A phyllosphere growth-promoting Geobacillus sp. strain for enhancing the resistance of rice to high temperature stress and its application

Abstract

The present invention discloses a leaf-sphere growth-promoting Geobacillus sp. for enhancing the resistance of rice to high temperature stress and its application. Geobacillus thermoparaffinivorans ) LspGeo, deposited with the General Microbiology Center of the China General Culture Collection Administration on February 28, 2025, with the deposit number CGMCC No. 33681. The application of the thermophilic Geobacillus sp. LspGeo in improving the heat stress tolerance and / or growth promotion of rice is of great significance for the development of superior bacterial strain resources that can enhance the heat tolerance and recovery of rice, as well as for the development of biological agents that specifically enhance the heat tolerance of rice.

Description

Technical Field

[0001] The invention relates to a phyllosphere growth-promoting Geobacillus sp. for enhancing the high temperature stress resistance of rice and application thereof. Background Art

[0002] Heat stress caused by global warming is one of the major abiotic stresses in rice production, second only to drought and salinity in its impact. Rice (Oryza sativa L.) is one of humanity's most important staple crops, and its heading and flowering periods coincide with periods of persistently high temperatures in my country. Heat damage can severely impact rice's heading, flowering, and grain filling processes. High temperatures can cause dry tips on rice leaves, incomplete glume closure, and shrunken grains, significantly reducing yields. Unlike the gradual annual warming associated with global warming, heat stress caused by extreme heat waves occurs when ambient temperatures rise above the normal growth threshold for a period of time. This has a greater impact on crop growth and yield, placing greater demands on crop tolerance and resilience. Therefore, given the increasing intensity and frequency of heat damage globally, research on the mechanisms and pathways for improving the heat stress tolerance of staple crops like rice is urgently needed to ensure food security.

[0003] In recent years, significant progress has been made in the study of plant heat stress, focusing on the molecular mechanisms of transcription factors, such as the heat shock transcription factor family, and their regulatory genes, as well as the use of modern biotechnology to breed heat-resistant and stress-resistant varieties. However, in addition to leveraging the plant's own heat stress response and cultivating heat-resistant rice varieties through breeding, plants can also alleviate heat stress by recruiting beneficial microorganisms.

[0004] Under abiotic stresses (such as drought, salinity, and high and low temperatures), crops can enhance their stress tolerance by altering the composition of their rhizosphere microbiome and selectively recruiting microbes with specific functions. Numerous studies have demonstrated that microbes can colonize within or on plant tissues such as roots and leaves, forming mutually beneficial symbiotic systems. These microbes play a crucial role in enhancing crop resistance and resilience to environmental stress. Therefore, studying the mechanisms and regulatory pathways by which the plant microbiome promotes plant tolerance to environmental stress has become a hot topic in the field of plant-microbe interactions. Several studies have demonstrated that beneficial microbes such as Pseudomonas, Bacillus, and Enterobacter, as well as fungi such as Paecilomyces, interact with crops through complex signaling networks, enhancing plant heat tolerance. In addition to enhancing plant heat tolerance indirectly through regulating hormone levels and promoting plant growth, plant probiotics can also enhance plant heat tolerance through direct regulatory pathways. High levels of ROS accumulation in plants can induce the colonization of microorganisms with high antioxidant activity or that can enhance the expression of plant antioxidant genes. Therefore, exploring the potential of plant probiotics in improving crop tolerance to extreme heat stress can reduce the non-productive consumption of photosynthetic products in crops under environmental stress and is also a key breakthrough in improving rice's tolerance and recovery from heat damage. Summary of the Invention

[0005] The purpose of the present invention is to provide a phyllosphere growth-promoting bacterium screened from rice leaves that can improve the heat damage resistance of rice, in response to the practical problem that rising global temperatures cause frequent extreme high temperature events, which in turn restrict the growth and metabolism of food crops and seriously reduce global food production.

[0006] Another object of the present invention is to provide a bacterial agent prepared by the strain.

