A composite microbial agent, its preparation method and application

Abstract

The present invention relates to a compound microbial agent, a preparation method thereof and an application thereof, belonging to the technical field of microorganisms. In the compound microbial agent, Peribacillus frigoritolerans The microbial agent of NMI04: the microbial agent of Bacillus amyloliquefaciens QZB03: the microbial agent of Bacillus subtilis NMC07 = (1-5):(1-5):(1-5); the effective viable count in the compound microbial agent is 1×10

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a compound microbial agent, a preparation method thereof, and an application thereof. Background Art

[0002] Bacillus is widely distributed in nature, with a large population, extremely strong reproductive ability, stable physical and chemical properties, and a very broad antibacterial spectrum, making it one of the most highly regarded and widely studied microbial biocontrol agents at present. As a dominant bacterial community in the microecological environment, Bacillus has strong disease prevention, growth promotion, and stress resistance functions. Therefore, Bacillus and its biocontrol preparations can be used for the prevention and treatment of diseases of the roots, leaves, fruits, and flowers of various crops, play a certain role in promoting the normal growth and development of plants, and at the same time can enhance the utilization rate of nutrients by crops, thereby greatly improving the stress resistance of plants. As a safe and effective microbial resource, Bacillus plays an increasingly important role in modern agricultural production. With technological progress and the growth of market demand, its application fields will be further broadened, making greater contributions to the goal of sustainable agricultural development.

[0003] Microbial agents use the life activities of microorganisms to increase the content of nitrogen, available phosphorus, or potassium in the soil, or convert some substances in the soil that crops cannot directly utilize into absorbable nutrients, or increase crop growth stimulants, or inhibit the activities of plant pathogens, thereby improving soil fertility, improving the nutritional conditions of crops, and increasing crop yields. Microbial agents can usually be rationally used according to their functions and characteristics, covering a variety of improvement functions and ensuring significant effects, being able to increase crop yields and significantly improve soil properties. In recent years, due to soil compaction and declining fertility caused by the large amount of chemical fertilizers applied, resulting in reduced crop yields, exploring new microbial agents to partially or gradually replace chemical products has become a development trend.

[0004] At present, the research and application of microbial inoculants have developed from single strains to multi-strain composites. The development from single-strain with single function to multi-functional composite microbial flora is an inevitable trend in line with the long-term development of microbial inoculants. Composite microbial inoculants refer to those that include one or more beneficial microorganisms and microbial carriers, contain a certain number of viable bacteria, and have the functions of rapidly supplementing beneficial bacteria in the soil, inhibiting harmful bacteria, regulating the soil pH value, improving soil fertility, enhancing the plant's ability to resist pests and diseases, and promoting plant growth. They can well overcome the shortcomings of the application of single inoculants. In the long run, compared with single strains, composite inoculants can ensure long-term and significant effects without changing with the external and soil environments. With the improvement of environmental awareness, people are increasingly concerned about the environmental pollution problems caused by the excessive use of chemicals in agricultural production. As a low-carbon, pure natural, pollution-free, non-toxic and harmless product, composite microbial inoculants can improve the soil ecological environment for crop growth through the metabolic activities of beneficial microorganisms, improve the soil fertility level, and can stimulate and induce the stress resistance of crops by secreting trace active substances, thereby promoting the efficient growth of crops and greatly increasing the yield and quality of crops. Composite microbial inoculants are one of the important tools for modern agriculture and ecological construction. They not only help improve crop productivity but also provide new ideas and technical means for solving global environmental problems. In the context of the current development of green agriculture, ecological agriculture and sustainable agriculture, composite microbial inoculants have broad application prospects. Summary of the Invention

[0005] Aiming at the problem that the existing single inoculant has poor effects in terms of plant disease resistance and low-temperature tolerance, the present invention provides a composite inoculant, its preparation method and application to solve the above problems. The present invention Peribacillus frigoritolerans compounds Bacillus amyloliquefaciens and Bacillus subtilis to obtain a composite inoculant, which improves the plant's disease resistance and low-temperature tolerance while also enhancing the plant's salt tolerance effect.

