A diazotroph composition and its use in enhancing resistance in sugar cane

Abstract

The application discloses a kind of nitrogen-fixing bacteria composition and its purposes in enhancing resistance of sugarcane, belong to microbial technical field.The application will salt inhabitant nitrogen-fixing bacteria ( Azotobacter salinestris ), he feather delford bacteria ( Delftia tsuruhatensis ) and sodium-demanding vibrio ( Vibrio natriegens ) respectively fermentation culture, centrifugal resuspension after bacteria liquid is mixed, and has been made to enhance the composite microbial inoculant of resistance of sugarcane.The composite microbial inoculant is applied to sugarcane, and experimental results show that the composite microbial inoculant can effectively enhance the resistance of sugarcane to bacterial disease and drought stress, and the three nitrogen-fixing bacteria have significant synergistic effect.The application provides new microbial materials and processing methods for improving drought resistance and disease resistance of sugarcane in sugarcane planting, simple steps, low cost, green and environmental protection, with outstanding application value and broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a nitrogen-fixing bacteria composition and its use in enhancing sugarcane resistance. Background Technology

[0002] Sugarcane (Saccharum spp.) is a tall plant belonging to the genus Saccharum in the subtribe of the family Gramineae. Sugarcane is a major sugar crop globally, characterized by its preference for high temperatures, high water requirements, high nutrient uptake, and long growing season, exhibiting very strict requirements for heat and water.

[0003] Sugarcane, due to its large plant volume and leaf area, exhibits vigorous transpiration, resulting in a high water requirement during its growth and development. Sugarcane yield is significantly positively correlated with water demand. In some regions, sugarcane is planted in hilly areas without irrigation, forming "rainfed" dryland sugarcane, whose water requirement depends entirely on rainfall. In recent years, intensified global warming and frequent high temperatures and low rainfall in summer and autumn have exacerbated drought, becoming a significant factor restricting sugarcane yield and quality improvement. To address the impact of drought on sugarcane growth, a series of experiments and studies have been conducted, primarily focusing on breeding drought-resistant varieties to enhance the sugarcane's drought resistance and mitigate the effects of drought stress. Significant efforts have also been made to improve cultivation management and farming techniques to alleviate drought stress and increase sugarcane yield, but this problem remains largely unresolved.

[0004] Besides drought, sugarcane yield reduction is often caused by microbial pathogens. Among these, sugarcane white streak disease, caused by Xanthomonas albilineans (Xa), is the most representative bacterial disease. Xa can invade the xylem, phloem, and parenchyma tissues of sugarcane. When Xa invades the xylem, it immediately produces Xanthomonas albilineans toxin, which blocks chlorophyll differentiation in the sugarcane, resulting in white streaks on the leaves. Currently, considerable research has been conducted on the pathogenic mechanism of sugarcane white streak disease and its control using chemical pesticides and plant hormones, but a highly effective control solution has yet to be found.

[0005] Nitrogen-fixing bacteria are a collective term for bacteria that can fix nitrogen from the air that plants cannot directly absorb and utilize, and convert it into nitrogen fertilizer. Rhizobia are the most representative example. They often live in symbiosis with legumes, providing the nitrogen fertilizer needed for plant protein synthesis. However, there are no reports on the role of nitrogen-fixing bacteria in enhancing sugarcane resistance. Summary of the Invention

[0006] The purpose of this invention is to provide a nitrogen-fixing bacteria composition and its use in enhancing sugarcane resistance, thereby addressing the problems existing in the prior art. The compound microbial agent of this invention can effectively enhance sugarcane's resistance to bacterial diseases and drought stress, leveraging the synergistic effect between different nitrogen-fixing bacteria. This invention provides new microbial materials and treatment methods for resisting drought and disease stress in sugarcane cultivation. The process is simple, low-cost, and environmentally friendly, possessing outstanding application value and broad application prospects.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] The present invention provides a nitrogen-fixing bacterial composition comprising Azotobacter salinestris, Delftia tsuruhatensis, and Vibrio natriegens.

[0009] The present invention also provides the application of the above-mentioned nitrogen-fixing bacteria composition in the preparation of a compound microbial agent that enhances sugarcane resistance.

