Bacillus tropicus KH15 and application thereof

Abstract

This invention discloses a tropical Bacillus KH15 and its applications. Its depository is the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC 35147. This invention can effectively improve the heat and drought resistance of plants. The product described in this invention can play an important role in drought resistance, yield increase, and desertification control, and has significant ecological and economic value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a tropical Bacillus KH15 and its applications. Background Technology

[0002] The arid, semi-arid, and sub-humid ecologically fragile zones are characterized by a prevailing southeast monsoon in summer, with rainfall and heat occurring simultaneously, and precipitation mainly occurring from April to September each year. While the rainy season also falls within April to September in some areas, there is significant spatial heterogeneity, with nine out of ten years experiencing drought. Due to climate change and unreasonable human disturbance, vegetation has severely degraded. Since the beginning of the 21st century, drought has intensified for several consecutive years, with consecutive spring, summer, and autumn droughts, which are extremely detrimental to the fragile ecosystem. The vast Northwest region experiences low precipitation and high evaporation, and drought is often accompanied by high temperatures. Drought is the most common adverse factor for agricultural production in the Northwest region, and mitigating the threats of high temperatures and drought to plants (especially food crops such as corn and wheat) is a global challenge.

[0003] Therefore, it is necessary to develop a product that can improve the heat resistance and drought resistance of plants. Summary of the Invention

[0004] The purpose of this invention is to provide a tropical Bacillus KH15 strain and its applications. This strain is a tropical Bacillus strain that can survive and reproduce in arid environments, and its applications can effectively improve the heat resistance and drought resistance of plants. The tropical Bacillus KH15 strain and its inoculant products described in this invention can play an important role in drought resistance, yield increase and desertification control, and have significant ecological and economic value.

[0005] The tropical spores provided by this invention ( Bacillus tropicus The strain KH15 was deposited on July 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC NO:35147.

[0006] The tropical Bacillus (Bacillus tropicalis) described in this invention Bacillus tropicus KH15 can significantly improve the heat and drought resistance of plants, promoting normal growth and yield increase under high temperature and drought conditions. The tropical Bacillus KH15 described in this invention can increase the heat resistance temperature of plants by about 5-8°C and extend the drought resistance time by about 10-15 days.

[0007] This invention provides a microbial inoculant, the active ingredient of which includes the aforementioned Bacillus tropicalis KH15.

[0008] In the above technical solution, the microbial agent also includes a carrier; the carrier includes a solid carrier and a liquid carrier.

[0009] In the above technical solutions, the dosage forms of the microbial agents include aqueous solutions, powders, granules, etc.

[0010] The present invention provides a method for using the aforementioned microbial inoculant, wherein the microbial inoculant comprising a solid carrier is applied to plants, and / or the microbial inoculant comprising a liquid carrier is applied to plants.

[0011] In the above technical solutions, the microbial agents are applied to plants through methods such as seed dressing, fertilization, drip irrigation, and spraying.

[0012] In the above technical solution, the plants include corn, oats, and wheat, etc.

[0013] This invention provides the application of the aforementioned Bacillus tropicalis KH15 or the aforementioned microbial inoculant in improving the heat resistance and drought resistance of plants.

[0014] This invention provides the application of the aforementioned Bacillus tropicalis KH15 or the aforementioned microbial agent in the preparation of agricultural formulations for improving the heat resistance and drought resistance of plants.

[0015] This invention provides a method for preparing the aforementioned microbial inoculant, wherein the *Bacillus tropicalis* KH15 is mixed with a dry powder matrix to form a microbial inoculant, namely a heat-resistant and drought-resistant inoculant, such that the effective viable count of *Bacillus tropicalis* KH15 in the heat-resistant and drought-resistant inoculant is greater than or equal to 1 billion CFU / g. In use, the heat-resistant and drought-resistant inoculant is inoculated onto the surface of plant (e.g., corn, oats, wheat, etc.) seeds; the amount of heat-resistant and drought-resistant inoculant inoculated onto the surface of plant (e.g., corn, wheat, etc.) seeds is 3-5 grams per kilogram of seeds.

[0016] In the above technical solution, the dry powder matrix comprises the following components by weight percentage: Attapulgite (300 mesh) 60%-70% Humic acid powder (300 mesh) 20%-29% Trehalose 0.1%-1%; Ferrous ammonium citrate 0.1%-0.5% Dinitrosalicylic acid 0.01%-0.1% o-Phenanthroline 0.01%-0.1%.

[0017] In the above technical solution, the effective viable count of Bacillus tropicalis KH15 in the heat-resistant and drought-resistant bacterial agent is 1 billion CFU / g to 1.5 billion CFU / g.

[0018] In the above technical solution, when using it, the heat-resistant and drought-resistant bacterial agent is inoculated onto the surface of plant (such as corn, oats, wheat, etc.) seeds.

[0019] In the above technical solution, the amount of heat-resistant and drought-resistant bacterial agent inoculated onto the surface of plant (e.g., corn, wheat, etc.) seeds is 3 to 5 grams per kilogram of seeds.

[0020] The combination of the dry powder drought-resistant strain (i.e., the tropical Bacillus KH15) and the carrier matrix (i.e., the dry powder matrix) of the present invention has the following characteristics: (1) The dry powder matrix can effectively protect the survival of tropical Bacillus KH15, making tropical Bacillus KH15 a stable and reliable microbial agent product; (2) The components contained in the dry powder matrix can synergistically enhance the effect of tropical Bacillus KH15 strain in stimulating plant heat resistance and drought resistance; (3) The dry powder carrier (i.e., the dry powder matrix) can be attached to the seed surface by electrostatic adsorption, making it convenient to use the microbial agent of the present invention for seed dressing; (4) The microbial agent of the present invention exists on the seed surface and can interact with it during seed germination, stimulating the plant's heat resistance and drought resistance, and improving the germination rate and normal growth ability of the plant in high temperature and drought environments.

