KR101185737B1 - Halophilic Bacillus licheniformis strain promoting salt stress tolerance in plant and uses thereof - Google Patents

Abstract

The present invention is a basophilic Bacillus rickenformis ( Bacillus ) to enhance the salt stress resistance of plants licheniformis ) RS656 Microbial agent for enhancing salt stress resistance of a plant containing the strain, the strain or a culture thereof as an active ingredient, and a method for enhancing salt stress resistance of a plant comprising immersing or irrigating the strain into a plant or a seed of the plant. It is about.

Description

Halophilic Bacillus licheniformis strain promoting salt stress tolerance in plant and uses according to the present invention.

The present invention relates to a basophilic Bacillus rickenformis strain for enhancing the salt stress resistance of plants, and more particularly, to the bacterium Bacillus rickenformis ( Bacillus ) for promoting salt stress resistance of plants. licheniformis ) RS656 Microbial agent for enhancing salt stress resistance of a plant containing the strain, the strain or a culture thereof as an active ingredient, and a method for enhancing salt stress resistance of a plant comprising immersing or irrigating the strain into a plant or a seed of the plant. It is about.

Ethylene is a plant hormone that exists in the gaseous state, and the optimum amount is present under normal conditions. It is involved in a wide range of growth and development processes such as seed germination, root hair development, root elongation, leaves and abscess, fruit maturation and organ aging (Bleecker and Kende, 2000, Annu). Rev Cell Dev Biol 16, 1-18). However, biotic and abiotic stress conditions cause imbalances in ethylene production and increase the level of ethylene, which significantly inhibits stem and root elongation and reduces plant growth and overall growth (Stephen et al., 2002, Crop Sci 42, 746-753). Salt stress has been reported to result in increased production of ethylene and a marked decrease in plant growth (Bacilio et al., 2004, Biol Fertil Soils 40, 188-193). Hormones fundamentally regulate growth factors in plants, and under stress conditions, ethylene may be one of the most important plant hormones (Jackson, 2002, J Expt). Botany 53, 175-181). Lowering the concentration of ethylene in plants reduces the level of ethylene that inhibits root elongation and growth of plants (Zahir et al., 2009, ArcH Microbiol 191, 415-424). Bacteria with ACC deaminase activity reduce the levels of stress ethylene, causing salt stress (Cheng et al., 2007, Can J Microbiol 53, 912-918), immersion stress (Gricko and Glick, 2001, Plant Physiol Biochem 39, 11-17), heavy metal stress (Burd et al., 1998, Appl Environ Microbiol 64, 3663-3668) and pathogen invasion (Wang et al., 2000, Can J Microbiol 46, 898-907) have been reported to be involved in plant resistance enhancement and growth maintenance (Glick et al. , 1998, J Theor Biol 190, 63-68). ACC, an ethylene biosynthetic precursor produced under stress conditions, is produced from plant roots (Bayliss et al., 1997, Can J Microbiol 43, 809-818) and is absorbed by bacteria and by the ACC dehydrogenase ACC deaminase. It was assumed to be hydrolyzed with ammonia and alpha-ketobutyrate. ACC is hydrolyzed by bacteria producing ACC deaminase, and a small amount of non-hydrolyzed ACC is converted to ethylene, which reduces the stress-inhibitory effect of ethylene on plant growth (Penrose and Glick, 2001, Can J Microbiol 47, 368-372). Therefore, ACC deaminase activity of plant growth promoting rhizobacteria (PGPR) under stress directly affects plant growth. In general, however, PGPR is resistant to salts up to certain limits, and in addition, increasing or decreasing salt concentrations may result in the reduction or loss of some PGP properties (Upadhyay et al., 2009, Curr). Microbiol 59, 489-496). While salt soil is a natural habitat for basophils (Lichfield, 2002, J Ind Microbiol Biotechnol 28, 21-22), Isolation of plant growth promoting basophils that produce ACC deaminase from the natural habitat can provide the best benefits for salt stressed plants.

Therefore, the present inventors have isolated basophils having ACC deaminase activity, and the basophils against growth of salt seedlings ( Capsicum annuum L.) and enhancement of salt resistance by controlling the synthesis of stress level ethylene with the basophils. The efficiency of the was evaluated.

Korean Patent No. 10-0477165 discloses a Bacillus rickenformis strain for controlling fungal diseases of plants, and Korean Patent No. 10-0747701 discloses Bacillus rickenformis for inhibiting plant pathogenic fungi and promoting plant growth. Strains are disclosed.

The present invention is derived from the above-mentioned demands, and has a basophilic Bacillus rickeniformis ( Bacillus) having ACC deaminase activity. licheniformis ) RS656 The present invention has been accomplished by confirming that the strain reduces the ethylene production of the plant to enhance resistance to salt stress.

