Bacillus marisflavi with high yield of iaa and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a strain of Caldibacillus kokeshiiformis and application thereof. The Caldibacillus kokeshiiformis is specifically Caldibacillus kokeshiiformis MT with a preservation number of CGMCC NO. 25356. The strain has the abilities of high indole-3-acetic acid (IAA) production, organic phosphorus decomposition and siderophore production, can be used for producing IAA, and can be used in the growth of plants, can obviously promote the growth of crops, shorten the cultivation time of crops, and improve the yield.

Description

Technical fields:

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus simius that produces high levels of IAA and has the ability to decompose organic phosphorus and produce ferrophosphate, and its applications. Background technology:

[0002] Crops are often influenced by rhizosphere microorganisms during their growth and development. Some rhizosphere microorganisms can metabolize substances that promote plant growth, such as indoleacetic acid (IAA) and gibberellins. IAA, as a plant hormone, can stimulate plant cell growth and, at appropriate concentrations, significantly promote the growth of roots, stems, and leaves, shortening cultivation time and increasing yield. Other rhizosphere microorganisms can decompose poorly soluble nutrients in the soil, such as insoluble organic phosphorus, metabolizing them into available phosphorus that plants can directly absorb and utilize, increasing phosphorus uptake, reducing phosphate fertilizer use, and promoting crop growth. Still other rhizosphere microorganisms can secrete heparin, which can specifically bind to Fe in the soil. 3+ chelates are formed when Fe in the soil 3+ Even at very low concentrations, it can still chelate, and the chelate can be directly absorbed and utilized by plants, thereby improving the iron deficiency problem in crops. Moreover, the iron chelate can compete with plant pathogens for limited iron ions, inhibiting the growth of pathogens and thus playing a role in preventing and controlling plant diseases.

[0003] Chinese patent document CN111484946A discloses a thermostable IAA-producing Bacillus strain and its application. This strain is *Bacillus belye*, which promotes seed germination, can solubilize phosphorus and fix nitrogen, improves soil fertility, provides sufficient nutrition for plant growth, enhances crop resistance, and has heat resistance, which is beneficial for the composting of agricultural waste. Chinese patent document CN112342162A discloses a phosphate-solubilizing bacterium producing IAA and its application. This phosphate-solubilizing bacterium is *Bacillus licheniformis*, which promotes plant growth through IAA production and phosphorus solubilization, achieving increased yield and income. High utilization rates of essential elements can reduce fertilizer use and protect the ecological environment, making it widely applicable in agricultural production. Chinese patent document CN111057665A discloses a cellulose-degrading bacterium n3 that produces IAA and its applications. Cellulose-degrading bacterium n3 has a strong ability to produce CMC enzymes and can also produce IAA. The CMC enzyme activity can reach up to 24.96 U / mL, and the IAA secretion can reach up to 19.07 mg / L. This bacterium can be used to prepare straw decomposition-promoting bacteria agents that produce IAA and / or CMC enzymes or have growth-promoting functions, thereby being applied to straw decomposition and crop growth promotion, achieving increased straw return efficiency and crop yield. Chinese patent document CN114517165A discloses a salt-tolerant growth-promoting bacterium KVP3 that produces IAA and EPS, and its preparation. The salt-tolerant growth-promoting bacterium KVP3 can promote the germination and growth of plant seeds under high salt concentrations and can secrete IAA and EPS. The maximum secretion of IAA can reach 305.9 mg / L, and the maximum secretion of EPS can reach 0.37 g / 100 mL. The salt-tolerant growth-promoting bacterium KVP3 can be used to prepare novel microbial inoculants such as salt-tolerant seed soaking agents that produce IAA and / or EPS or have growth-promoting functions, so as to promote crop growth and increase crop yield in saline soil.

[0004] The strains disclosed in the aforementioned patent documents all have the ability to synthesize IAA, but their IAA synthesis ability is limited. Therefore, screening for high-yield IAA strains is an urgent need for those skilled in the art. Summary of the Invention:

[0005] To solve the above-mentioned technical problems, the present invention obtained a strain of Bacillus pyrophorus from cow manure compost fermentation products. It has the ability to produce high levels of indoleacetic acid, decompose organic phosphorus, and produce iron phosphate, and has wide applications in the field of agricultural microbiology.

[0006] One of the technical solutions provided by this invention is a strain of *Caldibacillus skokeshiiformis* MT, which was deposited on July 20, 2022, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCCNO.25356.

[0007] The second technical solution provided by this invention is the application of *Caldibacillus kokeshiiformis* MT, particularly in the production of IAA; specifically, the method for producing IAA by fermentation using *Caldibacillus kokeshiiformis* MT is as follows:

[0008] Inoculate the Bacillus tinctoria MT seed culture into IAA-producing liquid medium at an inoculation rate of 5-15% (v / v) and incubate at 35-55℃ and 150-200rpm for 8-72h.

[0009] IAA-producing liquid culture medium: yeast extract 4.5-5.5g, tryptone 8-12g, sodium chloride 10-20g, L-tryptophan 0-500mg, distilled water 1L, pH 7.2;

[0010] Preferably, the fermentation temperature is 50°C;

[0011] Preferably, the incubation time is 8-48 hours;

[0012] Preferably, the L-tryptophan concentration is 400 mg / L.

