Saline-alkali-resistant phosphorus-solubilizing composting bacillus subtilis C007, microbial agent, microbial fertilizer and application of microbial agent and microbial fertilizer

Abstract

The invention discloses a strain of saline-alkali-resistant phosphorus-solubilizing compost bacillus subtilis C007, which can enhance the survival and colonization of beneficial microorganisms in saline-alkali soil farmland soil and plant root systems, also has the functions of solubilizing phosphorus and secreting auxin (IAA), and plays a role in promoting the growth of plants. The compound microbial fertilizer can improve the soil environment and the agricultural ecosystem on one hand, can promote the growth of plants, and can achieve the purposes of reducing the application of chemical fertilizers and improving the soil fertility of saline-alkali land farmlands to a certain extent.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to salt-alkali resistant phosphorus-dissolving composting Bacillus stercoris C007, a bacterial agent, a microbial fertilizer and applications thereof. Background Art

[0002] Bacillus stercoris is a subspecies of Bacillus subtilis. It is a type of Gram-positive bacteria that can produce resistant endospores. Its cells are rod-shaped and covered with a large amount of calcium pyridine dicarboxylate. It can produce a large amount of secondary metabolites. Currently, it is widely used in sludge harmless treatment, food, microbial deodorant, microbial composting agent and biological water-soluble organic fertilizer.

[0003] Compound microbial fertilizer is a new type of fertilizer in which microbial functions play a leading role. Compound microbial fertilizer is essentially a fertilizer whose purpose is to improve the efficiency of crop absorption of nutrients and increase yield. At the same time, as a new type of fertilizer, compound microbial fertilizer also plays a role in protecting and improving the environment. Compound microbial fertilizer contains a variety of microbial strains and organic and inorganic materials. Organic and inorganic nutrients can not only provide nutrients to plants, but also stabilize the microorganisms in fertilizers and improve the diversity of microorganisms in saline-alkali soils. Therefore, compound microbial fertilizers have advantages in terms of function and environmental protection, and are increasingly receiving attention in the process of agricultural production.

[0004] At present, microbial fertilizers have achieved certain effects in agricultural production. However, most microbial fertilizers have single functionality, low product technology integration, high prices, low specificity, certain limitations, poor regional adaptability and short product shelf life. Therefore, they have not been widely promoted. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a salt-alkali resistant phosphate-dissolving composting bacillus Bacillus stercoris C007. The salt-alkali resistant phosphate-dissolving composting bacillus Bacillus stercoris C007 not only has strong self-generation ability and high temperature resistance, but also has resistance to saline-alkali environment, especially has the function of activating inorganic phosphorus (P) in saline-alkali land, can promote cellulose degradation, and has a promoting effect on the growth of crops.

[0006] The present invention is achieved through the following technical solutions:

[0007] The invention discloses a salt-alkali resistant phosphate-dissolving composting bacillus Bacillusstercoris C007 separated and purified from compost and an application thereof. The strain has been preserved in China Center for Type Culture Collection (CCTCC), with a preservation address of Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, a preservation date of August 28, 2024, and a preservation number of CCTCCNO: M20241868.

[0008] Combining the strain's morphological characteristics, physiological and biochemical tests, and phylogenetic analysis, it was found that the strain had large, white, raised colonies, neat edges, a smooth and moist surface, was opaque, and produced spores. The strain had relatively loose requirements for growth conditions and low requirements for light conditions, and was able to use a variety of organic and inorganic substances as carbon and nitrogen sources for growth and reproduction. Its 16S rDNA sequence is shown in SEQ ID No. 1, which confirmed that it belonged to the composting Bacillus stercoris, and the sequence similarity with the closest strain Bacillus stercoris N6 was as high as 99.86%.

[0009] The salt-alkali resistant phosphate-dissolving composting bacillus Bacillus stercoris C007 has the ability to activate inorganic phosphorus (P), can grow with sparingly soluble phosphate as a phosphorus source, and convert it into soluble phosphate. Under laboratory shaking flask conditions, the composting bacillus Bacillus stercoris C007 can convert tricalcium phosphate to 189.17 mg / L.

[0010] The salt-alkali resistant and phosphate-dissolving composting Bacillus stercoris C007 can secrete indoleacetic acid (IAA) under the induction of L-tryptophan, and the secretion amount can reach 5.29 mg / L.

[0011] The salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 can grow in a saline-alkali environment, and its growth-tolerant NaCl concentration is greater than 14% (w / v, 140 g / L), and the tolerable pH value is 8.0-10.5.

[0012] The salt-alkali resistant and phosphate-dissolving composting Bacillus stercoris C007 has the ability to degrade cellulose. When sodium carboxymethyl cellulose is used as the sole carbon source, the cellulase activity of this strain is 6.12 U / mL.