In order to solve the above problems, the present invention is a basophilic Bacillus rickenformis ( Bacillus ) to improve the salt stress resistance of plants licheniformis ) RS656 Provide strains.

The present invention also provides a microbial agent for enhancing salt stress resistance of a plant containing the strain or its culture as an active ingredient.

The present invention also provides a method of enhancing salt stress resistance of a plant comprising the step of immersing or irrigating the strain into a plant or seed of the plant.

Bacillus licheniformis of the present invention ( Bacillus licheniformis ) RS656 Since the strain and the microbial agent for enhancing salt stress resistance containing the same may alleviate the effects of salt stress on plant growth, it is thought that this may contribute to an increase in the yield of plants affected by reclaimed land and salt.

1 shows a qualitative test of basophils against ACC deaminase enzyme activity. Basophils were plated in modified NFb medium (pH 7.2) with 3.0 mM ACC and 5% NaCl as a nitrogen source. Plates were incubated at 25 ° C. for 4 days and growth was classified as strong, mild, weak and absent. The ability of basophils to use ACC was demonstrated by confirming growth after incubation for 4 days in the same medium without any nitrogen source, confirming that ACC was used as the nitrogen source.
Figure 2 shows the effect of salt stress on (a) growth and (b) root extension of pepper seedlings under greenhouse conditions. The 21 day old seedlings were exposed to salt stress and symptoms were observed after 2 weeks (35 day old plants). A = control (0 NaCl), B = 100 mM, C = 150 mM, D = 200 mM, E = 250 mM NaCl
Figure 3 shows the effect of inoculation of ACC deaminase producing basophils on (a) growth and (b) root elongation of salt stress treated pepper seedlings under greenhouse conditions. A = seedlings of basophils and untreated salts; B = seedlings untreated and exposed to 150 mM NaCl stress for 2 weeks; C, D, E = seedlings treated with ACC deaminase producing basophil seed and exposed to 150 mM NaCl stress for 2 weeks

In order to achieve the object of the present invention, the present invention provides a bacteriostatic Bacillus rickeniformis ( Bacillus ) to enhance the salt stress resistance of plants licheniformis ) RS656 Provide strains. The strain is preferably Bacillus licheniformis RS656 Strain (KACC 91566P). In addition, the strain is characterized by having an ACC (1-aminocyclopropane-1-carboxylic acid) diminase activity that reduces ethylene production of plants.

The present invention also provides a microbial agent for enhancing salt stress resistance of a plant containing the strain or its culture as an active ingredient.

The microbial agent is Bacillus licheniformis RS656 Strains may be included as an active ingredient. The microbial preparation according to the present invention may be prepared in the form of a liquid fertilizer and may be used in the form of powdered powder by adding an extender thereto or granulated by formulating it. However, the formulation is not particularly limited. In other words, it can be formulated as a biofertilizer to overcome this in eco-friendly organic farming where the chemical fertilizer supply is limited.

The present invention also provides a method for enhancing salt stress resistance of a plant comprising the step of immersing or irrigating the strain into a plant or seed of the plant. The plant may preferably be pepper, but is not limited thereto.

As a method of improving the salt stress resistance of the plant may be carried out by immersing or irrigation, that is, spraying the seed culture or the culture medium cultured the strain and the microbial preparation using the strain. In the case of the immersion method, the culture solution and the preparation can be shaken inoculated with the irrigation or the seed in the culture solution and the preparation in the soil around the plant.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Isolation and Growth Conditions of Bacterial Strains

Basophilic strains used in the present invention were isolated from soil samples collected in salt-containing areas of the coast (West, Incheon, South Korea). The soil was transferred to the laboratory and filtered with a 2 mm sieve to screen out plant debris and visible fauna. The soil was diluted 10-fold using sterile saline (0.85% NaCl), shaken at 150 rpm for 15 minutes, and then Trptone Soy Agar medium (TSA, peptone 15g / L) with 10% NaCl; Tryptone 5 g / L; dextrose 2.5 g / L, pH 8.5). A total of 32 basophils were isolated and maintained in TSA slant (pH 7.2) with 5% NaCl and passaged every 3 months at 4 ° C.

