A Bacillus zanthoxyli strain SCUEC18 with potassium and phosphorus solubilizing and growth promoting functions and its application

Abstract

The present invention discloses a Bacillus zanthoxyli SCUEC18 strain with the functions of potassium and phosphorus solubilization and growth promotion, belonging to the technical field of microorganisms. The present invention isolates a Bacillus zanthoxyli strain ( Bacillus zanthoxyli ) SCUEC18, with the preservation number of CCTCC NO: M 20241197; the Bacillus zanthoxyli SCUEC18 strain has good salt tolerance, acid and alkali tolerance, and drought tolerance, and the Bacillus zanthoxyli SCUEC18 strain has good potassium solubilization, inorganic phosphorus solubilization, organic phosphorus solubilization, nitrogen fixation, indole acetic acid production, and the ability to inhibit pathogenic fungi of crops. The Bacillus subtilis ATCC6633 strain has a promoting effect on the potassium solubilization ability of the Bacillus zanthoxyli SCUEC18 strain. In addition, the Bacillus zanthoxyli SCUEC18 strain can significantly improve soil fertility and / or promote plant growth.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Bacillus zanthoxyli SCUEC18 strain with potassium-solubilizing, phosphorus-solubilizing and growth-promoting functions and its application. Background Art

[0002] Plant growth-promoting rhizobacteria (PGPR) are a class of microorganisms living near the rhizosphere of plants, including bacteria, fungi, actinomycetes, etc. These microorganisms can promote plant growth, enhance plant disease resistance and improve environmental quality. It is mainly manifested in the production of growth hormones such as indole acetic acid and gibberellin to promote the growth and development of plant roots; the production of antibiotics and antibacterial proteins to inhibit soil-borne pathogenic microorganisms and reduce the occurrence of plant diseases; reducing the use of nitrogen fertilizers by converting nitrogen in the air into nitrogen compounds that can be absorbed by plants; degrading organic pollutants in the soil, such as pesticide residues and heavy metals, etc., to reduce the harm to the ecological environment. For example, Sphingomonas and Nocardioidaceae can degrade soil heavy metals; Nitrospira can oxidize nitrite in the soil into nitrate; Methylotrophs and Pseudoxanthomonas participate in the decomposition of dinitrotoluene and methylated aromatic compounds. Therefore, in modern agricultural production, plant growth-promoting rhizobacteria are widely used in organic agriculture and sustainable agriculture models to increase crop yields, improve soil quality and protect the ecological environment.

[0003] Bacillus zanthoxyli ( Bacillus zanthoxyli ) is a bacterium colonized in the rhizosphere of plants. In the prior art, it has been reported that Bacillus zanthoxyli has various functions, such as being able to synthesize urea, produce plant growth hormones and organic acids, promoting the root growth of crops such as tomatoes and flue-cured tobacco, increasing their nutrient absorption capacity and improving their immune systems; producing the volatile ketone tridecanone, attracting root-knot nematodes and cooperating with the use of chemical pesticides to play a role in controlling root-knot nematode disease, producing antibacterial drug AgNPs, inhibiting and killing a variety of bacteria and viruses; inhibiting the degradation of chloroplasts under high salt stress, enhancing the salt stress tolerance of plants, and inhibiting the growth of soil-borne pathogenic microorganisms.

[0004] However, there are few reports on Bacillus zanthoxyli in terms of potassium solubilization, inorganic phosphorus solubilization, organic phosphorus solubilization, nitrogen fixation, indole acetic acid production, inhibition of pathogenic fungi of crops, growth promotion, etc. Summary of the Invention

[0005] The object of the present invention is to provide a Bacillus zanthoxyli SCUEC18 strain with the functions of potassium solubilization, phosphorus solubilization and growth promotion, and its application. The Bacillus zanthoxyli SCUEC18 strain provided by the present invention has good salt tolerance, acid-base tolerance and drought tolerance, and the Bacillus zanthoxyli SCUEC18 strain has good potassium solubilization, inorganic phosphorus solubilization, organic phosphorus solubilization, nitrogen fixation, indole acetic acid production, and the ability to inhibit crop pathogenic fungi. The Bacillus subtilis ATCC6633 strain has a promoting effect on the potassium solubilization ability of the Bacillus zanthoxyli SCUEC18 strain. In addition, the Bacillus zanthoxyli SCUEC18 strain can significantly improve soil fertility and / or promote plant growth.

[0006] In a first aspect, the present invention provides a Bacillus zanthoxyli ( Bacillus zanthoxyli )SCUEC18 strain with the functions of potassium solubilization, phosphorus solubilization and growth promotion. The preservation number of the Bacillus zanthoxyli ( Bacillus zanthoxyli )SCUEC18 strain is CCTCC NO:M 20241197.

[0007] In some embodiments, the Bacillus zanthoxyli SCUEC18 strain is a Gram-positive bacterium, rod-shaped, with an optimum growth pH value of 6.0-9.0, for example, it can be 6.0, 7.0, 8.0, 9.0 or other values within this range; the highest tolerated NaCl concentration is 6.0% (W / V); and the Bacillus zanthoxyli SCUEC18 strain has at least one of the following functions: 1) salt tolerance; 2) acid-base tolerance; 3) drought tolerance; 4) potassium solubilization; 5) inorganic phosphorus solubilization; 6) organic phosphorus solubilization; 7) nitrogen fixation; 8) indole acetic acid production; 9) inhibition of crop pathogenic fungi.