[0013] In another aspect of the present invention, the present invention provides a microbial agent, comprising the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 described in the above technical solution.

[0014] In another aspect of the present invention, the present invention proposes a culture medium for preparing the microbial agent described in the above technical scheme. According to an embodiment of the present invention, the culture medium includes: corn starch 8-15 g / L, soybean cake powder 5-10 g / L, K2HPO4 1-5 g / L, KH2PO4 1-5 g / L, ZnSO4 0.1-0.5 g / L, MgSO4 1-5 mg / L, CuSO4 1-5 mg / L, and Na2MoO4 1-3 mg / L.

[0015] In another aspect of the present invention, the present invention provides a composite microbial agent, comprising the microbial agent as described in the above technical solution. Optionally, the spore content of salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 in the microbial agent is not less than 100 billion cfu / g.

[0016] The composite microbial agent described in the above technical solution comprises a Bacillus Velez subtilis agent, wherein the Bacillus Velez subtilis is selected from Bacillus Velez subtilis with a deposit number of CCTCC NO: M 20221937;

[0017] Optionally, the Bacillus Velez spore content in the Bacillus Velez inoculant is not less than 130 billion cfu / g.

[0018] In another aspect of the present invention, the present invention provides a microbial fertilizer comprising at least one of the following:

[0019] The salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007, its culture or its processed product described in the above technical solution;

[0020] The microbial agent described in the above technical solution;

[0021] The composite microbial agent described in the above technical solution.

[0022] The microbial fertilizer described in the above technical solution further includes auxiliary materials or trace elements;

[0023] The auxiliary materials include at least one of the following: straw powder, organic fertilizer or potassium humate;

[0024] The trace elements include at least one of the following: ammonium molybdate, sodium molybdate or ferrous sulfate.

[0025] The microbial fertilizer described in the above technical solution has an effective bacterial count of not less than 200 million cfu / g.

[0026] In another aspect of the present invention, the present invention proposes the use of the microorganism, the microbial agent, the composite microbial agent or the microbial fertilizer in the above technical solution in at least one of the following:

[0027] Improve crop resistance to stress, especially resistance to salinity and alkali;

[0028] Promote crop growth.

[0029] In another aspect of the present invention, the present invention provides a method for improving crop stress resistance, in particular, salt and alkali resistance; and promoting crop growth. According to an embodiment of the present invention, the method comprises: adding at least one of the following to the soil where the crop is planted: the aforementioned microorganisms, microbial agents, composite microbial agents, and microbial fertilizers.

[0030] The advantages and beneficial effects of the present invention are:

[0031] Summary and description of the characteristics and performance of salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007. C007 not only has strong high temperature resistance, but also has resistance to saline-alkali environment. Its growth tolerance NaCl concentration is >14% (w / v, 140g / L), and the pH value it can tolerate is 8.0-10.5. It has the function of activating inorganic phosphorus (P), and the conversion amount of tricalcium phosphate is up to 189.17mg / L. At the same time, it has a promoting effect on the growth of crops, and can secrete indoleacetic acid (IAA) under the induction of L-tryptophan, and the secretion amount can reach 5.29mg / L. It can promote cellulose degradation, and the cellulase activity is 6.12U / mL. The composite bacterial agent and fertilizer prepared by combining this strain with the Bacillus Velez microbial agent (deposit number is CCTCC NO: M 20221937, for details, see the disclosed content in the patent application with application number 2023113257516), can cooperate with each other to achieve the effect of 1+1 greater than 2.

[0032] The composite microbial agent / fertilizer disclosed in the present invention can not only enhance the survival and colonization of beneficial microorganisms in saline-alkali farmland soil and plant roots, but also has the functions of nitrogen fixation, phosphorus solubilization and secretion of plant auxin (IAA), which promotes plant growth. The composite microbial agent / fertilizer can improve the soil environment and agricultural ecosystem on the one hand, and can also promote plant growth, and to a certain extent can achieve the purpose of reducing the application of chemical fertilizers and improving the fertility of saline-alkali farmland soil.

[0033] Collection Information

[0034] Composting Bacillus stercoris C007, the strain has been deposited, the depository unit: China Center for Type Culture Collection (CCTCC), the deposit address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the deposit date: August 28, 2024, the deposit number: CCTCC NO:M 20241868. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a colony observation diagram of the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 of the present invention;

[0036] Figure 2 It is a phylogenetic tree of the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 of the present invention based on 16S rDNA sequence analysis;

[0037] Figure 3 This is a comparison chart showing that the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 of the present invention promotes the growth of wheat in saline-alkali soil;

[0038] Figure 4 This is a schematic diagram of wheat growth under soil culture conditions using compound microbial fertilizer (in the figure: bacterial fertilizer);

[0039] Figure 5 This is a comparison chart of wheat plant height and root length under soil culture conditions using compound microbial fertilizer (in the picture: bacterial fertilizer);

[0040] Figure 6 This is a schematic diagram of wheat growth under hydroponic conditions using compound microbial fertilizer (in the picture: bacterial fertilizer);

[0041] Figure 7 This is a comparison chart of wheat plant height and root length under hydroponic conditions using compound microbial fertilizer (in the picture: bacterial fertilizer);

[0042] Figure 8 This is a statistical chart of wheat plant height and root length under hydroponic conditions using compound microbial fertilizer (in the picture: bacterial fertilizer);

[0043] For ordinary technicians in this field, other relevant drawings can be obtained based on the above drawings without any creative work. DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with specific embodiments.

