Thermophilic bacillus subtilis lsg20 and use thereof

Abstract

The application belongs to the field of agricultural microorganism technology, and particularly relates to a thermophilic bacillus subtilis LSG20 and application thereof. The strain is classified and named as bacillus subtilis Bacillus subtilis , is preserved in the China General Microbiological Culture Collection Center, and the preservation time is March 31, 2025, and the preservation number is CGMCC No. 34029. The strain has excellent high-temperature adaptability and rhizosphere colonization capacity. Application of LSG20 in the whole growth period of rice can alleviate the inhibition of high-temperature stress on the physiology of seedlings. Application of LSG20 in the heading stage can effectively improve the pollen activity, thousand-grain weight and yield per plant under high temperature, reduce the chalky kernel rate, restore the amylose and total starch content, and realize the dual stability of yield and quality. In addition, the strain also shows broad-spectrum growth promotion and heat tolerance enhancement on various crops, which provides a new way for using thermophilic microbial resources to improve the stress resistance of crops.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to Bacillus subtilis LSG20 and its applications. Background Technology

[0002] Microbial regulation, as a sustainable agricultural strategy, has been proven to enhance crop heat tolerance. Existing research largely understands the role of microorganisms as activating or amplifying existing plant heat responses, particularly HSP expression, antioxidant systems, and hormone signaling. These studies primarily focus on response intensity and upstream regulation, with limited attention paid to the regulation of downstream execution mechanisms in the heat response. Particularly concerning is the synergistic effect between different molecular chaperones in the crucial execution stage of protein homeostasis, where microbial regulation remains insufficient. Furthermore, most existing research focuses on beneficial microorganisms under ambient temperature conditions, but the persistence and effectiveness of these ambient-temperature beneficial microorganisms under high-temperature environments are relatively limited. In contrast, thermophilic microorganisms possess a natural advantage in long-term survival and maintaining biological activity in high-temperature environments, thus holding promise for providing stable heat tolerance support for rice under sustained high-temperature conditions. However, the mechanisms by which thermophilic microorganisms mediate rice heat tolerance and their application potential remain poorly understood.

[0003] Global warming has led to a significant increase in the frequency, duration, and intensity of extreme heat events. Rice, a staple crop for more than half the world's population, is highly sensitive to temperature. High-temperature stress not only inhibits rice growth but also reduces final yield and grain quality. Multiple forecasts indicate that the risk of extreme heat in major rice-growing regions of Asia will continue to rise. Therefore, improving the heat tolerance of rice is of great significance for ensuring food security and promoting sustainable agricultural development. Although traditional breeding and genetic engineering have made significant contributions to improving plant heat tolerance, they are generally limited by long cycles and complex genetic backgrounds.

[0004] Under high-temperature stress, crops can enhance their stress tolerance by selectively recruiting microorganisms with specific functions by altering the composition of the rhizosphere microbiome. Microorganisms can colonize within or on the surface of plant tissues such as roots and leaves, forming mutually beneficial symbiotic systems. These microorganisms play a crucial role in improving the resistance and resilience of crops under environmental stress. Currently, research on microorganisms that enhance plant heat tolerance mainly focuses on mesophilic strains of the thermotolerant genus *Bacillus* spp. For example, Brevibacterium linens RS16 significantly enhances the heat tolerance of rice by producing ACC deaminase, which reduces the accumulation of stress ethylene in the plant, thereby mitigating the inhibitory effect of heat stress on plant growth (Choi, Jeongyun, Aritra Roy Choudhury, Denver I. Walitang, Yi Lee, and Tongmin Sa. 2021. “ACC Deaminase‐producing Brevibacterium Linens RS16Enhances Heat‐stress Tolerance of Rice (Oryza Sativa L.).”PhysiologiaPlantarum 174 (1). https: / / doi.org / 10.1111 / ppl.13584.). These mesophilic growth-promoting bacteria can stably colonize rice rhizosphere or endophytic environment and exert their effects through various mechanisms, including producing reactive oxygen species scavenging enzymes, inducing heat shock protein expression, and regulating plant hormone balance. Bacillus subtilis, on the other hand, is a strictly thermophilic Gram-positive bacterium. This microorganism is usually isolated from high-temperature environments such as hot springs and compost. Its genome features show that it contains a large number of thermostable enzymes and metabolic pathways adapted to high temperatures. It is this strict thermophilic characteristic that makes it generally difficult for it to establish an effective symbiotic relationship with plants. There are currently no reports of it colonizing with plants and exerting biological functions. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems, the purpose of this invention is to provide Bacillus subtilis LSG20 and its applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides Bacillus subtilis LSG20, which is classified as Bacillus subtilis. Bacillus subtilis It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 31, 2025, with accession number CGMCC No. 34029.

