KR20240021574A - Novel peanibacillus tianmuensis strain and composition for controlling plant diseases using the strain or culture filtrate - Google Patents

Abstract

The present invention relates to the Paenibacillus tianmuensis YM002 strain (accession number: KACC 92433P), and to a composition for controlling plant diseases using the YM002 strain (accession number: KACC 92433P) and its culture filtrate. The strain or culture filtrate according to the present invention improves the stress resistance of plants, thereby enabling sustainable agriculture and eliminating or minimizing the use of chemical pesticides.

Description

Composition for controlling plant diseases using the new strain Pennybacillus tianmuensis and its culture filtrate {NOVEL PEANIBACILLUS TIANMUENSIS STRAIN AND COMPOSITION FOR CONTROLLING PLANT DISEASES USING THE STRAIN OR CULTURE FILTRATE}

The present invention relates to a composition for controlling plant diseases using a novel microorganism or its culture filtrate, and more specifically, to a composition for controlling plant diseases using a novel Pennybacillus tianmuensis strain or its culture filtrate.

In modern agriculture, in order to achieve food self-sufficiency and improve farm income through high yields, it is necessary to replace or supplement chemical pesticides and fertilizers due to problems such as environmental pollution caused by the continuous use of chemical pesticides and fertilizers, and quality degradation due to residual pesticides and residual toxic substances. The need to develop a biocontrol agent that can control the disease is emerging. The development of biological control agents using various microorganisms existing in nature is essential for sustainable agriculture by reducing the use of chemical pesticides and fertilizers.

There are 825 species of Cucurbitaceae family plants distributed around the world in 118 genera. In Korea, watermelons, melons, cucumbers, and pumpkins are mainly cultivated, and there are cases where it is not easy to suppress pathogens through chemical control. Taking cucumbers as an example, there are about 18 known diseases that occur in domestic cucumbers, and phytopathogenic bacteria and fungi are involved in them. Diseases that mainly occur in cucumbers and cause problems during cultivation include vine blight, vine scorch, powdery mildew, downy mildew, and gray mold. These diseases are a major cause of quality deterioration and reduced yield in cucumber cultivation, but they can be controlled by applying appropriate chemicals. Cucumber fruit rot is an important seed-borne disease that occurs widely in cucurbit plant growing areas across the country and causes a lot of damage. Cucumber fruit rot pathogens exist in the tissues of diseased plants or in infected seeds, and occur the following year after wintering. Infected seedlings show water-soaked lesions on the leaves. Contact transmission is possible through agricultural work such as grafting and vine attraction, and secondary transmission is mainly caused by rainwater or irrigation. Pathogens mainly move into the stomata about 3 weeks after flowering and infect the fruit. Afterwards, symptoms progress rapidly before fruit harvest. In particular, the cucumber fruit rot pathogen is a bacterium that grows vigorously in a high temperature and humidity environment above 30℃, and generally spreads rapidly when cucumber fruit rot disease occurs in nursery beds where dense cultivation is conducted under high temperature and humidity conditions. It is a very difficult disease to control because it occurs throughout the entire growing season and, like many other bacterial diseases, there is no control method, and even disease-resistant varieties have no apparent effect.

The purpose of the present invention is to provide a composition for controlling plant diseases using the new Paenibacillus tianmuensis YM002 strain (accession number: KACC 92433P) or its culture filtrate, which can be prescribed at the pre-growth stage.

In addition, the purpose of the present invention is to provide a method of eliminating or minimizing the use of chemical pesticides and fertilizers by improving the stress resistance of plants and thereby enabling sustainable agriculture.

The object of the present invention is not limited to what has been mentioned above, and other objects not mentioned will be clearly understood by those skilled in the art from the description below.

In order to solve the above technical problem, according to one aspect of the present invention, Paenibacillus tianmuensis YM002 strain (accession number: KACC 92433P) is provided.

Here, it may be provided in a composition for promoting cucumber plant growth containing the strain or the culture filtrate of the strain.

Here, a composition for inducing systemic resistance to cucumber plant pathogens containing the strain or a culture filtrate of the strain may be provided.

Here, a composition for controlling plant diseases for controlling cucumber fruit rot disease containing the strain or the culture filtrate of the strain as an active ingredient may be provided.