[0008] The SCUEC18 strain was isolated from the rhizosphere soil of healthy-growing cigars in the cigar planting area of Laifeng County, Enshi Tujia and Miao Autonomous Prefecture, Hubei Province. The indole test and methyl red test of the SCUEC18 strain were positive, and the citrate test, Voges-Proskauer test and hydrogen sulfide test were negative.

[0009] The 16S rDNA of the SCUEC18 strain was sequenced, and its 16S rDNA sequence was subjected to BLAST multiple sequence alignment in NCBI (https: / / www.ncbi.nlm.nih.gov / ), and the phylogenetic tree of the type strain and the test strain was constructed using MEGA7. The results showed that the SCUEC18 strain had a 99% similarity with Bacillus zanthoxyli . Combining the morphological, physiological and biochemical characteristics and molecular biology identification results, the SCUEC18 strain was named Bacillus zanthoxyli ( Bacillus zanthoxyli )SCUEC18 strain.

[0010] In a second aspect, the present invention provides a biological bactericide, comprising any one of the above-mentioned Bacillus zanthoxyli ( Bacillus zanthoxyli ) strain SCUEC18.

[0011] In some embodiments, the biological bactericide comprises at least one of a liquid bactericide and a solid bactericide; wherein, the content of Bacillus zanthoxyli ( Bacillus zanthoxyli ) strain SCUEC18 in the liquid bactericide is 1.0×10 9 cfu / mL to 1.0×10 11 cfu / mL, for example, it can be 1.0×10 9 cfu / mL, 5.0×10 9 cfu / mL, 1.0×10 10 cfu / mL, 5.0×10 10 cfu / mL, 1.0×10 11 cfu / mL or other values within this range; the content of Bacillus zanthoxyli ( Bacillus zanthoxyli ) strain SCUEC18 in the solid bactericide is 1.0×10 9 cfu / g to 1.0×10 11 cfu / g, for example, it can be 1.0×10 9 cfu / g, 5.0×10 9 cfu / g, 1.0×10 10 cfu / g, 5.0×10 10 cfu / g, 1.0×10 11 cfu / g or other values within this range.

[0012] In some embodiments, the liquid bactericide is obtained by inoculating the above-mentioned Bacillus zanthoxyli ( Bacillus zanthoxyli ) strain SCUEC18 into a culture medium for cultivation.

[0013] It can be understood that the culture medium can be a common culture medium in the art as long as it can satisfy the normal growth of the Bacillus zanthoxyli ( Bacillus zanthoxyli ) strain SCUEC18. In the present invention, the culture medium is preferably LB medium.

[0014] In some embodiments, the solid bactericide is obtained by drying the above-mentioned liquid bactericide.

[0015] In some preferred embodiments, the drying is spray drying, and the process conditions for spray drying include: using corn starch as a carrier, controlling the material concentration to be 10-25% (W / V), for example, it can be 10%, 15%, 20%, 25% or other values within this range; the inlet air temperature is 100-200 °C, for example, it can be 100 °C, 120 °C, 140 °C, 160 °C, 180 °C, 200 °C or other values within this range; the feeding rate is 10-30 mL / min, for example, it can be 10 mL / min, 15 mL / min, 20 mL / min, 25 mL / min, 30 mL / min or other values within this range.

[0016] In some more preferred embodiments, the process conditions for spray drying include: using corn starch as a carrier, controlling the material concentration to be 15% (W / V), the inlet air temperature to be 160 °C, and the feeding rate to be 20 mL / min.

[0017] It can be understood that the drying can be conventionally selected according to actual use, and spray drying is preferred in the present invention; and the carrier for spray drying can also be conventionally selected according to actual use, and corn starch is preferred in the present invention.

[0018] In a third aspect, the present invention provides a combined microbial agent, including any one of the above-mentioned Bacillus zanthoxyli SCUEC18 strain and Bacillus subtilis ATCC6633 strain; wherein, the Bacillus subtilis ATCC6633 strain has a promoting effect on the potassium-releasing ability of the Bacillus zanthoxyli SCUEC18 strain.

[0019] In a fourth aspect, the present invention provides the application of any one of the above-mentioned Bacillus zanthoxyli ( Bacillus zanthoxyli )SCUEC18 strain, any one of the above-mentioned biological microbial agents or the above-mentioned combined microbial agent in enhancing soil fertility.

[0020] In some embodiments, enhancing soil fertility specifically includes: enhancing at least one of the number of microorganisms in the soil, the content of available nitrogen in the soil, the content of available phosphorus in the soil, and the content of available potassium in the soil.

[0021] In some embodiments, the microorganisms include potassium-releasing microorganisms, and the available nitrogen includes nitrate nitrogen.

[0022] In a fifth aspect, the present invention provides the application of any one of the above-mentioned Bacillus zanthoxyli ( Bacillus zanthoxyli )SCUEC18 strain, any one of the above-mentioned biological microbial agents or the above-mentioned combined microbial agent in promoting plant growth and / or inhibiting pathogenic fungi of crops.