[0006] The technical solution of the present invention is as follows:

[0007] In the first aspect, the present invention provides a composite inoculant, including Peribacillus frigoritolerans NMI04, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), QZB03 and Bacillus subtilis ( Bacillus subtilis ), NMC07. Among them Peribacillus frigoritoleransNMI04 is deposited in the General Microbiological Center of the China General Microbiological Culture Collection Center, with the deposit number of CGMCC No 31153, the deposit date of July 3, 2024, and the address of the deposit institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Bacillus amyloliquefaciens QZB03 is deposited in the General Microbiological Center of the China General Microbiological Culture Collection Center, with the deposit number of CGMCC No 31155, the deposit date of July 3, 2024, and the address of the deposit institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Bacillus subtilis NMC07 is deposited in the General Microbiological Center of the China General Microbiological Culture Collection Center, with the deposit number of CGMCC No 31151, the deposit date of July 3, 2024, and the address of the deposit institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] Furthermore, in the compound bacterial agent, by mass, Peribacillus frigoritolerans The solid bacterial agent of NMI04: the solid bacterial agent of Bacillus amyloliquefaciens QZB03: the solid bacterial agent of Bacillus subtilis NMC07 = (1 - 5):(1 - 5):(1 - 5); the effective viable count in the compound bacterial agent is 1×10 8 ~1×10 11 CFU / g.

[0009] In the second aspect, the present invention provides a preparation method of the above compound bacterial agent, which is specifically as follows:

[0010] Mix the solid bacterial agent containing Peribacillus frigoritolerans NMI04, the solid bacterial agent containing Bacillus amyloliquefaciens QZB03, and the solid bacterial agent containing Bacillus subtilis NMC07 evenly to obtain the compound bacterial agent.

[0011] Furthermore, the preparation method of the solid bacterial agent containing Peribacillus frigoritolerans NMI04 is as follows:

[0012] Activate NMI04 on the NA plate medium, take a loop of the activated Peribacillus frigoritolerans NMI04 and inoculate it into the LB medium, culture it at 35°C and 180 rpm for 8 - 24 h to obtain Peribacillus frigoritolerans the NMI04 seed liquid; inoculate the seed liquid into the fermentation medium, and continue to ferment at an inoculation amount of 6%, 35°C, 300 rpm, and an aeration rate of 1:1.2 for 24 - 48 h until the spore rate is over 90% to complete the fermentation, and obtain the fermentation broth; then add 5% - 10% diatomaceous earth to the fermentation broth by volume ratio, and perform spray drying at 120°C to obtain the solid bacterial agent containing Peribacillus frigoritolerans NMI04. Peribacillus frigoritolerans

[0013] ​Furthermore, the preparation method of the solid bacterial agent containing Bacillus amyloliquefaciens QZB03 is as follows:

[0014] Activate Bacillus amyloliquefaciens QZB03 on an NA plate medium. Take a loop of the activated Bacillus amyloliquefaciens QZB03 and inoculate it into an LB medium. Culture it at 35°C and 180 rpm for 8 - 24 h to obtain a Bacillus amyloliquefaciens QZB03 seed solution; inoculate the seed solution into a fermentation medium and continue fermentation at an inoculation amount of 6%, 35°C, 300 rpm, and an aeration rate of 1:1.2 for 24 - 48 h until the spore rate is over 90% to complete fermentation and obtain a fermentation broth; then add 5% - 10% diatomaceous earth to the fermentation broth by volume ratio and perform spray drying at 120°C to obtain a solid bacterial agent containing Bacillus amyloliquefaciens QZB03.

[0015] Furthermore, the preparation method of the solid bacterial agent containing Bacillus subtilis NMC07 is as follows:

[0016] Activate Bacillus subtilis NMC07 on an NA plate medium. Take a loop of the activated Bacillus subtilis NMC07 and inoculate it into an LB medium. Culture it at 35°C and 180 rpm for 8 - 24 h to obtain a Bacillus subtilis NMC07 seed solution; inoculate the seed solution into a fermentation medium and continue fermentation at an inoculation amount of 6%, 35°C, 300 rpm, and an aeration rate of 1:1.2 for 24 - 48 h until the spore rate is over 90% to complete fermentation and obtain a fermentation broth; then add 5% - 10% diatomaceous earth to the fermentation broth by volume ratio and perform spray drying at 120°C to obtain a solid bacterial agent containing Bacillus subtilis NMC07.