As a single nitrogen source ACC  Qualitative analysis of the use ( ACC Diaminazine  Active qualitative analysis)

The ability to use ACC as a nitrogen source is the result of the enzymatic activity of ACC deaminase observed according to the modified method of Poonguzhali et al. (2006, Plant Soil 286, 167-180). Isolated basophils were first incubated in TSA (5% NaCl, pH 7.2). ACC solution (0.5 M) (Sigma Chemical Co., St. Louis, Missouri, USA) was filtered through a sterile filter (0.2 μm) and stored frozen at −20 ° C. (Penrose and Glick, 2001, Can J Microbiol 47, 368-372). Basophils were plated in NFb medium supplemented with 3.0 mM ACC instead of NH ₄ Cl as the nitrogen source. Prior to smearing, 150 μl of the ACC solution was dried by dispersing on the surface of NFb medium (modified with 5% NaCl, pH 7.2). After plating, the plates were incubated at 30 ° C. for 4 days. Growth status was checked every 12 hours and classified into strong, mild, weak and absent. The ability of basophils to use ACC was demonstrated by maintaining the same basophils for 4 days in control NFb solid medium without any nitrogen source, confirming no growth, confirming the use of ACC as a nitrogen source. Based on the ACC utilization, the effectiveness of the basophils was evaluated in reducing the production of ethylene induced by salt stress and in improving the effect of salt stress on pepper growth.

ACC Diaminazine  Active quantitative analysis

Preparation of Enzyme Extracts

Quantitative analysis of ACC deaminase activity (EC 4.1.99.4) shows Honma and Shimomura (1978, Agric) measuring the amount of alpha-ketobutyrate produced when ACC deaminase degrades ACC. Biol Chem 42, 1825-1831) was modified according to the method of Penrose and Glick (2003, Physiologia Plantarum 118, 10-15). ACC deaminase activity was measured in cell extracts of basophil bacteria prepared by the following procedure. The bacteria were first incubated in TSA (Tryptic Soybean Agar, Difco Laboratories) with 5% NaCl, and then the incubated bacteria were inoculated in 5 ml tryptic soy broth (modified with TSB-5% NaCl) at 30 ° C for 24 ～. Incubated for 36 hours. It was then collected by centrifugation at 4 ° C., 10,000 rpm for 10 minutes. The collected cells were washed with 5 ml NFb medium and centrifuged again (2 times) at 4 ° C., 10,000 rpm for 10 minutes. The cells are then added 5% NaCl and supplemented with NCC liquid medium supplemented with ACC as the only nitrogen source (NFb composition per liter: 5.0 g L-malic acid, 4.5 g KOH, 1.8 g KH ₂ PO ₄ , 0.6 g K ₂ HPO ₄ , 1.64% Fe EDTA with 0.2 g MgSO ₄ ? 7H ₂ O, 0.02 g CaCl? 2H ₂ O and trace elements and vitamins: 0.4 g CuSO ₄ ? 5 H ₂ O, 0.12 g ZnSO ₄ ? 7H ₂ O, 1.4 g H ₂ BO ₃ , 1.0 g NaMoO ₄ ? H ₂ O, 1.5 g MnSO ₄ ? 5H ₂ O, 100 mg biotin, 200 mg pyridoxal-HCl; pH 7.2). Immediately prior to inoculation, 30 μl of frozen 0.5 M ACC solution (filtered with 0.2 μm sterile filter and frozen at −20 ° C.) was added to the cell suspension with an ACC concentration of 3.0 mM final and 25 for 24 hours in a shake incubator. Incubation was carried out at 150 ° C condition. Cultured bacterial cells were collected by centrifugation at 10,000 rpm for 10 minutes at 4 ° C. The supernatant was removed and the cell pellet was washed with 0.1M Tris-HCl (pH 7.6). The pellet was stored at -20 ° C for 30 minutes. Cells were washed again by suspending the cell pellet in 1 ml of 0.1 M Tris-HCl (pH 7.6) and transferred to a 1.5 ml centrifuge tube. The suspension was recentrifuged at 10,000 rpm for 5 minutes and the supernatant was removed. The pellet was suspended in 600 μl of 0.1 M Tris-HCl, pH 8.5, 30 μl of toluene was added to the cell suspension and vortexed for 30 seconds. 200 μl of the toluene treated cell suspension was stored separately at 4 ° C. for the next protein quantification. The remaining toluene treated cell suspension was used for the analysis of ACC deaminase activity.

One- aminocyclopropane -One- carboxylic acid  ( ACC ) Diaminazine  Analysis of activity

200 μl of toluene treated cells were transferred to a new 1.5 mL centrifuge tube, and 20 μl of 0.5 M ACC was added to the cell suspension and shaken and incubated at 30 ° C. for 15 minutes. After addition of 1 ml of 0.56 M HCl, the mixture was vortexed and centrifuged at 10,000 rpm for 5 minutes at room temperature. Cell debris was removed and 1 ml of supernatant was transferred to a new glass test tube and vortexed with 800 μl of 0.56 M HCl. Thereafter, 300 μl of dinitro-phenyl hydrazine (DNPH) was added to the glass test tube. The mixture was vortexed again and incubated at 30 ° C. for 30 minutes. Prior to absorbance measurement, 2 mL of 2N NaOH was added and mixed, and the absorbance was measured at 540 nm. The amount of alpha-ketobutyrate produced by the activity of ACC deaminase was calculated by subtracting the absorbance values of the cell extract without ACC and the reagent mixture from the absorbance values of the cell extract with ACC.

