Growth-promoting rhizosphere bacillus for enhancing high-temperature stress resistance of rice and application of growth-promoting rhizosphere bacillus for enhancing high-temperature stress resistance of rice

Abstract

The invention discloses a rhizosphere growth-promoting geobacillus for enhancing high-temperature stress resistance of rice and application of the rhizosphere growth-promoting geobacillus. The invention relates to a bacillus thermophilus LspGeo, which is preserved in China General Microbiological Culture Collection Center (CGMCC) on February 28, 2025, and the preservation number of the bacillus thermophilus LspGeo is CGMCC No.33681. The bacillus thermophilus LspGeo has the advantages that the bacillus thermophilus LspGeo is preserved in the China General Microbiological Culture Collection Center (CGMCC) on February 28, 2025; the invention also discloses the application of the bacillus thermophilus LspGeo in improving the heat stress resistance of the rice and / or promoting the growth of the rice. The invention has important significance for developing excellent strain resources capable of improving the heat resistance and the recovery capability of the rice and developing biological preparations capable of improving the heat resistance of the rice in a targeted manner.

Description

Technical Field

[0001] The present invention relates to a phyllosphere-promoting Geobacillus sp. that enhances the high-temperature stress resistance of rice and its application. Background Art

[0002] High-temperature stress caused by global warming is one of the main abiotic stresses in rice production, and the harm it causes is second only to drought and salt stress. Rice (Oryza sativa L.) is one of the most important food crops for humans, and its booting and flowering stages coincide with the period when the temperature in China continues to rise. High-temperature heat damage will seriously affect the processes of rice booting, flowering, and filling. High temperature will cause phenomena such as dry tips of rice leaves, non-closure of glumes, and empty and shriveled grains, resulting in a significant reduction in yield. Different from the annual slow warming of global warming, the heat / high-temperature stress generated by extreme high-temperature heatwaves is caused by the ambient temperature rising above the normal growth temperature threshold level for a period of time, which has a greater impact on crop growth and yield and higher requirements for the stress tolerance and recovery ability of crops. Therefore, in the context of the increasing intensity and frequency of global high-temperature heat damage, studying how to improve the mechanisms and pathways of high-temperature heat damage tolerance in major crops such as rice is an urgent need to ensure food security.

[0003] In recent years, important progress has been made in the research on plants under high-temperature stress, mainly focusing on the exploration of the molecular mechanisms of transcription factors such as the heat shock transcription factor family and their regulatory genes, as well as related research on breeding stress-resistant varieties with high-temperature tolerance using modern biotechnology. However, to cope with heat stress, in addition to relying on the plant's own response to high-temperature stress and breeding high-temperature-tolerant rice varieties through breeding means, plants can also recruit beneficial microorganisms to alleviate high-temperature stress.

[0004] Under abiotic stress (such as drought, salinity, high and low temperature, etc.), crops can enhance their stress tolerance by selectively recruiting specific functional microbiomes by changing the composition of the rhizosphere microbiome. A large number of studies have shown that microorganisms can colonize inside or on the surface of tissues such as plant roots and leaves to form a mutually beneficial symbiotic system, and these microorganisms play an important role in improving the resistance and resilience of crops under environmental stress. Therefore, studying the mechanisms and regulatory pathways by which plant microbiomes promote plant tolerance to environmental stress has become a research hotspot in the current field of plant-microbe interactions. Some studies have confirmed the interaction between beneficial microorganisms such as bacteria of the genus Pseudomonas, Bacillus, Enterobacter, etc. and fungi such as Paecilomyces and crops through complex signal networks to improve plant heat tolerance. In addition to improving plant heat tolerance through indirect pathways such as regulating hormone levels and promoting plant growth, plant probiotics can also enhance plant heat tolerance through some direct regulatory pathways. The accumulation of high levels of ROS in plants can induce the colonization of microorganisms with high antioxidant activity or those that can increase the expression of plant antioxidant genes. Therefore, exploring the potential of plant probiotics in improving the ability of crops to tolerate extreme high temperature stress can reduce the non-productive consumption of photosynthates by crops under environmental stress, and is also an important breakthrough point for achieving improved tolerance and recovery ability of rice to heat damage. Summary of the Invention

[0005] The object of the present invention is to provide a phyllosphere growth-promoting bacterium screened from the rice phyllosphere that can improve the heat resistance of rice in view of the actual problem that the frequent occurrence of extreme high temperature events caused by the rising global temperature restricts the growth and metabolism of food crops and severely reduces the global food production.