[0017] Furthermore, the components of the LB medium are as follows: peptone 10 g / L, NaCl 5 g / L, yeast extract 10 g / L, sterile water, pH = 7.2.

[0018] In the third aspect, the present invention provides an application of the above composite bacterial agent in preventing and treating cucumber fusarium wilt.

[0019] In the fourth aspect, the present invention provides an application of the above composite bacterial agent in promoting the growth of Chinese flowering cabbage.

[0020] In the fifth aspect, the present invention provides an application of the above composite bacterial agent in enhancing the soil salinity tolerance of crops.

[0021] In the sixth aspect, the present invention provides an application of the above composite bacterial agent in enhancing the low-temperature tolerance of crops.

[0022] The beneficial effects of the present invention are as follows:

[0023] The compound microbial inoculant of the present invention is a compound inoculant composed of three beneficial microorganisms, which can well overcome the disadvantages of the application of single inoculants. Compared with single strains, the compound inoculant can ensure long-term effectiveness and significant effects, and is not easily affected by changes in the external and soil environments. The compound inoculant of the present invention has a significant ability to inhibit crop pathogens and has good biological control effects. It can significantly improve the growth of crops and has excellent growth-promoting ability. It can also induce the salt tolerance and cold tolerance of crops, thereby promoting the efficient growth of crops and greatly improving the yield and quality of crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 It is in Example 1 of the present invention Peribacillus frigoritolerans Growth graph of NMI04 in NA medium.

[0026] Figure 2 It is in Example 1 of the present invention Peribacillus frigoritolerans Gram staining graph and spore staining graph of NMI04; where A is the Gram staining graph and B is the spore staining graph.

[0027] Figure 3 Growth graph of Bacillus amyloliquefaciens QZB03 in NA medium in Example 1 of the present invention.

[0028] Figure 4 Gram staining graph and spore staining graph of Bacillus amyloliquefaciens QZB03 in Example 1 of the present invention; where A is the Gram staining graph and B is the spore staining graph.

[0029] Figure 5 Growth graph of Bacillus subtilis NMC07 in NA medium in Example 1 of the present invention.

[0030] Figure 6 Gram staining graph and spore staining graph of Bacillus subtilis NMC07 in Example 1 of the present invention; where A is the Gram staining graph and B is the spore staining graph.

[0031] Figure 7 It is the verification result graph of biocontrol ability in Example 3.

[0032] Figure 8 It is the verification result graph of growth-promoting ability in Example 4.

[0033] Figure 9It is the verification result graph of the salt tolerance ability of crops in Example 5 (0.2% saline soil).

[0034] Figure 10 It is the verification result graph of improving the salt tolerance ability of crops in Example 5 (0.4% saline soil).

[0035] Figure 11 It is the verification result graph of improving the low temperature tolerance ability of crops in Example 6. Detailed implementation manners

[0036] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0037] Example 1

[0038] 1、 Peribacillus frigoritolerans Isolation and identification of NMI04

[0039] (1) Sampling: On April 10, 2024, soil in a greenhouse growing tomatoes in Hohhot City, Inner Mongolia Autonomous Region (111.8548° east longitude, 40.8297° north latitude) was collected and stored in a self-sealing bag for later use.

[0040] (2) Isolation:

[0041] Take 5 g of rhizosphere soil collected in step (1) and add it to a conical flask containing 45 mL of sterilized pure water. Heat it in a water bath at 100 °C for 10 minutes. After standing, take the upper layer solution and dilute it into samples with different concentration gradients. Use a spreader to spread it on a nutrient agar (NA) plate medium and incubate it upside down at 35 °C for 24 h.

[0042] (3) Purification: Select single colonies with different morphologies. Streak and purify the strains of each single colony 3 times on an NA plate. Screen the strains according to different colony morphologies and determine their numbers. Perform test tube slant preservation and store them in a 4 °C refrigerator as test bacteria for later experiments. At the same time, place them in a sterilized glycerol tube with a concentration of 50% and store them at -20 °C for later use.

