Bacillus amyloliquefaciens and application thereof in relieving salt stress of scleroderma plants

Abstract

The invention relates to the technical field of microorganisms, and particularly provides bacillus amyloliquefaciens and application thereof in relieving salt stress of scleroderma plants. The preservation number of the bacillus amyloliquefaciens is CCTCC (China Center For Type Culture Collection) CNO: M 20242154; the nucleotide sequence of the bacillus amyloliquefaciens is as shown in SEQ ID NO. 1. Compared with the prior art, the bacillus amyloliquefaciens PG-4 provided by the invention can improve the osmotic regulation capability of the sclerodera plant seedlings under salt stress, enhance the tolerance to the salinized environment and promote the growth of the sclerodera plant seedlings under salt stress.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and more particularly to a bacillus amyloliquefaciens and application thereof in alleviating salt stress of plants of the genus Sclerotium. Background Art

[0002] At present, the problem of soil salinization is becoming increasingly prominent. The global saline soil area exceeds 833 million hectares (accounting for 8.7% of the earth's area). About 2,000 hectares of arable land are lost every day due to soil salinization, and the yield of many crops has decreased by 10-25%. Soil salinization has become a major resource and ecological problem that is prevalent in countries around the world. Excessive soil salt concentration affects all stages of plant growth and development, and seriously damages a series of physiological and biochemical processes of plants, including plant ion balance, photosynthesis, cell membrane permeability, osmotic regulating substances, antioxidant enzyme system, lipids and nitrogen metabolism, thereby inhibiting plant growth and even death. It is the main abiotic stress that affects crop yield and quality.

[0003] Rhizosphere microorganisms are microbial communities that are planted in the soil area around the plant roots and interact with the plant roots or form a symbiotic relationship. As the dominant bacteria in the rhizosphere microbial community, the root microorganisms (PGPR) interact with plants and environmental factors to form a complex network of interactions. Under saline conditions, PGPR improves plant growth by inducing plant stress response systems and synthesizing anti-stress biochemical substances. In addition, PGPR also alleviates plant salt stress damage by producing extracellular polysaccharides, regulating ion homeostasis, producing ACC deaminase, and synthesizing plant hormones.

[0004] The construction and function of soil microbial communities play an important role in maintaining the stability of soil ecosystems. In recent years, the use of beneficial soil microorganisms to improve saline land has received widespread attention from scholars at home and abroad. In the improvement of saline-alkali land, it is usually achieved by increasing the amount of beneficial soil microbial agents, changing the abundance of the bacterial community and the community structure, maintaining the nutrient balance in the saline soil, adjusting the proportion of each element, and improving the soil aggregate structure, thereby promoting the improvement of saline soil and ecological restoration. Compared with other soil salinization control measures, biological methods have lower application costs, less negative impact on soil fertility and metabolic activity, and are more conducive to the sustainable development of the ecological environment.

[0005] The salinization problem in coastal areas has always been an important factor that plagues agricultural production, and the supply of tropical leguminous forage microbial agents targeting this problem is relatively scarce. Microbial agents can promote the growth and reproduction of beneficial microorganisms in the soil, improve soil structure, reduce the adverse effects of salinity on crop growth, and improve the salt-alkali tolerance and yield of forage. However, the types of tropical leguminous forage microbial agents on the market for coastal saline-alkali areas are limited and cannot meet the needs of farmers and breeders.

[0006] Macrotyloma uniflorum (Lam.) Verdc., an annual dicotyledonous native grass species of the genus Macrotyloma in the Leguminosae family, is high in nitrogen content, rich in nutrients such as protein, and has strong insect resistance and drought tolerance. It has strong growth potential and can adapt to relatively harsh environmental conditions. It is an excellent feed and green manure crop. Macrotyloma is one of the important undeveloped edible legumes and is currently grown in many countries and regions, including tropical regions in Asia and Africa, China, India and Australia. 'Yazhou' Macrotyloma was established as a local variety by the National Grass Variety Approval Committee in 2015. It is a high-protein, low-fat edible legume crop widely grown in Hainan. 'Yazhou' Macrotyloma has a high germination rate, fast plant growth, and strong field competitiveness. It can be used as an excellent inter-row weed control crop in plantations and has broad application prospects. Like other legumes, hard-shelled bean is also susceptible to soil salinity. Therefore, ecological and cost-effective biological measures can be taken to improve its salt tolerance. However, based on current relevant research, there are no reports on the interaction between hard-shelled bean and microorganisms in response to salt stress, and little is known about the physiological and molecular mechanisms of its interaction with microorganisms and response to salt stress. Summary of the invention

