Application of composite phosphate-solubilizing bacterial agent to promotion of wheat growth

Abstract

The invention relates to the field of environment and agricultural biotechnology, in particular to an application of a composite phosphate-solubilizing bacterial agent in promoting wheat growth, which is characterized in that germinated wheat is cultivated firstly, then the germinated wheat is placed in soil mixed with the composite phosphate-solubilizing bacterial agent to be cultivated, and then the germinated wheat is cultivated in soil mixed with the composite phosphate-solubilizing bacterial agent. Furthermore, the growth of wheat is promoted by the cooperation of enterobacter hormaechei HS6, pantoea agglomerans CT3 and wheat bran in the compound phosphate-solubilizing bacterium agent. Wherein the enterobacter hormaechei HS6 and the pantoea agglomerans CT3 can efficiently convert insoluble phosphorus in the soil into soluble phosphorus, the utilization rate of phosphorus in the soil is effectively increased, the use amount of chemical phosphate fertilizer is reduced, nutritional ingredients are increased for the soil, the physical and chemical properties of the soil are improved, wheat growth can be well promoted, and the application prospect is good.

Description

Technical Field

[0001] The invention relates to the fields of environment and agricultural biotechnology, and in particular to application of a composite phosphate-dissolving bacteria agent in promoting wheat growth. Background Art

[0002] Phosphorus is one of the essential nutrients for plant growth and development. Organic phosphorus in soil generally accounts for 10% to 15% of the total phosphorus in the soil. As an important component of some compounds and enzymes in plants, phosphorus plays an indispensable role in the transformation, transportation and storage of substances in plants. It participates in the metabolism of plants (such as cell division, signal transduction rate, macromolecular biosynthesis and photosynthesis, etc.), and can also increase the stress resistance and adaptability of plants. It also plays an important role in promoting the division and proliferation of plant cells. The lack of phosphorus will directly affect the photosynthesis, respiration and metabolism of substances and energy of plants, thereby affecting the yield and quality of plants.

[0003] Soil microorganisms control key bio-geo-chemical processes in agricultural ecosystems and play a vital role in nutrient cycling and energy flow. Phosphate-dissolving bacteria is a general term for microorganisms that can decompose phosphorus-containing compounds and promote the effectiveness of phosphorus, which can improve the utilization efficiency of phosphate fertilizers and phosphate rock. Phosphate-dissolving bacteria are generally considered to be a high-quality alternative to chemical phosphate fertilizers. While promoting plant absorption of phosphorus, phosphate-dissolving bacteria not only do not harm the agricultural ecological environment, but also have the potential to repair the soil environment polluted by traditional commercial fertilizers. Phosphate-dissolving bacteria contain a large number of phosphate-dissolving microorganisms that can degrade sparingly soluble phosphorus, which have varying degrees of influence on crop growth, soil phosphorus conversion and utilization, and changes in soil fertility. With its advantages of no secondary pollution, sustainable development and environmental protection, it has become a green production method to replace phosphate fertilizers. It is an effective environmentally friendly strategy and has received widespread attention.

[0004] As an emerging environmentally friendly way to improve the soil environment, phosphate-dissolving bacteria improve the effective phosphorus content in the soil and promote crop growth with the green concept of sustainable development. However, the development of compound phosphate-dissolving bacteria in promoting crop growth needs to be further expanded to promote soil improvement and agricultural development. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide an application of a composite phosphate-dissolving bacteria agent to promote wheat growth. The application of the composite phosphate-dissolving bacteria agent to promote wheat growth can effectively improve the utilization rate of phosphorus in the soil and promote wheat growth because the composite phosphate-dissolving bacteria agent has an excellent phosphate-dissolving effect.

[0006] In order to achieve the purpose of the above invention, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides an application of a composite phosphate-dissolving bacteria agent to promote wheat growth, comprising the following steps:

[0008] S1. Cultivation of wheat seeds: Place the wheat seeds in a culture dish and cultivate them for 6-8 days until they sprout 2-3 cm to obtain germinated wheat;

[0009] S2, applying a composite phosphate-dissolving bacteria agent: after passing the soil through a 16-20 mesh sieve, put the composite phosphate-dissolving bacteria agent into the soil, mix the soil thoroughly and put it into a planting pot to obtain stand-by soil, and put the germinated wheat cultivated in step S1 into the stand-by soil at a depth of 1.5 cm to 2.5 cm;

[0010] S3. Cultivation of wheat: placing the planting pot with the germinated wheat in a sunny place, watering every 23h to 25h for 20d to 22d, harvesting the wheat to obtain wheat plants;

[0011] The composite phosphate-dissolving bacteria agent comprises Enterobacter holmesii HS6, Pantoea agglomerans CT3, culture medium and wheat bran;

[0012] The classification name of the Enterobacter hormaechei HS6 is Enterobacter hormaechei, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on October 19, 2023, with a deposit number of CGMCC NO.: 28680;

[0013] The classification name of the Pantoea agglomerans CT3 is Pantoea agglomerans, which was deposited in the General Microbiological Center of the China Culture Collection Administration on January 8, 2025, with the deposit number CGMCCNO.: 33335.