[0043] (4) Molecular identification

[0044] Pick a single colony of strain NMI04 and streak it on an NA plate. After culturing at 35 °C for 12 h, send the cultured NA plate to Sangon Biotech (Shanghai) Co., Ltd. and entrust it to perform strain identification. The 16S rDNA sequence is shown in SEQ ID NO.1.

[0045] Submit the sequenced sequence to Gen-Bank for homology comparison, and preliminarily determine that the strain is Peribacillus frigoritolerans . Peribacillus frigoritolerans NMI04 is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 31153, the deposit date is July 3, 2024, and the address of the deposit institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0046] 2. Isolation and identification of Bacillus amyloliquefaciens QZB03

[0047] (1) Sampling: On April 10, 2024, soil was collected from a ginger field in Qingzhou City, Shandong Province (118.5935°E, 36.8055°N) and placed in a self-sealing bag for preservation and standby.

[0048] (2) Isolation

[0049] Take 5 g of the rhizosphere soil collected in step (1) and add it to a conical flask containing 45 mL of sterilized pure water. Heat it in a water bath at 100 °C for 10 minutes. After standing, take the upper layer solution and dilute it into samples with different concentration gradients. Use a spreader to spread it on a nutrient agar (NA) plate medium and incubate it upside down at 35 °C for 24 h.

[0050] (3) Purification

[0051] Select single colonies with different morphologies. Streak-purify the strains of each single colony 3 times on an NA plate. Screen the strains according to different colony morphologies and determine their numbers. Conduct test tube slant preservation and store them in a 4 °C refrigerator as test bacteria for later experiments. At the same time, place them in a sterilized glycerol tube with a concentration of 50% and store them at -20 °C for standby.

[0052] (4) Molecular identification

[0053] Pick a single colony of strain QZB03 and streak it on an NA plate. After culturing at 35 °C for 12 h, send the cultured NA plate to Sangon Biotech (Shanghai) Co., Ltd. and entrust it to conduct strain identification. The 16S rDNA sequence is shown in SEQ ID NO. 2.

[0054] Submit the sequenced sequence to Gen-Bank for homology comparison, and preliminarily determine that the strain is Bacillus amyloliquefaciens. Bacillus amyloliquefaciens QZB03 is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No 31155, the deposit date is July 3, 2024, and the address of the deposit institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0055] 3. Isolation and Identification of Bacillus subtilis NMC07

[0056] (1)Sampling: On April 10, 2024, soil was collected from a lettuce-growing plot in Hohhot, Inner Mongolia Autonomous Region (111.7856° E, 40.5891° N) and stored in a self-sealing bag for later use.

[0057] (2)Isolation:

[0058] Take 5 g of rhizosphere soil collected in step (1) and add it to a conical flask containing 45 mL of sterilized pure water. Heat it in a water bath at 100 °C for 10 minutes. After standing, take the upper layer solution and dilute it into samples with different concentration gradients. Use a spreader to spread it on a nutrient agar (NA) plate medium and incubate it upside down at 35 °C for 24 h.

[0059] (3)Purification: Select single colonies with different morphologies. Streak-purify the strains of each single colony 3 times on an NA plate. Screen the strains according to different colony morphologies and determine their numbers. Perform tube slant preservation and store them in a 4 °C refrigerator as test bacteria for later experiments. At the same time, place them in a sterilized glycerol tube with a concentration of 50% and store them at -20 °C for later use.

[0060] (4)Molecular identification

[0061] Pick a single colony of strain NMC07 and streak it on an NA plate. After culturing at 35 °C for 12 h, send the cultured NA plate to Sangon Biotech (Shanghai) Co., Ltd. and entrust it to perform strain identification. The 16S rDNA sequence is shown in SEQ ID NO. 3.