[0045] In this document, the terms “salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007”, “composting Bacillus stercoris C007”, “Bacillus stercoris C007” and “C007” are interchangeable.

[0046] Example 1 Isolation and purification of salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007

[0047] Sample source: Compost samples from Nanpi pig farm in Cangzhou, Hebei Province, sampling time: August 2017.

[0048] Separation and purification process and steps.

[0049] Take 10 grams of the sample and put it in 90 ml of sterile water with glass beads, place it on a 55℃ shaker and shake it for 30 minutes to break up the bacterial mass as much as possible. After the sample is processed, it is immediately diluted with sterile water to obtain bacterial solutions of different concentrations. Use a sterile pipette to draw 100 microliters of diluted bacterial solution, and drip it on a sodium carboxymethyl cellulose medium (CMC-Na 20g, Na2HPO42.5g, KH2PO41.5g, Agar Powder20g, peptone 2.5g, deionized water 1000ml, pH 8.0) plate with aseptic operation, and spread it evenly with a sterile spreader. After inoculation, place the plate upside down in a constant temperature incubator and culture at 55℃. According to the morphological characteristics of the colony, pick a single colony with good growth, moderate size, and obvious appearance. Use an inoculation loop to pick a single colony and streak it on the plate for further separation and purification. The composition of the plate streaking medium is the same as that of the medium used for coating the plate. After repeated plate streaking, single colony C007 was purified.

[0050] Example 2 Identification of salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007

[0051] 1) Identification of biological characteristics

[0052] Bacillus stercoris C007, a salt-alkali resistant phosphate-dissolving composting bacillus, has large, white, raised colonies with neat edges, a smooth and moist surface, is opaque, and produces spores. It has relatively loose requirements for growth conditions and light conditions, and can use a variety of organic and inorganic substances as carbon and nitrogen sources for growth and reproduction. (See Figure 1 ).

[0053] 2) Identification of physiological and biochemical characteristics

[0054] The physiological and biochemical characteristics of the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 were studied using a bacterial microbiochemical identification tube. The physiological and biochemical characteristics of the composting Bacillus stercoris C007 are shown in Table 1.

[0055] Table 1 Physiological and biochemical characteristics of Bacillus stercoris C007

[0056]

[0057] 3) Systematic classification and identification

[0058] Primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R

[0059] (5'-TACGGYTACCTTGTTACGACTT-3') amplified the rRNA gene sequence of strain C00716S. The PCR reaction system was 25 μL, which contained 12.5 μL 2×GoldStar Best MasterMix, 0.5 μL primer 27F, 0.5 μL primer 1492R, 3 μL bacterial solution template, and finally added sterile ultrapure water to 25 μL. The PCR reaction program was: (i) 94℃5min pre-denaturation; (ii) 34 cycles of 94℃1min denaturation, 55℃1min annealing, 72℃1.5min extension; (iii) the final extension condition was 72℃10min. The PCR amplification product was submitted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0060] SeqMan 7.1.0.44 was used to splice the measured sequences to obtain the 16S rRNA gene sequence of strain C007. The spliced ​​sequence file was compared with the data in the NCBI 16S database using the NCBI Blast program to obtain the species information with the greatest similarity to the sequence of the species to be tested, which was the identification result. The obtained sequence was the 16S rRNA gene sequence of Bacillus stercoris C007, as shown in SEQ ID NO: 1.

[0061] The 16S rRNA gene sequence of Bacillus stercoris C007 was compared with the NCBI (National Center for Biotechnology Information) database. The results showed that the sequence similarity between the 16S rRNA gene sequence of Bacillus stercoris C007 and that of Bacillus stercoris N6 was as high as 99.86%. The phylogenetic tree was constructed using MEGA7.1 (see Figure 2 ), forming a stable evolutionary branch with the sequence of Bacillus stercorisN6.

[0062] Example 3 Determination of the Composting Capacity of Bacillus stercoris C007

[0063] (1) Self-generation ability and high temperature resistance

[0064] During the isolation of Bacillusstercoris C007, a culture condition of 55°C was used. C007 was able to survive and grow rapidly at this temperature, and the OD600 value could grow to above 2.5 within 12 hours. A 5L fermenter was used for expansion culture, and 3L of culture medium was prepared. When the fermentation temperature was 37°C, the inoculated culture medium needed to be sterilized at 118°C for 25 minutes, while at 55°C, the inoculated culture medium did not need to be sterilized.