[0023] In some embodiments, the plants include cigars.

[0024] The beneficial effects of the present invention are as follows: Different from the prior art, a Bacillus zanthoxyli strain ( Bacillus zanthoxyli ) SCUEC18 is isolated for the first time in the present invention. The Bacillus zanthoxyli strain SCUEC18 has good salt tolerance, acid and alkali tolerance, and drought tolerance. In addition, the Bacillus zanthoxyli strain SCUEC18 has good abilities of potassium solubilization, inorganic phosphorus solubilization, organic phosphorus solubilization, nitrogen fixation, indole-3-acetic acid production, and inhibition of crop pathogenic fungi. Moreover, the Bacillus zanthoxyli strain SCUEC18 can significantly improve soil fertility and / or promote plant growth. Therefore, it has good application prospects. Description of the Drawings

[0025] Figure 1 It is a potassium solubilization circle diagram of the SCUEC18 strain on the modified Aleksandrov solid medium in Example 1 of the present invention;

[0026] Figure 2 It is a Gram staining diagram of the SCUEC18 strain in Example 1 of the present invention;

[0027] Figure 3 It is a phylogenetic tree diagram of the SCUEC18 strain in Example 1 of the present invention;

[0028] Figure 4 It is a potassium solubilization diagram of the SCUEC18 strain in Example 2 of the present invention;

[0029] Figure 5 It is an inorganic phosphorus solubilization diagram of the SCUEC18 strain in Example 3 of the present invention;

[0030] Figure 6 It is an organic phosphorus solubilization diagram of the SCUEC18 strain in Example 4 of the present invention;

[0031] Figure 7 It is a growth diagram of the SCUEC18 strain on the Ashby solid medium in Example 5 of the present invention. Among them, A and C are negative control strains without nitrogen fixation ability, and B and D are the Bacillus zanthoxyli strain SCUEC18;

[0032] Figure 8 It is a qualitative detection result diagram of indole-3-acetic acid production by the SCUEC18 strain in Example 6 of the present invention. Among them, A is the LB medium without inoculation of bacteria, and B is the supernatant of the fermentation broth inoculated with the SCUEC18 strain;

[0033] Figure 9 It is a diagram of the indole-3-acetic acid content produced by the SCUEC18 strain in Example 6 of the present invention;

[0034] Figure 10 It is a diagram of the growth amount of the SCUEC18 strain in the LB medium with different NaCl concentrations in Example 7 of the present invention;

[0035] Figure 11 It is the growth quantity diagram of strain SCUEC18 in LB medium with different pH values in Example 8 of the present invention;

[0036] Figure 12 It is the growth quantity diagram of strain SCUEC18 in LB medium with different polyethylene glycol (PEG 6000 ) concentrations in Example 9 of the present invention.

[0037] Biological preservation

[0038] The strain provided by the present invention is preserved in the China Center for Type Culture Collection (CCTCC), with the preservation address: Wuhan University, Wuhan, China, preservation number: CCTCC NO: M 20241197, preservation time: June 11, 2024, taxonomic name: Bacillus zanthoxyli SCUEC18 ( Bacillus zanthoxyli SCUEC18). Specific implementation manners

[0039] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0040] For the experimental methods without specific conditions noted in the embodiments, they are usually carried out according to the conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in "Molecular Cloning: A Laboratory Manual" by M.R. Green, "Molecular Biology" by Robert·F·Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the embodiments can be obtained from commercial channels without special instructions.

[0041] In the present invention, the following media are used:

[0042] Modified Aleksandrov medium: sucrose 5.0 g, MgSO4·7H2O 0.5 g, Na2HPO4 2.0 g, CaCO3 0.1 g, FeCl3·6H2O 0.005 g, potassium feldspar powder 1.0 g, deionized water 1.0 L, pH 7.0, and 0.25% bromothymol blue is added to each liter of the medium.

[0043] LB medium: 10.0 g of NaCl, 10.0 g of tryptone, 5.0 g of yeast extract powder, 1.0 L of deionized water, pH 7.0.

[0044] LA medium: Add 15 - 20 g / L of agar to the LB medium.

[0045] Potassium - releasing medium: 10.0 g of sucrose, 0.5 g of MgSO4·7H2O, 1.0 g of CaCO3, 1.0 g of (NH4)2SO4, 0.1 g of NaCl, 0.5 g of yeast extract powder, 2.0 g of Na2HPO4, 10.0 g of potassium feldspar powder, 1.0 L of deionized water, pH 7.0.

[0046] Meng Jinna inorganic phosphorus medium: 10.0 g of glucose, 0.03 g of MnSO4·4H2O, 0.3 g of KCl, 0.3 g of NaCl, 0.5 g of (NH4)2SO4, 0.3 g of MgSO4·7H2O, 2.5 g of Ca3(PO4)2, 0.03 g of FeSO4·7H2O, 1.0 L of deionized water, pH 7.0.

[0047] Meng Jinna organic phosphorus medium: 10.0 g of glucose, 0.3 g of KCl, 0.3 g of NaCl, 0.5 g of (NH4)2SO4, 0.3 g of MgSO4·7H2O, 0.03 g of FeSO4·7H2O, 0.03 g of MnSO4·4H2O, 2.5 g of CaCO3, 0.2 g of lecithin, 1.0 L of deionized water, pH 7.0.