[0006] Another object of the present invention is to provide a microbial agent prepared from this strain.

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

[0008] The object of the present invention can be achieved by the following technical solutions: To solve the above technical problems, the present invention first provides a Geobacillus thermoalkaliphilus ( Geobacillus thermoparaffinivorans ) LspGeo, which was isolated from the rice phyllosphere with strong heat tolerance. The strain provided by the present invention was deposited at the China General Microbiological Culture Collection Center (abbreviation: CGMCC; address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; postal code: 100101) on February 28, 2025, and the preservation number of the strain LspGeo is CGMCC No. 33681.

[0009] A microbial agent prepared from Geobacillus thermoalkaliphilus LspGeo of the present invention.

[0010] The microbial agent is a bacterial suspension of Geobacillus thermoalkaliphilus LspGeo.

[0011] The microbial agent of the present invention is prepared by the following method: The Geobacillus thermoalkaliphilus ( Geobacillus thermoparaffinivorans ) LspGeo is activated and then inoculated into R2A liquid medium for fermentation culture. After the strain culture is completed, the obtained bacterial liquid is centrifuged and the cells are washed, and the cells are resuspended with an equal volume of sterile water to obtain the bacterial suspension of Geobacillus LspGeo.

[0012] The R2A liquid 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 it is sterilized at 115 °C for 30 min.

[0013] When performing the above-mentioned culture, the culture temperature is 45 °C, the rotation speed is 170 rpm, and the culture is carried out until the OD 600 = 1.0.

[0014] Use of Geobacillus thermoalkaliphilus LspGeo in improving the heat tolerance stress ability of rice and / or promoting the growth of rice.

[0015] As a preference of the present invention, the application of the growth-promoting bacteria is selected from the following (a), (b), (c) and / or (d): Improving the growth of rice seedlings under high temperature stress, Promoting the root development of rice seedlings under high temperature stress, Improving the activity of related antioxidant enzymes in rice seedlings under high temperature stress.

[0016] Reducing the accumulation of malondialdehyde in rice seedlings under high temperature stress.

[0017] In the above application, (a) is specifically manifested as that the growth indexes (plant height, SPAD value, above-ground fresh weight) of rice seedlings under high temperature stress are improved after inoculating the strain LspGeo compared with rice plants that are also under high temperature stress but not inoculated with the strain LspGeo.

[0018] In the above application, (b) is specifically manifested as that the indexes related to root growth and development (root length, root volume, root surface area, average root diameter, root tip number, root fork number) of rice seedlings under high temperature stress are improved after inoculating the strain LspGeo compared with rice plants that are also under high temperature stress but not inoculated with the strain LspGeo.

[0019] In the application, (c) is specifically embodied in that the antioxidant enzyme (SOD, POD, CAT) activities of rice seedlings under high temperature stress are increased after inoculation with strain LspGeo compared with those of rice plants under the same high temperature stress but without inoculation with strain LspGeo.

[0020] In the application, (d) is specifically embodied in that the content (accumulation amount) of malondialdehyde in rice seedlings under high temperature stress is reduced after inoculation with strain LspGeo compared with that of rice plants under the same high temperature stress but without inoculation with strain LspGeo.

[0021] Beneficial effects:

[0022] The present invention provides a Geobacillus thermoglucosidans LspGeo that enhances the high temperature stress resistance of rice. Making the strain into a microbial agent and spraying it on rice leaves can significantly promote the development levels of the aboveground parts and roots of rice plants under high temperature stress, and is accompanied by an increase in the activities of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as a decrease in the content of malondialdehyde (MDA). The present invention is of great significance for developing excellent bacterial species resources that can improve the heat tolerance and recovery ability of rice, and for developing biological agents targeting the improvement of rice heat tolerance. Description of the drawings