[0014] Furthermore, the 16S rDNA gene sequence of the Enterobacter holmesii HS6 is shown in SEQ ID No. 1;

[0015] The 16S rDNA gene sequence of the Pantoea agglomerans CT3 is shown in SEQ ID No.2.

[0016] Furthermore, the particle size of the wheat bran is a size that can pass through a 30-50 mesh sieve. Wheat bran contains rich nutrients, which can promote the growth of Enterobacter horneri HS6 and Pantoea agglomerans CT3 on the one hand, and can serve as a carrier of Enterobacter horneri HS6 and Pantoea agglomerans CT3 to form a solid composite phosphate-dissolving bacteria agent on the other hand, and can also increase nutrients for the soil and improve the physical and chemical properties of the soil, thereby better promoting wheat growth.

[0017] Furthermore, in step S1, the wheat seeds are placed in a culture dish, distilled water is added, and then placed in a constant temperature incubator at 28°C for 6 to 8 days until the seeds sprout to 2 cm to 3 cm, thereby obtaining germinated wheat.

[0018] Furthermore, in step S2, the mass percentage of the composite phosphate-dissolving bacteria agent in the soil is 3% to 9%.

[0019] Furthermore, the preparation method of the composite phosphate-dissolving bacteria agent comprises the following steps:

[0020] S1) Strain activation: Enterobacter HS6 and Pantoea agglomerans CT3 were streaked onto LB solid medium, cultured at 28°C for 48 h, and then single colonies were picked and inoculated into LB liquid medium. The culture was then placed in a constant temperature shaker at 28°C and shaken at 180 r / min until OD 600 is 0.6-0.8, and HS6 bacterial suspension and CT3 bacterial suspension are obtained;

[0021] S2), preparing a liquid bacterial agent: mixing the prepared HS6 bacterial suspension and the CT3 bacterial suspension to form a composite bacterial solution, then inoculating the composite bacterial solution into an NBRIP liquid culture medium, adding sodium glutamate, and then placing the mixture in a constant temperature shaker at 28° C. and shaking at 180 r / min to prepare a liquid bacterial agent;

[0022] S3) preparing a composite phosphate-dissolving bacterial agent: adding the prepared liquid bacterial agent to the sterilized wheat bran, and then culturing it at 28° C. for 2 to 4 days to obtain the composite phosphate-dissolving bacterial agent.

[0023] Further, in step S1), Enterobacter HS6 and Pantoea agglomerans CT3 stored in glycerol at -80°C were streaked on LB solid medium, cultured at 28°C for 48 hours, and then single colonies were picked and inoculated into LB liquid medium, and then placed in a constant temperature shaker at 28°C and shaken at 180 r / min for 23 hours to 25 hours until OD 600 is 0.6-0.8, and HS6 bacterial suspension and CT3 bacterial suspension are obtained.

[0024] Furthermore, the effective viable counts of the HS6 bacterial suspension and the CT3 bacterial suspension obtained in step S1) were both 1×10 9 CFU / mL~9×10 9 CFU / mL.

[0025] Furthermore, in step S2), the volume ratio of the HS6 bacterial suspension to the CT3 bacterial suspension is 1:1; the inoculation amount of the composite bacterial suspension in the NBRIP liquid culture medium is 1% of the volume percentage of the culture medium; the addition amount of sodium glutamate in the NBRIP liquid culture medium is 0.3% of the volume percentage of the culture medium; and the culture is placed in a constant temperature shaker at 28°C and shaken at 180 r / min for 72 h to 120 h.

[0026] Furthermore, in step S3), the mass percentage of the liquid bacterial agent in the wheat bran is 25% to 35%; and / or

[0027] In step S3), the wheat bran sterilized at 121° C. and 104 KPa for 30 min is placed in a sterile polyethylene bag, the liquid bacterial agent obtained in step S2) is added to the wheat bran in the sterile polyethylene bag, the bag is sealed after mixing, 5 to 8 ventilation holes are pierced on the surface of the polyethylene bag with a sterilized needle, and then a polyethylene bag is put in and 5 to 8 ventilation holes are pierced, and then the bag is cultured at 28° C. for 2 to 4 days to obtain the composite phosphate-dissolving bacterial agent.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The application of a composite phosphate-dissolving bacteria agent of the present invention to promote wheat growth is to first cultivate germinated wheat, then place the germinated wheat in soil mixed with the composite phosphate-dissolving bacteria agent for cultivation, and then use the Enterobacter hornii HS6, Pantoea agglomerans CT3 and wheat bran in the composite phosphate-dissolving bacteria agent to synergistically promote the growth of wheat. Among them, Enterobacter hornii HS6 and Pantoea agglomerans CT3 can efficiently convert the insoluble phosphorus in the soil into soluble phosphorus, effectively improve the utilization rate of phosphorus in the soil, reduce the use of chemical phosphorus fertilizers, increase the nutrients in the soil and improve the physical and chemical properties of the soil, thereby being able to well promote wheat growth, and having a good application prospect.

