Bacterial agent containing salt-tolerant strain trm45540 and application thereof

Abstract

The application discloses a bacterial agent containing a salt-tolerant and alkali-tolerant strain TRM45540 and application thereof. The bacterial agent containing the salt-tolerant and alkali-tolerant strain TRM45540 is a fermentation liquor obtained by inoculating the strain TRM45540 in a fermentation medium and performing fermentation; a preparation method of the bacterial agent containing the salt-tolerant and alkali-tolerant strain TRM45540, wherein an ISP4 medium with a pH of 7-12 and a sodium chloride content of 0.2wt%-9wt% is used as the fermentation medium to perform fermentation on the strain TRM45540; and application of the bacterial agent containing the salt-tolerant and alkali-tolerant strain TRM45540, which is used for preventing soil compaction and improving soil fixation capacity of a pepper root system, or used for promoting growth of the pepper, or used for improving soil microbial richness. The bacterial agent helps to reduce use of chemical fertilizers and promote effective use of microbial resources.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial inoculants. Specifically, it relates to inoculants containing the salt-tolerant strain TRM45540 and their applications. Background Technology

[0002] Currently, chili peppers are the most widely planted vegetable and the most consumed raw material for spicy condiments in China, with an annual planting area remaining stable at 2.1 million hectares. 2 The total output reached 64 million tons, accounting for 50% of the world's total chili pepper production.

[0003] Xinjiang, my country, has low rainfall, a dry climate, and abundant sunshine and heat resources, making it very suitable for growing chili peppers.

[0004] Currently, the main way to increase agricultural production in my country is through the application of chemical fertilizers. Chemical fertilizers easily cause ecological imbalance, increase soil salinity, reduce soil fertility, harm human and animal health, and cause serious pollution to the ecological environment. Farmland without chemical fertilizers has a high level of environmental safety, but its production efficiency is low and the quality of produce is poor. Although many farmers still use chemical fertilizers to increase yields, the resulting decline in soil fertility year after year, soil compaction, and environmental pollution are pressing problems. Therefore, there is an urgent need to find a new, environmentally friendly, high-yield, and efficient application method. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a bacterial agent containing salt-tolerant strain TRM45540 and its application, so as to improve the soil compaction problem in saline-alkali areas, promote the growth of pepper fruits, improve the ability of pepper roots to fix the soil, and prevent soil erosion.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] A microbial agent containing the salt-tolerant strain TRM45540, wherein the microbial agent is a fermentation broth obtained by inoculating strain TRM45540 into a fermentation substrate; the 16S rRNA sequence of strain TRM45540 is shown in SEQ ID NO.1, and this strain was deposited at the China Center for Type Culture Collection on November 12, 2015, with accession number CCTCC NO:M2015657.

[0008] The above-mentioned bacterial agent containing the salt-tolerant strain TRM45540 was fermented on ISP4 medium, which contained sodium chloride of 0.2 wt% to 9 wt% and had a pH of 7 to 12.

[0009] The above-mentioned method for preparing the inoculum containing the salt-tolerant strain TRM45540 involves using ISP4 medium with a pH of 7–12 and a sodium chloride content of 0.2 wt%–9 wt% as the fermentation substrate to ferment strain TRM45540. The fermentation temperature is 28–30℃, and during fermentation, the fermentation broth is stirred at a speed of 150–180 r / min until the OD600 value of the fermentation broth reaches 0.6–6. Under these fermentation conditions, when the OD600 value of the fermentation broth reaches 6, the number of strain TRM45540 in the fermentation broth is essentially saturated, and the nutrients in the ISP4 medium are basically fully utilized without waste. Alternatively, fermentation can be stopped when the OD600 value of the fermentation broth containing strain TRM45540 reaches 0.6, and the fermentation broth with an OD600 value of 0.6 can be directly used to irrigate the soil. This method can shorten the fermentation time.

[0010] ISP4 is a culture medium suitable for Streptomyces. Too high or too low sodium chloride content, or too high or too low pH of the culture medium, may affect the growth and proliferation of strain TRM45540.

[0011] Preferably, fermentation should be carried out using ISP4 medium with a sodium chloride content of 0.2 wt% and a pH of 7, at a fermentation temperature of 28°C, and with stirring at a rate of 160 r / min. Although strain TRM45540 can adapt to high salinity and alkalinity, it proliferates faster under these conditions of sodium chloride content, pH, temperature, and stirring rate, which is more conducive to saving fermentation time.

[0012] The above-mentioned application of the microbial agent containing the salt-tolerant strain TRM45540 can be described as follows: the application is to use the microbial agent containing the salt-tolerant strain TRM45540 to prevent soil compaction and enhance the soil-fixing ability of chili pepper roots; the application is to use the microbial agent containing the salt-tolerant strain TRM45540 to promote chili pepper growth; or the application is to use the microbial agent containing the salt-tolerant strain TRM45540 to increase soil microbial richness.

[0013] The application of the above-mentioned inoculant containing the salt-tolerant bacterial strain TRM45540 requires soil with a soluble sodium chloride content of 0.2wt%–9wt%, a soil pH of 7–12, and an available iron content greater than or equal to 1 mg / kg. The soil bulk density is generally 1–1.5 g / cm³. 3 The density is relatively close to that of the culture medium. The range of soluble sodium chloride content, pH, and available iron content in soils suitable for inoculants containing the salt-tolerant strain TRM45540 is approximately equal to the range of sodium chloride content, pH, and iron content in the culture medium.