[0065] (2) Resistance to saline-alkali environment

[0066] Under the premise that the pH value of Gibbson's modified medium (5g casein, 5g tryptone, 10g yeast extract, 3g trisodium citrate, 2g magnesium sulfate heptahydrate, 2g potassium chloride, 15g agar powder, 1000ml deionized water) is 7.5, the C007 strain can tolerate a medium with a salt content of up to 140g / L. Under the premise that the salt content of Gibbson's modified medium (5g casein, 5g tryptone, 10g yeast extract, 3g trisodium citrate, 2g magnesium sulfate heptahydrate, 2g potassium chloride, 15g agar powder, 1000ml deionized water) is 50g / L, the C007 strain can tolerate a pH value of 10.5.

[0067] (3) Determination of the ability to dissolve inorganic phosphorus (P)

[0068] Strain C007 was inoculated into NBRIP liquid culture medium (Ca3(PO4)25.0 g / L, glucose 10.0 g / L, (NH4)2SO40.15 g / L, KCl 0.2 g / L, MgCl2·6H2O 0.5 g / L, MgSO4·7H2O 0.25 g / L) at a 2% inoculation rate, and cultured in a constant temperature shaker at 30°C and 150 rpm in the dark for 24 hours. The culture medium was taken to determine the soluble phosphorus content.

[0069] Take an appropriate amount of bacterial suspension into a centrifuge tube, centrifuge at 25°C and 12000rpm for 5 minutes, mix the obtained supernatant with ammonium molybdate-ammonium metavanadate-sulfuric acid color development solution and distilled water in a volume ratio of 1:1:2, place the mixture at 65°C for 10 minutes, and measure the absorbance value at 420nm with a spectrophotometer. Set up a blank control at the same time.

[0070] Prepare a standard curve of soluble phosphorus solution: prepare soluble phosphorus solutions of different concentrations, mix them thoroughly with ammonium molybdate-ammonium metavanadate-sulfuric acid colorimetric solution and distilled water in a volume ratio of 1:1:2, let the mixed solution stand at 65°C for 10 minutes, and then measure the absorbance at 420nm with a spectrophotometer. The resulting curve is used as the standard curve.

[0071] The test results showed that strain C007 had the ability to dissolve inorganic phosphorus (P) into soluble phosphorus, and the phosphorus (P) solubilizing capacity of strain C007 could reach 189.17 mg / L.

[0072] (4) Determination of the ability to promote cellulose degradation

[0073] The activity of cellulase is defined as the amount of enzyme that catalyzes the hydrolysis of substrate to produce 1 micromole of glucose, which is 1 enzyme activity unit (U), and the unit is U / mL.

[0074] Prepare standard glucose (anhydrous glucose is dried to constant weight and dissolved in pure water), add standard glucose solutions of different concentrations into graduated test tubes, add distilled water to the scale line, then add DNS reagent, boil in boiling water bath for 5 minutes, cool and make up to volume with distilled water, measure OD value at 540 mm by spectrophotometry, and draw a standard curve.

[0075] Preparation of crude enzyme solution of target strain: The fermentation liquid of target strain was centrifuged at 10000r / min for 15min to obtain crude enzyme solution. The crude enzyme solution was added with 1% CMC-Na solution, hydrolyzed at 45℃ for 20min, and then DNS colorimetric solution was added in boiling water bath for 5min, cooled to constant volume, and the OD value was measured by spectrophotometry at 540nm to calculate the amount of glucose.

[0076] It was calculated that when sodium carboxymethyl cellulose was the sole carbon source, the cellulase activity of strain C007 was 6.12 U / mL.

[0077] (5) Determination of the ability to secrete indoleacetic acid (IAA)

[0078] The strain C007 was inoculated into LB liquid culture medium with an L-tryptophan concentration of 200 mg / L at a 2% inoculation rate, and cultured in a constant temperature shaker at 30° C. and 180 rpm for 24 hours in the dark. The culture medium was then taken to determine the IAA content.

[0079] Take an appropriate amount of bacterial suspension into a centrifuge tube, centrifuge at 25°C and 12000rpm for 5 minutes, mix the obtained supernatant with Salkowski colorimetric solution in a volume ratio of 1:1, keep the mixture at room temperature and away from light for 30 minutes, and measure the absorbance value at 530nm with a spectrophotometer. Set up a blank control at the same time.

[0080] Prepare a standard curve of indoleacetic acid (IAA) solution: prepare indoleacetic acid (IAA) solutions of different concentrations, mix them thoroughly with Salkowski colorimetric solution at a volume ratio of 1:1, let the mixed solution stand at room temperature in the dark for 30 minutes, measure the absorbance at 530 nm with a spectrophotometer, and use the resulting curve as the standard curve.

