A plant growth-promoting rhizobacterium PJ-3 capable of alleviating saline-alkali stress in maize and its application

Abstract

The present invention discloses a plant growth-promoting rhizobacterium PJ-3 capable of alleviating saline-alkali stress in maize and its application. The present invention provides Myroides odoratimimus with the strain number PJ-3, and its registration number in the General Microbiology Center of the China Microbial Culture Collection Center is CGMCC No. 29090. The strain PJ-3 provided by the present invention has potential application prospects in alleviating moderate saline-alkali stress in maize, promoting maize growth and increasing yield, and has good application prospects in developing microbial inoculants for the growth of crops in saline-alkali land and special microbial fertilizers for alleviating saline-alkali stress in maize.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural production, and relates to a plant growth promoting rhizobacterium PJ-3 capable of alleviating saline-alkali stress of maize and its application. Background Art

[0002] Soil salinization, as a type of land desertification, is the biggest problem in soil degradation. Among them, the northeastern, northwestern, northern and coastal areas of China are the main areas where saline-alkali soils are distributed. Due to the very low organic matter content, low soil fertility, poor physical and chemical properties, and the presence of many anions and cations harmful to crops in saline-alkali soils, it is difficult for crops to emerge, posing a great challenge to the sustainable development of agriculture. Therefore, alleviating saline-alkali stress of crops and promoting increased production and efficiency of saline-alkali land are of great significance to the needs of the country and the people.

[0003] Plant growth promoting rhizobacteria (PGPR) refer to a class of bacteria that can promote plant growth within the rhizosphere of plants. Rhizosphere soil microorganisms are the second genome of plants and play an important role in improving the growth and yield of crops in saline-alkali land and improving saline-alkali land. Many scholars have found that the application of salt-tolerant PGPR can reduce the growth stress of different crops such as peppers, tomatoes, soybeans, wheat, rapeseed and lettuce under saline-alkali conditions. PGPR can induce an increase in the antioxidant enzyme activity of crops under saline-alkali stress, which is an important mechanism for crops to reduce the harm of reactive oxygen species (ROS) under saline-alkali stress. The mechanisms by which PGPR helps crops improve salt tolerance and promote crop growth under saline-alkali stress mainly involve the synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, the biosynthesis of plant hormones, induced systemic tolerance and osmotic protection, etc.

[0004] Microbial organic fertilizers can increase the utilization rate of chemical fertilizers, improve soil properties, increase soil fertility, enhance the stress resistance of crops and increase crop yields. Strains are the basis for the production and application of microbial fertilizers. Exploring efficient and multifunctional microorganisms that can alleviate the saline-alkali stress of crops has potential application prospects in improving the salt tolerance of crops and the quality of crops in saline-alkali land, and is of great significance in promoting the sustainable development of saline-alkali land. Summary of the Invention

[0005] The purpose of the present invention is to provide a plant growth promoting rhizobacterium PJ-3 capable of alleviating saline-alkali stress of maize and its application.

[0006] In the first aspect, the present invention claims to protect a Myroides odoratimimus.

[0007] The Myroides odoratimimus strain PJ-3 claimed by the present invention has the deposit number CGMCC No. 29090 registered with the China General Microbiological Culture Collection Center. This strain has the ability to promote plant growth, such as siderophore production, IAA production, protease production, etc.

[0008] In a second aspect, the present invention claims a culture of the Myroides odoratimimus strain PJ-3 described in the first aspect above. The culture is a substance (all substances in the culture container) obtained by culturing the Myroides odoratimimus strain PJ-3 in a bacterial medium.

[0009] In the above culture, the substance includes the Myroides odoratimimus strain PJ-3 (the cells themselves) and its metabolites.

[0010] The term "metabolite" refers to primary metabolites and / or secondary metabolites produced during the metabolism of microorganisms. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the outside world and generate substances and energy for maintaining life activities through catabolism and anabolism. The products of primary metabolism are primary metabolites, such as monomers like monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, etc., and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, lipids, etc. Secondary metabolism refers to the process by which microorganisms synthesize some substances with no clear function for the life activities of microorganisms using primary metabolites as precursors during a certain growth period. The products of secondary metabolism are secondary metabolites, mostly compounds with relatively complex molecular structures. According to their functions, they can be classified into types such as antibiotics, hormones, alkaloids, toxins, etc.