[0014] The application of the above-mentioned inoculant containing the salt-tolerant strain TRM45540 prevents soil compaction by promoting the formation of soil aggregates with a particle size of 2 mm. Strain TRM45540 can absorb iron and produce UDPAG, which indirectly promotes the formation of soil aggregates and prevents soil compaction.

[0015] The application of the above-mentioned inoculant containing the salt-tolerant strain TRM45540, which promotes the growth of chili peppers, includes promoting the growth of root length, stem length, leaf width, and fruit yield.

[0016] The application of the aforementioned inoculant containing the salt-tolerant strain TRM45540 to improve soil microbial richness involves irrigating the soil with the inoculant containing TRM45540. These soil microorganisms include Flavobacterium species. Soil irrigated with the fermentation broth of strain TRM45540 exhibits a richer microbial population, particularly a higher number of Flavobacterium. A richer variety of soil microorganisms allows for the decomposition and utilization of a wider range of organic matter, resulting in more humus and better water retention. Furthermore, Flavobacterium can mineralize organic phosphorus in the soil, converting it into inorganic phosphorus, which is more readily absorbed by plants. This enhances the efficiency of phosphorus absorption by peppers, promoting root growth and overall plant development.

[0017] The above-mentioned application of the bacterial agent containing the salt-tolerant strain TRM45540 involves fermenting the bacterial agent containing the salt-tolerant strain TRM45540 by inoculating strain TRM45540 into ISP4 medium with a pH of 7 and a sodium chloride content of 0.2 wt%. The fermentation temperature is 28°C, and during the fermentation process, the fermentation broth is stirred at a speed of 160 r / min until the OD600 value of the fermentation broth reaches 6.

[0018] When the above-mentioned microbial agent containing the salt-tolerant strain TRM45540 is used to prevent soil compaction and enhance the soil-fixing ability of chili pepper roots, promote chili pepper growth and / or increase soil microbial richness, the diluted microbial agent containing the salt-tolerant strain TRM45540 is used to irrigate the soil where chili peppers are planted; the OD600 value of the diluted microbial agent containing the salt-tolerant strain TRM45540 is 0.6.

[0019] Preservation Information

[0020] Classification and nomenclature of biological materials: Mutable Streptomyces

[0021] Latin name of biological material: Streptomyces mutabilis

[0022] Biological material used for ginseng: TRM45540

[0023] Name of depositary institution: China Center for Type Culture Collection

[0024] Address of the depository: No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China (within Wuhan University)

[0025] Date of deposit: November 2, 2015

[0026] Accession number: CCTCC NO:M2015657

[0027] The technical solution of the present invention achieves the following beneficial technical effects:

[0028] 1. The microbial agent containing the salt-tolerant strain TRM45540 provided in this invention is suitable for use in saline-alkali areas, and is especially suitable for saline-alkali soils used for planting chili peppers. Strain TRM45540 can absorb available iron from the soil and secrete uridine diphosphate N-acetylglucosamine (UDPAG). UDPAG can be used by soil microorganisms to synthesize extracellular polysaccharides. When the extracellular polysaccharides produced by soil microorganisms increase, the content of soil aggregates at the 2mm level can be effectively increased, making the soil looser, which in turn helps to improve the soil's water and fertilizer retention capacity, and promotes plant growth and rooting. Furthermore, strain TRM45540 can effectively promote the proliferation of Flavobacterium microorganisms in the soil. Flavobacterium microorganisms can promote the mineralization of organic phosphorus in the soil. The mineralized phosphorus can be directly absorbed by peppers, which reduces the need for plant roots to secrete organic acids to activate phosphorus in the soil and improves the efficiency of pepper roots in absorbing phosphorus. Phosphorus itself has the effect of promoting plant (especially plant roots) growth. When pepper root length increases and the particle size distribution of soil aggregates changes, the pepper root system's soil-fixing capacity increases. This prevents irrigation water from carrying away a large amount of rhizosphere soil and further causing the loss of activated nutrients in the rhizosphere soil. At the same time, it also keeps the rhizosphere soil loose and porous, which is conducive to pepper root respiration and the proliferation of rhizosphere microorganisms. Moreover, the root system has a high water and fertilizer retention capacity.

[0029] 2. The salt-tolerant bacterial strain TRM45540 contains plant growth-promoting factors. Irrigating chili peppers with a microbial agent containing TRM45540 can promote root length, stem length, leaf width, and fruit yield. Secondary metabolites produced by strain TRM45540 can improve soil microbiota. This improved microbiota composition leads to increased humus content and soil structure, further promoting plant growth. Compared to chemical fertilizers, using this microbial agent helps prevent soil compaction, improves soil fertility in the long term, promotes sustainable agricultural development, and makes agricultural production greener and safer. This represents a breakthrough in developing green and ecological agriculture in Xinjiang, gradually perfecting the path of sustainable agricultural development in Xinjiang. This microbial fertilizer has broad application prospects, helping to reduce the use of chemical fertilizers and promote the effective utilization of microbial resources. Attached Figure Description

