KR102456371B1 - Method of increasing activity of a plat growth-promoting bacteria using non-thermal plasma - Google Patents

Abstract

The present invention relates to a method for increasing the vitality and activity of plant growth promoting bacteria using non-thermal plasma.

Description

Method of increasing activity of a plat growth-promoting bacteria using non-thermal plasma

The present invention relates to a method for increasing the activity of plant growth promoting bacteria using non-thermal plasma.

Rapidly growing populations, global warming and environmental pollution pose new threats to modern agriculture, leading to global food shortages. The world needs to develop sustainable and environmentally friendly ways to improve agricultural productivity. The use of plant growth promoting bacteria (PGPB) as a biological fertilizer has been proposed as an alternative to conventional methods such as pesticides, herbicides and fungicides. PGPB is a bacterium that lives in the rhizome of a plant or lives inside a plant, and improves plant productivity through various plant growth promoting activities such as nitrogen fixation, efficient use of nutrients by plants, hormonal regulation, and growth inhibition of harmful microorganisms. Bacillus, Enterobacter and Corynebacterium are known as nitrogen-fixing PGPBs that symbiotically improve plant growth and health.

The practical use and commercialization of PGPB as a biofertilizer in agriculture began worldwide in the 1950s. The current biofertilizer startups account for about 5% of the total chemical fertilizer market, and nitrogen-fixing microorganisms are a major component of biofertilizers. Biological fertilizers using PGPB have demonstrated several advantages in agricultural practices such as environmental evolutionary nutrition, improvement of soil fertility, and regulation of biotic and abiotic stress. However, efficient use of PGPB sometimes suffers from agricultural applications due to the inconsistent efficiency and vigor of PGPB. Since PGPB is not broadly efficient for many plant species, field application may lead to inconsistent results. In particular, the surrounding environment and the existing microbiome can influence the activity of PGPB. Cell formation of PGPB is not always stable, because the changeable environment and plant species influence the success of PGPB settlement. Finally, poor growth in culture systems compared to their natural habitats can make mass production of many PGPB species difficult. Although genetic engineering tools are being applied to improve the limitations of PGPB utilization, genetically engineered strains can have undesirable effects on soil microbial communities, and some genes are still problematic for successful plant colonization. .

Atmospheric pressure non-thermal plasma has been proposed as a potential tool to enhance the activity of PGPB. Plasma is known as the fourth state of matter, or ionized gas. Plasma is one of the environmentally friendly and sustainable new technologies and has been applied to various agricultural applications in recent years. Studies have shown that plasma has a positive effect on seed germination, plant growth and plant resistance. For example, Korean Application No. 10-2016-0018678 discloses a method for sterilizing seeds and promoting germination using non-thermal plasma. In addition, non-thermal plasma-activated water (PAW) can be used as a fertilizer by inactivating various microorganisms. Plasma exhibited a wide range of effects (activation to inactivation) in biological target samples, depending on the dose and reactive species generated from the plasma. However, the potential of plasma in the activation of beneficial microorganisms has been rarely explored. Improving the vitality and functional activity of isolated PGPB is essential for agricultural sustainability. Improving the activity of available microorganisms is more important than ever.

As a result of studies to improve the vitality and functional activity of PGPB, the present inventors confirmed that PGPB treated with plasma enhances plant growth development and disease resistance, and completed the present invention.

As a result of studies to improve the activity of PGPB, a microorganism useful for plants, the present inventors confirmed that PGPB treated with plasma promotes plant growth and development and disease resistance, and completed the present invention. In more detail, when plasma was treated with Bacillus subtilis CB-R05, a bacterium that promotes plant growth, the proliferation and motility of the bacteria were increased. As a result of inoculating rice with plasma-treated bacteria, rice growth and grain yield were significantly increased, and resistance to fungal pathogens was increased. As a result of plasma treatment with another plant growth promoting bacterium, Klebsiella pneumoniae KW7-S06, the bacterial growth rate increased, and as a result of inoculation on rice and barley, the germination rate and growth increased. Therefore, when plasma is treated with plant growth promoting bacteria, growth and motility of bacteria are promoted, and it was confirmed that bacteria whose activity was increased by plasma increased plant growth development, yield, and resistance to fungal pathogens.

In order to achieve the above object, the present invention may provide a method for increasing the activity of useful microorganisms comprising the following steps:

(a) generating plasma in a plasma generating device and treating useful microorganisms;

(b) exchanging the culture medium of the plasma-treated useful microorganisms; and

(c) culturing useful microorganisms in the exchanged medium.