[0011] In the above culture, the bacterial medium can be a solid medium or a liquid medium.

[0012] The term "culture" refers to the general term for liquid or solid media with microbial populations after artificial inoculation and cultivation. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biological pure culture of microorganisms or can contain a certain amount of medium, metabolites, or other components produced during the cultivation process. The term "culture" also includes subculture obtained by subculturing microorganisms, which can be a culture of a certain generation or a mixture of several generations.

[0013] In a third aspect, the present invention claims a bacterial agent.

[0014] The microbial agent claimed by the present invention contains Myroides odoratimimus PJ-3 described in the first aspect above, metabolites of the Myroides odoratimimus PJ-3, and / or the culture described in the second aspect above.

[0015] Among them, the microbial agent is a microbial agent for promoting plant growth.

[0016] In the above microbial agent, the active ingredient of the microbial agent can be the Myroides odoratimimus PJ-3, metabolites of the Myroides odoratimimus PJ-3, and / or the culture of the Myroides odoratimimus PJ-3. The active ingredient of the microbial agent can also contain other biological components or / and non-biological components. Those skilled in the art can determine other active ingredients of the microbial agent according to the effect of promoting plant growth.

[0017] In the above microbial agent, in addition to the active ingredient, the microbial agent also contains a carrier. The carrier can be a carrier commonly used in the pesticide field and biologically inert. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be at least one of mineral materials, plant materials, or polymer compounds; the mineral materials can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant materials can be at least one of corn flour, bean flour, and starch; the polymer compounds can be polyvinyl alcohol and / or polyglycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0018] In the above microbial agent, the dosage form of the microbial agent can be various dosage forms, such as liquid agent, emulsion, suspension, powder, granule, wettable powder, or water dispersible granule.

[0019] According to needs, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added to the microbial agent.

[0020] In the fourth aspect, the present invention claims any one of the following applications of the Myroides odoratimimus PJ-3 described in the first aspect above, metabolites of the Myroides odoratimimus PJ-3, the culture described in the second aspect above, or the microbial agent described in the third aspect above:

[0021] (A1) Producing protease;

[0022] (A2) Preparation of protease-producing products;

[0023] (A3) Siderophore production;

[0024] (A4) Preparation of siderophore-producing products;

[0025] (A5) IAA production;

[0026] (A6) Preparation of IAA-producing products.

[0027] In a fifth aspect, the present invention claims any one of the following applications of the Myroides odoratimimus PJ-3 described in the first aspect above, or the metabolite of the Myroides odoratimimus PJ-3, or the culture described in the second aspect above, or the microbial agent described in the third aspect above:

[0028] (B1) Promoting plant growth;

[0029] (B2) Preparation of products for promoting plant growth;

[0030] (B3) Increasing plant yield;

[0031] (B4) Preparation of products for increasing plant yield.

[0032] In a sixth aspect, the present invention claims any one of the following applications of the Myroides odoratimimus PJ-3 described in the first aspect above, or the metabolite of the Myroides odoratimimus PJ-3, or the culture described in the second aspect above, or the microbial agent described in the third aspect above:

[0033] (C1) Promoting plant seed germination;

[0034] (C2) Preparation of products for promoting plant seed germination;

[0035] (C3) Promoting the increase of plant bud length and / or root length;

[0036] (C4) Preparation of products for promoting the increase of plant bud length and / or root length;

[0037] (C5) Increasing the emergence rate of plants;

[0038] (C6) Preparation of products for increasing the emergence rate of plants;

[0039] (C7) Promoting the increase of plant plant height;

[0040] (C8) Preparation of products for promoting the increase of plant plant height.

[0041] In the fifth and sixth aspects described above, the promotion of plant growth can be the promotion of plant growth under salt stress and / or alkali stress conditions. The increase in plant yield can be the increase in plant yield under salt stress and / or alkali stress conditions. The promotion of plant seed germination can be the increase in the promotion of plant seed germination under salt stress and / or alkali stress conditions. The promotion of the increase in plant bud length and / or root length can be the promotion of the increase in plant bud length and / or root length under salt stress and / or alkali stress conditions. The increase in plant emergence rate can be the increase in plant emergence rate under salt stress and / or alkali stress conditions. The promotion of the increase in plant height can be the promotion of the increase in plant height under salt stress and / or alkali stress conditions.