[0030] (2) The present invention provides an application of a composite phosphate-dissolving bacteria agent to promote wheat growth. The composite phosphate-dissolving bacteria agent is formed by synergizing Enterobacter hullii HS6 and Pantoea agglomerans CT3, and utilizing a culture medium to promote growth and metabolism. Meanwhile, wheat bran further promotes the growth of the bacteria and serves as a carrier of the bacteria to form a composite phosphate-dissolving bacteria agent. The composite phosphate-dissolving bacteria agent can synergistically convert the insoluble phosphorus in the soil into soluble phosphorus, thereby adding rich nutrients to the soil and improving the physical and chemical properties of the soil, thereby promoting wheat growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 It is a scanning electron microscope image of Enterobacter hallii HS6 of the present invention.

[0033] Figure 2 It is a scanning electron microscope image of Pantoea agglomerans CT3 of the present invention.

[0034] Figure 3 This is a graph showing the wheat growth with different application rates of composite phosphate-dissolving bacteria.

[0035] Figure 4This is a graph showing the test results of the effects of different application rates of composite phosphate-dissolving bacteria on the fresh weight of wheat.

[0036] Figure 5 This is a test result chart showing the effect of different application rates of composite phosphate-dissolving bacteria on the chlorophyll content of wheat.

[0037] Figure 6 This is a graph showing the test results of the effect of different application rates of composite phosphate-dissolving bacteria on the total phosphorus content of wheat.

[0038] Figure 7 This is a graph showing the test results of the effects of different application rates of composite phosphate-dissolving bacteria on the content of mineral elements in the aboveground parts of wheat plants.

[0039] Figure 8 This is a graph showing the test results of the effects of different application rates of composite phosphate-dissolving bacteria on the content of mineral elements in the underground part of wheat plants. DETAILED DESCRIPTION

[0040] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0041] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. In the present invention, the singular forms "a", "said" and "the" used in the embodiments and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0042] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0043] The culture medium formula described in the following examples is as follows:

[0044] LB medium (g / L): peptone 10 g, yeast extract 5 g, NaCl 10 g, pH 7.0-7.2.

[0045] LB solid culture medium: Add 18g to 20g of agar powder per 1L of LB culture medium.

[0046] NBRIP medium (g / L): glucose 10 g, (NH 4 ) 2 SO 4 0.5g, NaCl 0.3g, MgSO 4 7H 2 O0.3g, FeSO 4 7H 2 O 0.03g, MnSO4 ·2H 2 O 0.03g, Ca 3 (PO 4 ) 2 5g, KCl 0.3g, lecithin 0.2g, pH 7.2~7.4.

[0047] NBRIP solid culture medium: Add 18g to 20g of agar powder to every 1L of NBRIP culture medium.

[0048] Example 1

[0049] An application of a composite phosphate-dissolving bacteria agent to promote wheat growth comprises the following steps:

[0050] S1. Cultivation of wheat seeds: Place the wheat seeds in a culture dish, add distilled water, and then place in a constant temperature incubator at 28° C. for 7 days until the seeds sprout to 2 cm to 3 cm, thereby obtaining germinated wheat;

[0051] S2, applying a composite phosphate-dissolving bacteria agent: after passing the soil through an 18-mesh sieve, put the composite phosphate-dissolving bacteria agent into the soil, mix the soil thoroughly and put it into a planting pot to obtain stand-by soil, and put the germinated wheat cultivated in step S1 into the stand-by soil at a depth of 2 cm; wherein the mass percentage of the composite phosphate-dissolving bacteria agent in the soil is 3%;

[0052] S3. Cultivation of wheat: Place the planting pots containing germinated wheat in a sunny place, water them every 24 hours for 21 days, harvest the wheat and obtain wheat plants.

[0053] The composite phosphate-dissolving bacteria agent includes Enterobacter huxleyi HS6, Pantoea agglomerans CT3, culture medium and wheat bran; wherein the particle size of the wheat bran is a size that can pass through a 40-mesh sieve.

[0054] Among them, the classification name of Enterobacter hormaechei HS6 is Enterobacter hormaechei, which has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on October 19, 2023, with the deposit number CGMCC NO.: 28680. Among them, the scanning electron micrograph of Enterobacter hormaechei HS6, such as Figure 1 shown.

[0055] Among them, the classification name of Pantoea agglomerans CT3 is Pantoea agglomerans, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on January 8, 2025, with the deposit number CGMCC NO.: 33335. Among them, the scanning electron microscopy image of Pantoea agglomerans CT3 is as follows: Figure 2 shown.

[0056] Among them, the 16S rDNA gene sequence of Enterobacter hallii HS6 is shown in SEQ ID No.1; the 16S rDNA gene sequence of Pantoea agglomerans CT3 is shown in SEQ ID No.2.