[0007] This invention obtained a thermophilic Bacillus subtilis strain LSG20 that can continuously and actively grow at high temperatures through targeted screening in hot spring environments. Utilizing its high-temperature adaptability, it regulates the heat tolerance of rice throughout its entire growth cycle through a root colonization system: strengthening the antioxidant and osmotic regulation systems during the seedling stage to maintain physiological homeostasis; and protecting pollen activity and grain-filling during the reproductive growth stage to ensure yield formation and starch accumulation. Simultaneously, its broad-spectrum cross-crop applicability was verified, forming an innovative technical route centered on "thermophilic inoculum source—rhizosphere colonization—systematic heat tolerance regulation—synergistic improvement of yield and quality," providing a flexible and sustainable microbial solution for crop stress-resistant cultivation under the background of climate warming.

[0008] This invention provides a thermoresistant bacterial preparation, with Bacillus subtilis LSG20 or its fermentation broth as the active ingredient.

[0009] This invention provides the application of the thermophilic Bacillus subtilis LSG20 or the thermotolerant bacterial preparation in alleviating high temperature stress in rice, wherein the alleviation of high temperature stress in rice is achieved by increasing the activity of antioxidant enzymes in rice and the content of osmotic regulators in rice cells.

[0010] Furthermore, the antioxidant enzymes include POD, CAT, and SOD.

[0011] Furthermore, the osmotic regulating substances include proline, glutathione, and soluble sugars.

[0012] This invention provides the application of the thermophilic Bacillus subtilis LSG20 or the thermotolerant bacterial preparation in improving rice yield, wherein the yield includes thousand-grain weight and yield per plant.

[0013] This invention provides the application of the thermophilic Bacillus subtilis LSG20 or the thermotolerant bacterial preparation in alleviating reproductive damage in rice caused by high temperature stress, wherein the alleviation of reproductive damage in rice caused by high temperature stress is achieved by improving pollen activity.

[0014] This invention provides the application of the thermophilic Bacillus subtilis LSG20 or the thermotolerant bacterial preparation in alleviating rice grain-filling abnormalities caused by high temperature stress, wherein the grain-filling abnormalities include a decrease in amylose and total starch content and an increase in chalky grain rate.

[0015] This invention provides the application of the thermophilic Bacillus subtilis LSG20 or the thermotolerant bacterial preparation in improving the biomass of crops under high temperature stress, including corn, wheat, tomato and cabbage.

[0016] Furthermore, the biomass includes plant height, fresh weight, and dry weight.

[0017] The present invention has the following beneficial effects: (0) This invention provides a thermophilic Bacillus subtilis strain LSG20 derived from a hot spring environment. This strain has good high-temperature adaptability and can maintain growth and biological activity under high temperature conditions. Compared with commonly used mesophilic and room-temperature beneficial microorganisms on the market, LSG20 can maintain its activity at high temperatures during production and transportation, and has advantages that other room-temperature microorganisms do not have in practical use.

[0018] (0) LSG20 can significantly alleviate the growth inhibition, oxidative damage and physiological damage caused by high temperature stress to two rice seedlings (including Japonica rice Nipponbare and Indica rice Jingliangyou Huazhan), improve antioxidant enzyme activity, enhance osmotic regulation capacity and reduce membrane lipid peroxidation level.

[0019] (0) LSG20 can improve pollen activity, thousand-grain weight and yield per plant during the reproductive growth period of rice, and improve chalkiness rate, amylose and total starch content, thereby simultaneously improving yield and quality stability. In summary, LSG20 has the function of improving the heat resistance of japonica rice and indica rice throughout the entire growth period, and the effect is significant.