[0013] This invention also provides applications of Caldibacillus kokeshiiformis MT, particularly in promoting plant growth. It can be used to increase the yield of vegetables such as cucumbers, tomatoes, and lettuce, as well as for the prevention and control of plant diseases and the high-temperature composting and fermentation of agricultural waste such as livestock and poultry manure.

[0014] The third technical solution provided by this invention is a bacterial agent containing *Caldibacillus skokeshiiformis* MT;

[0015] Furthermore, the bacterial agent is a Caldibacillus kokeshiiformis MT inoculant, specifically a fermentation broth obtained by fermenting Caldibacillus kokeshiiformis MT to produce IAA: the Caldibacillus kokeshiiformis MT seed liquid is inoculated into IAA-producing liquid culture medium at an inoculation rate of 5-15% (v / v), and cultured at 35-55℃ and 150-200rpm for 8-72h to obtain the fermentation broth;

[0016] IAA-producing liquid culture medium: yeast extract 4.5-5.5g, tryptone 8-12g, sodium chloride 10-20g, L-tryptophan 0-500mg, distilled water 1L, pH 7.2;

[0017] Furthermore, the MT activity of *Bacillus tinctoria* in the fermentation broth was 2.0-3.7 × 10⁻⁶. 8 cfu / mL, IAA content ranged from 38.83 to 469.21 mg / L.

[0018] Furthermore, the inoculant is a microbial inoculant prepared from *Bacillus thermophilus* MT, and the preparation method is as follows:

[0019] Sterilize the solid fermentation medium with moist heat. When the temperature drops to 50-60℃, add 0.05-1% (w / w) L-tryptophan. Inoculate the MT inoculum seed solution into the solid fermentation medium at 5-15% (v / w). Incubate at 35-55℃ for 1-3 days. After fermentation, dry at 35-55℃, grind and pulverize to obtain the microbial agent.

[0020] The microbial inoculant has a bacterial activity of 2.7-4.3 × 10⁻⁶. 9 cfu / g, IAA content is 139.76-301.74 mg / kg;

[0021] Furthermore, *Bacillus tinctoria* MT was streaked onto LB plates and activated by incubation at 50°C for 2 days.

[0022] Furthermore, after activating Bacillus thermophilus MT, bacterial cells were picked and inoculated into seed culture medium and cultured at 50°C for 12 hours to obtain seed culture.

[0023] Preferably, the solid fermentation medium is sterilized by moist heat at 121°C for 30 minutes. When the temperature drops to 50-60°C, 0.1% L-tryptophan is added. The MT inoculum seed solution is inoculated into the solid fermentation medium at 10% (v / w). The medium is cultured at 55°C for 2 days. After fermentation, the medium is dried at 55°C for 1 day. The inoculum is then ground and pulverized using a pulverizer and passed through a 20-mesh sieve to obtain the microbial agent.

[0024] The seed culture medium consists of 4.5-5.5g yeast extract, 8-12g tryptone, 10-20g sodium chloride, 1L distilled water, and pH 7.2.

[0025] The solid-state fermentation medium consists of: 200-500 parts rice husk, 350-600 parts wheat bran, 100-250 parts corn flour, 3.5-5 parts calcium sulfate, 0.65-1 part calcium oxide, 2-3 parts dipotassium hydrogen phosphate, 1.5-2.5 parts magnesium sulfate, 10-30 parts sodium chloride, and 600-900 parts water.

[0026] The fourth technical solution provided by this invention is the application of the bacterial agent containing *Caldibacillus skokeshiiformis* MT;

[0027] Furthermore, the application of the seed soaking agent, particularly in promoting plant growth;

[0028] Furthermore, the application involves soaking seeds after diluting the seed soaking agent;

[0029] Furthermore, the dilution factor is 500-2000 times; preferably, it is diluted 1000 times.

[0030] Furthermore, the application of the microbial inoculant, particularly in promoting plant growth, is described below:

[0031] It is used when transplanting fruits and vegetables. The application method is hole application or trench application, and the application rate is 2-5 kg / mu, preferably 2.5 kg / mu.

[0032] Beneficial effects:

[0033] The present invention provides Caldibacillus kokeshiiformis MT and the inoculant prepared therefrom, which can significantly promote crop growth, shorten crop cultivation time, and increase yield.

[0034] 1. This invention provides a strain of *Caldibacillus kokeshiiformis* MT that exhibits high production of indoleacetic acid, decomposition of organophosphates, and production of ferrophosphate. It can be used for the production of indoleacetic acid and also for plant growth processes, promoting yield increases.

[0035] 2. This invention uses the fermentation broth obtained by preparing indoleacetic acid from Bacillus kokeshiiformis MT as a seed soaking agent. Soaking seeds with this broth can increase the root length, root fresh weight, hypocotyl length, and hypocotyl fresh weight of crops.

[0036] 3. The microbial agent preparation method provided by this invention is simple, convenient, and has a high success rate. By utilizing the high temperature and salt tolerance of Bacillus pyriformis, the growth of other bacteria can be effectively avoided. Attached image description:

[0037] Figure 1 Phylogenetic tree.