[0021] Experiments showed that after 24 hours of treatment in a 50°C water bath, inoculation with the Bacillus tropicalis KH15 inoculant (i.e., microbial inoculant) described in this invention alone could improve the heat resistance of plants (such as corn, wheat, and oats): the survival rate of corn seedlings in the KH15 treatment group was 81.6%, while the survival rate of corn seedlings in the control group was 10.8%; the survival rate of wheat seedlings in the KH15 treatment group was 84.3%, while the survival rate of wheat seedlings in the control group was 45.7%; the survival rate of oat seedlings in the KH15 treatment group was 57.1%, while the survival rate of oat seedlings in the control group was 19.3%. Experiments showed that inoculation of plants (such as oats and wheat) with Bacillus tropicalis KH15 inoculum (i.e., the microbial inoculum of this invention) alone improved their heat resistance and drought resistance. Survival rates after 21 days under drought conditions of 22-30℃ and soil moisture of 3%-4% were as follows: oats treated with Bacillus tropicalis KH15 inoculum (i.e., the microbial inoculum of this invention) had a survival rate of 85.8%, while the control group had a survival rate of 69.5%; wheat treated with Bacillus tropicalis KH15 inoculum (i.e., the microbial inoculum of this invention) had a survival rate of 86.4%, while the control group had a survival rate of 70.1%.

[0022] This invention provides a method for preparing the aforementioned microbial inoculant, wherein the tropical Bacillus KH15 is mixed with organic fertilizer to form a microbial inoculant, namely a heat-resistant and drought-resistant bio-organic fertilizer, and the effective viable count of tropical Bacillus KH15 in the heat-resistant and drought-resistant bio-organic fertilizer is greater than or equal to 0.2 billion CFU / g.

[0023] The organic fertilizer mentioned refers to the product of high-temperature composting and fermentation of manure from cattle, sheep, chickens, pigs, etc., which is existing technology.

[0024] In the above technical solution, the effective viable count of Bacillus tropicalis KH15 in the heat-resistant and drought-resistant bio-organic fertilizer is 0.2 billion / gram to 0.5 billion CFU / gram.

[0025] The heat-resistant and drought-resistant bio-organic fertilizer of the present invention has the following characteristics: (1) The organic matter in the organic fertilizer can provide a favorable living environment for Bacillus tropicalis KH15, and the organic nutrients such as amino acids contained therein can also promote the growth and reproduction of Bacillus tropicalis KH15; (2) The heat-resistant and drought-resistant bio-organic fertilizer has low cost and large dosage. After being applied to the soil, it can create a rhizosphere environment containing a dominant community of drought-resistant strains, which helps to improve the plant's high temperature resistance and drought resistance; (3) The heat-resistant and drought-resistant bio-organic fertilizer can also improve the physical and chemical properties of the rhizosphere soil, improve the air permeability and water and fertilizer retention capacity, and further enhance the plant's high temperature resistance and drought resistance during the seedling growth stage.

[0026] The heat-resistant and drought-resistant bio-organic fertilizer was mixed into the soil at a rate of 0.3% of the soil weight, while the control soil used 0.3% conventional organic fertilizer. Each pot contained 1.5 kg of soil. After sowing, the plants (such as corn) were allowed to germinate and grow. After 35 days, they were treated with a 50°C water bath for 24 hours, and then transferred to a normal temperature (22-30°C) for continued cultivation. The results of the above pot experiment show that using the heat-resistant and drought-resistant bio-organic fertilizer described in this invention alone can improve the plant's high-temperature tolerance.

[0027] Heat-resistant and drought-resistant bio-organic fertilizer was mixed into the soil at a rate of 0.3% of the soil weight, while the control soil used 0.3% conventional organic fertilizer. Each pot was accurately filled with 1.5 kg of soil and the same amount of water, and the gross weight was recorded. Plants (e.g., corn) were sown and allowed to germinate and grow. For the first 30 days, soil moisture was maintained at the initial gross weight through watering to ensure uniform moisture levels. Watering was then stopped, and the soil was allowed to dry naturally. The number of surviving plants was recorded after 25 days. The results of this pot experiment indicate that using heat-resistant and drought-resistant bio-organic fertilizer alone can improve the drought tolerance of plants.

[0028] The present invention has the following advantages: 1. The tropical Bacillus KH15 of the present invention was isolated from soil samples taken from saline-alkali and sandy wastelands in Northwest China, and is particularly suitable for sandy wastelands in Northwest China and other regions. 2. The tropical Bacillus KH15 of the present invention can improve the drought resistance and heat tolerance of plants, enabling them to survive in hot and dry environments, especially plants that are not tolerant of hot and dry environments, and even more so plants that are not tolerant of hot and dry environments in hot and dry desert environments; the tropical Bacillus KH15 of the present invention can increase the heat tolerance temperature of plants by about 5-8°C and extend the drought tolerance time by about 10-15 days; 3. The tropical Bacillus KH15 of this invention has both ecological and economic benefits in desertification control: Traditional desertification control methods select drought-resistant wild plants such as reeds, which can grow in the desert, but have low economic benefits; this invention enables high-quality forage crops such as oats and alfalfa, which are not tolerant of high temperatures and drought, to grow in the desert, turning the desert into an oasis while obtaining high-quality forage, allowing the income from forage to offset the cost of desertification control, making desertification control a sustainable industry; at the same time, this invention can also improve the high temperature and drought resistance of grain crops such as corn and wheat, thereby increasing the yield of corn and wheat in arid areas and providing technical support for ensuring food security. Attached Figure Description

[0029] Figure 1 This is a growth diagram of corn plants inoculated with the strain of the present invention after heat shock treatment.