[0007] Another object of the present invention is to provide applications of the strain and the bacterial agent.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] In order to solve the above technical problems, the present invention first provides a thermophilic alkanobacillus ( Geobacillus thermoparaffinivorans ) LspGeo, isolated from the leaf sphere of a rice plant with strong heat tolerance. The strain provided by the present invention was deposited on February 28, 2025, at the General Microbiology Center of the China Culture Collection Administration (CGMCC; Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China). The LspGeo strain is deposited with CGMCC No. 33681.

[0010] A bacterial agent prepared by the thermophilic Geobacillus LspGeo of the present invention.

[0011] The bacterial agent is a bacterial suspension of thermophilic Geobacillus LspGeo.

[0012] The bacterial agent of the present invention is prepared by the following method: the thermophilic Geobacillus sp. Geobacillus thermoparaffinivorans ) After activation, LspGeo was inoculated into R2A liquid culture medium for fermentation. After the strain culture was completed, the bacterial liquid obtained was centrifuged and the bacteria were washed, and the bacteria were resuspended in an equal volume of sterile water to obtain the bacterial suspension of the Geobacillus LspGeo.

[0013] The R2A liquid culture medium contains 0.25 g of tryptone, 0.5 g of acid-hydrolyzed casein, 0.5 g of yeast extract powder, 0.5 g of soluble starch, 0.3 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.3 g of sodium pyruvate, 0.25 g of peptone, and 0.5 g of glucose per liter; after the medium is prepared, the pH value is adjusted to 7.2 ± 0.2 and sterilized at 115°C for 30 minutes.

[0014] During the culture, the culture temperature was 45°C, the rotation speed was 170 rpm, and the culture was continued until the bacterial solution OD 600 =1.0.

[0015] The application of the thermophilic Geobacillus sp. LspGeo in improving the heat stress resistance of rice and / or promoting the growth of rice.

[0016] As a preferred embodiment of the present invention, the application of the growth-promoting bacteria is selected from the following (a), (b), (c) and / or (d):

[0017] Improve the growth of rice seedlings under high temperature stress,

[0018] Promote root development of rice seedlings under high temperature stress,

[0019] Improve the activity of related antioxidant enzymes in rice seedlings under high temperature stress.

[0020] Reducing the accumulation of malondialdehyde in rice seedlings under high temperature stress.

[0021] In the application, (a) is specifically manifested in that the growth indicators (plant height, SPAD value, and aboveground fresh weight) of rice seedlings subjected to high temperature stress and inoculated with the strain LspGeo are improved compared with rice plants that are also subjected to high temperature stress but not inoculated with the strain LspGeo.

[0022] In the application, (b) is specifically manifested in that the root growth and development-related indicators (root length, root volume, root surface area, average root diameter, number of root tips, and number of root forks) of rice seedlings subjected to high temperature stress after inoculation with the strain LspGeo are improved compared with rice plants that are also subjected to high temperature stress but not inoculated with the strain LspGeo.

[0023] In the application, (c) is specifically manifested in that the activities of relevant antioxidant enzymes (SOD, POD, CAT) in rice seedlings subjected to high temperature stress and inoculated with the strain LspGeo are increased compared with rice plants that are also subjected to high temperature stress but not inoculated with the strain LspGeo.

[0024] In the application, (d) is specifically manifested in that the malondialdehyde content (accumulation amount) of rice seedlings subjected to high temperature stress and inoculated with the strain LspGeo is reduced compared with rice plants subjected to high temperature stress but not inoculated with the strain LspGeo.