The plasma includes a glow discharge, a corona discharge, an arc discharge, a townsend discharge, a dielectric barrier discharge, and a hollow cathode discharge. (hollow cathode discharge), radio-frequency discharge (radiofrequency (RF) discharge), microwave discharge (microwave discharge), and may be generated by any one selected from the group consisting of electron beams.

The plasma may be generated in a device having an energy of 1 to 60 mW/cm ² .

The conditions for the treatment include setting the distance of the bacteria from the plasma generating source of the plasma generating device to 0.5 to 5 cm, and treating the plasma for 1 minute to 10 minutes and 1 to 3 times.

The useful microorganism may be a plant growth promoting microorganism.

The plant growth promoting microorganism may be any one selected from the group consisting of plant growth promoting bacteria, plant growth promoting viruses, and plant growth promoting fungi.

The plant growth promoting bacteria are Lactobacillus ( Lactobacillus ), Oenococcus ( Oenococcus ), Acetobacter ( Acetobacter ), Chitinophaga ( Chitinophaga ), Ochrobactrum ( Ochrobactrum ), Pediococcus ( Pediococcus ), by Cella ( Weissella ), Gluconacetobacter ( Gluconacetobacter ), Xanthomonas ( Xanthomonas ), Bacillus ( Bacillus ), Sphingobacterium ( Sphingobacterium ), Pseudomonas ( Pseudomonas ), Sporolactobacillus , Sporolactobacillus ( Bacteria_uc_g ), Panibacillus ( Paenibacillus ), Streptomyces ( Streptomyces ) and Klebsiella ( Klebsiella ) may be any one selected from the group consisting of.

The present invention also comprises the steps of inoculating a plant seed with the activated microorganism prepared by the above method; It can provide a method of increasing plant disease resistance or promoting plant growth comprising a.

The plant may be any one selected from the group consisting of wheat, barley, Sudangrass, rice, Chinese cabbage, cucumber, tomato and ginseng.

In the present invention, as a result of treating the plasma with Bacillus subtilis CB-R05, which is a bacterium that promotes plant growth, there was an effect of increasing the proliferation and motility of the bacteria. Plasma-treated bacteria inoculated rice have the effect of significantly increasing plant growth development and grain yield. Also, rice plants inoculated with plasma-treated bacteria showed increased resistance to fungal pathogens. As a result of plasma treatment with another plant growth promoting bacterium, Klebsiella pneumonia KW7-S06, the bacterial growth rate increased, and as a result of inoculating rice and barley with plasma-treated bacteria, seed germination rate and plant growth were increased. When the plasma is treated with the plant growth promoting bacteria, the growth and motility of the bacteria are promoted, and the vitality of the bacteria is increased, thereby increasing the growth development and yield of the plant and the resistance to fungal pathogens.

1 is a schematic diagram of a method for increasing the activity of plant growth promoting microorganisms using plasma, (a) is a step of treating plant growth promoting microorganisms with plasma, (b) is plasma-treated plant growth promoting microorganisms It is a step of exchanging the culture medium of, (c) is a step of culturing plant growth promoting microorganisms in the exchanged medium.
2 is a result of confirming the proliferation of B. subtilis CB-R05 bacteria after plasma treatment, (a) is the result of confirming the number of CFU of bacteria after nitrogen and air plasma treatment, (b) is the cell concentration according to the culture time ( Absorbance at 600 nm) was measured to compare the growth of bacteria, and (c) shows bacteria cultured in Swarm assay medium (LB medium, 1% TTC solution containing 0.5% agar) at 30°C for the indicated time. (Left) is the result of comparing the diameter of the swimming zone (right).
3 is a result of confirming the germination of rice seeds using a suspension of plasma-treated bacteria, untreated bacteria or deionized water, (a) is the daily seed germination rate for 9 days, (b) is the germination after 9 days It is a result of comparing the number of seeds grown in percentage, and (c) is a result showing the length of roots grown from germinated seeds.
Figure 4 is the result of confirming the growth and grain yield of rice using a suspension of plasma-treated bacteria, untreated bacteria or deionized water, (a) is the average length result of each seed grown for 6 weeks from germinated seeds, (b) is the average dry weight result of each seed grown for 6 weeks from germinated seeds, and (c) is the result showing the average number of grains harvested from individual plants after 16 weeks.
5 is a result of comparing the resistance of rice plants from fungal pathogen infection, (a) is a control-related gene in seedlings (2 weeks old) grown in seeds inoculated with B. subtilis CB-R05 treated with plasma and untreated with plasma ( OsPR3, OsPR5 and LOX ) is the result of confirming the expression level, (b) is the result of confirming the average level of disease severity of individual seedlings inoculated with R. solani after 4 weeks, (c) is the result of confirming the expression level of R. solani after 4 weeks. Results showing the average number of dead leaves of individual seedlings inoculated with solani .
6 is a result of confirming the activity of Klebsiella pneumonia KW7-S06 bacteria according to the plasma treatment time, (a) is a result of comparing the number of CFU of bacteria after plasma treatment, (b) is the cell density during the culture time Bacterial growth is the result of measuring the comparison of, (c) is the result of comparing the survival rate of bacteria by checking the confocal laser scanning microscope image of KW7-S06 stained using LIVE / DEAD BacLight bacterial survival kit, (d) is Results of the relative proportions of the number of live and dead cells of each treatment, quantified from different microscopic images.
7 is a result of comparing the germination and growth of rice seeds inoculated with plasma-activated bacteria, (a) is the daily rice seed germination rate results tested daily for 7 days from each treatment, (b) is the leaf of rice seedlings and the total dry weight result of the roots.
8 is a result of comparing the germination of barley seeds inoculated with plasma-activated bacteria. (a) is the result of the germination rate of barley seeds every day, (b) is a stereoscopic microscope showing the change in the shape of barley seeds by germination period. This is the result of checking