[0010] This invention also provides a method for preparing a compound microbial agent to enhance sugarcane resistance, comprising the following steps:

[0011] After activating the above-mentioned nitrogen-fixing bacteria, Delfordia tsuruhata, and sodium-dependent Vibrio, they were fermented, centrifuged, the precipitates were collected and resuspended in sequence to obtain bacterial solutions.

[0012] The bacterial solution is mixed evenly to obtain the compound bacterial agent.

[0013] Furthermore, the ratio of the number of viable bacteria of the halophilic nitrogen-fixing bacteria, the Delfordia tsuruhata bacteria, and the sodium-dependent Vibrio bacteria is 1:1:1.

[0014] Furthermore, in the compound microbial agent, the total viable concentration of the *Azotobacter halophila*, *Delfordia tsuruha*, and *Vibrio natans* is not less than 1 × 10⁻⁶. 7 CFU / mL.

[0015] The present invention also provides a compound microbial agent obtained according to the above preparation method.

[0016] The present invention also provides the application of the above-mentioned nitrogen-fixing bacteria composition or the above-mentioned compound bacterial agent in enhancing sugarcane resistance.

[0017] Furthermore, the resistance includes resistance to bacterial diseases and resistance to drought.

[0018] The present invention also provides a method for enhancing sugarcane resistance, comprising the following steps:

[0019] Before planting sugarcane, soak the sugarcane seed stalks in the above-mentioned compound microbial agent;

[0020] During the sugarcane seedling stage, the above-mentioned compound microbial agent is evenly sprayed onto the surface of the sugarcane leaves.

[0021] Furthermore, the resistance includes resistance to bacterial diseases and resistance to drought.

[0022] The present invention discloses the following technical effects:

[0023] This invention involves the separate fermentation and centrifugation of *Azotobacter salinestris*, *Delftiatsuruhatensis*, and *Vibrio natriegens*, followed by resuspension, to create a compound microbial agent that enhances sugarcane resistance. Application of this compound agent to sugarcane demonstrates its effectiveness in enhancing resistance to bacterial diseases and drought stress, with the three nitrogen-fixing bacteria exhibiting a significant synergistic effect. This invention provides a novel microbial material and treatment method for resisting drought and disease stress in sugarcane cultivation. The process is simple, low-cost, and environmentally friendly, possessing significant application value and broad application prospects. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Existing technologies for promoting sugarcane tolerance to drought stress include: a drought-resistant and highly efficient compound fertilizer specifically for sugarcane cultivation, the raw materials of which include plant straw, humic acid, urea, diammonium phosphate, potassium sulfate, maifanite, animal manure, magnesium sulfate, zinc sulfate, fly ash, superabsorbent resin, traditional Chinese medicine powder, and Bacillus licheniformis. Specifically, the plant straw is one or a mixture of wheat straw, cotton stalks, and corn straw. The animal manure is one or a mixture of cow manure, horse manure, and chicken / duck manure. The superabsorbent resin is specifically poly(2-acrylamido-2-methylpropanesulfonic acid-acrylic acid). The raw materials of the traditional Chinese medicine powder include *Gnaphalium affine*, *Chrysanthemum indicum*, *Polygonum hydropiper*, *Curculigo orchioides*, *Smilax china*, *Sargentodoxa cuneata*, *Fructus Arundinaceae*, *Rhizoma Melilotifoliae*, *Cortex Corni*, *Radix Astragali*, and *Radix Rhei*. The preparation method includes: First, sun-drying plant straw for 6-10 days to obtain plant straw with a moisture content of no more than 10%, then crushing it in a crusher to obtain straw particles with a particle size of 1-2 cm; Second, piling animal manure layer by layer outdoors into a manure pile 2 meters wide and 2 meters high, and fermenting it under loose and aerated conditions. When the compost temperature rises to 60-70℃, continue composting for 3-4 days. After composting, dry the animal manure in the sun, and then crush the dried manure to obtain solid particles with a particle diameter of no more than 0.2-0.5 cm. The third step involves preparing traditional Chinese medicine powder, which is then sieved through a 100-200 mesh screen. The fourth step involves preparing a highly absorbent resin. The fifth step involves mixing and blending animal manure, plant straw, humic acid, and Bacillus licheniformis, maintaining a pH of 5-6. The adjusted mixture is then piled in a fermentation tank for wet fermentation. The fermentation pile is 4 meters wide at the bottom and 2-3 meters high, covered with a moisture-retaining and heat-insulating film. The fermentation temperature is 40-45℃, and the moisture content is 60-70%. After 60 hours, when the center temperature of the pile exceeds 65℃, the pile is turned over for the first time, and then turned over every 20-24 hours. Fermentation ends after 1-2 turnings. The sixth step involves drying and pulverizing fly ash and maifanite separately to obtain particles with a diameter of 0.2-0.5 mm. The solid powder particles are 1 cm in size. Step 7: The fermentation product obtained in step 5 is mixed with urea, potassium sulfate, diammonium phosphate, fly ash, maifanite, zinc sulfate and magnesium sulfate, and stirred evenly. Step 8: The mixture obtained in step 7 is mixed with Chinese herbal medicine powder and super absorbent resin, and then dried and granulated to obtain drought-resistant and high-efficiency compound fertilizer.