[0062] Submit the sequenced sequence to GenBank for homology comparison. It is preliminarily determined that this strain is Bacillus subtilis. Bacillus subtilis NMC07 is deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No 31151, the deposit date is July 3, 2024, and the address of the deposit institution is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0063] Example 2

[0064] Preparation method of the compound microbial agent, including the following steps:

[0065] (1)Place Peribacillus frigoritolerans NMI04 was activated on an NA plate medium. Take a loop of the activated Peribacillus frigoritolerans NMI04 and inoculate it into LB medium. The components of the LB medium are as follows: peptone 10 g / L, NaCl 5 g / L, yeast extract 10 g / L, sterile water, pH = 7.2; culture it at 35 °C and 180 rpm for 8 - 24 h to obtain Peribacillus frigoritoleransNMI04 seed liquid; inoculate the seed liquid into the fermentation medium, and continue fermentation at an inoculation amount of 6%, 35 °C, 300 rpm, and an aeration rate of 1:1.2 for 24 - 48 h until the spore rate reaches over 90% to complete fermentation and obtain the fermentation broth; then add 5% - 10% diatomaceous earth to the fermentation broth by volume ratio and perform spray drying at 120 °C to obtain a solid microbial agent containing Peribacillus frigoritolerans NMI04.

[0066] (2) Activate Bacillus amyloliquefaciens QZB03 on the NA plate medium, pick a loop of the activated Bacillus amyloliquefaciens QZB03 and inoculate it into the LB medium. The composition of the LB medium is as follows: peptone 10 g / L, NaCl 5 g / L, yeast extract 10 g / L, sterile water, pH = 7.2; culture at 35 °C and 180 rpm for 8 - 24 h to obtain the Bacillus amyloliquefaciens QZB03 seed liquid; inoculate the seed liquid into the fermentation medium, and continue fermentation at an inoculation amount of 6%, 35 °C, 300 rpm, and an aeration rate of 1:1.2 for 24 - 48 h until the spore rate reaches over 90% to complete fermentation and obtain the fermentation broth; then add 5% - 10% diatomaceous earth to the fermentation broth by volume ratio and perform spray drying at 120 °C to obtain a solid microbial agent containing Bacillus amyloliquefaciens QZB03.

[0067] (3) Activate Bacillus subtilis NMC07 on the NA plate medium, pick a loop of the activated Bacillus subtilis NMC07 and inoculate it into the LB medium. The composition of the LB medium is as follows: peptone 10 g / L, NaCl 5 g / L, yeast extract 10 g / L, sterile water, pH = 7.2; culture at 35 °C and 180 rpm for 8 - 24 h to obtain the Bacillus subtilis NMC07 seed liquid; inoculate the seed liquid into the fermentation medium, and continue fermentation at an inoculation amount of 6%, 35 °C, 300 rpm, and an aeration rate of 1:1.2 for 24 - 48 h until the spore rate reaches over 90% to complete fermentation and obtain the fermentation broth; then add 5% - 10% diatomaceous earth to the fermentation broth by volume ratio and perform spray drying at 120 °C to obtain a solid microbial agent containing Bacillus subtilis NMC07.

[0068] (4) Mix the solid microbial agent containing Peribacillus frigoritolerans NMI04, the solid microbial agent containing Bacillus amyloliquefaciens QZB03, and the solid microbial agent containing Bacillus subtilis NMC07 according to the following mass ratio. The mixing ratio is shown in Table 1 below.

[0069] Table 1 - Mixing method of the compound microbial agent

[0070]

[0071] Example 3

[0072] Verification of the biocontrol ability of the compound microbial agent

[0073] Taking cucumber fusarium wilt as the control target, the biocontrol experiment was carried out using the compound microbial agent provided by the present invention. The experimental time was from September 2024 to November 2024. Five parallel treatments were set up, namely QZB03 group, NMI04 group, NMC07 group, compound microbial agent group and blank group, with 10 replicates for each treatment. The specific application method was as follows: The solid microbial agents containing Peribacillus frigoritolerans NMI04, the solid microbial agent containing Bacillus amyloliquefaciens QZB03, the solid microbial agent containing Bacillus subtilis NMC07 and the compound microbial agent A were made into dilution solutions with viable cell counts of 1×10 8 CFU / mL. 100 mL was taken and irrigated at the roots of 4-week-old cucumber seedlings (the blank group was irrigated with clear water). After 24 hours of pretreatment, the pathogen of cucumber fusarium wilt (Fusarium oxysporum f. sp. cucumerinum) with a turbidity value of 1.0 MCF was inoculated by the root irrigation method, and the disease index was continuously recorded and the phenotype was observed.