[0042] Further, the salt stress is a salt concentration equivalent to an NaCl content greater than 0 and less than or equal to 90 g / L (such as an NaCl concentration greater than 0 and less than or equal to 70 g / L, or an NaCl concentration greater than 0 and less than or equal to 50 g / L, or an NaCl concentration of 3 g / L or 0.15 g / L).

[0043] Further, the alkali stress is an alkaline condition with a pH not higher than 12 (such as a pH greater than 7 and less than or equal to 12, or a pH greater than 7 and less than or equal to 11, or a pH greater than 7 and less than or equal to 10, or a pH of 9.5 or 8.5).

[0044] In one embodiment of the present invention, the salt stress and the alkali stress coexist, specifically NaCl 3 g / L, pH 9.5 (severe saline-alkali stress). In another embodiment of the present invention, the salt stress and the alkali stress coexist, specifically NaCl 0.15 g / L, pH 8.5 (moderate saline-alkali stress).

[0045] In the seventh aspect, the present invention claims any one of the following applications of the Myroides odoratimimus PJ-3 described in the first aspect above, or the metabolites of the Myroides odoratimimus PJ-3, or the culture described in the second aspect above, or the bacterial agent described in the third aspect above:

[0046] (D1) Alleviating plant saline-alkali stress;

[0047] (D2) Preparing a product for alleviating plant saline-alkali stress.

[0048] Wherein, the saline-alkali stress is salt stress and / or alkali stress.

[0049] Further, the salt stress is a salt concentration equivalent to an NaCl content greater than 0 and less than or equal to 90 g / L (such as an NaCl concentration greater than 0 and less than or equal to 70 g / L, or an NaCl concentration greater than 0 and less than or equal to 50 g / L, or an NaCl concentration of 3 g / L or 0.15 g / L).

[0050] Further, the alkali stress is an alkaline condition with a pH not higher than 12 (such as a pH greater than 7 and less than or equal to 12, or a pH greater than 7 and less than or equal to 11, or a pH greater than 7 and less than or equal to 10, or a pH of 9.5 or 8.5).

[0051] In one embodiment of the present invention, the salt stress and the alkali stress coexist, specifically with an NaCl concentration of 3 g / L and a pH of 9.5 (severe saline-alkali stress). In another embodiment of the present invention, the salt stress and the alkali stress coexist, specifically with an NaCl concentration of 0.15 g / L and a pH of 8.5 (moderate saline-alkali stress).

[0052] In the above aspects, the product can specifically be a microbial inoculant or a microbial fertilizer. Such as a microbial inoculant for the growth of plants (crops) in saline-alkali land or a special microbial fertilizer for alleviating the saline-alkali stress of plants.

[0053] In the above aspects, the plant can be any one of the following:

[0054] (E1) Maize;

[0055] (E2) A plant of the genus Zea;

[0056] (E3) A gramineous plant;

[0057] (E4) A monocotyledonous plant.

[0058] For the plant growth-promoting rhizobacteria provided by the present invention, soil greenhouse cultivation test studies show that the emergence rate and average plant height of the experimental group of maize plants inoculated with the PJ-3 strain in the rhizosphere are significantly better than those of the control group of maize plants not inoculated with the PJ-3 strain under moderate saline-alkali stress; at the same time, maize seed germination test studies show that the bud length and root length of the experimental group of maize seeds inoculated with the PJ-3 strain are significantly higher than those of the control group not inoculated with the PJ-3 strain under severe saline-alkali stress. Therefore, the strain PJ-3 provided by the present invention has potential application prospects in alleviating the saline-alkali stress of maize, promoting maize growth and increasing yield, and has good application prospects in developing microbial inoculants for the growth of crops in saline-alkali land and special microbial fertilizers for alleviating the saline-alkali stress of maize.

[0059] Depository Instructions

[0060] Taxonomic name: Myroides odoratimimus;

[0061] Biological material referred to: PJ-3;

[0062] Depository institution: General Microbiology Center of China Committee for Culture Collection of Microorganisms;

[0063] Abbreviation of depository institution: CGMCC;

[0064] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0065] Date of deposit: November 21, 2023;

[0066] Registration number in the depository center: CGMCC No. 29090. Description of the drawings

[0067] Figure 1 It is a colony morphology diagram of strain PJ-3 on the plate.