Preparation of Standards

The amount (nmol) of alpha-ketobutyrate produced by the reaction was determined by comparing the absorbance of the sample with a standard curve of alpha-ketobutyrate in the range between 0.1 and 1.0 nmol at 540 nm. 100 mmol / L alpha-ketobutyrate (Sigma-Aldrich Co.) solution was prepared using 0.1 M Tris-HCl (pH 8.5) and stored at 4 ° C. 200 μl of a series of published concentrations of alpha-ketobutyrate and 300 μl of 2,4-dinitrophenyl hydrazine reagent (0.2% 2,4-dinitrophenyl hydrazine in 2 M HCl) (Sigma-Aldrich Co.) The mixture was mixed and incubated at 30 ° C. for 30 minutes. Color development was achieved by addition of 2.0 ml 2 M NaOH and absorbance values were measured at 540 nm. To measure the specific activity of the cultures, protein quantification was performed according to the Lowry method (Lowry et al., 1951, J Biol). Chem 193, 265-275).

Of extracellular polysaccharide  produce

The production of extracellular polysaccharides is described by Nicolaus et al. (1999, J Ind Microbiol Biotechnol 23, 489-496) was qualitatively analyzed. Bacterial strains were cultured in 100 ml medium with 5% NaCl (10 yeast extracts 10 g / L), casamino acid 7.5 (g / L), trisodium citrate 3.0 (g / L), KCl 2.0 (g / L), _{MgSO 4? 7H 2 O 20 (} g / L), MnCl 2? 4H 2 O 0.36 (mg / L) , and _{FeSO 4? 7H 2 O 50 (} g / L)) in for 5 days in 25 ℃, 150 rpm condition Incubated. Supernatants were collected by centrifugation at 4 ° C., 1000 rpm for 10 minutes. Cold anhydrous ethanol (3 times) was then added under stirring, and the formation of precipitate was considered as a positive reaction to the production of extracellular polysaccharides.

Hoginess  Taxonomic Identification of Strains

For taxonomic identification, the collected basophil bacterial strains were analyzed for 16S rRNA gene sequences. The collected bacterial strains were cultured in TSA (Difco), and DNA was extracted according to the method of Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd edn. New York, USA: Cold Spring Harbor Laboratory Press). The 16S rRNA gene was amplified by PCR using forward primer (27F-AGAGTTTGATCCTGGCTCAG; SEQ ID NO: 1) and reverse primer (1492R-GGTTACCTTGTTACGACTT; SEQ ID NO: 2). 16S rRNA nucleotide sequences were identified by PCR-direct sequencing using the fluorescent dye terminator method (ABI prism ™ Bigdye ™ Terminator cycle sequencing ready reaction kit V.3.1), and the product was purified using the Millipore-Montage dye removal kit. Finally, the product was sequenced in ABI 3730XL capillary DNA sequencer (50 cm capillary). Strain sequences were registered in the NCBI database (Registration No .: GU968490).

Tree tree analysis

The sequence is then passed to the RDP analysis tool of Ribosomal Database Project-II Release 9 (http://rdp.cme.msu.edu/index.jsp) to determine the close relatives and taxonomic lineage of the sequence. Was processed by. Isolated basophils are Bacillus rickenformis ( Bacillus) licheniformis ) (RS656) showed the highest similarity. Phylogenetic analysis is performed by CLUSTAL W (Thompson et al., 1994, Nucleic Acids After multiple sequencing of data by Res 22, 4673-4680), it was performed using MEGA version 4.1 (Kumar et al., 2004, Brief Bioinform 5, 150-163). Distance is Kimura's two-parameter model (Kimura, 1980, J Mol Obtained using the options according to Evol 16, 111-120, and clustering was performed using the neighbor-joining method (Saitou and Nei, 1987, Mol). Biol Evol 4, 406-425). Statistical reliability of the intersection was calculated by bootstrapping using 100 replicates (Felsenstein, 1985, Evolution 39, 783-791).

