A synthetic functional microbial agent and application thereof in salt tolerance and growth promotion of wheat

Abstract

The application discloses a kind of synthetic functional microbial inoculants and its application in salt-tolerant growth promotion of wheat, adopt hydrophobic gordon's bacteria ( Gordonia hydrophobica ), odyssey lysine bacillus ( Lysinibacillus odysseyi ), sea bed motility microorganism ( Planomicrobium okeanokoites ), north China broussonetia yellow color bacterium ( Luteimonas huabeiensis ), epidermis brevibacterium ( Brevibacterium epidermidis ) five strains of bacteria, microbial inoculant has no antagonism between strains, salt-tolerant, and other significant characteristics such as viable count, can significantly promote spring wheat germination rate, seedling length and root length, and has significant promoting effect on seedling length and root length of winter wheat.Under salt stress conditions, the microbial inoculant can significantly improve the activities of catalase, peroxidase and superoxide dismutase of wheat, and the total chlorophyll content;after treatment with the microbial inoculant, the contents of malondialdehyde and proline of wheat are significantly reduced.The microbial inoculant plays a promoting and protecting role in the process of resisting salt stress of spring wheat and winter wheat, and has an impact on the development and sustainable development of arid region agriculture.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a synthetic functional bacterial agent and its preparation method, as well as its application in promoting salt tolerance in wheat. Background Technology

[0002] Soil salinization impacts soil health and environmental quality, posing a significant ecological and environmental problem for dryland agriculture worldwide and severely limiting and hindering agricultural development and sustainable development in arid regions. The global area of ​​saline-alkali soils is approximately 1.1 × 10⁻⁶. 9 hm 2 Approximately 36 billion hectares of soil in China are salinized to varying degrees, accounting for 4.9% of the country's arable land (Liu 2023). The Xinjiang Uygur Autonomous Region is known as a "museum of saline-alkali land," characterized by the coexistence of salinization and desertification, strong salt accumulation and surface aggregation, and complex soil salt composition. The area of ​​saline-alkali soil in Xinjiang Uygur Autonomous Region accounts for 9913 × 10⁻⁶% of the total saline-alkali soil area in China. 4 hm 2 22.01% of ) in Xinjiang Uygur Autonomous Region; 407×10 4 hm 2 Of the cultivated land in Xinjiang, 30.12% is affected by salinization to varying degrees (Zhang Wanyin et al. 2020). Meanwhile, Xinjiang Uygur Autonomous Region is rich in halophytes, with Chenopodiaceae being the largest family of halophytes in the region (Wang Lei et al., 2008). *Haloxylon ammodendron*, a rare-halogenated true halophyte in Chenopodiaceae (Kong Xianwu, 1979), can continuously absorb large amounts of salt ions from the environment in saline habitats, thereby improving soil properties, reducing soil salinity, and increasing soil organic matter. However, phytoremediation alone has many shortcomings, such as low remediation efficiency, lack of specificity, and applicability only to low to medium concentrations of environmental stress. Plant-microbe combined remediation is a more effective approach that successfully overcomes these shortcomings.

[0003] In production applications, the practice of improving the plant growth environment and thus increasing crop yield by inoculating beneficial microorganisms has a long history (Baha and Bekki, 2015). However, due to the influence of various environmental factors, the effect of using a single strain is often suppressed, leading to unstable functional effects. Therefore, the focus of current research on plant microbial function has shifted from single microbial strains to the community level of the microbiome. Synthetic microbial communities refer to artificially created microbial communities by co-culturing two or more species under known culture conditions (Großkopf et al., 2014; de Roy et al., 2014). The advantages of synthetic microbial communities over natural microbial communities and single strains are: reduced complexity of microbial communities, known species, relatively simple and controllable composition, stronger adaptability to the environment, ability to achieve more complex functions, and the ability to construct and prove more complex system models through mathematical models (Biliouris et al., 2012; Jagmann et al., 2014; Martins et al., 2023).