[0068] Figure 2 It is a microscopic examination diagram of Gram staining of strain PJ-3.

[0069] Figure 3 It is the result of salt tolerance determination of strain PJ-3.

[0070] Figure 4 It is the result of alkali tolerance determination of strain PJ-3.

[0071] Figure 5 It is a diagram of the protease-producing ability of strain PJ-3.

[0072] Figure 6 It is a diagram of the siderophore-producing ability of strain PJ-3.

[0073] Figure 7 It is the effect of strain PJ-3 on the germination of corn seeds under severe saline-alkali stress.

[0074] Figure 8 It is the effect of strain PJ-3 on the growth of corn under moderate saline-alkali stress. Detailed implementation manners

[0075] The present invention will be further described in detail below in conjunction with specific implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0076] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0077] Example 1. Isolation and Identification of Strain PJ-3

[0078] I. Isolation of Strains

[0079] Weigh 10 g of rhizosphere soil sample (from Guyuan County, Zhangjiakou City, Hebei Province), add it to 90 ml of sterile water containing glass beads, and shake it vigorously at 150 r / min for 30 min to obtain the rhizosphere soil mother liquor bacterial suspension; dilute the above bacterial suspension with sterile water to 10 -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5 gradients. Take 10 -3 , 10 -4 , 10 -5 three consecutive dilution degrees of 100 μL and spread them on the prepared nutrient agar medium (formula: 3 g of beef extract, 10 g of peptone, 5 g of sodium chloride, 15 - 20 g of agar, 1000 mL of distilled water, adjust pH = 7.0) plates, and set three replicates for each gradient. At the same time, use sterile distilled water as a blank control. All plates are placed in a constant temperature incubator at 30°C for cultivation. After 3 days, select bacterial single colonies with different colors and morphologies for streak plating, and transfer them to glycerol tubes for preservation after purification.

[0080] II. Bacterial Genetic Identification and Phylogeny

[0081] Select a bacterial genomic DNA extraction kit (TIANGEN) to extract the DNA of the above isolated strains. The PCR amplification primers are 16S rDNA universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′), 1492R (5′-TACGGTTACCTTGTTACGACTT-3′), and the size of its DNA fragment is about 1500 bp, which is synthesized by Shanghai Sangon Biotech Co., Ltd. The PCR amplification reaction system includes 3 μL of DNA template, 1 μL of each of the 27F and 1492R primers (10 μmoL·L -1 ), 12.5 μl of 2×mix Taq enzyme (product of GenStar Company), and 7.5 μL of ddH2O, and is made into a 25 μL system. The PCR amplification reaction program: pre-denaturation at 94°C for 10 min; denaturation at 94°C for 30 s, annealing at 55°C for 1 min, extension at 72°C for 1.5 min, 30 cycles; finally, extend at 72°C for 10 min and store at 4°C.

[0082] The 16S rDNA gene of the tested bacteria was amplified, and the sequencing results were uploaded to the database at https: / / www.ncbi.nlm.nih.gov / nuccore / / for online alignment. The MEGA7.0 (Molecular Evolutionary Genetics Analysis) software was used for cluster analysis by the Neighbor-Joining method, and a phylogenetic tree was constructed with a Bootstrap value of 1000.

[0083] After alignment with the NCBI database, it was determined that the strain PJ-3 was Myroides odoratimimus, with a maximum similarity of 99.86%. The 16S rDNA sequence of strain PJ-3 is shown in SEQ ID No.1.