[0038] Figure 2 Colony morphology of Bacillus tinctoria MT.

[0039] Figure 3 MT morphology of Bacillus tinctoria.

[0040] Figure 4 Acid and alkali resistance of Bacillus tinctoria MT.

[0041] Figure 5 The relationship between the concentration of IAA synthesized by Bacillus tinctoria MT and the initial concentration of L-tryptophan.

[0042] Figure 6 The ability of Bacillus tinctoria MT to degrade organic phosphorus.

[0043] Figure 7 The ability of Bacillus tinctoria MT to produce heptaphilin. Detailed implementation method:

[0044] The technical content of the present invention will be further described below with reference to the embodiments. However, the present invention is not limited to these embodiments, and the scope of protection of the present invention cannot be limited by the following embodiments.

[0045] Example 1: Identification of Caldibacillus kokeshiiformis MT

[0046] LB medium: 5g yeast extract, 10g tryptone, 10g sodium chloride, 20g agar, 1L distilled water, pH 7.2.

[0047] Strains of strain MT, selected from cow manure compost fermentation products, were streaked onto LB medium and incubated at 50°C for 2 days. Cells were collected, and genomic DNA was extracted from strain MT using a Trans Genomic DNA Extraction Kit. 16S rDNA was amplified using a PCR system consisting of: 5 μl 10× buffer, 1 μl dNTPs, 1 μl 27F, 1 μl 1492R, and 0.5 μl Taq enzyme. Primers for 27F were: 5'-AGAGTT TGA TCC TGG CTCA-3'; primers for 1492R were: 5'-GGT TAC CTT GTT ACG ACTT-3'. PCR conditions were: 94°C for 4 min, 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, with a final extension at 72°C for 10 min. The PCR amplification products were ligated into the Trans pEASY T3 vector, transformed into E. coli T1 competent cells, and positive clones were screened and sent to Beijing BGI Genomics Co., Ltd. for sequencing.

[0048] The 16S rDNA sequence of strain MT was compared on the EzBioCloud website, and the result was Caldibacillus kokeshiiformis, with a homology of 99.72%.

[0049] The 16S rDNA sequence is shown in SEQ ID NO: 1 of the sequence listing:

[0050]

[0051] Phylogenetic trees were constructed using MEGA software, based on 16S rDNA sequences, using the Neighbour-Joining method with a Btoostrap value of 1000. The phylogenetic trees are shown below. Figure 1 The strain MT is most closely related to Caldibacillus kokeshiiformis, therefore the strain was named Caldibacillus kokeshiiformis MT.

[0052] MT is a rod-shaped, spore-forming, facultative anaerobic bacterium. Its colony morphology on LB agar plates is as follows: Figure 2 As shown, the microscopic morphology is as follows Figure 3 As shown.

[0053] Caldibacillus kokeshiiformis MT has been deposited at the China General Microbiological Culture Collection Center (CGMCC), a designated depository of the State Intellectual Property Office. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is July 20, 2022, and the deposit number is CGMCC No. 25356.

[0054] Example 2: Determination of salt, acid, alkali and temperature resistance of Bacillus tinctoria MT

[0055] I. Salt Tolerance Test

[0056] 1. Detection Method

[0057] Bacillus tinctoria MT was inoculated into LB liquid medium at a volume of 100 mL / 500 mL and incubated on a shaker at 50 °C and 200 rpm for 1 day. Then, 1% of the culture was inoculated into LB liquid medium containing different salt concentrations at a volume of 50 mL / 150 mL. The salt concentrations were 0%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, and 12%, with three replicates for each treatment. All samples were incubated at 50 °C and 200 rpm for 1 day, and the OD was measured. 600nm Absorbance value.

[0058] 2. Test Results

[0059] The optimal salt concentration for Bacillus tinctoria thermophilus (MT) is 1%, and it can tolerate a salt concentration of 10%.

[0060] Table 1. Salt tolerance of Bacillus tinctoria (MT) (OD) 600nm )

[0061] Salt concentration 0 0.5％ 1％ 2％ 3％ 4％ 5％ OD 1.596±0.042 1.761±0.019 1.906±0.010 1.803±0.032 1.685±0.025 1.525±0.028 1.266±0.034 Salt concentration 6％ 7％ 8％ 9％ 10％ 11％ 12％ OD 0.927±0.015 0.807±0.025 0.721±0.012 0.435±0.022 0.106±0.017 0.008±0.001 0.003±0.001

[0062] II. Acid and Alkali Resistance Test

[0063] 1. Detection Method

[0064] Bacillus tinctoria MT was inoculated into LB liquid medium at a volume of 100 mL / 500 mL and incubated on a shaker at 50 °C and 200 rpm for 12 h. Then, 1% of the culture was inoculated into LB liquid medium at different pH values ​​(3, 4, 5, 6, 7, 8, 9, 10, and 11), with three replicates for each treatment. The culture was then incubated on a shaker at 50 °C and 200 rpm for 8 h, and the OD was measured. 600nm The absorbance value.

[0065] 2. Test Results

[0066] like Figure 4 As shown, with OD 600nm The vertical axis is denoted by 'x', and the horizontal axis is 'pH'. *Bacillus tinctoria* grows in a pH range of 5-9, with an optimum pH of 7.0, and exhibits good growth under alkaline conditions.