[0007] In view of this, the object of the present invention is to provide a Bacillus amyloliquefaciens and its use in alleviating salt stress of Scleroderma plants. The Bacillus amyloliquefaciens PG-4 of the present invention can improve the osmotic regulation ability of Scleroderma plant seedlings under salt stress, enhance tolerance to saline environment, and promote the growth of Scleroderma plant seedlings under salt stress.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] Application of Bacillus amyloliquefaciens or a bacterial agent containing Bacillus amyloliquefaciens in improving the salt stress tolerance of Scleroderma plants;

[0010] The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242154;

[0011] The nucleotide sequence of the Bacillus amyloliquefaciens is shown in SEQ ID NO.1.

[0012] The Bacillus amyloliquefaciens of the present invention has the following morphology: on an LB culture medium plate, it is milky white, translucent to opaque, nearly circular, the surface of the colony is slightly smooth to semi-rough, the edge is irregular, slightly wrinkled and convex, the texture is sticky, and it is easy to pick up. The strain is blue-purple in Gram staining, so the strain is a Gram-positive bacterium, the bacterial body is in the shape of a long rod, and can form oval spores.

[0013] Preferably, the temperature for subculturing the Bacillus amyloliquefaciens on LB solid culture medium is 28° C., and the time for subculturing is 48-72 hours.

[0014] Preferably, the components of the LB solid culture medium include 10 g of TRYPTONE (tryptone), 5 g of YEAST EXTRACT (yeast extract), and 10 g of NaCl.

[0015] Preferably, the Bacillus amyloliquefaciens has the following physiological and biochemical characteristics: gelatin liquefaction +, starch hydrolysis +, catalase +, VP.

[0016] Preferably, the genus Cerithidea includes Cerithidea serrata.

[0017] The present invention also provides the use of Bacillus amyloliquefaciens or a bacterial agent containing Bacillus amyloliquefaciens in promoting the seed germination of Scleroderma plants under salt stress;

[0018] The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242154;

[0019] The nucleotide sequence of the Bacillus amyloliquefaciens is shown in SEQ ID NO.1.

[0020] Preferably, the genus Cerithidea includes Cerithidea serrata.

[0021] The present invention also provides the use of Bacillus amyloliquefaciens or a bacterial agent containing Bacillus amyloliquefaciens in promoting the growth of Scleroderma plants at the seedling stage under salt stress;

[0022] The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242154;

[0023] The nucleotide sequence of the Bacillus amyloliquefaciens is shown in SEQ ID NO.1.

[0024] Preferably, the genus Cerithidea includes Cerithidea serrata.

[0025] The present invention also provides the use of Bacillus amyloliquefaciens or a bacterial agent containing Bacillus amyloliquefaciens in any one of (1) to (5);

[0026] (1) Improving the seedling length of sclerocarpon plants under salt stress;

[0027] (2) increasing root length of scleroderma plants under salt stress;

[0028] (3) increasing the fresh weight of sclerocarpon plants under salt stress;

[0029] (4) increasing the dry weight of Bean plants under salt stress;

[0030] (5) Improving the germination rate of seeds of the genus Pseudocarpus under salt stress;

[0031] The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242154;

[0032] The nucleotide sequence of the Bacillus amyloliquefaciens is shown in SEQ ID NO.1.

[0033] Preferably, the genus Cerithidea includes Cerithidea serrata.

[0034] The present invention also provides the use of Bacillus amyloliquefaciens or a bacterial agent containing Bacillus amyloliquefaciens in any one of (1) to (3);

[0035] (1) Producing pectinase;

[0036] (2) Producing starch hydrolyzing enzymes;

[0037] (3) Protease production;

[0038] The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242154;

[0039] The nucleotide sequence of the Bacillus amyloliquefaciens is shown in SEQ ID NO.1.

[0040] Preferably, the genus Cerithidea includes Cerithidea serrata.

[0041] The present invention also provides a salt-tolerant growth-promoting agent, comprising Bacillus amyloliquefaciens or a bacterial agent containing Bacillus amyloliquefaciens;

[0042] The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242154;

[0043] The nucleotide sequence of the Bacillus amyloliquefaciens is shown in SEQ ID NO.1.