[0081] The results showed that strain C007 could secrete indoleacetic acid (IAA) under the induction of L-tryptophan, and the secretion amount could reach 5.29 mg / L.

[0082] (6) Promote the growth of wheat in saline-alkali soil

[0083] Preparation of bacterial suspension: Pick a single colony of strain C007 and inoculate it into LB liquid medium for 24 hours. Then subculture it again to enrich the cells. After 24 hours of culture, transfer the C007 culture to a centrifuge tube and centrifuge it at 10000 rpm for 10 minutes. Resuspend the collected cells in sterile 1x phosphate buffer (PBS), and dilute the cell suspension of strain C007 to 10 8 cells / mL.

[0084] Preparation of wheat seeds: Select complete and disease-free wheat varieties "Jiemai 19" seeds, soak them in 75% ethanol for 3 minutes, then soak them in 5% (v / v) sodium hypochlorite solution for 3 minutes, and then rinse the wheat seeds three times with sterile distilled water to complete the surface disinfection and sterilization of the wheat seeds. After the surface disinfection and sterilization of the wheat seeds is completed, place the wheat seeds in a sterile culture dish, and fill the culture dish with sterile distilled water so that the wheat seeds are completely immersed in the sterile distilled water to break the dormancy of the wheat seeds.

[0085] Soil preparation: The saline-alkali farmland soil (soil conductivity 1.9 ms / cm) from Nandagang, Huanghua, Hebei was selected and sieved through a 2 mm sieve and packed in a pot of 8 cm x 8 cm x 8 cm.

[0086] Ten wheat seeds were planted in each pot (as the first day of planting); thinning was performed after the seedlings emerged more uniformly (as the fifth day of planting) and 25 mL of the prepared bacterial solution was poured around the roots of the wheat seedlings (as the first inoculation), and the negative control group was poured with the same volume of sterile 1x phosphate buffer (PBS); then the second and third inoculations were performed on the fifth and fourteenth days after thinning, respectively, and the potted plants were grown at room temperature.

[0087] After 30 days of cultivation, the wheat potted plants were observed, and various indicators of the potted plants were measured and analyzed. The growth of the wheat potted plants was observed, and it was found that the growth of the wheat seedlings inoculated with strain C007 was better than that of the negative control group ( Figure 3 The plant height, fresh weight, dry weight and root surface area of ​​potted wheat seedlings were measured. The measurement results and analysis showed that strain C007 promoted the growth of wheat seedlings, which was mainly manifested in a significant increase in plant height, fresh weight, dry weight and root surface area (see Table 2).

[0088] Table 2 Bacillus stercoris C007 promotes wheat growth

[0089]

[0090] Example 4 Functional determination of composite bacterial agent

[0091] In another aspect of the present invention, the present invention provides a composite microbial agent. According to an embodiment of the present invention, the composite agent includes the microbial composting Bacillus stercoris C007 agent as described in the above technical solution. The composite agent of the present invention has great application value in improving crop stress resistance, especially salt and alkali resistance; and promoting crop seedling growth.

[0092] According to an embodiment of the present invention, the composite microbial agent includes a Bacillus Velez subtilis agent, and the Bacillus Velez subtilis agent is selected from Bacillus Velez subtilis with a deposit number of CCTCC NO: M 20221937. According to this composite agent, composting Bacillus stercoris C007 and Bacillus Velez subtilis have great application value for the growth of wheat seedlings, and the synergistic effect of the two bacteria can increase the height of wheat plants by 8-10% compared with the single application.

[0093] According to an embodiment of the present invention, the composite microbial agent includes: 45 to 55 parts by weight (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or any two of the values ​​as the range of endpoint values) of the microbial composting Bacillus stercoris C007 agent; 45 to 55 parts by weight (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or any two of the values ​​as the range of endpoint values) of the Bacillus velez agent. Thus, the synergistic effect of promoting the growth of wheat seedlings can be better exerted.

[0094] The composting Bacillus stercoris C007 inoculant contains no less than 100 billion cfu / g of spores of Bacillus stercoris C007. The composting Bacillus stercoris C007 inoculant contains no less than 130 billion cfu / g of spores of Bacillus berlez.

[0095] The composite bacterial agent comprises composting Bacillus stercoris C007 and Bacillus velezii (the deposit number is CCTCC NO: M 20221937).