[0074] Disease grade: Grade 0, asymptomatic; Grade 1, cotyledons yellowed but not wilted; Grade 2, cotyledons wilted; Grade 3, cotyledons and true leaves wilted or the plant was dwarfed; Grade 4, dead.

[0075] Disease index = ∑(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × representative value of the highest level) × 100;

[0076] Control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100.

[0077] The experimental results are shown in Table 2 and Figure 7 as follows.

[0078] Table 2 - Results of biocontrol ability verification

[0079]

[0080] The disease conditions of cucumber seedlings treated in each of the above groups are shown in the photos and tables. In the blank group, almost all plants showed symptoms of cucumber fusarium wilt, with the lower part or the whole plant having yellow and withered leaves, and some plants showing dwarfing symptoms; in the single microbial agent treatment groups (QZB03 group, NMI04 group, NMC07 group), different degrees of fusarium wilt symptoms were also shown; while in the compound microbial agent group, more than half of the plants did not show symptoms and grew well, and the remaining plants showed yellowing of old leaves but did not wither. It shows that the compound microbial agent has an obvious control effect on cucumber fusarium wilt.

[0081] Example 4

[0082] Verification of the growth-promoting ability of the compound microbial agent

[0083] Taking pakchoi as the test object, a growth promotion experiment was carried out using a compound bacterial agent. The experimental time was from September 2024 to November 2024. Five parallel treatments were set up, namely the QZB03 group, the NMI04 group, the NMC07 group, the compound bacterial agent group, and the blank group. Each treatment had 10 replicates. For the QZB03 group, the NMI04 group, the NMC07 group, and the compound bacterial agent group, 100 mL of the corresponding bacterial agent dilution (prepared in the same way as in Example 3) was taken for root irrigation treatment. The blank group was applied with 100 mL of clear water. The pakchoi was transplanted after the third true leaf unfolded, and the first root irrigation treatment was carried out on the second day after transplantation. After that, the root irrigation treatment was carried out once every 7 days for a total of 3 times. Seven days after the third root irrigation treatment, the plant height of the pakchoi was measured with a ruler, the stem diameter was measured with a vernier caliper, and the fresh weight of the above-ground part was weighed with a balance. The results are shown in Table 3 and Figure 8 as follows.

[0084] Table 3 - Results of growth promotion ability verification

[0085]

[0086] As shown in Table 3 and Figure 8 the results indicate that when applying the compound bacterial agent solution, the plant height increased by 9.2%, the stem diameter increased by 19.9%, and the fresh weight increased by 19.1% compared with the blank group, and the growth promotion effect was significantly better than that of the single bacterial agent treatment group. Thus, it can be seen that the compound bacterial agent has good growth promotion performance on pakchoi.

[0087] Example 5

[0088] Verification of the ability of compound bacterial agent to improve the salt tolerance of crops

[0089] Taking Chinese cabbage as the test object, a growth promotion experiment was carried out using a compound bacterial agent. The experimental time was from September 2024 to November 2024. Five parallel treatments were set up, namely the QZB03 group, the NMI04 group, the NMC07 group, the compound bacterial agent group, and the blank group. Each treatment had 10 replicates.

[0090] Soil pretreatment: According to the salt content of medium-saline soil being 0.2% - 0.4%, the soil was pretreated. Two neutral salts, NaCl and Na 2 SO 4 , and two alkaline salts, NaHCO 3 and Na 2 CO 3 were mixed evenly in a ratio of 1:9:9:1. Weigh the mixed salts in proportions of 0.2% and 0.4% of the soil weight respectively, dissolve them, pour them into the soil respectively, and mix well to make saline soil with salt contents of 0.2% and 0.4%.

[0091] Experimental treatments: For the QZB03 group, NMI04 group, NMC07 group, and compound microbial agent group, 100 mL of the corresponding microbial agent dilution solution (prepared in the same way as in Example 3) was taken for root irrigation treatment; the blank group was treated with 100 mL of clear water. Pak choi was transplanted after the third true leaf unfolded, and the first root irrigation treatment was carried out on the second day after transplantation. Thereafter, root irrigation treatment was carried out once every 7 days for a total of 3 times. Seven days after the third root irrigation treatment, the plant height of the pak choi was measured with a ruler, the stem diameter was measured with a vernier caliper, and the fresh weight of the above-ground part was weighed with a balance. The results are shown in Table 4 and Figures 9 - 10 as follows.

