KR20150001241A - Novel Pseudomonas sp. JBCS1880, and Biological Control of Bacterial Grain Rot and Growth Promotion of Rice Plants Using the Same - Google Patents

Abstract

The present invention relates to novel Pseudomonas sp. JBCS1880 and a composition for controlling bacterial grain rot and growth promotion of rice plants using the same. Provided in the present invention is Pseudomonas sp. JBCS1880 (Accession No. KACC 91827P). Moreover, the strain is characterized by having an antibacterial activity for Burkholderia glumae and Xanthomonas oryzae.

Description

A novel Pseudomonas sp. JBCS1880 strain, a rice bacterium used for controlling the blight of rice bran, and a plant for promoting growth {Novel Pseudomonas sp. JBCS1880, and Biological Control of Bacterial Grain Rot and Growth Promotion of Rice Plants Using the Same}

The present invention relates to a novel Pseudomonas sp. JBC1880 strain, a rice bacterium and a composition for promoting growth of rice bran blight using the same.

Recently, the demand for environmentally friendly control is increasing due to the emergence of resistant bacteria to chemical pesticides, the destruction of ecosystem by chemical pesticides, and the risk of residual toxicity. Since bio-based crop protection is considered as an alternative to solve the problems of crop protection based on chemical synthesis, many biological pesticides using Bacillus and Pseudomonas spp. Are used to control pathogens of crops using microorganisms or natural substances worldwide Research and development, some of them are on the market, and the market of related microbial pesticides is gradually expanding. However, most of the microbial preparations used for biological control are concerned with the control of diseases caused by fungal pathogens. Therefore, researches on the development of agents for controlling diseases caused by bacterial pathogens and related products are limited.

Bacterial rice blight disease is caused by seed infectious disease, and the onset of the disease is promoted during the seedling process of rice, and the damage of rice seed weight and germination rate is reduced. In order to control rice blight disease, which is increasing in recent years, it is sterilized by using oxolinic acid. However, the development of alternative control methods is urgent due to the emergence of resistant bacteria and damages to non - target microorganisms, but there is no study on bacterial rice blight disease.

Disclosure of Invention Technical Problem [17] The present invention provides a novel pseudomonas JBCS1880 strain isolated from rice paddy, demonstrating the control effect against bacterial rice blight and promoting growth of rice, and developing a suitable treatment method thereof. Therefore, the present invention can be utilized not only as a seedling of healthy rice in a nursery, but also as an alternative means for reducing the use of chemical pesticides.

The present invention has developed a microbial agent for promoting the growth of rice in a rice seedling growing plant which is suffering from a great damage to rice in the domestic and foreign countries and which is expected to cause further damage due to the warming due to climate change in the future, To be used in related markets.

In order to achieve the above object, the present invention provides a novel microorganism, Pseudomonas sp. JBCS1880 (Accession No. KACC 91827P).

In one embodiment of the present invention, the strain is characterized by having a bacterial rice blight blight control effect. Further, the strain is characterized in that it has a growth promoting effect. Moreover, the strain is characterized by having antimicrobial activity against bacteria byeoal blight fungus (Burkholderia glumae) and fungal huinip blight (Xanthomonas oryzae pv. Oryzae).

In order to achieve the above object, the present invention provides a culture medium of Pseudomonas sp. JBCS1880. In addition, the present invention provides a composition for controlling bacterial rice blight disease and growth promotion comprising the above-mentioned culture liquid as an effective ingredient.

In one embodiment of the present invention, the JBCS1880 strain is characterized by a concentration of 10 ⁷ to 10 ⁸ CFU / ml.

The novel Pseudomonas sp. JBCS1880 strain inhibits the decay of rice seedlings caused by bacterial rice blast fungus which causes serious damage to rice. It also promotes the growth of root and stem of seedlings and enables healthy seedlings. This will not only help to nurture healthy seedlings in mass nursery, but also reduce the damage caused by bacterial rice blight which has low control effect by medicines.