[0025] Beneficial effects:

[0026] The present invention provides a strain of thermophilic Geobacillus sp. LspGeo that enhances rice's resistance to high-temperature stress. When formulated into a microbial agent and sprayed onto rice leaves, this strain significantly promotes the development of the aboveground and root systems of rice plants under high-temperature stress. This is accompanied by increased activity of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and decreased malondialdehyde (MDA) content. This invention is of great significance for the development of superior bacterial strains that can enhance rice's heat tolerance and resilience, as well as for the development of biological agents specifically designed to improve rice's heat tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Colony morphology of strain LspGeo grown on R2A solid medium;

[0028] Figure 2 Phylogenetic tree of 16S rDNA sequences of strain LspGeo;

[0029] Figure 3 Phenotypes of rice plants after inoculation with the first generation of heat-acclimated phyllosphere functional bacteria;

[0030] Figure 4 Phenotypes of rice plants after inoculation with the sixth generation of heat-acclimated phyllosphere functional bacteria;

[0031] Figure 5 Phenotypes of rice plants after inoculation with the twelfth generation of heat-acclimated phyllosphere functional bacteria;

[0032] Figure 6 This figure shows the effect of strain LspGeo on the growth phenotype of rice seedlings of Nanjing 46 under high temperature stress;

[0033] Figure 7 The effect of strain LspGeo on superoxide dismutase (SOD) activity in leaves of rice cultivar Nanjing 46 under high temperature stress was analyzed by analysis of variance (ANOVA) with p < 0.05. All data are mean ± SD, with 6 biological replicates.

[0034] Figure 8 The effect of strain LspGeo on peroxidase (POD) activity in leaves of rice cultivar Nanjing 46 under high temperature stress was analyzed by analysis of variance (ANOVA) with p < 0.05. All data are mean ± SD, with 6 biological replicates.

[0035] Figure 9 The effect of strain LspGeo on catalase (CAT) activity in leaves of rice cultivar Nanjing 46 under high temperature stress was analyzed by analysis of variance (ANOVA) with p < 0.05. All data are mean ± SD, with 6 biological replicates.

[0036] Figure 10 The effect of strain LspGeo on the malondialdehyde (MDA) content in the leaves of rice cultivar Nanjing 46 under high temperature stress was analyzed by analysis of variance (ANOVA) with p < 0.05. All data are mean ± SD, with 6 biological replicates.

[0037] Figure 11 The results of the effect of strain LspGeo on the plant height of rice cultivar Nanjing 46 under high temperature stress were shown in the figure. Statistical analysis was performed using analysis of variance (ANOVA) with p < 0.05. All data are mean ± standard deviation, with 6 biological replicates.

[0038] Figure 12 The effect of strain LspGeo on the SPAD value of chlorophyll content in rice seedlings of Nanjing 46 under high temperature stress was statistically analyzed using analysis of variance (ANOVA) with p < 0.05. All data are mean ± SD, with 6 biological replicates.

[0039] Figure 13 The effect of strain LspGeo on the aboveground fresh weight of rice seedlings of Nanjing 46 under high temperature stress was statistically analyzed using analysis of variance (ANOVA) with p < 0.05. All data are mean ± SD, with 6 biological replicates.

[0040] Figure 14 The effect of strain LspGeo on the total root length of rice seedlings of Nanjing 46 under high temperature stress was statistically analyzed using analysis of variance (ANOVA) with p < 0.05. All data are mean ± SD, with 6 biological replicates.

[0041] Figure 15 The effect of strain LspGeo on the root volume of rice cultivar Nanjing 46 seedlings under high temperature stress was statistically analyzed using analysis of variance (ANOVA) with p < 0.05. All data are mean ± SD, with 6 biological replicates.

[0042] Figure 16 The effect of strain LspGeo on the root surface area of rice seedlings of Nanjing 46 under high temperature stress was statistically analyzed using analysis of variance (ANOVA) with p < 0.05. All data are mean ± SD, with 6 biological replicates.

[0043] Figure 17 The effect of strain LspGeo on the average root diameter of rice seedlings of Nanjing 46 under high temperature stress was statistically analyzed using analysis of variance (ANOVA) with p < 0.05. All data are mean ± SD, with 6 biological replicates.

[0044] Figure 18 The effect of strain LspGeo on the root tip number of rice seedlings of Nanjing 46 under high temperature stress was statistically analyzed using analysis of variance (ANOVA) with p < 0.05. All data are mean ± SD, with 6 biological replicates.