[0092] Table 4 - Growth results of pak choi

[0093]

[0094] The experimental results show that in the 0.2% saline-alkali soil, the treatment group applying the compound microbial agent had a 14.6% increase in plant height, a 20% increase in stem diameter, and a 26% increase in fresh weight compared with the blank group. In the 0.4% saline-alkali soil, the treatment group applying the compound microbial agent had a 5% increase in plant height, a 30% increase in stem diameter, and a 38.2% increase in fresh weight compared with the blank group. Compared with the 0.2% saline-alkali soil, in the 0.4% saline-alkali soil, the plant height of pak choi in the blank group increased, but the stem diameter and fresh weight decreased significantly, and the growth vigor weakened significantly. However, in the compound microbial agent treatment group, there were no significant changes in plant height, stem diameter, or fresh weight. Among the single microbial agent treatment groups, the NMI04 group and NMC07 group also improved the salt stress tolerance of crops to a certain extent, but the performance was not as significant as that of the compound microbial agent group. Thus, it can be seen that applying the compound microbial agent significantly improved the salt tolerance of pak choi.

[0095] Example 6

[0096] Verification of the ability of the compound microbial agent to improve the low-temperature tolerance of crops

[0097] Using wheat as the experimental object, a growth promotion experiment was carried out with the compound microbial agent. The experimental time was from September 2024 to November 2024. Five parallel treatments were set, namely the QZB03 group, NMI04 group, NMC07 group, compound microbial agent group, and blank group, with 10 replicates for each treatment group. The treatment groups were made into 2000-fold dilution solutions (the viable bacteria count was 5×10 7 CFU / mL) with four groups of corresponding microbial agents as the nutrient solution for wheat hydroponics; the blank group used clear water as the nutrient solution. After germination, the seed tray was placed in an incubator at 7°C for cultivation. After 15 days, the plant height, root length, and single plant weight of the wheat were measured. The results are shown in Table 5 and Figure 11 as follows.

[0098] Table 5 - Growth results of wheat at 7°C

[0099]

[0100] The test results show that at 7°C, the application of the compound microbial inoculum solution did not significantly increase the plant height compared to the blank group, but the root length increased by 29.9% and the weight per plant increased by 77.1%. It can be seen that the compound microbial inoculum can significantly improve the low-temperature tolerance of wheat and enable it to grow robustly at low temperatures.

[0101] Although the present invention has been described in detail by referring to the accompanying drawings and in conjunction with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the present invention, and all such modifications or substitutions should be within the scope of the present invention. / Any person skilled in the art within the technical scope disclosed by the present invention can easily conceive of changes or substitutions, which should all be covered by the protection scope of the present invention.

Claims

1. An application of a composite bacterial agent in improving crop tolerance to soil salinity and alkali, characterized in that: The bacterial agent is Peribacillus frigoritolerans NMI04, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )QZB03 and Bacillus subtilis ( Bacillus subtilis )NMC07 composition; Peribacillus frigoritolerans NMI04 is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with a deposit number of CGMCC No 31153 and a deposit date of July 3, 2024. The address of the depository institution is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; Bacillus amyloliquefaciens QZB03 is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with a deposit number of CGMCC No 31155 and a deposit date of July 3, 2024. The address of the depository institution is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; Bacillus subtilis NMC07 is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with a deposit number of CGMCC No 31151 and a deposit date of July 3, 2024. The address of the depository institution is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; In the composite bacterial agent, by weight, Peribacillus frigoritolerans Solid bacterial agent of NMI04: solid bacterial agent of Bacillus amyloliquefaciens QZB03: solid bacterial agent of Bacillus subtilis NMC07 = (1-5): (1-5): (1-5); the number of effective live bacteria in the composite bacterial agent is 1×10 8 ~1×10 11 CFU / g.

