A novel salt-alkali tolerant Bacillus sp. N3-4 from ginseng soil and its application in improving the ability of plants to tolerate salt stress

Abstract

The invention discloses a novel bacillus N3‑4 from saline-alkali soil and its application in improving the salt stress tolerance of plants, belonging to the field of microbial technology. The novel bacillus N3‑4 from saline-alkali soil is isolated from saline-alkali soil, is salt-alkali tolerant and can improve the salt stress tolerance of plants, and is preserved in the General Microbiological Center of the China Microbiological Culture Collection Administration, and is classified and named Neobacillus ginsengisoli , the deposit number is CGMCC No. 32574. The benefits of the present invention are: the novel Bacillus N3‑4 from ginseng soil has a high degree of salt-alkali tolerance and is suitable for growth and reproduction in saline-alkali land; it has the ability to degrade inorganic phosphorus and produce iron carriers, can improve the salt stress tolerance of plants (cucumber seedlings), can be applied to the cultivation of saline-alkali plants, and promote the comprehensive utilization of saline-alkali land.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a novel salt-alkali tolerant ginseng soil Bacillus N3-4 and an application thereof in improving the salt stress tolerance of plants. Background Art

[0002] At present, soil salinization has become a global ecological and food security issue that cannot be ignored. Improving saline-alkali land, breeding salt-alkali tolerant economic species and improving the salt-alkali tolerance of economic species are important means to solve the problem.

[0003] Salt-alkali land improvement includes physical, chemical, biological and other methods, among which biological improvement is the most economical, effective and environmentally friendly. Salt-alkali tolerant microorganisms and plant growth promoting rhizosphere bacteria (PGPR) are widely used in salt-alkali land improvement and improving plant tolerance to salt-alkali stress. PGPR refers to microorganisms that can promote plant growth, prevent and control diseases, increase crop yields and produce metabolites that are beneficial to plant growth, thereby promoting plant growth and development. Some PGRPs with strong salt-alkali tolerance can be used to improve salt-alkali land and improve plant salt-alkali tolerance. Their main mechanisms of action include:

[0004] (1) Secretion of osmotic substances such as proline, betaine, polyamines, trehalose, and soluble sugars. These osmotic substances work synergistically with osmotic substances produced by plants to increase the osmotic pressure of plant cells, thereby enhancing the salt-alkali tolerance of plants;

[0005] (2) Secrete plant growth hormones such as indoleacetic acid to promote the development of plant roots, increase the total volume and surface area of ​​plant roots, and increase the absorption and utilization rate of water by plant roots, thereby alleviating the osmotic stress caused by salinity and alkali stress on plants;

[0006] (3) Secretion of extracellular polysaccharides, which can regulate the water microenvironment around the rhizosphere soil and thus improve soil properties;

[0007] (4) Synthesize high-affinity potassium transporters (volatile compounds), which can promote plant absorption of K + , increasing plant K + / Na + , thereby enhancing the salt-alkali tolerance of plants;

[0008] (5) Secrete acidic organic matter such as oxalic acid, tartaric acid, and succinic acid, which can neutralize the alkalinity of the soil and thus improve soil properties;

[0009] (6) Secrete antioxidant enzymes such as catalase and peroxidase, which can improve the antioxidant capacity of plants and thus improve their ability to cope with stress;

[0010] (7) Provide nutrients such as iron carriers, soluble phosphorus, and soluble potassium. These nutrients are beneficial for plant absorption, which can promote plant growth and thus improve the plant's ability to cope with stress.

[0011] At present, the screening and research of PGPR are mostly focused on plant growth promotion (growth promotion mechanism, growth promotion effect). The screening and research of PGPR that can improve the plant's tolerance to salt-alkali stress are relatively few. Existing studies show that most PGPR cannot adapt to high-salt-alkali environments and cannot grow and reproduce in saline-alkali lands. Although some PGPR can adapt to high-salt-alkali environments, there are very few PGPR that can improve the plant's tolerance to salt-alkali stress. Summary of the invention

[0012] The purpose of the present invention is to provide a PGPR which has strong salt-alkali tolerance and can improve the salt stress tolerance of plants, and the application of the PGPR in improving the salt stress tolerance of plants.

[0013] In order to achieve the above object, the present invention adopts the following technical solution:

[0014] A novel Bacillus ginseng soil N3-4, which is isolated from saline-alkali soil in Kenli District, Dongying City, Shandong Province, is salt-alkali tolerant and can improve the ability of plants to tolerate salt stress, and is preserved in the General Microbiological Center of the China Microbiological Culture Collection Administration, and is classified and named Neobacillus ginsengisoli The deposit number is CGMCC No. 32574, the deposit date is November 11, 2024, and the deposit address is Beijing, China.

