KR20240150821A - Composition for controlling bacterial grain rot of rice plants using Cytobacillus firmus JBRS159 and silicon - Google Patents

Abstract

The present invention relates to a composition for controlling rice seed infectious diseases using the Cytobacillus firmus JBRS159 strain and silicon, and more specifically, to a composition that exhibits the effect of controlling bacterial rice grain wilt and leg disease and promoting plant growth.
A composition for controlling rice seed infectious diseases using the Cytobacillus firmus JBRS159 strain according to the present invention exhibits excellent controlling effects against bacterial grain wilt and leg blight, and can promote plant growth.

Description

{Composition for controlling bacterial grain rot of rice plants using Cytobacillus firmus JBRS159 strain and silicon}

The present invention relates to a composition for controlling rice seed infectious diseases using the Cytobacillus firmus JBRS159 strain and silicon, and more specifically, to a composition that exhibits the effect of controlling bacterial rice grain wilt and leg disease and promoting plant growth.

In rice farming, bacterial seed diseases transmitted through seeds include rice grain wilt, rice leg disease, bacterial streak disease, and rice white leaf blight. Since these rice seed diseases directly reduce crop yields and have a significant impact on quality, chemical pesticides, which are organic synthetic pesticides, have been developed to prevent these diseases. However, as problems such as toxicity to livestock, damage to natural enemies such as bees, and residues in agricultural products and the environment have arisen due to the use of chemical pesticides, research is being conducted on eco-friendly pesticides that can replace chemical pesticides, and in particular, the development of eco-friendly materials using agriculturally useful microorganisms is actively underway.

Siderophores are substances that specifically bind to iron ions, which are essential elements for plant growth, and directly help plant growth. In addition, siderophores can competitively inhibit the uptake of iron ions by plant pathogens, thereby inhibiting the growth of plant pathogens. Therefore, the Cytobacillus firmus JBRS159 strain can help plant growth by producing siderophores, and at the same time, it can also exhibit the effect of controlling rice seed disease.

Silicon was known to have no important function in plants, but some plant species have the characteristic of absorbing silicon from the soil in the form of Si(OH) ₄ and accumulating more than other major nutrients. In particular, the absorption rate of silicon in rice is known to be very high compared to nitrogen. The effects of silicon on plant growth, development, yield, and disease resistance have been observed in various plant species. In addition, it is known to provide the potential to withstand biotic and abiotic stresses and to enhance disease resistance from insect and other pest attacks.

Korean Patent Publication No. 10-2006-0114038 (2006.11.03)

The problem of the present invention is to provide Cytobacillus firmus JBRS159 strain (Accession Number: KACC 92494P) for controlling rice seed infectious diseases.

In addition, the problem to be solved by the present invention is to provide a composition for controlling rice seed infectious diseases, which comprises Cytobacillus firmus JBRS159 strain (Accession No.: KACC 92494P), a culture thereof, or a mixture thereof as an effective ingredient.

In addition, the problem solved by the present invention is to provide a method for controlling rice seed infectious diseases, which comprises a step of treating rice seeds or soil with the composition for controlling rice seed infectious diseases.

The problems to be solved by the present invention are not limited to the problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

In order to achieve the above technical task, according to one aspect of the present invention, Cytobacillus firmus JBRS159 strain (Accession Number: KACC 92494P) for controlling rice seed infectious diseases is provided.

In one embodiment, the rice seed infectious disease may be at least one of bacterial grain rot caused by the bacterial rice grain wilt fungus Burkholderia glumae and rice leg disease caused by the bacterial rice leg disease fungus Gibberella fujikuroi .

In one embodiment, the strain may comprise the base sequence of SEQ ID NO: 1.

According to another aspect of the present invention, a composition for controlling rice seed infectious diseases is provided, comprising Cytobacillus firmus strain JBRS159 (Accession No.: KACC 92494P), a culture thereof, or a mixture thereof as an effective ingredient.

In one embodiment, the composition may further comprise a silicon compound.

