Drought-resistant growth-promoting plant endophytic bacillus strain and application thereof

Abstract

The invention provides a drought-resistant growth-promoting plant endophytic bacillus strain, which is named as Bacillus sp.AB513 and preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M20242072, the preservation date is September 25, 2024, and the nucleotide sequence of 16S rRNA (Ribosomal Ribonucleic Acid) is as shown in SEQ ID NO.1. The drought-resistant growth-promoting plant endophytic bacillus strain has the advantages that the drought-resistant growth-promoting plant endophytic bacillus strain can be applied to drought-resistant growth-promoting plants; the invention further provides application of the strain. The strain is used for promoting plant growth under drought conditions. The strain provided by the invention can promote germination of alfalfa under drought conditions, increase root length and bud length of alfalfa seedlings, increase fresh weight and dry weight of the alfalfa seedlings under drought conditions, increase chlorophyll content of alfalfa cotyledons and increase soluble protein content of the alfalfa cotyledons.

Description

Technical Field

[0001] The invention belongs to the technical field of drought-resistant growth-promoting strains, and specifically relates to a drought-resistant growth-promoting plant endophytic Bacillus strain and an application thereof. Background Art

[0002] Bacillus is a common non-pathogenic bacterium found in soil, water and plants. It is safe for the ecological environment and humans and has the effect of promoting plant growth. Therefore, it has been widely used as a growth-promoting microbial agent in agricultural production. However, most of the microbial agents currently on the market are applied to the soil in the form of fertilizers. Their growth-promoting effect must meet three key conditions: (1) the strains must be able to survive and form a dominant population in a complex soil environment; (2) the amount of growth-promoting substances produced by the agents must be sufficient and stable; (3) the growth-promoting effect of the agents must not gradually decay over time and with changes in the soil environment. In addition, in terms of the production of microbial agents, the amount of bacteria used in such microbial fertilizers is large, the production cost is high, the process is complex and the effect is unstable. Therefore, traditional dosage forms of agricultural microbial agents are gradually unable to meet the needs of crop resistance and green sustainable development, so the development of new microbial agents is imminent.

[0003] Research on drought-resistant alfalfa cultivation mainly focuses on improving irrigation and seeding methods, and research on microbial agents is also mainly focused on soil improvement to adapt to alfalfa cultivation. Therefore, the development of drought-resistant growth-promoting strains for drought-resistant alfalfa cultivation will solve the above-mentioned problem of poor drought resistance of alfalfa, promote the industrialization of dryland forage, improve the yield and quality of forage, reduce land desertification, and help the green and sustainable development of agriculture in arid areas. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide: a drought-resistant and growth-promoting plant endophytic Bacillus strain and its application, which can be used to promote the growth of alfalfa under drought conditions, promote the germination of alfalfa, increase the root length and shoot length of alfalfa seedlings, increase the fresh weight and relative water content of alfalfa seedlings under drought conditions, increase the chlorophyll content of alfalfa cotyledons, and increase the soluble protein content of alfalfa cotyledons.

[0005] To solve the above technical problems, the technical solution adopted by the present invention is: a drought-resistant and growth-promoting plant endophytic Bacillus strain, the drought-resistant and growth-promoting plant endophytic Bacillus strain is named Bacillus sp. AB513, preserved in the China Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: M 20242072, and the preservation date is September 25, 2024; the nucleotide sequence of 16S rRNA of the drought-resistant and growth-promoting plant endophytic Bacillus strain is shown in SEQ ID NO.1.

[0006] Preferably, the drought-resistant and growth-promoting endophytic Bacillus strain is used to promote the growth of plants under drought conditions.

[0007] Preferably, the plant is alfalfa.

[0008] Preferably, the drought-resistant and growth-promoting endophytic Bacillus strain is used to promote alfalfa germination under drought conditions.

[0009] Preferably, the drought-resistant and growth-promoting endophytic Bacillus strain is used to increase the root length and shoot length of alfalfa seedlings under drought conditions.

[0010] Preferably, the drought-resistant and growth-promoting endophytic Bacillus strain is used to increase the fresh weight and relative water content of alfalfa seedlings under drought conditions.

[0011] Preferably, the drought-resistant and growth-promoting endophytic Bacillus strain is used to increase the chlorophyll content of alfalfa cotyledons under drought conditions.