[0044] Preferably, the preparation method of the salt-tolerant growth-promoting agent comprises:

[0045] Inoculate Bacillus amyloliquefaciens or a bacterial culture containing Bacillus amyloliquefaciens into LB liquid medium and culture at 28°C and 200 r / min until OD 600 is 0.6.

[0046] The present invention also provides a method for improving the growth and salt tolerance of Scleroderma plants at the seedling stage, comprising treating the seeds or seedling roots of Scleroderma plants with Bacillus amyloliquefaciens or a bacterial agent containing Bacillus amyloliquefaciens, or the salt tolerance growth promoter, and then sowing the seeds;

[0047] The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242154;

[0048] The nucleotide sequence of the Bacillus amyloliquefaciens is shown in SEQ ID NO.1.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] (1) The Bacillus amyloliquefaciens PG-4 provided by the present invention can improve the osmotic regulation ability of Scleroderma plant seedlings under salt stress, enhance tolerance to saline environment, and promote the growth of Scleroderma plant seedlings under salt stress; Bacillus amyloliquefaciens alleviates stress in an environmentally friendly manner, providing a theoretical basis and technical support for the study of the salt tolerance mechanism of tropical legumes and the comprehensive development and utilization of related microbial agents.

[0051] (2) In the present invention, compared with the non-inoculated treatment under salt stress, PG-4 seed soaking can promote the germination of seeds of the genus Scleroderma under salt stress, and increase the length of the radicle and plumule, which has a significant promoting effect on the seed germination process.

[0052] (3) Through potted experiments, the present invention further verified the effect of Bacillus amyloliquefaciens PG-4 on the growth of Yazhou hard bean seedlings under salt stress. The results showed that compared with the uninoculated seedlings under salt stress, the plant height of Yazhou hard bean seedlings increased by 1.0 times after PG-4 strain inoculation, the fresh weight of the aboveground part and the underground part increased by 2.7 times and 1.7 times, respectively, and the dry weight of the aboveground part and the underground part increased by 2.9 times and 1.5 times, respectively.

[0053] Biological Deposit Description

[0054] The amyloliquefaciens of the present invention is named Bacillus amyloliquefaciens PG-4, and its classification name is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). It was deposited in the China Center for Type Culture Collection on October 10, 2024, with the address: Wuhan University, Wuhan, China, and the deposit number is CCTCCNO: M 20242154. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 : Phylogenetic tree of Bacillus amyloliquefaciens PG-4 constructed in Example 2;

[0056] Figure 2 : Schematic diagram of the colony morphology of Bacillus amyloliquefaciens PG-4 on LB solid medium;

[0057] Figure 3 : Schematic diagram of Gram staining results of Bacillus amyloliquefaciens PG-4;

[0058] Figure 4 : Schematic diagram of the catalase reaction of Bacillus amyloliquefaciens PG-4;

[0059] Figure 5 : Schematic diagram of starch hydrolysis reaction of Bacillus amyloliquefaciens PG-4;

[0060] Figure 6 : Schematic diagram of the cellulase production capacity determination of Bacillus amyloliquefaciens PG-4;

[0061] Figure 7 : Schematic diagram of the assay of protease production capacity of Bacillus amyloliquefaciens PG-4;

[0062] Figure 8 : Schematic diagram of the determination of the pectinase production capacity of Bacillus amyloliquefaciens PG-4;

[0063] Fig. 9 : Schematic diagram of the determination of starch production ability of Bacillus amyloliquefaciens PG-4;

[0064] Fig.10 : Schematic diagram of the experiment of Bacillus amyloliquefaciens PG-4 on Stylosanthes under salt stress;

[0065] Fig.11 : Schematic diagram of the experiment of Bacillus amyloliquefaciens PG-4 on pigeon pea under salt stress;

[0066] Fig.12 :The phenotype of growth of Yazhou hard bean plants under salt stress by Bacillus amyloliquefaciens PG-4;

[0067] Fig.13:Effects of Bacillus amyloliquefaciens PG-4 on physiological parameters of Yazhou hard bean under salt stress. DETAILED DESCRIPTION

[0068] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0069] In order to further illustrate the present invention, the following examples are used to explain it in detail. The raw materials used in the following examples and comparative examples of the present invention are all commercially available products.