[0084] III. Biological characteristics analysis of strain PJ-3

[0085] 1. Morphological observation and Gram staining

[0086] The strain PJ-3 obtained by secondary screening was streaked on a nutrient agar medium using an inoculation loop and incubated in an inverted position in a constant temperature incubator at 30 °C for 24 h to observe the basic morphology of the colonies. Take a sterilized glass slide and operate in a laminar flow hood. Pipette a drop of sterile water onto the glass slide, dip a small amount of the bacteria, and spread it into a uniform thin layer with an inoculation loop. Place the specimen side up, hold one end of the glass slide, and carefully heat it slightly above the alcohol lamp to evaporate the water. After cooling, start staining. Drop 1-2 drops of ammonium oxalate crystal violet on the smear film to cover the smear, and stain for about 1 min. Tilt the glass slide and rinse it with a small stream of water under the tap until the water rinsed off is colorless; Pipette 300 μL of iodine solution and drop it on the smear film to cover the smear, and stain for about 1 min. Tilt the glass slide and rinse it with a small stream of water under the tap until the water rinsed off is colorless; Tilt the glass slide, drop 95% ethanol for decolorization until the ethanol flowing out is no longer purple, and then wash it with water after about 20-30 s; Drop 1-2 drops of safranin stain on the smear film to cover the smear, and stain for about 1 min. Tilt the glass slide and rinse it with a small stream of water under the tap until the water rinsed off is colorless; Blot the water droplets with absorbent paper, and place the specimen slide under the microscope for observation after it is dry.

[0087] The results showed that the single colonies of strain PJ-3 on the nutrient agar medium were round and flat, with neat edges, yellow, smooth and opaque ( Figure 1 ). Under the microscope, the colonies were red, indicating that it was a Gram-negative bacterium ( Figure 2 ).

[0088] 2. Determination of the Physiological and Biochemical Characteristics of Strain PJ

[0089] (1) Inoculate strain PJ-3 into tryptone soy agar medium (TSA, formula: 15 g of tryptone, 5 g of soy peptone, 5 g of sodium chloride, 15 g of agar powder, 1000 mL of distilled water, pH = 7.3) and culture it for no more than 16 h to avoid spore formation.

[0090] (2) Place the turbidimeter steadily, calibrate and adjust the turbidimeter reading to 100. Use an Inoculatorz cotton swab to pick up a colony with a diameter of about 3 mm from the streaked plate and inoculate it into the IF-A inoculation solution (product of Biolog). During the inoculation process, gently smear the cotton swab on the wall of the inoculation bottle, shake the inoculation tube several times to evenly inoculate the strain, repeat several times, and adjust the turbidity of the inoculation solution to 90-98%.

[0091] (3) Pour the bacterial suspension inoculated with the colony into a sterile V-shaped sample-loading water tank. Use an 8-channel pipette to carefully add the bacterial suspension to all wells in an amount of 100 μL per well in sequence. Cover the lid of the microplate, taking care not to touch the upper and lower surfaces to avoid inaccurate measurement.

[0092] (4) Place the loaded Biolog GENEIII 96-well plate directly into the OmniLog incubation and reading instrument. After incubating at a constant temperature of 33 °C for 24-48 h in the instrument, read the data results for identification. The results are shown in Table 1.

[0093] Table 1. Results of the Physiological and Biochemical Characteristics Determination of Strain PJ-3

[0094]

[0095]

[0096]

[0097]

[0098] Note: +, positive; -, negative; w, weakly positive.

[0099] Through the above identification, strain PJ-3 was identified as Myroides odoratimimus. This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on November 21, 2023, with the deposit number CGMCC No. 29090. Hereinafter, it is referred to as strain PJ-3 for short.

[0100] Example 2: Determination of Salt Tolerance and Alkalinity Tolerance of Strain PJ-3

[0101] I. Determination of Salt Tolerance

[0102] Using the beef extract peptone medium (with 0.5% NaCl, % means g / 100 mL) as a control, change the NaCl content in the beef extract peptone medium to 1.0%, 3.0%, 5.0%, 7.0%, 9% (all % mean g / 100 mL), and set aside. Inoculate 14 μL of the single bacterial suspension of strain PJ-3 into 330 mL of the above-prepared media with different salinities respectively, and measure the OD at this time 600 . Set three replicates for each treatment, and culture them in the dark with shaking at 30 °C and 200 rpm for 12 h, and measure the OD every 12 hours 600 value to evaluate the salt tolerance of strain PJ-3

[0103] The salt tolerance of strain PJ-3 is shown in Table 2 and Figure 3 as follows. The results show that: Strain PJ-3 can grow normally in the NA medium containing 0.5%-5% NaCl, and can still grow in the NA medium containing 7% NaCl and the OD 600 reaches 0.373 ± 0.069 at 48 h, and grows slowly in the NA medium containing 9% NaCl and the OD 600 reaches 0.236 ± 0.003 at 48 h

[0104] Table 2: Salt Tolerance of Strain PJ-3

[0105]

[0106] Note: Different lowercase letters in the table indicate significant differences (P < 0.05).