Hereinafter, the present invention will be described in more detail.

Improving the vitality and functional activity of isolated PGPB is essential for agricultural sustainability, and research to improve the vitality and activity of plant growth promoting bacteria is becoming more important than ever. As a result of research to improve the vitality and functional activity of PGPB, the inventors confirmed that PGPB treated with plasma enhances plant growth development and disease resistance, and completed the present invention.

In order to achieve the above object, the present invention may provide a method for increasing the activity of useful microorganisms comprising the following steps:

(a) generating plasma in a plasma generating device and treating useful microorganisms;

(b) exchanging the culture medium of the plasma-treated useful microorganisms; and

(c) culturing useful microorganisms in the exchanged medium.

The plasma referred to in this specification is a non-thermal plasma, and the term “non-thermal atmospheric pressure plasma” is because it has a large chemical reactivity to a target object without thermal change, is relatively stable in energy, and acts only on the surface of the reacting material. It has the advantage of not changing the state of the interacting substances or damaging them.

The present inventors used a relatively low energy DBD (dielectric barrier discharge) plasma device of 1 to 60 mW/cm ² or less ( FIG. 1A ).

The plasma includes a glow discharge, a corona discharge, an arc discharge, a townsend discharge, a dielectric barrier discharge, and a hollow cathode discharge. (hollow cathode discharge), radio-frequency discharge (radiofrequency (RF) discharge), microwave discharge (microwave discharge) and may be generated by any one selected from the group consisting of electron beams (electron beams), preferably For example, it can be generated by a dielectric barrier discharge.

Dielectric barrier discharge (DBD) is a high-voltage sinusoidal wave or short pulse width applied between insulated electrodes using a specialized AC power source to generate low-temperature atmospheric plasma under air or other types of gas. It is an annular type (see Fig. 1a).

The plasma may be generated in a device having an energy of 1 to 60 mW/cm ² .

Plasma may be generated by supplying frequencies of 0.1 to 10 lpm (Liter Per Minute), 0.1 to 10 kV, and 0.1 kHz to 100 kHz to the plasma generating device, more preferably 0.1 to 3 lpm (Liter Per Minute), It may be 0.1 to 3 kV and 0.1 kHz to 30 kHz, and most preferably 1.5 lpm (Liter per minute), 1.2 kV input voltage, and a frequency of 15 kHz may be supplied to generate plasma.

The conditions for the treatment include setting the distance of the bacteria from the plasma generating source of the plasma generating device to 0.5 cm to 5 cm, and treating the plasma for 1 minute to 10 minutes and 1 to 3 times.

The carrier gas may be any one or more selected from the group consisting of nitrogen, helium, argon, air and oxygen, and most preferably air or nitrogen.

The useful microorganism may be a plant growth promoting microorganism. Useful microorganisms are the names of microbial materials developed for the purpose of soil improvement and natural organic farming. In general, microorganisms such as yeast, lactic acid bacteria, yeast, photosynthetic bacteria, and actinomycetes that have been used by mankind for a long time are included in the fermentation of food. These microorganisms produce antioxidants, or antioxidants, through which they coexist and inhibit spoilage.