[0030] Figure 2 This is a diagram of the wheat root system in the control group and experimental group in Example 5 of the present invention.

[0031] Figure 3 This is a survival chart of oats in the control group in Example 6 of the present invention.

[0032] Figure 4 This is a survival chart of oats in the experimental group in Example 6 of the present invention. Detailed Implementation

[0033] The present invention will be further described in conjunction with the following embodiments.

[0034] 1. Isolation of strains: This invention uses the thermal shock bioassay method to screen heat-resistant and drought-resistant strains.

[0035] Soil samples were collected from deserts in Ningxia and Xinjiang, regions characterized by prolonged high temperatures and drought. Bacteria were isolated and purified from these soil samples and stored at 4°C. Corn seeds were disinfected with a freshly prepared 6% sodium hypochlorite solution for 10 minutes, rinsed five times with sterile water, and germinated on water agar plates (28°C). When the taproot of the seedlings reached approximately 5-10 mm in length, they were transplanted into seedling trays. The seedling trays were disinfected with potassium permanganate solution, and the seedling substrate was sterilized with high-temperature steam for 50 minutes. After cooling, the substrate was placed into the seedling trays. Germinated corn seeds were added to the substrate in the seedling trays, with 1 ml of the test bacterial strain added to each cell, resulting in a bacterial concentration of approximately 100 million CFU / ml. The plants were cultured in a light incubator for 21 days, with 16 hours of light at +28°C and 8 hours of darkness at +20°C. After 21 days of culture, the seedling trays were placed in a 58°C water bath to completely immerse the roots—this was a heat shock. After 12 hours of heat shock, the seedling trays were removed and returned to the light incubator for further cultivation. Plants subjected to heat shock begin to die. Extensive screening is conducted to identify strains that enhance plant resistance to heat shock. After repeated heat shock experiments, a microbial strain that improves plant tolerance to heat shock is selected and designated KH15 (e.g., Figure 1 As shown, from Figure 1 It can be seen that after heat shock treatment, corn plants inoculated with ineffective strains withered, while corn plants inoculated with effective strains survived.

[0036] The applicant discovered a strong correlation between plant heat shock tolerance and drought resistance. Therefore, the applicant tested whether microbial strains that enhance heat shock tolerance could also improve drought resistance. The specific method involved cultivating plants in large seedling trays (32 cells, overall dimensions 58cm x 34cm x 5.5cm, single cell diameter 3.8cm) using normal field soil as the substrate. Sixteen cells were inoculated with the microbial strain, and 16 cells served as a control. Five replicates were performed to eliminate moisture errors through extensive replication. Watering was stopped, allowing the moisture in the culture medium to gradually decrease. Finally, seven microbial strains that improved drought resistance in maize were obtained. Through trials on oats and wheat, *Bacillus tropicalis* KH15 was ultimately selected as a strain that improved drought resistance in maize, oats, and wheat. Field plot trials demonstrated that *Bacillus tropicalis* KH15 significantly enhanced the heat and drought resistance of maize, wheat, and oats. Oats inoculated with the tropical Bacillus KH15 of this invention grew well in a desert control experiment. This shows that the tropical Bacillus KH15 of this invention can not only promote the growth of desert plants, but also promote the normal growth of desert economic crops, and has great ecological and economic value.

[0037] 2. Strain identification Targeted DNA sequencing for microbial gene identification: Result analysis: Gene amplification showed a single, clear target band, sequence attached. Seq:>16S-KH15.34685483 Sequencing results, analyzed by BLAST alignment, showed sequence similarity to other strains as follows: 1. Bacillus tropicus (NR157736.1) 99.928% Tropical Bacillus; 2. Bacillus nitratireducens (NR157732.1) 99.928%; Based on the homology analysis of the 16S rRNA sequence, KH15 has a similarity of 99.928% with Bacillus tropicalis. At the same time, this strain has the ability to stimulate heat resistance and drought resistance in plants, so it was identified as Bacillus tropicalis.

[0038] Example The present invention will now be described in detail by taking examples of its application to certain plants to improve their heat and drought resistance. This invention will also provide guidance for applying the invention to other plants to improve their heat and drought resistance.

[0039] Example 1: Heat Resistance Test of Maize Induced by Bacillus Tropicalis KH15 Experimental Methods: The corn variety used was "Dika 159". The test soil was sterilized at 121℃ for 2 hours and tested sterile on a PDA plate. The soil was placed in plastic square boxes with the following dimensions: top opening side length 12cm, box depth 15cm, and bottom side length 10cm. After surface sterilization, corn seeds were placed in the plastic square boxes with 10 seeds per box, and the experiment was repeated twice. Treatment 1 was the control group, which received no bacterial solution but 5 ml of sterile water. Treatment 2 was the group treated with KH15 bacterial solution of this invention alone, with 5 ml (1 x 10) of KH15 bacterial solution (i.e., the heat-resistant and drought-resistant bacterial agent described in this invention) added to each box. 8 CFU / ml), 0.5 ml per seed; Treatment 3 is the treatment of KH15 bacterial solution of the present invention + bio-organic fertilizer (i.e., simultaneously using the heat-resistant and drought-resistant bacterial agent and the heat-resistant and drought-resistant bio-organic fertilizer described in the present invention), with 5 ml (1x10) of KH15 bacterial solution of the present invention (i.e., the heat-resistant and drought-resistant bacterial agent of the present invention) added to each box. 8 Each seed was given 0.5 ml of water (CFU / ml), and 0.3% heat-resistant and drought-resistant bio-organic fertilizer was added to the soil. The soil moisture was adjusted to 60%. On the 10th day, 15 ml of water was added to each box, on the 15th day, 25 ml of water was added, and on the 20th day, 50 ml of water was added to maintain normal plant growth. After culturing in a plant light incubator for 25 days, the plants were taken out and subjected to heat treatment in water baths at different temperatures for 24 hours. Then, they were put back into the plant light incubator for continued cultivation, and the survival rate of the plants was observed. The survival rate of corn after the above heat treatment is shown in Table 1.