[0004] Currently, synthetic functional microbial communities have been widely applied in agricultural production and biological control. For example, Hu et al. (2016) used eight strains of Pseudomonas to form a synthetic microbial community, which reduced the density of pathogenic bacteria in the rhizosphere soil of tomatoes. Niu et al. (2017) used seven bacteria from the roots of maize to form a community that inhibited the colonization of plant pathogenic fungi. In promoting plant growth, Durán et al. (2018) and Zhuang et al. (2020) screened bacterial strains that promoted plant growth from the rhizosphere microbial communities of Arabidopsis thaliana and garlic, respectively. Regarding stress resistance, Catrillo et al. (2017) constructed a synthetic microbial community using 35 bacterial strains from the roots of Arabidopsis thaliana and other cruciferous plants, enhancing the activity of PHR1, a major regulator of phosphate stress response, and promoting the integration of phosphate stress and immunity. Li et al. (2021) constructed a synthetic community of 13 bacteria to rescue root rot in Astragalus membranaceus by activating plant-induced systemic resistance. Schmitz et al. (2022) used five bacterial strains from the roots of the desert plant Indigofera argentea to construct a synthetic microbial community, thereby protecting tomatoes from high salt stress. However, there are few reports on its application in promoting wheat's resistance to salt stress. Summary of the Invention

[0005] To address the lack of reports in existing technologies regarding the use of synthetic microbial agents to promote wheat resistance to salt stress, this invention provides a synthetic functional microbial agent and its preparation method. This invention also provides the application of this synthetic functional microbial agent in promoting wheat growth under salt stress conditions. By implementing the technical solution of this invention, the germination rate, seedling length, and root length of spring wheat can be significantly improved, and it also has a significant promoting effect on the seedling length and root length of winter wheat. Under salt stress conditions, this microbial agent can significantly promote and protect spring and winter wheat in resisting salt-alkali stress, which has an impact on agricultural development and sustainable development in arid areas.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] This invention provides a synthetic functional bacterial agent, wherein the bacterial agent is composed of *Gordonella hydrophobicum* (…). Gordonia hydrophobic Odyssey Lysine Bacillus ( Lysinibacillus odysseyi ), seabed biogenic microbes ( Planomicrobium okeanokoites ), North China glucosinolates ( Luteimonas huabeiensis ), Epidermal short bacillus ( Brevibacterium epidermidis It is obtained by mixing resuspended solutions.

[0008] Preferably, the bacterial agent is composed of *Gordonella hydrophobicum* (…). Gordonia hydrophobica Odyssey Lysine Bacillus ( Lysinibacillus odysseyi ), seabed biogenic microbes ( Planomicrobium okeanokoites ), North China glucosinolates ( Luteimonas huabeiensis ), Epidermal short bacillus ( Brevibacterium epidermidis The resuspension was obtained by mixing the resuspensions in a volume ratio of 1:1:1:1:1.

[0009] Preferably, the optical density (OD600) of the resuspension of each strain in the bacterial agent is 0.2.

[0010] Preferably, the optical density OD600 of the bacterial agent is 0.02.

[0011] Furthermore, the present invention also provides a method for preparing a synthetic functional bacterial agent, specifically using the following steps: The bacterial strain is inoculated into TSA containing 2% NaCl and cultured to the exponential growth phase; the cells are collected by centrifugation, washed once with 1×PBS, and resuspended in 10 mM MgCl2 solution; the optical density (OD600) of each cell solution is adjusted to 0.2; the selected bacterial strains are mixed at a ratio of 1:1:1:1:1 to obtain the synthetic bacterial agent, and then the agent is diluted to an OD600 of 0.02 for later use.

[0012] Furthermore, this application also provides the application of a synthetic functional microbial agent in promoting wheat growth under salt stress conditions.

[0013] The application involves the following steps: Select wheat seeds and sterilize them three times with 3% NaClO solution for 1 minute each time, followed by washing them three times with sterile water; pre-line sterile filter paper in a 100 mm × 100 mm sterile petri dish and soak the filter paper with 1.2% NaCl; soak the wheat seeds in a synthetic functional microbial agent for 8 hours, then place the seeds in petri dishes containing 1.2% NaCl, with 20 seeds in each petri dish, and allow them to germinate at room temperature.

