Bacillus altitudinis and application thereof

Abstract

The invention relates to the technical field of agricultural biology, in particular to bacillus altitudinis and application thereof, and is particularly suitable for improving the growth performance of tropical corn in a low absorbable iron stress environment. The bacillus altitudinis has been preserved in Institute of Microbiology, Academy of Sciences, Guangdong Province on June 3, 2025, and the address of the bacillus altitudinis is the fifth floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou City; the preservation number of the strain is GDMCC NO: 66398. The secretion of the bacillus altitudinis can promote the absorption of insoluble iron ions and other important nutrient elements by the tropical corn, so that the biological generation of chlorophyll is promoted, and the growth of the tropical corn is promoted.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and in particular to an aerial bacillus and its application, which is particularly suitable for improving the growth performance of tropical corn under a low-absorbable iron stress environment. Background Art

[0002] The plant growth-promoting effects of growth-promoting rhizobacteria (PGPR) have been extensively studied, but their specific effects on crops in different climatic zones remain unclear. Tropical maize is significantly more dependent on soil microorganisms than temperate maize, and traditional methods have been unable to effectively address the growth limitations of tropical maize due to insufficient bioavailability. Therefore, the development of specific growth-promoting rhizobacteria for tropical maize is of great production significance. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides an alpine Bacillus and its application. The strain can promote the adaptation and improvement of tropical corn to low-absorbable iron ion soil, enhance the expression of corn chlorophyll synthesis-related genes, thereby increasing the chlorophyll content in leaves and promoting plant growth, effectively overcoming the related defects in the existing technology.

[0004] A rhizosphere growth-promoting bacterial agent for promoting the growth of tropical corn and its application, comprising the following steps:

[0005] 1. Propagating the high-altitude Bacillus;

[0006] 2. Make soil fertilizer through nutrient matrix;

[0007] 3. Colonization of the high-altitude Bacillus;

[0008] 4. Maintain an environment for the growth of the strain;

[0009] More specific: High-altitude Bacillus saltitudinis propagation process

[0010] Activation of bacterial strains: Take the -80℃ frozen high-altitude Bacillus subtilis GDMCC NO: 66398 strain, inoculate it into LB solid medium containing 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, and 20 g / L agar, and culture it upside down at 37℃ for 24 hours. Pick a single colony and transfer it to 50 mL seed medium containing 3 g / L beef extract, 10 g / L peptone, and 5 g / L NaCl. Culture it at 37℃ and 200 rpm with shaking for 12 hours to obtain an activated seed solution with OD600 ≥ 1.0.

[0011] Secondary propagation: Transfer the activated liquid into a 500L fermenter at a 2% inoculum size. The culture medium consists of 15 g / L corn flour, 10 g / L soybean meal, 5 g / L glucose, and 2 g / L K2HPO4. Control parameters: temperature 37°C, initial pH 7.2, aeration 8 L / min, increased to 15 L / min after 7 hours, stirring speed 200 rpm. Ferment for 48 hours until the spore formation rate is ≥90% and observed under a microscopy.

[0012] Bacterial concentration control: centrifuge the fermentation broth at 4000 rpm for 15 min to collect the bacteria, adjust the viable bacterial count to 1×10^10 CFU / mL with sterile saline, add 3% mineral potassium fulvate + 0.2% potassium sorbate as a protective agent, and store at 4°C for use within 7 days.

[0013] 2. Preparation of tropical rhizosphere fertilizer

[0014] Matrix formula: 60% of decomposed sugarcane bagasse with particle size less than 2mm, 20% of coconut bran, 10% of cassava flour, 5% of bone meal, and 3% of brown sugar to provide carbon source. After mixing, sterilize with wet heat at 121℃ for 30 minutes.

[0015] Bacterial agent loading: Spray 1×10^10 CFU / mL of the expanded bacterial solution onto the sterilized substrate at a ratio of 1:10. Control the moisture content to 55%-60% so that the solution forms a ball when held by hand and falls apart when dropped to the ground. Add 0.1% complex enzyme preparation cellulase + protease to promote bacterial colonization. Ferment aerobically at 28°C for 72 hours, turning the pile twice a day.

[0016] Finished product standards: viable bacteria count ≥ 2×10^8 CFU / g, pH 6.0-7.0, organic matter content ≥ 45%, moisture ≤ 30%. After packaging, store at room temperature away from light and have a shelf life of 3 months.

