence homology. It was identified as a strain showing high molecular lineage relationship. Therefore, based on the above results, Probio-23 was identified as a strain of the genus Lactobacillus , and the strain of the present invention was deposited to the Korea Microorganism Conservation Center on October 27, 2001 (Accession Number: KCCM-10327).

Example 2 Toxicity of Root-knot Nematodes of the Microorganism Probio-23 in Lactobacillus Species exam

In order to investigate the activity of killing the root-knot nematode of the microorganism Probio-23 of the genus Lactobacillus identified in Example 1, put about 15-20 root-knot nematodes 2 larvae on a 96 well plate, and then culture the day strain of the above strain. 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-23 showed effect in 1/100 fold dilution and 1/200 fold dilution (Table 1).

Example 4 Anti-nematode Activity Field Experiment of Microbial Probio-23 Strains of the Genus Lactobacillus

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 heating water also decreased similarly to root root water, soaking and irrigation treatments were 141.7, which was reduced by 62.1% compared to the non-treated, and irrigation treatments were reduced to 85.7% by 55.7 (Fig. 3).

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.

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).

As described above, the microorganism Probio-23 strain of the genus Lactobacillus of the present invention kills the larvae and eggs of the nematodes, which are severely damaged by crop roots, and have a characteristic of antagonistic action against nematode infection. Treatment with plant cultivation area has the excellent effect of reducing environmental pollution and natural ecosystem destruction caused by pesticides and producing pollution-free natural agricultural products.

Claims (6)

Microorganisms of the genus Lactobacillus that act antagonistically against phytoparasitic nematode infections and have nematicidal activity against root-knot nematodes. The microorganism of claim 1, wherein the microorganism is Lactobacillus sp. Probio-23 strain (KCCM-10327). Plant parasitic nematode infection prevention or treatment comprising a strain having the nematode activity of claim 1 or 2 and an agrochemically acceptable carrier. The method according to claim 3, wherein the composition has antagonistic activity against nematodes Bacillus cereus Probio-32 (KCCM-10297), Bacillus microorganisms Probio-33 (KCCM-10298), Bacillus microorganisms Probio- 35 (KCCM-10300), Enterococcus microorganism Probio-36 (KCCM-10302), and mixtures thereof. A method for preventing or treating an infection of phytoparasitic nematodes by treating the microbial cells of claim 1 or 2, the culture medium thereof, or the composition of claim 3 or 4 with a plant part, soil, or seed. The method of claim 5, wherein the nematode is a root-knot nematode.