Bacillus amyloliquefaciens and application thereof in promoting growth of plants in salinized land

Abstract

The invention discloses bacillus amyloliquefaciens and application of the bacillus amyloliquefaciens in promoting growth of plants in salinized land. The strain is preserved in Guangdong Microbial Culture Collection Center on June 24, 2025, and the preservation number is GDMCC NO: 67014. The bacillus amyloliquefaciens R1 obtained by screening is a high-yield IAA (Indoleacetic Acid), is a bacterial strain capable of remarkably promoting the growth of plants in the salinized land, and has good development potential and application value for promoting the growth of the plants in the salinized land. The problem that in the prior art, microbial strains with the rhizosphere growth-promoting and saline-alkaline-tolerant functions are lacked is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural microorganism technology, and in particular to a Bacillus amyloliquefaciens and its application in promoting the growth of plants in saline land. BACKGROUND

[0002] The problem of salinization significantly reduces the effective utilization rate of land, leading to large-scale farmland abandonment; farmland planting is mostly carried out by flooding irrigation and irrigation without drainage, and the overuse of chemical fertilizers and pesticides further promotes the increasing salinization of soil, thereby seriously restricting the development of regional agricultural productivity and affecting the balance of social economic structure and ecological safety. Containing the continuous expansion of saline land and improving its ecological function have become one of the core tasks to promote the sustainable development of agriculture.

[0003] Using biological methods to relieve salt stress and improve crop yield is an important way to develop green agriculture. Microbial measures for salinization land improvement are to improve the growth potential and induce salt tolerance of plants on the premise of respecting the original genetic background of plants. Plant growth-promoting rhizobacteria (PGPR) are microorganisms that grow in the rhizosphere of plants and can secrete special bioactive substances, which promote plant growth and improve stress resistance through direct mechanisms (phosphorus and potassium release, IAA (indole acetic acid) synthesis) and indirect mechanisms (osmotic regulation, antioxidant defense, etc.). Among them, IAA-type PGPR bacteria are a kind of efficient growth-promoting bacteria, which have the advantages of low cost, high efficiency, quick effect and environmental protection in improving the rhizosphere microenvironment of crops and further improving saline-alkali land. At present, the research and development of rhizosphere growth-promoting salt-tolerant functional microorganisms have the problems of low salt-tolerant ability of the strains, more application of composite microbial agents, and complex process. Therefore, it is of great social significance and economic value to screen beneficial microorganisms with better salt-tolerant ability, develop and prepare efficient microbial agents with simple process for plant growth promotion and saline-alkali land improvement. SUMMARY

[0004] To solve the above technical problems, the purpose of the present application is to provide a Bacillus amyloliquefaciens and its application in promoting the growth of plants in saline land, so as to solve the problem of lack of rhizosphere growth-promoting salt-tolerant functional microbial strains in the prior art.

[0005] The technical scheme for solving the above technical problems of the present application is as follows: a Bacillus amyloliquefaciens is provided. Bacillus amyloliquefaciens The strain belongs to the genus Bacillus, and is named R1. The strain was preserved in the Guangdong Microbial Culture Collection Center on June 24, 2025, with the preservation number GDMCC NO: 67014 and the address of the preservation unit being No. 100, Martyrs Road, Guangzhou, China.

[0006] On the basis of the above technical scheme, the present application can be further improved as follows: Further, the 16S rRNA gene sequence of Bacillus amyloliquefaciens is shown as SEQ ID No: 1.

[0007] Further, when Bacillus amyloliquefaciens R1 grows on R2A medium, the colony is milky white, and the edge is smooth without wrinkles.

[0008] The application also provides the use of the above-mentioned Bacillus amyloliquefaciens in promoting the growth of plants in saline land.

[0009] Further, the temperature tolerance range of Bacillus amyloliquefaciens is 10-40℃, the NaCl tolerance range is 0-11 w / v%, the pH value tolerance range is 6-10, and the 24h IAA yield is more than 30μg / mL.

[0010] Further, the application includes promoting the growth of plant seedling roots and stems.

[0011] The application also provides the use of the above-mentioned Bacillus amyloliquefaciens in preparing an indole-3-acetic acid product.

