Plant rhizosphere growth-promoting bacteria capable of effectively improving alfalfa growth and application thereof

Abstract

The present invention belongs to the field of agricultural microbial application technology and relates to plant rhizosphere growth-promoting bacteria and their applications that can effectively increase alfalfa growth. The rhizosphere growth-promoting bacteria is Burkholderia MCWP-A3-15. The Burkholderia MCWP-A3-15 is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 31562. The Burkholderia of the present invention can promote alfalfa growth, increase alfalfa yield, improve soil physical and chemical properties, and increase soil fertility.

Description

Technical Field

[0001] The present invention belongs to the technical field of application of agricultural microorganisms, and is mainly directed to plant rhizosphere growth-promoting bacteria, specifically Burkholderia and its use in regulating alfalfa growth and improving soil physical and chemical properties. Background Art

[0002] The Inner Mongolia Autonomous Region is one of my country's major alfalfa production bases. With the development of modern animal husbandry, strong national policy support, and the rapid growth of the dairy industry, alfalfa has gradually become an irreplaceable and reliable forage. However, although alfalfa is a salt- and alkali-tolerant crop, the amount of nutrients directly available to it in the soil decreases significantly with increasing soil salinization, still insufficient to meet its growth needs. Plant growth-promoting rhizobacteria can promote plant growth under salinity and alkali stress through various mechanisms. Therefore, screening and identifying plant growth-promoting rhizobacteria in Inner Mongolia has great potential and research value for maintaining soil productivity and promoting plant growth.

[0003] Rhizobacteria can significantly improve alfalfa's nutrient acquisition efficiency. Some bacteria, such as Rhizobium meliloti, possess the ability to fix nitrogen. These bacteria, like Rhizobium medicagogue, invade alfalfa's roots, forming nodules that convert nitrogen from the air into ammoniacal nitrogen that the plant can use. This provides a stable and inexpensive nitrogen source for alfalfa growth, effectively promoting lush stems and leaves—a key factor in high alfalfa yields. Furthermore, many growth-promoting bacteria secrete organic acids, such as oxalic and citric acid, which dissolve insoluble phosphorus and potassium minerals in the soil, releasing these elements for alfalfa to absorb. Adequate phosphorus promotes alfalfa's well-developed root system, enhances stress resistance, and promotes flower bud differentiation and seed formation. Potassium ensures strong stems, improves photosynthesis efficiency, and reduces the risk of lodging. These two factors work together to boost alfalfa yields. Rhizobacteria help alfalfa thrive in adverse environments such as drought, high temperatures, and salinity. When faced with drought, growth-promoting bacteria encourage alfalfa to synthesize more osmotic regulating substances such as proline and betaine, reducing intracellular osmotic pressure and helping the plant to absorb water from the dry soil. This helps maintain cell turgor and ensures the normal functioning of physiological processes such as photosynthesis, preventing drought-induced growth stagnation or even death, and maintaining stable yields. In saline-alkali soils, rhizosphere growth-promoting bacteria secrete special substances to regulate the pH of the rhizosphere soil, reducing the toxicity of salt and alkali to alfalfa roots. This also enhances the root system's ability to reject salt, allowing alfalfa to take root and grow in saline-alkali soils, expanding the range of alfalfa cultivation and indirectly opening up new avenues for increasing overall yield. In summary, rhizosphere growth-promoting bacteria have a comprehensive impact on alfalfa growth and development, from nutrient supply, hormone regulation, disease prevention, to stress resistance assistance, playing a significant role in promoting alfalfa yield. Rational utilization of rhizosphere growth-promoting bacteria is expected to become a key strategy for high-yield alfalfa cultivation. Summary of the Invention

[0004] The present invention provides a Burkholderia MCWP-A3-15 strain. The strain provided by the present invention was collected from a Leymus chinensis and alfalfa cultivation area in Inner Mongolia. Rhizosphere soil was first excavated from the Leymus chinensis and alfalfa cultivation area for isolation, purification, and 16S rDNA identification and classification. Further indoor preliminary screening and field rescreening were performed. By measuring indices such as alfalfa plant height, root length, aboveground biomass, and soil physical and chemical properties after inoculation, strains with significant alfalfa yield-increasing effects were analyzed and selected, providing a basis for high-yield alfalfa cultivation.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The invention provides a plant rhizosphere growth-promoting bacterium capable of effectively improving the growth of alfalfa. The rhizosphere growth-promoting bacterium is Burkholderia MCWP-A3-15.