1 shows a phylogenetic analysis result using 16S rDNA of Pseudomonas JBCS1880 according to an embodiment of the present invention.
Figure 2 shows the results of treatment of bacterial rice grain blight at different concentrations of Pseudomonas JBCS1880 according to one embodiment of the present invention.
Figures 3 and 4 illustrate the effect of Pseudomonas JBCS1880 on growth of rice in accordance with one embodiment of the present invention.
Figures 5 and 6 show the control effect of bacterial rice grain blight with Pseudomonas JBCS1880 according to one embodiment of the present invention.
Figure 7 shows the effect of Pseudomonas JBCS1880 on growth of rice according to one embodiment of the present invention.
Figure 8 shows the effect of JBCS1880 on the growth of plant pathogens according to one embodiment of the present invention. (A) B. glumae, (B) X. a . pv. glycines, (C) B. cinerea, (D) B. oryzae, (E) C. acutatum, (F) F. oxysporum , (G) F. graminearum, (H) F. moniliforme.
FIG. 9 shows (A) Siderophore formation, (B) solubilization ability of phosphoric acid, and (C) protease activity according to an embodiment of the present invention.
Figure 10 shows the production of salicylic acid by JBCS1880 according to one embodiment of the present invention.
Figure 11 shows the drag force of the crude extract obtained from JBCS1880 according to an embodiment of the present invention.
Figure 12 shows the yield strength of the ethyl acetate layer from the supernatant of JBCS1880 according to one embodiment of the present invention.
Figure 13 shows RP-HPLC chromatograms of antibiotic compounds produced by JBCS1880 according to one embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Example 1: Pseudomonas sp. Selection and identification of JBCS1880 strain

(1) Isolation and identification of rhizosphere microorganisms

A soil sample (5 g) or rice paddy (1 g) was taken in sterilized distilled water of 10 times the volume and cultured at 28 ° C for 10 minutes with shaking at 180 rpm. Then, the suspension was decanted by 0.1 mL trypic soy agar (TSA) and cultured at 28 ° C for 24 hours. The grown colonies were cultured in fresh TSA medium and stored at -70 ° C in LB medium containing 20% glycerol.

The separated microorganisms were cultured in Luria-Bertani (LB) medium at 30 ° C for 24 hours. Then, gDNA was extracted using Genomic DNA Prep Kit (Solgent Co. Ltd, Korea) and 27F (5'-AGA GTT TGA TCC TGG CTC AG-3 ') and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3') primers were used to analyze the 16S rDNA sequence of the selected microorganism. Then, BLAST program (http://www.ncbi.nlm microbes were identified using .nih.gov/BLAST/). After confirming the Gram reaction using 3% KOH, the nutrient availability was examined using Easy 24E kit (Hanil Komed Co. Ltd, Korea) and identified using the EASY View (Hanil Komed Co. Ltd, Korea) program .

As a result, the JBCS1880 isolate was selected as the final isolate from gram-negative bacteria (Table 1) and was identified as Pseudomonas spp. By analysis of 16S rDNA sequence. On the other hand, in order to investigate the flexible relationship with the 16S rDNA sequence, the neighboring joining method using the MEGA5 program showed that Pseudomonas sp. It has been confirmed that there is a high degree of flexibility with ICMP17674 (NCBI accession number FJ643465.1) (Fig. 1).

Isolate name Region Origin Gram reaction JBCS1880 Buan, Jeonbuk Rice, seeds -

Analysis of nutrient availability using Easy 24E kit (Hanil Komed, Korea) showed similar pattern to that of Pseudomonas spp. Using lactose, rhamnose, lysine, arginine, ornithine. (Table 2).

Nutritional Sources Results Nutritional Sources Results Arabitol - Glucopyranoside - Lactose + Esculin - Sucrose - ONPG (beta-galactosidase) - Mannitol - Tryptophan - Dulcitol - Phenylalanine - Adonitol - Potassiumnitrate - Sorbitol - Urea + Cellobiose - Sodium thiosulfate - Raffinose - Lysine + Rhamnose + Arginine + Inositol - Ornithine + Maltose - Glucose -

Experimental Example: Pseudomonas sp. Effect of JBCS1880 concentration on control of bacterial rice blight

(1) Preparation of the seedling seedlings by inoculation with bacterial rice blast fungus

Rice seeds (cv. Shannuri) were purchased from the rice paddy fields of the National Institute of Food Science and Technology and used in this experiment. First, we used a slightly modified method of Fang et al. (2009) to make artificially seeded seeds. Only healthy seeds were selected and surface sterilized with 2% sodium hypochlorite for 2 minutes and washed with distilled water. B. glumae was cultured on TSB medium at 180 rpm for 24 hours at 28 ° C and then adjusted to a concentration of 1 × 10 ⁸ CFU / mL. Then, carboxymethyl cellulose (CMC) was added at a concentration of 0.2% . The surface-sterilized seeds (10 g / 100 ml) were inoculated with the bacterium, and then dried at room temperature (12 hr) at 25 ° C and 100 rpm for 12 hours.