[0012] The application also provides a plant growth promoting agent for saline land, which comprises the above-mentioned Bacillus amyloliquefaciens. Further, the Bacillus amyloliquefaciens is a fermentation strain or fermentation product of the strain.

[0013] The application has the following beneficial effects: 1. The Bacillus amyloliquefaciens R1 of the application has strong environmental tolerance and can grow in a saline-alkali environment with a salt concentration of ≤11 w / v% and a pH value of 10.0; the 24h IAA yield is more than 30μg / mL, which is a high-IAA-yield bacterial strain that can significantly promote the growth of plants in saline land. The potting experiment application example shows that, after being treated with the strain R1 agent, the potting wheat seedling rate under heavy salt stress is increased by more than 25%, the wheat plant height increase rate is 75.48%, the root length growth rate is 61.31%, and the wheat fresh weight increase is 17.00%, compared with the control group.

[0014] 2. The strain R1 of the application has simple culture conditions, is easy to preserve, has good growth-promoting efficiency, has higher salt-tolerant growth-promoting performance (>7g / kg), can be prepared into a single agent, has a simplified process, and can solve salt stress in a targeted manner. Therefore, the strain R1 of the application has good development potential and application value in promoting the growth of plants in saline land. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The figure is a growth-promoting effect diagram of the strain R1 in a potting experiment for strain rescreening; Figure 2 The figure is a culture characteristic of the strain R1 on R2A medium; Figure 3A phylogenetic tree of strain R1; Figure 4 A IAA production curve of strain R1; Figure 5 A pot-grown wheat growth effect diagram after applying R1 microbial agent; Figure 6 A pot-grown wheat seedling rate result diagram after applying R1 microbial agent; Figure 7 A pot-grown wheat plant height result diagram after applying R1 microbial agent; Figure 8 A pot-grown wheat root length result diagram after applying R1 microbial agent; Figure 9 A pot-grown wheat fresh weight result diagram after applying R1 microbial agent; Figure 10 A pot-grown wheat chlorophyll content result diagram after applying R1 microbial agent; Figure 11 A pot-grown wheat malondialdehyde content result diagram after applying R1 microbial agent. DETAILED DESCRIPTION

[0016] The principles and features of the present application are described below in conjunction with the accompanying drawings, and the examples are only used to explain the present application and are not intended to limit the scope of the present application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be purchased on the market.

[0017] Example 1: Screening and identification of strain R1 I. Isolation of strains The method for screening the salt-tolerant growth-promoting bacterial strain R1 of the present application is as follows.

[0018] 1. Collection of soil samples Five soil samples were randomly collected from farmland in Wuyuan County, Bayannur City, Inner Mongolia Autonomous Region (41.08 °N, 108.41 °E) at a sampling depth of 0-20 cm, labeled and taken back to the laboratory.

[0019] 2. Isolation and purification of strains Prepare TSA medium (Tryptone 15 g, Soybean Peptone 5 g, Sodium Chloride 5 g, Agar 20 g, Distilled Water 1000 mL), Beef Extract Peptone medium (Beef Extract 3.0 g, Peptone 10.0 g, NaCl 5.0 g, Agar 20 g, Distilled Water 1000 mL), LB medium (Tryptone 10.0 g, Yeast Extract 5.0 g, NaCl 10.0 g, Agar 20 g, Distilled Water 1000 mL), R2A medium (Yeast Extract 0.5 g, Peptone 0.5 g, Casein Hydrolysate 0.5 g, Glucose 0.5 g, Soluble Starch 0.5 g, Potassium Dihydrogen Phosphate 0.3 g, Magnesium Sulfate Anhydrous 0.024 g, Sodium Pyruvate 0.3 g, Agar 20 g, Distilled Water 1000 mL), adjust the pH and salt content of the medium to the same as the soil sample, prepare the culture plate for standby.