[0030] Figure 1 Photographs of colonies formed by strain TRM45540 on the surface of ISP4 culture medium in this embodiment of the invention;

[0031] Figure 2 Scanning electron microscope image of strain TRM45540 grown on ISP4 medium in this embodiment of the invention;

[0032] Figure 3 Scanning electron microscope image of strain TRM45540 grown on ISP4 medium supplemented with ferrous sulfate in this embodiment of the invention;

[0033] Figure 4a The growth of strain TRM45540 on ISP4 medium with a salt concentration of 3wt% in the embodiments of the present invention;

[0034] Figure 4b The growth of strain TRM45540 on ISP4 medium with a salt concentration of 5 wt% in the embodiments of the present invention;

[0035] Figure 4c The growth of strain TRM45540 on ISP4 medium with a salt concentration of 7wt% in the embodiments of the present invention;

[0036] Figure 4d The growth of strain TRM45540 on ISP4 medium with a salt concentration of 9 wt% in the embodiments of the present invention;

[0037] Figure 5a The growth of strain TRM45540 on ISP4 medium at pH 9 in this embodiment of the invention;

[0038] Figure 5b The growth of strain TRM45540 on ISP4 medium at pH 10 in this embodiment of the invention;

[0039] Figure 5c The growth of strain TRM45540 on ISP4 medium at pH 11 in this embodiment of the invention;

[0040] Figure 5d The growth of strain TRM45540 on ISP4 medium at pH 12 in this embodiment of the invention;

[0041] Figure 6 Analysis results of siderophore gene clusters of strain TRM45540 in the embodiments of the present invention;

[0042] Figure 7The plant enzyme factor scanning results of strain TRM45540 in the embodiments of the present invention;

[0043] Figure 8a The culture results of strain TRM45540 on Assumption medium in the embodiments of the present invention;

[0044] Figure 8b The culture results of strain TRM45540 on Alexandrov medium in the embodiments of the present invention;

[0045] Figure 8c The culture results of strain TRM45540 on soybean casein agar medium in the embodiments of the present invention;

[0046] Figure 8d The culture results of strain TRM45540 on Congo red cellulose medium in the embodiments of the present invention;

[0047] Figure 8e The culture results of strain TRM45540 on PKO inorganic phosphorus medium in the embodiments of the present invention;

[0048] Figure 9a The measurement results of chili root length after 2 weeks of seedling pot experiment in this embodiment of the invention;

[0049] Figure 9b The measurement results of chili root length after 3 weeks of seedling pot experiment in this embodiment of the invention;

[0050] Figure 9c The measurement results of chili root length after 4 weeks of seedling pot experiment in this embodiment of the invention;

[0051] Figure 10a The measurement results of pepper stem length after 2 weeks of seedling pot experiment in this embodiment of the invention;

[0052] Figure 10b The measurement results of chili stem length after 3 weeks of seedling pot experiment in this embodiment of the invention;

[0053] Figure 10c The measurement results of pepper stem length after 4 weeks of seedling pot experiment in this embodiment of the invention;

[0054] Figure 11a The measurement results of pepper leaf width after 2 weeks of seedling pot experiment in this embodiment of the invention;

[0055] Figure 11b The measurement results of pepper leaf width after 3 weeks of seedling pot experiment in this embodiment of the invention;

[0056] Figure 11c The measurement results of pepper leaf width after 4 weeks of seedling pot experiment in this embodiment of the invention;

[0057] Figure 12a The measurement results of fresh weight of pepper plants after 2 weeks of seedling pot experiment in this embodiment of the invention;

[0058] Figure 12b The measurement results of fresh weight of pepper plants after 3 weeks of seedling pot experiment in this embodiment of the invention;

[0059] Figure 12c The measurement results of fresh weight of pepper plants after 4 weeks of seedling pot experiment in this embodiment of the invention;

[0060] Figure 13a The measurement results of the dry weight of pepper plants after 2 weeks of seedling pot experiment in this embodiment of the invention;

[0061] Figure 13b The measurement results of the dry weight of pepper plants after 3 weeks of seedling pot experiment in this embodiment of the invention;

[0062] Figure 13c The measurement results of the dry weight of pepper plants after 4 weeks of seedling pot experiment in this embodiment of the invention;

[0063] Figure 14a Photographs of the growth status of 80-day-old peppers in this embodiment of the invention;

[0064] Figure 14b Photographs of 100-day-old chili peppers in this embodiment of the invention;

[0065] Figure 14c Comparison of plant height of 100-day-old peppers in this invention embodiment;

[0066] Figure 15a Comparison of iron content in chili pepper fruits from different treatment groups in this invention embodiment;

[0067] Figure 15b Comparison of nitrogen content in chili pepper fruits from different treatment groups in this invention embodiment;

[0068] Figure 15c Comparison of phosphorus content in chili pepper fruits from different treatment groups in this invention embodiment;

[0069] Figure 15d Comparison of potassium content in chili pepper fruits from different treatment groups in this invention embodiment;

[0070] Figure 16a Results of determination of available iron content in soil two months after planting chili peppers in this embodiment of the invention;

[0071] Figure 16b The results of measuring the total iron content in the soil two months after planting chili peppers in this embodiment of the invention;