[0023] Figure 1 Colony morphology of strain LspGeo growing on R2A solid medium; Figure 2 Phylogenetic tree of the 16S rDNA sequence of strain LspGeo; Figure 3 Phenotype of rice plants after inoculation with the first-generation heat-acclimated phyllosphere functional microbiota; Figure 4 Phenotype of rice plants after inoculation with the sixth-generation heat-acclimated phyllosphere functional microbiota; Figure 5 Phenotype of rice plants after inoculation with the twelfth-generation heat-acclimated phyllosphere functional microbiota; Figure 6 It is a result diagram of the effect of strain LspGeo on the growth phenotype of Nanjing 46 rice seedlings under high temperature stress; Figure 7 Effect of strain LspGeo on the activity of superoxide dismutase (SOD) in the leaves of Nanjing 46 rice seedlings under high temperature stress. Statistical analysis was performed using analysis of variance p < 0.05 (ANOVA). All data are mean ± standard deviation, and 6 biological replicates were carried out; Figure 8Effects of strain LspGeo on the peroxidase (POD) activity in the leaves of Nanjing 46 rice seedlings under high-temperature stress. Statistical analysis was performed using analysis of variance with p < 0.05 (ANOVA). All data are presented as mean ± standard deviation, and six biological replicates were conducted. Figure 9 Effects of strain LspGeo on the catalase (CAT) activity in the leaves of Nanjing 46 rice seedlings under high-temperature stress. Statistical analysis was performed using analysis of variance with p < 0.05 (ANOVA). All data are presented as mean ± standard deviation, and six biological replicates were conducted. Figure 10 Effects of strain LspGeo on the malondialdehyde (MDA) content in the leaves of Nanjing 46 rice seedlings under high-temperature stress. Statistical analysis was performed using analysis of variance with p < 0.05 (ANOVA). All data are presented as mean ± standard deviation, and six biological replicates were conducted. Figure 11 Results graph of the effects of strain LspGeo on the plant height of Nanjing 46 rice seedlings under high-temperature stress. Statistical analysis was performed using analysis of variance with p < 0.05 (ANOVA). All data are presented as mean ± standard deviation, and six biological replicates were conducted. Figure 12 Effects of strain LspGeo on the SPAD value of chlorophyll content in the leaves of Nanjing 46 rice seedlings under high-temperature stress. Statistical analysis was performed using analysis of variance with p < 0.05 (ANOVA). All data are presented as mean ± standard deviation, and six biological replicates were conducted. Figure 13 Effects of strain LspGeo on the fresh weight of the aboveground parts of Nanjing 46 rice seedlings under high-temperature stress. Statistical analysis was performed using analysis of variance with p < 0.05 (ANOVA). All data are presented as mean ± standard deviation, and six biological replicates were conducted. Figure 14 Effects of strain LspGeo on the total root length of Nanjing 46 rice seedlings under high-temperature stress. Statistical analysis was performed using analysis of variance with p < 0.05 (ANOVA). All data are presented as mean ± standard deviation, and six biological replicates were conducted. Figure 15 Effects of strain LspGeo on the root volume of Nanjing 46 rice seedlings under high-temperature stress. Statistical analysis was performed using analysis of variance with p < 0.05 (ANOVA). All data are presented as mean ± standard deviation, and six biological replicates were conducted. Figure 16 Effects of strain LspGeo on the root surface area of Nanjing 46 rice seedlings under high-temperature stress. Statistical analysis was performed using analysis of variance with p < 0.05 (ANOVA). All data are presented as mean ± standard deviation, and six biological replicates were conducted. Figure 17Effect of strain LspGeo on the average root diameter of Nanjing 46 rice seedlings under high temperature stress. Statistical analysis was performed using analysis of variance with p < 0.05 (ANOVA). All data are presented as mean ± standard deviation, and six biological replicates were conducted. Figure 18 Effect of strain LspGeo on the number of root tips of Nanjing 46 rice seedlings under high temperature stress. Statistical analysis was performed using analysis of variance with p < 0.05 (ANOVA). All data are presented as mean ± standard deviation, and six biological replicates were conducted. Figure 19 Effect of strain LspGeo on the number of root forks of Nanjing 46 rice seedlings under high temperature stress. Statistical analysis was performed using analysis of variance with p < 0.05 (ANOVA). All data are presented as mean ± standard deviation, and six biological replicates were conducted.