The plant growth promoting microorganism may be any one or more selected from the group consisting of plant growth promoting bacteria, plant growth promoting viruses, and plant growth promoting fungi.

Bacillus subtilis CB-R05, a plant growth-promoting bacteria (PGPB) isolated from rice, by generating plasma using air or nitrogen gas having a relatively low energy of 1 to 60 mW/cm2 or less, and It was treated with Klebsiella pneumoniae KW7-S06. Colony forming units (CFU) of Bacillus subtilis CB -R05 treated with plasma were significantly increased compared to Bacillus subtilis CB-R05 treated with plasma ( FIG. 2A ). As a result of monitoring the concentration of bacterial cells over the incubation time, it was confirmed that the concentration of plasma-treated bacteria increased more rapidly over time ( FIG. 2b ). Flagellar motility of plasma-treated bacteria was significantly increased in low concentration agar medium compared to untreated bacteria (Fig. 2c). It was confirmed that the treatment of plasma promotes the growth of plant growth promoting bacteria. The colony forming unit (CFU) of Klebsiella pneumoniae KW7-S06 treated with plasma had the highest CFU of bacteria treated with nitrogen plasma for 3 minutes ( FIG. 6 ). However, KW7-S06 bacteria treated with plasma for 10 minutes showed a decrease in bacteria compared to KW7-S06 bacteria treated with plasma for 3 minutes. Through this, it was confirmed that there is a threshold value at which bacteria are activated or deactivated depending on the energy delivered by the plasma.

The plant growth promoting bacteria are Lactobacillus ( Lactobacillus ), Oenococcus ( Oenococcus ), Acetobacter ( Acetobacter ), Chitinophaga ( Chitinophaga ), Ochrobactrum ), Pediococcus ( Pediococcus ), Weissella ( Weissella ), Gluconacetobacter ( Gluconacetobacter ), Xanthomonas , Bacillus ( Bacillus ), Sphingobacterium , Pseudomonas ( Pseudomonas ), Sporolactobacillus ( Sporolactobacillus ) g), Panibacillus ( Paenibacillus ), Streptomyces ( Streptomyces ) and Klebsiella ( Klebsiella ) may be any one selected from the group consisting of, preferably any one selected from the group consisting of Bacillus, Enterobacter, Klebsiella and Corynebacterium It may be one, and most preferably may be Bacillus or Klebsiella pneumonia .

The present invention may also provide a method for promoting plant growth and promoting plant disease resistance, comprising the step of inoculating plant seeds with the bacteria prepared by the above-described method.

As a result of inoculating rice with plasma-treated Bacillus subtilis CB-R05, there was no significant difference in germination rate for each treatment group ( FIGS. 3a and 3b ). The length of the primary root was significantly greater in the seeds inoculated with the bacteria untreated with plasma and the seeds inoculated with the bacteria treated with plasma than the water-treated control seeds or the seeds directly treated with plasma, but the No significant difference was observed between the inoculated seeds and the seeds inoculated with plasma-treated bacteria ( FIG. 3c ).

Plasma-treated Klebsiella pneumoniae KW7-S06 was inoculated into rice and barley. As a result of inoculation on rice, the germination rate and dry weight of rice seeds were higher in the case of seeds inoculated with bacteria treated with nitrogen plasma for 3 minutes. After 7 days, about 98% of the seeds inoculated with bacteria treated with nitrogen plasma for 3 minutes germinated while the untreated control seeds germinated about 81% (Fig. 7a). The highest dry weight was observed in the seedlings of the bacterial group treated with plasma for 3 min ( FIG. 7b ). The seed germination rate by plasma-treated bacteria was also improved for barley seeds. Morphological observations of barley seeds were performed on days 3 and 5 after each treatment ( FIG. 8B ). As shown in Fig. 8a, the barley seed germination rate showed the greatest difference at 3 days and 5 days after each treatment.

As a result of analyzing the growth and yield of individual rice after 6 and 16 weeks, respectively, the rice seeds grown in the seeds inoculated with bacteria treated with plasma showed the highest height, dry weight and grain yield during the treatment (FIG. 4) . The height of seeds inoculated with nitrogen and air plasma-treated bacteria was higher than that of seeds inoculated with untreated bacteria and directly plasma-treated (Fig. 4a). The average dry weight of seeds inoculated with nitrogen and air plasma-treated bacteria was significantly greater than that of the other treated groups (Fig. 4b). The use of nitrogen (550 seeds/plant) and air (624 species/plant) plasma treated bacteria significantly increased the grain yield (number of seeds harvested per plant) ( FIG. 4c ).