[0096] Preparation of composite bacterial agent: The culture medium used for C007 is 8g / L corn starch, 8.5g / L bean cake powder, 2 g / L K2HPO4, 1 g / L KH2PO4, 0.1 g / L ZnSO4, 2 mg / L MgSO4, 2 mg / L CuSO4, 1 mg / L Na2MoO4. The culture medium used for Bacillus Velezii is 6g / L corn starch, 7g / L bean cake powder, 1.5g / L rice flour, 1.5g / L bran, 1g / L corn steep liquor, 0.5g / L calcium carbonate, 0.2g / L potassium nitrate, 0.3g / L magnesium sulfate, 0.2 g / L K2HPO4, 0.2 g / L Na2HPO4. Both strains are fermented in a 20L fermenter, sampling every 4h, and stopping the tank when the spore maturity is 50%. The obtained bacterial liquid is directly sprayed onto the bacterial powder by high-temperature centrifugal drying, and the two are mixed in a ratio of 1:1 by mass to obtain a composite bacterial agent.

[0097] Preparation of wheat seeds: Select complete and disease-free wheat varieties "Jiemai 19" seeds, soak them in 75% ethanol for 3 minutes, then soak them in 5% (v / v) sodium hypochlorite solution for 3 minutes, and then rinse the wheat seeds three times with sterile distilled water to complete the surface disinfection and sterilization of the wheat seeds. After the surface disinfection and sterilization of the wheat seeds is completed, place the wheat seeds in a sterile culture dish, and fill the culture dish with sterile distilled water so that the wheat seeds are completely immersed in the sterile distilled water to break the dormancy of the wheat seeds.

[0098] Preparation of saline-alkali soil: The saline-alkali farmland soil (soil conductivity 1.9ms / cm) from Nandagang, Huanghua, Hebei Province was selected and sieved through a 2mm screen and placed in a 8cm x 8cm x 8cm pot. The amount of microbial agent applied was based on the recommended amount for field use (15kg / hm 2 ), convert the content of bacterial fertilizer in each kilogram of soil, and apply the biological agent at a dosage of 0.02g / kg. Mix the soil and biological agent evenly, then pot and water.

[0099] Ten wheat seeds were planted in each pot (as the first day of planting); after the seedlings emerged more evenly, thinning was performed (as the fifth day of planting) and 25 mL of the prepared bacterial solution was poured around the roots of the wheat seedlings (as the first inoculation), and the negative control group was poured with the same volume of sterile 1x phosphate buffer (PBS), and the potted plants were grown at room temperature.

[0100] After 17 days of cultivation, the wheat potted plants were observed, and various indicators of the potted plants were measured and analyzed. The growth of the wheat potted plants was observed, and the plant height, root length and other indicators of the potted wheat seedlings were measured. It was found that the growth of the wheat seedlings inoculated with the composite microbial agent was better than that of the negative control group and the wheat seedlings inoculated with a single microbial agent (see Table 3).

[0101] Table 3 C007 and Bacillus velezine synergistically promote wheat growth

[0102]

[0103] Experimental data show that compound microbial agents can better enhance the synergistic effect of beneficial microorganisms and promote plant growth better.

[0104] Example 5 Functional determination of microbial fertilizer

[0105] In another aspect of the present invention, a microbial fertilizer is provided. According to an embodiment of the present invention, the microbial fertilizer comprises at least one of the following: the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007, its culture or processed product, the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 agent, and the composite microbial agent. Therefore, the microbial fertilizer of the embodiment of the present invention has great application value in improving crop stress resistance, especially salt-alkali resistance; and promoting crop growth.

[0106] According to an embodiment of the present invention, the microbial fertilizer described in the technical solution has an effective bacterial count of not less than 200 million cfu / g. In some embodiments, the effective bacterial count of the microbial fertilizer is not less than 200 million cfu / g, not less than 500 million cfu / g, not less than 800 million cfu / g, and not less than 1 billion cfu / g. Thus, the effect of microbial action can be further improved.

[0107] According to the microbial fertilizer described in the embodiment of the present invention, the microbial fertilizer also includes auxiliary materials or trace elements;

[0108] The auxiliary materials include at least one of the following: straw powder, organic fertilizer or potassium humate;

[0109] The trace elements include at least one of the following: ammonium molybdate, sodium molybdate or ferrous sulfate.

[0110] According to an embodiment of the present invention, the organic fertilizer is a plant-derived organic fertilizer, which can be an organic fertilizer suitable for crop planting as known in the art, and the specific composition can be flexibly selected according to actual needs. In certain embodiments, the plant-derived organic fertilizer main component is mushroom residue, decomposed straw, soybean meal, Chinese medicine residue, etc.

[0111] Components and preparation process of compound microbial fertilizer

[0112] Provided is a composite microbial fertilizer, comprising the following raw materials: straw powder, organic fertilizer, potassium humate, trace elements and a combination of beneficial microorganisms; the beneficial microorganisms are salt-alkali resistant phosphorus-dissolving composting Bacillus stercoris C007 and Bacillus velezensis (with a deposit number of CCTCCNO: M 20221937). The trace elements are ammonium molybdate, sodium molybdate and ferrous sulfate, and the proportion of each component is 1:1:1.