[0017] 3. Root planting technology at seedling stage

[0018] Transplanting time: 7-10 days after sowing in tropical areas when corn is in the 2-leaf 1-heart stage. Choose cloudy days or evenings to avoid ultraviolet damage.

[0019] Point inoculation: dig a V-shaped hole 8-10 cm deep 5 cm away from the root system, apply 20 g of bacterial fertilizer containing 4×10^9 CFU of live bacteria to each hole, cover with soil, and then pour 50 mL of clean water containing 0.1% alginate to guide the migration of bacteria by utilizing the hydrotaxis of the root system.

[0020] Efficiency enhancement measures: Simultaneously insert a 1cm diameter bamboo charcoal stick for pretreatment: soak in 1% rice vinegar for 24 hours. The microporous structure of bamboo charcoal can adsorb the bacterial agent and slowly release it, extending the colonization period; for acidic soil pH <5.5, premix 2% wood ash into the bacterial fertilizer to adjust the microenvironment.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The high-altitude Bacillus (Bacillus saltitudinis) in the present invention has been deposited in the Institute of Microbiology, Guangdong Academy of Sciences on June 3, 2025, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the deposit number is GDMCC NO: 66398.

[0023] Application of rhizosphere exudates of an aerial Bacillus sp. in promoting the growth of tropical maize.

[0024] The invention comprises at least the high-altitude Bacillus with the deposit number of GDMCC NO: 66398 as claimed in claim 1; the viable bacterial concentration of the high-altitude Bacillus is ≥ 1×10 8 CFU / g, the absorbance of bacterial solution was 1.0±0.1.

[0025] The invention also comprises an iron carrier, which is a secretion of the strain and has a concentration of 0.5 to 2 mmol / L.

[0026] The high-altitude Bacillus promotes the tropical corn to absorb iron ions, thereby enhancing the expression of chlorophyll synthesis-related genes of the tropical corn and promoting the production of chlorophyll in the plant.

[0027] The present invention effectively promotes the absorption and utilization of nutrients in the soil, especially low-absorbability iron, by tropical corn through the application of high-altitude Bacillus. The high-altitude Bacillus can secrete iron carriers, enhance the absorption capacity of the corn root system for iron elements, improve the root system's iron uptake efficiency, and thus promote the growth and development of the corn root system. The application method of the microbial agent is simple and easy to operate. It is only necessary to evenly apply the microbial agent containing the high-altitude Bacillus around the corn root system. Through practical application verification, the present invention is significantly effective in increasing tropical corn yield and improving corn quality, and has broad application prospects and promotion value.

[0028] At the same time, the application of this high-altitude Bacillus can also reduce the use of chemical fertilizers and reduce agricultural production's environmental pollution, which is in line with the concept of green and sustainable development of modern agriculture. As a biofertilizer, it can not only promote crop growth, but also improve soil structure and fertility, contributing to the sustainable development of agriculture.

[0029] In summary, the high-altitude Bacillus of the present invention and its application in promoting the root growth of tropical corn not only have significant economic and social benefits, but also can promote the development of agriculture in a greener and more sustainable direction. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Effects of rhizospheric bacteria on corn growth in different climatic zones (10d).

[0031] Figure 2 Effects of rhizospheric bacteria on corn growth in different climatic zones (60d).

[0032] Figure 3 To investigate the effects of rhizospheric bacteria on plant height, root length, and chlorophyll content of maize in different climatic zones (10 days and 60 days), chlorophyll content was measured in the middle of the first and second leaves (10 days) and the sixth leaf (60 days).

[0033] Figure 4 The effect of rhizospheric bacteria on the expression of chlorophyll synthesis-related genes in maize in different climatic zones.

[0034] Figure 5 Effects of rhizospheric bacteria on the growth of other temperate and tropical maize (10d).

[0035] Figure 6 Comparison of plant height, root length, and chlorophyll content between temperate maize varieties B73 and MO17 and tropical maize varieties YML32 grown in normal soil, sterilized soil, and inoculated soil on day 10. Asterisks indicate significant differences (*P < 0.1, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns indicates not significant; two-tailed unpaired t-test).

[0036] Figure 7 The figure shows three sets of X-ray fluorescence spectrometry analysis from left to right, representing normal soil (Normal), sterilized soil (Sterilized), and added soil (Add).

[0037] Figure 8 、 Figure 9 is the chlorophyll content of tropical corn YML in different types of nutrient solutions.

[0038] Figure 10 This is an analysis chart of the effects of high-altitude Bacillus on the iron elements in the leaves of tropical corn YML.