Here, cucumber fruit rot may be caused by Acidovorax citrulli .

Here, a method of inducing systemic resistance to a cucumber plant pathogen is provided, comprising treating the strain or the culture filtrate of the strain to a cucumber plant, an area around the cucumber plant, or both.

Here, the method for enhancing systemic induced resistance may be treating one or more selected from the group consisting of seeds, roots, stems, leaves, and the entire plant of the cucumber plant.

Here, a method for promoting cucumber plant growth is provided, including the step of treating the strain or the culture filtrate of the strain to the cucumber plant, the area around the cucumber plant, or both.

According to the present invention, a composition for controlling cucumber fruit rot disease using the new Paenibacillus tianmuensis YM002 (Accession number: KACC 92433P), which can be prescribed at the pre-growth stage, and its culture filtrate can be provided.

According to another embodiment of the present invention, the stress resistance of plants is improved, thereby enabling sustainable agriculture, thereby eliminating or minimizing the use of chemical pesticides and fertilizers.

The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

Figure 1 is a diagram showing the activity of Paenibacillus tianmuensis YM002 strain to inhibit the growth of phytopathogenic fruit rot bacteria ( Acidovorax citrulli ) on a medium.
Figure 2 is a diagram showing that Paenibacillus tianmuensis YM002 belongs to Paenibacillus tianmuensis molecularly through 16s rRNA sequencing.
Figure 3 is a diagram showing the plant growth promotion-related activity of the Paenibacillus tianmuensis YM002 strain on a solid medium.
Figure 4 is a diagram showing the plant growth promotion-related activity of Paenibacillus tianmuensis YM002 strain on a liquid medium.
Figure 5 is a diagram showing the plant growth promotion-related activity of Paenibacillus tianmuensis YM002 strain in cucumber plants.
Figure 6 shows that Paenibacillus tianmuensis YM002 strain suppresses cucumber fruit rot caused by Acidovorax citrulli in cucumber plants.
Figure 7 is a diagram showing the induction of systemic acquired resistance by Paenibacillus tianmuensis YM002 strain in cucumber plants.
Figure 8 is a diagram showing the inhibition of biofilm formation of Acidovorax citrulli by Paenibacillus tianmuensis YM002 strain.
Figure 9 is a diagram showing the decrease in motility of Acidovorax citrulli by Paenibacillus tianmuensis YM002 strain.

The purpose and effects of the present invention, and technical configurations for achieving them, will become clear by referring to the embodiments described in detail below along with the accompanying drawings. In describing the present invention, if it is determined that a detailed description of a known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted. And the terms described below are terms defined in consideration of donation in the present invention, and may vary depending on the intention or custom of the user or operator.

However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms. These embodiments are merely provided to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the present invention of the scope of the invention, and that the present invention is defined by the scope of the claims. It just becomes. Therefore, the definition should be made based on the contents throughout this specification.

Hereinafter, embodiments of the present invention will be described in detail.

As used herein, “plant” refers to any living organism belonging to the plant kingdom (i.e., any genus/species within the plant kingdom), such as, but not limited to, trees, herbs, shrubs, grasses, vines, ferns, mosses, and green algae. does not Therefore, representative plants to which the composition can be applied include brassicas, bulb vegetables, grains, citrus, cotton, cucurbits, fruit vegetables, leafy vegetables, legumes, oil seed crops, peanuts, pomegranates, root vegetables, tuber vegetables, and corm vegetables. , stone fruits, tobacco, strawberries and other berries, and various ornamental plants. In this specification, the plant may be cucumber.

In this specification, “culture medium” or “culture filtrate” refers to the result obtained by inoculating a strain into a medium and culturing it for a certain period of time. The culture medium itself is obtained by culturing the strain according to the present invention in a suitable liquid medium, and the culture medium is filtered or centrifuged to isolate the strain. A filtrate (filtrate or centrifuged supernatant) from which Including but not limited to this. In addition, the probiotic or antibacterial composition may contain a culture such as a solid medium culture or a dried product or extract of the culture, but is not limited thereto, and may further include a suitable excipient or carrier.