[0012] Preferably, the drought-resistant and growth-promoting plant endophytic Bacillus strain is used to increase the soluble protein content of alfalfa cotyledons under drought conditions. Compared with the prior art, the present invention has the following advantages:

[0013] 1. The drought-resistant and growth-promoting plant endophytic Bacillus strain of the present invention is an endophytic bacterium isolated and purified from the Xinjiang-specific desert plant Sophora japonica, which cooperates with the host plant to adapt to the desert environment, has the characteristics of colonizing in the plant and accompanying the plant growth to play a long-term role in synergistically absorbing water and nutrients to promote the growth of the plant root system. The drought-resistant and growth-promoting plant endophytic Bacillus strain can be used to promote the growth of alfalfa under drought conditions, promote the germination of alfalfa, increase the root length and bud length of alfalfa seedlings; increase the fresh weight and relative water content of alfalfa seedlings, and increase the chlorophyll content and soluble protein content of alfalfa cotyledons.

[0014] 2. The present invention uses drought-resistant and growth-promoting plant endophytic Bacillus agents to treat alfalfa seeds, and promotes plant seed germination and drought-resistant growth through the symbiosis between the strain and the plant. The required strain dosage is small, and the drought-resistant and growth-promoting effect is significant.

[0015] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a Gram staining identification diagram of the strain in Example 1 of the present invention.

[0017] Figure 2 The figure is a colony morphology diagram on the beef extract peptone plate of Example 1 of the present invention.

[0018] Figure 3 This is the 16s rRNA phylogenetic tree of the strain in Example 1 of the present invention.

[0019] Figure 4 It is the germination rate of alfalfa under different treatments in Example 2 of the present invention.

[0020] Figure 5 It is the alfalfa germination rate on the fourth day in Example 2 of the present invention.

[0021] Figure 6 Root length (A) and shoot length (B) of alfalfa under different treatments in Example 2 of the present invention.

[0022] Figure 7 The fresh weight of alfalfa (left figure) and relative water content of seedlings (right figure) under different treatments of Example 2 of the present invention are shown.

[0023] Figure 8 The chlorophyll content of alfalfa cotyledons under different treatments in Example 2 of the present invention.

[0024] Fig. 9 It is the soluble protein content of alfalfa cotyledons under different treatments of Example 2 of the present invention.

[0025] Fig.10 It is the soluble sugar content of alfalfa cotyledons under different treatments of Example 2 of the present invention. DETAILED DESCRIPTION

[0026] Example 1

[0027] This example is about the separation, purification and identification of Bacillus sp. AB513 strain.

[0028] (1) Isolation and purification of Bacillus sp. AB513 strain

[0029] Bacillus sp.AB513 strain was isolated from the root of Sophora japonica of the Psoralea corylifolia family, which was collected from the Takermohur Desert in Huocheng County, Yili River Valley, Xinjiang. The specific method is as follows: the root of Sophora japonica was sterilized on the surface and put into a sterile mortar with 10 mL of sterilized water to grind into a paste, and 200 μL of the original solution, 100-fold dilution and 1000-fold dilution were respectively applied to beef extract peptone plates, with 5 replicates for each gradient. Cultured at 37°C until a single colony grew, and streaked repeatedly until a pure culture strain was obtained.

[0030] (2) Morphological identification

[0031] Purified strains were observed using conventional Gram staining:

[0032] ① Wipe the slide with alcohol, dry it with an alcohol lamp, drop a drop of physiological saline on the slide, use a burned and cooled inoculation loop to take a small amount of bacteria and smear it in the water to form a coating with a diameter of about 1 cm, and burn the inoculation loop with an alcohol lamp to sterilize. Let the smear dry naturally at room temperature / heat and dry it at a high temperature about 15 cm away from the flame, and then pass it back and forth over the flame of the alcohol lamp 3 times to inactivate and fix it.

[0033] ② Add 1-2 drops of crystal violet staining solution, stain for 1 minute, and gently rinse with distilled water.

[0034] ③ After drying, add Gram's iodine solution for 1 minute, and rinse gently with distilled water.

[0035] ④ Add a drop of decolorizer (95% alcohol), shake the slide from time to time until no purple color falls off (about 15 seconds), and rinse gently with distilled water.

[0036] ⑤ After drying, add safranin counterstaining solution and counterstain for 1 minute. Rinse gently with distilled water and examine under a microscope after the slide is dry.

[0037] ⑥ Oil immersion microscopy: Purple bacteria are Gram-positive bacteria, and red bacteria are Gram-negative bacteria.

[0038] The results are as follows Figure 1 , Figure 2 Gram staining showed that Bacillus sp.AB513 strain was short rod-shaped and was a Gram-positive bacterium. Figure 1 As shown. On beef extract peptone solid medium, the colonies formed by Bacillus sp.AB513 strain are round, with wavy edges, milky white, rough surface, no protrusions, and sticky texture, as shown. Figure 2 shown.