[0067] III. Temperature Resistance Test

[0068] 1. Detection Method

[0069] Bacillus tinctoria MT was inoculated into LB liquid medium at a volume of 100 mL / 500 mL and cultured on a shaker at 50 °C and 200 rpm for 12 h. Then, 1% of the culture was inoculated into LB liquid medium and cultured at 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 60 °C, and 65 °C at 200 rpm for 1 day. OD was then measured. 600nm The absorbance value.

[0070] 2. Test Results

[0071] The growth temperature range of Bacillus tinctoria MT is 30-60℃, with the optimal growth temperature being 50℃.

[0072] Table 2. Growth Temperature of Bacillus pyridae

[0073]

[0074] Example 3: Determination of IAA Production Capacity of Bacillus tinctoria MT

[0075] I. Effects of different temperatures on the IAA production capacity of Bacillus thuringiensis MT

[0076] 1. Preparation of IAA fermentation broth

[0077] IAA-producing liquid culture medium: 5g yeast extract, 10g tryptone, 10g sodium chloride, 200mg L-tryptophan, 1L distilled water, pH 7.2.

[0078] Streak *Bacillus tinctoria* MT onto LB plates and incubate at 50°C for 2 days. Inoculate into LB liquid medium and incubate at 50°C for 12 hours. Transfer 10% of the culture into IAA-producing liquid medium at a loading volume of 100 mL / 500 mL and incubate at 35°C, 40°C, 50°C, and 55°C for 2 days at 200 rpm.

[0079] 2. Methods for determining IAA concentration

[0080] The IAA content was determined using the Salkowski method.

[0081] Determination of IAA standard curve: Prepare IAA standard solutions with concentrations of 0, 10, 20, 30, 40, and 50 μg / mL, and mix them with Salkowski colorimetric solution (50 mL 35% HClO4 + 1 mL 0.5 mol / L FeCl3) at a volume ratio of 1:1. Incubate at room temperature in the dark for 30 min, and then measure the OD. 530nm Absorbance. Plotting IAA concentration on the x-axis, OD... 530nm Plot the absorbance values ​​on the ordinate to obtain the IAA standard curve.

[0082] Determination of IAA concentration in the test solution: Centrifuge the fermentation broth at 6000 rpm for 15 min. Using uninoculated IAA-producing liquid medium as a control, mix the supernatant with an equal volume of Salkowski colorimetric solution, incubate at room temperature in the dark for 30 min, and measure the OD. 530nm Absorbance. Calculate the IAA concentration of the test solution based on the IAA standard curve.

[0083] 3. Test Results

[0084] Bacillus tinctoria MT can synthesize IAA at temperatures ranging from 35 to 55°C, with the highest IAA concentration (241.73 mg / L) achieved at 50°C, as shown in Table 3.

[0085] Table 3. IAA production capacity of Bacillus tinctoria MT at different temperatures (mg / L)

[0086] Incubation temperature 35℃ 40℃ 50℃ 55℃ IAA content 63.31±2.08 181.40±2.04 241.73±1.80 237.51±2.03

[0087] II. Relationship between fermentation time and IAA synthesis by Bacillus tinctoria masculinus MT

[0088] IAA-producing liquid culture medium: 5g yeast extract, 10g tryptone, 10g sodium chloride, 200mg L-tryptophan, 1L distilled water, pH 7.2.

[0089] Bacillus tinctoria MT was streaked onto LB agar plates and incubated at 50°C for 2 days. It was then inoculated into LB liquid medium and incubated at 50°C for 12 hours. Finally, it was transferred at a 10% concentration to IAA-producing liquid medium (100 mL / 500 mL) and incubated at 50°C and 200 rpm for 4 days. The IAA content was determined using the Salkowski method.

[0090] Thermoplastic Bacillus tinctoria MT synthesized 25.91 mg / L of IAA after 8 hours of fermentation, 217.59 mg / L after 24 hours, and 241.73 mg / L after 48 hours. Thereafter, the IAA content did not increase significantly (Table 4). This indicates that the IAA synthesis rate was fastest during the first 0-24 hours of fermentation, decreased between 24-48 hours, and was completely eliminated after 48 hours, at which point IAA synthesis ceased. Therefore, IAA synthesis in *Bacillus tinctoria MT* mainly occurred within the first 48 hours.

[0091] Table 4. Correspondence between fermentation time and conversion rate of Bacillus tinctoria MT (IAA, mg / L)

[0092] Training time 8h 24h 48h 72h 96h IAA 25.91±1.39 217.59±1.47 241.73±1.80 242.17±1.04 241.34±0.59

[0093] III. Effect of initial L-tryptophan concentration on IAA synthesis by *Bacillus tinctoria* MT

[0094] IAA-producing liquid culture medium: 5g yeast extract, 10g tryptone, 10g sodium chloride, 0-500mg / L L-tryptophan, 1L distilled water, pH 7.2.