[0107] II. Determination of Alkalinity Tolerance

[0108] Using the beef extract peptone medium (pH = 7.0) as a control, change the pH of the beef extract peptone medium to 8.0, 9.0, 10.0, 11.0, 12.0, and set aside. Inoculate 14 μL of the single bacterial suspension of strain PJ-3 into 330 mL of the above-prepared media with different pH values respectively, and measure the OD at this time 600 . Set three replicates for each treatment, and culture them in the dark with shaking at 30 °C and 200 rpm for 12 h, and measure the OD every 12 hours 600 value to evaluate the alkalinity tolerance of strain PJ-3

[0109] The alkalinity tolerance of strain PJ-3 is shown in Table 3 and Figure 4As shown in the figure. The results showed that the strain PJ-3 could grow in the beef extract peptone medium with pH = 12, showing good alkali tolerance. Especially, it grew well between pH 7-11.

[0110] Table 3. Alkali tolerance performance of strain PJ-3

[0111]

[0112] Note: Different lowercase letters in the table indicate significant differences (P<0.05).

[0113] Example 3. Determination of the growth-promoting ability of strain PJ-3

[0114] I. Determination of protease production ability

[0115] Determination medium: 5 g of skim milk powder was dissolved in 50 mL of distilled water, and another 1.5 g of agar was dissolved in 50 mL of distilled water, sterilized separately. When cooled to 45-50 °C, the two solutions were mixed and poured into plates.

[0116] The strain PJ-3 was inoculated onto the above milk medium (i.e., the determination medium), cultured at 28 °C for 3 days, observed for the appearance of a clear zone around the colonies, and photographed. The ability of the strain to produce protease was judged according to the size of the clear zone.

[0117] II. Determination of siderophore production ability

[0118] Determination medium: Chrome azurol S (CAS) 60.5 mg; Hexadecyltrimethylammonium bromide (HDTMA) 72.9 mg; Ferrous chloride hexahydrate 2.645 mg; Agar 9.0 g; 0.1 mol / L phosphate buffer 50 mL. The following are the reagent contents in 50 mL of buffer: Sodium dihydrogen phosphate dihydrate 295.25 mg; Disodium hydrogen phosphate dodecahydrate 1213.5 mg; Ammonium chloride 125 mg; Potassium dihydrogen phosphate 37.5 mg; Sodium chloride 62.5 mg; dissolved in 1000 mL of distilled water; pH 6.6-7.0.

[0119] The strain PJ-3 was inoculated onto the above CAS detection medium (i.e., the determination medium), cultured at 28 °C for 3 days, observed for the appearance of a clear zone around the colonies, and photographed. The ability of the strain to produce siderophores was judged according to the size of the clear zone.

[0120] III. Determination of IAA production ability

[0121] Drawing of the standard curve: Prepare standard IAA solutions with concentrations of 10, 20, 30, 40, and 50 μg / mL in volumetric flasks. Add 100 μL of standard IAA solutions with different concentrations to 96-well plates (previously added with an equal volume of Salkowski colorimetric solution) in triplicate, quickly place them in a dark environment for 30 min, and add 100 μL of NA medium (i.e., beef extract peptone medium, formula: 3 g of beef extract, 10 g of peptone, 5 g of sodium chloride, 15 - 20 g of agar, 1000 mL of distilled water, adjust pH = 7.0) to 100 μL of Salkowski colorimetric solution as a negative control, and add 100 μL of standard IAA solution with a concentration of 50 μg / mL to 100 μL of Salkowski colorimetric solution as a positive control, and measure OD 530 absorbance, take the average three times, use the IAA concentration as the abscissa, and OD 530 value as the ordinate to draw the standard curve.

[0122] Inoculate strain PJ-3 in NA liquid medium (containing 500 mg / L of L-tryptophan) at 180 r / min and culture at 30 °C for 48 h. Take 1 mL of the bacterial solution into a centrifuge tube, centrifuge at 8000 r / min for 5 min, then take 100 μL of the supernatant and add it to a 96-well plate, repeat three times, add 100 μL of Salkowski colorimetric solution, quickly place it in the dark for 30 min, measure the OD530 absorbance, take the average three times and substitute it into the standard curve to calculate the IAA content.