[0039] (3) Analysis of the evolutionary status of AB513

[0040] Refer to the "Succinct Molecular Biology Experiment Guide" to extract AB513 strain genomic DNA, perform PCR amplification, PCR products are purified and recovered, connected to the pTA2 vector, transformed into DH5α cells, sequenced after blue-white screening, and constructed a phylogenetic tree, such as Figure 3 The results showed that the drought-resistant and growth-promoting endophytic bacteria of the present invention belonged to the genus Bacillus, and the sequence similarity with the Bacillus subtilis strain DSM 10 was 99.80%.

[0041] (4) Propagation and preservation of bacterial strains

[0042] ① Prepare beef extract peptone liquid, solid culture medium and 50% glycerol, and sterilize at 121℃ for 20 minutes.

[0043] ② Take 1 μL of bacterial solution from the original storage tube and add it to 1000 μL of sterile water. Vortex and shake for 30 seconds. Take 50 μL and spread it on beef extract peptone solid culture medium. Invert and culture at 37℃ for 1-2 days.

[0044] ③Pick a single colony and place it in 20 mL of fresh beef extract peptone liquid culture medium, shake and culture at 37°C, 200 rpm until OD 600 It is 0.8-1.0.

[0045] ④ Take the culture described in ③ and transfer it to fresh beef extract peptone liquid medium at a 1% inoculation rate, and culture it at 37℃, 200r / min shaking in a shaker until OD 600 is 1.0.

[0046] ⑤ Add equal volumes of 50% glycerol and the culture medium described in ④ to a sterile freezing tube, mix well, and store at -80°C.

[0047] The drought-resistant and growth-promoting plant endophytic Bacillus strain isolated in this example is named Bacillus sp. AB513 and deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M20242072 and the deposit date being September 25, 2024; the nucleotide sequence of 16S rRNA of the drought-resistant and growth-promoting plant endophytic Bacillus strain is shown in SEQ ID NO.1.

[0048] Example 2

[0049] This example shows the effect of the Bacillus sp. AB513 strain in Example 1 on promoting the growth of alfalfa under extreme drought conditions.

[0050] The drought-resistant and growth-promoting plant endophytic Bacillus strain (Bacillus sp. AB513) is used to promote the growth of alfalfa under drought conditions, promote the germination of alfalfa, increase the root length and shoot length of alfalfa seedlings, increase the fresh weight and dry weight of alfalfa seedlings under drought conditions, increase the chlorophyll content of alfalfa cotyledons, and increase the soluble protein content of alfalfa cotyledons.

[0051] The alfalfa variety used in the experiment in this embodiment is the domestically cultivated variety "Zhongmu No. 3". The treatment method of the alfalfa seed treatment group involved in this embodiment is as follows:

[0052] (1) Water treatment group (CK):

[0053] ①Put a double layer of sterilized filter paper on the sterilized culture dish;

[0054] ②Evenly add 4.2 mL of sterile water to the filter paper in each culture dish;

[0055] ③ Evenly spread 40 alfalfa seeds on each dish and set up 5 replicates;

[0056] ④ Add 0.3-0.7 mL of sterile water every 2 days until the filter paper is saturated. When the culture dish is tilted, liquid will slowly flow out.

[0057] The number of seeds germinating was observed and recorded every 24 hours, and the standard for seed germination was when the embryo broke through the seed coat by 2 mm.

[0058] (2) Drought group (D):

[0059] ① Prepare 20% PEG 6000 solution and filter it with a 0.22μm filter membrane for sterilization;

[0060] ② Cover the sterilized culture dish with double-layer sterilized filter paper;

[0061] ③Evenly add 2.8mL PEG + 1.4mL sterile water to the filter paper in each culture dish;

[0062] ④ Evenly spread 40 alfalfa seeds on each dish and set up 5 replicates;

[0063] ⑤ Add 0.3-0.7 mL of sterile water every 2 days until the filter paper is saturated and liquid slowly flows out when the culture dish is tilted.

[0064] The number of seeds germinating was observed and recorded every 24 hours, and the standard for seed germination was when the embryo broke through the seed coat by 2 mm.

[0065] (3) Drought-adding bacteria group (DE-AB513):

[0066] ① Take Bacillus sp.AB513 OD 600 The culture was centrifuged at 4°C, 5000 rpm, for 5 min, the supernatant was discarded, and an equal volume of sterile PBS was added to resuspend the culture for later use.

