KR100860726B1 - Bacillus thuringiensis Probio-35 strain for controlling parasitic nematode and method of treating nematode infections in plants - Google Patents

Abstract

The present invention relates to Bacillus thuringiensis Probio-35 and an effective control method of nematodes using the same, the present invention kills the larvae and eggs of nematodes, which are parasitic on the roots of crops and cause severe damage to nematode infections. Because of its antagonistic properties, it is very effective to reduce environmental pollution and natural ecosystem destruction caused by pesticides and produce pollution-free natural agricultural products when treated in plant cultivation areas with severe nematode damage.

Nematodes, compositions, plant diseases, microbial pesticides, Bacillus

Description

Bacillus thuringiensis Probio-35 strain for controlling parasitic nematode and method of treating nematode infections in plants}

Figure 1 shows the 16S rRNA sequence of Bacillus thuringiensis Probio-35 of the present invention.

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 present invention relates to Bacillus thuringiensis Probio-35 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. By using Serengenesys Probio-35 to treat and prevent nematode infections, it is possible to reduce the use of chemical pesticides, thereby reducing the risk of human toxicity, killing soil microorganisms and environmental pollution, and enabling the production of safe natural produce. Nematode bacteria, compositions for preventing or treating nematode infections containing the same, 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 pathogenic nematodes, it is generally possible to divide into ectoparasites, which take nutrients from cells outside the roots, and endoparasites, which invade and host food, 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. However, since the 1950s, 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. 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 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 in order 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, and thus, Bacillus thuringiensis . Probio-35 was newly isolated and nematode control experiment with the strain was 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 thuringiensis Probio-35 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.

The above objects of the present invention are achieved by providing Bacillus thuringiensis Probio-35 showing an activity of nematicids in an effective amount thereof, and a method for controlling plant diseases by nematodes using the strain or composition. It became.

The present invention isolates Bacillus thuringiensis Probio-35, a microbial antagonistic and killing bacterium against plant pests, from the soil, and formulated the microbial pesticide composition and the root prevention nematode using the same. To a method of treatment.

The present invention isolated from the natural world salseon loyalty strain 16S rDNA nucleotide sequence (SEQ ID NO: 1) a molecular Phylogenetic analysis of Bacillus (Bacillus) were 100% and 99.7% of the 16S rDHA nucleotide sequence homology as a strain belonging to the genus in based on Bacillus the type strain of the write Lindsay N-Sys type strain and Bacillus cereus (Bacillus cereus) of (Bacillus thuringiensis) was identified as a strain showing the highest molecular phylogenetic relationship flexible. The isolated strain was named Bacillus thuringiensis Probio-35 by identifying microorganisms according to physiological, morphological and biochemical results, and the Korean Federation of Culture Colleftions (361, Hongje-dong, Seodaemun-gu, Seoul). -Korean Culture Center of Microorganisms (hereinafter referred to as "KCCM", Korean Culture Center of Microorganisms, 2nd Floor, Yurim Building, 2nd Floor, Accession No. KCCM-10300 (KCCM-10300) identification reference: deposited as Bacillus thuringiensis Probio-35).

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 anti-nematode microorganism of the present invention shows nematicidal activity against nematodes, it can be used as a microbial pesticide for the treatment and prevention of plant diseases caused by an elected infection. Here, the "biocontrol agent" is a pesticide used to control insects, mites, nematodes, and other phytopathogens or weeds that directly harm the crops by using the microorganisms themselves or using a preparation 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 in general, root-knot nematodes ( Meloidogyne spp. ) Exhibiting 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 thuringiensis microbial Probio-35 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 thuringiensis ( Bacillus thuringiensis ) Probio-35 strains in order to find out the effect on the direct packaging in the plastic house that has been infected with root-knot nematodes in Seongju region divided into treated strains and untreated spheres As a result of investigating the root weight, the number of humps per egg and the number of egg sacs, it was found that the root weight was much increased when the strain of the present invention was treated. Moreover, the nematicidal microorganism formulation 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 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 thuringiensis Probio-35 of the present invention and 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 obtained by adding surface activators, inert carriers, preservatives, wetting agents, feed promoters, attractants, encapsulating agents, binders, emulsifiers, dyes, UV protectors, buffers, etc. can do. 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 Lactobacillus microorganism Probio-23 (KCCM-10327), Bacillus cereus, isolated from the soil by the inventors Microorganisms that exhibit antagonistic activity against nematode activity or nematodes, such as Probio-32 (KCCM-10297), Bacillus serringiensis Probio-33 (KCCM-10298), Enterococcus microorganism Probio-36 (KCCM-10302), or The nematode may be effectively removed by a combination of chemical nematicides (eg chitosan, etc.) which are not toxic to the mixture or nematode microorganisms and which have 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 treating and protecting a plant and its environment by treating an effective amount of the present invention with 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 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 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 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-35 was incubated at 37 ° C. in MRS medium (Difco).

In order to identify strain Probio-35, sequencing and analysis of 16S rDNA gene was performed. Base sequence determination and analysis of the 16s rDNA gene was performed by Yoon et al., Int. J. Syst. Bacteriol., 47, 933, 1997.