[0057] The preparation method of the composite phosphate-dissolving bacteria agent comprises the following steps:

[0058] S1) Strain activation: Enterobacter HS6 and Pantoea agglomerans CT3 stored in glycerol at -80°C were streaked onto LB solid culture medium, cultured at 28°C for 48 h, and then single colonies were picked and inoculated into LB liquid culture medium, and then placed in a constant temperature shaker at 28°C and cultured at 180 r / min for 24 h until OD600 was 0.6-0.8 to obtain HS6 bacterial suspension and CT3 bacterial suspension; wherein the effective viable counts of HS6 bacterial suspension and CT3 bacterial suspension were both 1×10 9 CFU / mL~9×10 9 CFU / mL;

[0059] S2), preparing a liquid bacterial agent: mixing the prepared HS6 bacterial suspension and the CT3 bacterial suspension in a volume ratio of 1:1 to form a composite bacterial solution, and then inoculating the composite bacterial solution into an NBRIP liquid culture medium at an inoculum amount of 1% by volume, and adding 0.3% by volume of sodium glutamate, and then placing the mixture in a constant temperature shaker at 28°C and shaking at 180 r / min for 96 hours to obtain a liquid bacterial agent;

[0060] S3), prepare a composite phosphate-dissolving bacteria agent: put the wheat bran sterilized at 121°C, 104KPa for 30 minutes into a sterile polyethylene bag, add the liquid bacterial agent prepared in step S2) into the wheat bran in the sterile polyethylene bag at an application amount of 30% by mass, seal the bag after mixing, pierce 5 ventilation holes on the surface of the polyethylene bag with a sterilized needle, put another layer of polyethylene bag and pierce 5 ventilation holes, and then culture at 28°C for 3 days to obtain the composite phosphate-dissolving bacteria agent.

[0061] Example 2

[0062] The present embodiment is different from the first embodiment in that the mass percentage of the composite phosphate-dissolving bacteria agent in the soil is 6%. The remaining conditions and methods of the present embodiment are the same as those of the first embodiment.

[0063] Example 3

[0064] The present embodiment is different from the first embodiment in that the mass percentage of the composite phosphate-dissolving bacteria agent in the soil is 9%. The remaining conditions and methods of the present embodiment are the same as those of the first embodiment.

[0065] Example 4

[0066] The invention discloses an application of a composite phosphate-dissolving bacteria agent to promote wheat growth. The difference between this embodiment and embodiment 1 is that in this embodiment, in step S1, the wheat is placed in a constant temperature incubator at 28°C for 6 days until the wheat sprouts 2cm to 3cm, thereby obtaining germinated wheat; in step S2, the soil is passed through a 16-mesh sieve and then placed in the composite phosphate-dissolving bacteria agent, and the germinated wheat cultivated in step S1 is placed in the stand-by soil at a depth of 1.5cm; in step S3, watering is performed every 23 hours for 20 days; and the particle size of the wheat bran is a size that passes through a 30-mesh sieve.

[0067] The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0068] Example 5

[0069] The invention discloses an application of a composite phosphate-dissolving bacteria agent to promote wheat growth. The difference between this embodiment and embodiment 1 is that in this embodiment, in step S1, the wheat is placed in a constant temperature incubator at 28°C for 8 days until the wheat sprouts 2cm to 3cm, thereby obtaining germinated wheat; in step S2, the soil is passed through a 20-mesh sieve and then placed in the composite phosphate-dissolving bacteria agent, and the germinated wheat cultivated in step S1 is placed in the stand-by soil at a depth of 2.5cm; in step S3, watering is performed every 25 hours for 22 days; and the particle size of the wheat bran is a size that passes through a 50-mesh sieve.

[0070] The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0071] Example 6

[0072] The invention discloses an application of a composite phosphate-dissolving bacteria agent for promoting wheat growth. The difference between this embodiment and embodiment 1 is that, in this embodiment, in step S1), the mixture is placed in a constant temperature shaker at 28°C and cultured at 180 r / min for 23 hours until the OD600 is 0.6-0.8; in step S2), the mixture is placed in a constant temperature shaker at 28°C and cultured at 180 r / min for 72 hours to obtain a liquid bacteria agent; in step S3), the liquid bacteria agent is added to wheat bran in a sterile polyethylene bag at an application amount of 25% by mass; 6 ventilation holes are pierced on the surface of the polyethylene bag with a sterile needle, a polyethylene bag is then put on and 7 ventilation holes are pierced, and then cultured at 28°C for 2 days to obtain the composite phosphate-dissolving bacteria agent.