[0040] Table 1. Survival rate of maize after heat treatment

[0041] As shown in Table 1 above: When the heat treatment temperature is 35℃, the survival rate of corn in treatments 2 and 3 is higher than that in treatment 1; when the heat treatment temperature increases from 35℃ to 40℃, the survival rate of corn in treatment 1 decreases significantly, while the survival rates of corn in treatments 2 and 3 do not decrease significantly and remain above 92%, with treatment 3 having a better survival rate than treatment 2; when the heat treatment temperature increases from 40℃ to 45℃, the survival rate of corn in treatment 1 decreases to less than half, while the survival rates of corn in treatments 2 and 3 decrease somewhat, but not significantly, remaining above 87%, more than double that of treatment 1, with treatment 3 having a better survival rate than treatment 2; when the heat treatment temperature increases from 45℃ to 50℃, corn in treatment 1 is basically unable to survive, while the survival rates of corn in treatments 2 and 3 decrease somewhat, but not significantly, remaining above 81%, with treatment 3 having a better survival rate than treatment 2. Therefore, the heat resistance of corn treated with treatment 2 (using the heat-resistant and drought-resistant bacterial agent of this invention) and treatment 3 (using a combination of the heat-resistant and drought-resistant bacterial agent and the heat-resistant and drought-resistant bio-organic fertilizer) was much higher than that of treatment 1. This demonstrates that this invention can significantly improve the heat resistance of plants. The heat resistance of corn treated with treatment 3 (using a combination of the heat-resistant and drought-resistant bacterial agent and the heat-resistant and drought-resistant bio-organic fertilizer) was better than that of treatment 2 (using the heat-resistant and drought-resistant bacterial agent of this invention). This shows that combining the heat-resistant and drought-resistant bacterial agent with the heat-resistant and drought-resistant bio-organic fertilizer is more beneficial for improving the heat resistance of plants. Therefore, this invention can significantly improve the heat resistance of plants.

[0042] Example 2: Heat Resistance Test of Wheat Activated by Bacillus Tropicalis KH15 Experimental Methods: The wheat variety used was "Cangmai 6005". The test soil was sterilized at 121℃ for 2 hours and tested sterile on a PDA plate. The soil was placed in plastic square boxes with the following dimensions: top opening side length 12cm, box depth 15cm, bottom side length 10cm. After surface sterilization, wheat seeds were placed in the plastic square boxes with soil, 30 seeds per box, and the experiment was repeated twice. Treatment 1 was the control group, without bacterial solution, with 5 ml of sterile water added; Treatment 2 was the group inoculated with KH15 bacterial solution alone, with 5 ml (1 x 10) of KH15 bacterial solution (i.e., the heat-resistant and drought-resistant bacterial agent described in this invention) added to each box. 8 CFU / ml), 0.5 ml per seed; Treatment 3 is KH15 bacterial solution + bio-organic fertilizer treatment (i.e., simultaneously using the heat-resistant and drought-resistant bacterial agent and the heat-resistant and drought-resistant bio-organic fertilizer described in this invention), with 5 ml (1x10) of KH15 bacterial solution (i.e., the heat-resistant and drought-resistant bacterial agent described in this invention) added to each box. 8Each seed was given 0.5 ml of water (CFU / ml), and 0.3% heat-resistant and drought-resistant bio-organic fertilizer was added to the soil. The soil moisture was adjusted to 60%. On the 10th day, 15 ml of water was added to each box, on the 15th day, 25 ml of water was added, and on the 20th day, 50 ml of water was added to maintain normal plant growth. After culturing in a plant light incubator for 25 days, the plants were taken out and subjected to heat treatment in water baths at different temperatures for 24 hours. Then, they were put back into the plant light incubator for continued cultivation, and the survival rate of the plants was observed. The survival rate of wheat after the above heat treatment is shown in Table 2.

[0043] Table 2 Wheat survival rate after heat treatment

[0044] As shown in Table 2 above: When the heat treatment temperature is 35℃, the wheat survival rate of treatments 2 and 3 is higher than that of treatment 1; when the heat treatment temperature increases from 35℃ to 40℃, the wheat survival rate of treatment 1 decreases significantly, while the wheat survival rates of treatments 2 and 3 do not decrease significantly and remain above 93%, with treatment 3 having a better survival rate than treatment 2; when the heat treatment temperature increases from 40℃ to 45℃, the wheat survival rate of treatment 1 decreases to nearly half, while the wheat survival rates of treatments 2 and 3 decrease somewhat but not significantly, remaining around 90%, with treatment 3 having a better survival rate than treatment 2; when the heat treatment temperature increases from 45℃ to 50℃, the wheat survival rate of treatment 1 decreases significantly to less than half, while the wheat survival rates of treatments 2 and 3 decrease somewhat but remain above 84%, approximately twice that of treatment 1, with treatment 3 having a better survival rate than treatment 2. Therefore, the wheat treated with treatment 2 (using the heat-resistant and drought-resistant bacterial agent described in this invention) and treatment 3 (using the heat-resistant and drought-resistant bacterial agent in combination with heat-resistant and drought-resistant bio-organic fertilizer) exhibited significantly higher heat resistance than treatment 1. This demonstrates that this invention can significantly improve the heat resistance of plants. Furthermore, the wheat treated with treatment 3 (using the heat-resistant and drought-resistant bacterial agent in combination with heat-resistant and drought-resistant bio-organic fertilizer) showed better heat resistance than treatment 2 (using the heat-resistant and drought-resistant bacterial agent described in this invention). This indicates that combining the heat-resistant and drought-resistant bacterial agent with the heat-resistant and drought-resistant bio-organic fertilizer is more beneficial for improving the heat resistance of plants. Therefore, this invention can significantly improve the heat resistance of plants.