[0015] The aforementioned novel Bacillus ginsengii N3-4 is used to enhance the salt stress tolerance of plants, wherein the plants are cucumber seedlings.

[0016] The present invention is beneficial in that:

[0017] (1) The novel ginseng soil Bacillus N3-4 obtained by the present invention is isolated from saline-alkali soil, has a high degree of salt-alkali tolerance, and is suitable for growth and reproduction in saline-alkali soil;

[0018] (2) The novel Bacillus N3-4 obtained from the ginseng soil of the present invention has the ability to degrade inorganic phosphorus and produce iron carriers, which can improve the salt stress resistance of plants (cucumber seedlings), and can be applied to the cultivation of saline-alkali plants and promote the comprehensive utilization of saline-alkali land. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a colony morphology of the novel Bacillus sp. N3-4 from Ginseng soil on a high-salt inorganic phosphorus solid medium;

[0020] Figure 2 This is a phylogenetic tree analysis diagram of the novel Bacillus sp. N3-4 from Ginseng soil;

[0021] Figure 3 This is a graph showing the results of the test of the phosphorus solubilization ability of the novel Bacillus sp. N3-4 from ginseng soil;

[0022] Figure 4 This is a graph showing the siderophore production capacity test results of the novel Bacillus sp. N3-4 from Ginseng soil;

[0023] Figure 5 This is a comparison chart of the growth of cucumber seedlings in each group after 14 days of cultivation;

[0024] Figure 6 This is the statistical result of the fresh weight of cucumber seedlings in each group after 14 days of cultivation;

[0025] Figure 7 It is a statistical graph of the plant height of cucumber seedlings in each group after 14 days of cultivation. DETAILED DESCRIPTION

[0026] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] 1. Screening of strain N3-4

[0028] Soil samples were collected from saline-alkali land in Kenli District, Dongying City, Shandong Province (37.60446941°N, 118.81132062°E). Then 2 g of soil sample was added to 100 mL of high-salt inorganic phosphorus liquid culture medium and cultured in a constant temperature incubator at 30°C and 140 rpm for 5 days. After that, 1 mL of turbid culture solution was aspirated and graded diluted with sterile 0.9% sodium chloride solution. The solution was evenly spread on a high-salt inorganic phosphorus solid culture medium and cultured in an inverted incubator at 30°C for 48 h. After the culture was completed, round, 2.3 mm-6.4 mm in diameter, and white single colonies ( Figure 1 ), the bacteria in this single colony were recorded as strain N3-4.

[0029] The formula of high-salt inorganic phosphorus liquid culture medium: 50g sodium chloride, 10g glucose, 0.5g ammonium sulfate, 0.5g yeast extract powder, 0.3g potassium chloride, 0.3g magnesium sulfate, 0.03g ferrous sulfate, 0.03g manganese sulfate, 5.0g tricalcium phosphate, and 1L ultrapure water.

[0030] The formula of high-salt inorganic phosphorus solid culture medium: add 15g of agar to the formula of high-salt inorganic phosphorus liquid culture medium.

[0031] 2. Identification of strain N3-4

[0032] A single colony was picked from the plate, and the DNA of strain N3-4 was extracted and its 16S rDNA sequence was amplified using a conventional bacterial total DNA extraction method. The forward primer was 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 2), and the reverse primer was 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 3). The PCR amplification system was 20 μL, including 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 10 μL of 2×Taq PCR premix, 1 μL of bacterial DNA, and 7 μL of ddH2O. The PCR amplification procedure was conventional.

[0033] After the PCR amplification was completed, 2 μL of the PCR amplification product was taken and electrophoresed using 1.2% agarose gel. The DNA fragments separated by electrophoresis were then sequenced. The sequencing results are shown in SEQ ID NO: 1.

[0034] The sequencing results were first analyzed by BLAST search on the NCBI website, then homology analysis was performed with the GenBank database, and finally a phylogenetic tree was constructed using MEGA7.0 software. Neobacillus ginsengisoli Closely related ( Figure 2 ), so the strain N3-4 was named as Bacillus ginseng Neobacillus ginsengisoli .