[0073] The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0074] Example 7

[0075] The invention discloses an application of a composite phosphate-dissolving bacteria agent for promoting wheat growth. The difference between this embodiment and embodiment 1 is that, in this embodiment, in step S1), the mixture is placed in a constant temperature shaker at 28°C and cultured at 180 r / min for 25 hours until the OD600 is 0.6-0.8; in step S2), the mixture is placed in a constant temperature shaker at 28°C and cultured at 180 r / min for 120 hours to obtain a liquid bacteria agent; in step S3), the liquid bacteria agent is added to wheat bran in a sterile polyethylene bag at an application amount of 35% by mass; 8 ventilation holes are pierced on the surface of the polyethylene bag with a sterile needle, a polyethylene bag is then put on and 8 ventilation holes are pierced, and then cultured at 28°C for 4 days to obtain the composite phosphate-dissolving bacteria agent.

[0076] The remaining conditions and methods of this embodiment are the same as those of embodiment 1.

[0077] Experimental testing:

[0078] The design of the experimental test is shown in Table 1 below.

[0079] Table 1 Design table of experimental detection

[0080] Experimental Group Soil / Quality Compound phosphate-dissolving bacteria agent / application amount CK 1kg 0 T1 1kg 3％ T2 1kg 6％ T3 1kg 9％

[0081] As shown in Table 1, in the CK group, the soil used for growing wheat was not applied with the compound phosphate-dissolving bacteria agent; in the T1 group, the soil used for growing wheat was applied with 3% of the compound phosphate-dissolving bacteria agent, that is, the mass percentage of the compound phosphate-dissolving bacteria agent in the soil was 3%; in the T2 group, the soil used for growing wheat was applied with 6% of the compound phosphate-dissolving bacteria agent, that is, the mass percentage of the compound phosphate-dissolving bacteria agent in the soil was 6%; in the T3 group, the soil used for growing wheat was applied with 9% of the compound phosphate-dissolving bacteria agent, that is, the mass percentage of the compound phosphate-dissolving bacteria agent in the soil was 9%.

[0082] (I) Detection of the effects of different application rates of composite phosphate-dissolving bacteria on wheat growth

[0083] The experimental test design table in Table 1 was used to cultivate wheat according to the application method of a composite phosphate-dissolving bacteria agent to promote wheat growth in Example 1, Example 2 and Example 3. After the cultivation was completed, the growth of the potted plants was photographed and recorded, the wheat was harvested, the excess soil was removed, the soil at the roots was washed, the surface moisture was dried with phosphorus-free filter paper, and the plant height was measured, and the growth of the wheat plants was observed.

[0084] The plant heights of wheat at the end of cultivation in the CK, T1, T2 and T3 groups were measured by laying the wheat plants flat on a black background cloth, measuring the plant heights with a soft tape measure, and taking photos to record. Figure 3 As shown by Figure 3It can be seen that in the blank control group, i.e., the CK group, the lodging situation was more serious than that in the T1 group, T2 group, and T3 group, and the tip of the leaves was yellowed. Although a few lodgings occurred in the T1 group, T2 group, and T3 group, the overall growth was uniform and upright, and the plants were stronger than those in the CK group. It can be seen that the growth of wheat cultivated in the T1 group, T2 group, and T3 group increased to varying degrees compared with the CK group. This shows that in the application of the composite phosphate-dissolving bacteria agent of the present invention to promote wheat growth, the composite phosphate-dissolving bacteria agent used can well promote wheat growth.

[0085] (II) Detection of the effects of different application rates of composite phosphate-dissolving bacteria on wheat plant height and fresh weight

[0086] The experimental test design table in Table 1 was used to cultivate wheat according to the application method of a composite phosphate-dissolving bacteria agent to promote wheat growth in Example 1, Example 2 and Example 3. After the cultivation was completed, the growth of the potted plants was photographed and recorded, the wheat was harvested, the excess soil was removed, the soil at the roots was washed, and the surface moisture was dried with phosphorus-free filter paper and the fresh weight of the wheat was measured.

[0087] Method for weighing fresh weight of wheat: Place a small beaker on a balance, place the dried wheat plant sample in the beaker, weigh it, and record the data. The fresh weight data analysis results of wheat cultivated in the CK group, T1 group, T2 group, and T3 group are as follows: Figure 4 As shown. Figure 4 It can be seen that the plant height of the T1 group, T2 group and T3 group increased slightly compared with the CK group, and increased by 3.13%, 2.72% and 3.34% respectively compared with the CK group. In addition, the total fresh weight of the plants in the T1 group, T2 group and T3 group increased compared with the CK group, and increased by 6.05%, 23.96% and 46.97% respectively compared with the CK group, among which the T3 group increased significantly compared with the CK group. It shows that the application of a composite phosphate-dissolving bacteria agent of the present invention to promote wheat growth has a significant growth-promoting effect on the growth of wheat. That is, the application of the composite phosphate-dissolving bacteria agent of the present invention has a promoting effect on the growth of wheat, and the effect of the application of the composite phosphate-dissolving bacteria agent on the fresh weight of wheat is more significant, and the greater the application amount, the more significant the increase, indicating that the composite phosphate-dissolving bacteria agent applied by the present invention can promote the growth of wheat quality, and then promote the growth of wheat.