The composition of the present invention can be prepared in the form of a liquid fertilizer, and can be used in the form of powder by adding an extender, or can be formulated and granulated. However, there is no particular limitation to the formulation.

The composition for inducing systemic induced resistance in plants of the present invention can increase plant resistance to one or more stresses selected from the group consisting of moisture, temperature, salt, pH, nutrients, and pathogens, inhibit disease development, and promote growth.

As used herein, “stress” may be biotic, abiotic, or biotic stress on plants, and specifically, abiotic stress may be dryness, high temperature, low temperature, high salinity, or nutrient deficiency. The term "abiotic stress" is used herein in its usual sense as the negative effect of abiotic factors on living organisms in a particular environment, and therefore in relation to plants, the negative effects of abiotic factors on plants in a particular environment. It means influence. Biotic stresses include living disturbances such as fungi or harmful insects, while abiotic stressors can be naturally occurring or man-made and include temperature, dry soil, osmotic stress, drought, salt or nutrient deficiencies. These include, all of which can cause harm to plants in the affected area. For example, salt stress may include increased salt concentrations or drought. Nutrient deficiency may be a lack of soil nutrients such as potassium, phosphate or iron in the soil area surrounding the plant. (Soil) Increased stress resistance to nutrient deficiencies, such as phosphate, can be provided by improved solubilization of nutrients lacking in the soil.

The term “resistance (or tolerance) to salt” is used herein in its ordinary sense, which refers to the resistance (tolerance) of plants to salt concentrations. Thus, “increasing the salt tolerance or tolerance of a plant” usually refers to the practice of increasing salt concentrations (in their environment, such as soil or water) when a plant is exposed to salt concentrations higher than what is physiologically acceptable for the plant. It means increasing or improving a plant's ability to endure or endure.

The term “moisture resistance (or tolerance)” or “desiccation resistance (or tolerance)” refers to the ability of a plant to avoid stress and its consequences by maintaining a desirable moisture balance and swelling even when exposed to normally dry conditions. It is also used herein in the meaning of. Therefore, “increasing the water (or desiccation) resistance” of a plant will maintain a more desirable water balance and expansion when exposed to drought conditions where the plant does not receive the required amount of water on a regular basis to maintain water balance and expansion. It means increasing or improving a plant's ability to

The term “plant pathogen” refers to pathogens that invade plants and cause loss or damage to the leaves, stems, roots or fruits of plants, such as fungi, bacteria, viruses and phytoplasma. ) includes.

The term "plant pathogen resistance" or "bacterial resistance" generally refers to the ability to enhance the growth of plants by avoiding infection and growth of pathogens, and enhancing resistance to such pathogens refers to the control, sterilization, and other methods used in the field. Includes the meaning of insect repellent.

The composition for inducing systemic resistance in plants according to the present invention is a plant's resistance to pathogen stress that causes one or more plant diseases selected from the group consisting of green blight fungus ( Ralstonia solanacearum ) and late blight fungus ( Phytophthora capsici ), and disease occurrence. It can inhibit and promote growth.

The composition for inducing systemic resistance in plants of the present invention is effective for inhibiting resistance, disease development, and It can promote growth.

The present invention promotes or promotes plant growth by inducing systemic resistance in plants. In the above, 'plant growth' refers to the germination of plants, differentiation of each organ (roots, stems, leaves, fruits, etc.), flowering or development, and increase in size and weight, and increase in yield.

The composition for inducing systemic induced resistance of plants of the present invention includes the strain of the present invention, the strain culture medium, or a mixture thereof, and any other ingredients used in the field to enhance systemic induced resistance or promote growth of plants. may additionally be included. For example, it may further include one or more selected from the group consisting of plant growth regulators, plant growth stimulators, and plant nutrients.

The plant growth regulator, plant growth stimulant or plant nutrient may be a chemical or another strain of bacteria, a herbicide, fungicide, pesticide, fertilizer or any combination thereof.

The composition of the present invention that promotes plant growth may include other beneficial microorganisms, microbial active substances, organic fertilizers, and chemical fertilizers. Other beneficial microorganisms include Bacillus spp., Azotobacter spp., Trichoderma spp., and Saccharomyces spp. This may be included.