[0024] Biological material preservation information

[0025] LspGeo, taxonomically named Geobacillus thermoalkaliphilus Geobacillus thermoparaffinivorans was deposited in the China General Microbiological Culture Collection Center. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. The deposit date was February 28, 2025, and the deposit number is CGMCC No. 33681. Detailed implementation mode

[0026] The following is a detailed description of the embodiments of the present invention. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments. At the same time, the reagent products and experimental methods used in the following embodiments, unless otherwise specified, are conventional reagents or methods in the art and will not be elaborated further.

[0027] The heat / high temperature stress described in the present invention refers to an environment exceeding the optimal growth temperature of crops. For example, for rice, the optimal growth temperature is a daily average temperature of 25 - 30°C. If the daily maximum 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 regarded as the entire protection scope of the present invention.

[0028] The crops described in the present invention are preferably gramineous crops, more preferably rice crops. In the embodiments, the rice variety Nanjing 46 is used as an example for illustration, but it cannot be regarded as the entire protection scope of the present invention.

[0029] The present invention provides a screening and preparation method for Geobacillus thermoalkaliphilus LspGeo.

[0030] The present invention provides a Geobacillus thermoalkaliphilus ( Geobacillus thermoparaffinivorans)LspGeo, the Bacillus geothermicus LspGeo has a deposit number of CGMCC No. 33681.

[0031] The Bacillus geothermicus LspGeo of the present invention was isolated from the phyllosphere of heat-tolerant rice plants under 45 °C heat stress, and was identified as Bacillus geothermicus by colony characteristics and 16S rDNA sequence. The colonies of the Bacillus geothermicus LspGeo of the present invention on R2A solid medium are small, convex and smooth on the surface, and milky white ( Figure 1 ). The 16S rDNA sequence of strain LspGeo was analyzed by online BLAST in NCBI, and a phylogenetic tree was constructed based on the top 6 related bacteria with the highest similarity. According to the results of the phylogenetic tree, the strain LspGeo of the present invention has the closest genetic relationship with strain Geobacillus thermoparaffinivorans strain A1 (KC252966.1) with a relatively high confidence level, and was identified as Geobacillus thermoparaffinivorans ( Figure 2 ).

[0032] After the seeds of Nanjing 46 rice were surface-sterilized and germinated, the germinated rice seeds with consistent germination status were selected and transferred to a rice seedling box filled with ultrapure water. The ultrapure water or nutrient solution was changed every three days, and the international standard rice nutrient solution (IRRI) with 1 / 4, 1 / 3, 1 / 2, and standard concentrations was changed in turn according to the growth status of the rice. When the rice grew to seven leaves and one heart, a soil culture growth experiment under high temperature stress was carried out.

[0033] Each liter of the international standard rice nutrient solution (IRRI) contains 165.175 g of ammonium sulfate, 40.83 g of potassium dihydrogen phosphate, 60.9 g of potassium sulfate, 147 g of calcium chloride dihydrate, 246.5 g of magnesium sulfate heptahydrate, 142.1 g of sodium metasilicate nonahydrate, 133.11 g of L-aspartic acid, 1.781 g of manganese chloride tetrahydrate, 0.0944 g of sodium molybdate dihydrate, 1.2366 g of boric acid, 0.2214 g of zinc sulfate heptahydrate, 0.0799 g of copper sulfate pentahydrate, and 5.5604 g of sodium ferric ethylenediaminetetraacetate. The pH value of the international standard rice nutrient solution (IRRI) needs to be adjusted to about 5.8.

[0034] In the examples of the present invention, by spraying the bacterial agent LspGeo on rice seedlings, after heat stress, the experimental group sprayed with the bacterial agent LspGeo had stronger antioxidant enzyme activity and a lower accumulation of malondialdehyde (the content of which can be used to evaluate the degree of membrane damage in plants) compared with the control group. Therefore, the phyllosphere growth-promoting bacteria can improve the high-temperature tolerance of rice. And the above-ground part and root development status of the rice are better.

[0035] The present invention also provides a method for improving the heat tolerance of rice, comprising the following steps: before high-temperature stress, spraying the bacterial suspension of LspGeo on the rice leaves to ensure that the inoculation amount of the strain on the surface of each fully expanded leaf of the rice plant is 10 5 cfu.