[0095] Bacillus tinctoria MT was streaked onto LB agar plates and incubated at 50°C for 2 days. It was then inoculated into LB liquid medium and incubated at 50°C for 12 hours. Finally, it was transferred at 10% to IAA-producing liquid medium containing different concentrations of tryptophan (100 mL / 500 mL) and incubated at 50°C and 200 rpm for 3 days. The IAA content in the fermentation broth was measured at 8, 24, 48, and 72 hours of fermentation using the Salkowski method.

[0096] from Figure 5 It can be seen that within the tryptophan concentration range (0-500 mg / L) in this experiment, no IAA was generated after 48 hours. The ability of *Thermoplastic Acid MT* to synthesize IAA was positively correlated with the initial L-tryptophan concentration; the ability to synthesize IAA increased with increasing initial L-tryptophan concentration. When the initial L-tryptophan concentration was 0-400 mg / L, the IAA concentration increased significantly with increasing initial L-tryptophan concentration; when the initial L-tryptophan concentration was 450-500 mg / L, the IAA concentration increased with increasing initial L-tryptophan concentration, but the increasing trend slowed down.

[0097] After 48 hours of fermentation, the IAA yield was 448.10 mg / L when the initial L-tryptophan concentration was 450 mg / L, and 469.21 mg / L when the initial L-tryptophan concentration was 500 mg / L. When no L-tryptophan was added, Bacillus tinctoria MT could still synthesize IAA (38.83 mg / L), possibly because the culture medium contained free tryptophan or it came from protein decomposition.

[0098] Example 4: Determination of the ability of *Bacillus tinctoria* MT to hydrolyze organic phosphorus and produce ferrophosphate.

[0099] I. Determination of Organophosphorus Solubility

[0100] 1. Qualitative detection of organophosphorus compounds

[0101] 1.1 Detection Method

[0102] Mongkina Organic Phosphorus Medium: 10g glucose, 0.5g (NH4)2SO4, 0.3g NaCl, 0.3g KCl, 0.3g MgSO4·7H2O, 0.03g FeSO4·7H2O, 0.03g MnSO4·4H2O, 0.2g lecithin, 1g CaCO3, 0.5g yeast extract, 20g agar, 1L water, pH 7.0-7.2, sterilized at 115℃ for 20min.

[0103] Bacillus tinctoria MT was inoculated into LB liquid medium at a volume of 100 mL / 500 mL and cultured on a shaker at 50 °C and 200 rpm for 3 days. 60 μl of the culture was added to one well of a perforated Monkina organophosphate plate, and the same volume of uninoculated LB liquid medium was added to the other well as a control (CK). The plates were then incubated at 50 °C for 3 days, and the size of the zona pellucida was measured.

[0104] 1.2 Test Results

[0105] like Figure 6 As shown, the left well is *Bacillus tinctoria* MT, with a zona pellucida size of 2.5 mm; the right well is CK, with no zona pellucida. *Bacillus tinctoria* MT has the ability to solubilize organophosphates.

[0106] 2. Quantitative Detection of Organophosphorus Compounds

[0107] 2.1 Detection Method

[0108] 2.1.1 Preparation of reagents

[0109] Meng Jinna's organophosphorus liquid culture medium: glucose 10g, (NH4)2SO4 0.5g, NaCl 0.3g, KCl 0.3g, MgSO4·7H2O 0.3g, FeSO4·7H2O 0.03g, MnSO4·4H2O 0.03g, lecithin 0.2g, CaCO3 1g, yeast powder 0.5g, water 1L, pH 7.0-7.2, sterilized at 115℃ for 20min.

[0110] Phosphorus standard stock solution: Accurately weigh 0.2195 g of potassium dihydrogen phosphate dried at 105℃ for 2 h, dissolve it in distilled water, add 5 ml of sulfuric acid solution (ρ 1.84), cool, and then standardize to 1000 ml. At this point, the phosphorus (P) concentration is 50 mg / L.

[0111] Molybdenum-antimony sulfate stock solution: Solution A: Weigh 0.5g of potassium antimony tartrate and dissolve it in 100mL of water. Solution B: Weigh 10g of ammonium molybdate and dissolve it in 450mL of water. Slowly add 153mL of concentrated H₂SO₄ while stirring. Then add solution A to solution B, and finally add water to a final volume of 1L. Shake thoroughly and store in a brown bottle.

[0112] Molybdenum-antimony anti-colorimetric reagent: Add 1.50g of L-ascorbic acid to 100mL of molybdenum-antimony sulfate stock solution. This reagent is effective for 24 hours and should be prepared before use.

[0113] 2.1.2 Determination of Phosphorus Standard Curve

[0114] Pipette 0, 200, 400, 600, 800, and 1000 μL of 50 mg / L phosphorus standard stock solution into 50 mL volumetric flasks, corresponding to concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1 mg / L, respectively. Add 5 mL of molybdenum-antimony anti-chromic reagent, dilute to the mark with ddH₂O, shake well, and let stand for 30 min. Measure the absorbance at 660 nm. Plot a standard curve.