Resistance to fungal diseases caused by Rhizoctonia solani of 2 week-old seeds grown in the treated seeds (plasma-treated bacteria, non-plasma-treated bacteria or deionized water) was confirmed. Transcription levels of three representative PR (Pathogen Resistant) genes in rice, OsPR3, OsPR5 and LOX , were significantly increased in rice plants inoculated with air plasma-treated bacteria compared with rice plants inoculated with untreated bacteria (FIG. 5a) . In particular, the expression levels of PR3 and PR5 genes were increased 10-fold or more in rice plants inoculated with plasma-treated bacteria compared to rice plants inoculated with untreated bacteria ( FIG. 5A ). After a week, rice plants inoculated with plasma-treated bacteria had less severe disease infection compared to rice plants inoculated with untreated bacteria ( FIG. 5b ). Untreated control plants (water inoculation) showed the most severe disease severity of the three treatments ( FIG. 5B ). The number of dead leaves was reduced in plants inoculated with plasma-treated bacteria compared to plants inoculated with untreated bacteria and untreated controls ( FIG. 5c ). As such, when inoculated with plasma-treated bacteria, it was confirmed that rice had resistance to the R. solani pathogen.

The plant may be any one selected from the group consisting of wheat, barley, Sudangrass, rice, Chinese cabbage, cucumber, tomato or ginseng. Most preferably, it may be rice.

Hereinafter, the present invention will be described in more detail through examples. The following examples are only preferred specific examples of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

<Example 1> Confirmation of the effect of Bacillus subtilis CB-R05

1.1 Plasma device

A relatively low energy DBD (dielectric barrier discharge) plasma device of 1 to 60 mW/cm ² or less was used (FIG. 1A).

1.2 Bacterial treatment of plasma and measurement of bacterial growth

Bacillus subtilis CB-R05, a plant growth-promoting bacteria (PGPB) isolated from rice roots, was treated with DBD plasma. To treat the bacteria with DBD plasma, the bacterial suspension was incubated in tryptic soy broth (TSB) at 37°C for 16 hours and then centrifuged at 3,134×g for 5 minutes. The bacterial cell pellet was suspended in fresh TSB and the concentration was adjusted to an optical density of 1.0 at 600 nm. The bacterial suspension (5 ml) was placed in a 35 mm Petri dish and exposed to DBD plasma. The plasma treated bacterial suspension was centrifuged at 3,134 x g for 5 minutes and the bacterial pellet was washed with sterile distilled water. The bacterial cell pellet was resuspended in 5 ml of fresh TSB and regenerated for 1 hour. To measure bacterial growth, regenerated bacteria were spread on tryptic soy agar (TSA) plates and the number of CFUs was counted.

Colony forming units (CFU) of B. subtilis CB-R05 were regenerated in fresh medium for 1 hour, then the CFU of bacteria treated with nitrogen plasma for 3 and 10 minutes and air plasma treated for 1 minute and 3 minutes were It was significantly higher than the CFU of plasma untreated bacteria (Fig. 2a). As such, the proliferation of bacteria was promoted by plasma treatment of nitrogen and air. When monitoring the concentration of bacterial cells over incubation time, the concentration of plasma-treated bacterial cells increased more rapidly over time than that of untreated bacteria in air plasma for 3 min (Fig. 2b). The pinkish halo zone on the plate of B. subtilis CB-R05 is not observed in the non-motile bacterium S. aureus, indicating movement of B. subtilis CB-R05 (Fig. 2c). The diameter of the pinkish halo regions was slightly larger in the plate of plasma-treated bacteria than in plasma-untreated bacteria 16 h after inoculation (Fig. 2c). After 36 hours, the diameter of the halo diameter was significantly larger than that of the plasma-treated bacterial plate than that of the non-plasma-treated plate ( FIG. 2c ). The longer the incubation period, the greater the difference in the size of the halo zone between the plasma-treated microorganisms and the untreated bacteria. The flagellar motility of the plasma-treated bacteria was significantly increased in the low concentration agar medium compared to the untreated bacteria.