[0088] (III) Detection of the effects of different application rates of composite phosphate-dissolving bacteria on wheat chlorophyll content

[0089] The experimental detection design table in Table 1 was used to cultivate wheat according to the application method of a composite phosphate-dissolving bacteria agent to promote wheat growth in Example 1, Example 2 and Example 3. After the cultivation was completed, the growth of the potted plants was photographed and recorded, the wheat was harvested, and then the chlorophyll content of the wheat harvested from different experimental groups was detected.

[0090] The method for determining the chlorophyll content of wheat is as follows: take 0.1 g of fresh leaves from the same height of the plant, cut them into pieces and put them into a 10 mL centrifuge tube, add 10 mL of 95% ethanol, wrap them in tin foil and place them away from light, shake them evenly during the process to allow the chlorophyll to be fully extracted, take 3 mL to 4 mL into a 1 cm colorimetric dish after 24 hours, compare the colors at wavelengths of 665 nm and 645 nm, use 95% ethanol as a blank reference, and calculate the chlorophyll a and chlorophyll b contents according to formulas (1) and (2).

[0091] Chla=(12.72D 665 - 2.59D 645 )V×N×(1000×W) -1 (1)

[0092] Chlb=(22.88D 645 - 4.67D 665 )V×N×(1000×W) -1 (2)

[0093] Where: Chla, Chlb—chlorophyll a, chlorophyll b content, mg·L -1 ; V—volume of photosynthetic pigment extract, mL; N—dilution multiple; W—sample weight, g.

[0094] Among them, the chlorophyll content of wheat cultivated in the CK group, T1 group, T2 group and T3 group was determined as follows Figure 5 As shown. Figure 5 It can be seen that the chlorophyll a and chlorophyll b contents of wheat cultivated in the T1 group, T2 group and T3 group are slightly increased compared with the chlorophyll a and chlorophyll b contents of the CK group. Among them, the chlorophyll a of wheat cultivated in the T1 group, T2 group and T3 group increased by 6.2%, 20% and 15% respectively compared with the CK group; the chlorophyll b of wheat cultivated in the T1 group, T2 group and T3 group increased by 4.4%, 8.9% and 9.7% respectively compared with the CK group. It shows that the application of a composite phosphate-dissolving bacteria agent of the present invention to promote wheat growth can improve the chlorophyll content of wheat and promote the photosynthesis of wheat.

[0095] (IV) Detection of the effects of different application rates of composite phosphate-dissolving bacteria on total phosphorus content in wheat

[0096] The experimental detection design table in Table 1 was used to cultivate wheat according to the application method of a composite phosphate-dissolving bacteria agent to promote wheat growth in Example 1, Example 2 and Example 3. After the cultivation was completed, the growth of the potted plants was photographed and recorded, the wheat was harvested, and then the total phosphorus content of the wheat harvested from different experimental groups was detected.

[0097] Among them, the method for determining the phosphorus content of wheat is as follows: take a fresh wheat sample and dry it in a 70℃ oven for 48h. After confirming that the moisture is completely dried, grind it in a mortar and pass it through a 40-mesh sieve for later use. Weigh 0.1g of dried wheat sample (accurate to 0.0001g), add 9mL nitric acid, digest it at 100℃ for 1h, then heat it to 170℃ and digest it for 3h until the sample is completely digested and the solution becomes clear and transparent. Rinse the digestion tube with distilled water and transfer all the digestion solution to a colorimetric tube, make it dilute to 25mL, shake it well, dilute it 1-fold with distilled water, and inject it into an inductively coupled plasma optical emission spectrometer (ICP-OES) to determine the phosphorus element in wheat.

[0098] Among them, the results of ICP-OES determination of total phosphorus content in wheat are as follows Figure 6 As shown. Figure 6 It can be seen that the wheat cultivated by the T1 group, the T2 group and the T3 group has increased the total phosphorus content of the aboveground part and the underground part of the plant due to the application of the composite phosphate-dissolving bacteria agent of the present invention. On the whole, the growth degree of the total phosphorus content of the aboveground part is greater than the growth of the total phosphorus content of the underground part. Among them, the total phosphorus content of the aboveground part shows an upward and then gentle trend with the increase of the application amount of the composite phosphate-dissolving bacteria agent. The total phosphorus content of the T1 group, the T2 group and the T3 group is increased by 0.3%, 1.55% and 1.46% respectively compared with the CK group, and the total phosphorus content of the T2 group and the T3 group is significantly increased compared with the CK group. In addition, the total phosphorus content of the underground part shows an upward trend with the application amount of the composite phosphate-dissolving bacteria agent. The total phosphorus content of the T1 group, the T2 group and the T3 group is significantly different from that of the CK group, which is increased by 0.5%, 0.72% and 0.86% respectively compared with the CK group. In general, the application of the composite phosphate-dissolving bacteria agent of the present invention to promote wheat growth can greatly increase the total phosphorus content of wheat, thereby reducing the use of chemical phosphate fertilizers, increasing the nutrients in the soil and improving the physical and chemical properties of the soil, thereby promoting wheat growth and having a good application prospect. (V) Detection of the effects of different application amounts of composite phosphate-dissolving bacteria agent on the mineral element content of the aboveground part of wheat plants

[0099] The experimental detection design table in Table 1 was used to cultivate wheat according to the application method of a composite phosphate-dissolving bacteria agent to promote wheat growth in Example 1, Example 2 and Example 3. After the cultivation was completed, the growth of the potted plants was photographed and recorded, the wheat was harvested, the excess soil was removed, the soil at the roots was washed, and the surface moisture was dried with phosphorus-free filter paper to measure the mineral element content of the aboveground part of the wheat plant.