In one embodiment, the silicon compound can be potassium silicate (K ₂ SiO ₃ ) or silicon nanoparticles (SiO ₂ ).

According to another aspect of the present invention, a method for controlling rice seed disease is provided, comprising the step of treating rice seeds or soil with the composition for controlling rice seed disease.

The composition for controlling rice seed infectious diseases using the Cytobacillus firmus JBRS159 strain according to the present invention exhibits excellent controlling effects against bacterial grain wilt and leg blight, and has the effect of promoting plant growth.

The attached drawings are intended to explain the contents of the present invention in more detail to a person skilled in the art, but the technical idea of the present invention is not limited thereto.
Figure 1 is a graph showing the bacterial rice blight control effect of Cytobacillus firmus JBRS159 strain according to concentration.
Figure 2 is a photograph showing the effect of Cytobacillus firmus JBRS159 strain on bacterial rice blight.
Figure 3 is a graph showing the effect of silicon compounds on bacterial rice blight control at different concentrations.
Figure 4 is a photograph showing the effect of silicon compounds on controlling bacterial rice wilt.
Figure 5 is a graph comparing the bacterial rice grain wilt control effects when treated with Cytobacillus firmus JBRS159 strain alone, with silicon compound alone, and with simultaneous treatment with JBRS159 strain and silicon compound.
Figure 6 is a photograph showing the effect of controlling bacterial rice grain wilt when treated with Cytobacillus firmus JBRS159 strain alone, with a silicon compound alone, and with simultaneous treatment with JBRS159 strain and silicon compound.
Figure 7 is a graph comparing the control effects of Cytobacillus firmus JBRS159 strain alone, silicon compound alone, and simultaneous treatment of JBRS159 strain and silicon compound on leg disease.
Figure 8 is a photograph showing the effect of controlling leg disease when treated with Cytobacillus firmus JBRS159 strain alone, treated with a silicon compound alone, and treated simultaneously with JBRS159 strain and silicon compound.
Figure 9 is a graph comparing the changes in the fresh weight and number of lateral roots of Arabidopsis thaliana when treated with Cytobacillus firmus JBRS159 strain alone, with a silicon compound alone, and with simultaneous treatment with JBRS159 strain and silicon compounds.
Figure 10 is a graph comparing the fresh weight and dry weight of rice when treated with Cytobacillus firmus JBRS159 strain alone, silicon compound alone, and simultaneous treatment with JBRS159 strain and silicon compound.
Figure 11 is a photograph showing the growth status of rice when treated with Cytobacillus firmus JBRS159 strain alone, silicon compound alone, and simultaneous treatment with JBRS159 strain and silicon compound.
Figure 12 is a photograph showing the results of experiments on siderophore production ability (Figure 12A), phosphate solubilization ability (Figure 12B), silicon solubilization ability (Figure 12C), and protease production ability (Figure 12D) of Cytobacillus firmus JBRS159 strain.

Hereinafter, the present invention will be described in more detail. However, this is presented as an example, and the present invention is not limited thereby, and the present invention is defined only by the scope of the claims described below.

When rice is infected with the bacterial grain wilt fungus and suffers from bacterial rice grain wilt, rice seeds fail to germinate and show symptoms of rot, or leaves fail to unfold, leaf sheaths turn brown, growth is poor, and the plant dies. The above fungus overwinters in the seed and is transmitted to healthy seeds during sowing, and overwinters in rice straw, etc. and becomes a source of infection in the field the following year. In addition, when rice is infected with the leg-leg disease fungus and suffers from leg-leg disease, the leg-leg disease fungus easily multiplies by utilizing the nutrients secreted by rice seeds as they germinate and secretes gibberellin, causing the rice to grow 1.5 times taller than normal during the seedling period, and then shrivel and die within 1-2 weeks. Leg-leg disease is also a rice seed-borne infectious disease transmitted through seeds.

Chemical agents such as oxolinic acid or gasgamycin can be used to control bacterial rice wilt, and chemical agents such as prochloraz can be used to control leg disease. However, due to issues such as residues and toxicity, research on environmentally friendly pesticides that can replace these chemical pesticides is actively being conducted.