[0045] Example 3: Heat Resistance Test of Oats Induced by Bacillus Tropicalis KH15 Experimental Methods: The oat variety used was "Baiyan No. 2". The test soil was sterilized at 121℃ for 2 hours and tested sterile on a PDA plate. The soil was placed in plastic square boxes with the following dimensions: top opening side length 12cm, box depth 15cm, and bottom side length 10cm. After surface sterilization, the oat seeds were placed in the plastic square boxes with soil, 30 seeds per box, and the experiment was repeated twice. Treatment 1 was the control group, without bacterial solution, with 5 ml of sterile water added; Treatment 2 was the group inoculated with KH15 bacterial solution alone, with 5 ml (1 x 10) of KH15 bacterial solution (i.e., the heat-resistant and drought-resistant bacterial agent described in this invention) added to each box.8 CFU / ml), 0.5 ml per seed; Treatment 3 is KH15 bacterial solution + bio-organic fertilizer treatment (i.e., simultaneously using the heat-resistant and drought-resistant bacterial agent and the heat-resistant and drought-resistant bio-organic fertilizer described in this invention), with 5 ml (1x10) of KH15 bacterial solution (i.e., the heat-resistant and drought-resistant bacterial agent described in this invention) added to each box. 8 Each seed was given 0.5 ml of water (CFU / ml), and 0.3% heat-resistant and drought-resistant bio-organic fertilizer was added to the soil. The soil moisture was adjusted to 60%. On the 10th day, 15 ml of water was added to each box, on the 15th day, 25 ml of water was added, and on the 20th day, 50 ml of water was added to maintain normal plant growth. After culturing in a plant light incubator for 25 days, the plants were taken out and subjected to heat treatment in water baths at different temperatures for 24 hours. Then, they were put back into the plant light incubator for continued cultivation, and the survival rate of the plants was observed. The survival rate of oats after the above heat treatment is shown in Table 3.

[0046] Table 3 Oat survival rate after heat treatment

[0047] As shown in Table 3 above: when the heat treatment temperature is 35℃, the oat survival rate of treatments 2 and 3 is higher than that of treatment 1; when the heat treatment temperature increases from 35℃ to 40℃, the oat survival rate of treatment 1 decreases significantly, while the oat survival rates of treatments 2 and 3 do not decrease significantly and remain above 88%, with treatment 3 having a better survival rate than treatment 2; when the heat treatment temperature increases from 40℃ to 45℃, the oat survival rate of treatment 1 decreases to nearly half, while the oat survival rates of treatments 2 and 3 decrease somewhat but remain above 79%, with treatment 3 having a better survival rate than treatment 2; when the heat treatment temperature increases from 45℃ to 50℃, the oat survival rate of treatment 1 decreases significantly to 35.3%, while the oat survival rates of treatments 2 and 3 remain above 67%, more than double that of treatment 1, with treatment 3 having a better survival rate than treatment 2. Therefore, the heat resistance of oats treated with treatment 2 (using the heat-resistant and drought-resistant bacterial agent described in this invention) and treatment 3 (using the heat-resistant and drought-resistant bacterial agent in combination with heat-resistant and drought-resistant bio-organic fertilizer) is much higher than that of treatment 1. This demonstrates that this invention can significantly improve the heat resistance of plants. The heat resistance of oats treated with treatment 3 (using the heat-resistant and drought-resistant bacterial agent in combination with heat-resistant and drought-resistant bio-organic fertilizer) is superior to that of treatment 2 (using the heat-resistant and drought-resistant bacterial agent of this invention), and the combination of the heat-resistant and drought-resistant bacterial agent and the heat-resistant and drought-resistant bio-organic fertilizer in this invention is more conducive to improving the heat resistance of plants. Therefore, this invention can significantly improve the heat resistance of plants.

[0048] Example 4: Experiment on drought resistance in corn, wheat, and oats induced by Bacillus tropicalis KH15 After completing the heat resistance test, an experiment was conducted to test the drought resistance of plants stimulated by strain KH15. The tested plant varieties were: wheat variety Cangmai 6005, oat variety Baiyan 2, and maize variety Dika 159.

[0049] Take soil from the cornfield, crush and mix it, then put it into a pot with an upper diameter of 11cm, a lower diameter of 8cm, and a height of 14cm.

[0050] Control treatment: Seeds of the tested plants were not treated with KH15 inoculant. After sowing, the soil in each pot was moistened with the same amount of water and placed in a plant cultivation box to allow the seeds to germinate and grow normally. Water was supplemented during the growth period to maintain normal plant growth. After 35 days of cultivation, the soil moisture of all potted plants was adjusted to 55%, and then they were moved outdoors for open-air cultivation without supplementing water, allowing the soil to dry naturally. The survival rate of the control group plants was recorded when the mortality rate reached about 50%.