[0039] Figure 11 Under normal soil conditions, different treatments (Normal: no bacteria added to the soil;

[0040] AddBacteriaSoil(2): Add Bacillussaltitudinis to the soil; AddBacteriaSoil(4): Add Streptomycesfilamentosus to the soil;

[0041] AddBacteriaSoil(2+4): Comparison of the effects of adding Bacillus subtilis and Streptomyces filamentosus to the soil at the same time on the plant height, root length and chlorophyll content of tropical corn YML.

[0042] Figure 12 This is the effect of high-altitude Bacillus on the growth of tropical corn YML. DETAILED DESCRIPTION

[0043] The following embodiments of the present invention are described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0044] The present invention provides a high-altitude Bacillus and its application.

[0045] The high-altitude Bacillus (Bacillus saltitudinis) in the present invention has been deposited in the Institute of Microbiology, Guangdong Academy of Sciences on June 3, 2025, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the deposit number is GDMCC NO: 66398.

[0046] The 16S rRNA sequence is as follows:

[0047]

[0048] Application of high-altitude Bacillus in promoting root growth of tropical corn.

[0049] The invention comprises at least the high-altitude Bacillus with the deposit number of CGMCC No. 2 as claimed in claim 1; the viable bacterial concentration of the high-altitude Bacillus is ≥ 1×10 8 CFU / g, the absorbance value of bacterial solution is 1.0.

[0050] The invention also comprises an iron carrier, which is ferric hydroxide and has a concentration of 0.5 to 2 mmol / L.

[0051] High-altitude Bacillus can promote the production of chlorophyll in plants.

[0052] Example 1:

[0053] Set up a soil experiment with three different treatments:

[0054] ① Nutrient soil that has not been sterilized with high-pressure steam (normal soil);

[0055] ② Nutrient soil sterilized three times with high-pressure steam (sterilized soil);

[0056] ③ After three times of high-pressure steam sterilization, nutrient soil (Add) containing five bacteria unique to tropical corn (including Bacillus saltitudinis, Bacillus subtilis, Streptomyces filamentosus, Enterobacter ludwigii and Priestia aryabhattai) was added.

[0057] Figure 1 The phenotypic differences between temperate maize YE, JH and tropical maize CML, YML after 10 days of growth in three soil treatments are shown.

[0058] Figure 2 Figure 2 shows the growth performance of tropical maize (CML) and corn (YML) under different treatments over a 60-day growth period. *The plant on the left side of each pot is a CML plant, and the plant on the right side is a YML plant.

[0059] Figure 3 To quantitatively analyze plant height, root length, and chlorophyll content at 10 and 60 days, chlorophyll content was measured at the middle of the first and second leaves (10 days) and the sixth leaf (60 days).

[0060] The results of plant growth index observation showed that on the 10th day after corn germination and transplanting, the plant height, root length and chlorophyll content of tropical corn in normal soil and sterilized soil were significantly higher than those in the sterilized soil group, and the normal soil treatment had the best effect ( Figure 1 、 Figure 3 This trend persists over the 60-day observation period ( Figure 2 ). However, there was no significant difference in the growth of temperate maize YE and JH under different treatments, indicating that tropical maize is more dependent on rhizospheric bacteria.

[0061] Example 2:

[0062] To further verify the differences in the responses of corn in different climate zones to PGPR, this example selected temperate corn B73 and MO17 and tropical corn ThumbMaize and YML32 for comparative experiments.

[0063] The results are as follows Figure 5 、 Figure 6 As shown in the figure: the plant height, root length and chlorophyll content of tropical corn in normal soil and sterilized soil were significantly better than those in the sterilized soil treatment group (except for the plant height of ThumbMaize in sterilized soil), while there was no significant difference among the three treatments of temperate corn.

[0064] Figure 5 The growth phenotypes of B73, MO17, ThumbMaize, and YML32 at 10 days after seed germination and transplanting are shown;

[0065] Figure 6 The study compared the differences in plant height, root length, and chlorophyll content between temperate maize varieties B73 and MO17 and tropical maize varieties YML32 grown in normal soil, sterilized soil, and inoculated soil on day 10. The results further support the idea that tropical maize has a stronger growth-promoting effect under the action of PGPR.