[0014] Preferably, the application of a synthetic functional microbial agent in promoting wheat growth under salt stress conditions uses wheat seeds of varieties Xinchun 26 and Xindong 18.

[0015] Compared with the prior art, the present invention has the following features:

[0016] (1) The synthetic functional bacterial agent provided by this invention uses hydrophobic Gordon's bacteria ( Gordonia hydrophobica Odyssey Lysine Bacillus ( Lysinibacillus odysseyi ), seabed biogenic microbes ( Planomicrobium oceano-ites ), North China glucosinolates ( Luteimonas huabeiensis ), Epidermal short bacillus ( Brevibacterium epidermidis The five strains of bacteria have significant characteristics such as no antagonism between strains, salt and alkali tolerance, and high viable count.

[0017] (2) The synthetic functional microbial agent provided by the present invention can significantly promote the germination rate, seedling length and root length of spring wheat by soaking wheat seeds, and also has a significant promoting effect on the seedling length and root length of winter wheat.

[0018] (3) The synthetic functional microbial agent provided by the present invention can significantly increase the activity of catalase, peroxidase, superoxide dismutase and total chlorophyll content in wheat under salt stress conditions. After treatment with the microbial agent, the malondialdehyde and proline content in wheat is significantly reduced. The microbial agent plays a promoting and protective role in the process of spring wheat and winter wheat resisting salt and alkali stress, and has an impact on agricultural development and sustainable development in arid areas. Attached Figure Description

[0019] Figure 1 The results show the colony morphology of five bacterial strains.

[0020] Figure 2 The effects of synthetic microbial agents on the morphology of hydroponic wheat plants.

[0021] Figure A shows an observation of the morphology of spring wheat hydroponically grown plants; Figure B shows an observation of the morphology of winter wheat hydroponically grown plants.

[0022] Figure 3The image shows the effects of synthetic inoculants on wheat germination rate, seedling length, and root length.

[0023] Figure A shows the change in germination rate of spring wheat; Figure B shows the change in germination rate of winter wheat; Figure C shows the change in seedling length of spring wheat; Figure D shows the change in seedling length of winter wheat; Figure E shows the change in root length of spring wheat; and Figure F shows the change in root length of winter wheat.

[0024] Figure 4 The graph shows the changes in the activity of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), total chlorophyll content, malondialdehyde (MDA) content, and proline (PRO) content of wheat affected by the synthetic inoculant.

[0025] Figure A shows the changes in catalase (CAT) activity in spring wheat; Figure B shows the changes in catalase (CAT) activity in winter wheat; Figure C shows the changes in superoxide dismutase (SOD) activity in spring wheat; Figure D shows the changes in superoxide dismutase (SOD) activity in winter wheat; Figure E shows the changes in peroxidase (POD) activity in spring wheat; Figure F shows the changes in peroxidase (POD) activity in winter wheat; Figure G shows the changes in chlorophyll content in spring wheat; Figure H shows the changes in chlorophyll content in winter wheat; Figure I shows the changes in malondialdehyde (MDA) content in spring wheat; Figure J shows the changes in malondialdehyde (MDA) content in winter wheat; Figure K shows the changes in proline (PRO) content in spring wheat; and Figure L shows the changes in proline (PRO) content in winter wheat. Detailed Implementation

[0026] The following examples are provided to further illustrate the content of this invention, but should not be construed as limiting the invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and essence of the invention are within the scope of this invention.

[0027] The hydrophobic Gordon's bacterium used in this invention ( Gordonia hydrophobica Odyssey Lysine Bacillus ( Lysinibacillus odysseyi ), seabed biogenic microbes ( Planomicrobium okeanokoites ), North China glucosinolates ( Luteimonas huabeiensis ), Epidermal short bacillus ( Brevibacterium epidermidis All five strains were harmless and safe, including *Gordonella hydrophobicum*. Gordonia hydrophobica Odyssey Lysine Bacillus ( Lysinibacillus odysseyi ), Epidermal short bacillus ( Brevibacterium epidermidis Seabed biogenic microorganisms ( ) can be purchased through other public channels such as the China General Microbiological Culture Collection Center (CGMCC). Planomicrobium oceanokoitesIt can be purchased through other public channels such as the Shandong University Weihai Marine Microbial Resource Center, and *Gastrodinium glabra* (North China spp.) Luteimonas huabeiensis It can be purchased through other public channels such as Beijing Biowell Biotechnology Co., Ltd.