(2) Pseudomonas sp. Cultivation and seed treatment of JBCS1880 strain

Pseudomonas sp. The strain JBCS1880 was inoculated into TSA and cultured at 28 ° C for 24 hours. (10 g / 100 ml) was incubated at 25 ° C and 100 rpm for 1 hour in a suspension of JBCS1880 (0.2% CMC added) to obtain JBCS1880 And then dried for 1 hour at room temperature.

 (3) Effect of treatment concentration on control of bacterial rice blight and growth promotion (test using roll towel)

To investigate the effect of the JBCS1880 strain on the control of bacterial rice blight disease, the strain JBCS1880 was treated with bacterial rice blight germs at a concentration of 1 × 10 ⁶ , 10 ⁷ , 10 ⁸ CFU / ml, . ≪ / RTI > (ISTA, 2003), and then 30 ml of distilled water was added to maintain a sufficient amount of water. Then, the seeds were treated at 30 ° C (light / dark, 16 / 8 hr). After 14 days of sowing, the incidence rate was calculated as 0 = no disease, 1 = light yellow leaf, 2 = severe yellowing and atrophy, 3 = seed after seed germination and 4 = non germinated seed. ; Onset Index _{= (0n 0 + 1n 1 +} 3n 2 + 5n 3 + 7n 4) / 7N × 100. In this equation, n _{0 ?? 4} indicates the number of leaves showing the onset of each degree (0 ~ 4), N Represents the total number of seedlings surveyed. On the other hand, seeds treated with distilled water containing only CMC without adding bacteria were used as a control.

When JBCS1880 was treated at the concentrations of 10 ⁷ and 10 ⁸ CFU / ml, germination rates were increased by 11.1% and 25%, respectively, and the onset index was decreased by 24.9% and 54.6%, respectively (Fig. 2). On the other hand, germination rate tended to be slightly inhibited at high concentrations.

In order to investigate the effect of rice seedlings on the growth of rice, germination rate was investigated 3 days after cultivation by inoculating different kinds of JBCS1880 strains to the disinfected healthy seeds in the same manner as above, And root length were measured. The plant growth promoting degree was calculated by using a vigor index, a calculation formula, a vitality index [(average length of stem + average length of root) × germination rate (%)] (Abdul-Baki and Anderson, 1973). Each experiment was treated with 3 replicates using 10 seeds, and each experiment was repeated 3 times.

After a review of concentrations the growth promoting effect of JBCS1880 strain compared to non-treated length of the roots and enjoy at 10 ⁸ CFU / ml it was increased by respectively 21.2% as compared to non-treatment, 44.5%, 10 ⁷ and 10 ⁸ CFU / ml concentration of Live weight was increased by 12.5% and 48.0%, respectively. The viability index, which reflects the germination, root and stem elongation, increased by 6.8% and 95.1% at concentrations of 10 ⁷ and 10 ⁸ CFU / ml, respectively (FIGS. 3 and 4).

As shown above, 10 ⁸ CFU / ml was judged to be the optimal concentration in the control of bacterial rice blight disease prevention and growth promotion using a paper towel, and the greenhouse (pot) experiment was conducted based on this concentration.