[0020] (4) LSG20 also has growth-promoting and heat-resistant enhancement effects on various crops such as corn, wheat, and tomatoes, and has good potential for broad-spectrum application.

[0021] (5) This invention provides new microbial resources and application methods for improving crop heat resistance by utilizing thermophilic microorganisms, and has agricultural application development value. Attached Figure Description

[0022] Figure 1 The images show the morphological characteristics and physiological and biochemical identification of Bacillus subtilis LSG20. A shows the colony morphology of LSG20 on LB agar plates; B shows the cell morphology of LSG20 observed under transmission electron microscopy; C shows the growth curves of LSG20 at different temperatures (28℃, 37℃, 50℃, 60℃); D shows the growth status of LSG20 in media containing different mass fractions (1%, 2%, 3%, 5%, 8%, 10%) of NaCl; E shows the growth status of LSG20 in liquid media under different pH conditions (pH 4, 5, 6, 7, 8, 9, 10, 11); F shows the enzyme activity test results of LSG20; and G shows the physiological and biochemical test results of LSG20.

[0023] Figure 2 Phenotypic images of Nipponbare and Kyohoku Hanazono under normal temperature (NS) and high temperature stress (HS) conditions, with and without LSG20 inoculation. Scale bar: 13cm. A represents Nipponbare, and B represents Kyohoku Hanazono.

[0024] Figure 3The graphs show the effects of Bacillus subtilis LSG20 on the growth and physiological parameters of two rice varieties under high-temperature stress. A represents plant height, B represents fresh weight, C represents dry weight, D represents ascorbate peroxidase (APX) activity, E represents peroxidase (POD) activity, F represents catalase (CAT) activity, G represents superoxide dismutase (SOD) activity, H represents proline content, I represents glutathione (GSH) content, J represents soluble sugar content, K represents chlorophyll content, and L represents malondialdehyde (MDA) content. ns indicates no significant difference; *p<0.05, **p<0.01, ***p<0.001.

[0025] Figure 4 These images depict the effects of *Bacillus subtilis* LSG20 on the heat tolerance of rice during its reproductive growth stage. A shows the pollen morphology and staining results for *Nipponbare*, B shows the pollen morphology and staining results for *Jingliangyou Huazhan*, C shows the mature plant size and yield per pot for *Nipponbare*, D shows the mature plant size and yield per pot for *Jingliangyou Huazhan*, E shows the chalky appearance of the grains in *Nipponbare*, and F shows the chalky appearance of the grains in *Jingliangyou Huazhan*. The scale bars for *Nipponbare* are 30cm and 6cm, and for *Jingliangyou Huazhan* they are 30cm and 15cm.

[0026] Figure 5 The charts show the statistical indicators of rice reproductive indicators. A represents pollen viability, B represents chalkiness rate, C represents thousand-grain weight, D represents amylose content, E represents single-plant seed setting, and F represents total starch content.

[0027] Figure 6 This is an electron microscope image of the colonized tissue.

[0028] Figure 7 The graphs show the heat resistance of wheat and corn. In the graphs, A is the appearance of wheat, B is the appearance of corn, C is the wheat plant height, D is the wheat fresh weight, E is the wheat dry weight, F is the corn plant height, G is the corn fresh weight, and H is the corn dry weight.

[0029] Figure 8 The graphs show the heat resistance of tomatoes and bok choy. A represents the appearance of tomatoes, B represents the appearance of bok choy, C represents the tomato plant height, D represents the fresh weight of tomatoes, E represents the dry weight of tomatoes, F represents the bok choy plant height, G represents the fresh weight of bok choy, and H represents the dry weight of bok choy. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0031] Example 1: Isolation and identification of thermophilic bacteria.

[0032] I. Experimental Methods.