[0067] ② Soak 5-10g of alfalfa seeds in 10mL of inoculant, shaking every half an hour to allow them to soak for 4-6 hours;

[0068] ③ Cover the sterilized culture dish with double-layer sterilized filter paper;

[0069] ④ Add 2.8 mL PEG to each culture dish and add 1.4 mL sterile water, and evenly disperse on the filter paper;

[0070] ⑤ Evenly spread 40 alfalfa seeds on each dish, and set up 5 replicates;

[0071] ⑥ Add 0.3-0.7 mL of sterile water every 2 days until the filter paper is saturated and liquid slowly flows out when the culture dish is tilted.

[0072] The number of seeds germinating was observed and recorded every 24 hours, and the standard for seed germination was when the embryo broke through the seed coat by 2 mm.

[0073] (I) Results of drought-resistant germination of alfalfa seeds

[0074] In order to study the effect of Bacillus sp.AB513 on the drought resistance of alfalfa, the number of germination seeds was recorded every day from 1 to 4 days after the alfalfa seeds germinated, and the germination rate of alfalfa seeds was plotted. Figure 4 As shown in the figure, on the fourth day of seed germination, the germination rate of alfalfa in the water treatment group reached an average of 100%, the germination rate in the drought and bacteria treatment group reached an average of 68.3%, and the germination rate in the drought treatment group was only 22.5%. Figure 5 As shown, compared with drought treatment alone, the seed germination rate of the bacteria-added group significantly increased by more than 3 times, an increase of 45.8%.

[0075] (II) Results of root growth and sprout growth of alfalfa seedlings

[0076] In order to study the drought resistance and growth promotion effect of Bacillus sp. AB513 on alfalfa, the seedlings were taken on the 4th day of alfalfa seed germination, and the root length and shoot length were measured with a ruler (6 plants in each treatment, repeated 3 times), and the results were counted and plotted. Figure 6 As shown, the root length of drought plus bacteria group ( Figure 6 A) and bud length ( Figure 6 B) was significantly higher than the drought group; under 20% PEG6000 drought stress, the root length and shoot length of the bacteria-added group were 0.88 cm and 0.47 cm, respectively, which were significantly higher than the root length (0.19 cm) and shoot length (0.12 cm) of the drought control group. The results showed that strain AB513 could significantly promote the drought-resistant growth and development of plant seedlings (p<0.01).

[0077] (III) Test results of fresh weight and relative water content of alfalfa seedlings

[0078] In order to study the effect of Bacillus sp.AB513 on drought resistance and growth promotion of alfalfa, seedlings were taken on the 6th day of alfalfa seed germination. Ten plants were taken for each treatment, repeated 3 times, and weighed 3 times. The weighed seedlings were dried at 105℃ for 15 minutes, dried at 80℃ to constant weight (about 8 hours), cooled to room temperature and weighed, which is the dry weight. Figure 7 Left) and relative water content of seedlings ( Figure 7 Right), such as Figure 7As shown, the average fresh weight of seedlings in the water treatment group was 0.28g, the fresh weight of the drought plus bacteria group was 0.2g, and the fresh weight of the drought group was 0.165g. After the T single-tail test, it can be seen that the fresh weight of the drought plus bacteria group was significantly higher than that of the drought group (p<0.05). At the same time, the relative water content of seedlings in the drought plus bacteria group was 86.5%, which was significantly higher than the relative water content of the drought group (74.5%). The results show that strain AB513 can significantly increase the water content of seedlings, thereby promoting their resistance to drought stress and maintaining growth and development (p<0.05).

[0079] (IV) Test results of chlorophyll content in alfalfa cotyledons

[0080] Chlorophyll content is related to the intensity of plant photosynthesis and can be used as an indicator to measure the growth status of plants and the degree of damage caused by drought stress. In order to study the drought-resistant and growth-promoting effects of Bacillus sp.AB513 on alfalfa, on the 6th day after the germination of alfalfa seeds in different treatment groups, 0.05 g of fresh alfalfa leaves were weighed and placed in a centrifuge tube wrapped in aluminum foil. 5 mL of 95% ethanol was added to completely soak the leaves in ethanol. The leaves were placed in a 4°C refrigerator for 72 hours (until the leaves turned white). After sufficient extraction, the absorbance was measured with a spectrophotometer (repeated 3 times for each treatment). The chlorophyll content of alfalfa was counted and plotted, such as Figure 8 As shown: The total chlorophyll content of the water treatment group is 0.944 mg g -1 The total chlorophyll content of the drought-plus-bacteria group was 0.414 mg·g -1 , while the total chlorophyll content in the drought group was only 0.058 mg·g -1 The results showed that strain AB513 could significantly increase the total chlorophyll content of plants, thereby promoting plant growth and coping with drought stress (p<0.01).