[0020] Measure 45 mL of normal saline in a 100 mL conical flask, add dozens of glass beads, seal with sealing film, and then steam sterilize at 121°C for 20 min. Weigh 5 g of treated soil sample into the sterilized normal saline conical flask, shake in a shaking table at 28°C and 160 rpm for 30 min. Gradient dilute the soil suspension in a clean bench to obtain 10 -3 , 10 -4 , 10 -5 gradient dilutions in turn. Use the coating method to evenly coat 100 µL of the diluent on the above prepared medium, set three parallels for each dilution, and place in a 28°C constant temperature incubator for 3-5 d. Obtain single colony strains, then multiple streak inoculation on the corresponding medium plate to obtain purified strains.

[0021] 3. Screening of IAA-producing strains Prepare TSA liquid medium, beef extract peptone liquid medium, LB liquid medium and R2A liquid medium, divide into test tubes, 9 mL per tube, after steam sterilization, add 1 mL of 0.1% tryptophan solution by filtering sterilization. Use a loop to pick single colonies from TSA, beef extract peptone, LB and R2A medium plates into the corresponding liquid medium, and place in a shaking table (28°C, 160 rpm) for 5 d. Use Salkowski method to determine IAA content, take 1 mL of bacterial liquid into a 1.5 mL centrifuge tube, centrifuge at 10000 rpm for 10 min, take 0.5 mL of supernatant, add 2 mL of Salkowski's colorimetric solution, color for 30 min in the dark, and then colorimetric at 530 nm with a spectrophotometer. Select strains with OD 530 values above 0.3, i.e. high IAA-producing strains with IAA production above 30 µg / mL, use a loop to pick and plate streak culture, continuously transfer for 2 times, observe and record, and obtain high IAA-producing strains.

[0022] 4. Re-screening of salt-tolerant growth-promoting bacteria (pot experiment) The wheat variety used in the experiment was Zhongmai 86. The seeds were screened by removing blackened and damaged grains. The seeds were soaked in 70% ethanol for 5 min, then in 2% (w / v) sodium hypochlorite solution for 2 min, and then rinsed with distilled water. The seeds were then soaked in warm water at 28°C for 6 h. Filter paper was used to absorb the water and placed in a petri dish. Three pieces of filter paper were placed in each dish to create a paper bed. The wheat seeds were placed evenly in the paper bed, leaving a certain distance between them. The petri dishes were placed in a 28°C incubator for 24 h.

[0023] The initial screening of the bacterial strain was inoculated into beef extract peptone liquid medium and placed in a shaking bed (28°C, 160 rpm) for 48 h. The OD value of the bacterial suspension was measured by spectrophotometry. The bacterial suspension was diluted to an OD value of 0.6 with the culture medium. 600 600

[0024] The experimental soil was sieved through a 2 mm sieve to remove plant roots, rocks, and other debris. A portion of the treated soil was dried in an electric drying oven at 80°C for about 6 h to a constant weight. 5.0 g of the dried soil was placed in a dry 100 mL conical flask, 45.0 mL of boiled distilled water was added, and the flask was sealed and placed in a shaking bed at 160 rpm for 1 h. The soil pH was measured using a pH meter (FE20), and the conductivity of the supernatant was measured using a conductivity meter (DDS-307). The salt content of the experimental soil was calculated. Based on the original salt content of the soil, the salt content was adjusted to 3.2 g / kg, 5.2 g / kg, and 7.5 g / kg by adding sodium chloride solution, respectively, and the treatments were designated as light salt, medium salt, and heavy salt, respectively. Each treatment had three replicates and one blank. The flower pots used had a mouth diameter of 7 cm, a base diameter of 5 cm, and a height of 8 cm, and the soil capacity was 150 g. 120 g of the prepared soil was placed in the flower pots, and each pot was watered with 20 mL of water. The germinated wheat seeds were placed on the soil in the flower pots, with 10 seeds per pot. 10 mL of diluted bacterial suspension (3.6 x 10 8 cfu) was added to each pot, and 10 mL of liquid medium was added to the blank group. A thin layer of soil was added on top of the wheat seeds, and the pots were sprayed with an appropriate amount of tap water every day. After 21 days of incubation at 26-28°C, the bacterial strains that promoted the growth of wheat seeds in different salt concentrations were selected as salt-tolerant growth-promoting bacteria. The growth-promoting effect of R1 in the bacterial strain re-screening pot experiment is shown in Figure 1 .