[0051] KH15 inoculant (i.e., the heat-resistant and drought-resistant inoculant described in this invention) treatment: Seeds of the tested plants were treated with KH15 inoculant (100 million CFU / mL). After sowing, the soil in each pot was moistened with the same amount of water and placed in a plant cultivation box to allow the seeds to germinate and grow normally. Water was supplemented during the growth period to maintain normal plant growth. After 35 days of cultivation, the soil moisture of all potted plants was adjusted to 55%, and then they were moved outdoors for open-air cultivation. No further watering was provided, allowing the soil to dry naturally. The plant survival rate was recorded.

[0052] KH15 bio-organic fertilizer (i.e., the heat-resistant and drought-resistant bio-organic fertilizer described in this invention) treatment: 0.3% of bio-organic fertilizer containing the KH15 strain (i.e., the heat-resistant and drought-resistant bacterial agent described in this invention) was added to the soil. The test plant seeds were not treated with the KH15 bacterial agent. After sowing, the soil in each pot was moistened with the same amount of water and placed in a plant cultivation box to allow the test plant seeds to germinate and grow normally. Water was supplemented during the growth period to maintain normal plant growth. After 35 days of cultivation, the soil moisture in all pots was adjusted to 55%, and then the plants were moved outdoors for open-air cultivation without further watering, allowing the soil to dry naturally. The plant survival rate was recorded.

[0053] Treatment with KH15 inoculant + bio-organic fertilizer (i.e., simultaneously using the heat-resistant and drought-resistant inoculant and bio-organic fertilizer described in this invention): Seeds of the tested plants were treated with KH15 inoculant (100 million / mL), and 0.3% bio-organic fertilizer containing KH15 strain was added to the soil. After sowing, each pot was moistened with the same amount of water and placed in a plant cultivation box to allow the seeds to germinate and grow normally. Water was supplemented during the growth period to maintain normal plant growth. After 35 days of cultivation, the soil moisture in all pots was adjusted to 55%, and then the plants were moved outdoors for open-air cultivation without further watering, allowing the soil to dry naturally. The plant survival rate was recorded. The experimental results after the above treatment are shown in Table 4.

[0054] Table 4. Results of drought resistance tests (survival rate) of Bacillus tropicalis KH15 stimulating maize, wheat, and oats.

[0055] As shown in Table 4 above, under the same drought conditions, when the wheat survival rate in the control group reached 51.3%, the wheat survival rate treated with this invention was all above 80%. Wheat treated with bio-organic fertilizer had a higher survival rate, wheat treated with KH15 microbial agent had a better survival rate than wheat treated with bio-organic fertilizer, and wheat treated with KH15 microbial agent + bio-organic fertilizer had the highest survival rate. Similarly, when the oat survival rate in the control group reached 50.6%, the oat survival rate treated with this invention was all above 80%. Oat treated with KH15 microbial agent had a higher survival rate, oat treated with bio-organic fertilizer had a better survival rate than oat treated with KH15 microbial agent, and oat treated with KH15 microbial agent + bio-organic fertilizer had the highest survival rate. When the corn survival rate in the control group was 48.4%, the corn survival rate treated with this invention was all above 75%. Corn treated with KH15 microbial agent had a higher survival rate, corn treated with bio-organic fertilizer had a better survival rate than corn treated with KH15 microbial agent, and corn treated with KH15 microbial agent + bio-organic fertilizer had the highest survival rate. It is evident that this invention can significantly improve the drought resistance of plants.

[0056] Example 5: Field trial of Bacillus tropicalis KH15 in winter wheat In a certain region, most winter wheat is grown in areas without irrigation, where drought stress is common. Experiments were conducted in a village in a certain county, a village A in a certain city, a village B in a certain city, and a farm in a certain city to investigate the microorganisms of this invention that enhance wheat drought resistance. In this embodiment, during the experiment, the wheat in the experimental areas experienced five days of high temperatures of 38-41°C, and more seriously, 33 days without rainfall. The high temperature and drought conditions in the fields where winter wheat was grown were quite severe; under normal circumstances, winter wheat would struggle to grow normally under such conditions.

[0057] Since field trial designs should not be too complex, this experiment used the combined application of the heat- and drought-resistant bacterial agent and the heat- and drought-resistant bio-organic fertilizer of this invention as the experimental group, with conventional cultivation as the control group. This embodiment used the same experimental and control groups in a village in a county, a village A in a city, a village B in a city, and a farm in a city. The specific experimental methods are as follows: The field trial implementation method for the control group was as follows: Step (1) When leveling the land, apply conventional organic fertilizer (excluding KH15) at a rate of 100 kg per mu, and apply compound fertilizer (NPK18-10-18) at a rate of 30 kg per mu as base fertilizer; Step (2) Sow wheat seeds into the soil without mixing the seeds with any inoculant; Step (3) Watering, covering with soil, covering with film and other cultivation management measures are the same as usual.

[0058] The experimental group's field experiment on microbial drought resistance was conducted as follows: Step (1) Leveling the experimental plot and applying organic fertilizer. Before sowing, 100 kg of organic fertilizer containing drought-resistant microorganism KH15 strain (i.e., the heat-resistant and drought-resistant bio-organic fertilizer described in this invention) was applied to the soil as base fertilizer, forming a relatively dominant drought-resistant microbial community in the soil. Step (2) The seeds were mixed with a heat-resistant and drought-resistant agent containing KH15 strain (i.e., the heat-resistant and drought-resistant agent described in this invention), at a dosage of 5 g / kg of seeds. The mixed wheat seeds were then sown in the soil, followed by watering, covering with soil, and covering with film. Through the implementation of steps (1) and (2), a high concentration of KH15 microbial community was formed in a small area on the seed surface, and a lower concentration of KH15 microbial community was formed in a larger area around the seeds. This method of application can give full play to the role of KH15 in inducing drought resistance in plants. 30 kg of compound fertilizer (NPK18-10-18) was applied as base fertilizer per mu, and other cultivation and management measures were the same as conventional methods.