[0072] Figure 17aThe UDPAG standard curve used in this embodiment of the invention to measure the UDPAG production of strain TRM45540;

[0073] Figure 17b The measurement results of UDPAG production of strain TRM45540 cultured in a culture medium without added ferrous sulfate in this embodiment of the invention;

[0074] Figure 17c The measurement results of UDPAG production of strain TRM45540 cultured in a ferrous sulfate concentration of 1 mg / L in the embodiments of the present invention;

[0075] Figure 17d The measurement results of UDPAG production of strain TRM45540 cultured in a ferrous sulfate medium with a ferrous sulfate concentration of 100 mg / L in this embodiment of the invention;

[0076] Figure 17e The measurement results of UDPAG production of strain TRM45540 cultured in a ferrous sulfate medium with a ferrous sulfate concentration of 1000 mg / L in this embodiment of the invention;

[0077] Figure 17f Measurement results of UDPAG standard samples in embodiments of the present invention;

[0078] Figure 18a The composition of soil aggregates in soil irrigated with fermentation broth of strain TRM45540 in this embodiment of the invention;

[0079] Figure 18b The composition of soil aggregates in the soil used for irrigating the culture medium in this embodiment of the invention;

[0080] Figure 18c The composition of soil aggregates in the soil irrigated with water in this embodiment of the invention;

[0081] Figure 19 Comparison of soil microbial community composition in different treatment groups in this invention embodiment;

[0082] Figure 20 A schematic diagram illustrating the mechanism by which strain TRM45540 promotes pepper growth;

[0083] Figure 21 A comparison of the soil-fixing capacity of the root system of 100-day-old peppers in this embodiment of the invention. Detailed Implementation

[0084] 1. Morphological characteristics of strain TRM45540

[0085] Strain TRM45540 was inoculated onto solid ISP4 medium and solid ISP4 medium supplemented with 10 mg / L ferrous sulfate, and cultured at 28°C until colonies grew. Colony morphology was observed visually, and the morphological characteristics of the strain were observed using a scanning electron microscope. The solid ISP4 medium was formulated as follows: 10 g / L soluble starch, 2 g / L dipotassium hydrogen phosphate, 2 g / L sodium chloride, 4 g / L ammonium sulfate, 1 g / L calcium carbonate, 2 g / L magnesium sulfate heptahydrate, and 1.8 wt% agar powder, with the pH adjusted to 7–7.4. This ISP4 medium is the optimal growth medium for strain TRM45540; that is, strain TRM45540 can rapidly and massively proliferate on this ISP4 medium. The morphology of colonies and the morphological characteristics of strain TRM45540 observed on this medium are relatively accurate.

[0086] 2. Salt and alkali tolerance test of the strain

[0087] Using ISP4 as the basal medium, salt (sodium chloride) concentrations were increased to prepare salt media with mass fractions of 3 wt%, 5 wt%, 7 wt%, and 9 wt%, as well as alkaline media with pH values ​​of 9, 10, 11, and 12 for salt tolerance experiments. Strains TRM45540 were inoculated onto these salt media and cultured at 28°C for 7 days before observation to investigate the strain's tolerance to salt and alkali.

[0088] 3. Scanning the plant growth-promoting genes and gene clusters in the strain.

[0089] Plant growth-promoting enzyme sequences were searched in the NCBI database and compared with HMMER (Hidden Markov Model) to validate and refine the search results. Plant growth-promoting factor sequences were further identified and scanned from these enzyme sequences. The HMMER model used contained information on known gene families associated with plant growth-promoting factors; these gene families' HMMER models were obtained from the pfam database (https: / / www.ebi.ac.uk / interpro / ) and used as reference sequences. Furthermore, HMM search software was used to specifically target proteins produced by TRM45540 from the scanned plant growth-promoting factor sequences, and the gene families to which the specifically targeted proteins belonged were searched. In addition, antiSMASH v.6.0 was used to identify secondary metabolite biosynthesis gene clusters (smBGCs) in TRM45540. Simultaneously, the presence of siderophore-related gene clusters in the strain was analyzed; if present, the potential for plant growth promotion was further verified experimentally.

[0090] 4. Plate confrontation experiment to verify the potential of the strain to promote plant growth and metabolism.

[0091] The nitrogen fixation, phosphorus solubilization, potassium solubilization, and cellulase-producing protease capabilities of strain TRM45540 were assessed using the plate confrontation method to screen for superior strains. Nitrogen fixation was detected using Ashby's medium, phosphorus solubilization using PKO inorganic phosphorus medium (Pikovaskaia's, PKO), potassium solubilization using Alexandrov medium, cellulase production using Congo red cellulose medium, and protease production using soybean casein agar (TSA). The size of the hydrolysis zone was used to determine the activity level of the strains in these differential media.

[0092] 5. Pot experiment verification

[0093] Using the pepper variety 1998 as the research object, a seedling pot experiment and a pot experiment were set up. In both the seedling pot experiment and the pot experiment, three treatments were set up: water, ISP4 medium and fermentation broth of strain TRM45540, in order to explore the growth-promoting effect of fermentation broth of strain TRM45540 on pepper 1998.