[0028] In this application, the following embodiments use the term "hydrophobic Gordon's bacteria ( Gordonia hydrophobica ")" is abbreviated as "Hydrophobic Gordon's bacillus"; "Odyssey Lysine Bacillus ( Lysinibacillus odysseyi "Odyssey Lysine Bacillus" is abbreviated as "Odyssey Lysine Bacillus"; "Seabed Motile Microbes ( Planomicrobium okeanokoites ")" is abbreviated as "seabed motile microbes"; "North China glaucoma ( Luteimonas huabeiensis Both are abbreviated as "Glaucoma glabra" and "Short-lived epidermal bacteria". Brevibacterium epidermidis The abbreviation for "epidermal short bacillus" is recorded as "epidermal short bacillus".

[0029] The Xinchun 26 and Xindong 18 wheat seeds used in this application were donated by the Institute of Nuclear Technology and Biotechnology of the Xinjiang Academy of Agricultural Sciences. The general public can purchase them from the Institute of Nuclear Technology and Biotechnology of the Xinjiang Academy of Agricultural Sciences or seed companies.

[0030] The soybean broth agar medium (TSA) used in this application is a conventional culture medium in this technical field.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0032] Example 1: A synthetic functional microbial agent

[0033] This invention provides a synthetic functional bacterial agent, which is obtained by mixing hydrophobic Gordon's bacterium, Odyssey lysine spores, seabed biogenic microbes, North China luteomonas, and epidermal short rods resuspension.

[0034] The hydrophobic Gordon's bacteria used ( Gordonia hydrophobica Odyssey Lysine Bacillus ( Lysinibacillus odysseyi ), seabed biogenic microbes ( Planomicrobium okeanokoites ), North China glucosinolates ( Luteimonas huabeiensis ), Epidermal short bacillus ( Brevibacterium epidermidis See Appendix for colony morphology. Figure 1 As shown.

[0035] The present invention also provides a method for preparing a synthetic functional bacterial agent, which is specifically prepared by the following steps: the bacterial strain is inoculated into TSA containing 2% NaCl and cultured to the exponential growth phase, the cells are collected by centrifugation, washed once with 1×PBS, and resuspended in 10 mM MgCl2 solution; the optical density OD600 of each cell solution is adjusted to 0.2; the selected bacterial strains are mixed at a ratio of 1:1:1:1:1 to obtain the synthetic bacterial agent, and then the bacterial agent is diluted to an OD600 of 0.02 for later use.

[0036] Furthermore, this application also provides the application of a synthetic functional microbial agent in promoting wheat growth under salt stress conditions. The application comprises the following steps: selecting wheat seeds, sterilizing them three times with 3% NaClO solution for 1 minute each time, and then washing them three times with sterile water; pre-laying sterile filter paper in a 100 mm × 100 mm sterile petri dish and adding 1.2% NaCl to saturate the filter paper; soaking the wheat seeds with the synthetic functional microbial agent for 8 hours, and then placing the seeds in petri dishes containing 1.2% NaCl, with 20 seeds in each petri dish, and germinating at room temperature.

[0037] Example 2: Hemolytic Detection

[0038] Hydrophobic *Gordonella hydrophobicis*, *Bacillus oryzae*, *Microorganisms cephalomycin*, *Glaucoma chinensis*, and *B. epidermolysis* strains were inoculated onto hemolytic medium and cultured at 30°C for 3-7 days. A 1-2 mm translucent hemolytic ring was observed around the colony, indicating that the strain possessed hemolytic activity. The test results showed that the selected *Gordonella hydrophobicis*, *Bacillus oryzae*, *Microorganisms cephalomycin*, *Glaucoma chinensis*, and *B. epidermolysis* strains did not exhibit hemolytic reactions, indicating that these five strains are biosafe and can be used for the construction of subsequent synthetic bacterial agents.