[0115] 2.1.3 Quantitative Determination of Organic Phosphorus Hydrolysates by Bacillus tinctoria (MT)

[0116] Bacillus tinctoria MT was inoculated into LB liquid medium and cultured at 50℃ and 200 rpm for 2 days. The bacterial suspension was centrifuged at 6000 rpm for 10 min, and the cells were collected. The cells were washed three times with 0.9% physiological saline and resuspended in Munkina organophosphate liquid medium. The suspension was cultured at 35℃ and 200 rpm with shaking for 7 days. The bacterial suspension was centrifuged at 10000 rpm for 10 min, diluted a certain factor, and 5 mL of molybdenum anti-antimony reagent was added. ddH2O was added to bring the volume to the mark, and the mixture was shaken well and allowed to stand for 30 min. The absorbance was measured at 660 nm. Uninoculated Munkina organophosphate liquid medium was used as a blank control.

[0117] 2.2 Test Results

[0118] Bacillus tinctoria MT can decompose insoluble organic phosphorus (lecithin), increasing the soluble phosphorus content by 2.89 mg / L compared to the blank control.

[0119] II. Determination of Ferrophilic Production Capacity

[0120] 1. Detection Method

[0121] Ferrophilic production detection medium: ① CAS chromaine 60.5 mg dissolved in 50 mL water; ② 10 mL 10 mmol / L FeCl3 solution (containing 10 mmol / L HCl); ③ 72.9 mg HDTMA dissolved in 40 mL water; ④ 750 mL water + 100 mL salt solution (3 g KH2PO4 + 5 g NaCl + 10 g NH4Cl to 100 mL water) + 20 g agar + 30.24 g PIPES, adjust pH to 6.8 with NaOH; ⑤ 30 mL 10% acid-hydrolyzed casein solution; ⑥ 10 mL 20% glucose solution. Mix ① and ② thoroughly and add them to ③ while stirring; sterilize ③, ④, ⑤, and ⑥ separately at 115℃ for 20 min; after sterilization, cool to 50℃, add ⑤ and ⑥ to ④ first, then slowly add ③ and pour the mixture onto the plate.

[0122] Streak *Bacillus tinctoria* MT onto an LB agar plate. Pick a single colony from the LB agar plate and attach it to one side of a ferophile production test plate. Pick a blank LB agar plate from the other side and attach it as a control (CK). Incubate at 37°C for 3 days (too high a temperature will affect the ferophile production plate). Measure the colony diameter (d) and the diameter of the ferophile zone (D). The D / d value represents the ferophile production capacity of the strain.

[0123] 2. Test Results

[0124] like Figure 7 As shown, the left side represents *Bacillus tinctoria* MT, which has a siderophilic ring and a D / d value of 2.5; the right side represents CK, which does not have a siderophilic ring. *Bacillus tinctoria* MT has the ability to produce siderophiles.

[0125] Example 5: Preparation of Bacillus tinctoria MT inoculant

[0126] IAA-producing liquid culture medium: 5g yeast extract, 10g tryptone, 10g sodium chloride, 400mg L-tryptophan, 1L distilled water, pH 7.2.

[0127] Streak *Bacillus tinctoria* MT onto LB agar plates and incubate at 50°C for 2 days. Inoculate into LB liquid medium and incubate at 50°C for 12 hours. Transfer 10% of the culture medium to IAA-producing liquid medium at a loading of 100 mL / 500 mL and incubate at 50°C and 200 rpm for 2 days to obtain *Bacillus tinctoria* MT inoculant.

[0128] The bacterial activity of the *Bacillus thermophilus* MT inoculum was 2.1 × 10⁻⁶. 8 The cfu / mL concentration and the IAA content were 431.3 mg / L.

[0129] Example 6: Application of Bacillus tinctoria thermophilus MT inoculant

[0130] I. Growth-promoting effect of Bacillus tinctoria MT seed soaking agent on cucumber seeds

[0131] Select plump cucumber seeds (Andy No. 4), wash the surface of the seeds with clean water and pat dry. Then, soak the seeds in the Bacillus tinctoria MT seed soaking agent prepared in Example 5 (2.1 × 10⁻⁶ live bacteria). 8 Cucumber seeds were diluted with water at concentrations of CFU / mL and IAA content of 431.30 mg / L, respectively, at ratios of 500, 1000, and 2000. The seeds were soaked in *Bacillus simonii* MT at different dilutions for 7 hours, with a control group soaked in water for 7 hours. After soaking, the seeds were rinsed five times with water and placed in petri dishes lined with filter paper, 15 seeds per dish, with three replicates per treatment. The dishes were incubated in a 28℃ artificial climate chamber in the dark for 7 days, with water added periodically. Seed germination rate, hypocotyl length, root length, and root fresh weight were measured. The data were analyzed using Duncan's new multiple range test in DPS software, and the results are shown in Table 5.

[0132] A 1000-fold dilution of *Bacillus pyriformis* MT seed soaking agent significantly increased cucumber seed root length, root fresh weight, hypocotyl length, and hypocotyl fresh weight, increasing them by 11.32%, 38.83%, 19.12%, and 21.30%, respectively, compared to the control. Furthermore, 500-fold and 2000-fold dilutions of *Bacillus pyriformis* MT seed soaking agent also increased root length, root fresh weight, hypocotyl length, and hypocotyl fresh weight to varying degrees. Therefore, soaking cucumber seeds in *Bacillus pyriformis* MT seed soaking agent significantly promotes cucumber seed growth, with the 1000-fold dilution showing the best effect.