Experimental Example: Pseudomonas sp. JBCS1880 strain Yonghoon Bacterium blight prevention and growth promotion effect

(1) Effect of bacterial rice blight prevention and growth promotion in a greenhouse (pot)

The coated seeds were seeded in a pot containing rice seedlings (Pungnong, Korea) at a concentration of JBCS1880 (1 × 10 ⁸ CFU / ml), which was excellent. Each pot was grown in a plant culture room (light / dark, 16/8 hr, 25 캜), and the same amount of water was supplied every day. Seed treated with distilled water containing only CMC without treating the bacterium was used as an untreated control and seed treated with benomyl was used as a chemical control. After 14 days of sowing, 0 = no disease, 1 = light yellow leaf, 2 = severe yellowing and atrophy, 3 = seed after seed germination, and 4 = non germinated seed. Two treatments were carried out using 100 seeds (50 seeds / pot) per treatment and three replicates were conducted. On the other hand, the growth promoting activity of JBLS1880 strain was examined for the root length and stem length, viability and viability index after 30 days of seeding.

As a result, the onset index was 45.2 and 30.4 in the untreated and benomyl treated groups, respectively, whereas the onset index of the JBCS1880 strain was 25.9, which was 42.7% and 14.8% (FIG. 5 and FIG. 6). As a result, the JBCS1880 strain was considered to be superior to the benomyltihal hydrating agent in controlling the bacterial blight of rice paddy, so that it could be substituted for the chemical pesticide do.

On the other hand, when JBCS1880 strain was treated at 1 × 10 ⁸ CFU / ml, the length of shoots increased by 17.4%, 10.0%, and the length of roots increased by 67.0% and 14.8%, respectively, , 6.9%, and the fresh weight was increased by 49.6% and 1.2%, respectively (FIG. 7). On the other hand, it seems to have a positive effect on the increase of the number of tillers. The above results suggest that the treatment with JBCS1880 strain can be expected to have a better controlling effect than the treatment with the chemical pesticide, benomyltylam, as well as the growth promotion effect.

Experimental Example: Pseudomonas sp. JBCS1880, a bactericidal rice blight prevention mechanism

(1) Antimicrobial activity test for major bacterial and fungal pathogens of rice

Bacterial blight bacterium byeoal of JBCS1880 strain (B. glumae) and the antimicrobial activity of such huinip blight fungi (X. o. Pv. Oryzae) were investigated by the inoculation method-layer (dual inoculation technique). The sterilized LB agar broth was cooled, and then bacterial cells such as bacterial rice blast fungus and blighted blight were mixed at a concentration of 1 × 10 ⁷ CFU / mL, and then dispensed onto the plate. The paper disk was placed on the plate, the JBCS1880 strain was inoculated thereon, and the size of the inhibition zone was measured 2 days after incubation at 28 ° C.

The resistance of JBCS1880 strain to fungal pathogens was determined by confluent culture. First, the fungal pathogens cultured on the PDA medium for 7-10 days were removed with a cork borer (8 mm) along with the medium. The hyphae were placed in the center of the PDA medium, and the paper disks (8 mm ) Was inoculated with 10 μl of JBCS1880 (1 × 10 ⁸ CFU / ml). After incubation at 25 ° C for 7-10 days, the following equation was calculated: RI = (D ₀ -D _a ) / D ₀ × 100 (RI; relative degree of mycelial growth inhibition, D ₀ ; Mycelial growth inhibition was measured by mycelial elongation on a medium inoculated with antagonist D _a . Each experiment was repeated three times.

As a result JBCS1880 strains showed antimicrobial activity against bacteria byeoal blight bacterium (B. glumae), huinip blight fungi (X. o. Pv. Oryzae) . This suggests that it is possible to control the bacterial pathogen, which is the most serious problem in rice, against bacterial rice blight and blight of blight. On the other hand, no pathogenic fungi were observed against fungal fungal pathogens such as Bipolaris oryzae , Fusarium moniliforme , Pyricularia grisea , Rhizoctonia solani (Table 3, Fig. 8). On the other hand, addition is determined that the antimicrobial activity even in the bean spot pathogen (Pseudomonas cichorii) and fire blight bacterium (Xanthomonas axnopodis pv. Glycines), was investigated as mainly with a high antifungal activity only on bacterial pathogens.