[0113] Preparation of beneficial microorganisms: The culture medium used for C007 is 8g / L corn starch, 8.5g / L bean cake powder, 2 g / L K2HPO4, 1 g / L KH2PO4, 0.1 g / L ZnSO4, 2 mg / L MgSO4, 2 mg / L CuSO4, and 1 mg / L Na2MoO4. The culture medium used for Bacillus Velezii is 6g / L corn starch, 7g / L bean cake powder, 1.5g / L rice flour, 1.5g / L bran, 1g / L corn steep liquor, 0.5g / L calcium carbonate, 0.2g / L potassium nitrate, 0.3g / L magnesium sulfate, 0.2 g / L K2HPO4, and 0.2 g / L Na2HPO4. Both strains were fermented in a 20L fermenter, with samples taken every 4h, and the tank was stopped when the spore maturity was 50%. The obtained bacterial liquid is directly sprayed onto the bacterial powder by high-temperature centrifugal drying, and the two are mixed in a mass ratio of 1:1 to obtain a composite bacterial agent.

[0114] The preparation and use process of compound microbial fertilizer is as follows: stir the cultivated compound bacteria, straw powder, organic fertilizer, potassium humate, and trace elements, add appropriate amount of distilled water, stir evenly and in appropriate size. Then, store at room temperature after drying. Mix the compound bacteria, straw powder, organic fertilizer, potassium humate, and trace elements in a mass ratio of 3:1.5:1.5:0.5:0.1. The trace elements include equal proportions of ammonium molybdate, sodium molybdate, and ferrous sulfate.

[0115] a. Effect of compound microbial fertilizer on the growth of wheat seedlings in saline-alkali soil

[0116] Preparation of wheat seeds: Select intact and disease-free wheat seeds, soak them in 75% ethanol for 3 minutes, then soak them in 5% (v / v) sodium hypochlorite solution for 3 minutes, and then rinse the seeds three times with sterile distilled water to complete the surface disinfection and sterilization of the seeds. After the surface disinfection and sterilization of the seeds is completed, place the seeds in a sterile culture dish and fill the culture dish with sterile distilled water so that the seeds are completely immersed in the sterile distilled water to break the dormancy of the seeds.

[0117] Soil preparation: The saline-alkali farmland soil (soil conductivity 1.9 ms / cm) from Nandagang, Huanghua, Hebei was selected and sieved through a 2 mm sieve and packed in a pot of 8 cm x 8 cm x 8 cm.

[0118] The application amount of compound microbial fertilizer was converted into potted application amount of 2g / kg, 1g / kg, and 0.5g / kg according to the field application amount (100, 50, and 25 kg / mu), and 5 replicates were set for each treatment. After mixing the test soil and compound microbial fertilizer, the soil was irrigated with sterile distilled water and wheat was planted. Five seeds were planted in each pot (as the first day of planting); after the seedlings emerged more uniformly, thinning was carried out (as the fifth day of planting), and the potted plants were placed at room temperature for growth.

[0119] After 14 days of cultivation, the plant pots were observed, and various result indicators of the pots were measured and analyzed.

[0120] 1) Observe the growth of wheat potted plants and find that the growth of wheat seedlings with compound microbial fertilizer is better than that of the negative control group, and the seedling height is significantly higher than that of the negative control group ( Figure 4 , Figure 5 );

[0121] 2) The plant height and root length of potted wheat seedlings were measured. The measurement results and analysis showed that the application of compound microbial fertilizer promoted the growth of wheat seedlings, mainly manifested in the increase of plant height and root length of wheat seedlings;

[0122] Data collection and analysis results: Based on the statistical method, the single-factor ANOVA test of SPSS software (P<0.05 indicates significant difference; P<0.01 indicates extremely significant difference) was used to conduct statistical analysis on the various measurement indicators of wheat potted seedlings. The analysis results showed that the plant height of wheat seedlings with 0.5g of compound microbial fertilizer increased by 23.05% compared with the negative control group, the plant height of wheat seedlings with 1.0g of compound microbial fertilizer increased by 23.20% compared with the negative control group, and the plant height of wheat seedlings with 2.0g of compound microbial fertilizer increased by 27.60% compared with the negative control group, all of which reached significant differences. The root length of wheat seedlings with 0.5g of compound microbial fertilizer increased by 61.93% compared with the negative control group, and the root length of wheat with 1.0g of compound microbial fertilizer increased by 33.94% compared with the negative control group, all of which reached significant differences. The analysis results showed that the application amount of different compound microbial fertilizers was significantly increased compared with the negative control group. The plant height of wheat seedlings treated with compound microbial fertilizer increased by 23.20% to 27.60%. The root length of wheat seedlings treated with compound microbial fertilizer increased by 6.42% to 61.93%.