[0094] The preparation method of fermentation broth for strain TRM45540 is as follows: First, strain TRM45540 is inoculated with ISP4 liquid medium as the seed culture. Then, 70 L of ISP4 liquid medium is prepared as the fermentation substrate, and fermentation is carried out at a constant temperature of 28°C and a stirring rate of 160 r / min for 7 days to obtain the fermentation broth of strain TRM45540. Under the temperature and ISP4 medium volume conditions of this embodiment, after 7 days of fermentation, the OD600 value of the fermentation broth reached 6 and basically stopped increasing, indicating that the fermentation entered the stationary phase. In other embodiments, if different fermentation temperatures or different volumes of fermentation substrate are used, the time to reach the stationary phase may vary, but the fermentation process should be completed before the OD600 value exceeds 6 to avoid over-fermentation leading to microbial death.

[0095] For ease of use, the fermentation broth after 7 days of fermentation is diluted with sterile water at a volume ratio of 1:10. The OD600 value of the diluted fermentation broth is 0.6, and it can be used directly for irrigation.

[0096] The seedling pot experiment was conducted as follows: First, seeds of the chili pepper 8819 were planted in seedling pots and watered until germination. Subsequently, the plants were watered three times a week with 100 mL of a 10-fold diluted fermentation broth of strain TRM45540 (containing bacterial cells), water, or ISP4 medium (diluted 10-fold with sterile water). During the experiment, the plants were observed weekly, and the tallest plants were selected for measurements of root, stem, leaf, fresh weight, and dry weight. To ensure the accuracy of the results, nine samples were collected from each of the fermentation broth, water, and medium groups, and the average values ​​were analyzed.

[0097] One month after the seedling pot experiment, the plants were transplanted with soil from the seedling pots into larger pots for a pot experiment. The pot experiment also included three treatments: water, ISP4 medium, and fermentation broth from strain TRM45540. Watering was done three times a week, 200 mL each time. Nine plants were used for each treatment in pots for verification. After the peppers reached 100 days of growth, the fruits were harvested and weighed to accurately assess fruit yield. The iron, nitrogen, phosphorus, and potassium content of the pepper fruits was measured by Yangling Biotechnology Co., Ltd., and the collected data were analyzed and visualized using the gene cloud platform of Paisennong Company.

[0098] 6. Determine the effect of soil structure improvement

[0099] Two months after planting potted peppers, soil samples were taken from the pepper-grown areas for amplicon sequencing to investigate changes in soil microbial communities in different treatment groups. Soil aggregate content at different pore sizes was measured to evaluate soil structure changes, and the available iron content in the pepper-grown soil was determined. Furthermore, to investigate the effect of soil iron content on the amount of UDPAG produced by strain TRM45540 (UDPAG production indirectly affects soil aggregate structure), different concentrations of ferrous sulfate (1 mg, 100 mg, and 1000 mg ferrous sulfate per liter of ISP4 medium) were added to liquid ISP4 medium. Strain TRM45540 was cultured in these media at 28℃, pH 7, and a salt content of 1 wt%. The fermentation broth was stirred at 160 rpm during cultivation. After 7 days, the bacterial cells were filtered out, and the amount of UDPAG produced by strain TRM45540 was determined using quantitative mass spectrometry.

[0100] 7. Experimental Results

[0101] 7.1 Morphological characteristics of strain TRM45540

[0102] After being inoculated into ISP4 medium and cultured for 7 days, strain TRM45540 produced white spores and yellow pigment. Colonies of strain TRM45540 on the surface of the ISP4 medium appeared as follows: Figure 1 As shown in the figure. Scanning electron micrographs of strain TRM45540 on ordinary solid ISP4 medium and solid ISP4 medium supplemented with 10 mg / L ferrous sulfate are shown in the figure. Figure 2 and Figure 3 As shown. From Figure 3 It can be observed that after adding 10 mg / L of ferrous sulfate to the culture medium, strain TRM45540 produced extracellular polymers.

[0103] 7.2 Salt and alkali tolerance of strain TRM45540

[0104] Depend on Figures 4a to 4d It can be seen that strain TRM45540 can grow in culture media with salt concentrations ranging from 3 wt% to 9 wt%. Figures 5a-5d It can be seen that strain TRM45540 can grow in culture media with pH values ​​ranging from 9 to 12. These results indicate that strain TRM45540 possesses a certain degree of salt and alkali tolerance. This, combined with the ISP4 medium formulation in section "1. Morphological Characteristics of Strain TRM45540" and... Figure 1 The observation results of the bacterial colonies showed that strain TRM45540 can survive normally in an environment with a soluble sodium chloride content of 0.2wt% to 9wt% and a pH of 7 to 12.

[0105] 7.3 Genes and gene clusters promoting plant growth in strain TRM45540

[0106] The antiSMASH prediction results showed that strain TRM45540 (Query sequence in the figure) and Streptomyces coelicolor A3(2) [Streptomyces coelicolor A3(2) in the figure] had 83% similarity in gene clusters on deferoxamine B / deferoxamine E. Only one transferred gene was missing, and the gene clusters composed of other genes were complete (e.g., Figure 6 As shown in the image, this indicates that strain TRM45540 has the ability to produce siderophores. Siderophores can be used to provide iron to plants, thereby promoting their growth, indicating that strain TRM45540 has the potential to promote plant growth.