[0048] Ashby medium: 10.0 g of mannitol, 0.2 g of NaCl, 0.2 g of MgSO4·7H2O, 1.0 g of CaCO3, 0.2 g of KH2PO4, 0.2 g of CaSO4·2H2O, 1.0 L of deionized water, pH 7.0.

[0049] In the above media, if you want to prepare a solid medium, you need to add 15 - 20 g / L of agar. If you want to prepare a liquid medium, you don't need to add agar.

[0050] In the present invention, the Salkowski colorimetric solution is: 1 mL of 0.5 mol / L FeCl3, 30 mL of H2SO4, 50 mL of deionized water.

[0051] Example 1 Isolation, purification and identification of strain SCUEC18

[0052] 1.1 Directed enrichment and isolation of strain SCUEC18

[0053] The soil sample was taken from the rhizosphere soil of healthy-growing cigars in the cigar-growing area of Laifeng County, Enshi Tujia and Miao Autonomous Prefecture, Hubei Province. 10 g of the above-mentioned soil sample was accurately weighed into a triangular flask, 90 mL of sterile water was added, and after culturing at 37 °C and 150 rpm for 12 h, 1 mL of the bacterial solution was then aspirated and mixed with 9 mL of sterile water to obtain 10 -1 diluted bacterial solution. Then, 1 mL was aspirated from the 10 -1 diluted solution and added to 9 mL of sterile water to obtain 10 -2 diluted bacterial solution. The above operation was repeated to obtain 10 -3 、10 -4 、10 -5 、10 -6 diluted bacterial solutions. 200 μL of the bacterial solutions with different dilution concentrations were respectively spread on the modified Aleksandrov solid medium and cultured at 37 °C for 48 h, and then streaked and isolated and purified multiple times to obtain 20 microorganisms with potassium-dissolving halos. Among them, the strain with a relatively large potassium-dissolving halo ( Figure 1 ), was named strain SCUEC18.

[0054] 1.2 Identification of strain SCUEC18

[0055] Physiological and biochemical identification: Referring to the "Manual for Systematic Identification of Common Bacteria", indole test, methyl red test, Voges-Proskauer test, citrate test, and hydrogen sulfide test were carried out to determine the physiological and biochemical characteristics of strain SCUEC18. The results are shown in Table 1.

[0056] Table 1 Physiological and biochemical characteristics of strain SCUEC18

[0057]

[0058] It can be seen from the results in Table 1 that the indole test and methyl red test of strain SCUEC18 were positive, and the citrate test, Voges-Proskauer test, and hydrogen sulfide test were negative.

[0059] Gram staining was performed on strain SCUEC18, and the results are as Figure 2 shown.

[0060] From Figure 2 it can be seen that strain SCUEC18 was rod-shaped, purple after Gram staining, and was a Gram-positive bacterium.

[0061] Molecular biology identification: The genomic DNA of strain SCUEC18 was extracted, and using the total DNA of strain SCUEC18 as a template, the 16S rDNA sequence was amplified using primers 16S rDNA-F and 16S rDNA-R. Among them, the nucleotide sequences of 16S rDNA-F and 16S rDNA-R are as follows:

[0062] 16S rDNA-F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2);

[0063] 16S rDNA-R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 3);

[0064] The amplified product was sent to Tsingke for sequencing. Its 16S rDNA sequence is as shown in SEQ ID NO: 1, and the sequence length is 1425 bp. This sequence was subjected to BLAST multiple sequence alignment in NCBI (https: / / www.ncbi.nlm.nih.gov / ), and MEGA7 was used to construct the phylogenetic tree of the type strain and the test strain. The results are as Figure 3 shown.

[0065] It can be seen from Figure 3 that the SCUEC18 strain has 99% similarity with Bacillus zanthoxyli . Combining the morphological, physiological and biochemical characteristics and the results of molecular biological identification, the strain SCUEC18 was named Bacillus zanthoxyli ( Bacillus zanthoxyli ) SCUEC18 strain.

[0066] Example 2 Determination of potassium-dissolving ability of Bacillus zanthoxyli SCUEC18 strain

[0067] The Bacillus zanthoxyli SCUEC18 strain was inoculated on an LA plate for activation for 12 h. A single colony was picked and inoculated into an LB medium, and cultured at 30 °C and 150 rpm for 12 h. This was the seed solution. After washing the seed solution three times with sterile water, it was inoculated into the potassium-dissolving medium at an inoculation amount of 0.5%, and cultured at 30 °C and 150 rpm for 336 h. Samples were taken at 0, 24, 120, 216, and 312 h, and the soluble potassium content in the samples was detected. The control group added an equal amount of sterile water. The experiment was set up in three parallels. The results are as Figure 4 shown.