[0035] 3. Salt and alkali tolerance test of strain N3-4

[0036] 1. Test of salt tolerance of strains

[0037] The strain N3-4 was inoculated into LB liquid culture medium and cultured in a constant temperature incubator at 30°C and 140rpm for 12h. Then the bacterial solution was collected and diluted 10 times with a sterile 0.9% sodium chloride solution. 10μL of the dilution was applied to LB solid culture medium containing different concentrations of sodium chloride (0g / L, 10g / L, 30g / L, 50g / L, 70g / L, 90g / L, 100g / L, 120g / L, 150g / L) and a pH value of 8. Three spots were applied on each plate (3 spots were distributed in a triangle, 10μL of the dilution was applied at each spot). After that, the plate was placed in an inverted culture in a constant temperature incubator at 30°C for 24h. The growth of the colonies was observed to determine the salt tolerance of the strain N3-4.

[0038] The growth conditions of strain N3-4 on the above-mentioned LB solid culture media are shown in the table below.

[0039] Table 1 Growth of strain N3-4 on LB solid medium with different salinities

[0040]

[0041] It can be seen from Table 1 that strain N3-4 can tolerate a maximum salinity of 10% (sodium chloride concentration is 100 g / L) and has a strong salt tolerance.

[0042] 2. Test of strain alkali resistance

[0043] The strain N3-4 was inoculated into LB liquid culture medium and cultured in a constant temperature incubator at 30°C and 140rpm for 12h. The bacterial liquid was then collected and diluted 10 times with a sterile 0.9% sodium chloride solution. 10μL of the dilution was spot-applied on LB solid culture media with different pH values ​​(7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, adjusted with 1M sodium hydroxide solution). Three spots were spot-applied on each plate (3 spots were distributed in a triangle, 10μL of the dilution was applied at each spot). After that, the plate was placed in an inverted culture incubator at 30°C for 24h. The growth of the colonies was observed to determine the alkali resistance of the strain N3-4.

[0044] The growth conditions of strain N3-4 on the above-mentioned LB solid culture media are shown in the table below.

[0045] Table 2 Growth of strain N3-4 on LB solid medium with different alkalinity

[0046]

[0047] It can be seen from Table 2 that the strain N3-4 can tolerate a maximum pH value of 9.5, and has a strong alkali resistance.

[0048] In summary, strain N3-4 has strong salt-alkali tolerance and is suitable for growth and reproduction in saline-alkali land.

[0049] 4. Mechanism of strain N3-4 in improving plant salt stress tolerance

[0050] 1. Detection of the phosphorus solubilizing ability of strain N3-4

[0051] The strain N3-4 was inoculated into LB liquid culture medium and cultured in a constant temperature incubator at 30°C and 140 rpm for 12 h. The bacterial liquid was then collected and 10 μL of the bacterial liquid was streaked on an inorganic phosphorus solid culture medium and cultured inverted in a constant temperature incubator at 30°C for 5 days.

[0052] like Figure 3 As shown, an obvious phosphate-dissolving zone can be observed on the plate, indicating that strain N3-4 has the ability to dissolve phosphate.

[0053] The formula of inorganic phosphorus solid culture medium: 10g glucose, 0.5g ammonium sulfate, 0.5g yeast extract powder; 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate, 0.03g ferrous sulfate, 0.03g manganese sulfate, 5.0g tricalcium phosphate, 15g agar, and 1L ultrapure water.

[0054] 2. Detection of the siderophore-producing ability of strain N3-4

[0055] The strain N3-4 was inoculated into LB liquid culture medium and cultured in a constant temperature incubator at 30°C and 140 rpm for 12 h. The bacterial liquid was then collected and 10 μL of the bacterial liquid was spot-smeared on CAS detection culture medium at 3 locations (3 points distributed in a triangle, 10 μL of bacterial liquid at each point), and placed in a constant temperature incubator at 30°C for inversion culture for 48 h.

[0056] like Figure 4 As shown, an obvious orange siderophore halo can be observed on the plate, indicating that strain N3-4 has the ability to produce siderophores.

[0057] Formula of CAS detection medium: chrome azuro blue S (CAS) 60.5 mg, hexadecyltrimethylammonium bromide (HDTMA) 72.9 mg, ferric chloride hexahydrate 2.645 mg, sodium dihydrogen phosphate dihydrate 295.25 mg, disodium hydrogen phosphate dodecahydrate 1213.5 mg, ammonium chloride 125 mg, potassium dihydrogen phosphate 37.5 mg, sodium chloride 62.5 mg, agar 9000 mg, ultrapure water 1 L, pH 6.8±0.1.

[0058] Strain N3-4 has the ability to dissolve phosphorus, which can convert inorganic phosphorus in the soil into effective phosphorus that is easier for plants to absorb, thereby improving the plant's nutritional status and thus enhancing the plant's ability to tolerate salt stress. At the same time, strain N3-4 has the ability to produce iron carriers, which can increase the plant's intake of iron and thus improve the plant's ability to tolerate salt stress.