[0030] Existing technologies for promoting sugarcane tolerance to drought and high-temperature stress include: the application of *Bacillus bellis* JB-23 in promoting sugarcane bud germination under stress conditions; stress conditions include, but are not limited to, high-temperature stress and / or drought stress. *Bacillus bellis* JB-23 can mitigate the decrease in bud germination rate caused by high-temperature stress and, to a certain extent, ensure the development of sugarcane buds. It promotes sugarcane bud germination; *Bacillus bellis* JB-23 and its prepared inoculants can promote sugarcane bud germination under stress conditions; it promotes sugarcane growth. *Bacillus bellis* JB-23 promotes sugarcane plant height growth; promotes stem diameter growth; promotes the biomass of both aboveground and underground parts of sugarcane; and increases the relative chlorophyll content (SPAD value) of sugarcane. *Bacillus bellis* JB-23 can secrete IAA, protease, cellulase, and amylase, which can increase sugarcane biomass in the field, thus benefiting sugarcane yield.

[0031] Existing technical solutions for controlling sugarcane white stripe disease include: an agent for the prevention and / or treatment of sugarcane white stripe disease, comprising: *Bacillus spp.*, *Bacillus megaterium*, *Bacillus laterosporus*, *Alternaria alternata* activator protein, calcium acetate, magnesium sulfate, and glucose. The combination of *Bacillus spp.*, *Bacillus megaterium*, and *Bacillus laterosporus* can inhibit the growth of *Xanthomonas spp.*, and *Alternaria alternata* activator protein can stimulate plant growth, increase chlorophyll content, and thus enhance sugarcane's resistance to the pathogen. Simultaneously, calcium acetate and magnesium sulfate, under the action of glucose, can activate the enzyme system within the sugarcane, promoting plant growth and development, and also helping to prevent sugarcane cracking. This agent specifically includes the following concentrations of raw materials: *Bacillus spp.* (1-3) × 10 9 cfu / mL, Bacillus megaterium (1-3) × 10 9 cfu / mL, Bacillus retrograde (1-3) × 10 9 The formulation contains cfu / mL, Alternaria faecium activator protein 20-40 mg / L, calcium acetate 15-20 mg / L, magnesium sulfate 10-13 mg / L, and glucose 10-15 mg / L. This formulation is prepared as a pesticide for sugarcane and also includes pesticide-acceptable excipients, including at least one of excipients, lubricants, antioxidants, preservatives, binders, fillers, or thickeners; the pesticide formulation is a powder, wettable powder, suspension concentrate, dry suspension concentrate, or granules.

[0032] The *Azotobacter salinestris* strain of this invention was purchased from the China Industrial Microbiological Culture Collection Center (CICC) under accession number CICC 10309; the *Delftia tsuruhatensis* strain was purchased from the same center under CICC under accession number CICC 10492; and the *Vibrio natriegens* strain was purchased from the same center under CICC under accession number CICC 10908. The public can purchase these strains from the aforementioned collection centers.

[0033] In this invention, *Azotobacter halophilus* is cultured on a nitrogen-fixing medium, which comprises the following components: 0.5 g yeast extract, 20.0 g mannitol, 0.2 g KH₂PO₄, 0.8 g K₂HPO₄, 0.2 g MgSO₄·7H₂O, 0.1 g CaSO₄·2H₂O, 1.0 mg FeCl₃, 1.0 mg Na₂MoO₄·2H₂O, 15.0 g agar, and 1000.0 mL distilled water, pH 7.2; all components are sterilized at 121°C for 15 min. Without agar, the medium is liquid.