[0039] Example 3: Strain Antagonism Experiment

[0040] For the obtained *Gordonella hydrophobicae*, *Bacillus oryzae*, *Microorganisms flocculation*, *Glaucoma chinensis*, and *Bacillus epidermidis*, pairwise antagonism experiments were conducted on the selected strains. One strain was selected for liquid culture, while the others were cultured on solid media. The cultured bacterial solutions were evenly spread on solid media. A perforator was used to punch holes in the colonies of the solid-cultured strains. Agar blocks containing colonies were picked up with tweezers and inverted onto a plate containing indicator bacteria. The plate was then covered and incubated in an incubator. The formation of transparent inhibition zones around the agar blocks was observed, and the diameter of the inhibition zones was measured. The measurements showed that no transparent inhibition zones formed between the selected strains, indicating that there was no antagonistic reaction between them, and they could be used for the construction of subsequent synthetic agents.

[0041] Example 4: Construction of Synthetic Functional Microbial Agents

[0042] Based on the descriptions in Examples 1 through 3 above, strains that are tolerant to 5% NaCl, do not exhibit hemolytic activity, and have no antagonistic interactions were selected from the above results. These strains were inoculated into TSA containing 2% NaCl and cultured until the exponential growth phase. Cells were collected by centrifugation, washed once with 1×PBS, and resuspended in 10 mM MgCl2 solution. The optical density (OD) of each cell solution was measured. 600 Adjust to 0.2. Mix the selected strains at a ratio of 1:1:1:1:1 to obtain the synthetic inoculum, and then dilute the inoculum to OD. 600 0.02 is reserved.

[0043] Example 5: The effect of synthetic microbial agents on wheat growth

[0044] Based on the synthetic functional microbial agents obtained in the four examples described above, wheat seeds of the commonly planted varieties Xinchun 26 and Xindong 18 in Xinjiang Uygur Autonomous Region were selected. The seeds were surface-sterilized three times with 3% NaClO solution for 1 minute each time, followed by three washes with sterile water. The experimental group was soaked in a mixed culture of synthetic microbial flora, while the control group was soaked in sterile water. Sterile filter paper was pre-lined in 100 mm × 100 mm sterile petri dishes, and 1.2% NaCl was added to saturate the filter paper. After soaking for 8 hours, the seeds were placed in petri dishes containing 1.2% NaCl, with 20 seeds in each dish, and germination was carried out at room temperature. Approximately 10 days later, when the seedlings had their third true leaf, germination rate, root length, and seedling length were measured to preliminarily evaluate growth. Each treatment in this experiment had three technical replicates and three biological replicates. The results of the hydroponic plant morphology observations of spring and winter wheat are attached. Figure 2 As shown, the growth momentum of spring and winter wheat treated with the synthetic inoculant (CM group) was significantly better than that of the salt stress treatment groups (CK+NaCl and CK+H2O). See the appendix for measurements of germination rate, seedling length, and root length of spring and winter wheat. Figure 3 The results showed that under salt stress, the mixed microbial agent significantly promoted the germination rate, seedling length, and root length of spring wheat. It also had a significant promoting effect on the seedling length and root length of winter wheat.

[0045] Example 6: Detection of the effect of synthetic microbial agents on the activity of wheat plant enzymes

[0046] Based on the treatment with the synthetic inoculant described in Example 5 above, wheat leaves were collected 10 days later and stored in a liquid nitrogen tank. Plant enzyme activity was detected using the BCA protein assay kit, catalase (CAT) kit, superoxide dismutase (SOD) kit, peroxidase (POD) kit, malondialdehyde (MDA) content kit, proline (PRO) content assay kit, and chlorophyll content kit from Suzhou Keming Biotechnology Co., Ltd.