[0036] The rice phyllosphere growth-promoting bacterial liquid is prepared by the following method: Streaking the Geobacillus thermoalkaliphilus LspGeo strain on a solid R2A solid medium and culturing it at 45°C. After single colonies are formed, pick a single colony and inoculate it into 4 ml of R2A liquid medium, and culture it overnight with shaking at 170 rpm. Then, pipette 500 μl of the bacterial liquid into a 50 ml liquid R2A culture medium, and culture it with shaking at 170 rpm and 45°C until the OD600nm of the bacterial liquid is 1.0. Centrifuge the bacterial liquid with an OD600nm of 1.0 at 1000 rpm for 10 minutes, discard the supernatant, and resuspend it with an equal volume of sterile water.

[0037] Each liter of the R2A liquid medium contains 0.25 g of tryptone, 0.5 g of acid hydrolysate 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; after the medium is prepared, adjust the pH value to 7.2 ± 0.2 and sterilize it at 115°C for 30 min.

[0038] Since the daily maximum temperature in this field is higher than 35°C and lasts for more than three days, it is considered to be under high-temperature stress. In the examples of the present invention, an extreme high-temperature stress (45°C) was set for the experiment.

[0039] To further illustrate the present invention, a method for improving the high-temperature tolerance of rice seedlings provided by the present invention will be described in detail below in conjunction with the drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0040] In the examples of the present invention, unless otherwise specified, the methods and materials used are all conventionally conceivable. The test materials in the examples of the present invention are Nanjing 46, which can be purchased from the Jiangsu Academy of Agricultural Sciences and is the main rice variety in the middle and lower reaches of the Yangtze River.

[0041] For the result data involved in the examples, the data are expressed as mean ± SE, and statistical analysis is performed using SPSS (version 25.0, SPSS Inc., USA). One-way analysis of variance is used for data analysis, and Duncan's multiple variance test is used for mean comparison, with P < 0.05. Histograms are drawn using GraphPad Prism (8.0.2).

[0042] Example 1

[0043] Set up a heat acclimation experiment to domesticate the phyllosphere growth-promoting microbial community. Different temperature conditions were set in a temperature-controlled light incubator. The normal temperature treatment was 28 °C, and the high-temperature stress temperature was 45 °C. Rice seedlings were cultivated in soil at normal temperature until they had seven leaves and one heart, and then were treated under normal temperature and high-temperature stress conditions respectively. During the high-temperature stress treatment, the treatment groups were placed in a high-temperature incubator from 10:00 to 16:00 every day, and after the stress was completed, they were returned to grow under normal temperature conditions. The stress treatment lasted for 2 weeks. After 2 weeks of heat stress treatment, phyllosphere microbial community samples were collected, which were the first-generation domesticated samples; the phyllosphere microbial community samples were inoculated onto the phyllosphere of the next batch of rice seedlings (with seven leaves and one heart) at an inoculation amount of 10 5 cfu / leaf for the next generation of domestication. The domestication process was the same as that of the previous generation, and successive generations of domestication (HS+Com) were carried out in this way. Two control groups were set up during the domestication process of each generation. One control group was treated at normal temperature throughout the process (CK), and the other control group was treated with only stress but without inoculation of the phyllosphere domesticated microbial community (HS).

[0044] Results and Analysis As Figure 3 、 Figure 4 、 Figure 5 respectively show the phenotypes of rice plants at the initial stage (the 1st generation), middle stage (the 6th generation), and late stage (the 12th generation) of heat domestication. Among them, CK was the normal temperature treatment, HS was the high-temperature stress treatment without inoculation of the microbial community, and HS+Com was the high-temperature stress treatment with inoculation of the domesticated microbial community.

[0045] As Figure 3 shown, the growth of rice plants treated with heat stress by spraying the 1st generation domesticated microbial community was basically the same as that of rice plants treated with heat domestication without inoculation of the bacteria.

[0046] As Figure 4 shown, the growth of rice plants treated with heat stress by spraying the 6th generation domesticated microbial community was better than that of rice plants treated with heat domestication without inoculation of the bacteria.

[0047] As Figure 5 shown, the growth of rice plants treated with heat stress by spraying the 12th generation domesticated microbial community was significantly better than that of rice plants treated with heat domestication without inoculation of the bacteria.