Accordingly, the present invention provides a Cytobacillus firmus JBRS159 strain (Accession Number: KACC 92494P) for controlling rice seed infectious diseases, which exhibits a control effect against rice seed infectious diseases such as bacterial grain wilt and leg disease.

The above JBRS 159 strain can be obtained by isolation and identification from rice grains.

In the present invention, the rice seed infectious disease may be at least one of bacterial grain rot caused by the bacterial rice grain wilt fungus ( Burkholderia glumae ) and rice leg disease caused by the rice leg pathogen ( Gibberella fujikuroi ).

In the present invention, the strain may include the base sequence of sequence number 1.

[Sequence number 1]

AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACGGATGGGAGCTTGCTCCCAGACCATCAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAATTCTTTCCCTCACATGAGGGAAAGCTGAAAGATGGCATCTCGCTATCACTTACAGATGGGCCCGCGGCGCATTAGCTAGTTGG TGAGGTAACGGCTCACCAAGGCCACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAG TCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTCAGGGAAGAACAAGTACCGGAGTAACTGCCGGTACCTTGACGGTACCTGACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTCCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATT GGAAACTGGGGAACTTGAGTGCAGAAGAGAAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTTTGGTCTGTAACTGACGCTGAGGCGCGAAAG CGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTCCTGACAACCCTAGAGATAGGGCGTTC CCCTTCGGGGGACAGGATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAA GGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTTAAGCGAATCCCATAAAACCATTCTCAGTTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCA CACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCT

The entire base sequence of the above strain can be confirmed in the gene locus tag No. PUS82_04120 of NCBI GenBank (Acc. No. JAQZDS000000000), and the base sequence of 16S rRNA is as sequence number 1.

The present invention also provides a composition for controlling rice seed infectious diseases, comprising Cytobacillus firmus JBRS159 strain (Accession No.: KACC 92494P), a culture thereof, or a mixture thereof as an effective ingredient.

In this specification, “culture” refers to the result obtained by inoculating bacteria into a medium and culturing it for a certain period of time, or the result obtained by filtering the same using a physical method.

In the present invention, the composition may additionally contain a silicon compound.

In the present invention, the silicon compound may be potassium silicate (K ₂ SiO ₃ ) or silicon nanoparticles (SiO ₂ ).

The present invention also provides a method for controlling rice seed disease, comprising the step of treating rice seeds or soil with the composition for controlling rice seed disease.

Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, the following examples and experimental examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

[Example 1] Production of diseased seeds inoculated with rice grain wilt fungus

To artificially create diseased seeds, the method of a previous literature (Fang, Y., Xu, LH, Tian, WX, Huai, Y., Yu, SH, Lou, MM, Xie, GL, 2009. Real-time Fluorescence PCR Method for Detection of Burkholderia glumae from Rice. Rice Sci. 16, 157-160.) was slightly modified. First, only healthy rice seeds were selected, surface-sterilized with 2% sodium hypochlorite for 2 minutes, and then washed three times with distilled water. Meanwhile, the bacterial rice grain wilt fungus (B. glumae ) was cultured in LB broth at 28℃ for 24 h with shaking at 180 rpm, and then diluted to a concentration of 1× ¹⁰⁸ CFU/mL in sterilized distilled water containing 0.2% carboxymethyl cellulose (CMC). Surface-sterilized seeds were placed in the bacterial rice grain wilt suspension (10 g/100 mL) and cultured at 25℃ and 150 rpm for 12 h to allow bacterial cells to attach to the seeds. The seeds were then dried at room temperature for 12 h and used to investigate the control effect.

[Example 2] Cytobacillus firmus ( Cytobacillus firmus ) Isolation and identification of JBRS159 strain

Rhizosphere soil collected from rice fields was placed in 10-fold sterilized distilled water, shaken at 180 rpm at 28℃ for 10 minutes, and the suspension was diluted 10-fold, spread on LB, and cultured at 28℃ for 24 h. The colonies grown here were subcultured on fresh LB medium, then placed on LB medium containing 20% glycerol, stored at -70℃, and used in experiments as needed. It was identified as Cytobacillus firmus CfJBRS159 after whole genome sequencing. The virus sequence was registered in NCBI (Acc. No. JAQZDS000000000), and the strain was deposited in the Korean Agricultural Culture Collection (KACC), National Institute of Agricultural Sciences, Rural Development Administration (strain number KACC 92494P).