The microbial active substances may include enzyme precursors, microbial metabolites, organic acids, carbohydrates, enzymes, and/or trace elements. For example, microbial active substances may include processed enzyme products such as yeast autolysates, humic materials, seaweed extracts, starch, amino acids, and trace elements such as zinc, iron, copper, manganese, bromine, and molybdenum. .

Other beneficial microorganisms, microbial active substances and organic fertilizers may be the same as described above. Chemical fertilizers can contain a variety of chemicals that can provide nitrogen, phosphorus and potassium nutrients to support plant growth. For example, chemical fertilizers may include urea, calcium phosphate, potassium phosphate, and mixed nitrogen-phosphate-potassium (N-P-K) fertilizers. Chemical fertilizers may also include other materials known in the art.

The present invention relates to one or more selected from the group consisting of Paenibacillus tianmuensis YM002 strain (Accession number: KACC 92433P), a culture medium of the strain, a composition containing the strain, and a composition containing the culture medium of the strain. It provides a method of inducing systemic resistance in a plant comprising treating the plant, the area surrounding the plant, or both.

The parts of the plant applied to the embodiments of the present invention are not limited. Accordingly, it includes both the whole plant as well as parts of the plant, such as seeds, shoots, stems, roots, leaves, and fruits, as well as the whole plant and parts thereof.

Examples of the present invention can be carried out by immersing or drenching, that is, spraying, the strain, the culture medium culturing the strain, and a composition using the strain into seeds or plants. In the case of the immersion method, the culture solution and preparation can be irrigated into the soil around the plant or seeds can be immersed in the culture solution and composition and shaken for inoculation.

The surrounding area applied by embodiments of the present invention refers to the range in which the composition of the present invention can exert its effect, and includes both above ground and underground areas. For example, it may be an area within about 2 m, within about 1 m, within about 70 cm, within about 50 cm, within about 25 cm, within about 10 cm, or within about 5 cm around the plant, plant part, or fruit.

The method according to an embodiment of the present invention may be performed by applying a composition comprising the Paenibacillus tianmuensis YM002 strain at any time during the life cycle of the plant, during one or more stages of the life cycle of the plant, or in the life cycle of the plant. It involves application at regular intervals or continuously throughout the life of the plant. Therefore, the composition can be applied as needed. The composition may, for example, be applied to the plant during growth, before and/or during flowering and/or before and/or during seed development. In one example, the composition may be applied before, during and/or immediately after plants are transplanted from one location to another location, such as from a greenhouse or hotbed to the field. In another example, each composition may be applied to the plant multiple times at desired intervals.

The method for inducing systemic resistance of the present invention can improve plant resistance, disease development, and growth against one or more stresses selected from the group consisting of moisture, temperature, salt, pH, nutrients, and pathogens.

The method for inducing systemic resistance in plants according to the present invention can suppress the development of cucumber fruit rot ( Acidovorax citrulli ). Specific details about pathogens are as described above.

The method for enhancing the stress resistance of plants of the present invention is to inhibit the resistance, disease development, or growth of any one or more plants selected from the group consisting of trees, herbs, shrubs, grasses, vines, ferns, moss, green algae, monocots, and dicots. can promote.

The present invention relates to one or more selected from the group consisting of Paenibacillus tianmuensis strain (Accession number: KACC 92433P), a culture medium of the strain, a composition containing the strain, and a composition containing the culture medium of the strain. A method for promoting plant growth is provided, comprising treating the plant, the area around the plant, or both.

The plant growth promotion method of the present invention can promote the growth of any one or more plants selected from the group consisting of trees, herbs, shrubs, grasses, vines, ferns, moss, green algae, monocots, and dicots.

For the present invention, soil located on the mountain behind Andong National University (Songcheon-dong, Andong-si, Gyeongsangbuk-do) was collected, various bacteria present in the soil were isolated, and among these, a study was conducted to discover strains showing antibacterial activity against plant pathogenic bacteria ( Acidovorax citrulli ). As a result , the Paenibacillus tianmuensis strain of the present invention was newly discovered.

The Paenibacillus tianmuensis strain of the present invention showed a direct antagonistic effect against three strains of plant pathogenic bacteria ( Acidovorax citrulli (cucumber fruit rot)) on the medium ( Fig. 1 ).