[0059] 5. Application of strain N3-4 in improving plant tolerance to salt stress

[0060] The strain N3-4 was inoculated into LB liquid medium and placed in a constant temperature incubator at 30°C and 140 rpm for 12 h. The bacterial liquid was then collected and centrifuged at 8000 rpm to obtain the bacterial cells. The bacterial cells were then washed three times with a sterile 0.9% sodium chloride solution and resuspended with a sterile 0.9% sodium chloride solution to prepare an OD value. 600 =0.25 of bacterial suspension.

[0061] Select plump cucumber seeds with no damage on the surface, soak them in 5% sodium hypochlorite solution for 8 minutes, and wash them with sterile distilled water for 3 times. Place the treated cucumber seeds in a petri dish covered with a layer of cotton, pour deionized water to soak the seeds, cover the petri dish with plastic wrap, and place them in a 30°C shaker until they germinate and grow two cotyledons (it takes 2-3 days).

[0062] The nutrient soil and vermiculite were mixed evenly at a mass ratio of 3:2, and then sterilized and packaged into culture boxes, 250g per box. 30mL of sterile sodium chloride solution of different concentrations (0g / L, 2.9g / L, 8.8g / L, 17.5g / L) was sprayed into each culture box and mixed well, so that the culture boxes were divided into non-salinization group, mild salinization group, moderate salinization group and severe salinization group. After testing, the conductivity of each group was 42.3μs / cm, 414.0μs / cm, 2028.0μs / cm and 3286.7μs / cm, respectively.

[0063] 20 mL of bacterial suspension (bacterial group) or 20 mL of sterile 0.9% sodium chloride solution (sterile group) were added to the above-mentioned culture boxes to obtain: no-salinization-bacterial group, no-salinization-sterile group, mild-salinization-bacterial group, mild-salinization-sterile group, moderate-salinization-bacterial group, moderate-salinization-sterile group, severe-salinization-bacterial group and severe-salinization-sterile group, and then 7 cucumber seedlings with two cotyledons were planted in each of the above-mentioned culture pots, and cultured at room temperature with a 16h / 8h light-dark cycle. Each culture pot was sprayed with an appropriate amount of sterile water every day, and each culture pot was sprayed with 30 mL of sterile sodium chloride solution of the corresponding concentration every 4 days. After 14 days of culture, the fresh weight and plant height of the cucumber seedlings in each group were measured.

[0064] After 14 days of cultivation, the growth of cucumber seedlings in each group was shown in Figure 5 , fresh weight statistics are shown in Figure 6 , the plant height statistics are shown in Figure 7 .

[0065] Depend on Figure 5 It can be seen that strain N3-4 can not only promote the growth of cucumber seedlings, but also significantly improve the survival rate of cucumber seedlings in severely salinized soil. Among them, the mortality rate of the severe salinization-sterile group was 57.1%, and the mortality rate of the severe salinization-bacterial group was 0, which significantly improved the salt tolerance of cucumber seedlings.

[0066] Depend on Figure 6 and Figure 7It can be seen that after adding strain N3-4 to the non-salinization group, the fresh weight of cucumber seedlings increased by 34.6%, and the plant height increased by 22.5%; after adding strain N3-4 to the mild salinization group, the fresh weight of cucumber seedlings increased by 41.6%, and the plant height increased by 10.6%; after adding strain N3-4 to the moderate salinization group, the fresh weight of cucumber seedlings increased by 38.1%, and the plant height increased by 12.7%; after adding strain N3-4 to the severe salinization group, the fresh weight of cucumber seedlings increased by 48.2%, and the plant height increased by 13.4%.

[0067] In summary, the novel Bacillus N3-4 from ginseng soil screened from saline-alkali soil in the present invention has both strong salt-alkali tolerance and can improve the salt stress tolerance of plants (cucumbers).

[0068] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A novel strain of Bacillus ginsengii N3-4, characterized in that: The novel Bacillus ginseng soil N3-4 was isolated from saline-alkali soil in Kenli District, Dongying City, Shandong Province, is salt-alkali tolerant, and can improve the ability of plants to tolerate salt stress. It is preserved in the General Microbiological Center of the China Microbiological Culture Collection Administration and is classified and named Neobacillus ginsengisoli The deposit number is CGMCC No.32574, the deposit date is November 11, 2024, and the deposit address is Beijing, China.

2. Use of the novel Bacillus ginsengii N3-4 according to claim 1 in improving the salt stress tolerance of plants, wherein the plant is cucumber.

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