[0025] II. Bacterial strain identification 1. Culture morphological characteristics ​​The morphological structure of the bacteria was observed under a microscope. Strain R1 was a gram-positive bacterium, the cells were short rods, generally single, spore-forming, facultative anaerobic, and the colonies on R2A medium plates cultured at 28°C for 4 days were round, convex, smooth in surface, with a relatively neat edge, opaque ivory in color, moist and delicate in texture, and easy to pick up. The characteristics of the plate culture were as follows Figure 2 .

[0026] 2. Physiological and biochemical tests After the purified strain was activated and cultured, physiological and biochemical tests were performed, and the genus and species of R1 were preliminarily identified according to the Bergey's Manual of Determinative Bacteriology.

[0027] The results showed that R1 could hydrolyze starch and gelatin, and the utilization tests of glucose, sucrose, maltose and mannose were positive, while the utilization tests of lactose, xylose, rhamnose and galactose were negative; the contact enzyme test, V-P test, nitrate reduction test and citrate utilization test were positive, and R1 could be preliminarily classified into the genus Bacillus.

[0028] 3. 16S rRNA gene sequence identification The 16S rRNA gene sequence of the high-IAA-producing strain R1 was determined to determine its genus and species.

[0029] The total DNA of the strain was extracted by a DNA kit, and the 16S rDNA was amplified by using universal primers. The forward primer Primer A was 27f: 5'-AGAGTTTGATCCTGGCTCAG-3', and the reverse primer Primer B was 1525r: 5'-AGAAAGGAGGTGATCCAGCC-3'.

[0030] The PCR amplification system was as follows: Primer A 1.0 μL; Primer B 1.0 μL; DNA template 2.0 μL; ddH2O 21 μL; 2×Mastermix 25 μL.

[0031] The PCR reaction condition parameters were as follows: 94°C, 3 min, 1 cycle; 94°C, 1 min, 55°C, 1 min, 72°C, 3 min, 30 cycles; 72°C, 5 min, 1 cycle.

[0032] The PCR product obtained by amplification was subjected to 16S rRNA gene sequence determination by Huada Gene Sequencing Company (Beijing Genomics Institute), and the sequence was 1472 bp. The gene sequence is shown in SEQ ID No: 1.

[0033] The 16S rRNA gene sequences of the strains obtained from sequencing were used for similarity searches on the EzBioCloud server (www.ezbiocloud.net). Using MEGA 11 software, the Neighbor-Joining (NJ) method was selected, and a two-parameter model was used to calculate the evolutionary distance. A bootstrap value of 1000 replications was chosen to construct a phylogenetic tree of the 16S rRNA gene sequences. Figure 3 ).

[0034] Salt-tolerant growth-promoting strain R1 and Bacillus amyloliquefaciens Located on the same subbranch, it is the most similar species to strain R1 in the 16S rRNA gene sequence determination results. Bacillus amyloliquefaciens Consistent (99.82% similarity).

[0035] Based on the comprehensive morphological characteristics, physiological and biochemical tests, and 16S rRNA gene sequence analysis, strain R1 was identified as Bacillus amyloliquefaciens (Bacillus). Bacillus amyloliquefaciens ).

[0036] 4. Measurement of the ability to produce alpha-1,4-acetylene (IAA) To prepare a standard curve for IAA concentration: Prepare a 100 μg / mL stock solution of IAA, and prepare IAA standard solutions of 0, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. Each group should be tested in triplicate, and the determination should be performed using the Salkowski method. Pipette 0.5 mL of the test solution, add 2 mL of Salkowski's colorimetric solution, and develop the color for 30 min under dark conditions. Then, quickly measure the color using a spectrophotometer (530 nm). Plot the statistical data to create a standard curve.

[0037] Seed culture of strain R1 was inoculated at a rate of 1% (v / v) into liquid medium containing 100 mg / L tryptophan and cultured at 28°C for 2 days. Each experimental group was run in triplicate, with a blank control consisting of uninoculated culture medium. Culture medium was collected every 4 hours and centrifuged at 10,000 rpm for 10 min. 0.5 mL of the supernatant was collected, and 2 mL of Alkowski's colorimetric solution was added. After developing the color under dark conditions for 30 min, the colorimetric result was quickly measured using a spectrophotometer (530 nm). An IAA yield curve was plotted. Figure 4 Strain R1 produced over 30 μg / mL of IAA at 24 h and 38.44 μg / mL at 48 h, making it a high-IAA-producing bacterial strain.