[0070] Example 1 Source of strain PG-4

[0071] The Bacillus amyloliquefaciens PG-4 used in this example was isolated from the rhizosphere soil of Yazhou hard-shelled beans (Danzhou, Hainan, 19°31'N, 109°34'E, average altitude 150m), and the deposit number of Bacillus amyloliquefaciens is CCTCC NO: M 20242154. The turbid bacterial liquid of the isolated and purified strain was mixed with an equal volume of 50% glycerol and stored in a -80°C refrigerator for later use.

[0072] Example 2 Physiological and biochemical characteristics of strains and identification of their taxonomic status

[0073] 2.1 Physiological and biochemical test materials and methods

[0074] The culture medium used for pathogen culture and related physiological and biochemical experiments is:

[0075] VP liquid medium: 5 g peptone, 5 g glucose, 5 g potassium dihydrogen phosphate, pH adjusted to 7.0-7.2, sterilized at 115°C for 20 min;

[0076] Gelatin liquefaction medium: 3g beef extract, 5g peptone, 20g gelatin powder, adjust pH to about 7.2, sterilize at 115℃ for 20min;

[0077] Cellulose-sodium (CMC-Na) medium: Na2HPO4 2.5g, KH2PO4 1.5g, peptone 2.5g, CMC-Na20g, agar 20g, pH adjusted to 7.0-7.5, sterilized at 121℃ for 20min;

[0078] Skim milk LB agar medium: 10 g tryptone, 10 g NaCl, 5 g yeast extract, 20 g agar, 20 g sterile skim milk, pH adjusted to 7.0-7.5, sterilized at 115°C for 20 min;

[0079] Pectinase-producing bacteria screening medium: pectin 4g, K2HPO4 0.1g, MgSO4 0.5g, NaNO3 3g, FeSO4·7H2O 0.01g, Congo red 0.4g, agar powder 12g, pH adjusted to 7.0, sterilized at 121℃ for 20min;

[0080] Catalase test: add 3% H2O2 solution to the center of the slide, pick a single PG-4 colony cultured for 24 hours and spread it evenly. If bubbles are generated, the catalase reaction is positive, otherwise it is negative. Figure 4 shown.

[0081] VP test: inoculate the strain to be tested into VP liquid culture medium, culture on a shaker (37°C, 180r / min) for 48h, add an equal amount of 40% NaOH solution (containing 0.3% creatine) into the fermentation liquid, observe the color change of the liquid culture medium after leaving it for 20min, if the color is red, it is positive, otherwise it is negative.

[0082] Gelatin liquefaction test: The strain to be tested is inoculated into the center of the gelatin liquefaction medium plate by puncture method, and the uninoculated medium plate is used as control, and cultured in a 28℃ constant temperature incubator for 5 days. After taking out the culture dish, put it in a 4℃ refrigerator and let it stand for 30 minutes. If the gelatin block on the surface of the culture medium is sunken and becomes liquid, it means that the bacteria can liquefy gelatin. If the surface is intact without sunken, it is considered that the gelatin liquefaction reaction is negative.

[0083] Cellulase production: Inoculate the strain into CMC-Na medium, culture at 28℃ for 5-7 days, pour Congo red solution (1g / L) into the medium, react for 15 minutes and then pour out the solution, then add 1mol / L NaCl solution, react for 15 minutes and then pour out the solution. If a transparent circle is formed around the strain, it indicates that the strain can produce cellulase, and the result is positive, such as Figure 6 shown.

[0084] Pectinase production: Inoculate the strain into the pectinase bacterial screening medium and culture it at 28°C for 5-7 days. If a transparent circle is observed around the strain, it indicates that the strain can produce cellulase. The result is positive, such as Figure 7 shown.

[0085] Starch hydrolyzing enzyme production: streak the strain onto a starch medium plate, culture at 28°C for 5-7 days, and then spray Lugol's iodine solution evenly on the surface of the medium. If a colorless transparent circle is formed around the colony, it means that the strain can produce starch hydrolyzing enzymes, and the starch hydrolysis test is positive. Figure 8 shown.

[0086] Protease production: Inoculate the strain into a skim milk LB agar medium plate and culture at 28°C for 5-7 days. If a halo is observed around the colony, it means that the strain can produce protease and the protease production test is positive. Fig. 9 shown.