[0081] (V) Test results of soluble protein in alfalfa cotyledons

[0082] In order to study the drought resistance and growth-promoting effect of Bacillus sp.AB513 on alfalfa, on the 10th day of alfalfa seed germination, about 0.05g of fresh alfalfa leaves were weighed (repeated 3 times for each treatment group), 5mL of distilled water was added, and then ground into a homogenate, transferred to a centrifuge tube, centrifuged at 3000r / min for 10min, and the supernatant was aspirated for later use. Take 1mL of the sample extract, put it into a test tube, add 5mL of Coomassie Brilliant Blue reagent, shake well, and measure the absorbance at 595nm after 6min, and check the protein content through the standard curve. The soluble protein content is as follows Fig. 9 As shown, the soluble protein content of the water treatment group was 3.7%, the drought group was 1.2%, and the drought plus bacteria group could reach 4.3%, indicating that strain AB513 can significantly increase the soluble protein content of cotyledons under drought stress to promote plant growth (p<0.05).

[0083] (VI) Test results of soluble sugar content in alfalfa cotyledons

[0084] Soluble sugar is an osmotic regulating substance. Under drought stress, plants will accumulate osmotic regulating substances to alleviate the water loss caused by drought in order to maintain normal growth and development. Therefore, drought can cause an increase in the soluble sugar content of plants. In order to study the drought-resistant and growth-promoting effects of Bacillus sp.AB513 on alfalfa, about 0.05 g of fresh leaves were weighed on the 10th day of alfalfa seed germination, and each treatment group was repeated 3 times. After adding 5 mL of distilled water to grind into a homogenate, centrifuged at 3000 r / min for 10 minutes, the supernatant was taken for testing. Take 1 mL of the sample extract in a centrifuge tube, add 5 mL of anthrone reagent, boil in a boiling water bath for 10 minutes, then ice-bathe the centrifuge tube to room temperature, and measure OD with a spectrophotometer at 20 minutes. 625 The absorbance value at nm was measured 3 times for each group. A standard curve was drawn using glucose as the standard substance, and the soluble sugar content of alfalfa was obtained through the curve. The results were statistically analyzed. Fig.10 As shown in the results, under 20% PEG6000 drought treatment, the soluble sugar content in the cotyledons was 3.2%, which was significantly higher than that in the water treatment group (1.0%), while the soluble sugar content in the drought-added bacteria group (2.6%) was significantly lower than that in the drought group. This result shows that compared with the drought stress group, the strain AB513 treatment alleviated the osmotic substances increased by drought stress, resulting in a decrease in soluble sugar content, and the strain promoted the plant to use soluble sugar as an energy substance for growth (p<0.05).

[0085] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A drought-resistant and growth-promoting plant endophytic Bacillus strain, characterized in that: The drought-resistant and growth-promoting plant endophytic Bacillus strain is named Bacillus sp.AB513, deposited in the China Center for Type Culture Collection, with a deposit number of CCTCCNO: M 20242072 and a deposit date of September 25, 2024; the nucleotide sequence of 16SrRNA of the drought-resistant and growth-promoting plant endophytic Bacillus strain is shown in SEQ ID NO.

1.

2. An application of the drought-resistant and growth-promoting plant endophytic Bacillus strain according to claim 1, characterized in that: The drought-resistant and growth-promoting plant endophytic Bacillus strain is used for promoting the growth of plants under drought conditions.

3. The use according to claim 2, characterized in that: The plant is alfalfa.

4. The use according to claim 3, characterized in that: The drought-resistant and growth-promoting plant endophytic Bacillus strain is used for promoting the germination of alfalfa under drought conditions.

5. The use according to claim 4, characterized in that: The drought-resistant and growth-promoting plant endophytic Bacillus strain is used for increasing the root length and shoot length of alfalfa seedlings under drought conditions.

6. The use according to claim 4, characterized in that: The drought-resistant and growth-promoting plant endophytic Bacillus strain is used for increasing the fresh weight and relative water content of alfalfa seedlings under drought conditions.

7. The use according to claim 4, characterized in that: The drought-resistant and growth-promoting plant endophytic Bacillus strain is used for increasing the chlorophyll content of alfalfa cotyledons under drought conditions.

8. The use according to claim 4, characterized in that: The drought-resistant and growth-promoting plant endophytic Bacillus strain is used for increasing the soluble protein content of alfalfa cotyledons under drought conditions.

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