[0100] Among them, the determination of mineral element contents in the aboveground parts of wheat plants cultivated in the CK, T1, T2 and T3 groups was as follows: Figure 7 As shown. Figure 7It can be seen that the Ca content in the aboveground part of wheat plants in the T1 group, T2 group and T3 group was significantly increased compared with the CK group. The T1 group, T2 group and T3 group increased by 54%, 38% and 91.9% respectively compared with the CK group, which was significantly improved compared with the CK group, indicating that the present invention has a more obvious promoting effect on the increase of Ca content in wheat plants through the application of the composite phosphate-dissolving bacteria agent. In addition, the Mg content in the aboveground part of wheat showed an upward trend with the increase in the application amount of the composite phosphate-dissolving bacteria agent. Compared with the T1 group, T2 group and T3 group, it increased by 40%, 52.7% and 52.6%, respectively, indicating that the composite phosphate-dissolving bacteria agent of the present invention has a significant promoting effect on the increase of Mg in the aboveground part of wheat. The aluminum content and magnesium content of the T3 group were significantly improved compared with the CK group. It shows that the composite phosphate-dissolving bacteria agent of the present invention promotes the absorption of aluminum and iron in the aboveground part of wheat.

[0101] (VI) Detection of the effects of different application rates of composite phosphate-dissolving bacteria on the content of mineral elements in the underground part of wheat plants

[0102] The experimental detection design table in Table 1 was used to cultivate wheat according to the application method of a composite phosphate-dissolving bacteria agent to promote wheat growth in Example 1, Example 2 and Example 3. After the cultivation was completed, the growth of the potted plants was photographed and recorded, the wheat was harvested, the excess soil was removed, the soil at the roots was washed, and the surface moisture was dried with phosphorus-free filter paper to measure the mineral element content of the aboveground part of the wheat plant.

[0103] Among them, the determination of mineral element contents in the underground parts of wheat plants cultivated in the CK, T1, T2 and T3 groups was as follows: Figure 8 As shown. Figure 8 It can be seen that the content of Ca in the underground part of wheat plants in the T1 group and the T3 group was significantly higher than that in the CK group, increasing by 41.6% and 48.2% respectively. In addition, the content of Mn in the underground part of wheat plants increased by 26.5%, 30.8% and 30% respectively in the T1 group, the T2 group and the T3 group compared with the CK group. The content of Mn in the underground part of wheat plants increased with the increase of the application amount of the composite phosphate-dissolving bacteria agent. The appropriate increase of the content of Mn is beneficial to the absorption of nitrogen and phosphorus by wheat and reduces the transpiration rate of wheat leaves. In addition, Sr is an indispensable trace element in the human body. Strontium-enriched wheat can provide certain nutrition for the human body, but the country has not yet clarified the standard of strontium-enriched wheat. There was no significant difference in the content of Sr among the CK group, the T1 group, the T2 group and the T3 group. Compared with the CK group, the content of Sr in the T1 group, the T2 group and the T3 group was lower, but the content of Sr in the T1 group, the T2 group and the T3 group was greater than 2.0 mg / kg. In summary, the composite phosphate-dissolving bacteria agent applied by the present invention has a relatively good promoting effect on the enrichment and utilization of Ca and Mn in the underground part of wheat, thereby adding rich nutrients to wheat and improving the physical and chemical properties of the soil, thereby promoting wheat growth.

[0104] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

[0105]

[0106] .

Claims

1. An application of a composite phosphate-dissolving bacteria agent to promote wheat growth, characterized in that: The following steps are involved: S1. Cultivation of wheat seeds: Place the wheat seeds in a culture dish and cultivate them for 6-8 days until they sprout 2-3 cm to obtain germinated wheat; S2, applying a composite phosphate-dissolving bacteria agent: after passing the soil through a 16-20 mesh sieve, put the composite phosphate-dissolving bacteria agent into the soil, mix the soil thoroughly and put it into a planting pot to obtain stand-by soil, and put the germinated wheat cultivated in step S1 into the stand-by soil at a depth of 1.5 cm to 2.5 cm; S3. Cultivation of wheat: placing the planting pot with the germinated wheat in a sunny place, watering every 23h to 25h for 20d to 22d, harvesting the wheat to obtain wheat plants; The composite phosphate-dissolving bacteria agent comprises Enterobacter holmesii HS6, Pantoea agglomerans CT3, culture medium and wheat bran; The classification name of the Enterobacter hormaechei HS6 is Enterobacter hormaechei, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on October 19, 2023, with a deposit number of CGMCC NO.: 28680; The classification name of the Pantoea agglomerans CT3 is Pantoea agglomerans, which was deposited in the General Microbiological Center of the China Culture Collection Administration on January 8, 2025, with the deposit number CGMCCNO.: 33335.