[0045] Figure 19 The effect of strain LspGeo on the number of root forks in rice seedlings of Nanjing 46 under high temperature stress was statistically analyzed using analysis of variance (ANOVA) with p < 0.05. All data are mean ± SD, with 6 biological replicates.

[0046] Biomaterial deposit information

[0047] LspGeo, classified as Geobacillus thermophilus Geobacillus thermoparaffinivorans , deposited in the General Microbiology Center of China Culture Collection Administration, the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is February 28, 2025, and the deposit number is CGMCC No.33681. DETAILED DESCRIPTION

[0048] The following embodiments of the present invention are described in detail. These embodiments are implemented based on the technical solutions of the present invention, and detailed implementation methods and specific operating procedures are given. However, the scope of protection of the present invention is not limited to the following embodiments. At the same time, the reagents and experimental methods used in the following examples are conventional reagents or methods in the art unless otherwise specified, and will not be repeated here.

[0049] The heat / high temperature stress mentioned in the present invention refers to an environment that exceeds the optimal growth temperature of the crop. For example, for rice, the optimal growth temperature is 25-30°C. If the maximum daily temperature is higher than 35°C for more than three days, it is considered to be under high temperature stress. In the embodiments, 45°C is used as an example for illustration, but it cannot be considered to be the entire protection scope of the present invention.

[0050] The crops described in the present invention are preferably gramineous crops, more preferably rice crops. In the embodiments, rice variety Nanjing 46 is used as an example for illustration, but it cannot be considered as the only crop in the entire protection scope of the present invention.

[0051] The present invention provides a method for screening and preparing thermophilic Geobacillus sp. LspGeo.

[0052] The present invention provides a thermophilic alkanobacillus ( Geobacillus thermoparaffinivorans ) LspGeo, the preservation number of the Geobacillus LspGeo is CGMCC No. 33681.

[0053] The thermophilic Geobacillus sp. LspGeo was isolated from the leaves of heat-tolerant rice plants subjected to 45°C heat stress and was confirmed to be a thermophilic Geobacillus sp. based on colony characteristics and 16S rDNA sequence identification. The thermophilic Geobacillus sp. LspGeo colonies grown on R2A solid culture medium were small, convex, smooth, and milky white. Figure 1 The 16S rDNA sequence of strain LspGeo was compared and analyzed by NCBI online BLAST, and a phylogenetic tree was constructed based on the top 6 related strains with the highest similarity. Geobacillus thermoparaffinivorans strain A1 (KC252966.1) is the closest relative with a high confidence level and is identified as Geobacillus thermoparaffinivorans ( Figure 2 ).

[0054] After surface disinfection and accelerated germination of Nanjing 46 rice seeds, white rice seeds with consistent germination status were selected and transferred to rice seedling boxes filled with ultrapure water. The ultrapure water or nutrient solution was replaced every three days, with 1 / 4, 1 / 3, 1 / 2, and then the standard concentration of International Standard Rice Reagent (IRRI) solution being replaced, depending on the rice's growth status. When the rice reached seven leaves and one heart, soil culture growth tests under high temperature stress were conducted.

[0055] The international standard rice nutrient solution (IRRI) contains 165.175g of ammonium sulfate, 40.83g of potassium dihydrogen phosphate, 60.9g of potassium sulfate, 147g of calcium chloride dihydrate, 246.5g of magnesium sulfate heptahydrate, 142.1g of sodium metasilicate nonahydrate, 133.11g of L-aspartic acid, 1.781g of manganese chloride tetrahydrate, 0.0944g of sodium molybdate dihydrate, 1.2366g of boric acid, 0.2214g of zinc sulfate heptahydrate, 0.0799g of copper sulfate pentahydrate, and 5.5604g of sodium ferric EDTA per liter. The pH value of the international standard rice nutrient solution (IRRI) needs to be adjusted to approximately 5.8.

[0056] In the present invention, by spraying rice seedlings with the bacterial agent LspGeo, after heat stress, the experimental group sprayed with LspGeo showed stronger antioxidant enzyme activity and lower accumulation of malondialdehyde (the content of which can be used to assess the degree of membrane damage in plants) than the control group. Therefore, the phyllosphere-growing bacteria can improve the heat tolerance of rice. Furthermore, the aboveground part and root system of the rice were better developed.