[0107] 7.4 Results of plant enzyme factor scanning of strain TRM45540

[0108] By consulting the NCBI database and comparing it with the HMMER model, 22 plant growth promoting factors were identified. Further analysis was conducted to determine whether strain TRM45540 contained these 22 plant growth promoting factors. Figure 7 Based on the scan results, strain TRM45540 contains at least 20 enzymatic factors related to plant growth.

[0109] 7.5 Results of the plate confrontation experiment of strain TRM45540

[0110] like Figures 8a-8e As shown, TRM45540 colonies on Assumption agar exhibited colony growth with a small nitrogen-fixing zone, while TRM45540 colonies on Alexandrov agar showed a larger potassium-solubilizing zone. TRM45540 colonies on soybean casein agar produced a yellow transparent zone, while TRM45540 colonies on Congo red cellulose agar showed no transparent zone. TRM45540 colonies on PKO inorganic phosphorus agar showed a phosphorus-solubilizing zone. These results indicate that strain TRM45540 possesses nitrogen-fixing, potassium-solubilizing, protein-degrading, and inorganic phosphorus-degrading functions.

[0111] 7.6 Seedling Pot Experiment Results

[0112] Figures 9a-9c , Figures 10a-10c , Figures 11a-11c , Figures 12a-12c and Figures 13a-13c The figures show the measurement results of root length, stem length, leaf width, fresh weight, and dry weight of peppers during the seedling pot experiment. The results indicate that during the one-month experimental period in the seedling pots, peppers watered with the fermentation broth of strain TRM45540 showed better root length, stem length, leaf width, and biomass than those watered with water or culture medium. This suggests that the fermentation broth of strain TRM45540 promotes pepper growth.

[0113] 7.7 Results of the pot experiment

[0114] Figure 14a From left to right, the peppers in the image represent the following groups: the group irrigated with water, the group irrigated with fermentation broth of strain TRM45540, and the group irrigated with culture medium. Figure 14a It was found that, compared with irrigation with water and irrigation with culture medium, peppers irrigated with the fermentation broth of strain TRM45540 showed more vigorous growth at 80 days of age. When the peppers were harvested at 100 days of age, the total yield of peppers irrigated with the fermentation broth of strain TRM45540 was 110.15 grams, while the total yields of peppers irrigated with water and culture medium were 7.60 grams and 3.40 grams, respectively. This indicates that the yield of peppers irrigated with the fermentation broth of strain TRM45540 was significantly higher than that irrigated with culture medium and water. Figure 14b As shown in the figure, from left to right, the pepper fruits are the fermentation broth group of strain TRM45540, the culture medium group, and the water group. Figure 14cThe image shows the plant height comparison results of 100-day-old peppers. From left to right, the pepper plants in the water irrigation group, the group irrigated with fermentation liquid of strain TRM45540, and the group irrigated with culture medium are: 73.8±0.23cm, 50.1±0.45cm, and 52.5±0.41cm, respectively. That is, the pepper plants in the group irrigated with fermentation liquid of strain TRM45540 were taller than the other two groups. Figure 21 From left to right, the images show a comparison of soil fixation in pepper roots after irrigation with fermentation broth of strain TRM45540, irrigation with culture medium, and irrigation with water. Figure 21 It can be seen that the pepper roots of the group irrigated with the fermentation liquid of strain TRM45540 have a higher soil-fixing capacity than the other two groups.

[0115] The strong soil-binding ability of the pepper roots in the group irrigated with fermentation broth of strain TRM45540 is the result of the combined effect of multiple factors. First, irrigating with fermentation broth of strain TRM45540 promotes the growth of pepper roots ( Figures 9a-9c ), longer root systems result in better soil-fixing ability; secondly, the strain TRM45540 in this embodiment can absorb and utilize iron and secrete UDPAG ( Figures 17c-17e As supporting evidence, UDPAG can indirectly improve the composition of soil aggregates. Figures 18a-18c This evidence suggests that the composition of soil aggregates altered after irrigation with the fermentation broth of strain TRM45540. Specifically, it improved soil particle size and structure, thereby changing soil cohesion and making it more suitable for fixation by chili pepper roots. Furthermore, the optimal soil particle size and structure are more conducive to chili pepper (and its roots) growth. Thirdly, the soil irrigated with the fermentation broth of strain TRM45540 is richer in microorganisms, theoretically allowing for the full decomposition of organic matter (i.e., increased microbial abundance enables the decomposition of organic matter that was previously indestructible). The soil contains more humus, which forms a thin film around the soil particles, altering the properties of the clay-particle contact surface and improving soil cohesion and water retention. This prevents excessive water loss during irrigation, avoiding the loss of active nutrients from the rhizosphere. Simultaneously, it maintains a loose and porous structure in the rhizosphere, promoting root respiration and the proliferation of rhizosphere microorganisms, while also enhancing the root system's water and fertilizer retention capacity.

[0116] 7.8 Measurement results of iron, nitrogen, phosphorus and potassium content in pepper fruits

[0117] Depend on Figures 15a-15dIt was found that there were no significant differences in the iron, nitrogen, phosphorus, and potassium content per unit weight of pepper fruits when irrigated with the fermentation broth of strain TRM45540, the culture medium, and the water group. However, the pepper fruit yield in the group irrigated with the fermentation broth of strain TRM45540 was significantly higher than that in the other two groups. This means that irrigating with the fermentation broth of strain TRM45540 increased the total amount of iron, nitrogen, phosphorus, and potassium in the pepper fruits, indicating that the fermentation broth of strain TRM45540 has a significant positive impact on pepper growth.