2. The use of a composite phosphate-dissolving bacteria agent to promote wheat growth as claimed in claim 1, characterized in that: The 16S rDNA gene sequence of the Enterobacter holmesii HS6 is shown in SEQ ID No. 1; The 16S rDNA gene sequence of the Pantoea agglomerans CT3 is shown in SEQ ID No.

2.

3. The use of a composite phosphate-dissolving bacteria agent to promote wheat growth as claimed in claim 1, characterized in that: The particle size of the wheat bran is a size that can pass through a 30-50 mesh sieve.

4. The use of a composite phosphate-dissolving bacteria agent to promote wheat growth as claimed in claim 1, characterized in that: In step S1, wheat seeds are placed in a culture dish, distilled water is added, and then placed in a constant temperature incubator at 28° C. for 6 to 8 days until sprouts grow to 2 cm to 3 cm, thereby obtaining germinated wheat.

5. The use of a composite phosphate-dissolving bacteria agent to promote wheat growth as claimed in claim 1, characterized in that: In step S2, the mass percentage of the composite phosphate-dissolving bacteria agent in the soil is 3% to 9%.

6. The use of a composite phosphate-dissolving bacteria agent to promote wheat growth as claimed in claim 1, characterized in that: The preparation method of the composite phosphate-dissolving bacteria agent comprises the following steps: S1) Strain activation: Enterobacter HS6 and Pantoea agglomerans CT3 were streaked onto LB solid medium, cultured at 28°C for 48 h, and then single colonies were picked and inoculated into LB liquid medium. The culture was then placed in a constant temperature shaker at 28°C and shaken at 180 r / min until OD 600 is 0.6-0.8, and HS6 bacterial suspension and CT3 bacterial suspension are obtained; S2), preparing a liquid bacterial agent: mixing the prepared HS6 bacterial suspension and the CT3 bacterial suspension to form a composite bacterial solution, then inoculating the composite bacterial solution into an NBRIP liquid culture medium, adding sodium glutamate, and then placing the mixture in a constant temperature shaker at 28° C. and shaking at 180 r / min to prepare a liquid bacterial agent; S3) preparing a composite phosphate-dissolving bacterial agent: adding the prepared liquid bacterial agent to the sterilized wheat bran, and then culturing it at 28° C. for 2 to 4 days to obtain the composite phosphate-dissolving bacterial agent.

7. The use of a composite phosphate-dissolving bacteria agent to promote wheat growth as claimed in claim 6, characterized in that: In step S1), Enterobacter HS6 and Pantoea agglomerans CT3 stored in glycerol at -80°C were streaked onto LB solid medium, cultured at 28°C for 48 h, and then single colonies were picked and inoculated into LB liquid medium, and then placed in a constant temperature shaker at 28°C and shaken at 180 r / min for 23 h to 25 h until OD 600 is 0.6-0.8, and HS6 bacterial suspension and CT3 bacterial suspension are obtained.

8. The use of a composite phosphate-dissolving bacteria agent to promote wheat growth as claimed in claim 6, characterized in that: The effective viable counts of the HS6 bacterial suspension and the CT3 bacterial suspension obtained in step S1) were both 1×10 9 CFU / mL~9×10 9 CFU / mL.

9. The use of a composite phosphate-dissolving bacteria agent to promote wheat growth as claimed in claim 6, characterized in that: In step S2), the volume ratio of the HS6 bacterial suspension to the CT3 bacterial suspension is 1:1; the inoculation amount of the composite bacterial suspension in the NBRIP liquid culture medium is 1% of the volume percentage of the culture medium; the addition amount of sodium glutamate in the NBRIP liquid culture medium is 0.3% of the volume percentage of the culture medium; and the culture is placed in a constant temperature shaker at 28°C and shaken at 180 r / min for 72 h to 120 h.

10. The use of a composite phosphate-dissolving bacteria agent for promoting wheat growth as claimed in claim 6, characterized in that: In step S3), the mass percentage of the liquid bacterial agent to the wheat bran is 25% to 35%; and / or In step S3), the wheat bran sterilized at 121° C. and 104 KPa for 30 min is placed in a sterile polyethylene bag, the liquid bacterial agent obtained in step S2) is added to the wheat bran in the sterile polyethylene bag, the bag is sealed after mixing, 5 to 8 ventilation holes are pierced on the surface of the polyethylene bag with a sterilized needle, and then a polyethylene bag is put in and 5 to 8 ventilation holes are pierced, and then the bag is cultured at 28° C. for 2 to 4 days to obtain the composite phosphate-dissolving bacterial agent.

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