[0033] Bacillus subtilis LSG20 was isolated from the roots of a heat-resistant plant found in a hot spring in Lingshui, Hainan. Fresh rhizosphere soil samples were collected and serially diluted (10⁻¹ to 10⁻¹) after being shaken with sterile water. 6 The bacteria were spread on LB agar plates and incubated at 60°C. After 48 hours of incubation, colonies of different types were picked and single colonies were further purified to obtain *Bacillus subtilis* LSG20. For long-term preservation, the strain was stored in a 40% glycerol solution at -80°C. For routine experiments, the bacteria were streaked from glycerol tubes and incubated on LB agar plates at 45°C to obtain single colonies, which were then used as experimental materials.

[0034] The physiological and biochemical characteristics of *Bacillus subtilis* LSG20 were determined according to Bergey's Manual of Bacterial Systematics and standard methods, including morphological observation, growth temperature range, pH range, and enzyme activity assays, as detailed below. Cell morphology was observed using Gram staining and transmission microscopy. Growth characteristics were assessed under different temperature (20–65 °C), pH (4.0–10.0), and NaCl concentrations (0%–10%, w / v). Oxidase activity was detected using the oxidase test strip colorimetric method, catalase activity was detected using the hydrogen peroxide decomposition method, starch hydrolysis capacity was detected using the starch agar plate-iodine solution colorimetric method, gelatin liquefaction capacity was detected using the gelatin liquefaction method, and citric acid utilization capacity was detected using the Simon's citrate medium colorimetric method.

[0035] II. Experimental Results.

[0036] LSG20 colonies are grayish-white, opaque, with a rough surface, and the cells are columnar. Figure 1 (A and B). Simultaneously, this strain can grow normally in 1%–10% NaCl solution, at pH 5–9 and 20–60°C, and possesses the ability to secrete various extracellular hydrolases and grow without nitrogen (Table 1). Figure 1 ).

[0037] Table 1: Physiological and Biochemical Characteristics The overall physiological characteristics of LSG20 are basically consistent with those of typical Bacillus subtilis, but it exhibits stronger stress tolerance. Notably, LSG20 maintains its growth state under high temperatures rather than entering spore dormancy, which is consistent with common mesophilic conditions. Bacillus The strategies relying on spore tolerance at high temperatures are significantly different.

[0038] Example 2: Effect of Bacillus subtilis thermophilus on the heat resistance of rice throughout its entire growth period.

[0039] I. LSG20 alleviates oxidative damage and physiological injury to rice seedlings caused by high temperature stress.

[0040] 1. Experimental Methods Preparation of fermentation broth: Bacillus subtilis LSG20 was inoculated into LB liquid medium and cultured with shaking at 180 rpm / min and 37℃ for 12 h to adjust to the logarithmic growth phase (OD). 600 =0.6) Reserved. The same applies below.

[0041] Experimental design: included 4 groups: (1) NS group: under normal temperature conditions, LSG20 was not inoculated (the control treatment was the same amount of culture medium); (2) NS-LSG20 group: under normal temperature conditions, LSG20 was inoculated; (3) HS group: under high temperature stress conditions, LSG20 was not inoculated; (4) HS-LSG20 group: under high temperature stress conditions, LSG20 was inoculated.

[0042] Treatment of rice seedlings with inoculum: On day 14, rice seedlings were treated with inoculum by immersing the roots in a thermophilic bacteria fermentation broth for 1 minute (the control treatment consisted of an equal volume of sterile culture medium). Immediately after treatment, the seedlings were returned to their original hydroponic conditions and cultured for another 7 days. On day 21, high-temperature stress treatment was initiated. The high-temperature stress conditions were: 14 hours of light, 42℃, and 90% relative humidity. Phenotypic data were recorded after high-temperature treatment.

[0043] Considering the differences in heat tolerance among different types of rice, the japonica rice variety Nipponbare and the indica rice variety JLYHZ were selected as representatives to evaluate the application potential of LSG20 under different genetic backgrounds. Chlorophyll content was determined colorimetrically at 645 nm and 663 nm using the ethanol extraction method; proline content was determined using the acidic naphthol colorimetric method, with absorbance read at 520 nm; soluble sugar content was determined using the anthrone method at 620 nm. Malondialdehyde (MDA) content was determined using the TBA method at 532 nm and 600 nm; glutathione (GSH) content was determined using the 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) colorimetric method, with absorbance measured at 412 nm. Regarding antioxidant enzyme activities, CAT activity was calculated by monitoring the H2O2 decomposition rate (240 nm); SOD activity was calculated based on the ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) reaction (560 nm); POD activity was measured at 470 nm using o-phenylenediamine as a substrate; and APX activity was calculated by measuring the ascorbic acid (AsA) oxidation rate (290 nm).