[0007] The Burkholderia MCWP-A3-15 ( Burkholderia sp. ), deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No. 31562, the deposit date is August 12, 2024, and the deposit address is No. 3, Yard 1, Beichen Road, Chaoyang District, Beijing.

[0008] The present invention also provides the use of the Burkholderia MCWP-A3-15 in promoting the growth of alfalfa. The Burkholderia MCWP-A3-15 can increase the plant height, root length and dry weight of alfalfa.

[0009] The present invention also provides application of the Burkholderia MCWP-A3-15 in improving alfalfa yield.

[0010] The present invention also provides the use of the Burkholderia MCWP-A3-15 in improving the physical and chemical properties of soil, wherein the physical and chemical properties include total nitrogen content and available phosphorus content.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] 1. The plant rhizosphere growth-promoting bacteria strain of the present invention is an indigenous rhizosphere growth-promoting bacteria collected and separated from the Leymus chinensis and alfalfa planting areas in the eastern part of Inner Mongolia. It is low-cost and pollution-free.

[0013] 2. The present invention screened and identified plant rhizosphere growth-promoting bacteria, and verified in pot and field experiments that Burkholderia can effectively increase the growth of alfalfa.

[0014] 3. The strain obtained by the present invention can effectively improve the physical and chemical properties of soil.

[0015] In summary, the Burkholderia MCWP-A3-15 provided by the present invention can promote alfalfa growth, increase alfalfa yield, and improve soil physical and chemical properties, significantly increase the nitrogen, phosphorus and potassium contents in the soil, and increase soil fertility. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Neighbor-joining phylogenetic tree of the 16S rDNA gene sequence of Burkholderia MCWP-A3-15.

[0017] Figure 2 : Effects of inoculation with different rhizospheric growth-promoting bacterial strains on alfalfa plant height and root length under sterile conditions.

[0018] Figure 3 : Effects of inoculation of different rhizosphere growth-promoting bacterial strains on dry weight of alfalfa plant under sterile conditions.

[0019] Figure 4 : Effects of inoculation of different rhizospheric growth-promoting bacteria on plant height and root length of alfalfa in field.

[0020] Figure 5 : Effects of inoculation of different rhizospheric growth-promoting bacteria on dry weight of alfalfa plant in field.

[0021] Figure 6 : Effects of rhizosphere growth-promoting bacteria on soil pH.

[0022] Figure 7 : Effects of rhizospheric growth-promoting bacteria on soil organic matter content.

[0023] Figure 8 : Effects of rhizospheric growth-promoting bacteria on soil total nitrogen content.

[0024] Figure 9 : Effects of rhizosphere growth-promoting bacteria on available phosphorus content in soil.

[0025] In the figure, abcde represents P The difference was significant at the <0.05 level. DETAILED DESCRIPTION

[0026] In order to make the purpose and advantages of the present invention more clear, the following is a complete description of the specific embodiments of the present invention in conjunction with the examples. Unless otherwise specified, the experimental methods described in the examples of the present invention are all conventional methods.

[0027] Example 1. Isolation, screening and identification of strains

[0028] 1. Sample collection

[0029] Plant rhizosphere soil was collected from alfalfa and leymus planting areas in eastern Inner Mongolia. The five-point sampling method was used during the initial flowering period of the plants. Tall and healthy plants were selected, and the above-ground parts of the plants were removed. The roots and root soil were placed in a sterile bag and placed in a foam box with ice packs. The samples were processed within 24 hours.

[0030] 2. Bacteria isolation

[0031] Bring the plant rhizosphere soil back to the laboratory, gently shake off the soil attached to the roots, weigh 10 g of the roots with a small amount of soil, and place them in a 90 mL sterile saline flask. Place 5 sterilized glass beads in the flask, shake on a shaker for 30 minutes (180 rpm), and then let it stand for 10 minutes. Next, prepare a sterile test tube and add 9 mL of saline. Add 1 mL of soil suspension to it to prepare 10 mL of saline. -2 Soil gradient dilution solution; then draw 1 mL of suspension from the test tube in the previous step and add it to another 9 mL test tube of the same size, repeat this process, and prepare 10 -3 , 10 -4 and 10 -5 Serial dilutions.