[0068] It can be seen from Figure 4It can be seen that in the control group, the soluble potassium contents in the samples at 0, 24, 120, 216, and 312 h were 0.26, 0.39, 0.13, 0.52, and 0.39 μg / mL, respectively. For Bacillus zanthoxyli SCUEC18 strain, during the first 24 h of cultivation, the soluble potassium content increased rapidly and reached 21.53 μg / mL at 24 h. During the period of 24 - 216 h of cultivation, the soluble potassium content increased steadily, and the soluble potassium content was 32.51 μg / mL at 216 h. During the period of 216 - 312 h of cultivation, the increase in soluble potassium content tended to be stable, and the soluble potassium content was 33.09 μg / mL at 312 h. The results show that Bacillus zanthoxyli SCUEC18 strain has high potassium-solubilizing ability.

[0069] Example 3 Determination of inorganic phosphorus-solubilizing ability of Bacillus zanthoxyli SCUEC18 strain

[0070] Inoculate Bacillus zanthoxyli SCUEC18 strain on LA plate for activation for 12 h, pick a single colony and inoculate it into LB medium, culture at 30 °C and 150 rpm for 12 h. This is the seed liquid. After washing the seed liquid three times with sterile water, inoculate it into Mengjinna inorganic phosphorus medium at an inoculation amount of 0.5%, culture at 30 °C and 150 rpm for 168 h, and take samples at 0, 24, 48, 96, and 144 h, and detect the soluble inorganic phosphorus content in the samples. Three parallel experiments are set, and the results are as Figure 5 shown.

[0071] From Figure 5 it can be seen that within the cultivation period of 0 - 96 h, the soluble inorganic phosphorus content in the medium of Bacillus zanthoxyli SCUEC18 strain increased from 0 g / mL to 28.04 g / mL; at 144 h, the soluble inorganic phosphorus content in the medium decreased slightly to 23.57 g / mL. The results show that Bacillus zanthoxyli SCUEC18 strain has good inorganic phosphorus-solubilizing ability.

[0072] Example 4 Determination of organic phosphorus-solubilizing ability of Bacillus zanthoxyli SCUEC18 strain

[0073] Inoculate Bacillus zanthoxyli SCUEC18 strain on LA plate for activation for 12 h, pick a single colony and inoculate it into LB medium, culture at 30 °C and 150 rpm for 12 h. This is the seed liquid. After washing the seed liquid three times with sterile water, inoculate it into Mengjinna organic phosphorus medium at an inoculation amount of 0.5%, culture at 30 °C and 150 rpm for 240 h, and take samples at 0, 24, 72, 120, 168, and 216 h, and detect the soluble organic phosphorus content in the samples. Three parallel experiments are set, and the results are as Figure 6 shown.

[0074] FromFigure 6 It can be seen that within the range of 0 - 168 h of culture time, the soluble organic phosphorus content in the medium of Bacillus zanthoxyli SCUEC18 strain increased from 0 g / mL to 29.83 g / mL; when cultured to 216 h, the soluble organic phosphorus content decreased slightly to 28.12 g / mL. The results show that Bacillus zanthoxyli SCUEC18 strain has good ability to decompose organic phosphorus.

[0075] Example 5 Determination of nitrogen fixation ability of Bacillus zanthoxyli SCUEC18 strain

[0076] Inoculate Bacillus zanthoxyli SCUEC18 strain on LA plate for activation for 12 h, then use toothpick to spot-inoculate on Ashby solid medium, culture at 30 °C for 24 h, after continuous subculture for three times, observe the plate results, and the results are as Figure 7 shown.

[0077] From Figure 7 it can be seen that compared with the negative control strain which cannot grow, SCUEC18 strain can still grow normally after three times of subculture. The results show that Bacillus zanthoxyli SCUEC18 strain has good nitrogen fixation ability.

[0078] Example 6 Determination of indole - 3 - acetic acid production ability of Bacillus zanthoxyli SCUEC18 strain

[0079] Inoculate Bacillus zanthoxyli SCUEC18 strain on LA plate for activation for 12 h, pick a single colony and inoculate it into LB liquid medium containing 0.1 g / L L - tryptophan, culture in a shaking incubator at 30 °C and 150 rpm for 24 h. Take 400 μL of the bacterial suspension and drop it into a 12 - well plate, at the same time add 400 μL of Salkowski colorimetric solution, add 400 μL of non - inoculated LB medium as a blank control, and observe after placing in the dark for 30 min. The results are as Figure 8 shown.

[0080] From Figure 8 it can be seen that compared with the blank control whose color has no change, the supernatant color of the fermentation broth inoculated with SCUEC18 strain turns red. The results show that Bacillus zanthoxyli SCUEC18 strain has the ability to produce indole - 3 - acetic acid.

[0081] Furthermore, inoculate SCUEC18 strain into LB medium containing 0.1 g / L L - tryptophan, control the OD 600 of the bacterial liquid to be 0.1, culture at 30 °C and 150 rpm for 120 h, and sample at 0, 24, 48, 72, 96 h, and determine the content of indole - 3 - acetic acid. Set three parallel experiments, and the results are as Figure 9 shown.