[0057] The present invention also provides a method for improving the heat damage resistance of rice, comprising the following steps: before high temperature stress, spraying the rice leaves with a suspension of LspGeo bacteria, ensuring that the inoculum amount of the strain on the surface of each fully expanded leaf of the rice plant is 10 5 cfu.

[0058] The rice leaf growth-promoting bacterial liquid was prepared by the following method: the thermophilic Geobacillus sp. LspGeo strain was streaked and cultured on solid R2A solid culture medium at 45°C. After a single colony was generated, a single colony was picked and placed in 4 ml of R2A liquid culture medium, shaken at 170 rpm and cultured overnight. Then, 500 μl of the bacterial liquid was aspirated and inoculated into 50 ml of liquid R2A culture medium, shaken at 170 rpm and 45°C until the OD600nm of the bacterial liquid was 1.0. The bacterial liquid with OD600nm=1.0 was centrifuged at 1000 rpm for 10 minutes, the supernatant was discarded, and the suspension was resuspended with an equal volume of sterile water.

[0059] The R2A liquid culture medium contains 0.25 g of tryptone, 0.5 g of acid-hydrolyzed casein, 0.5 g of yeast extract powder, 0.5 g of soluble starch, 0.3 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.3 g of sodium pyruvate, 0.25 g of peptone, and 0.5 g of glucose per liter; after the medium is prepared, the pH value is adjusted to 7.2 ± 0.2 and sterilized at 115°C for 30 minutes.

[0060] Since the highest daily temperature in the art is higher than 35°C and lasts for more than three days, it is considered to be high temperature stress. In the examples of the present invention, extreme high temperature stress (45°C) was set for the experiment.

[0061] To further illustrate the present invention, a method for improving the high temperature resistance of rice at the seedling stage provided by the present invention is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0062] In the examples of the present invention, unless otherwise specified, the methods and practices used are all conventionally conceivable. The test material in the examples of the present invention is Nanjing 46, which can be purchased from Jiangsu Academy of Agricultural Sciences and is a major rice variety grown in the middle and lower reaches of the Yangtze River.

[0063] The data presented in the examples are presented as mean ± SE and were statistically analyzed using SPSS (version 25.0, SPSS Inc., USA). Data were analyzed using one-way analysis of variance, and means were compared using Duncan's multiple variance test, with P < 0.05. Histograms were plotted using GraphPad Prism (8.0.2).

[0064] Example 1

[0065] A heat acclimation experiment was set up to acclimate the leaf-sphere growth-promoting bacteria community. Different temperature conditions were set in a temperature-controlled light incubator. The normal temperature treatment temperature was 28°C, and the high temperature stress temperature was 45°C. The rice seedlings were cultured in soil at room temperature until they had seven leaves and one heart, and then treated under normal temperature and high temperature stress conditions. During the high temperature stress treatment, the treatment group was placed in a high-temperature incubator from 10:00 to 16:00 every day. After the stress was completed, it was returned to normal temperature conditions for growth. The stress treatment lasted for 2 weeks. After 2 weeks of heat stress treatment, the leaf microbial community samples were collected. This sample was the first generation of acclimation sample; the leaf microbial community samples were collected at 10 5 The next batch of rice seedlings (seven leaves and one heart) were inoculated with the same inoculum of 100 cfu / leaf to acclimate the next generation. The acclimatization process was the same as the previous generation, and each generation was acclimated (HS+Com). Two control groups were set up during each generation of acclimatization: one control group was treated with constant temperature throughout (CK), and the other control group was treated with stress alone without the foliar acclimatization bacterial inoculation (HS).

[0066] Results and Analysis

[0067] like Figure 3 、 Figure 4 、 Figure 5 The phenotypes of rice plants at the early (1st generation), mid-term (6th generation), and late (12th generation) stages of heat acclimation are shown. CK represents normal temperature treatment, HS represents high temperature stress without bacterial inoculation, and HS+Com represents high temperature stress with inoculation of the acclimated bacterial community.