[0032] The prepared gradient dilution solution was spread on LB solid medium and streaked three times, and cultured at 37°C for 24 hours. Single colonies with obvious differences in colony morphology were selected for further screening.

[0033] Preparation of LB solid medium: 5 g yeast extract, 10 g tryptone, 10 g NaCl, 15 g agar powder, 1000 ml dH2O, and adjust the pH to 7 in a 1000 ml reagent bottle.

[0034] 3. Purification of strains

[0035] Purify using the plate streak method. Dip a small amount of bacterial solution into an inoculating loop and begin streaking along one edge of the plate. Hold the loop at a 30-40 degree angle to the plate surface, gently touch the plate, and streak with appropriate speed and force, keeping the initial streak as small as possible. Sterilize the loop by calcining it. After cooling, begin a second streak from the end of the first streak, partially overlapping the lines. Repeat this process three to four times for each gradient. Once all contaminants are clear, begin storage and identification.

[0036] 4. Identification of strains

[0037] DNA extraction: A bacterial genomic DNA extraction kit (brand: Tiangen; product number: DP302) was used to extract strain DNA.

[0038] The obtained bacterial DNA was used as a template to amplify the 16S rDNA sequence using the universal primer pair 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO. 1) / 1492R (5'-TACGGTTACCTTGTTACGACTT-3, SEQ ID NO. 2) and detected by electrophoresis.

[0039] PCR reaction system: 20 μL: DNA template 1 μL, 2× EasyTaq ® 10 μL of PCR SuperMix, 1 μL of upstream and downstream primers, and ddH2O to make up to 7 μL.

[0040] Amplification program: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s; annealing at 54 °C for 30 s, extension at 72 °C for 1 min 30 s; extension at 72 °C for 10 min; 35 cycles.

[0041] PCR products were sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for 16S rDNA gene sequencing. Successfully sequenced sequences were concatenated into a single sequence, and txt and FASTA files were created for Blast homology search and alignment. A phylogenetic tree was then constructed using the Neighbor-Joining method in MEGA (10.2.4) software to determine the phylogenetic status of the strain.

[0042] Table 1 Taxonomic status of 8 strains

[0043]

[0044] Finally, 8 excellent strains from 3 genera were isolated (Table 1). Sinorhizobium fredii strain DB23-1 has the highest similarity, reaching 99%; strain number 16S-GU-C6-12 has the highest similarity with strain Sinorhizobium sp. XGL154 has the highest similarity, reaching 99%; both are Sinorhizobium ( Sinorhizobium ). Strain numbers 16S-WP-A3-17 and 16S-YP-A3-7 are similar to strains Burkholderia sp.YP-AN-3 has the highest similarity, reaching 99%; strain number MCWP-A3-15 has the highest similarity with strain Burkholderia sp.DCY113 has the highest similarity, reaching 99%; both are Burkholderia ( Burkholderia ). Strain number 16S-YP-2 and strain Paenibacillus illinoisensis strain LB170 has the highest similarity, reaching 99%; strain number 16S-WP-B4-13 has the highest similarity with strain Paenibacillus agarexedensstrainCJ64 has the highest similarity, reaching 99%; strain number 16S-WP9-2 has the highest similarity with strain Paenibacillus polymyxa strain JK1 has the highest similarity, reaching 99%; both are Paenibacillus ( Paenibacillus ).

[0045] Example 2. Growth-promoting effect of rhizosphere growth-promoting bacteria on sterile potted seedlings indoors

[0046] 1. Seed treatment

[0047] Select plump, uniform-sized alfalfa seeds, disinfected with 6% sodium hypochlorite for 5 min, and rinsed three times with distilled water. Germination was performed in sterile Petri dishes in a 28°C constant-temperature incubator. After the embryo broke through the seed coat, seeds with uniform germination were selected and placed in small pots (7 cm in diameter, 10 cm in height) containing 100 g of sterilized substrate (vermiculite and sand in a 1:1 ratio). One seed was placed in each pot. The small pots were placed in an artificial climate chamber to maintain the substrate water holding capacity at 70%.