[0118] 7.9 Content of available iron and total iron in the soil for chili pepper cultivation

[0119] Depend on Figure 16a It can be seen that the available iron content in the soil irrigated with the fermentation broth of strain TRM45540 was significantly lower than that irrigated with water and culture medium, and also significantly lower than that in the control group (soil not planted with peppers). Figure 16b The measurement results of total iron in the soil showed that the total iron content of the soil irrigated with water, the soil irrigated with culture medium, and the soil irrigated with fermentation broth of strain TRM45540 were relatively similar, which indicates that strain TRM45540 can absorb and utilize available iron in the soil.

[0120] Results of an investigation into the ability of strain TRM45540 to absorb and utilize iron ions and produce UDPAG (uridine diphosphate N-acetylglucosamine).

[0121] This study used ISP4 as the basal culture medium, TRM45540 as the research object, and UDPAG to determine the standard curve. The obtained standard curve is shown below. Figure 17a As shown in the figure, the horizontal axis represents the concentration of UDPAG, and the vertical axis represents the signal intensity measured by the quantitative mass spectrometer. Figures 17b-17e The figures show the measurement results of UDPAG production of strain TRM45540 cultured in media without ferrous sulfate, in media with 1 mg / L ferrous sulfate, in media with 100 mg / L ferrous sulfate, and in media with 1000 mg / L ferrous sulfate (the horizontal axis represents the acquisition time, and the vertical axis represents the signal intensity measured by the quantitative mass spectrometer). Figure 17f The measurement results for the standard sample (horizontal axis represents acquisition time, vertical axis represents signal intensity measured by the quantitative mass spectrometer) are consistent with the labeling of the standard sample, indicating that the results obtained by quantitative mass spectrometry in this embodiment are true and reliable. Table 1 shows the data obtained from the test.

[0122] Table 1

[0123] Grouping compound Content (μg / mL) No ferrous sulfate added UDPAG 0.0131 1 mg / L ferrous sulfate UDPAG 0.0279 100 mg / L ferrous sulfate UDPAG 2.6501 1000 mg / L ferrous sulfate UDPAG 3.0663 Standard Sample UDPAG 0.0080

[0124] The data in the table shows that when ferrous sulfate is added to the culture medium of strain TRM45540, strain TRM45540 will utilize the ferrous sulfate to produce UDPAG. Furthermore, the amount of UDPAG produced gradually increases with the increase in the concentration of added ferrous sulfate, proving that strain TRM45540 utilizes iron to synthesize UDPAG. In other words, when an inoculant containing strain TRM45540 is used for soil irrigation, strain TRM45540 can utilize the iron in the soil to synthesize UDPAG. Even when the available iron content in the soil is only 1 mg / kg (approximately equal to 1 mg / L of ferrous sulfate added to the culture medium), strain TRM45540 can still utilize it and produce UDPAG. And when the ferrous sulfate content in the culture medium is equal to 1000 mg / L, strain TRM45540 can still survive normally.

[0125] UDPAG, as an important precursor in the synthesis of biomacromolecules such as polysaccharides and glycoproteins, may promote the secretion of extracellular polysaccharides (EPS) by microorganisms in the soil. EPS plays a crucial role in the formation and stabilization of soil aggregates, acting as a cementing agent to tightly bind fine soil particles together, forming structurally stable and erosion-resistant aggregates. Therefore, increased UDPAG production indirectly promotes the increase in both the quantity and quality of soil aggregates, helping to improve soil physical structure and enhance soil water and fertilizer retention capacity.

[0126] 7.11 Soil aggregate composition

[0127] The extracellular polysaccharides produced by strain TRM45540 can effectively bind soil particles, causing them to form larger soil aggregates. Figures 18a-18c The figures show the composition of soil aggregates under the conditions of irrigation with fermentation broth of strain TRM45540, irrigation with culture medium, and irrigation water, respectively. As can be seen from the figures, the soil irrigated with fermentation broth of strain TRM45540 showed a significant increase in aggregates with a particle size of 2 mm. The formation and increase in the number of soil aggregates of this particle size can prevent soil compaction, making the soil structure loose and porous, facilitating the infiltration of water and nutrients, promoting root absorption of nutrients and water, promoting root development, and ultimately promoting plant growth.

[0128] 7.12 Soil microbial community composition

[0129] Amplicon sequencing was performed on chili pepper soil two months after different treatments to detect the composition of the soil microbial community. The results are as follows: Figure 19As shown in the figure, relative abundance (i.e., relative richness) refers to the ratio of the number of 16S rRNA sequences of a certain microorganism in a sample to the total number of 16S rRNA sequences of all microorganisms in the sample. The figure shows that Flavobacterium (a microorganism of the genus Flavobacterium) was the dominant bacterium in the soil irrigated with the fermentation broth of strain TRM45540. Compared with irrigation water, the abundance of Flavobacterium in the soil irrigated with the fermentation broth of strain TRM45540 was significantly increased. According to literature reports, this bacterium can mineralize organic phosphorus in the soil. After being mineralized into inorganic phosphorus, it is more easily absorbed by plants, thus promoting plant growth. Increased mineralization of organic phosphorus can reduce the secretion of organic acids by pepper roots to activate phosphorus in the soil, improving the efficiency of phosphorus absorption by peppers. Furthermore, phosphorus itself promotes root growth, and longer roots are also beneficial for plant fixation in the soil.