[0034] In this invention, *Delfordia tsurugi* is cultured on LB medium, which comprises the following components: 10.0 g peptone, 5.0 g yeast extract, 10.0 g NaCl, 15.0 g agar, and 1000.0 mL distilled water, pH 7.0; all components are sterilized at 121°C for 15 min. Without agar, it is a liquid culture medium.

[0035] In this invention, *Vibrio natriureticis* is cultured on Bacto marine agar (2216), which comprises the following components: 1.0 g yeast extract, 5.0 g peptone, 0.1 g ferric citrate, 19.45 g NaCl, 5.9 g MgCl2, 0.55 g KCl, 3.24 g Na2SO4, 1.8 g CaCl2, 0.16 g Na2CO3, 0.08 g KBr, 34.0 mg SrCl2, 22.0 mg H3BO3, 4.0 mg NaSiO3, 2.4 mg NaF, 1.6 mg NH4NO3, 8.0 mg Na2HPO4, 15.0 g agar, and 1000.0 mL distilled water, pH 7.6. All components are sterilized at 121°C for 15 min. Without agar, the culture medium is liquid.

[0036] The sugarcane white stripe disease pathogen strain LB-1 (Xanthomonas white stripe LB-1) of the present invention has been disclosed in "Isolation and Identification of Pathogens of Sugarcane White Stripe Disease in Guangxi" (Wei Chunyan, Wei Jinju, Zhang Xiaoqiu, Zhang Baoqing, Song Xiupeng, Li Dewei, Qin Zhenqiang, Li Yangrui. Plant Quarantine, 2019, 33(01): 19-23); the applicant promises to distribute the above-mentioned biological material to the public within 20 years from the date of application of this invention.

[0037] Example 1

[0038] Preserved *Azotobacter halois*, *Delfordia tsuruha*, and *Vibrio natans* were inoculated into their respective culture media and activated at 28°C for 2 days. 100 μL of the activated bacterial solution was then inoculated into 50 mL of the corresponding liquid culture medium and cultured at 150 rpm at 28°C for 2 days. The culture medium was collected, centrifuged at 3000 rpm for 15 min, and the precipitate was collected and resuspended in liquid culture medium to a viable bacterial concentration of 1 × 10⁻⁶. 7 Collect bacterial solutions at CFU / mL. Mix equal volumes of bacterial solutions of *Azotobacter halojicus*, *Delfordia tsuruha*, and *Vibrio natriureticis* to create a compound microbial agent for enhancing sugarcane resistance.

[0039] Comparative Example 1

[0040] The only difference from Example 1 is that equal volumes of bacterial solutions of *Delfordia tsurugi* and *Vibrio natans* are mixed to create a compound microbial agent that enhances sugarcane resistance.

[0041] Comparative Example 2

[0042] The only difference from Example 1 is that equal volumes of bacterial solutions of *Hydrogentodoxa cuneata* and *Vibrio natans* are mixed to create a compound microbial agent that enhances sugarcane resistance.

[0043] Comparative Example 3

[0044] The only difference from Example 1 is that equal volumes of bacterial solutions of *Hydrogentodoxa cuneata* and *Delfordia tsuruha* are mixed to create a compound microbial agent that enhances sugarcane resistance.

[0045] Experimental Example 1: Sugarcane Planting Methods and Resistance to Bacterial Diseases

[0046] 1. Sugarcane cultivation

[0047] The test material was the sugarcane variety Xintai Sugar 20, which is susceptible to white streak disease. The experimental treatment was divided into 8 groups, namely the control group, Example 1 group, Comparative Examples 1-3 groups, Natrii halophilus group, Delfordia tsurugi group, and Vibrio natriureticis group. Each group was set up with 3 replicates, and each replicate had 40 sugarcane plants. The plants were grown in barrels in the experimental greenhouse of Guangxi Academy of Agricultural Sciences.