[0068] like Figure 3 As shown in the figure, the growth of rice plants sprayed with the first generation of acclimated bacteria for heat stress treatment was basically the same as that of rice plants not inoculated with bacteria for heat acclimation treatment.

[0069] like Figure 4 As shown in the figure, the growth of rice plants sprayed with the 6th generation acclimated bacteria for heat stress treatment is better than that of rice plants not inoculated with bacteria for heat acclimation treatment.

[0070] like Figure 5 As shown in the figure, the growth of rice plants sprayed with the 12th generation acclimated bacteria and subjected to heat stress was significantly better than that of rice plants not inoculated and subjected to heat acclimation.

[0071] Example 2

[0072] The rice phyllosphere microbial community sample was prepared by the following method: approximately 10 g of rice plant leaves were placed in a 100 mL sterile conical flask, 100 mL of sterile PBS buffer (0.02 mM, pH 7.0, 0.1% tween-80) was added, and the flask was shaken at 200 r / min for 40 min and ultrasonicated for 5 min (output frequency 40 kHz, power 200 W, 60 Hz). The flask was transferred to a 50 mL centrifuge tube and centrifuged at 1,000 r / min to collect the precipitate, which was the phyllosphere microbial community sample.

[0073] The rice phyllosphere growth-promoting bacteria were screened by the following method: culturable bacteria under high temperature conditions were isolated from the 12th generation of heat-acclimated rice phyllosphere microbial community samples, the phyllosphere microbial community samples were spread on R2A solid medium, and cultured in a 45°C incubator to select high temperature resistant culturable strains. The strains were cultured in R2A liquid medium until OD 600nm = 1.0, collect the cells and use 10 5 The inoculum was inoculated at a dose of 100 cfu / leaf into the leaf margins of rice seedlings (seven leaves and one heart), and the growth of the rice was observed. This resulted in the identification of the superior strain LspGeo, which significantly enhances the heat tolerance of rice. LspGeo was then identified by 16S rDNA sequence analysis.

[0074] Results and Analysis

[0075] like Figure 1 As shown, the colonies of the Geobacillus sp. LspGeo provided by the present invention on the R2A solid culture medium are small, convex, smooth and milky white.

[0076] The 16S rDNA sequence of strain LspGeo was compared and analyzed by NCBI online BLAST, and a phylogenetic tree was constructed based on the top 6 related strains with the highest similarity. Figure 2 As shown, the strain LspGeo of the present invention is different from the strain Geobacillus thermoparaffinivorans strain A1 (KC252966.1) is the closest relative with a high confidence level and is identified as Geobacillus thermoparaffinivorans .

[0077] like Figure 6 As shown, the strain LspGeo provided by the present invention can improve the growth of rice seedlings in soil culture under high temperature stress, and the growth of rice seedlings sprayed with the strain LspGeo is better than that of the control treatment without inoculation at high temperature.

[0078] Example 3

[0079] When plants are subjected to heat stress, reactive oxygen species (ROS) accumulate in large quantities, leading to increased membrane peroxidation. To overcome the damage caused by excessive ROS and regulate their redox state, plants activate a variety of defense strategies involving enzymatic and non-enzymatic antioxidants. The production of antioxidant enzymes such as catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) is an important plant coping strategy for stress. Malondialdehyde (MDA) is an indicator of membrane lipid peroxidation, and its content is positively correlated with the degree of peroxidation. To determine whether ROS scavenging and membrane lipid antioxidant capacity are altered in plants inoculated with LspGeo, this example measured the activities of relevant antioxidant enzymes and MDA content in rice tissues under heat stress, both inoculated with LspGeo and uninoculated rice, as well as uninoculated rice without heat treatment.