[0123] b. Effects of compound microbial fertilizer on the growth of wheat seedlings in aqueous solutions with different salt contents

[0124] After germination in a petri dish, the wheat seeds were transferred to a 50ml centrifuge tube containing an aqueous solution with different salt contents, and the plants were fixed with a sponge. The treatments were 75mM salt solution, 75mM salt solution + compound microbial fertilizer, 150mM salt solution, 150mM salt solution + compound microbial fertilizer, 300mM salt solution, and 300mM salt solution + compound microbial fertilizer. The dosage of compound microbial fertilizer was 0.02g. The hydroponic wheat was grown at room temperature. After 15 days of cultivation, the plant pots were observed, and the plant height of the hydroponic wheat was measured and analyzed.

[0125] 1) Observe the growth of wheat and find that the growth of wheat seedlings with compound microbial fertilizer is better than that of the negative control group, and the seedling height is significantly higher than that of the negative control group under the same salt concentration ( Figure 6 , Figure 7 );

[0126] 2) The plant height of wheat seedlings was measured. The results and analysis showed that the application of compound microbial fertilizer promoted the growth of wheat seedlings, which was mainly reflected in the increase of plant height of wheat seedlings in aqueous solutions with different salt contents ( Figure 8 );

[0127] 3) After measuring the height of wheat seedlings, the measurement results were statistically analyzed using SPSS.

[0128] Data collection and analysis results: Based on statistical methods, the plant height of wheat potted seedlings was statistically analyzed using the one-way ANOVA test of SPSS software (P<0.05 indicates a significant difference; P<0.01 indicates an extremely significant difference). The analysis results showed that in aqueous solutions with different salt contents, the increase in plant height of wheat seedlings applied with compound microbial fertilizers ranged from 5.83% to 34.86%, all of which were significant differences.

[0129] In summary, the application of compound microbial fertilizer can promote the growth of wheat seedlings.

[0130] The salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 in the present application can enhance the survival and colonization of beneficial microorganisms in saline-alkali farmland soil and plant roots, and also has the function of dissolving phosphate and secreting plant growth hormone (IAA), which promotes the growth of plants. The composite microbial fertilizer can improve the soil environment and agricultural ecosystem on the one hand, and can also promote plant growth, and to a certain extent can achieve the purpose of reducing the application of chemical fertilizers and improving the fertility of saline-alkali farmland soil.

[0131] SEQ ID NO:1

[0132] >C007_16S rDNA

[0133]

[0134] The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive effort falls within the protection scope of the present invention.

Claims

1. A salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 was deposited in the China Center for Type Culture Collection on August 28, 2024, and its deposit number is: CCTCC NO: M 20241868.

2. A microbial agent, characterized in that: It comprises the salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 according to claim 1.

3. A culture medium for preparing the microbial agent according to claim 2, characterized in that: The culture medium comprises: 8-15 g / L corn starch, 5-10 g / L bean cake powder, 1-5 g / L K2HPO4, 1-5 g / L KH2PO4, 0.1-0.5 g / L ZnSO4, 1-5 mg / L MgSO4, 1-5 mg / L CuSO4, and 1-3 mg / L Na2MoO4.

4. A composite microbial agent, characterized in that: The method comprises the microbial agent as claimed in claim 2.

5. The composite microbial agent according to claim 4, characterized in that: The composite microbial agent includes a Bacillus Velez subtilis agent, and the Bacillus Velez subtilis is selected from the Bacillus Velez subtilis with a deposit number of CCTCC NO: M 20221937.

6. The composite microbial agent according to claim 5, characterized in that: The spore content of salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007 is not less than 100 billion cfu / g; and / or, The Bacillus Velez spore content in the Bacillus Velez inoculant is not less than 130 billion cfu / g.

7. A microbial fertilizer, characterized in that: Contains at least one of the following: The salt-alkali resistant phosphate-dissolving composting Bacillus stercoris C007, its culture or its processed product as described in claim 1; The microbial agent according to claim 2; The composite microbial agent according to claim 4, claim 5 or claim 6.

8. The microbial fertilizer according to claim 7, characterized in that The microbial fertilizer also includes auxiliary materials or trace elements; The auxiliary materials include at least one of the following: straw powder, organic fertilizer or potassium humate; The trace elements include at least one of the following: ammonium molybdate, sodium molybdate or ferrous sulfate.

9. The microbial fertilizer according to claim 7, characterized in that: The effective bacterial count in the microbial fertilizer is not less than 200 million cfu / g.

10. Use of the microorganism according to claim 1, the microbial agent according to claim 2, the composite microbial agent according to claim 4, 5 or 6, or the microbial fertilizer according to claim 7, 8 or 9 in at least one of the following: Improve crop resistance to stress, especially resistance to salinity and alkali; Promote crop growth.

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