Experimental example 1. Cytobacillus firmus ( Cytobacillus firmus ) Test to confirm the effect of JBRS159 strain on rice grain blight control

To investigate the control effect of the CfJBRS159 strain on bacterial rice grain wilt, the CfJBRS159 strain was cultured in LB broth at 28℃ for 24 hours, and the obtained cells were used for the control effect assay. Infected seeds prepared by inoculating the bacterial rice grain wilt fungus in Example 1 were added to CfJBRS159 suspension (1× ¹⁰⁶ , ¹⁰⁷ , and ¹⁰⁸ CFU/ml) (10 g/100 ml) and cultured at 28℃ and 100 rpm for 1 hour to allow good attachment of the bacteria to the surface of the infected seeds, and then sown in pots containing rice seedling medium (Pungnong, Korea). Each pot was grown in a plant growth room (light/dark, 16/8 hr, 28℃) and supplied with the same amount of water every day. Seeds treated with distilled water containing only CMC without fungus treatment were used as a control group, and procloraz-copper chloride-tebuconazole suspension, which is used for controlling bacterial grain blight of rice, was used as a chemical control group for comparison. The disease incidence 21 days after sowing was investigated as 0 = no disease, 1 = pale yellow leaves, 2 = severe chlorosis and stunting, 3 = seedlings that died after germination, and 4 = seeds that did not germinate, and the following equation was used to calculate it; Disease index = (0n ₀ + 1n ₁ + 3n ₂ + 5n ₃ + 7n ₄ )/7N × 100. In this equation, n _0-4 represents the number of leaves showing each disease severity (0 to 4), and N represents the total number of seedlings investigated.

In order to find the optimal treatment concentration of the strain, CfJBRS159 was treated at concentrations of 1×10 ⁶ , 10 ⁷ , and 10 ⁸ CFU/ml to seeds artificially inoculated with the pathogen, and seeds treated with distilled water only and seeds treated with prochloraz-copper chloride-tebuconazole liquid wettable agent were used as untreated and chemically treated controls, respectively. As a result, the control effect generally tended to increase as the treatment concentration increased to 1×10 ⁸ CFU/ml. When CfJBRS159 was treated at concentrations of 1×10 ⁶ , 10 ⁷ , and 10 ⁸ CFU/ml, the disease indices were 46.7%, 43.3%, and 26.7%, respectively, which were lower than those treated with only the pathogen (58.0%) (Fig. 1). Meanwhile, when prochloraz-copper chloride-tebuconazole liquid solution was treated, the disease index was 8.0%. According to the above experimental results, the bacterial grain blight control effect of CfJBRS159 strain was relatively excellent, and the greatest control effect was shown when 1× ¹⁰⁸ CFU/ml concentration was treated.

Experimental Example 2. Test to confirm the effect of silicon compounds on bacterial rice wilt

The effect of silicon on bacterial grain blight of rice was investigated using water-soluble potassium silicate (K ₂ SiO ₃ ; Samchun chemicals, Seoul, South Korea) solutions and silicon dioxide nanoparticles (SiO ₂ nanoparticles, <50 nm; Sigma-Aldrich, USA). First, to confirm the appropriate concentration for control, the diseased seeds of Example 1 were treated (5 g/20 ml) with each silicon suspension (50 mg/L, 100 mg/L, 200 mg/L, and 400 mg/L) for 4 h. The treated seeds were placed on sterilized filter paper and dried for about 15-20 min. The seeds were placed in pots containing seedling medium (Pungnong Co., Korea) and grown in a plant growth room (light/dark, 16/8 hr, 27°C), supplying the same amount of water every day. Seeds treated only with distilled water without fungus treatment were used as a control group, and those treated with prochloraz-copper chloride-tricyclazol were used as a chemical treatment group for comparison. The disease incidence 21 days after sowing was investigated in the same manner as in Experimental Example 1, and then the control effect was investigated. The experiment was performed twice with 3 replicates using 100 seeds per treatment (100 seeds/pot).