[0044] 0. Experimental Results like Figure 2 and Figure 3 As shown, rice exhibits typical oxidative stress, physiological metabolic disorders, and photosynthetic system damage under high-temperature stress. First, high-temperature stress significantly induces the accumulation of reactive oxygen species (ROS), leading to enhanced cell membrane lipid peroxidation, manifested as a significant increase in malondialdehyde (MDA) content. Second, in the antioxidant system, the contents of peroxidase (POD) and ascorbate peroxidase (APX) are significantly increased, while the contents of catalase (CAT) and superoxide dismutase (SOD) decrease dramatically. Furthermore, in the osmotic regulation system, the contents of soluble sugars significantly increase, while the contents of proline and glutathione (GSH) significantly decrease. Simultaneously, high-temperature stress significantly inhibits the photosynthetic capacity of rice, specifically manifested as a significant decrease in chlorophyll content.

[0045] In contrast, LSG20 treatment alleviated the physiological damage to rice seedlings caused by high-temperature stress. Specifically, plant height, fresh weight, and dry weight increased by 16.97%, 16.84%, and 26.03% in Nipponbare rice, respectively; while in Jingliangyou and Huazhanzhong, these figures increased by 16.48%, 16.33%, and 20.73%, respectively. Simultaneously, LSG20 significantly reduced the MDA content in rice seedlings under high-temperature stress (Nip decreased by 65.62%, and JLYHZ by 33.03%), effectively alleviating membrane lipid peroxidation damage caused by high-temperature stress. In the antioxidant enzyme system, LSG20 significantly increased the activities of POD, CAT, and SOD, resulting in a significant enhancement of overall antioxidant capacity, although APX activity did not show a significant change (…). Figure 2 Furthermore, LSG20 significantly increased the contents of Proline and GSH, further promoted the accumulation of soluble sugars, and enhanced the cell osmotic regulation capacity of rice under high-temperature conditions. Chlorophyll content also increased significantly after LSG20 treatment, indicating that LSG20 effectively alleviated the inhibition of photosynthesis by high temperatures.

[0046] These results indicate that high-temperature stress in rice seedlings primarily leads to physiological imbalance by inducing oxidative damage, disrupting antioxidant and osmotic regulation systems, and inhibiting photosynthesis. The LSG20 treatment synergistically alleviates these damages at multiple physiological levels, suggesting that it helps maintain overall physiological homeostasis in rice seedlings under high-temperature conditions.

[0047] II. LSG20 plays a role in the entire growth period of rice and improves yield and grain quality.

[0048] 1. Experimental Methods To assess the heat tolerance of rice during its reproductive growth stage, rice seedlings treated as described above were transferred from the laboratory to a glass greenhouse for natural growth (each pot of rice in the treatment group was supplemented with 200ml of LSG20 bacterial solution once a month, and the OD of the bacterial solution was [not specified]). 600 =0.6), the high temperature stress group during the heading stage was treated in a heated greenhouse (42℃, 12h light / 12h dark, natural temperature) in a glass greenhouse until maturity.

[0049] Rice pollen activity was determined using the potassium iodide (I2-KI) staining method. Stamens were collected on the morning of the flowering day, crushed to release pollen, and then 1% (w / w) I2-KI staining solution was added and observed under a microscope. Thousand-grain weight and yield per plant were measured by collecting mature panicles at harvest. After seed hulling, 100 plump seeds were randomly selected from each treatment, and the chalkiness was observed under a backlight; the chalky grain rate was recorded and calculated. Total starch and amylose content in the seeds were determined using an enzymatic-colorimetric method.

[0050] 2. Experimental Results like Figure 4 and Figure 5 As shown, under normal temperature conditions, inoculation with LSG20 promoted yield formation in rice, increasing both the thousand-grain weight and the yield per plant compared to the uninoculated control group, indicating that LSG20 has a growth-promoting effect under normal temperature conditions.