[0087] Observation of bacterial morphology and colony characteristics: PG-4 was inoculated on LB medium plates by the three-zone streak method, and incubated at 28°C for 2-3 days. When complete and clear single colonies grew on the plates, the bacterial morphology and colony characteristics of the strain were observed. Single colonies on the plates were picked and stained with Galanz. The steps were as follows: (1) Strain smear, dried and fixed; (2) Crystal violet staining for 1 minute, washed with water; (3) Iodine staining for 1 minute, washed with water; (4) Decolorization solution was added, decolorization for 20-60 seconds, washed with water; (5) Safranin staining solution was used for 1 minute, washed with water; (6) The lens was dried and observed with a 100x oil immersion lens.

[0088] Molecular biological identification of strain PG-4: Genomic DNA was extracted from the PG-4 strain using a genomic DNA extraction kit, and then the 16S rDNA fragment was PCR amplified using the bacterial universal primer 27F / 1492R. After amplification, the size and specificity of the amplified fragment were tested by 1% agarose gel electrophoresis, and the PCR amplification product was sent to Nanshan Biotechnology (China) Co., Ltd. for purification and sequencing. After sequencing, the BLAST method (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) was used to analyze the 16S rDNA sequencing results of the PG-4 strain, and related sequences with high homology were selected for relationship analysis, and the phylogenetic tree was constructed by the neighbor-joining method using MEGA6.0 software.

[0089] 2.2 Experimental Results

[0090] The strain PG-4 isolated in Example 1 has the following morphological, physiological and biochemical characteristics:

[0091] a. Morphological characteristics of the bacteria: milky white, translucent to opaque, nearly round, with a slightly smooth to semi-rough surface, irregular edges, slightly wrinkled and raised, sticky texture, easy to pick up, such as Figure 2 shown.

[0092] like Figure 3As shown, the Gram stain of PG-4 strain is blue-purple, so this strain is a Gram-positive bacterium, the bacteria are long rod-shaped, and can form oval spores.

[0093] b. Physiological and biochemical characteristics: gelatin liquefaction +, starch hydrolysis +, catalase +, VP - ("+" indicates a positive reaction; "-" indicates a negative reaction). The results show that the PG-4 strain has good contact enzyme, gelatin liquefaction and starch hydrolysis tests. Figure 5 As shown, all of them were positive, and the VP (acetylmethylcarbinol) test results were negative. This result showed that the PG-4 strain had the ability to liquefy gelatin and could produce starch hydrolyzing enzymes and catalase.

[0094] c. Biological characteristics: PG-4 strain has the ability to secrete a variety of hydrolytic enzymes under different salt concentration conditions, including cellulase, pectinase, amylase and protease ( Figure 6-9 The results showed that the PG-4 strain had potential application value in enhancing plant resistance to salt stress.

[0095] d. Molecular biological identification of strain PG-4: After sequencing, the nucleotide sequence of the strain is shown in SED ID NO.1. Figure 1 As shown, PG-4 has a similarity of 99.37% with B. amyloliquefaciens NBRC 15535 (NCBI accession number: NR041455.1), and has a high similarity with other Bacillus amyloliquefaciens isolated strains (≥99%). Therefore, the strain was determined to be Bacillus amyloliquefaciens and was named B. amyloliquefaciens.

[0096] Example 3 Comparison of salt tolerance of strains

[0097] The plate streaking method and the spotting of equal amounts of bacterial solution (2 μL, OD 600 =0.6) inoculation method to compare the salt tolerance of five bacteria (all of which were Bacillus amyloliquefaciens screened from the rhizosphere soil of Yazhou hard bean). LB solid culture medium with salt contents of 0.3, 0.6, 0.9, 1.2 and 1.5 mol / L was prepared respectively, and single colonies of different strains were picked and inoculated on LB culture medium with different salt concentrations by three-zone streak method (three parallel replicates were set for each concentration), or 2 μL was drawn, OD 600 = 0.6 were inoculated into LB medium with different salt concentrations, and cultured at 28°C for 2-3 days. The growth of each strain on the medium was observed to determine its salt tolerance. The results are shown in Tables 1 and 2.