[0048] Before planting sugarcane, healthy seed stalks of New Taiwan Sugar No. 20 sugarcane were selected, cut into 3-5 cm single-bud segments, and placed in nylon seed bags. They were then treated in a 50℃ constant-temperature circulating water bath for 2 hours for detoxification (bacterial treatment). After rinsing with clean water, the segments were soaked in the corresponding compound microbial agent or bacterial solution for each group for 3 hours. The control group was soaked in a mixed liquid culture medium. After soaking, the bacterial solution was drained, and sugarcane was planted in buckets with one bud per bucket (32 cm × 38 cm diameter × height). Each bucket contained 20 kg of mixed soil (soil: organic fertilizer: sand = 70:20:10, W / W), and holes were drilled in the bottom of the bucket to enhance aeration. All treatments maintained the same light, temperature, and water and fertilizer management using conventional methods. During the sugarcane seedling stage (when 3-5 leaves were fully expanded), the corresponding compound microbial agent or bacterial solution for each group was evenly sprayed onto the surface of the sugarcane leaves at a rate of 20 mL per plant, until the leaves were thoroughly wetted without dripping. The control group was sprayed with an equal volume of mixed liquid culture medium.

[0049] 2. Pathogen culture

[0050] XAS solid medium comprises the following components: 10 g / L sucrose, 5 g / L bacterial grade peptone, 5 g / L yeast extract, 0.5 g / L potassium dihydrogen phosphate, 0.25 g / L magnesium sulfate heptahydrate, 0.05 g / L sodium sulfite, 5 g / L potassium bromide, 0.01 g / L benomyl, 15 g / L agar powder, 25 mg / L cephalexin, 30 mg / L neomycin, 50 mg / L kasugamycin, and 100 mg / L actinomyceteone. Without agar powder, it is XAL liquid medium.

[0051] Thaw the cryopreserved Xanthomonas aureus strain LB-1 stored at -80℃. Take 100 μL of the bacterial culture and add it to 1 mL of XAL liquid medium. Incubate at 28℃ and 200 rpm with shaking for 18-24 h. Then, take another 100 μL of the incubated bacterial culture and add it to sterile XAS solid medium. Spread the culture evenly and incubate at 28℃ for 5 days. Then, using an inoculation loop, aseptically pick a single colony of Xanthomonas aureus from the XAS solid medium and inoculate it into fresh XAS solid medium. Incubate at 28℃ for 5 days to obtain purified Xanthomonas aureus. Use a 10 μL sterile pipette tip to pick a single colony of the purified Xanthomonas aureus and add it to 50 mL of XAL liquid medium. Incubate at 28℃ and 200 rpm with shaking for 36-48 h. Prepare a bacterial concentration of 1×10⁻⁶ using sterile XAL liquid medium. 8 The pathogenic bacterial solution at cfu / mL was then used for inoculation.

[0052] 3. Pathogen inoculation

[0053] Five days after foliar spraying of the bacterial solution during the sugarcane seedling stage, 30 healthy, disease-free, and uniformly growing sugarcane plants were selected from each replicate in each group. The sugarcane was inoculated with the pathogen using the leaf-cutting inoculation method, where sterilized surgical scissors were dipped in the aforementioned pathogen solution and the tip of the leaves was cut off. After each inoculated plant, the surgical scissors were re-dipped in the bacterial solution. Finally, the sugarcane was sprayed with the pathogen solution once. Each group had three replicates.

[0054] 4. Disease incidence survey and resistance evaluation

[0055] After leaf inoculation, the plants were cultured routinely for 21 days. The severity of white streak disease on each sugarcane plant was observed and recorded. The disease index was calculated based on the incidence and severity of the disease.

[0056] The severity of white streak disease in sugarcane plants is determined using the following criteria based on plant symptoms:

[0057] Level 0: Asymptomatic;

[0058] Grade 1: 1-2 pencil-like white stripes;

[0059] Level 2: More than two pencil-like white stripes;

[0060] Grade 3: Leaves turn white or yellow;

[0061] Grade 4: Leaf necrosis;

[0062] Level 5: Plant dead.

[0063] The disease index is calculated using the following formula:

[0064] Disease index (%) = [∑(Disease level number × Number of diseased plants at the corresponding level) / (Highest disease level number × Total number of plants surveyed)] × 100.