[0080] Nanjing 46 rice was selected as the material. The rice germination, cultivation, and heat treatment processes were the same as those described in the above embodiment. Rice seedlings with consistent growth status were transplanted and soil-grown, and the LspGeo bacterial suspension was sprayed onto the rice leaves. A control treatment was sprayed with sterile water alone. The potted plants, sprayed with the growth-promoting bacteria and sterile water, were placed in a 45°C incubator for 6 hours of continuous heat stress. After the heat stress treatment, the plants were returned to a 28°C artificial climate chamber for 18 hours of constant temperature. This cycle lasted 14 days to complete the soil-grown growth experiment under high-temperature stress. Rice plants cultured in a 28°C artificial climate chamber for 24 hours served as a control.

[0081] After the heat treatment, 5 g of fresh leaves from each treatment were taken to determine the activities of relevant antioxidant enzymes and the content of malondialdehyde.

[0082] The aforementioned antioxidant enzyme activity indicators include superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and the aforementioned relevant antioxidant enzyme activities were measured using a kit produced by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

[0083] The malondialdehyde (MDA) content was determined using a kit produced by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

[0084] Results and Analysis

[0085] The experimental results are as follows Figures 7-10As shown, normal temperature refers to no inoculation and no high temperature treatment, high temperature stress refers to high temperature treatment without inoculation, and high temperature stress+LspGeo refers to inoculation and heat stress treatment.

[0086] like Figure 7 As shown, the strain LspGeo provided by the present invention can significantly improve the related antioxidant enzyme activities of rice seedlings under high temperature stress. The superoxide dismutase (SOD) activity of rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation at high temperature.

[0087] like Figure 8 As shown, the strain LspGeo provided by the present invention can significantly improve the related antioxidant enzyme activities of rice seedlings under high temperature stress. The peroxidase (POD) activity of rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0088] like Figure 9 As shown, the strain LspGeo provided by the present invention can significantly improve the related antioxidant enzyme activities of rice seedlings under high temperature stress. The catalase (CAT) activity of rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0089] like Figure 10 As shown, the strain LspGeo provided by the present invention can significantly reduce the accumulation of malondialdehyde (MDA) in rice seedlings under high temperature stress. The malondialdehyde (MDA) content of rice seedlings sprayed with the strain LspGeo is significantly lower than that of the control treatment without inoculation at high temperature.

[0090] Example 4

[0091] After heat treatment, growth indicators including plant height, aboveground fresh weight, and SPAD value were measured. Plant height was measured using a tape measure, and aboveground fresh weight was measured using an electronic balance. SPAD value was measured using a portable chlorophyll meter (SPAD-502 Plus).

[0092] Results and Analysis

[0093] like Figure 11-13 As shown, normal temperature refers to no inoculation and no high temperature treatment, high temperature stress refers to high temperature treatment without inoculation, and high temperature stress+LspGeo refers to inoculation and heat stress treatment.

[0094] like Figure 11 As shown, the strain LspGeo provided by the present invention can significantly improve the growth of rice seedlings in soil culture under high temperature stress. The plant height of rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation at high temperature.

[0095] like Figure 12As shown, the strain LspGeo provided by the present invention can significantly improve the growth of rice seedlings in soil culture under high temperature stress, and the SPAD value of rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation at high temperature.

[0096] like Figure 13 As shown, the strain LspGeo provided by the present invention can significantly improve the growth of rice seedlings in soil culture under high temperature stress, and the fresh weight of the aboveground part of rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0097] Example 5

[0098] After the heat treatment, rice roots were scanned. The roots were placed in a transparent sample tray and, after adding an appropriate amount of ultrapure water, spread out and stretched. Scanning was performed using a ScanMaker i800 Plus Scanner (China) and analyzed using LA-S software (Hangzhou, China). Root length, root surface area, root volume, average root diameter, and number of root tips were measured. Three biological replicates were performed for each treatment, and the average value was calculated.

[0099] The experimental results are as follows Figures 14 to 19 As shown, normal temperature refers to no inoculation and no high temperature treatment, high temperature stress refers to high temperature treatment without inoculation, and high temperature stress+LspGeo refers to inoculation and heat stress treatment.