[0048] 2. Inoculation treatment

[0049] When the alfalfa seedlings grew the first true leaf, they were inoculated with the 8 strains obtained in Example 1. The specific inoculation was: irrigate the roots once every 2 days with 1 mL of bacterial solution each time (bacterial concentration was 10 8 CFU / mL), with a total of 8 treatment groups. The first inoculation was used as the starting time, and the plant height, root length, and dry weight of each seedling were measured 45 days later.

[0050] 3. Control treatment

[0051] When the alfalfa seedlings grew the first true leaf, they were not irrigated with the fungus solution, but were treated with an equal amount of sterile water to serve as the control (CK).

[0052] 4. Promoting growth

[0053] Plant height is a direct reflection of alfalfa's growth vitality. A higher plant height usually means that the plant performs well in longitudinal growth, reflecting that the elongation and division activities of plant cells are relatively active. At the same time, alfalfa plant height is generally positively correlated with hay yield. A higher plant height usually means more above-ground biomass accumulation, which can result in higher yields when making hay. Alfalfa with a higher plant height has a relatively large total amount of stems and leaves, and after harvesting, drying and other processes, more hay products can be obtained. Therefore, conducting research on alfalfa plant height is of great significance to alfalfa yield. Figure 2As shown, different strains exhibited varying degrees of growth-promoting abilities, with alfalfa plant height increasing to varying degrees under each treatment (P < 0.01). Plant height inoculated with all eight growth-promoting rhizobacteria was significantly higher than that in the control (P < 0.01), increasing by 9.6% to 60.36% compared to the control. MCWP-A3-15 exhibited the greatest growth-promoting effect, increasing plant height by 60.36%. The order of effect on alfalfa plant height under the remaining strains was: 16S-GU-C6-12 > 16S-YP-A3-7 > 16S-WP-B4-13 > 16S-YP-8 > 16S-WP9-2 > 16S-WP-A3-17 > 16S-YP-2.

[0054] Root length is a key indicator of alfalfa's adaptation to soil conditions. The growth of its root system directly affects the growth and yield of the aboveground part. Longer root length means a more developed root system, which can better absorb water and nutrients from the soil, thereby providing sufficient material basis for the growth of the aboveground stems and leaves. Vigorous growth of stems and leaves can effectively increase alfalfa yield. Studying the root length of alfalfa helps to understand its nitrogen fixation ability and its effect on improving soil fertility under different soil conditions. Longer roots can shuttle through the soil, improve soil aeration and water permeability, promote the growth and reproduction of soil microorganisms, and benefit the ecological restoration of degraded soils. Therefore, conducting research on the root length of alfalfa plants has an important impact on alfalfa yield. Such as Figure 2 As shown in the results, alfalfa root length increased when inoculated with different rhizosphere growth-promoting bacteria under sterile conditions. The root length of alfalfa treated with MCWP-A3-15 was significantly longer than that of the CK (P<0.01), increasing by 67.71%. The root lengths of alfalfa treated with the other strains showed no significant difference from those in the control group (P<0.01).

[0055] The dry weight of alfalfa plants directly reflects the total amount of organic matter accumulated by the plants during their growth and is a key indicator for measuring their nutrient accumulation. A higher dry weight per plant indicates that alfalfa synthesizes and stores a large amount of organic matter such as carbohydrates, proteins, and fats through photosynthesis. Therefore, it is necessary to study the effects of inoculating different rhizosphere growth-promoting bacteria on alfalfa yield. Figure 3 As shown, compared to the control, the dry weight of alfalfa plants inoculated with different growth-promoting rhizobacteria increased. MCWP-A3-15 exhibited the greatest increase in dry weight, with a 61% increase. The order of effect of the remaining strains on alfalfa plant dry weight was: 16S-WP-A3-17 > 16S-YP-2 > 16S-YP-8 > 16S-YP-A3-7 > 16S-GU-C6-12 > 16S-WP-B4-13 > 16S-WP9-2.