1.4 Bacterial Inoculation on Rice Seeds and Evaluation of Plant Vitality

In order to inoculate the rice seeds with plasma-treated bacteria, the rice seeds were first sterilized by soaking them in 70% ethanol for 1 minute and then shaken in 1.2% (w/v) sodium hypochlorite (NaOCl) solution for 15 minutes. The rice seeds were shaken and washed 3 times with sterile deionized water (15 min each). For inoculation, 30 surface-sterilized rice seeds (cultivar Ilpum) were immersed in 10 ml of a suspension of plasma treated bacteria, untreated bacteria or deionized water at 30° C. for 1 hour. Deionized water was used as an inoculum for untreated controls. Also, for comparison, 50 rice seeds were directly treated with only gas and plasma (nitrogen and air). To evaluate the germination rate of rice seeds, 50 seeds from each treatment were placed on 2 layers of wet filter paper in a 90 mm Petri dish, and the Petri dish was incubated in a plant growth chamber (25 °C, 50% moisture, 16 h light and 8 h dark). did. The number of germinated seeds was recorded daily for 9 days. Germination rate (GR) was calculated as follows: GR (%) = h (number of germinated seeds per dish/total number of seeds)×100. Three replicate measurements were performed in each experiment, and the experiment was repeated three times. To measure plant growth and yield, germinated seeds (3 days after treatment) were planted in soil placed in plug trays (5 x 10 holes, 5 cm diameter, 57 mm x 37 mm) and in a growth chamber (25 ° moisture). 50%, light 16 hr, dark 8 hr). Plants were harvested after 6 weeks and the height of individual plants was measured. The harvested plants were dried in an oven (60° C.) for 3 days and the dry weight of the individual plants was measured. Rice yield was determined by harvesting aged grains after 16 weeks and counting the number of grains harvested per individual plant. Each experiment included 50 plants per treatment and was repeated 3 times.

Germination of rice seeds increased over time in all treatments. On day 9, a germination rate of about 50.77% was observed in all treatments, and the difference was not significant between treatments ( FIG. 3 ). The length of the primary root was determined from the seed inoculated with bacteria (B) that was not treated with plasma and the seed inoculated with the bacteria treated with plasma (PB), the control seed (UC) treated with water or the seed directly treated with plasma (P). was significantly larger than that (Fig. 3c). However, no significant difference was observed between the seed inoculated with the bacteria untreated with plasma and the seed inoculated with the bacteria treated with plasma ( FIG. 3c ).

Rice growth and yield were analyzed after 6 and 16 weeks, respectively. The rice plants grown on the plasma-treated bacteria-inoculated seeds showed the highest height, dry weight and grain yield during the treatment ( FIG. 4 ). The height of plants inoculated with nitrogen and air plasma-treated bacteria was higher than that of plants inoculated with untreated bacteria and plants directly plasma-treated (FIG. 4a). The average dry weight of plants inoculated with nitrogen and air plasma-treated bacteria was significantly greater than that of the other treated groups (Fig. 4b). nitrogen The use of (550 seeds/plant) and air (624 species/plant) plasma treated bacteria significantly increased the grain yield (number of seeds harvested per plant) ( FIG. 4c ). The average yield increased by more than 10% when plasma was treated with nitrogen and air plasma compared with bacteria that were not treated with plasma (FIG. 4c).

1.5 Disease resistance analysis

Two-week-old seeds grown in the treated seeds (plasma-treated bacteria, untreated bacteria or deionized water) were used to examine resistance to fungal pathogens caused by Rhizoctonia solani . Disease resistance was assessed by measuring disease severity and defense gene expression levels. To measure the severity of the disease, barley seeds infected with R. solani AG-1 were inserted into the soil, and two-week-old rice seeds were inoculated with the fungus. After growing for 4 weeks, the severity of infection and disease in the rice was evaluated. The severity of disease symptoms was determined by visual examination 2 weeks after inoculation. Disease severity was determined on the following scale: 1 = less than 10% of leaf area with lesions, 2 = 10-25% of area with lesions, 3 = 25-50% of area with lesions, 4 = 50-75% of leaf area with lesions, 5 = more than 75% of severe lesions or dead leaves. Analysis was performed for a total of 60 plants per treatment in 3 replicate batches (20 reps/replica). Expression of pathogenic related genes (PR) in rice plants was analyzed by RT-qPCR (Table 1).