[0066] also, Figure 4 This study demonstrates the effects of rhizosphere bacteria on the expression of genes related to chlorophyll synthesis. Samples were taken from the middle of the second leaf to measure the expression of four key chlorophyll synthesis genes: ZmGluTR, ZmGluTR-POR, ZmIX-Mg, and ZmCAO. The results showed that in temperate maize (YE), the expression levels of these genes did not differ significantly between the three soil treatments. However, in JH maize, the expression levels of some genes actually increased in sterilized soil. In contrast, in tropical maize (CML) and YML, the expression of these genes decreased significantly in sterilized soil.

[0067] Example 3:

[0068] This example conducts in-depth verification around the iron absorption mechanism. Rhizobacteria can significantly improve the bioavailability of iron in the soil by secreting siderophores and promoting iron reduction reactions. In contrast, high-temperature sterilization treatment easily leads to the loss of soil microorganisms and related nutrients, thereby reducing the absorbability of iron. Previous studies have shown that tropical corn bacteria promote the growth of tropical corn YML, but do not show significant effects on temperate corn.

[0069] Further X-ray fluorescence spectroscopy (XRF) analysis ( Figure 7 ). The figure shows three groups of atlases from left to right, representing normal soil (Normal), sterilized soil (Sterilized) and added bacteria soil (Add). Lines of different colors represent different elements. In each group of atlases, the iron (Fe) content peak is marked with a red frame, and the approximate iron content value is marked below the atlas. The results showed that: after 60 days, the iron content of leaves in different treatment groups was significantly different, specifically: normal soil> added bacteria soil> sterilized soil Figure 7 . This indicates that rhizospheric bacteria can improve the bioavailability of iron.

[0070] A nutrient solution experiment was further set up: tropical corn YML was cultured under four conditions: complete nutrient solution (MS), iron-deficient nutrient solution (MS-Fe), MS-Fe+Fe(OH)3, and MS-Fe+Fe(OH)3+tropical PGPR flora.

[0071] The results are as follows Figure 8 、 Figure 9 As shown, chlorophyll content decreased significantly in iron-deficient nutrient solutions; supplementation with Fe(OH)3 partially alleviated seedling chlorosis, with the effect being even more pronounced when combined with PGPR inoculation. In particular, the combination of Bacillus saltitudinis and Streptomyces filamentosus significantly restored chlorophyll content, outperforming either iron supplementation alone or the control group (containing the siderophore enterobactin).

[0072] In order to verify whether Bacillus altaica can promote the conversion of iron to meet the growth needs of tropical corn, the iron content of tropical corn YML cultured for 10 days in complete nutrient solution (MS), iron-deficient nutrient solution (MS-Fe), iron-deficient nutrient solution with added Fe(OH)3 and Bacillus altaica (MS-Fe+Fe(OH)3+Bacillus altaica) was analyzed. The results showed that the iron content of tropical corn YML treated with iron-deficient nutrient solution (MS-Fe) was lower than that of the complete nutrient solution (MS) treatment group. However, when Fe(OH)3 was supplemented in the iron-deficient nutrient solution and Bacillus altaica was added, the iron content of tropical corn YML was restored. Figure 10 This result indicates that Bacillus altaica can improve the bioavailability of iron in the maize rhizosphere soil. Figure 11 、 Figure 12 The results of the potted experiment are as follows: after inoculating AddBacteriaSoil(2) in normal soil, the plant height, root length and chlorophyll content were significantly better than those of the untreated group, further verifying its growth-promoting function.

[0073] The embodiments of the present invention are provided for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A high altitude Bacillus, characterized in that: The high altitude Bacillus, Bacillus altitudinis , was deposited on June 3, 2025 at the Institute of Microbiology, Guangdong Academy of Sciences, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the deposit number is GDMCC NO: 66398.

2. A high altitude Bacillus, characterized in that: The gene sequence of the Bacillus altaica is shown below as 16SrRNA.

3. Application of secretions of high-altitude Bacillus in promoting the growth of tropical corn.

4. The use of an alpine Bacillus sp. in promoting the growth of tropical corn according to claim 2, characterized in that: At least comprising the high-altitude Bacillus with the deposit number GDMCC NO: 66398 as claimed in claim 1; the viable bacterial concentration of the high-altitude Bacillus is ≥ 1×10 8 CFU / g, the absorbance of bacterial solution was 1.0±0.

1.

5. The use of an airborne Bacillus sp. in promoting root growth of tropical corn according to claim 3, characterized in that: The high-altitude Bacillus can promote the absorption of insoluble trivalent iron ions of tropical corn, thereby promoting the synthesis of chlorophyll in the plant.

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