Plant pathogenic bacteria and fungi Inhibition of growth (mm) Burkholderia glumae (Bacterial grain rot) 22.0 Xanthomonas oryzae pv. oryzae (Bacterial leaf blight) 18.0 Pseudomonas cichorii (Soybean bacterial leaf spot) 15.0 Xanthomonas axnopodis pv. glycines (Soybean bacterial pustule) 20.0 Bipolaris oryzae (Brown spot) 0.0 Fusarium moniliforme (Bakanae disease) 0.0 Fusarium graminearum (Fusarium blight) 0.0 Pyricularia grisea (Blast) 0.0 Rhizoctonia solani (Sheath blight) 0.0

(2) Determination of phosphoric acid solubilization, siderophore and HCN production

After the culture of JBCS1880 was performed on LB agar medium at 30 ° C for 24 hours, the PVK agar medium [Glucose 1%, (NH ₄ ) ₂ SO ₄ 0.05%, NaCl 0.02%, KCl 0.02%, MgSO ₄ .7H ₂ 0.05% of MnSO ₄ .7H ₂ O, 0.05% of FeSO ₄ .7H ₂ O, 0.05% of yeast extract, 0.5% of Ca ₃ (PO ₄ ) ₂ , 15% of agar] and incubated at 30 ° C for 5 days After that, the presence of clear zone was confirmed and the solubilization of phosphoric acid was confirmed.

To determine whether the selected microorganism produces siderophore, dissolve CAS (chrome azurol S) medium [60.5 mg CAS in 50 ml of distilled water, add 10 ml of 1 mM FeCl ₃ · 6H ₂ O dissolved in 10 mM HCl solution, 72.0mg HDTMA 750ml of distilled water, 10 × MM9 salts (60g / L Na 2 HPO 4, 0.009g / L KH 2 PO 4, 5g / L NaCl) in a sterile 100ml then added with a solution obtained by dissolving (Hexadecyltrimethyl-ammoiumbromide) , 15 g agar, 30.24 g pipes, 12 ml of 50% NaOH, 30 ml of 10% casamino acids solution, 10 ml of 20% glucose, 1 ml of 0.2% thiamine.HCl and the like were mixed and cooled to 50 ° C, The solution was slowly mixed to prevent foaming. First, JBCS1880 strain was inoculated on CAS agar medium and cultured at 30 ° C for 3 days. The presence of sialophore was confirmed by the formation of an orange halo zone.

The production of HCN was carried out according to the method of Miller and Higgins (1970). The strain cultured for 48 hours was streaked on LB medium supplemented with glycine (4.4 g / L), and 0.5% (w / v) picric acid was added to filter paper and treated on the lid of the plate. Changes in color were observed after incubation at 28 ° C.

As a result, the JBCS1880 strain was found to form an orange halo zone in the CAS medium to produce siderophore, and the ability of solubilizing phosphoric acid was weak but not HCN (Table 4, FIG. 9). These results suggest that the supply of siderophore and the solubilization of phosphoric acid contributed to the growth of seedlings.

Mechanism Production  Siderophore production +  Phosphate solubilization (+)  HCN production -  IAA production -  Salicylic acid +  Protease production +

(3) Plant hormone IAA and salicylic acid production assay

The pre-cultured JBCS1880 was inoculated into LB liquid medium (10 mL) at a concentration of 2% (v / v) and cultured at 25 ° C and 180 rpm for 24 hours. The degree of growth was measured at OD600 and the degree of IAA production was measured at OD535nm. The degree of IAA production was determined by centrifuging the culture with 1 ml of each concentration at 14000 rpm for 10 minutes to recover the supernatant. The supernatant and Salkowski's reagent : 4, the reaction was carried out at room temperature for 20 minutes, and the absorbance was measured at 535 nm.

To investigate the production of salicylic acid, JBCS1880 strain was cultured in casamino acid liquid medium for 24 hours (200 rpm, 28 ℃, cancer). 100 μl of this culture was transferred to 25 ml of fresh casamino acid medium and incubated for 36 hours under the same conditions. After centrifugation at 4000 rpm for 15 minutes, the supernatant was extracted with ethyl acetate. After concentrating it in a vacuum state to 1/3, 5 μl of 2M FeCl ₃ and 3 ml of distilled water were added per ml, and the degree of change to a purple iron-SA complex was measured by absorbance at 527 nm. .

Indole acetic acid (IAA), a typical auxin associated with plant growth promotion, was not produced (data not shown), but salicylic acid, a hormone associated with resistance, was found to be formed 24 hours after incubation, (Table 4, Fig. 10). These results suggest that the resistance of the seedlings may be enhanced in addition to the direct sterilization effect of antimicrobial agents.