2. An application of a composite bacterial agent in improving the low temperature tolerance of crops, characterized in that: The bacterial agent is Peribacillus frigoritolerans NMI04, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )QZB03 and Bacillus subtilis ( Bacillus subtilis )NMC07 composition; Peribacillus frigoritolerans NMI04 is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with a deposit number of CGMCC No 31153 and a deposit date of July 3, 2024. The address of the depository institution is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; Bacillus amyloliquefaciens QZB03 is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with a deposit number of CGMCC No 31155 and a deposit date of July 3, 2024. The address of the depository institution is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; Bacillus subtilis NMC07 is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with a deposit number of CGMCC No 31151 and a deposit date of July 3, 2024. The address of the depository institution is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; In the composite bacterial agent, by weight, Peribacillus frigoritolerans Solid bacterial agent of NMI04: solid bacterial agent of Bacillus amyloliquefaciens QZB03: solid bacterial agent of Bacillus subtilis NMC07 = (1-5): (1-5): (1-5); the number of effective live bacteria in the composite bacterial agent is 1×10 8 ~1×10 11 CFU / g.

3. The use according to claim 1 or 2, characterized in that: The preparation method of the composite bacterial agent is as follows: will contain Peribacillus frigoritolerans The solid bacterial agent containing NMI04, the solid bacterial agent containing Bacillus amyloliquefaciens QZB03 and the solid bacterial agent containing Bacillus subtilis NMC07 are uniformly mixed to obtain a composite bacterial agent.

4. The use according to claim 3, characterized in that contain Peribacillus frigoritolerans The preparation method of the solid inoculant of NMI04 is as follows: Will Peribacillus frigoritolerans NMI04 was activated on NA plate medium and a ring of activated Peribacillus frigoritolerans NMI04 was inoculated into LB medium and cultured at 35°C and 180 rpm for 8-24 h. Peribacillus frigoritolerans NMI04 seed liquid; inoculate the seed liquid into the fermentation medium, continue to ferment for 24-48 hours under the conditions of inoculation amount 6%, 35°C, 300rpm, and ventilation volume 1:1.2, until the spore rate is more than 90% and the fermentation is completed to obtain a fermentation liquid; then add 5%-10% diatomaceous earth by volume to the fermentation liquid, spray dry it at 120°C, and obtain a fermentation liquid containing Peribacillus frigoritolerans Solid inoculant of NMI04.

5. The use according to claim 3, characterized in that The preparation method of the solid bacterial agent containing Bacillus amyloliquefaciens QZB03 is as follows: Bacillus amyloliquefaciens QZB03 was activated on a NA plate culture medium, and a loop of the activated Bacillus amyloliquefaciens QZB03 was inoculated into an LB culture medium, and cultured at 35°C and 180rpm for 8-24h to obtain a seed solution of Bacillus amyloliquefaciens QZB03; the seed solution was inoculated into a fermentation culture medium, and fermentation was continued for 24-48h under the conditions of an inoculation amount of 6%, 35°C, 300rpm, and an aeration volume of 1:1.2, until the spore rate was more than 90% and the fermentation was completed to obtain a fermentation liquid; then, 5%-10% diatomaceous earth was added to the fermentation liquid by volume, and spray-dried at 120°C to obtain a solid bacterial agent containing Bacillus amyloliquefaciens QZB03.

6. The use according to claim 3, characterized in that The preparation method of the solid bacterial agent containing Bacillus subtilis NMC07 is as follows: Bacillus subtilis NMC07 was activated on a NA plate culture medium, and a loop of the activated Bacillus subtilis NMC07 was inoculated into an LB culture medium, and cultured at 35°C and 180rpm for 8-24h to obtain a seed solution of Bacillus subtilis NMC07; the seed solution was inoculated into a fermentation culture medium, and fermentation was continued for 24-48h under the conditions of an inoculation amount of 6%, 35°C, 300rpm, and an aeration volume of 1:1.2, until the spore rate was more than 90% and the fermentation was completed to obtain a fermentation liquid; then, 5%-10% diatomaceous earth was added to the fermentation liquid by volume, and spray-dried at 120°C to obtain a solid bacterial agent containing Bacillus subtilis NMC07.

7. The use according to any one of claims 4 to 6, characterized in that: The LB medium had the following components: 10 g / L peptone, 5 g / L NaCl, 10 g / L yeast extract, sterile water, pH=7.2.

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