Leaves were harvested from plants inoculated with plasma-treated bacteria, non-plasma-treated bacteria, or deionized water. Total RNA was extracted from collected leaves using TRIZOL (Invitrogen) and 5 μg RNA was used for the synthesis of cDNAs. cDNA was synthesized using SMART(TM) MMLV Reverse Transcriptase (Clontech). 1 μl of the synthesized cDNA was used as a template for qPCR. qPCR was performed using iQ SYBR Green Supermix (Biorad) and Real-Time PCR detection system (CFX96). The ubiquitin gene was used as a housekeeping gene (GenBank accession number CA763279). The primer sequences of the ubiquitin gene RT-PCR are as follows: forward 5'-CTCCCTGAGATTGCCCACAT-3' and reverse 5'-CACGACTGGCAGCAACAAAT-3'. The cycle for PCR was performed in a two-step protocol; After activation at 95°C for 3 minutes, denaturation at 95°C for 10 seconds, and annealing/extension at 60°C for 30 seconds were repeated 40 times.

3 The transcription level of the PR gene was significantly increased in two-week-old plants inoculated with air plasma-treated bacteria compared to plants inoculated with untreated bacteria ( FIG. 5A ). In particular, the PR3 and PR5 genes were expressed more than 10-fold in plants inoculated with plasma-treated bacteria compared to plants inoculated with non-plasma-treated bacteria (FIG. 5a). After inoculation with the fungal pathogen R. solani AG-1 into the roots of two-week-old seeds, the disease development of the plants was analyzed. After 4 weeks, rice plants inoculated with plasma-treated bacteria had less severe disease development compared to plants inoculated with plasma-untreated bacteria ( FIG. 5b ). Control plants untreated with bacteria (inoculated with water) showed the most severe disease severity among the three treatments (Fig. 5b). The number of dead leaves was reduced in the plants inoculated with the bacteria treated with plasma compared to the control group and the plants inoculated with the bacteria not treated with the bacteria (FIG. 5c). As such, when inoculated with plasma-treated bacteria, it was confirmed that rice had resistance to the R. solani pathogen.

Example 2. Confirmation of the effect of Klebsiella pneumonia KW7-S06

2.1 Confirmation of the increase rate of bacteria by plasma treatment

KW7-S06 ( Klebsiella pneumoniae ) was isolated as PGPB from various Korean rice cultivars in a previous study. The KW7-S06 bacterial suspension was adjusted to an optical density of 1.0 at 600 nm. Using the plasma apparatus described in Example 1.1, 5 ml from each bacterial suspension were plated onto a 35 mm Petri dish and then exposed to DBD plasma. To determine the optimal conditions for activating bacteria by plasma, by varying the plasma treatment time (1, 3 and 10 min), the degree of bacterial activation was compared. Plasma treatment was performed only once. After washing with sterile distilled water, the plasma-treated bacterial suspension was centrifuged at 4,000 rpm for 5 minutes, and then transferred to a new TSB medium. The bacterial suspension was incubated for 1 hour in a shaking incubator at 30°C. Plasma was used as an untreated bacterial suspension as a control. Bacterial activity was measured by CFU (Colony Forming Units). nitrogen for 3 minutes Plasma-treated bacteria had the highest CFU ( FIG. 6 ). The optimal treatment time for activating bacteria by plasma treatment was confirmed to be 3 minutes. nitrogen Plasma increased the proliferation rate of KW7-S06. In addition, it was confirmed that the KW7-S06 treated with plasma for 10 minutes showed a decrease in bacteria compared to KW7-S06 treated with plasma for 3 minutes. was presumed to be KW7-S06 activated by plasma treatment increased the proliferation rate compared to the control group not treated with plasma for the same incubation time. The proliferation of bacteria treated with plasma for 3 minutes was increased, whereas the proliferation of bacteria treated with plasma for 10 minutes was inhibited. This means that there is a threshold at which bacteria are activated or deactivated depending on the energy delivered by the plasma. Excessive levels above a certain threshold can inhibit bacterial activity.