[0056] In conclusion, Burkholderia MCWP-A3-15 can promote the growth of alfalfa and increase plant height, root length and dry weight per plant.

[0057] Example 3. Growth-promoting effect of rhizosphere growth-promoting bacteria in the field

[0058] 1. Soil profile of the test site

[0059] The physical and chemical properties of the soil in the experimental site are shown in Table 2.

[0060] Table 2 Physical and chemical properties of soil in the experimental site

[0061]

[0062] 2. Field seeding treatment

[0063] The experiment adopted a completely randomized block design with 9 treatments, including 8 bacterial treatments and 1 control treatment; each treatment was repeated 3 times, with a total of 27 plots, each with an area of ​​20 m 2 (4m×5m), with plots spaced 1m apart and covered with weed cloth. Plants were spaced 50cm apart in rows and holes. Sow seeds in holes, with 3-5 seeds per hole. After seedlings emerge, leave one plant per hole for single sowing and one plant per hole for mixed sowing. Irrigation was by spray irrigation. When the alfalfa seedlings grew their first true leaf, root inoculation was performed with 5 mL of bacterial solution (bacterial concentration 10 8 CFU / mL); the control treatment was root irrigation with equal amount of sterile water.

[0064] 3. Agronomic trait index determination and results

[0065] The following indicators were measured during the initial flowering period of alfalfa:

[0066] Plant height: 20 alfalfa plants were randomly selected from each plot and their vertical heights were measured. Figure 5 shown.

[0067] Root length: Dig out the 20 alfalfa plants whose heights have been measured from the soil. Carefully dig out the alfalfa plants completely from the soil, trying to keep the root system intact. Gently rinse the roots with clean water to remove the attached soil. Place the cleaned roots flat on a piece of white paper and use tweezers to stretch the roots as much as possible to avoid bending and overlapping of the roots. Then, use a ruler to measure the length of the roots section by section along the main root and lateral roots, starting from the base of the roots. Add up the lengths of each section to get the root length of a single alfalfa plant. The results are as follows: Figure 4 shown.

[0068] Dry weight of a single plant: 20 alfalfa plants were randomly selected from each plot and carefully dug out from the soil, trying to ensure that the plants were intact, including the stems, leaves, flowers, fruits of the above-ground parts and the roots of the underground parts. Rinse the collected alfalfa plants with clean water to remove the soil, impurities, etc. attached to the surface. For the root system, wash it gently to avoid damage. After washing, place the plant on absorbent paper and gently absorb excess water on the surface. Put the pretreated alfalfa plants in the oven and set the appropriate temperature and time for drying. Generally, the temperature is about 105°C for about 30 minutes. The purpose is to quickly stop the physiological and biochemical reactions in the plant body and prevent further decomposition or transformation of the substance. Then adjust the temperature to 70-80°C and continue drying until the weight difference between two consecutive weighings does not exceed the specified range. The results are as follows Figure 5 shown.

[0069] Depend on Figure 4 As shown, the MCWP-A3-15 inoculation treatment significantly increased alfalfa plant height compared to the control, while the other strains did not significantly increase alfalfa plant height. The order of influence of different strains on alfalfa plant height was: MCWP-A3-15 > 16S-WP9-2 > 16S-GU-C6-12 > 16S-YP-A3-7 > 16S-WP-B4-13 > 16S-YP-8 > 16S-YP-2 > 16S-WP-A3-17. MCWP-A3-15 was more effective in increasing root length than the other strains.

[0070] like Figure 5 As shown, field inoculation with different bacterial strains all increased alfalfa plant dry weight to a certain extent, with different strains exhibiting varying growth-promoting effects. Treatment with MCWP-A3-15 had the most significant effect, increasing alfalfa plant dry weight by approximately 33%. Treatments with strains 16S-WP-B4-13, 16SYP-2, 16S-WP9-2, 16S-YP-8, and 16S-WP-A3-17 increased alfalfa plant dry weight by 4%, 7%, 8%, 13%, and 15%, respectively.