[0051] Sustained high temperatures during the heading stage negatively impacted the reproductive development of rice. Firstly, pollen activity was significantly reduced. This decline indicates that high-temperature stress directly impaired the reproductive function of rice, thereby limiting grain filling and resulting in a significant decrease in thousand-grain weight and yield per plant. LSG20 treatment significantly alleviated the reproductive damage caused by high temperatures. Specifically, pollen activity (14.51% increase in Nipponbare, 18.15% increase in JLYHZ), thousand-grain weight (8.75% increase in Nipponbare, 17.11% increase in JLYHZ), and yield per plant (236.92% increase in Nipponbare, 36.52% increase in JLYHZ) were all significantly higher than in the uninoculated group under high temperatures. It should be noted that the increase is partly due to the significantly lower yield base in the uninoculated treatment under high-temperature stress, but it still clearly demonstrates that LSG20 plays a significant role in alleviating high-temperature-induced reproductive damage and maintaining yield stability.

[0052] Besides yield, seed quality is also affected by high-temperature stress. High-temperature stress leads to abnormal grain filling, disordered starch granule arrangement, and the formation of numerous chalky grains, resulting in a significant increase in the chalky grain rate. Inoculation with LSG20 significantly reduced the chalkiness of grains in both varieties. High-temperature stress also interferes with starch synthesis and accumulation in grains, with amylose content being a crucial indicator of rice quality. In this study, high-temperature treatment significantly reduced both amylose and total starch content in the grains of both rice varieties, reflecting limited carbohydrate transport and starch synthesis during the grain filling period. Notably, inoculation with LSG20 significantly alleviated the inhibition of starch synthesis caused by high temperatures, and both amylose and total starch content in both varieties recovered to varying degrees, indicating that it helps maintain carbon metabolism balance during the grain filling period and improves grain quality stability. LSG20 not only alleviated the decline in rice yield caused by high-temperature stress but also ensured stable grain quality.

[0053] Example 3: Planting capacity.

[0054] I. Experimental Methods.

[0055] The roots of 14-day-old rice seedlings were immersed in LSG20 fermentation broth for 1 minute (the strain was incubated overnight at 37°C with shaking, and the OD of the fermentation broth was adjusted). 600=0.6), with LSG20 inoculation as the treatment group and no inoculation as the control group. Rice seedlings after root soaking were allowed to continue growing in hydroponic boxes for 3 days, after which root samples were collected. The rice root samples were fixed by soaking in a 2.5% glutaraldehyde solution at 4℃ for 24 hours. The fixed samples were then dried overnight in a 35℃ oven. The dried samples were then fixed to a thin metal sheet using double-sided conductive tape. Finally, the root systems of rice seedlings inoculated and uninoculated with LSG20 at room temperature were observed using a scanning electron microscope (JOEL JSM 5600, JAPAN).

[0056] II. Experimental Results.

[0057] To verify the colonization ability of LSG20 in rice roots, this study used scanning electron microscopy to observe the root systems of rice inoculated with Bacillus subtilis. Figure 6 As shown, in the control treatment without LSG20 inoculation, the root surface structure of Nipponbare and Jingliangyou Huazhan rice was intact and clear, with no obvious microbial attachment observed. After inoculation with LSG20, typical rod-shaped bacteria were observed on the root surface of both types of rice, with the bacteria mostly attached to the root folds and gaps. LSG20 has good host adaptability and can form a stable colonization state in the rice rhizosphere, providing a physical basis for its growth-promoting and stress-resistance effects.

[0058] Example 4: LSG20 enhances the heat resistance of other crops.

[0059] I. Experimental Methods.

[0060] Preparation of fermentation broth: Bacillus subtilis LSG20 was inoculated into LB liquid medium and cultured with shaking at 180 rpm / min and 37℃ for 12 h to adjust to the logarithmic growth phase (OD). 600 =0.6) for later use. The wheat variety tested was Jimai 22 (winter wheat); the maize variety was Xianyu 335 (hybrid maize); the tomato variety was MoneyMaker (indeterminate); and the bok choy variety was Jingyannong Shanghaiqing (bok choy-type rapeseed).