[0082] From Figure 9 It can be seen that within 0 - 48 h of cultivation of Bacillus zanthoxyli SCUEC18 strain, the indole - 3 - acetic acid content in the SCUEC18 strain culture medium increased rapidly. The indole - 3 - acetic acid content was 12.80 mg / L at 48 h. From 48 - 72 h, the indole - 3 - acetic acid content in the Bacillus zanthoxyli SCUEC18 strain culture medium increased slowly and reached 13.01 mg / L at 72 h. When cultured to 96 h, the indole - 3 - acetic acid content decreased to 10.23 mg / L. The results show that Bacillus zanthoxyli SCUEC18 strain has good indole - 3 - acetic acid production ability.

[0083] Example 7 Antagonistic activity of Bacillus zanthoxyli SCUEC18 strain against pathogenic bacteria

[0084] 7.1 Antibacterial activity of SCUEC18 strain against plant pathogenic bacteria

[0085] Taking Fusarium oxysporum f. sp. niveum and Alternaria solani as indicator bacteria, the antibacterial activity of SCUEC18 strain against different plant pathogenic bacteria was studied.

[0086] Specifically, take Bacillus zanthoxyli SCUEC18 strain, inoculate it on LA plate and activate for 12 h, conduct antibacterial experiment with bacterial blocks, measure the diameter of the antibacterial zone, and use the conventional Bacillus subtilis ATCC6633 strain as a control. The measurement results are shown in Table 2.

[0087] Table 2 Antibacterial effects of SCUEC18 strain and ATCC6633 strain

[0088]

[0089] It can be seen from Table 2 that the diameters of the antibacterial zones of Bacillus zanthoxyli SCUEC18 strain against Fusarium oxysporum f. sp. niveum and Alternaria solani are 32.66 and 47.2 mm respectively. While the conventional Bacillus subtilis ATCC6633 strain has no inhibitory effect. The results show that the Bacillus zanthoxyli SCUEC18 strain screened in the present invention has a significant inhibitory effect on Fusarium oxysporum f. sp. niveum and Alternaria solani compared with the conventional Bacillus subtilis, and can be widely applied to the prevention and control of various different crop diseases.

[0090] Example 8 Determination of the promoting effect of Bacillus subtilis ATCC6633 strain on the potassium - releasing ability of Bacillus zanthoxyli SCUEC18 strain

[0091] The Bacillus zanthoxyli SCUEC18 strain and the Bacillus subtilis ATCC6633 strain were respectively inoculated on LA plates and activated for 12 h. Single colonies were picked and inoculated into LB medium, and cultured at 30 °C and 150 rpm for 12 h. This was the seed solution. After washing the seed solution three times with sterile water, the Bacillus subtilis ATCC6633 strain, the Bacillus zanthoxyli SCUEC18 strain, and their mixture of the SCUEC18 strain and the ATCC6633 strain were inoculated into potassium-solubilizing medium and cultured at 30 °C and 150 rpm for 144 h. Samples were taken at 0, 24, 48, 72, and 120 h to detect the soluble potassium content in the culture solution. Three parallel experiments were set up, and the results are shown in Table 3.

[0092] Table 3 Potassium-solubilizing amounts of the SCUEC18 strain, the ATCC6633 strain, and their mixed inoculation

[0093]

[0094] As can be seen from Table 3, the soluble potassium contents in the culture solution of the Bacillus subtilis ATCC6633 strain at 0, 24, 48, 72, and 120 h were 0.00, 1.26, 2.89, 2.92, and 1.60 μg / mL respectively. The soluble potassium contents in the culture solution of the Bacillus zanthoxyli SCUEC18 strain at 0, 24, 48, 72, and 120 h were 0.00, 21.84, 24.31, 25.86, and 27.17 μg / mL respectively. While the soluble potassium contents in the culture solution of the mixed inoculation of the SCUEC18 strain and the ATCC6633 strain at 0, 24, 48, 72, and 120 h were 0.00, 29.88, 35.56, 41.04, and 30.80 μg / mL respectively, which were higher than the sum of the soluble potassium contents of the separate cultures of the Bacillus zanthoxyli SCUEC18 strain and the Bacillus subtilis ATCC6633 strain. The results showed that the Bacillus subtilis ATCC6633 strain had a promoting effect on the potassium-solubilizing ability of the Bacillus zanthoxyli SCUEC18 strain.

[0095] Example 9 Determination of the salt tolerance of the Bacillus zanthoxyli SCUEC18 strain

[0096] Based on LB medium as the basal medium, different NaCl concentrations (2.0%, 4.0%, 6.0%, 8.0% (W / V)) were added to the LB medium. The Bacillus zanthoxyli SCUEC18 strain was spread on LA plates and activated for 12 h. Single colonies were picked and inoculated into LB medium containing different concentrations of NaCl, and cultured at 30 °C and 150 rpm for 24 h. Three parallel experiments were set up, and the results are as Figure 10 shown.

[0097] From Figure 10It can be seen that when the NaCl concentration is 0.0 - 6.0%, the OD of Bacillus zanthoxyli SCUEC18 strain 600 increases to between 2.079 and 1.900. When the NaCl concentration increases to 8.0%, the OD 600 value drops to 0.078. The results show that the maximum NaCl concentration tolerated by Bacillus zanthoxyli SCUEC18 strain is 6.0%.