[0130] The above experimental results show that strain TRM45540 absorbs and utilizes iron ions to produce polysaccharide signaling molecules, altering soil structure and preventing soil compaction. It also improves soil fertility in the long term, making nutrients more readily absorbed and utilized by plants. Furthermore, it improves soil microbial composition and promotes organophosphate mineralization, making it easier for plants to absorb and utilize. In addition, strain TRM45540 is an actinomycete that produces substances with antibacterial effects, playing a role in antibacterial and disease-preventing properties in peppers to a certain extent. The mechanism by which strain TRM45540 promotes pepper growth is as follows: Figure 20 As shown.

[0131] In this embodiment, the fermentation broth containing strain TRM45540, which is directly used to irrigate the soil for planting chili peppers, has an OD600 value of 0.6. It is obtained by diluting the fermentation broth with an OD600 value of 6. In other embodiments, fermentation can be stopped when the OD600 value of the fermentation broth containing strain TRM45540 reaches 0.6, and the fermentation broth can be directly used to irrigate the soil, which can shorten the fermentation time.

[0132] The 16S rRNA sequence (SEQ ID NO.1) of strain TRM45540 is as follows:

[0133]

[0134] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. The application of a bacterial agent containing the salt- and alkali-tolerant strain TRM45540, characterized in that, The bacterial agent containing the salt-tolerant and alkali-tolerant strain TRM45540 is a fermentation liquor obtained by inoculating the strain TRM45540 in a fermentation medium and then fermenting; the strain TRM45540 is Streptomyces variabilis (Streptomyces variabilis) Streptomyces mutabilis ), the 16S rRNA sequence of the strain TRM45540 is shown as SEQ ID NO. 1, the strain was preserved in China Center for Type Culture Collection on November 12, 2015, and the preservation number is CCTCC NO: M2015657; the fermentation medium is ISP4 medium, the sodium chloride content in the ISP4 medium is 0.2wt%-9wt%, and the pH of the ISP4 medium is 7-12. The application is to use the inoculant containing the salt-tolerant strain TRM45540 to prevent soil compaction and enhance the soil-fixing ability of chili pepper roots; or the application is to use the inoculant containing the salt-tolerant strain TRM45540 to promote chili pepper growth; or the application is to use the inoculant containing the salt-tolerant strain TRM45540 to increase soil microbial richness.

2. The application of the bacterial agent containing the salt-tolerant strain TRM45540 according to claim 1, characterized in that, The TRM45540 strain was fermented using ISP4 medium with a pH of 7–12 and a sodium chloride content of 0.2 wt%–9 wt%. The fermentation temperature was 28–30 °C. During the fermentation process, the fermentation broth was stirred at a speed of 150–180 r / min until the OD600 value of the fermentation broth reached 0.6–6.

3. The application of the bacterial agent containing the salt-tolerant strain TRM45540 according to claim 1, characterized in that, The soluble sodium chloride content in the soil is 0.2wt% to 9wt%, the pH of the soil is 7 to 12, and the available iron content in the soil is greater than or equal to 1 mg / kg.

4. The application of the bacterial agent containing the salt-tolerant strain TRM45540 according to claim 3, characterized in that, Inoculants containing the salt-tolerant strain TRM45540 prevent soil compaction by promoting the formation of soil aggregates with a particle size of 2 mm.

5. The application of the bacterial agent containing the salt-tolerant strain TRM45540 according to claim 3, characterized in that, The promotion of chili pepper growth includes: promoting the growth of root length, stem length, leaf width, and fruit yield.

6. The application of the bacterial agent containing the salt-tolerant strain TRM45540 according to claim 3, characterized in that, When used to improve soil microbial richness, soil is irrigated with a microbial agent containing the salt-tolerant strain TRM45540, wherein the soil microorganisms include Flavobacterium genus.

7. The application of the bacterial agent containing the salt-tolerant strain TRM45540 according to claim 3, characterized in that, The inoculum containing the salt-tolerant strain TRM45540 was obtained by inoculating strain TRM45540 into ISP4 medium with pH 7 and sodium chloride content of 0.2wt% and fermenting it. The fermentation temperature was 28℃, and the fermentation broth was stirred at 160 r / min during the fermentation process until the OD600 value of the fermentation broth reached 6.

8. The application of the bacterial agent containing the salt-tolerant strain TRM45540 according to claim 7, characterized in that, When using a microbial agent containing the salt-tolerant strain TRM45540 to prevent soil compaction and enhance the soil-fixing ability of chili pepper roots, promote chili pepper growth, and / or increase soil microbial richness, the diluted microbial agent containing the salt-tolerant strain TRM45540 is used to irrigate the soil where chili peppers are planted; the OD600 value of the diluted microbial agent containing the salt-tolerant strain TRM45540 is 0.6.

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