[0047] See the appendix for the results of plant enzyme activity analysis on hydroponically grown wheat. Figure 4 As shown, under salt stress conditions, this microbial agent can significantly increase the activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and total chlorophyll content in wheat. At the same time, after treatment with this microbial agent, the contents of malondialdehyde (MDA) and proline (PRO) in wheat are significantly reduced, indicating that this microbial agent plays a promoting and protective role in the process of spring wheat and winter wheat resisting salt and alkali stress.

[0048] Comparative Example 1: Comparison of growth-promoting effects of different strains and combinations

[0049] Based on the synthetic functional bacterial agent obtained in Example 4, five selected strains were prepared as single strains and in pairs at a 1:1 ratio for comparison in wheat germination experiments. Seeds of Xinchun 26 and Xindong 18 wheat varieties, commonly grown in Xinjiang Uygur Autonomous Region, were selected and surface-sterilized three times with 3% NaClO solution for 1 minute each time, followed by three washes with sterile water. The wheat seeds were soaked in the mixed culture of the synthetic bacterial consortium. Sterile filter paper was pre-lined in 100 mm × 100 mm sterile petri dishes, and the filter paper was soaked with 1.2% NaCl. After soaking for 8 hours, the seeds were placed in petri dishes containing 1.2% NaCl, with 20 seeds in each dish, and germination was carried out at room temperature, with three replicates per group. The seed germination rate was measured approximately 10 days later, when the seedlings showed their third true leaf. The group treatments and wheat germination rates are shown in Table 1 below.

[0050] Table 1: Grouping Treatments and Wheat Germination Rate

[0051]

[0052] By comparing the growth-promoting effects of the different strains and combinations mentioned above, it can be seen that the growth-promoting effect of individual strains on spring wheat and winter wheat is lower than that obtained by combining the selected strains in pairs. The synthetic functional bacterial agent composed of the five strains selected in this application has the best effect on promoting the growth of spring wheat and winter wheat under salt-alkali stress. The germination rate of spring wheat and winter wheat is significantly higher than that of other comparative treatment groups, which also confirms that the combination of the five strains selected in the technical solution provided in this application can play an excellent synergistic effect and has a significant impact on agricultural development and sustainable development in arid areas.

[0053] The above embodiments are merely examples to clearly illustrate the present invention 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 this invention.

Claims

1. A synthetic functional microbial agent, characterized in that, The bacterial agent was obtained by mixing resuspensions of *Gordonia hydrophobica*, *Lysinibacillus odysseyi*, *Planomicrobium okeanokoites*, *Luteimonas huabeiensis*, and *Brevibacterium epidermidis* in a volume ratio of 1:1:1:1:

1.

2. The method for preparing a synthetic functional microbial agent as described in claim 1, characterized in that, The specific preparation method is as follows: The strain was inoculated into TSA containing 2% NaCl and cultured to the exponential growth phase. The cells were collected by centrifugation, washed once with 1×PBS, and resuspended in 10 mM MgCl2 solution. The optical density (OD600) of each cell solution was adjusted to 0.

2. The selected strains were mixed in a ratio of 1:1:1:1:1 to obtain a synthetic bacterial agent, which was then diluted to an OD600 of 0.02 for later use.

3. The application of a synthetic functional microbial agent as described in any one of claims 1 to 2 in promoting wheat growth under salt stress.

4. The application of the synthetic functional microbial agent as described in claim 3 in promoting wheat growth under salt stress, characterized in that, The application uses the following steps: Select wheat seeds and sterilize them three times with 3% NaClO solution for 1 minute each time, then wash them three times with sterile water. Pre-line sterile filter paper in a 100 mm × 100 mm sterile petri dish and add 1.2% NaCl to soak the filter paper. Soak the wheat seeds with synthetic functional bacterial agent for 8 hours. Then place 20 seeds in each petri dish containing 1.2% NaCl and germinate at room temperature.

5. The application of the synthetic functional microbial agent as described in claim 3 in promoting wheat growth under salt stress, characterized in that, Selected from wheat seeds of either Xinchun No. 26 or Xindong No. 18.

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