Ethylene producing monitoring

The release of ethylene from pepper seedlings was measured according to the protocol of Madhaiyan et al. (2007, Planta 226, 867-876) with some modifications. The surface of the pepper seeds was sterilized with 70% ethanol for 1 minute and 2% sodium hypochlorite (NaOCl) for 30 seconds, followed by rinsing with sterile distilled water (5 times). Basophils were cultivated in TSA medium and cultured basophils were inoculated in Nfb medium with ACC 5 mM and 5% NaCl as the only nitrogen source, and then at 120 rpm at 120 rpm to induce ACC deaminase activity. Incubate for 24 hours with stirring. Cultured strains were centrifuged and washed after cell collection and resuspended in sterile 0.03 M magnesium sulfate (MgSO ₄ ). Seeds were treated for 4 hours in basophils suspension (1 × 10 ⁸ cfu / ml) or sterile water. The seeds were sown in seedling trays (40 holes / tray and 1 seed / hole) containing 150 g of sterile mixed soil and grown at 25 ° C. with a 12 hour photoperiod in a growth chamber. The seedlings (8 days old) were exposed to different levels of salt stress (100, 150 and 200 mM NaCl). Basophils and salt-free seedlings were used as negative controls, and salt-treated seedlings were used as positive controls. Pepper seedlings (12 seedlings for each treatment) were rooted, washed with saline at each concentration to remove soil from the roots and transferred to 120 ml bottles. The bottle was left open for 30 minutes to allow air to escape, then sealed for 4 hours with a rubber septum, and air samples were collected from the bottle top space. 1 ml of the collected air sample was injected by gas chromatography (dsCHROM 6200, Donam Instruments Inc., Republic of Korea) equipped with a Poropak-Q column and equipped with a flame ionization detector. The amount of ethylene released was expressed as pmol ethylene / g live weight / h compared to a standard curve made from pure ethylene (Praxair, Praxair Korea Co., Ltd). Three iterations were used for each treatment.

Greenhouse experiment

Determination of growth inhibition level of salt concentration

Greenhouse experiments were performed with minor modifications to the protocol described by Cheng et al. (2007, Can J Microbiol 53, 912-918). Pepper sterilized seeds were sown in seedling trays (1 seed per hole) filled with 150 g of sterile mixed soil, and grown in a growth chamber under constant conditions (25 ° C., 12 hours dark / light cycle). On day 8 after germination, each seedling was transplanted into a pollen (7.5 x 7.5 cm) filled with 200 g of mixed soil treated with 10 ml of nitrogen deficient Hoagland's nutrient solution and the pot was transferred to the greenhouse. To investigate the salt stress effect and to determine the level of inhibition of salt concentration on pepper growth, the seedlings were treated with 100 mM, 150 mM and 200 mM NaCl 2 weeks later (21 day old seedlings). The plant was harvested 5 weeks after growth (including the time of germination), and then the length and dry weight of the root and stem of the red pepper seedlings were recorded. Dry weight was determined by drying the plant at 70 ° C.

At growth inhibitory salt levels ACC Diaminazine  produce Basophils  Effect on Red Pepper Growth Promotion

As described above, the seed surface of the pepper was germinated by sterile and basophils. On day 8 after germination, each seedling was transplanted into a pot filled with 200 g of mixed soil (7.5 x 7.5 cm), then treated with 10 ml of nitrogen-deficient Hoagland's nutrient solution and the pot was transferred to the greenhouse. The average percentage growth in pepper growth was 52.68% under 150 mM NaCl stress (Table 3). Therefore, to investigate the basophil inoculation effect on salt stressed plants, additionally 150 mM NaCl was added to the nutrient solution and treated two weeks after transfer to the greenhouse. Seedlings untreated and untreated with salt were used as negative controls and salt treated seedlings as positive controls. The plants were harvested after 5 weeks and data recorded. Based on the data obtained, the salt resistance index (STI) of basophils and uninoculated plants was determined by Shetty et al. (1995, Environ). Pollution 88, 308-314) was determined as follows: STI = DWS or DWH / DWC (DWS = dry matter of plants grown under salt stress, DWH = basophil inoculation and plant growth under salt stress Dry matter, DWC = dry matter of plants grown in control conditions without salt stress and basophils inoculation).