[0098] Table 1 Effects of five PGPR strains on the growth of Yazhou hard bean seedlings under different concentrations of salt stress

[0099]

[0100]

[0101] Table 2 Effects of five PGPR strains on the fresh weight of Yazhou hard bean seedlings under different concentrations of salt stress

[0102]

[0103] As shown in Tables 1 and 2, with the increase of NaCl concentration, the fresh weight of seedlings in each treatment gradually decreased; however, at the same NaCl concentration, the PGPR strain inoculation treatment could significantly (P<0.05) increase the fresh weight of seedlings. Under low salt stress of 50 and 100 mmol / L NaCl, the fresh weight of seedlings inoculated with PG-4 strain increased most significantly compared with uninoculated seedlings; under high salt stress of 200 and 300 mmol / L NaCl, the fresh weight of seedlings inoculated with the strain increased significantly (P<0.05) by 11.9% to 22.9%. Among the five Bacillus PGPR strains, the PG-4 strain had the best effect on promoting the growth of Yazhou hard bean seedlings under salt stress conditions, and had great potential in improving plant salt stress resistance.

[0104] Example 4 Yazhou hard bean seed germination test

[0105] The culture medium used for bacterial culture and seed germination experiments was:

[0106] Water agar medium: agar 7 g / L, pH adjusted to 7.0, sterilized at 121°C for 15 min.

[0107] LB medium: TRYPTONE 10 g / L, YEAST EXTRACT 5 g / L, NaCl 10 g / L, pH adjusted to 7.0-7.3, sterilized at 121°C for 20 min.

[0108] Preparation of bacterial suspension: Inoculate the bacteria into LB liquid culture medium under sterile conditions and culture in a constant temperature shaker (28°C, 200r / min). Prepare bacterial suspension after the culture solution becomes turbid and measure the OD of the bacterial suspension using a spectrophotometer. 600 value, and adjust to OD 600 =0.6, culture at 28℃ for 2-3 days, and observe the growth of each strain on the culture medium to determine its salt tolerance.

[0109] NaCl was added to the water agar medium to obtain water agar medium containing 0 (no NaCl) and 100 mmol / L NaCl, respectively. After sterilization, the medium was poured into a sterile square culture dish (10 cm × 10 cm), sealed by condensation and set aside.

[0110] Select an appropriate amount of Yazhou hard bean seeds with full grains. Wash the surface of the seed coat with distilled water, soak it in 75% (v / v) ethanol for 50 seconds, then wash it with sterile water for 3 to 5 times, soak it in 3% (v / v) sodium hypochlorite for 3 minutes, and then wash it with sterile water for 5 to 8 times. Place the sterilized seeds in a clean bench and use absorbent paper to absorb excess water on the surface of the seeds.

[0111] The disinfected seeds were placed in sterilized centrifuge tubes and sterile water and the corresponding bacterial suspension were added respectively. Each treatment was placed in a constant temperature shaker (28°C, 200r / min) and shaken and soaked for 2h.

[0112] The soaked seeds were neatly placed in square culture dishes filled with water agar medium with different salt concentrations (5 replicates were set for each treatment), sealed with sealing film quickly after sowing, cultured in the dark at 25°C for 2 days, and then transferred to light for growth. The seed germination rate was calculated at different germination times, the root length and seedling length were measured, and 5 seedlings were randomly selected from each dish and their fresh weight was weighed. The results are shown in Tables 3 and 4.

[0113] The experimental treatment groups included: no bacterial inoculum and no salt stress (CN), bacterial inoculum but no salt stress (BN), no bacterial inoculum but salt stress (CS), and bacterial inoculum and salt stress (BS).

[0114] Table 3 Effects of Bacillus amyloliquefaciens PG-4 on the germination rate of Yazhou hard bean seeds under salt stress

[0115]

[0116] Table 4 Effect of PG-4 strain on biomass during seed germination under salt stress

[0117]

[0118]

[0119] As shown in Tables 3 and 4, salt stress had a serious negative impact on seeds during germination, significantly reducing radicle length, embryo length and fresh weight (P<0.05). However, under 100mmol / L NaCl stress, the radicle length, embryo length and fresh weight of seeds inoculated with PG-4 strain increased significantly (P<0.05) compared with those without inoculation. The results of this study showed that PG-4 strain inoculation could alleviate the salt stress damage suffered by hard bean seeds during germination to a certain extent and improve the salt stress resistance of seeds.

[0120] Example 5 Pigeon pea and Stylosanthes seed germination test

[0121] After the seeds of pigeon pea and stylosanthes were treated and inoculated with the same method as above, the PG-4 strain was co-cultured with the pigeon pea seeds and stylosanthes under 0 and 100 mmol / L NaCl stress. The preparation of bacterial solution, the configuration of salt-containing culture medium, seed treatment, inoculation method and culture conditions were the same as those in Example 4 above.