[0098] Example 10 Determination of acid and alkali tolerance of Bacillus zanthoxyli SCUEC18 strain

[0099] Using LB medium as the basal medium, LB media with pH values of 4.0, 6.0, 7.0, 8.0, 9.0, and 10.0 were prepared respectively. The Bacillus zanthoxyli SCUEC18 strain was spread on the LA plate for activation for 12 h, and single colonies were picked and inoculated into LB media with different pH values. It was cultured at 30 °C and 150 rpm for 24 h. Three parallel experiments were set up to measure the OD 600 value, and the results are as Figure 11 shown.

[0100] From Figure 11 it can be seen that when the pH value of the medium for Bacillus zanthoxyli SCUEC18 strain is between 6.0 and 9.0, the OD 600 value is between 1.686 and 2.079. When the pH value is 4.0, the OD 600 value is 0.215. When the pH value of the medium is 10.0, the OD 600 value is 0.314. The results show that Bacillus zanthoxyli SCUEC18 strain can grow well when the pH value of the medium is between 6.0 and 9.0.

[0101] Example 11 Determination of drought tolerance of Bacillus zanthoxyli SCUEC18 strain

[0102] Using LB medium as the basal medium, LB media containing polyethylene glycol (PEG 6000 ) at concentrations of 0.0%, 10.0%, 20.0%, 25.0%, and 30.0% were prepared respectively. The Bacillus zanthoxyli SCUEC18 strain was spread on the LA plate for activation for 12 h, and single colonies were picked and inoculated into LB media containing different concentrations of polyethylene glycol (PEG 6000 ). It was cultured at 30 °C and 150 rpm for 24 h. Three parallel experiments were set up to measure the OD 600 value, and the results are as Figure 12 shown.

[0103] From Figure 12 it can be seen that with the increase of PEG 6000With the increase in concentration, the OD of Bacillus zanthoxyli SCUEC18 strain 600 value gradually decreases. When the PEG 6000 concentration is 0.0%, the OD 600 value is 2.079; when the PEG 6000 concentration is 10.0%, the OD 600 value is 1.244; when the PEG 6000 concentration is 20.0%, the OD 600 value is 0.918; when the PEG 6000 concentrations are 25.0% and 30.0%, the OD 600 values of Bacillus zanthoxyli SCUEC18 strain decrease significantly, being 0.640 and 0.590 respectively. The results show that Bacillus zanthoxyli SCUEC18 strain has good drought tolerance performance.

[0104] Example 12 Application test of Bacillus zanthoxyli SCUEC18 strain in improving soil fertility

[0105] 12.1 Preparation of liquid and solid bacterial agents of Bacillus zanthoxyli SCUEC18 strain

[0106] The Bacillus zanthoxyli SCUEC18 strain preserved on the slant was activated and inoculated on LA medium, and incubated at a constant temperature of 30 °C for 12 h; the bacteria on the activated medium were picked and inoculated into LB medium, and the culture temperature was 30 °C, and cultured for 24 h to obtain the culture solution, which is the liquid bacterial agent of Bacillus zanthoxyli SCUEC18 strain.

[0107] Furthermore, part of the liquid bacterial agent of Bacillus zanthoxyli SCUEC18 strain was spray-dried to obtain a solid bacterial agent. Among them, the preferred process conditions for spray drying are: corn starch as the carrier for spray drying, the material concentration is 15%, the inlet air temperature is 160 °C, and the feeding speed is 20 mL / min, so that the viable count of Bacillus zanthoxyli SCUEC18 strain in the solid bacterial agent is 1.0×10 9 cfu / g~1.0×10 11 cfu / g.

[0108] 12.2 Test on the effect of Bacillus zanthoxyli SCUEC18 strain in improving soil fertility

[0109] The test soil is the soil in the cigar planting area of Enshi Tujia and Miao Autonomous Prefecture, Hubei Province. The sand and gravel are sieved out with a 2 mm sieve, and sterilized at 121 °C for 1 h. After cooling, the soil is filled in a 1000 mL beaker.

[0110] Inoculate 100 mL of Bacillus zanthoxyli SCUEC18 liquid inoculant into the above-mentioned soil-filled beakers. For the control group, mix 100 mL of LB liquid medium with the soil. Set 5 replicates for each treatment group. After 30 days, measure the soil microbial quantity and the contents of soil nitrate nitrogen, available phosphorus, and available potassium. The results are shown in Table 4 below.

[0111] Table 4 Microbial quantity and the contents of soil nitrate nitrogen, available phosphorus, and available potassium

[0112]

[0113] As can be seen from Table 4, compared with the control group, the soil bacteria, potassium-solubilizing bacteria, and the contents of soil nitrate nitrogen, available phosphorus, and available potassium in the soil with the addition of strain SCUEC18 are: 12.61×10 8 CFU / g, 11.02×10 7 CFU / g, 35.72 mg / kg, 45.08 mg / kg, 277.21 mg / kg, which are significantly higher than those in the control group: 9.22×10 8 CFU / g, 1.86×10 7 CFU / g, 29.67 mg / kg, 36.87 mg / kg, 260.72 mg / kg. The results show that Bacillus zanthoxyli SCUEC18 strain has the effect of increasing the contents of soil nitrate nitrogen, available phosphorus, and available potassium, and can significantly improve soil fertility.