[0133] Table 5. Growth-promoting effect of Bacillus tinctoria MT seed soaking agent on cucumber.

[0134]

[0135] Note: abcd indicates significance at the 5% level.

[0136] II. Field Trial of Bacillus tinctoria MT Seed Soak for Promoting Lettuce Growth

[0137] The Bacillus tinctoria MT inoculum prepared in Example 5 (2.1 × 10⁻⁶ live bacteria) was used for inoculation. 8 Lettuce seeds (containing CFU / mL and IAA content of 431.30 mg / L) were diluted 500, 1000, and 2000 times with water, respectively. Lettuce seeds (soft-tailed lettuce) were soaked in the diluted Bacillus thuringiensis MT seed soaking solution for 7 hours, with water soaking serving as a control. After soaking, the seeds were rinsed with water, dried, and then sown. The lettuce was sown in seedling trays and transplanted when it had 4-6 true leaves. This experiment was conducted in a cold greenhouse, with 4 treatments and 4 replicates, using a completely randomized block design. Each plot was 4.6 m × 7.00 m = 32.2 m². 2 The row spacing is 25cm × 20cm.

[0138] Field management followed conventional methods, namely, applying 1500 kg / mu of farmyard manure and 15 kg / mu of urea as basal fertilizer before sowing. Topdressing was applied every 10 days, for a total of 3 topdressings. The first topdressing was applied with irrigation water at 15 kg / mu of ammonium nitrate, and the second and third topdressings were applied with irrigation water at 18 kg / mu of compound fertilizer (16-16-16). Throughout the growing season, metalaxyl and agricultural streptomycin were used to control downy mildew, soft rot, and other diseases, and imidacloprid and pirimicarb were used to control aphids and other pests.

[0139] The yield of each plot was statistically analyzed, and the data were analyzed using Duncan's new multiple range test in DPS software. The results are shown in Table 6.

[0140] Table 6. Statistics on Lettuce Yield in Each Plot (Unit: kg)

[0141]

[0142] As shown in the table above, Bacillus tinctoria MT seed soaking agent diluted 500, 1000, and 2000 times significantly increased lettuce seed yield, with the 1000-fold dilution showing the best effect, increasing yield by 9.76% compared to the control (CK). Bacillus tinctoria MT seed soaking agent significantly promotes lettuce growth and has good application prospects.

[0143] Example 7: Preparation of Microbial Inoculants

[0144] Seed culture medium: 5g yeast extract, 10g tryptone, 10g sodium chloride, 1L distilled water, pH 7.2.

[0145] Solid-state fermentation medium: 300 parts rice husk, 500 parts wheat bran, 200 parts corn flour, 5 parts calcium sulfate, 0.75 parts calcium oxide, 3 parts dipotassium hydrogen phosphate, 2 parts magnesium sulfate, 10 parts sodium chloride, and 800 parts water.

[0146] Bacillus tinctoria MT was streaked onto LB agar plates and incubated at 50°C for 2 days. Cells were then picked and inoculated into seed culture medium, and incubated at 50°C for 12 hours to obtain the seed culture. The solid-state fermentation medium was sterilized by moist heat at 121°C for 30 minutes. When the temperature dropped to 50-60°C, 0.1% (w / w) L-tryptophan was added. The seed culture was then inoculated into the solid-state fermentation medium at a rate of 10% (v / w), with a sample volume of 300g / tray. The medium was incubated at 55°C for 2 days. After fermentation, the medium was dried at 55°C for 1 day, ground into a fine powder, and passed through a 20-mesh sieve to obtain the microbial inoculum. The viability of the microbial inoculum was 3.0 × 10⁻⁶. 9 The cfu / g and IAA content is 230.84 mg / kg.

[0147] Example 8: Application of Microbial Agents

[0148] The growth-promoting effect of the microbial inoculant prepared in Example 7 on tomatoes was tested in a greenhouse. Four treatments were included, with four replicates, using a completely randomized block design. Each plot was 4.20m × 7.00m = 29.4m². 2 The rows are spaced 80cm apart, the smaller rows 60cm apart, and the plant spacing is 40cm. Field management follows conventional methods, and the tomato variety is Yuansheng. Water on the day of transplanting, and water again 6 days later to help the seedlings establish. After the first fruit set, water every 12 days throughout the fruiting period. During the growing season, use aphid-killing agent to control whiteflies and other pests, and use a fungicide to control leaf mold.

[0149] Treatment 1. Conventional fertilization;

[0150] Treatment 2: Conventional fertilization + microbial inoculants;

[0151] Treatment 3. Conventional fertilization + carrier;

[0152] Treatment 4. Blank control;

[0153] Treatment 4 was not fertilized.

[0154] The conventional fertilization for treatments 1, 2, and 3 is as follows: apply 45 kg / mu of diammonium hydrogen phosphate as a base fertilizer before transplanting; apply 7 kg / mu of diammonium hydrogen phosphate as a top dressing after the seedlings have recovered; apply 12 kg / mu of compound fertilizer (16-6-22) in conjunction with irrigation when each ear of fruit begins to swell; stop applying top dressing 30 days before the vines are pulled up.