2.2 Measurement of germination rate and plant growth of seeds infected with plasma-treated bacteria

Rice and barley seeds were surface sterilized with 70% ethanol for 1 minute. Shake in 1.2% (w/v) sodium hypochlorite (NaClO) solution for 15 minutes. Rice and barley seeds were shaken 3 times with sterile distilled water (15 min each). Surface-sterilized Ilpum rice and seeds at 50° C. were inoculated into 10 ml of DBD plasma-treated bacterial suspension in an incubator at 30° C. for 1 hour. Distilled water was used as the inoculum for the untreated control group. To compare the germination rate of seeds inoculated with DBD plasma-treated bacteria, 50 seeds were placed at regular intervals on two layers of dried filter paper on a 90 mm Petri dish and placed in a plant growth chamber (25 °C, 50% humidity, 16 h light and 8 h dark cycle). Each experiment included 50 seeds per bacterium treated with DBD plasma in triplicate. To compare the germination rates of rice and barley seeds infected with plasma-treated bacteria, the number of germinated seeds was recorded daily for 7 days from the day of plasma treatment. The germinated rice seeds were placed in a plug tray with soil (5 x 10 holes, 5 cm in diameter, 57 x 37 mm), grown in a chamber at 25 °C with a 14 h light/10 h dark cycle, and seedling growth was monitored. To determine the dry weight of the harvested plants after 6 weeks, the harvested plants were dried in an oven (60° C.) for 3 days, and the dry weight of each individual plant was measured. Each experiment included 50 plants per treatment and was repeated 3 times. Morphological changes of barley seeds during germination were monitored and photographed for 5 days with a stereomicroscope (S8APO, Leica, Germany). Seeds inoculated with bacteria treated with nitrogen plasma for 3 minutes had higher rice seed germination rate and dry weight. After 7 days, about 98% of the seeds inoculated with the bacteria treated with nitrogen plasma for 3 minutes germinated while about 81% of the non-plasma-treated control seeds germinated ( FIG. 7A ). To compare growth development for each treatment, seedling growth after germination was assessed by dry weight. The highest dry weight was observed in the seedlings of the bacterial group treated with plasma for 3 min ( FIG. 7b ). The average dry weight of seedlings was increased in the plasma-treated bacterial group than in the untreated bacterial group. The seed germination rate by plasma-treated bacteria was also improved for barley seeds. Morphological observations of barley seeds were performed on days 3 and 5 after each treatment ( FIG. 8B ). As shown in Fig. 8a, the barley seed germination rate showed the greatest difference at 3 days and 5 days after each treatment. As a result, with the exception of untreated control seeds, primary roots appeared in the corners of the seed coats of bacteria-infected seeds treated with bacteria and plasma. Primary roots were observed in all treatments of the 5-day-old seeds, and in particular, the primary roots of the plasma-treated bacteria-infected seeds were most developed. The germination rates of the control, plasma bacteria untreated and plasma treated bacteria groups were 16%, 28% and 38%, respectively. The final germination rates were 74%, 91%, and 100% in the control group, bacteria not treated with plasma, and bacteria treated with plasma, respectively. In bacteria treated with plasma, the number of seeds germinated each day was increased compared to other germination treatments. That is, the bacteria activated by plasma treatment increased the seed germination rate and germination rate. KW7-S06 activated by plasma treatment promoted seed germination and growth development of seedlings, and this effect showed the same pattern in barley seed germination.

Claims (10)

(a) 1 to 60 mW/cm ² By generating plasma in a plasma generating device having an energy of Bacillus ( Bacillus ) and / or Klebsiella ( Klebsiella ), which are bacteria promoting plant growth, 1 minute to 10 minutes and 1 to 3 times treating the ash plasma;
(B) exchanging the culture medium of the plasma-treated Bacillus ( Bacillus ) and / or Klebsiella ( Klebsiella ); and
(c) culturing Bacillus and/or Klebsiella in the exchanged medium;
A method for enhancing the survival and growth of Bacillus and / or Klebsiella comprising a. The method of claim 1, wherein the plasma is a glow discharge, a corona discharge, an arc discharge, a townsend discharge, a townsend discharge, the electric barrier discharge (dielectric barrier discharge) ), hollow cathode discharge, radio-frequency discharge (radiofrequency (RF) discharge), microwave discharge (microwave discharge) and electron beams by any one selected from the group consisting of A method of enhancing the survival and growth of the resulting, Bacillus and/or Klebsiella . delete According to claim 1, wherein the treatment condition is to set the distance of the bacteria from the plasma generating source of the plasma generating device to 0.5 cm to 5 cm, Bacillus ( Bacillus ) and / or Klebsiella ( Klebsiella ) To promote survival and growth Way. delete delete delete The method of claim 1, wherein the Bacillus ( Bacillus ) and / or Klebsiella ( Klebsiella ) prepared by the method comprising the step of inoculating the plant seeds; Plant disease resistance or method of promoting plant growth. The method of claim 8, wherein the plant is any one selected from the group consisting of wheat, barley, Sudangrass, rice, Chinese cabbage, cucumber, tomato or ginseng. The method of claim 8, wherein the plant disease resistance or plant growth promotion is effective during the entire plant cycle.

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