The base sequences of the 16S rRNA genes of Probio-35 are each set forth in SEQ ID NO: 1 (FIG. 1). Type strain of strain Probio-35 is a Bacillus (Bacillus) Lindsey N-Sys (Bacillus thuringiensis), each written Bacillus as 100% and 99.7% of the 16S rDNA sequence homology as a strain belonging to the genus in Molecular Phylogenetic analysis based on 16S rDNA base sequence the type strain of the Bacillus cereus (Bacillus cereus) was identified as a strain showing the highest molecular phylogenetic relationship flexible. Therefore, the Probio-35 was deposited on the basis of the above results as identified by Lindsey N-Sys (Bacillus thuringiensis) strains of Bacillus written, and July 25, 2001 the Korea Culture Center of Microorganisms (accession number: KCCM-10300).

Example 2 Toxicity Test of Root-knot Nematodes of Bacillus thuringiensis Probio-35

In order to investigate the activity of killing the root-knot nematode of Bacillus thuringiensis Probio-35 identified in Example 1, put about 15-20 root-knot nematode 2 larvae on a 96 well plate. One day, two days after the addition of the culture medium was directly examined under a microscope and the mobility of the second larva larva was considered dead nematode (Fig. 2.) As a result, Probio-35 1/10 times dilution solution, 1 The effect was shown in the / 20-fold diluent (Table 1).

Example 4: Anti-nematode activity field experiment of Bacillus thuringiensis Probio-35 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 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 Bacillus thuringiensis Probio-35 strain of the present invention parasitic on the roots of crops, which kills the larvae and eggs that are causing serious damage, and has an antagonistic effect on nematode infection. Treatment of plants with severe damage to plants reduces the environmental pollution and natural ecosystem destruction caused by pesticides, and has a very good effect of producing pollution-free natural agricultural products.

<110> Probionic Co. <120> Bacillus thuringiensis Probio-35 strain for controlling parasitic          nematode and method of treating nematode infections in plants <130> probio-35 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1507 <212> DNA <213> Bacillus thuringiensis Probio-35 (KCCM-10300) <220> <221> rRNA (222) (1) .. (1507) <223> 16S rRNA nucleotide sequence <400> 1 gatgaacgct ggcggcgtgc ctaatacatg caagtcgagc gaatggattg agagcttgct 60 ctcaagaagt tagcggcgga cgggtgagta acacgtgggt aacctgccca taagactggg 120 ataactccgg gaaaccgggg ctaataccgg ataacatttt gaactgcatg gttcgaaatt 180 gaaaggcggc ttcggctgtc acttatggat ggacccgcgt cgcattagct agttggtgag 240 gtaacggctc accaaggcaa cgatgcgtag ccgacctgag agggtgatcg gccacactgg 300 gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac 360 gaaagtctga cggagcaacg ccgcgtgagt gatgaaggct ttcgggtcgt aaaactctgt 420 tgttagggaa gaacaagtgc tagttgaata agctggcacc ttgacggtac ctaaccagaa 480 agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttatccgg 540 aattattggg cgtaaagcgc gcgcaggtgg tttcttaagt ctgatgtgaa agcccacggc 600 tcaaccgtgg agggtcattg gaaactggga gacttgagtg cagaagagga aagtggaatt 660 ccatgtgtag cggtgaaatg cgtagagata tggaggaaca ccagtggcga aggcgacttt 720 ctggtctgta actgacactg aggcgcgaaa gcgtggggag caaacaggat tagataccct 780 ggtagtccac gccgtaaacg atgagtgcta agtgttagag ggtttccgcc ctttagtgct 840 gaagttaacg cattaagcac tccgcctggg gagtacggcc gcaaggctga aactcaaagg 900 aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga 960 accttaccag gtcttgacat cctctgaaaa ccctagagat agggcttctc cttcgggagc 1020 agagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080 cgcaacgagc gcaacccttg atcttagttg ccatcattaa gttgggcact ctaaggtgac 1140 tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc 1200 tgggctacac acgtgctaca atggacggta caaagagctg caagaccgcg aggtggagct 1260 aatctcataa aaccgttctc agttcggatt gtaggctgca actcgcctac atgaagctgg 1320 aatcgctagt aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca 1380 ccgcccgtca caccacgaga gtttgtaaca cccgaagtcg gtggggtaac ctttttggag 1440 ccagccgcct aaggtgggac agatgattgg ggtgaagtcg taacaaggta gccgtatcgg 1500 aaggtgc 1507

Claims (5)

Bacillus thuringiensis Probio-35 strain having a nematicidal activity (KCCM-10300). Root-knot nematode infection prevention or treatment composition comprising the strain having the nematicidal activity of claim 1 and an agrochemically acceptable carrier. delete A method for preventing or treating an infection of root-knot nematodes by treating the plant parts, soil, or seeds with one selected from the microbial cells of claim 1, their culture solution, and the composition of claim 2. delete

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