(5) Protease production assay

Protease production was determined by inoculating a bacterial suspension (10 μl) into a paper disk placed on a skim milk agar medium supplemented with 2% casein, incubating at 28 ° C for 72 hours, and then forming a clearing zone , Which was experimented with three iterations.

As a result, the JBCS1880 strain formed a zona pellucida in a medium supplemented with casein protease activity (Table 4, Fig. 9), which is thought to be related to the expression of the pathway resistance to bacterial pathogens.

(5) Antibacterial activity and protease activity of supernatant of JBCS1880

After culturing the JBCS1880 strain, only the supernatant was taken to determine the antimicrobial activity. First, the bacteria were inoculated on TSB medium, cultured at 28 ° C and 180 rpm for 48 hours, transferred to a 10-fold volume of fresh medium, and further cultured for 48 hours. The culture broth was centrifuged at 13,000 rpm for 30 minutes to remove cells, and then filtered through a 0.22 μm filter paper to obtain a cell free supernatant (CFS). This supernatant was added to the paper disk in the same manner as above, and 50 μl of the supernatant was added to the paper. Ammonium sulphate was added to the culture supernatant to a concentration of 60%, and the mixture was allowed to stand overnight at 4 ° C. The mixture was centrifuged at 6,000 g for 15 minutes, and 100 mM Tris-HCl buffer (pH 8.0) crude purified compound.

As a result, the supernatant obtained after the culture of JBCS1880 did not show any pathway resistance against the bacterium, but protease activity was also shown when using crude extract. This means that the JBCS1880 strain secretes the antimicrobial substance or protease externally, so that the antimicrobial activity of the antimicrobial substance or protease can be confirmed after the pure antagonist and the protease are separated from the supernatant. (FIG. 11).

(6) Separation and purification of antimicrobial substances

The suspension of JBCS1880 strain in LB medium overnight was inoculated into fresh LB medium at a concentration of 1/50 and cultured at 25 ° C and 200 rpm for 48 hours. It was centrifuged at 8,000 rpm for 30 minutes and then filtered with filter paper (Whatman No. 4). Antibacterial compounds were extracted from the supernatant (CFS) thus obtained twice with ethyl acetate (Yazgan et al. 2001). Ethyl acetate was completely evaporated and dissolved in methanol. Gel-filtration chromatography was performed at a flow rate of 1.0 ml / min using Sephadex LH-20 resin (Amersham Bioscience AB, SE-75184 Uppsala, Sweden). On the other hand, the fractions which were active through the paper disk assay were traced in the process of separation, and only the fractions with pathway were collected and analyzed by reverse-phase high performance liquid chromatography (RP-HPLC) on a Zorbax C18 column (4.6 × 150 mm (V / V) methanol / water (0.04% trifluoroacetic acid) at a flow rate of 1.0 ml / min. The elution pattern was fractionated while observing at 210 nm, and the activity was assayed.

As a result, two fractions with antimicrobial activity were obtained (FIG. 12 and FIG. 13), and the separation, purification and identification of the natural antimicrobial substance will be continuously carried out.

It is to be understood that the invention is not limited to the disclosed embodiment, but is capable of many modifications and variations within the scope of the appended claims. It is self-evident.

National Institute of Agricultural Science KACC91827P 20130621

Claims (7)

Pseudomonas sp (Pseudomonas sp.) JBCS1880 (Accession No. KACC 91827P). The Pseudomonas sp. JBCS1880 according to claim 1, which is characterized by having a bacterial rice blight blight control effect. The Pseudomonas sp. JBCS1880 according to claim 1, which has a growth promoting effect. The Pseudomonas sp. JBCS1880 according to claim 1, characterized by having antibacterial activity against B. glumae and X. o.v. oryzae. A culture medium for a strain according to any one of claims 1 to 4. A composition for controlling bacterial rice blight disease and growth promotion comprising the culture liquid according to claim 5 as an active ingredient. 7. The composition of claim 6, wherein the JBCS1880 strain has a concentration of 10 ⁷ to 10 ⁸ CFU / ml.

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