[0061] All seedling experiments were conducted in a light-climate chamber. On day 14, seedlings underwent inoculum treatment by immersing their roots in a thermophilic bacteria fermentation broth for 1 minute (the control treatment consisted of an equal volume of sterile culture medium). Immediately after treatment, the seedlings were returned to their original hydroponic conditions for continued cultivation. The growth conditions for wheat and corn seedlings were 25℃ / 20℃ (light / dark), a photoperiod of 16h / 8h (light / dark), and a relative humidity of 70%; the growth conditions for tomato and bok choy seedlings were 25℃ (light / dark), a photoperiod of 16h / 8h (light / dark), and a relative humidity of 70%. Seedlings cultured for 21 days were subjected to heat treatment (14h light, 42℃, 90% relative humidity). Phenotypic data were recorded after the high-temperature treatment.

[0062] II. Experimental Results.

[0063] like Figure 7 and Figure 8 As shown, this study further validated the application potential of LSG20 in maize, wheat, tomato, and Chinese cabbage to assess its broad applicability in agricultural production. Under normal temperature conditions, LSG20 treatment significantly promoted root development and plant growth in all crops, with significant increases in plant height, fresh weight, and dry weight. However, under high-temperature stress, uninoculated plants generally exhibited stunted growth, leaf curling, yellowing, scorching edges, and even death. In contrast, LSG20-inoculated plants showed upright leaves and reduced yellowing. Even under high-temperature stress, the plant height, fresh weight, and dry weight of all crops after LSG20 inoculation remained significantly higher than the control group. In conclusion, LSG20 demonstrates broad-spectrum growth-promoting and heat-resistance potential in various crops, providing theoretical support for its development and promotion as a microbial intervention resource for heat-sensitive crops.

[0064] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0065] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0066] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Bacillus subtilis LSG20, characterized in that, The taxonomic name of the thermophilic Bacillus subtilis LSG20 is Bacillus subtilis Bacillus subtilis , and it was preserved in the China General Microbiological Culture Collection Center on March 31, 2025, with a preservation number of CGMCC No. 34029.

2. A thermoresistant bacterial preparation, characterized in that, The active ingredient is Bacillus subtilis LSG20 or its fermentation broth as described in claim 1.

3. The application of the thermophilic Bacillus subtilis LSG20 of claim 1 or the thermotolerant bacterial preparation of claim 2 in alleviating high-temperature stress in rice, characterized in that, The method of alleviating high-temperature stress in rice is achieved by increasing the activity of antioxidant enzymes in rice and the content of osmotic regulators in rice cells.

4. The application according to claim 3, characterized in that, The antioxidant enzymes include POD, CAT, and SOD.

5. The application according to claim 3, characterized in that, The osmotic regulators include proline, glutathione, and soluble sugars.

6. The application of the thermophilic Bacillus subtilis LSG20 of claim 1 or the thermoresistant bacterial preparation of claim 2 in improving rice yield, characterized in that, The yield includes the weight of a thousand seeds and the yield per plant.

7. The application of the thermophilic Bacillus subtilis LSG20 of claim 1 or the thermotolerant bacterial preparation of claim 2 in alleviating reproductive damage in rice caused by high-temperature stress, characterized in that, The method of alleviating rice reproductive damage caused by high temperature stress is achieved by increasing pollen activity.

8. The application of the thermophilic Bacillus subtilis LSG20 of claim 1 or the thermotolerant bacterial preparation of claim 2 in alleviating abnormal grain filling in rice caused by high temperature stress, characterized in that, The abnormal grouting included a decrease in amylose and total starch content, and an increase in chalky grain rate.

9. The application of the thermophilic Bacillus subtilis LSG20 of claim 1 or the thermotolerant bacterial preparation of claim 2 in improving crop biomass under high temperature stress, characterized in that, The crops mentioned include corn, wheat, tomatoes, and cabbage.

10. The application according to claim 9, characterized in that, The biomass includes plant height, fresh weight, and dry weight.

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