[0038] 5. Determination of R1 growth conditions (1) Temperature tolerance test Strain R1 was inoculated on beef extract peptone solid medium and placed in constant temperature incubators at 0°C, 4°C, 10°C, 20°C, 25°C, 28°C, 30°C, 37°C, 40°C, and 45°C. The growth of the strain was observed. At 0°C, 4°C, and 10°C, the strain grew slowly and the growth cycle was relatively long. Therefore, the growth of the strain was observed every 2 or 4 weeks at 0°C, 4°C, and 10°C. The growth of the strain was recorded at 7d and 14d at other temperatures. The results showed that the growth temperature tolerance range of R1 was 10-40°C.

[0039] (2) NaCl tolerance experiment 40mL of liquid culture medium was prepared in a 100mL triangular flask and sterilized. After cooling, it was ready for use. A single colony of the strain was picked and placed in beef extract peptone liquid medium. The seed liquid was prepared by placing it in a shaking bed (28°C, 160rpm) for 48h. The OD value of the bacterial suspension was determined by spectrophotometry at 600nm. The OD600 value of the bacterial liquid was adjusted to 0.6. The inoculation amount was 1% (v / v). The inoculum was transferred to beef extract peptone liquid medium with salt content of 0%, 1%, 3%, 5%, 7%, 9%, 11%, 13%, and 15% (all w / v). Three parallel experimental groups were set up, and the medium without inoculation was set as the control group. The culture was placed in a constant temperature incubator at 28°C and shaken. After 3d of culture, the light absorption value of the bacterial suspension at 600nm was determined to determine the NaCl tolerance range of the strain. The results showed that the NaCl tolerance range of R1 was 0-11w / v% (0-11g / 100mL).

[0040] (3) pH tolerance experiment 40mL of beef extract peptone liquid medium was prepared in a 100mL triangular flask and sterilized. After cooling, it was ready for use. A single colony of R1 was picked and placed in beef extract peptone liquid medium. The seed liquid was prepared by placing it in a shaking bed (28°C, 160rpm) for 48h. The OD value of the bacterial suspension was determined by spectrophotometry at 600nm. The OD value was adjusted to 0.6. The inoculation amount was 1% (v / v). The inoculum was transferred to beef extract peptone liquid medium with pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0. Three parallel experimental groups were set up, and the medium without inoculation was set as the control group. The culture was placed in a constant temperature incubator at 28°C and shaken. After 3d of culture, the light absorption value of the bacterial suspension at 600nm was determined and analyzed. The results showed that the pH tolerance range of R1 was 6.0-10.0.

[0041] Example 2: Application of R1 bacterial agent Strain R1 was applied to pot-grown wheat under different salt stress soil conditions. The pot experiment method was consistent with that of Example 1.

[0042] Take 5 mL of bacterial suspension and add 45 mL of sterile normal saline. Gradient dilution is obtained in turn 10 -4 , 10 -5 , 10 -6 Gradient dilution. Using the coating method, 100 µL of the diluent is evenly coated on the beef extract protein peptone plate, and three parallel plates are set under each dilution, and placed at 28°C for 3-5 days. The plates with 20-300 bacterial colony counts are used as the counting standard to count the number of viable bacteria (cfu). The fermented liquid with cfu≥2×10 8 of the national standard for agricultural microbial inoculants (GB 20287-2006) is used to prepare R1 inoculant.

[0043] R1 inoculant is applied to the potting soil. The experimental soil is divided into light salt group, medium salt group, and heavy salt group (the soil salt content is consistent with the complex screening potting experiment of salt-tolerant growth-promoting bacteria), and three parallel treatments are set for different salt concentrations. After 14 days of incubation at 26-28°C, the agronomic traits and physicochemical parameters of wheat are determined.