[0100] like Figure 14 As shown, the strain LspGeo provided by the present invention can significantly improve the root growth and development of rice seedlings under high temperature stress. The total root length of rice seedlings sprayed with the strain LspGeo is significantly longer than that of the control treatment without inoculation under high temperature.

[0101] like Figure 15 As shown, the strain LspGeo provided by the present invention can significantly improve the root growth and development of rice seedlings under high temperature stress. The root volume of rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0102] like Figure 16 As shown, the strain LspGeo provided by the present invention can significantly improve the root growth and development of rice seedlings under high temperature stress. The root surface area of rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0103] like Figure 17As shown, the strain LspGeo provided by the present invention can significantly improve the root growth and development of rice seedlings under high temperature stress. The average root diameter of rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0104] like Figure 18 As shown, the strain LspGeo provided by the present invention can significantly improve the root growth and development of rice seedlings under high temperature stress. The number of root tips of rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0105] like Figure 19 As shown, the strain LspGeo provided by the present invention can significantly improve the root growth and development of rice seedlings under high temperature stress. The number of root forks of rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0106] From the above examples, it can be concluded that the thermophilic Geobacillus sp. LspGeo strain of the present invention can effectively improve the high temperature tolerance of rice and promote the development of the aboveground part and root system of rice.

[0107] Although the above example provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A thermophilic alkanobacillus ( Geobacillus thermoparaffinivorans ) LspGeo, characterized in that It was deposited in the General Microbiology Center of China Culture Collection Administration on February 28, 2025, with the deposit number CGMCC No. 33681.

2. The thermophilic Geobacillus sp. according to claim 1 Geobacillus thermoparaffinivorans ) Bacterial agent prepared by LspGeo.

3. The microbial agent according to claim 2, characterized in that The bacterial agent is a thermophilic alkanobacillus ( Geobacillus thermoparaffinivorans ) LspGeo bacterial suspension.

4. The microbial agent according to claim 3, characterized in that The bacterial agent is prepared by the following method: the thermophilic alkanobacillus ( Geobacillus thermoparaffinivorans ) After activation, LspGeo is inoculated into R2A liquid culture medium for fermentation. After the strain culture is completed, the bacterial liquid obtained is centrifuged and the bacteria are washed, and the bacteria are resuspended in an equal volume of sterile water to obtain the bacterial agent.

5. The microbial agent according to claim 4, characterized in that The R2A liquid culture medium contains 0.25 g of tryptone, 0.5 g of acid-hydrolyzed casein, 0.5 g of yeast extract powder, 0.5 g of soluble starch, 0.3 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.3 g of sodium pyruvate, 0.25 g of peptone, and 0.5 g of glucose per liter; after the medium is prepared, the pH value is adjusted to 7.2 ± 0.2 and sterilized at 115°C for 30 minutes.

6. The microbial agent according to claim 4, characterized in that During fermentation, the culture temperature was 45°C and the rotation speed was 170 rpm. The culture was continued until the bacterial solution OD 600 =1.

0.

7. The thermophilic Geobacillus according to claim 1 ( Geobacillus thermoparaffinivorans ) Use of LspGeo in improving the heat stress tolerance of rice and / or promoting the growth of rice, characterized in that, The application is selected from the following (a), (b), (c) and / or (d): (a) Improve the growth of rice seedlings under high temperature stress, (b) Promote root development of rice seedlings under high temperature stress, (c) Improve the activity of related antioxidant enzymes in rice seedlings under high temperature stress, (d) Reduced the accumulation of malondialdehyde in rice seedlings under high temperature stress.

8. Use of the bacterial agent according to any one of claims 2 to 6 for improving the heat stress resistance of rice and / or promoting the growth of rice, characterized in that: The application is selected from the following (a), (b), (c) and / or (d): (a) Improve the growth of rice seedlings under high temperature stress, (b) Promote root development of rice seedlings under high temperature stress, (c) Improve the activity of related antioxidant enzymes in rice seedlings under high temperature stress, (d) Reduced the accumulation of malondialdehyde in rice seedlings under high temperature stress.

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