As a result of investigating the control effect against bacterial rice grain blight using suspensions of potassium silicate and silicon dioxide nanoparticles, when neither potassium silicate nor silicon dioxide nanoparticles were treated at a concentration of 50 mg/L, there was no control effect, but when 100 mg/L of potassium silicate or silicon dioxide nanoparticles were treated, it was confirmed that the incidence of bacterial rice grain blight was significantly reduced compared to the untreated case. In addition, when silicon dioxide nanoparticles were treated, it was observed that not only the control effect but also the growth increased (Fig. 2 and Fig. 3). Meanwhile, the control effect of potassium silicate or silicon dioxide nanoparticles against bacterial rice grain blight was expressed to a similar degree as the control effect of 100 mg/L up to a concentration of 400 mg/L, and it was confirmed that the growth of rice was slightly delayed when each compound was treated at a concentration of 200 mg/L or higher.

Experimental example 3. Cytobacillus firmus ( Cytobacillus firmus ) Test to confirm the effect of simultaneous treatment of JBRS159 strain and silicon compound on bacterial rice grain blight disease

To confirm the effect of simultaneous treatment of strains and silicon compounds, potassium silicate or silicon dioxide nanoparticles were added to each bacterial suspension to a final concentration of 100 mg/L and treated in the same manner as in Experimental Example 1. After 21 days of cultivation, the degree of disease development was compared to that of bacteria, potassium silicate, or silicon dioxide nanoparticles treated alone.

In order to confirm the control effect of bacterial rice grain wilt when treating with bacteria and silicon compounds simultaneously, potassium silicate or silicon dioxide nanoparticles were added to each bacterial suspension and treated with the diseased seeds. The disease severity after 21 days of cultivation was compared with that when bacteria, potassium silicate, or silicon dioxide nanoparticles were treated alone. The disease severity was 65.3% in the untreated case where only the pathogen was treated, 11.3% when prochloraz copper chloride-tebuconazole liquid suspension was treated, and 23.5%, 32.0%, and 32.8% were treated alone when bacteria, potassium silicate, or silicon dioxide nanoparticles were treated, and 21.0% and 22.5% were found when potassium silicate or silicon dioxide nanoparticles were mixed with the CfJBRS159 suspension (Figs. 5 and 6). As a result, when the CfJBRS159 strain was treated with a mixture of silicon compounds, the effect of controlling bacterial rice grain wilt increased.

Experimental example 4. Cytobacillus firmus ( Cytobacillus firmus ) Test to confirm the effect of JBRS159 strain and silicon compound on the control of leg disease

To create seeds artificially inoculated with the pathogen, Gibberella fujikuroi was inoculated into an Erlenmeyer flask containing potato sucrose broth and cultured at 25°C and 120 rpm for 4 to 5 days. The culture solution was filtered through two layers of gauze to collect the Gibberella fujikuroi spores, centrifuged at 1,600 g for 10 minutes, and the spore concentration was adjusted to 10 ⁵ spores/ml. 5 g of rice grains (approximately 120) were soaked in 10 ml of the spore suspension and treated under vacuum for 1 hour. These seeds artificially inoculated with the pathogen were used in the experiment, and those treated only with distilled water were used as an untreated control for comparison. After artificially infected seeds were cultured as described above, potassium silicate or silicon dioxide nanoparticles were added to the CfJBRS159 suspension (10 ml) to a final concentration of 100 mg/L or a suspension prepared solely of the fungus, potassium silicate, or silicon dioxide nanoparticles, and the diseased seeds were immersed in the suspension and used for the control effect test in the same manner as in Experimental Example 1. The seeds treated with sterile distilled water or fludioxonil dispersant were used as an untreated control group or a chemical pesticide-treated group for comparison of the control effects. Each treated seed was cultured at 15°C for 24 hours and then at 30°C for 24 hours to promote germination. These seeds were sown in pots containing seedling soil, cultured at 30°C and 100% relative humidity for 3 days, and then re-cultured in a greenhouse to investigate the incidence of leg disease.