As shown in the results of the replacement culture (agar diffusion assay), the growth of plant pathogenic bacteria ( Acidovorax citrulli ) was suppressed when three strains of plant pathogenic bacteria (Acidovorax citrulli) were cultured simultaneously with the YM002 strain.

The Paenibacillus tianmuensis YM002 strain of the present invention was identified through 16S ribosomal RNA (16S rRNA) sequencing and phylogenetic analysis ( Fig. 2 ).

The Paenibacillus tianmuensis YM002 strain of the present invention exhibited PGP (zinc solubilization, siderophore, protease and amylase production, ACC deaminase) activity on solid media ( Fig. 3 ).

The Paenibacillus tianmuensis YM002 strain of the present invention exhibited PGP (IAA and ammonia production) activity in liquid medium ( Fig. 4 ).

When inoculated with a suspension (OD _600nm = 0.05 or 0005) of the Paenibacillus tianmuensis YM002 strain of the present invention, it was found to have a growth promoting effect on cucumber plants ( Fig. 5 ).

When cucumber plants were inoculated with Acidovorax citrulli strain KACC 17001, 17005, and 17909, water-soaked lesions occurred on cucumber plant leaves, but the suspension (1x10 ⁶ cfu/ml) of the Pennybacillus tianmuensis YM002 strain of the present invention and 10% of YM002 When simultaneously inoculated with the culture filtrate of the strain, cucumber plants showed an effect of reducing water damage spots caused by Acidovorax citrulli ( Fig. 6 ).

RT-PCR was performed to confirm the expression of genes related to induced systemic resistance of the Paenibacillus tianmuensis YM002 strain of the present invention. When cucumber plants were treated with a suspension of YM002 (10 ⁶ cfu/ml) and 10% culture filtrate, the expression levels of PAL1, PR1-1a, and CTR1 genes increased. This indicates that strain AY001 has the effect of increasing plant disease resistance in tomato plants ( Fig. 7 ).

In order to confirm the inhibition of biofilm formation of the cucumber fruit rot pathogen ( Acidovorax citrulli ) of the Paenibacillus tianmuensis YM002 strain of the present invention, cucumber fruit rot disease was cultured in a liquid medium diluted with the culture filtrate of the YM002 strain. When culturing Acidovorax citrulli, the formation of biofilm was suppressed.

In order to confirm the inhibition of motility of the cucumber fruit rot pathogen ( Acidovorax citrulli ) of the Paenibacillus tianmuensis YM002 strain of the present invention, the culture filtrate of the YM002 strain was diluted on a solid medium and the cucumber fruit rot pathogen ( When culturing Acidovorax citrulli, an effect of suppressing motility was observed.

Hereinafter, the present invention will be described in more detail through examples. However, these are only for explaining the present invention in more detail and the scope of the present invention is not limited thereto.

Example 1: Selection and identification of YM002 strain

To classify and identify the YM002 strain, total gDNA was isolated using a gDNA isolation kit. Afterwards, the 16S rRNA portion was amplified through polymerase chain reaction (PCR) using primers 27F (5'-AGTTTGATCCTGGCTCAG-3') and 1492R (5'-GTTACCTTGTTACGACTT-3').

PCR conditions were 95°C once for 2 minutes, repeated 31 times at 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, and then maintained at 72°C for 5 minutes. After analyzing the base sequence of the amplified PCR part, species with similar base sequences were identified using the BLAST network service at NCBI (National Center for Biotechnology Information).

As a result of phylogenetic analysis, strain YM002 was found to have 98.91% similarity to Pennybacillus tianmuensis B27 in the 16S rRNA base sequence (1091bp), confirming that it is a species of Pennybacillus tianmuensis ( Fig. 2 ).

Example 2 Evaluation of cucumber plant growth promotion effect of YM002 strain

The cucumber plant growth promotion effect of the YM002 strain was evaluated. To evaluate the effect of the YM002 strain on cucumber plant growth promotion, 2-week-old cucumber plants were treated with a suspension of the YM002 strain (OD _600nm = 0.05 or 0005) ( Fig. 5 ). In cucumber plants treated with a suspension of the YM002 strain (OD _600nm = 0.005), a growth promotion effect was observed in growth indicators except height and stem fresh weight when compared to the untreated group ( Figure 5 , Mock). In plants treated with a suspension of the YM002 strain (OD _600nm = 0.05), a growth promotion effect was observed in growth indicators except height when compared to the untreated group ( Figure 5 , Mock). Looking at the experimental results, there was a difference as shown in Figure 5 after 2 weeks of treatment with the suspension of the YM002 strain (OD _600nm = 0.05 or 0005), confirming that there was a growth promotion effect when the YM002 strain was treated on the plant.