[0122] The experimental treatment groups included:

[0123] C1: sterile water treatment; C2: LB liquid culture medium treatment; T1: PG-4 bacterial suspension treatment; C3: 100mmol / NaCl treatment; C4: 100mmol / NaCl treatment in LB liquid culture medium; T2: 100mmol / NaCl+PG-4 bacterial suspension treatment.

[0124] Table 5 Effect of PG-4 strain on biomass during germination of pigeon pea seeds under salt stress

[0125]

[0126] Table 6 Effect of PG-4 strain on biomass of Stylosanthes germination under salt stress

[0127]

[0128]

[0129] The morphology of Stylosanthes and Pigeon pea under all treatments was as follows Fig.10 , Fig.11 As shown. Under non-salt stress conditions, inoculation with PG-4 can significantly promote the growth of pigeon pea and stylosanthes. The seedling length, root length, seedling fresh weight and root dry weight of pigeon pea and stylosanthes inoculated with PG-4 strains are significantly greater than those of the uninoculated control group. Under salt stress conditions, compared with the uninoculated control group, the root length, seedling length and fresh weight of the seedlings inoculated with PG-4 strains were significantly increased.

[0130] Example 5 Potted Plant Test

[0131] After seed treatment and inoculation, the seeds were germinated at room temperature for 3 days, and the seedlings with consistent growth were selected and transplanted into pots filled with sterile potting soil (nutrient soil: vermiculite = 1:1). There were 8 treatments in total, 8 replicates (pots) in each treatment, and 4 seedlings were planted in each pot. The pots were arranged in a randomized block design. Root inoculation was performed 4 days after transplanting, and the OD was prepared as described above. 600= 0.6 of Bacillus amyloliquefaciens suspension, and 1 mL of bacterial solution was injected into the root of each seedling with a syringe. 1 mL of sterile water was injected into the root of each seedling in the sterile water-treated potted plant of the control group. The second additional root irrigation was carried out 7 days after inoculation, and 1 mL of the corresponding solution was also injected into the root of each seedling. Salt stress was applied 5 days after the first root irrigation and inoculation. 20 mL of 100 mmol / L NaCl solution was poured into the flower pots treated with salt stress every two days, and 20 mL of water was poured into the flower pots not treated with salt stress. Water once every 3 days to keep the soil moist.

[0132] After 14 days of salt stress, the plants were removed from each treatment, and the roots were rinsed with sterile water to remove the attached soil, and then the water on the surface of the plants was absorbed with filter paper. The plant height and root length of the Yazhou hard bean plants were directly measured with a millimeter ruler. The aboveground part and the root of the plant were cut with scissors at the base of the plant stem, and their fresh weight (Fresh weight, FW) was measured respectively. Then, they were placed in an 80°C oven to dry to constant weight, and their dry weight (Dry weight, DW) was measured respectively. Each treatment was repeated 4 times, such as Fig.12 shown.

[0133] Table 7 Analysis of the growth of Yazhou hard bean plants under salt stress by Bacillus amyloliquefaciens PG-4

[0134]

[0135] From Table 7 and Fig.12 It can be seen that under non-salt stress conditions, inoculation with PG-4 can significantly promote the growth of Yazhou hard bean plants. The seedling length, root length, seedling fresh weight and root dry weight of Yazhou hard bean inoculated with PG-4 strains were significantly (P<0.05) greater than those of the uninoculated control group. Under salt stress conditions, PG-4 strains reduced the inhibition of salt stress on the growth of Yazhou hard bean plants. The seedling length of uninoculated plants was about 9.65 cm, and the seedling length of PG-4 strain-inoculated plants was about 10.05 cm, while there was no significant difference in root length between strain-inoculated and uninoculated plants (P>0.05). In addition, under salt stress, compared with the uninoculated control group, the fresh weight of the aboveground and underground parts of the PG-4 strain-inoculated treatment increased by 2.7 times and 1.7 times, respectively, and the dry weight of the aboveground and underground parts increased by 2.9 times and 1.5 times, respectively.

[0136] Example 6 Plant physiological index detection

[0137] method:

[0138] After the plant samples obtained from the above experiment were rinsed with sterile water, the surface moisture was absorbed by filter paper and placed in a mortar, and liquid nitrogen was added for grinding. After grinding, 0.1 g of plant samples were weighed and homogenized in 1 mL of extract solution in an ice bath, and then the tissue homogenate was poured into a 2 mL centrifuge tube and centrifuged at 8000 g for 10 min at 4 ° C. The samples were tested for plant proline, total soluble sugar, malondialdehyde content, superoxide dismutase, and peroxidase activity using BC0290 PRO, BC0030 TSS, BC0025 MDA, BC0170 SOD, and BC0090 POD kits (Solarbio, Beijing, China) according to the kit instructions.