[0059] Experimental results: 1. Root growth For plants growing in arid environments, a well-developed root system is beneficial for survival. In the winter wheat experimental field, the wheat roots treated with the aforementioned heat-resistant and drought-resistant bacterial agent in the experimental group were significantly larger than those in the control group. Figure 2 As shown, Figure 2 The plant on the left in the image represents the root of winter wheat in the control treatment (fresh weight 1.47 g / plant), while the plant on the right represents the root system of winter wheat in the experimental group (fresh weight 3.51 g / plant). It is evident that the experimental group significantly promoted root growth.

[0060] 2. Number of tillers Tillering is an important phenomenon in wheat growth, and the number of tillers reflects, to some extent, the robustness of individual wheat plants. Generally speaking, wheat with more tillers usually has a more developed root system and more vigorous above-ground growth, indicating that it can make full use of soil nutrients, water, and light resources in the early stages of growth, and is growing well.

[0061] Field surveys showed that the number of wheat tillers in the experimental groups was higher than that in the control groups in all different experimental areas. Among them, the wheat tiller number in the treatment group of a certain village in a certain county was the most outstanding among all areas. Specifically, in Village A of a certain city, the number of wheat tillers in the experimental group increased by 17% compared to the control group in 2024; in 2025, this figure increased to 23% in the experimental group of Village A of the same city. Furthermore, the number of wheat tillers in Village B of a certain city increased significantly, by 57% compared to the control group; and the number of wheat tillers in the experimental group of a certain farm in the same city increased by 10% compared to the control group. It is evident that the experimental groups can significantly increase the number of tillers per plant.

[0062] 3. Plant height Plant height is a direct indicator of wheat growth and development. Except for Village A in a certain city in 2024, the plant height of wheat in the experimental groups in other regions was higher than that of the control group, with the experimental group from a certain farm in a certain city showing the most significant increase. Specifically, data shows that in Village A of a certain city in 2025, the plant height of wheat in the experimental group increased by 8% compared to the control group, while the increase in plant height in the experimental group from the farm in the same city was even more significant, increasing by 17% compared to the control group. This demonstrates that the experimental groups can significantly increase plant height.

[0063] 4. Number of ears The number of ears is an important indicator of optimized wheat population structure, signifying even plant distribution and coordinated growth between individuals and the overall population. In all experimental areas, the number of wheat ears in the experimental groups was significantly higher than that in the control groups, with Village B in a certain city showing the most outstanding performance, reaching the highest value. Specifically, data showed that in Village A of a certain city, the number of wheat ears in the treatment group increased by 20% in 2024 compared to the control group, and by 86% in 2025; the number of ears in Village B of a certain city increased by 73% compared to the control group; and the number of ears in the experimental group of a certain farm in a certain city increased by 13% compared to the control group. This demonstrates that the experimental groups significantly increased the number of ears per plant.

[0064] 5. Thousand-grain weight Thousand-grain weight is a key indicator for evaluating wheat seed quality. A higher thousand-grain weight indicates that the wheat seeds are fuller and firmer, and that they have accumulated more nutrients. In all experimental areas, the thousand-grain weight of the experimental treatment groups was better than that of the control group. Among them, the experimental group in Village A of a certain city had the highest thousand-grain weight in 2024. Specific increases were as follows: the experimental group in Village A of the city increased by 0.8% compared to the control group in 2024; by 2025, the increase in the experimental group in Village A of the city had expanded to 8%; the thousand-grain weight improvement effect in Village B of the city was significant, increasing by 24% compared to the control group; and the thousand-grain weight of the experimental group in a certain farm of the city also achieved a 7% increase compared to the control group. It is evident that the experimental groups can significantly increase the thousand-grain weight of the plant seeds.

[0065] 6. Yield Measurement The dry weight per square meter of wheat is directly related to yield; multiplying the dry weight per square meter by 667 gives the yield per mu (unit of land area). In all regions, the dry weight per square meter of wheat in the treatment groups was higher than that in the control group. The experimental group in Village A of a certain city showed the most outstanding performance in 2024, with the highest dry weight value. Specific increases are as follows: in Village A of the city in 2024, the experimental group increased by 12% compared to the control group, and the increase further expanded to 17% in 2025; the experimental group in a certain farm of the city showed a significant increase, exceeding the control group by 34%. It is evident that the experimental groups significantly increased yield.

[0066] The above experimental data show that the drought-resistant microorganism (i.e., tropical Bacillus KH15) described in this invention has a significant drought resistance and yield-increasing capacity in winter wheat production in arid areas, and can play an important role in drought resistance and yield increase, and has great ecological and economic value.

[0067] Example 6: Tropical Bacillus KH15 used for desert control Desertification control projects aim not only to transform deserts into oases but also to ensure that the vegetation growing there has economic value. Oats are excellent forage crops that can be used for desertification control, but enabling oats to grow in the hot and arid desert environment is a significant challenge. A major stressor in desertification control is that high desert temperatures restrict plant growth and survival; therefore, finding microorganisms that can stimulate plant heat and drought tolerance is a key breakthrough in desertification control.