[0155] In addition, treatment 2 was administered with 2.5 kg / mu of the microbial agent prepared in Example 7 at the time of transplanting; treatment 3 was administered with 2.5 kg / mu of the carrier at the time of transplanting. The carrier was a solid fermentation medium (300 parts rice husk, 500 parts wheat bran, 200 parts corn flour, 5 parts calcium sulfate, 0.75 parts calcium oxide, 3 parts dipotassium hydrogen phosphate, 2 parts magnesium sulfate, 10 parts sodium chloride, and 800 parts water) which was sterilized by moist heat at 121℃ for 30 min. When the temperature dropped to 50-60℃, 0.1% (w / w) L-tryptophan was added, and the mixture was dried.

[0156] The yield of each plot was statistically analyzed, and the data were analyzed using Duncan's new multiple range test in DPS software. The results are shown in Table 7.

[0157] Table 7. Tomato Plot Yield Statistics (Yield Unit: Kilograms)

[0158]

[0159] As shown in the table above, conventional fertilization combined with microbial inoculants (treatment 2) was significantly better than other treatments, increasing yield by 5.95% compared to conventional fertilization (treatment 1) and by 28.34% compared to the control (treatment 4). These microbial inoculants significantly promoted tomato growth and have promising application prospects.

[0160] Example 9: Preparation of Microbial Inoculants

[0161] Seed culture medium: 5g yeast extract, 10g tryptone, 10g sodium chloride, 1L distilled water, pH 7.2.

[0162] Solid-state fermentation medium: 200 parts rice husk, 350 parts wheat bran, 100 parts corn flour, 3.5 parts calcium sulfate, 0.65 parts calcium oxide, 2 parts dipotassium hydrogen phosphate, 1.5 parts magnesium sulfate, 10 parts sodium chloride, and 600 parts water.

[0163] Bacillus tinctoria MT was streaked onto LB agar plates and incubated at 50°C for 2 days. Cells were then picked and inoculated into seed culture medium, and incubated at 50°C for 12 hours to obtain the seed culture. The solid-state fermentation medium was sterilized by moist heat at 121°C for 30 minutes. When the temperature dropped to 50-60°C, 1% (w / w) L-tryptophan was added. The seed culture was then inoculated into the solid-state fermentation medium at a rate of 15% (v / w), with a sample volume of 300g / tray. The medium was incubated at 50°C for 2 days. After fermentation, the medium was dried at 55°C for 1 day, ground into a fine powder, and passed through a 20-mesh sieve to obtain the microbial inoculum. The viability of the microbial inoculum was 4.3 × 10⁻⁶. 9 The cfu / g and IAA content is 301.74 mg / kg.

[0164] Example 10 Preparation of Microbial Inoculants

[0165] Seed culture medium: 5g yeast extract, 10g tryptone, 10g sodium chloride, 1L distilled water, pH 7.2.

[0166] Solid-state fermentation medium: 500 parts rice husk, 600 parts wheat bran, 250 parts corn flour, 5 parts calcium sulfate, 1 part calcium oxide, 3 parts dipotassium hydrogen phosphate, 2.5 parts magnesium sulfate, 30 parts sodium chloride, and 900 parts water.

[0167] Bacillus tinctoria MT was streaked onto LB agar plates and incubated at 50°C for 2 days. Cells were then picked and inoculated into seed culture medium, and incubated at 50°C for 12 hours to obtain the seed culture. The solid-state fermentation medium was sterilized by moist heat at 121°C for 30 minutes. When the temperature dropped to 50-60°C, 0.05% (w / w) L-tryptophan was added. The seed culture was then inoculated into the solid-state fermentation medium at a rate of 5% (v / w), with a sample volume of 300g / tray. The medium was incubated at 55°C for 2 days. After fermentation, the medium was dried at 55°C for 1 day, ground into a fine powder, and passed through a 20-mesh sieve to obtain the microbial inoculum. The viability of the microbial inoculum was 2.7 × 10⁻⁶. 9 The cfu / g and IAA content is 139.76 mg / kg.

[0168] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these modifications and combinations all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims

1. A strain of Bacillus pyridae ( Caldibacillus kokeshiiformis )MT, with accession number CGMCCNO.25356.

2. The claim 1 Caldibacillus kokeshiiformis Application of MT in the production of IAA.

3. The application as described in claim 2, characterized in that, The method for producing IAA is as follows: Inoculate the Bacillus thermophilus MT seed culture into the IAA-producing liquid culture medium at an inoculation rate of 5-15%, and culture at 35-55℃ and 150-200rpm for 8-72h. IAA-producing liquid culture medium: yeast extract 4.5-5.5g, tryptone 8-12g, sodium chloride 10-20g, L-tryptophan 0-500mg, distilled water 1L, pH 7.

2.

4. The claim 1 Caldibacillus kokeshiiformis Application of MT in plant growth promotion.

5. The claim 1 Caldibacillus kokeshiiformis The application of MT in increasing vegetable yield is characterized by, The vegetables mentioned are: lettuce or tomatoes.

6. The claim 1 Caldibacillus kokeshiiformis Application of MT in the field of high-temperature composting and fermentation of agricultural waste.

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