[0071] 4. Determination of soil physical and chemical properties

[0072] Adequate soil nutrients such as nitrogen, phosphorus, and potassium, as well as trace elements such as iron and zinc, are crucial for plant growth and development. Nitrogen is one of the key nutrients affecting alfalfa plant height, as it is an important component of plant protein and nucleic acid. A sufficient supply of nitrogen in the soil promotes vigorous plant growth and significant increases in plant height. Conversely, a lack of nitrogen leads to stunted alfalfa growth and reduced plant height. Phosphorus is involved in plant processes such as energy metabolism and cell division. An adequate supply of phosphorus fertilizer promotes root growth and increases the height of aboveground parts of the plant.

[0073] Soil pH determination: Weigh a certain amount of air-dried soil sample, place it in a conical flask, add carbon dioxide-free distilled water at a soil-water ratio of 1:2.5 (mass ratio), shake for a certain period of time, and then let it stand. Insert a glass electrode and a calomel electrode into the supernatant and use a pH meter to measure the pH value of the solution. The results are as follows: Figure 6 shown.

[0074] Determination of soil organic matter content: Accurately weigh a certain amount of air-dried soil sample, add excess potassium dichromate-sulfuric acid solution, oxidize the organic matter in the soil under oil bath conditions, titrate the remaining potassium dichromate with ferrous sulfate standard solution, and calculate the soil organic matter content based on the amount of ferrous sulfate consumed. The results are as follows Figure 7 shown.

[0075] Determination of total nitrogen content in soil: The soil sample is heated and digested with concentrated sulfuric acid and a catalyst to convert organic nitrogen into ammonium sulfate. Then, alkali is added for distillation to release ammonia, which is then absorbed by boric acid solution. The ammonia absorbed by the boric acid solution is then titrated with a standard acid solution, and the total nitrogen content in the soil is calculated based on the amount of acid used. The results are as follows: Figure 8 shown.

[0076] Determination of available phosphorus content in soil: Weigh 2.50g of air-dried soil sample that has passed through a 2mm sieve into a 250ml plastic bottle. Add 50.0ml of 0.5mol / L sodium bicarbonate solution (pH 8.5), shake at 20-25°C for 30min, and then filter with phosphorus-free filter paper. Pipette 10.0ml of the filtrate into a 50ml volumetric flask, add 5ml of ammonium molybdate-sulfuric acid solution, then add 5ml of ascorbic acid solution, dilute to volume with water, and shake well. After standing for 30min, perform colorimetric determination at a wavelength of 700nm. The results are as follows: Figure 9 shown.

[0077] Depend on Figure 6 It can be seen that the soil pH value of each treatment was 8.5-8.7, and there was no significant difference among the treatments (P>0.01). The content of organic matter, total nitrogen and available phosphorus was positively correlated with soil fertility. Figure 7 It can be seen that MCWP-A3-15 has little effect on the organic matter content. Figure 8 It can be seen that the total nitrogen content of MCWP-A3-15 treatment increased significantly compared with CK, while there was no significant difference between 16S-WP-A3-17 and 16S-GU-C6-12 treatments and CK. Figure 9 Available phosphorus increased to varying degrees after treatment with the different strains (P < 0.01). MCWP-A3-15 treatment showed a significant increase, reaching 1.3 times that of CK. In summary, Burkholderia MCWP-A3-15 can promote alfalfa growth, increasing plant height, root length, and dry weight per plant. It also improves soil physical and chemical properties, increasing total nitrogen and available phosphorus content, and enhancing soil fertility.

Claims

1. A plant rhizosphere growth-promoting bacterium capable of effectively increasing the growth of alfalfa, characterized in that: The rhizosphere growth-promoting bacteria is Burkholderia MCWP-A3-15; the preservation number of Burkholderia MCWP-A3-15 is CGMCC No.31562, and it is preserved in the General Microbiology Center of China Culture Collection Administration.

2. Use of the Burkholderia MCWP-A3-15 according to claim 1 in promoting the growth of alfalfa.

3. The use of Burkholderia MCWP-A3-15 in promoting alfalfa growth according to claim 2, characterized in that: Burkholderia MCWP-A3-15 can increase the plant height, root length and dry weight of alfalfa.

4. The use of Burkholderia MCWP-A3-15 according to claim 1 in improving soil physical and chemical properties, characterized in that: The physicochemical properties are total nitrogen content and available phosphorus content.

Images