Nutrients Analysis

Pepper seedlings were harvested and dried at 70 ° C. for nutrient analysis. Nitrogen, phosphorus, potassium, magnesium, calcium and sodium concentrations (mg / g) were ground to dry pepper grounds to prepare samples. Plant nitrogen concentrations were measured on a Kjeldahl Auto1030 analyzer after digestion with sulfuric acid and potassium sulfate. Phosphorus concentration was determined by dissolving the plant sample with concentrated sulfuric acid and perchloric acid, and then using Jackson (1973, Soil Chemical Analysis. Prentice-Hall of India Pvt. Ltd., New Delhi, Korea) using ammonium metavanadate reagent. India, pp 54-56). Standard solutions were prepared using potassium dihydrogen phosphate (Sigma, USA). For the analysis of other nutrients, the plant sample (0.5 g) was decomposed into a mixture of perchloric acid, sulfuric acid and distilled water (10: 1: 2) and then filtered twice (No. 6, Advantec Toyo) and the volume was Set to 100 ml. 10 ml of sample was used for analysis by inductively coupled plasma optical emission spectroscopy (ICP-OES, Optima 5300DV, Perkin Elmer, USA). A Quality Control Standard 21 stock solution (100 μg / ml of 5% HNO ₃ /tr.Tart./tr.HF) purchased from PerkinElmer Life and Analytical Sciences (710 Bridge Port Avenue, Shelton, CT 06484, USA) was used. . The working solution was prepared from the primary stock solution using deionized distilled water.

Statistical analysis

Data were statistically analyzed by analysis of variance (ANOVA) using SAS general linear model version 9.1 (SAS Institute Inc., Cary, NC, USA), and the mean was compared using the least significant difference test (LSD) ( P ≦ 0.05) (SAS 2009).

Example  1: classification system and PGP  characteristic

The taxonomic identification and properties of basophil strains are shown in Table 1. 16S rRNA gene sequencing was performed by Bacillus rickenformis licheniformis ) strains were identified. The Bacillus rickenformis strain was deposited with the Institute of Agricultural Biotechnology (KACC) on June 1, 2010 (accession number: KACC 91566P).

Bacillus The ACC deaminase activity of licheniformis was determined to be 0.86 μm alpha-ketobutyrate / mg protein / h (Table 1). The basophils were able to produce extracellular polysaccharides.

Details of basophils used Strain
Separation
General characteristics
Colony
shape
Gram
dyeing
PGP Characteristics Registration Number
Classification
ACC Deaminase Activity
(Μmol alpha-ketobutyrate / mg protein / h Extracellular polysaccharide RS656 Coastal soil Can grow at 10% NaCl and pH 6.5 to 8.5 yellow + 0.86 ± 0.06 + GU968490 Bacillus lichenifor mis RS656 (99%) ^a

Strains, isolates of strains, morphology of colonies on agar plates, and response of the strains to Gram staining are shown. ACC deaminase activity data are expressed as mean ± standard error of 3 replicates. The isolated strain from coastal soil was identified according to the published 16S rRNA sequence (RDP) of the strain, with the highest concordance expressed as a percentage in parentheses.

^a % sequence identity in RDP

Example  2: ethylene production monitoring

The measurement of ethylene release by gas chromatography detected significantly higher amounts of ethylene released from inoculated and uninoculated plants at each level of salt stress (Table 2). Ethylene synthesis increased 44.03, 64.04 and 73.86% (pmol ethylene / g FW / h) compared to negative control (untreated NaCl) in pepper plants stressed with 100 mM, 150 mM and 200 mM NaCl, respectively, but ACC deaminase Inoculation of production basophils reduced ethylene release to 24.25, 44.45 and 49.30%, respectively, compared to the positive control (strain untreated) (Table 2).

Effect of Inoculation of PGPH on Ethylene Production of Pepper Seedlings at Different Salt Concentrations process
Ethylene Release (pmol Ethylene / g FW / h) ^* 100 mM NaCl 150 mM NaCl 200 mM NaCl Positive control 353.54 ± 4.45 550.19 ± 8.87 756.97 ± 3.72 Negative control group 197.85 ± 1.11 197.85 ± 1.11 197.85 ± 1.11 Bacillus licheniformis 267.80 ± 2.57 305.66 ± 4.19 383.81 ± 9.10 LSD (P ≤ 0.05) 18.98 33.57 21.63

Data is expressed as mean ± standard error of 3 replicates.

Negative control: basophils and salt-free seeds and seedlings, positive control: seed not basophils, seedlings exposed to 100 mM, 150 mM and 200 mM NaCl stress for 2 hours, inoculation group: seed Bacillus Treated with licheniformis , seedlings exposed to 100 mM, 150 mM and 200 mM NaCl stress for 2 hours.

* Significance test at 0.05% with LSD.