[0065] The resistance of sugarcane to white stripe disease in each group was evaluated based on the disease index. The evaluation criteria are as follows:

[0066] High resistance: Disease index ≤ 5.0;

[0067] Disease resistance: 5.0 < Disease index ≤ 15.0;

[0068] Anti-inflammatory response rate: 15.0 < Disease index ≤ 30.0;

[0069] Sickness index: 30.0 < Sickness index ≤ 50.0;

[0070] High susceptibility: Severity index > 50.0.

[0071] 5. Experimental Results

[0072] Table 1. Experimental results on the effects of each treatment group on resistance to sugarcane white stripe disease.

[0073] Group Disease index Resistance evaluation control group 80.00 High sensitivity Example 1 Group 3.56 High resistance Comparative Example 1 31.78 Illness Comparative Example 2 28.89 China Anti Comparative Example 3 42.44 Illness Salt-dwelling nitrogen-fixing bacteria group 56.67 High sensitivity Tsuruhata Delford Bacterial Group 67.11 High sensitivity Sodium-dependent Vibrio group 48.22 Illness

[0074] The results are shown in Table 1. Compared with the control group, the compound microbial agent prepared based on the nitrogen-fixing bacteria composition (Azotobacter halois, Delfordia tsuruha, and Vibrio natophilia) significantly enhanced the resistance of sugarcane to the pathogen of sugarcane white stripe disease (Xanthomonas leucocephala). Furthermore, the enhancing effect of this nitrogen-fixing bacteria composition on sugarcane resistance was significantly better than that of any single nitrogen-fixing bacteria solution or any compound microbial agent prepared from two nitrogen-fixing bacteria. The combined use of Azotobacter halois, Delfordia tsuruha, and Vibrio natophilia showed a significant synergistic effect in improving sugarcane resistance to bacterial diseases.

[0075] Experimental Example 2: Drought Resistance of Sugarcane

[0076] 1. Sugarcane cultivation

[0077] The drought-intolerant sugarcane variety Xintai Sugar 16 was used as the test material. The sugarcane planting and treatment methods were the same as in Experiment 1. Each treatment was set up with 3 replicates, and 60 sugarcane plants were planted in each replicate.

[0078] 2. Drought Treatment and Indicator Detection

[0079] When sugarcane entered the elongation stage, each group of sugarcane plants was divided into two parts: a water-controlled treatment (drought stress) and a normal water supply treatment. The soil moisture content of the sugarcane plants in the water-controlled treatment was reduced to 8-10% as the starting point for drought stress, while the soil moisture content in the normal water supply treatment was maintained at 20-25%. After 30 days of drought stress treatment, the plants were re-irrigated to restore the soil moisture content to 20-25%, which was maintained until the end of the elongation stage. During the drought stress period, drought-related phenotypes were observed in all tested sugarcane plants. At the end of the elongation stage, the sugarcane was harvested, and the stalk yield of each group of tested sugarcane was measured.

[0080] Based on the sugarcane stalk yield of each group of tested sugarcane, the drought resistance index was calculated using the following formula:

[0081] ;

[0082] In the formula:

[0083] DRI: Drought Resistance Index of the tested sugarcane;

[0084] Y a Average sugarcane stalk yield (kg) under each drought treatment group.

[0085] Y m Average sugarcane stalk yield (kg) under normal water supply in each group.

[0086] Y A Average sugarcane stalk yield (kg) under drought treatment in the control group.

[0087] Y M Average yield of sugarcane stalks (kg) under normal water supply treatment in the control group.

[0088] The drought resistance of sugarcane should be evaluated according to the following criteria:

[0089] Extremely strong: Drought resistance index ≥ 1.20;

[0090] Strong: Drought resistance index 1.00-1.19;

[0091] Moderate: Drought resistance index 0.80-0.99;

[0092] Weak: Drought resistance index 0.60-0.79;

[0093] Extremely weak: drought resistance index ≤ 0.59.

[0094] 3. Experimental Results

[0095] Before drought stress, all sugarcane plants in the control group grew healthily with green leaves. After drought stress, the sugarcane plants in the control group showed significant wilting and yellowing of leaves. Compared with the control group, the sugarcane plants in the experimental group showed less wilting under drought stress, with very little yellowing of leaves, similar to the experimental group under normal water supply. The sugarcane plants in the control groups 1-3, the *Haloxylon ammodendron* group, the *Delfordia tsuruga* group, and the *Vibrio natans* group all showed obvious yellowing and wilting of leaves under drought stress.