[0048] Example 2

[0049] The rice phyllosphere microbial community sample was prepared by the following method: About 10 g of rice plant leaves were placed into a 100 mL sterile conical flask, 100 mL of sterilized PBS buffer (0.02 mM, pH 7.0, 0.1% tween-80) was added, and it was oscillated at 200 r / min for 40 min and sonicated for 5 min (output frequency 40 kHz, power 200 w, 60 Hz), then transferred into a 50 mL centrifuge tube, and the precipitate was collected by centrifugation at 1,000 r / min, which was the phyllosphere microbial community sample.

[0050] The rice phyllosphere growth-promoting bacteria were screened by the following method: Cultivable bacteria under high temperature conditions were isolated from the 12th generation rice phyllosphere microbial community sample after heat acclimation. The phyllosphere bacterial community sample was spread on R2A solid medium and cultured in an incubator at 45 °C, and the culturable strains with high temperature tolerance were selected. The strain was cultured in R2A liquid medium until OD 600nm = 1.0, the bacterial cells were collected and inoculated onto the phyllosphere of rice seedlings (with seven leaves and one heart) at an inoculum concentration of 10 5 cfu / leaf, and the growth status of rice was observed. Thus, the excellent strain LspGeo that could significantly enhance the heat tolerance of rice was screened, and LspGeo was identified by 16S rDNA sequence analysis.

[0051] Results and Analysis As Figure 1 shown, the colony of the Bacillus licheniformis LspGeo provided by the present invention on R2A solid medium was small, convex and smooth on the surface, and milky white.

[0052] The 16S rDNA sequence of strain LspGeo was analyzed by online BLAST in NCBI, and a phylogenetic tree was constructed based on the top 6 related bacteria with the highest similarity. As Figure 2 shown, the strain LspGeo of the present invention had the closest genetic relationship with the strain Geobacillus thermoparaffinivorans strain A1 (KC252966.1) with a relatively high confidence level, and was identified as Geobacillus thermoparaffinivorans .

[0053] As Figure 6 shown, the strain LspGeo provided by the present invention could improve the growth of rice seedlings under high temperature stress in soil culture, and the growth of rice seedlings sprayed with strain LspGeo was better than that of the control treatment without inoculation under high temperature.

[0054] Example 3

[0055] When plants are subjected to heat stress, a large amount of ROS (reactive oxygen species) accumulates, leading to an increased degree of membrane lipid peroxidation. To overcome the damage caused by excessive ROS and regulate the redox state, plants activate various 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 strategy for plants to cope with stress. Malondialdehyde (MDA) is one of the indicators of membrane lipid peroxidation, and its content is positively correlated with the degree of peroxidation. To determine whether the ROS scavenging level and the degree of membrane lipid antioxidant of the inoculated LspGeo plants have changed, in this example, the activities of relevant antioxidant enzymes and the content of malondialdehyde were measured in the rice tissues inoculated with LspGeo and non-inoculated and non-heat-treated under heat stress treatment.

[0056] Nanjingjing 46 was selected as the material, and the processes of rice germination, cultivation, and heat treatment were the same as the specific descriptions in the above embodiments. Rice seedlings with consistent growth status were selected for transplanting and soil cultivation, and the LspGeo bacterial suspension was sprayed onto the rice leaves, and only sterile water was sprayed as the control treatment. The potted plants sprayed with the growth-promoting bacteria and sterile water were placed in a 45 °C high-temperature incubator for continuous heat stress treatment for 6 h, and after the heat stress treatment was completed, they were restored to the normal temperature of 28 °C in the artificial climate chamber for 18 h, and this cycle was repeated for 14 days to complete the soil cultivation growth experiment under high-temperature stress. The rice cultivated at normal temperature (28 °C) in the artificial climate chamber for 24 h was used as the control.

[0057] After the heat treatment was completed, 5 g of fresh leaves from each treatment were taken to measure the activities of relevant antioxidant enzymes and the content of malondialdehyde.