Example  3: salt Under stress Basophil  Inoculation or Growth of Uninoculated Plants

Pepper seedlings (21 days old) exposed to different levels of salt stress for 2 weeks showed a pronounced effect of growth reduction with increasing salt compared to the negative control (Table 3 and FIG. 2). The overall growth reduction of pepper seedlings was about 50.0% under 150 mM NaCl treatment, so the next experiment was considered to handle salt stress of 150 mM NaCl. Salt stress treated plants inoculated with ACC deaminase producing basophils confirmed significantly higher growth in length and dry weight of root and ground (stem) than uninoculated plants (Table 4 and FIG. 3). Stem and root lengths in the positive control plants (150 mM) were 43.04 and 65.0% lower than in the negative control, whereas inoculations were 38.78 to 40.49% and 58.56 to 62.52% higher than the positive control. Dry weight decreased by 45.16% in stems and 57.73% in roots compared to negative control, while inoculation increased 27.66-37.04% in stems and 30.25-43.49% in roots compared to positive control. The results indicate that ACC deaminase producing basophils have the ability to mitigate the effects of salt stress on the growth of pepper plants.

Effect of different levels of salt concentration on the growth of red pepper process Length (cm) ^a Dry weight (g / plant) ^a Average growth decrease (%) ^b stem Root stem Root Control group
(0 NaCl) 24.91 ± 0.79 23.27 ± 1.18 2.48 ± 0.02 0.97 ± 0.03 100 mM NaCl 18.05 ± 0.52 15.10 ± 0.15 1.72 ± 0.03 0.59 ± 0.01 33.10 150 mM NaCl 14.19 ± 0.02 ^c 8.14 ± 0.20 1.36 ± 0.02 0.41 ± 0.01 52.68 200 mM NaCl 12.44 ± 0.23 ^c 6.31 ± 0.15 1.12 ± 0.01 0.31 ± 0.01 62.41 LSD
(P = 0.05) 2.23 1.09 0.14 0.06

The 21 day old seedlings were exposed to 100 mM, 150 mM and 200 mM NaCl stress, and data were recorded after 2 weeks, ie in 35 day old pepper seedlings. Data is expressed as mean ± standard error of 3 replicates.

^a The average length and dry weight of the stems and roots of ^a 35 day old pepper plant were measured in 9 plants for each treatment (3 plants / repeat experiment).

^b Reduced overall growth average of pepper at each level of saline stress

Effect of PGP Basophils in Greenhouse Conditions on Growth of Salt-Stressed Peppers process Length (cm) ^a Dry weight (g / plant) ^a stem Root stem Root Control (0 NaCl) 24.91 ± 0.79 ^a 23.27 ± 1.18 ^a 2.48 ± 0.02 ^a 0.97 ± 0.03 ^a Saline stress
(150 mM NaCl) 14.19 ± 0.02 ^c 8.14 ± 0.02 ^c 1.36 ± 0.02 ^c 0.41 ± 0.01 ^c Bacillus licheniformis
+ 150 mM NaCl 23.18 ± 0.60 ^b 19.93 ± 2.55 ^b 1.88 ± 0.03 ^b 0.78 ± 0.05 ^b LSD (P = 0.05) 1.27 2.89 0.06 0.06

The 21 day old seedlings were exposed to 150 mM salt stress and data were measured after 2 weeks, ie in 35 day old pepper seedlings. Data is expressed as mean ± standard error of 3 replicates.

Negative control: basophils and salt-free seeds and seedlings, positive control: seed not basophils, seedlings exposed to 150 mM NaCl stress for 2 weeks, inoculation group: seed Bacillus Seedlings treated with licheniformis , exposed to 150 mM NaCl stress for 2 weeks.

^a The average length and dry weight of the stems and roots of ^a 35 day old pepper plant were measured in 9 plants for each treatment (3 plants / repeat experiment). The values of the same letter did not differ significantly from each other at 0.05% (LSD).

Example  4: Biomass  Comparison of Distribution and Salt Resistance Indices

Imbalances of dry biomass allocation to roots and stems were found in stressed plants compared to negative control (untreated salts and basophils) and stressed plants inoculated with basophils. In control plants, dry weight was about 71.88% in stems and 28.12% in roots. In contrast, the stressed plants were 76.84% and 23.16%. Inoculation of basophils maintained growth in stressed plants (150 mM NaCl). In other words, biomass distribution and growth were nearly similar to control plants (Table 5 and FIG. 3). Salinization also reduced the root / stem ratio in dry matter (1.30-fold) compared to negative control plants (Table 5), while Bacillus The inoculation of licheniformis was increased 1.36 times compared to the positive control. The higher root / stem ratio of basophilic inoculated plants compared to unvaccinated stressed plants indicates that the distribution of biomass to the roots is increased by the inoculation of basophils. In addition, the salt tolerance index of pepper is significantly increased in basophil inoculated plants compared to salt stress treated pepper plants (Table 5). ACC Deaminase Production Bacillus Plants inoculated with licheniformis showed a 1.50-fold higher resistance index compared to the positive control (150 Mm NaCl).