[0096] Table 2. Experimental results of the effects of each treatment group on the drought resistance of sugarcane.

[0097] Group Drought Resistance Index drought resistance assessment control group 0.786 weak experimental group 1.295 Extremely strong Comparative Example 1 0.916 medium Comparative Example 2 0.953 medium Comparative Example 3 0.887 medium Salt-dwelling nitrogen-fixing bacteria group 0.788 weak Tsuruhata Delford Bacterial Group 0.792 weak Sodium-dependent Vibrio group 0.801 medium

[0098] Based on the combined phenotypic observations and drought resistance index results (Table 2), it is evident that, compared to the control group, the compound microbial agent prepared according to this invention, based on a nitrogen-fixing bacteria composition (Azotobacter halois, Delftobacter tsuruha, and Vibrio natophilia), significantly enhances the drought stress resistance of sugarcane. Furthermore, the enhancing effect of this nitrogen-fixing bacteria composition on sugarcane drought resistance is significantly superior to any single nitrogen-fixing bacteria inoculum or any compound microbial agent prepared from two nitrogen-fixing bacteria. The combined use of Azotobacter halois, Delftobacter tsuruha, and Vibrio natophilia exhibits a significant synergistic effect in improving the drought stress resistance of sugarcane.

[0099] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A nitrogen-fixing bacteria composition, characterized in that, Including halogenated nitrogen-fixing bacteria with a live count ratio of 1:1:1 ( Azotobacter salinestris ), Tsuruhada Delford bacteria ( Delftia tsuruhatensis ) and sodium-dependent Vibrio ( Vibrio natriegens ); The aforementioned nitrogen-fixing bacteria is deposited at the China Industrial Microbial Culture Collection Center, with accession number CICC 10309; The *Delfordia tsurugi* strain described is deposited at the China Industrial Microbial Culture Collection Center, with accession number CICC10492. The sodium-dependent Vibrio species is deposited at the China Industrial Microbial Culture Collection Center, with accession number CICC 10908.

2. The application of the nitrogen-fixing bacteria composition as described in claim 1 in the preparation of a compound microbial agent to enhance sugarcane resistance, characterized in that, The resistance mentioned refers to resistance to bacterial diseases or drought resistance; The bacterial disease mentioned is sugarcane white stripe disease caused by Xanthomonas leucocephala.

3. A method for preparing a compound microbial agent to enhance sugarcane resistance, characterized in that, Includes the following steps: After activating the *Hydrozotrophus*, *Delfordia tsuruhata* and *Vibrio natans* as described in claim 1, they were sequentially fermented, centrifuged, precipitated and resuspended to obtain bacterial solution. The bacterial solution is mixed evenly to obtain the compound bacterial agent; The ratio of viable counts of the *Hydrozotrophus*, *Delfordia tsuruha*, and *Vibrio natriureticis* is 1:1:

1.

4. The preparation method according to claim 3, characterized in that, In the compound microbial agent, the total viable concentration of the *Hydroxypyr*, *Delfordia tsuruha*, and *Vibrio natriureticis* is not less than 1 × 10⁻⁶. 7 CFU / mL.

5. A compound microbial agent obtained by the preparation method according to claim 3 or 4.

6. The application of the nitrogen-fixing bacteria composition of claim 1 or the compound microbial agent of claim 5 in enhancing sugarcane resistance, characterized in that, The resistance mentioned refers to resistance to bacterial diseases or drought resistance; The bacterial disease mentioned is sugarcane white stripe disease caused by Xanthomonas leucocephala.

7. A method for enhancing sugarcane resistance, characterized in that, Includes the following steps: Before planting sugarcane, the sugarcane seed stalks are soaked in the compound microbial agent described in claim 5; During the sugarcane seedling stage, the compound microbial agent described in claim 5 is evenly sprayed onto the surface of the sugarcane leaves; The resistance mentioned refers to resistance to bacterial diseases or drought resistance; The bacterial disease mentioned is sugarcane white stripe disease caused by Xanthomonas leucocephala.