The possibility of controlling rice leguminous wilt, a fungal disease that causes serious damage due to seed-borne wilt, was investigated using the CfJBRS15 fungus and silicon compounds. When CfJBRS15 or potassium silicate was treated, the incidence of leguminous wilt was 35.6% and 38.8%, respectively, which showed a significant reduction compared to the untreated group (53.2%) (Figs. 7 and 8). On the other hand, when potassium silicate was mixed with CfJBRS15, the incidence was reduced to 32.2%, showing a greater control effect than when treated alone.

Test Example 5. Cytobacillus firmus ( Cytobacillus firmus ) Test to confirm the growth promotion effect of JBRS159 strain and silicon compounds on Arabidopsis thaliana

To investigate the effects of CfJBRS159 strain and potassium silicate or silicon dioxide nanoparticles on the growth of Arabidopsis thaliana, surface-sterilized A. thaliana Col-0 seeds were treated with a mixture of each silicon solution (100 mg/L) and a bacterial suspension (1 × 10 ⁸ CFU/ml) containing 0.2% sterilized CMC, and shaken at 150 rpm for 30 min. The seeds were placed on a sterilizing filter, concentrated, and allowed to dry on the surface for 30 min at room temperature (25°C). The treated seeds were then sown on the surface of a medium containing 50% Murashige and Skoog (1/2MS) solution containing 1.5% sucrose and 0.8% (w/v) agar in a Petri dish. The seed-treated plates were sealed with parafilm and placed in a plant growth chamber at an angle of approximately 70º, and cultured under conditions of 23 ± 1°C and light (16-h light/8-h dark). The lengths of the roots and stems were measured after 10 days of culture. Meanwhile, seeds treated with sterilized distilled water (0.2% CMC) were used as a control. The experiment was conducted in triplicate with three plates containing five seeds each. The vigor index was calculated by the following formula, (average root length + average stem length) × germination rate (%). Meanwhile, surface-sterilized rice seeds were placed in CfJBRS159 and silicon compound suspensions containing 0.2% sterilized CMC, treated as described above, and grown in a plant growth room. Seeds treated in distilled water containing only CMC without fungus treatment were used as a control group, and the growth status (fresh weight, dry weight) of rice seedlings was investigated 21 days after plant culture.

In order to investigate the effects on plant growth, Arabidopsis was treated with each CfJBRS159 strain, potassium silicate and silicon dioxide nanoparticles, or a mixture of the strain and each silicon compound, and the growth degree was investigated. When treated with the strain, potassium silicate, or silicon dioxide nanoparticles (100 mg/L), the fresh weight of Arabidopsis thaliana significantly increased compared to the untreated control. When treated with a mixture of the strain and each silicon compound, the fresh weight of Arabidopsis thaliana increased compared to the untreated control, but did not increase significantly compared to when treated individually (Figs. 9 and 10). It was confirmed that the number of lateral roots also increased significantly when treated with the strain and silicon compounds. When the CfJBRS159 strain and potassium silicate were treated simultaneously, the number of lateral roots increased by more than twice, and it was confirmed that the roots were much more numerous and grew healthily (Fig. 11).

It was confirmed that when the CfJBRS159 strain was treated with potassium silicate or silicon dioxide nanoparticles, the fresh weight and dry weight of rice significantly increased compared to the untreated group. On the other hand, when the CfJBRS159 strain was mixed with potassium silicate or silicon dioxide nanoparticles and treated, the fresh weight increased compared to when treated alone, but there was no significant difference in the dry weight between the mixed treatment and the strain alone treatment. It was confirmed that when the strain and potassium silicate were mixed and treated, the growth of rice increased significantly.