[0044] The number of surviving wheat plants is counted, and the seedling rate is calculated. The height and root length of the wheat are measured using a ruler. The fresh weight of the wheat is measured using a balance.

[0045] Chlorophyll content determination: Mix anhydrous ethanol and acetone in a volume ratio of 1:2 to prepare the extraction solution. Take about 0.1 g of wheat leaves, accurately weigh, and place in a mortar. Use scissors to cut into small pieces, add 1 mL of distilled water and a small amount of calcium carbonate, and grind thoroughly in the dark. Rinse the mortar with the extraction solution, and pour the wash into the 5 mL volumetric flask. Make up to 5 mL. Extract for about 3 hours in the dark until the wheat leaf residue is white. Centrifuge the extract at 4000 rpm for 10 min, and take the supernatant for measurement. Adjust the spectrophotometer to zero with the extraction solution, and measure the OD values at 645 nm and 663 nm, respectively. Calculate the chlorophyll content in the wheat leaf plant tissue according to the following formula: Chlorophyll content = (20.21A 645 + 8.02A 663 ) × extraction solution volume / sample mass / 1000 MDA content determination: Use the MDA kit to determine the MDA content in the wheat leaves, and select the test solution for the experiment. Adjust the spectrophotometer to zero with distilled water, and measure the absorbance value at 532 nm with a 1 cm light path. Calculate the MDA content according to the kit instructions.

[0046] The growth effect of pot-grown wheat after applying R1 inoculant to the soil is as follows Figure 5 , The seedling rate of wheat is as follows Figure 6 , The plant height results of the wheat are as follows Figure 7 , The root length results of the wheat are as follows Figure 8 , The fresh weight weighing results of the wheat are as follows Figure 9 , The chlorophyll content results of the wheat are as follows Figure 10 , The malondialdehyde content of the wheat is as follows Figure 11 .

[0047] The results show that the R1 microbial agent treatment of the light salt group has no significant difference (seedling rate, plant height, root length, fresh weight, chlorophyll content) compared with the control group; Under the medium salt stress, after applying the microbial agent R1, the seedling rate, plant height, root length and fresh weight of the wheat are increased by 18.19%, 63.31%, 24.58% and 49.12% respectively, the chlorophyll content is increased by 43.73%, and the malondialdehyde content is reduced by 44.15%; Under the heavy salt stress, the seedling rate of the potted wheat is increased by 25.01% compared with the control group, the plant height of the potted wheat is increased by 75.48%, the root length is increased by 61.31%, the fresh weight of the wheat is increased by 17.00%, the chlorophyll content is increased by 75.61%, and the malondialdehyde content is reduced by 27.95%.

[0048] Therefore, under the medium salt and heavy salt stress, the microbial agent R1 treatment has a significant growth-promoting effect on the potted wheat, can effectively increase the chlorophyll content of the wheat, improve the photosynthetic capacity of the wheat, and effectively prevent the damage of the plant membrane.

[0049] The above only describes the preferred embodiments of the present application, and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A type of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens This strain belongs to the genus Bacillus, named R1, and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 24, 2025, with accession number GDMCC NO: 67014.

2. The Bacillus amyloliquefaciens according to claim 1, characterized in that, The Bacillus amyloliquefaciens has a colony with a milky white color and a smooth edge when grown on R2A medium.

3. The Bacillus amyloliquefaciens of claim 1 or 2 for use in promoting the growth of plants in saline land.

4. Use according to claim 3, characterized in that, The Bacillus amyloliquefaciens has a temperature tolerance range of 10-40℃, a NaCl tolerance range of 0-11 w / v%, a pH tolerance range of 6-10, and a 24h IAA yield of ≥30μg / mL.

5. Use according to claim 3, characterized in that, The use includes promoting the root and stem growth of plant seedlings.

6. The Bacillus amyloliquefaciens of claim 1 or 2 for use in the preparation of an indole-3-acetic acid product.

7. A microbial inoculum for promoting plant growth in saline soils, characterized in that, The Bacillus amyloliquefaciens of claim 1 or 2.

8. The growth promoting agent for plants in saline soil according to claim 7, characterized by, The Bacillus amyloliquefaciens is a fermentation strain or a fermentation product of the strain.

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