[0123] IV. Results and Analysis

[0124] The determination results of the protease-producing ability, siderophore-producing ability, and IAA-producing ability of strain PJ-3 are shown in Table 4, Figure 5 and Figure 6 as shown. Figure 5 This is the determination result of the protease-producing ability of strain PJ-3. Figure 6 This is the determination result of the siderophore-producing ability of strain PJ-3. The results show that: Strain PJ-3 has the ability to produce protease and siderophore, and the IAA production content is about 1.3491 ± 0.3254 μg / mL.

[0125] Table 4. Growth-promoting ability of strain PJ-3

[0126]

[0127] Example 4. Effect of strain PJ-3 on the germination of maize seeds under saline-alkali stress

[0128] The materials and reagents used in this example are as follows:

[0129] (1) Maize variety: Jingke 968.

[0130] (2) Disposable petri dish (9 cm in diameter).

[0131] (3) Sterile filter paper (8.5 cm in diameter).

[0132] (4) Severe saline-alkali stress solution: NaCl (3 g / L), Na2SO4 (1.5 g / L), NaHCO3 (4.5 g / L), Na2CO3 (0.6 g / L), pH = 9.5.

[0133] (5) Test strain: Strain PJ-3 isolated in Example 1.

[0134] (6) Beef extract peptone medium (NA medium): 3 g of beef extract, 10 g of peptone, 5 g of sodium chloride, 15 - 20 g of agar, 1000 mL of distilled water, adjust pH = 7.0.

[0135] First, clean the purple coating on the seed surface with clear water, then disinfect with 75% alcohol for 15 s, wash three times with sterile water, soak in 5% sodium hypochlorite solution for 30 min, and finally rinse three times with clear water to complete the disinfection work.

[0136] Pick a small amount of strain PJ-3 and inoculate it into 330 mL of NA liquid medium, culture at 200 rpm and 30 °C, and end the culture when the turbidity of the strain reaches OD600 = 1.3. Take 2 ml of the bacterial liquid in a centrifuge tube and centrifuge to obtain the cell precipitate, and add 2 ml of the severe saline-alkali stress solution to resuspend the cells. Place the sterile filter paper flat on the disposable petri dish, evenly place 6 corn seeds on it, and evenly drip 2 mL of the bacterial liquid on each seed to ensure soaking. At the same time, set a control group using an equal amount of sterile water instead of the bacterial liquid. 72 h after the treatment, detect the germination of corn seeds in each group, including measuring the bud length and root length.

[0137] The results are shown in Table 5 and Figure 7 As shown, in the control group without inoculating strain PJ-3 (using an equal amount of sterile water instead of the bacterial liquid), the average bud length of corn was 0.94 ± 0.36 cm, and the average root length was 2.02 ± 0.59 cm. In the experimental group inoculating strain PJ-3, the average bud length of corn was 2.52 ± 1.03 cm, and the average root length was 3.82 ± 1.62 cm. It can be seen that inoculating strain PJ-3 can improve the salt tolerance of corn seeds and promote seed germination and growth.

[0138] Table 5. Effects of Strain PJ-3 on the Germination of Corn Seeds under Saline-alkali Stress

[0139]

[0140] Note: "*" and "**" respectively indicate significant differences from the control group without inoculating strain PJ-3 at the 0.05 and 0.01 levels.

[0141] Example 5: Experiment on strain PJ-3 alleviating saline-alkali stress in maize

[0142] The materials and reagents used in this example are as follows:

[0143] (1) Maize variety: Jingke 968.

[0144] (2) Potting substrate: German Floragard peat soil, (pH = 6.6, EC = 3630 μs / cm).

[0145] (3) Disposable plastic pots (diameter 17 cm).

[0146] (4) Moderate saline-alkali stress solution: NaCl (0.15 g / L), Na2SO4 (1.5 g / L), NaHCO3 (3 g / L), Na2CO3 (0.45 g / L), pH = 8.5.

[0147] (5) Test strain: Strain PJ-3 isolated in Example 1.

[0148] (6) Beef extract peptone medium (NA medium): 3 g of beef extract, 10 g of peptone, 5 g of sodium chloride, 15 - 20 g of agar, 1000 mL of distilled water, adjust pH = 7.0.