Test Example 6. Test to confirm the siderophore production, phosphate solubilization, silicon solubilization, and protease production of Cytobacillus firmus JBRS159 strain

Siderophore production investigation: The siderophore production ability of strain CfJBRS159 was investigated using a modified chrome azurol S (CAS) agar method (Schwyn, B., Neilands, J.B., 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160, 47-56.). Briefly, the bacterial culture (20 μl) grown overnight in LB liquid medium was inoculated onto a paper disk placed on a CAS plate. After culturing at 30°C for 4 days, the strain showing an orange color was regarded as a siderophore-producing strain.

Phosphate solubilization assay: The phosphate solubilization ability of microorganisms was measured by inoculating them onto Pikovskaya's agar medium (Pikovskaya, R.I., 1948. Mobilization of phosphorus in soil in connection with vital activities by some microbial species. Microbiologia 17, 362-370.). After inoculating the microorganisms, the colonies were cultured at 28°C for 3 days. The formation of a clear zone around the colonies was evaluated as having the phosphate solubilization ability.

Silicon solubilization assay: The silicon utilization ability of microorganisms was investigated using glucose agar medium (Lee, K.-E., Adhikari, A., Kang, S.-M., You, Y.-H., Joo, G.-J., Kim, J.-H., Kim, S.-J., Lee, I.- J., 2019. Isolation and characterization of the high silicate and phosphate solubilizing novel strain Enterobacter ludwigii GAK2 that promotes growth in rice plants. Agronomy 9, 144.). First, microorganisms were inoculated into glucose agar medium and cultured at 28°C for 3 days. If a clear zone was formed around the colony, it was judged to have silicon utilization ability.

Protease productivity assay: Protease production was examined using skim milk agar (Smibert, R.M., Krieg, N.R., 1994. Phenotypic characterization, in: Gerhardt, P., Murray, R.G.E., Wood, W.A., Krieg, N.R. (Eds), Methods for general and molecular bacteriology. American Society of Microbiology, Washington, DC, pp. 607-654.). Bacterial cells cultured overnight were spotted onto a paper disk spread over the medium, and the formation of a clear zone around the colonies was confirmed.

To understand the mechanism of plant growth promotion and disease control of the strain in more detail, various characteristics related to the plant growth promotion and disease control effects of CfJBRS159 were investigated. As a result, it was confirmed that CfJBRS159 produced siderophores and protease through growth in CAS medium and skim milk agar medium, respectively, but did not produce hydrogen cyanide (HCN). Meanwhile, it was confirmed that it had the ability to solubilize phosphate but could not solubilize silicon, and it was investigated to produce indole-3-acetic acid (IAA), a plant hormone (Fig. 12).

The above description of the present invention is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single component may be implemented in a distributed manner, and likewise, components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

National Institute of Agricultural Sciences, Rural Development Administration, Microbial Bank (KACC) KACC92494P 20230222

Attach electronic file of sequence list

Claims (7)

Cytobacillus firmus strain JBRS159 for controlling rice seed rot disease (Accession number: KACC 92494P).
In paragraph 1,
Cytobacillus firmus strain JBRS159 (Accession Number: KACC 92494P), characterized in that the above rice seed infectious disease is at least one of rice grain wilt caused by the bacterium Burkholderia glumae and rice leg disease caused by the bacterium Gibberella fujikuroi .
In paragraph 1,
The above strain is Cytobacillus firmus JBRS159 strain (Accession Number: KACC 92494P) containing the base sequence of sequence number 1.
A composition for controlling rice seed infectious diseases, comprising Cytobacillus firmus JBRS159 strain (Accession No.: KACC 92494P) of Article 1, a culture thereof, or a mixture thereof as an effective ingredient.
In paragraph 4,
A composition for controlling rice seed disease, characterized in that the composition additionally contains a silicon compound.
In paragraph 4,
A composition for controlling rice seed disease, characterized in that the silicon compound is potassium silicate (K ₂ SiO ₃ ) or silicon nanoparticles (SiO ₂ ).
A method for controlling rice seed infectious diseases, comprising the step of treating rice seeds or soil with the composition of claim 4.