Effect of Basophil Inoculation on Distribution, Root / Stem Ratio and Salt Tolerance in Salt-Stressed Red Peppers process Distribution of Buildings (DM) ^a Root / stem ^b Salt tolerance index ^b stem (%) Root (%) Dry weight Control (0 NaCl) 71.88 28.12 0.39 ± 0.03 ^b Saline stress
(150 mM NaCl) 76.84 23.16 0.30 ± 0.02 ^c 0.51 ± 0.01 ^b Bacillus licheniformis
+ 150 mM NaCl 70.68 29.32 0.41 ± 0.04 ^a 0.77 ± 0.01 ^a LSD ( P = 0.05) 0.025 0.035

The 21 day old seedlings were exposed to 150 mM salt stress and the data were measured after 2 weeks, ie in 35 day old pepper seedlings. Data is expressed as mean ± standard error of 3 replicates.

Negative control: basophils and salt-free seeds and seedlings, positive control: seed not basophils, seedlings exposed to 150 mM NaCl stress for 2 weeks, inoculation group: seed Bacillus Seedlings treated with licheniformis , exposed to 150 mM NaCl stress for 2 weeks.

^The distribution in the building was calculated from the mean value of 9 plants (35 days old) for each treatment (3 plants / repeat experiment).

^b The root / stem dry weight average ratio and salt tolerance index of 35 day old pepper plants were determined from 9 plants for each treatment (3 plants / repeat experiment). The values of the same characters in the columns do not differ significantly from each other at 0.05% (LSD).

Example  5: nutrient absorption Basophil  effect

The contents of nitrogen, phosphorus, potassium, calcium, magnesium and sodium in the ground portions of pepper seedlings treated with different treatments were measured after 2 weeks of salt stress treatment. Plant nutrient analysis showed that nitrogen (13.24%), phosphorus (61.45%), potassium ions (70.93%), calcium ions (66.50%) and magnesium ions (65.53%) were significantly lower in salt stressed plants than negative controls. Showed that. In contrast, the sodium ion (67.66%) content was higher (Table 6). That is, salt stress negatively affects the absorption of essential nutrients from pepper seedlings. However, inoculation of basophil strains showed nitrogen (11.82-18.50%), phosphorus (16.30-38.62%), potassium ions (34.81-48.58%), calcium ions (30.58-55.44%) and magnesium ions of pepper seedlings under salt stress. (34.20 to 50.83%) content was increased. In contrast, sodium ions (46.64-54.70%) content was reduced compared to the unvaccinated positive control (150 mM) (Table 6).

Effect of Basophil Inoculation on Nutrients Accumulation in Salt-Stressed Pepper Grounds process Nutrient Content in Red Pepper Ground (mg / g DW) ^a nitrogen sign potassium calcium magnesium salt Negative control
no NaCl 56.79 ±
0.74 ^a 5.58 ±
0.27 ^a 28.91 ±
0.82 ^a 5.95 ±
0.23 ^a 3.53 ±
0.10 ^a 1.67 ±
0.04 ^c Salt stress (150 mM NaCl) 49.27 ±
2.25 ^b 2.15 ±
0.00 ^c 8.41 ±
0.12 ^c 1.99 ±
0.06 ^c 1.22 ±
0.02 ^c 5.15 ±
0.04 ^a Bacillus licheniformis + 150 mM NaCl 55.88 ±
0.68 ^a 2.57 ±
0.16 ^d 12.89 ±
0.71 ^b 2.87 ±
0.19 ^b 1.85 ±
0.08 ^b 2.75 ±
0.02 ^b LSD (P ≤ 0.05) 2.76 0.13 0.38 0.11 0.04 0.06

Twenty one day old seedlings were exposed to 150 mM salt stress and analyzed two weeks later, in 35 day old pepper seedlings. Data is expressed as mean ± standard error of 3 replicates.

Negative control: basophils and salt-free seeds and seedlings, positive control: seed not basophils, seedlings exposed to 150 mM NaCl stress for 2 weeks, inoculation group: seed Bacillus Seedlings treated with licheniformis , exposed to 150 mM NaCl stress for 2 weeks.

^a nutrient accumulation was calculated from the average value of three replicates for each treatment. The values of the same characters in the columns do not differ significantly from each other at 0.05% (LSD).

Agricultural Biotechnology Research Institute KACC91566 20100601

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Claims (5)

delete delete delete Basophilic Bacillus licheniformis RS656 Microbial agent for enhancing salt stress resistance of pepper plants containing strain (KACC 91566P) or a culture thereof as an active ingredient. Basophilic Bacillus licheniformis RS656 A method of enhancing salt stress tolerance of a pepper plant, comprising the step of immersing or irrigating the strain (KACC 91566P) in the pepper plant or seeds of the pepper plant.

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