[0114] Example 13 Pot experiment on the promotion of cigar growth by Bacillus zanthoxyli SCUEC18 strain

[0115] Use the solid inoculant of Bacillus zanthoxyli SCUEC18 prepared in Example 12 (1 g inoculant per plant, dissolved in 100 mL of water for root irrigation) to treat at the transplanting stage and the rosette stage respectively. Use water for root irrigation as the control. There are 80 plants for each treatment, with 3 replicates. Measure the plant height, stem girth, leaf number, leaf length, and leaf width at the rosette stage and the vigorous growth stage. The results are shown in Table 5.

[0116] Table 5 Statistical results of cigar plant height, stem girth, leaf number, leaf length, and leaf width

[0117]

[0118] As can be seen from Table 5, the stem girth, number of leaves, leaf length and leaf width of the cigar plants with the Bacillus zanthoxyli SCUEC18 strain inoculum irrigated at the rosette stage were 6.45 cm, 12.40, 38.99 cm and 24.27 cm respectively, all higher than those of the control group of cigar plants; the plant height, stem girth, number of leaves, leaf length and leaf width of the cigar plants with the Bacillus zanthoxyli SCUEC18 strain inoculum irrigated at the vigorous growth stage were 118.84 cm, 7.87 cm, 22.60, 46.83 cm and 29.19 cm respectively, all significantly higher than those of the control group of cigar plants. The results show that the Bacillus zanthoxyli SCUEC18 strain has a promoting effect on the growth of cigar plants.

[0119] In summary, the Bacillus zanthoxyli SCUEC18 strain provided by the present invention has good salt tolerance, acid and alkali tolerance and drought tolerance, and the Bacillus zanthoxyli SCUEC18 strain has good potassium solubilizing ability, inorganic phosphorus solubilizing ability, organic phosphorus solubilizing ability, nitrogen fixation ability, indole acetic acid production ability. In addition, the Bacillus zanthoxyli SCUEC18 strain can significantly improve soil fertility and / or promote plant growth.

[0120] It should be noted that the above embodiments all belong to the same inventive concept. The descriptions of the embodiments have their own emphases. For the parts not described in detail in individual embodiments, reference can be made to the descriptions in other embodiments.

[0121] The above embodiments only represent the implementation modes of the present invention. The descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

[0122] 16S rDNA sequence of the SCUEC18 strain (SEQ ID NO: 1)

[0123]

Claims

1. A Bacillus zanthoxyli SCUEC18 strain with the functions of potassium and phosphorus dissolution and growth promotion, characterized in that, The preservation number of the Bacillus zanthoxyli SCUEC18 strain is CCTCC NO: M 20241197.

2. The Bacillus zanthoxyli strain SCUEC18 according to claim 1, characterized in that, The Bacillus zanthoxyli SCUEC18 strain is a Gram-positive bacterium, rod-shaped, with an optimum growth pH value of 6.0 - 9.0 and a maximum tolerated NaCl concentration of 6.0%; and the Bacillus zanthoxyli SCUEC18 strain has at least one of the following functions: 1) salt tolerance; 2) acid and alkali tolerance; 3) drought tolerance; 4) potassium solubilization; 5) inorganic phosphorus solubilization; 6) organic phosphorus solubilization; 7) nitrogen fixation; 8) indole acetic acid production; 9) inhibition of crop pathogenic fungi.

3. A biological bactericide, characterized in that, It includes the Bacillus zanthoxyli SCUEC18 strain according to any one of claims 1 - 2.

4. The biological bacterial agent according to claim 3, characterized in that, The biological bactericide includes at least one of a liquid bactericide and a solid bactericide; Among them, the content of Bacillus zanthoxyli SCUEC18 strain in the liquid microbial inoculum is 1.0×10 9 cfu / mL to 1.0×10 11 cfu / mL; the content of Bacillus zanthoxyli SCUEC18 strain in the solid microbial inoculum is 1.0×10 9 cfu / g to 1.0×10 11 cfu / g.

5. A combined microbial inoculum, characterized in that, It includes the Bacillus zanthoxyli SCUEC18 strain according to any one of claims 1 - 2 and the Bacillus subtilis ATCC6633 strain; Among them, the Bacillus subtilis ATCC6633 strain has a promoting effect on the potassium solubilization ability of the Bacillus zanthoxyli SCUEC18 strain.

6. Use of the Bacillus zanthoxyli SCUEC18 strain according to any one of claims 1 - 2, the biological bactericide according to any one of claims 3 - 4, or the combined bactericide according to claim 5 in improving soil fertility.

7. The application according to claim 6, wherein The improvement of soil fertility specifically includes: improving at least one of the number of microorganisms in the soil, the content of available nitrogen in the soil, the content of available phosphorus in the soil, and the content of available potassium in the soil.

8. The application according to claim 7, characterized in that, The microorganisms include potassium-solubilizing microorganisms, and the available nitrogen includes nitrate nitrogen.

9. Use of the Bacillus zanthoxyli SCUEC18 strain according to any one of claims 1 - 2, the biological bactericide according to any one of claims 3 - 4, or the combined bactericide according to claim 5 in inhibiting crop pathogenic fungi; The crop pathogenic fungi are Fusarium oxysporum f. sp. niveum and Alternaria solani.

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