[0149] First, clean the purple coating on the seed surface with clean water, then disinfect it with 75% alcohol for 15 s, wash it three times with sterile water, soak it in 5% sodium hypochlorite solution for 30 min, and finally rinse it three times with clean water to complete the disinfection work.

[0150] Pick a small amount of strain PJ-3 and inoculate it into 330 mL of NA liquid medium, culture it at 200 rpm and 30 °C, and end the culture when the turbidity of the strain reaches OD600 = 1.3. Weigh 330 g of peat soil, add 300 mL of moderate saline-alkali stress solution and mix evenly, then load it into disposable plastic pots, repeat twice, with one pot as the control group and the other as the experimental group. Select maize seeds with as uniform size as possible, sow 5 seeds in each pot, and the sowing depth is 2 cm. Drop 10 mL of strain PJ-3 solution evenly on the maize seeds in the experimental group and ensure it soaks through. Replace the strain solution with an equal amount of sterile water in the control group. Observe and record the growth of maize every day, and collect the plants after 20 d to measure the emergence rate and plant height of the plants.

[0151] The results are shown in Table 6 and Figure 8As shown, the emergence rate of corn in the control group without inoculation of strain PJ-3 (replacing the bacterial solution with an equal amount of sterile water) was 3 / 5, and the average plant height was 32.00 ± 7.94 cm. The emergence rate of corn in the experimental group inoculated with strain PJ-3 was 5 / 5, and the average plant height was 37.80 ± 13.31 cm. It can be seen that inoculation with strain PJ-3 can effectively alleviate the saline-alkali stress of corn, improve the salt tolerance of corn plants and promote their growth.

[0152] Table 6. Experimental results of strain PJ-3 alleviating saline-alkali stress of corn

[0153]

[0154] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application but are made by conventional techniques known in the art.

Claims

1. A flavor-like aroma bacterium, characterized in that: The pseudo-aromatic flavor bacteria is Myroides odoratimimus , with the strain number of PJ-3 and the registration number in the China General Microbiological Culture Collection Center being CGMCC No. 29090.

2. Bacterial agent, characterized in that: The microbial agent contains the Myroides odoratimimus as claimed in claim 1.

3. The microbial agent according to claim 2, characterized in that: The microbial agent is a microbial agent for promoting plant growth.

4. Any one of the following applications of the Myroides odoratimimus as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3: (A1) Producing protease; (A2) Preparing a product for producing protease; (A3) Producing siderophore; (A4) Preparing a product for producing siderophore; (A5) Producing IAA; (A6) Preparing a product for producing IAA.

5. Any one of the following applications of the Myroides odoratimimus as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3: (B1) Promoting plant growth; (B2) Preparing a product for promoting plant growth; (B3) Increasing plant yield; (B4) Preparing a product for increasing plant yield; The promoting plant growth is promoting plant growth under salt stress and / or alkali stress conditions; The increasing plant yield is increasing plant yield under salt stress and / or alkali stress conditions; The plant is maize.

6. Any one of the following applications of the Myroides odoratimimus as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3: (C1) Promoting plant seed germination; (C2) Preparing a product for promoting plant seed germination; (C3) Promoting the increase of plant bud length and / or root length; (C4) Preparing a product for promoting the increase of plant bud length and / or root length; (C5) Increasing plant emergence rate; (C6) Preparing a product for increasing plant emergence rate; (C7) Promoting the increase of plant height; (C8) Preparing a product for promoting the increase of plant height; The promoting plant seed germination is promoting the increase of plant seed germination under salt stress and / or alkali stress conditions; The promoting the increase of plant bud length and / or root length is promoting the increase of plant bud length and / or root length under salt stress and / or alkali stress conditions; The increasing plant emergence rate is increasing plant emergence rate under salt stress and / or alkali stress conditions; The promoting the increase of plant height is promoting the increase of plant height under salt stress and / or alkali stress conditions; The plant is maize.

7. Any one of the following applications of the Myroides odoratimimus as claimed in claim 1 or the microbial agent as claimed in claim 2 or 3: (D1) Alleviating plant salt-alkali stress; (D2) Preparing a product for alleviating plant salt-alkali stress; The plant is maize.

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