Example 3 Evaluation of the effect of the YM002 strain on suppressing cucumber fruit rot disease

The inhibitory effect of the YM002 strain on cucumber fruit rot disease was evaluated. To evaluate the effectiveness of the YM002 strain in suppressing fruit rot, 2-week-old cucumber plants were treated with fruit rot bacterial suspension (1×10 ⁷ cfu/ml), YM002 suspension (1×10 ⁶ cfu/ml), and 10% culture filtrate. was processed ( Figure 6 ). In plants treated with cucumber fruit rot bacterial suspension (1×10 ⁷ cfu/ml), cucumber fruit rot occurred and the plants died ( Figure 6 ), but with cucumber fruit rot bacterial suspension (1×10 ⁷ cfu/ml), YM002 In plants treated simultaneously with the suspension (1×10 ⁶ cfu/ml) and 10% culture filtrate, water-soaked lesions were shown to be suppressed ( Fig. 6 ). Looking at the experimental results, in the case of cucumber fruit rot disease, there was a difference as shown in Figure 6 after 1 week of treatment, confirming that there was a disease suppressing effect when the YM002 strain was treated on the plant.

To confirm whether cucumber fruit rot disease was suppressed by induced systemic resistance (ISR) of the YM002 strain, the expression of the induced systemic resistance (ISR) marker gene was confirmed. Reverse transcription-polymerase chain reaction was performed to confirm the expression of PAL1 , PR1-1a , Apox1 , Lox1 , and CTR1 genes. It was confirmed that PR1-1a and CTR1 genes were expressed statistically significantly 12 hours after inoculation. It was confirmed that the PAL1 gene was expressed statistically significantly 24 hours after inoculation. Apox1 and Lox1 genes were not expressed ( Figure 7 ). Based on these results, it was confirmed that strain YM002 is effective in increasing disease resistance to fruit rot disease in cucumber plants.

In this specification and drawings, preferred embodiments of the present invention are disclosed, and although specific terms are used, these are merely used in a general sense to easily explain the technical content of the present invention and aid understanding of the present invention. There is no intention to limit the scope. It is obvious to those skilled in the art that in addition to the embodiments disclosed herein, other modifications based on the technical idea of the present invention can be implemented.

Rural Development Administration National Academy of Agricultural Sciences Microbial Bank (KACC) KACC92433P 20220803

Claims (9)

Paenibacillus tianmuensis YM002 strain (accession number: KACC 92433P).
A composition for promoting cucumber plant growth comprising the strain according to claim 1 or a culture filtrate of the strain.
A composition for inducing systemic resistance to cucumber plant pathogens, comprising the strain according to claim 1 or a culture filtrate of the strain.
According to paragraph 3,
A composition for inducing systemic resistance to cucumber plant pathogens, wherein the cucumber plant pathogen is a cucumber fruit rot pathogen.
A composition for controlling plant diseases for controlling cucumber fruit rot, comprising the strain according to claim 1 or a culture filtrate of the strain as an active ingredient.
According to paragraph 3,
A composition for controlling plant diseases, wherein the cucumber plant pathogen is caused by Acidovorax citrulli .
A method of inducing systemic resistance to a cucumber plant pathogen comprising treating a cucumber plant, an area around the cucumber plant, or both with the strain according to claim 1 or a culture filtrate of the strain.
In clause 7,
The resistance enhancement method is,
A method of inducing systemic resistance to cucumber plant pathogens, comprising treating at least one selected from the group consisting of seeds, roots, stems, leaves, and the entire plant of the cucumber plant.
A method for promoting growth of a cucumber plant comprising the step of treating the strain according to claim 1 or a culture filtrate of the strain on a cucumber plant, an area around the cucumber plant, or both.