[0058] The above antioxidant enzyme activity indicators include superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and the relevant antioxidant enzyme activities were measured using a kit produced by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

[0059] The content of the above malondialdehyde (MDA) was measured using a kit produced by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

[0060] Results and Analysis The experimental results are as Figures 7 - 10 shown, where normal temperature is non-inoculated and non-high-temperature-treated, high-temperature stress is high-temperature non-inoculated treatment, and high-temperature stress + LspGeo is the treatment of inoculated and heat-stressed.

[0061] As Figure 7 shown, the strain LspGeo provided by the present invention can significantly improve the activities of relevant antioxidant enzymes of rice seedlings under high-temperature stress, and the superoxide dismutase (SOD) activity of the rice seedlings sprayed with the strain LspGeo is significantly higher than that of the non-inoculated control treatment under high temperature.

[0062] As Figure 8 shown, the strain LspGeo provided by the present invention can significantly improve the activities of related antioxidant enzymes in 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.

[0063] As Figure 9 shown, the strain LspGeo provided by the present invention can significantly improve the activities of related antioxidant enzymes in 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.

[0064] As Figure 10 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 content of malondialdehyde (MDA) in rice seedlings sprayed with the strain LspGeo is significantly lower than that of the control treatment without inoculation under high temperature.

[0065] Example 4

[0066] After the heat treatment, the growth indexes including plant height, fresh weight of the above-ground part and SPAD value were detected. The plant height was measured using a tape measure, and the fresh weight of the above-ground part was measured using an electronic balance. The SPAD value was measured using a portable chlorophyll meter (SPAD-502 Plus).

[0067] Results and Analysis As Figures 11 - 13 shown, where normal temperature is the treatment without inoculation and without high temperature treatment, high temperature stress is the treatment without inoculation under high temperature, and high temperature stress + LspGeo is the treatment with inoculation and heat stress.

[0068] As Figure 11 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 under high temperature.

[0069] As Figure 12 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 SPAD value of rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0070] As Figure 13 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 fresh weight of the above-ground part of rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0071] Example 5

[0072] After the heat treatment, the rice roots were scanned. The roots were placed in a transparent sample dish, and an appropriate amount of ultrapure water was added to disperse the roots and keep them in a stretched state. They were scanned using a root scanner ScanMaker i800 Plus Scanner (China), and analyzed using LA-S software (Hangzhou, China) to measure indexes such as root length, root surface area, root volume, root average diameter, and number of root tips. Three biological replicates were measured for each treatment, and the average value was taken.

[0073] The experimental results are as Figures 14 - 19 shown, where normal temperature means no inoculation and no high-temperature treatment, high-temperature stress means high-temperature and no inoculation treatment, and high-temperature stress + LspGeo means inoculation and heat stress treatment.

[0074] As Figure 14 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 the rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0075] As Figure 15 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 the rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0076] As Figure 16 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 the rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0077] As Figure 17 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 the rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0078] As Figure 18 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 the rice seedlings sprayed with the strain LspGeo is significantly higher than that of the control treatment without inoculation under high temperature.

[0079] As Figure 19As 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.

[0080] From the above examples, it can be concluded that the Geobacillus thermoglucosidans strain LspGeo of the present invention can effectively improve the high-temperature tolerance of rice and promote the development of the above-ground part and roots of rice at the same time.

[0081] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope 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 Microorganism Culture Collection Administration on February 28, 2025, with the deposit number CGMCC No. 33681.

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

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

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

5. The bacterial 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 preparation of the culture medium, the pH value is adjusted to 7.2±0.2, and sterilized at 115°C for 30 minutes.

6. The bacterial 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 ) Application of LspGeo in improving the heat stress tolerance of rice and / or promoting the growth of rice.

8. The use according to claim 7, 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) increasing the activity of relevant antioxidant enzymes in rice seedlings under high temperature stress, (d) Reduced the accumulation of MDA in rice seedlings under high temperature stress.

9. Use of the bacterial agent according to any one of claims 2 to 6 in improving the heat stress resistance of rice and / or promoting the growth of rice.

10. The use according to claim 9, characterized in that: The application is selected from the following (a), (b), (c) and / or (d): (e) improving the growth of rice seedlings under high temperature stress, (f) Promote root development of rice seedlings under high temperature stress, (g) increasing the activity of relevant antioxidant enzymes in rice seedlings under high temperature stress, (h) Reduced the accumulation of MDA in rice seedlings under high temperature stress.

Images