[0139] result:

[0140] Chlorophyll content is an important indicator of plant health and photosynthetic capacity under salt stress. The chlorophyll content in the leaves of Yazhou hard bean plants was detected to explore the effect of PG-4 on the photosynthetic capacity of plants under salt stress. Fig.13 As shown in Figure 2, under salt stress, the chlorophyll content of Yazhou hard bean was significantly reduced (P < 0.05). However, under salt stress or no salt stress, the chlorophyll content of PG-4 inoculated was significantly higher than that of the uninoculated control group ( Fig.13 , A). In addition, the TSS content of plants inoculated with PG-4 under salt stress also increased significantly compared with the control group ( Fig.13 , B).

[0141] Salt stress caused a significant increase in the MDA content in leaves (P < 0.05). However, under salt stress, inoculation with PG-4 strain significantly reduced the MDA content in leaves of C. yam compared with the uninoculated control plants ( Fig.13 , C). This result shows that PG-4 inoculation can effectively alleviate the oxidative stress damage of plants under salt stress, thereby improving the salt tolerance of plants. In addition, the study found that PG-4 inoculation increased the POD activity of plant leaves under salt stress, among which the POD activity of leaves treated with BS was the highest ( Fig.13 , D).

[0142] Analysis of the sodium and potassium ion content in plant leaves showed that under salt stress, the plant's absorption of Na+ increased significantly, while reducing the absorption of K+. However, under salt stress, PG-4 inoculation treatment significantly (P<0.05) increased the accumulation of potassium in plant leaves, while reducing the sodium ion content ( Fig.13 , E and F). These results indicate that under salt stress, inoculation with PG-4 has a positive effect on plant potassium absorption and sodium excretion.

[0143] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of Bacillus amyloliquefaciens or a bacterial agent containing Bacillus amyloliquefaciens in improving the salt stress tolerance of Scleroderma plants; The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242154.

2. The use according to claim 1, characterized in that: The plants of the genus genus Ceratonia include Ceratonia serrata.

3. Application of Bacillus amyloliquefaciens or a bacterial agent containing Bacillus amyloliquefaciens in promoting seed germination of Scleroderma plants under salt stress; The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242154.

4. The use according to claim 3, characterized in that: The plants of the genus genus Ceratonia include Ceratonia serrata.

5. Application of Bacillus amyloliquefaciens or a bacterial agent containing Bacillus amyloliquefaciens in promoting the growth of Scleroderma plants at the seedling stage under salt stress; The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242154.

6. Use of Bacillus amyloliquefaciens or a bacterial agent containing Bacillus amyloliquefaciens in any one of (1) to (5); (1) Improving the seedling length of sclerocarpon plants under salt stress; (2) increasing root length of scleroderma plants under salt stress; (3) increasing the fresh weight of sclerocarpon plants under salt stress; (4) increasing the dry weight of Bean plants under salt stress; (5) Improving the germination rate of seeds of the genus Pseudocarpus under salt stress; The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242154.

7. Use of Bacillus amyloliquefaciens or a bacterial agent containing Bacillus amyloliquefaciens in any one of (1) to (3); (1) Producing pectinase; (2) Producing starch hydrolyzing enzymes; (3) Protease production; The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242154.

8. A salt-tolerant growth promoter, characterized in that Including Bacillus amyloliquefaciens or a bacterial preparation containing Bacillus amyloliquefaciens; The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242154.

9. The salt-tolerant growth promoter according to claim 8, characterized in that The preparation method thereof comprises: Inoculate Bacillus amyloliquefaciens or a bacterial culture containing Bacillus amyloliquefaciens into LB liquid medium and culture at 28°C and 200 r / min until OD 600 is 0.

6.

10. A method for improving the growth and salt tolerance of scleroderma plants at the seedling stage, characterized in that: Treating seeds or seedling roots of plants of the genus Sclerotium with Bacillus amyloliquefaciens or a bacterial agent containing Bacillus amyloliquefaciens, or the salt-tolerant growth-promoting agent according to claim 8 or 9, and then sowing; The Bacillus amyloliquefaciens is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20242154.

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