[0068] In this embodiment, the ecological environment of a certain desert is extremely harsh, specifically manifested in: the highest temperature reaching 67.2℃, with a diurnal temperature range exceeding 40℃; the average annual precipitation not exceeding 100 mm, with a minimum of only 4-5 mm; while the average evaporation is as high as 2500-3400 mm. It is evident that the high summer temperatures in the desert are the greatest stressor in this region, necessitating a significant improvement in the heat tolerance of plants for their survival and growth. This invention stimulates the heat and drought resistance of plants through microbial stimulation, enabling them to grow in the arid environment of this desert.

[0069] The control group consisted of uninoculated conventional planting: On May 15, 2025, oat variety "Baiyan No. 2" was planted in the desert of a desert control experimental area of ​​a forestry and grassland bureau in an autonomous prefecture. Two tons of conventional organic fertilizer (excluding KH15) were applied per mu to provide organic matter to the desert. There is groundwater under the desert in the experimental area, which was desalinated and used as irrigation water, and the plants were watered through drip irrigation.

[0070] The experimental group adopted the drought-resistant microbial application method of this invention: On May 15, 2025, oat "Baiyan No. 2" was planted in the desert of a desert control experimental area of ​​a forestry and grassland bureau in an autonomous prefecture. Tropical Bacillus KH15 drought-resistant microbial agent (i.e., the heat-resistant and drought-resistant microbial agent described in this invention) was mixed onto the oat seeds. After row sowing, a KH15-containing aqueous solution (i.e., the microbial agent described in this invention) was drip-irrigated around the roots, forming a dominant drought-resistant microbial community in the seeds and roots. Simultaneously, the microbial agent of this invention was mixed with organic fertilizer, straw, and other organic materials to create microbial organic fertilizer. Two tons of microbial organic fertilizer were applied per acre to provide organic matter to the desert, forming a "microbial-fertilizer complex" which was applied to the sand layer. This provided nutrition for the microorganisms and improved water retention capacity, preventing the microbial agent from being directly exposed to high temperatures and drought and becoming inactive. Other cultivation and management measures were the same as the control group.

[0071] The test results after the above treatment are shown in Table 5 and Figure 3 , Figure 4 As shown. From Figure 3 It can be seen that the oats in the control area of ​​this embodiment could not survive. From Figure 4 It can be seen that the survival rate of oats in the experimental group was higher.

[0072] Table 5. Results of an oat planting experiment at a forestry and grassland experimental base in a certain autonomous prefecture in 2025.

[0073] Table 5 shows that, under the same conditions, the oat germination rate in the desert of a certain autonomous prefecture's forestry and grassland bureau's desert control experimental area was much higher in the control group, more than four times that of the control group. The survival rate of oats in the control group was 0%, while the survival rate in the experimental group was 86.1%. This demonstrates that the *Bacillus tropicalis* KH15 of this invention can significantly improve the heat tolerance and drought resistance of plants, enabling heat-sensitive economic crops to survive and grow normally in the extremely harsh desert ecological environment where the maximum temperature reaches 67.2℃, the diurnal temperature range exceeds 40℃, the average annual precipitation is less than 100 mm, and the average evaporation is as high as 2500-3400 mm. Therefore, this invention can play an important role in desert control and has significant ecological and economic value.

[0074] In summary, the Bacillus tropicalis KH15 strain and its inoculant products of this invention can improve the drought resistance of plants, extend their survival time under arid conditions, and provide them with opportunities to grow in subsequent rainfall. This strain can also significantly improve the heat resistance of plants, a characteristic that is highly suitable for desertification control.

[0075] All other unspecified parts belong to the prior art.

Claims

1. A tropical Bacillus KH15, characterized in that: Its depository is the China General Microbiological Culture Collection Center, with accession number CGMCC 35147.

2. A microbial inoculant, characterized in that: The active ingredient includes Bacillus tropicalis KH15 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that: The microbial agent also includes a carrier; the carrier includes a solid carrier and a liquid carrier.

4. The method of using the microbial agent according to claim 3, characterized in that: Applying microbial agents, including solid carriers, to plants, and / or applying microbial agents, including liquid carriers, to plants.

5. The method of using the microbial agent according to claim 4, characterized in that: The plants mentioned include corn, oats, and wheat.

6. The application of the tropical Bacillus KH15 of claim 1 or the microbial agent of claim 2 in improving the heat resistance and drought resistance of plants.

7. The use of the tropical Bacillus KH15 of claim 1 or the microbial agent of claim 2 in the preparation of agricultural formulations for improving the heat resistance and drought resistance of plants.

8. A method for preparing the microbial inoculant according to claim 2, characterized in that: The tropical Bacillus KH15 is mixed with a dry powder matrix to form a microbial agent, namely a heat-resistant and drought-resistant agent, such that the effective viable count of tropical Bacillus KH15 in the heat-resistant and drought-resistant agent is greater than or equal to 1 billion CFU / g.

9. The method for preparing microbial inoculants according to claim 8, characterized in that: The dry powder matrix comprises the following components by weight percentage: 300-mesh attapulgite clay 60%-70% 300-mesh humic acid powder 20%-29% Trehalose 0.1%-1%; Ferrous ammonium citrate 0.1%-0.5% Dinitrosalicylic acid 0.01%-0.1% o-Phenanthroline 0.01%-0.1%.

10. A method for preparing the microbial inoculant according to claim 2, characterized in that: The tropical Bacillus KH15 is mixed with organic fertilizer to form a microbial inoculant, namely a heat-resistant and drought-resistant bio-organic fertilizer, so that the effective viable number of tropical Bacillus KH15 in the heat-resistant and drought-resistant bio-organic fertilizer is greater than or equal to 0.2 billion CFU / gram.

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