Application of slow-growing rhizobium G94 in acid soil improvement

Abstract

This invention discloses the application of *Staphylococcus aureus* G94 in acidic soil improvement, relating to the fields of soil improvement and plant growth promotion. The application of *Staphylococcus aureus* G94 in acidic soil improvement, regulation of crop nutrient accumulation, and enhancement of crop stress resistance is demonstrated by this invention. Inoculation with *Staphylococcus aureus* G94 under acid stress significantly increased the pH of non-heat-prone soils of maize and soybean; significantly reduced the total exchangeable acidity of maize and soybean soils in heat-prone areas; and increased the exchangeable hydrogen ions in maize soils in both heat-prone and non-heat-prone areas. + The content of H in soybean hot soil was reduced. + The content of Al2O3 was significantly reduced in hot soils of maize and soybean; 3+ The content of TN, TC and TP in the roots of both crops was significantly reduced, and the fresh and dry weight of the aboveground parts was increased. The content of TN, TC and TP in the roots was significantly increased, and the content of TC and TP in the aboveground parts of soybean and the TN and TC in the roots were significantly increased.

Description

Technical Field

[0001] This invention belongs to the field of soil improvement and plant growth promotion technology, and more specifically, relates to the application of slow-growing rhizobium G94 in acid soil improvement. Background Technology

[0002] Wood ash has multiple benefits for soil, primarily including soil improvement, nutrient provision, and pest and disease control. First, wood ash is a natural alkaline substance rich in potassium, calcium, and trace elements, making it suitable as a soil conditioner and fertilizer in agriculture. Applying wood ash can neutralize acidic soil, increase its pH value, improve soil structure, increase permeability, and provide plants with the necessary nutrients, thereby improving crop yield and quality. Second, wood ash has the ability to control pests and diseases. It can be dispersed in the field, forming a fine powder that deters many pests such as aphids and ants. Furthermore, wood ash can reduce fungi and bacteria in the soil, helping to prevent the spread of soil-borne diseases. Wood ash also promotes root growth. Spreading wood ash in the field can promote crop root growth, supplement the mineral elements needed by crops, improve the crop's water and fertilizer absorption capacity, and enhance plant robustness and lodging resistance. Finally, wood ash also has bactericidal properties. It can be used to control aphids by blocking their spiracles with its alkaline properties and fine particles, causing physiological abnormalities and death. However, wood ash has some problems in soil improvement, including: reaction with acidic fertilizers leading to nitrogen volatilization; unsuitability for alkaline soils; potential for seedling and root burn; heavy metal contamination and nutrient loss; potential impact on soil microbial communities; and excessive use of wood ash may also affect groundwater quality, as some soluble minerals and salts may seep into the groundwater. In addition, nutrients in wood ash are easily lost in humid environments.

[0003] Bradyrhizobium is a group of Gram-negative bacteria that form root nodules in symbiosis with legumes and perform biological nitrogen fixation within these nodules. They live in the soil or within root nodules formed on the roots of legumes, providing nitrogen nutrition to the plant by converting atmospheric nitrogen into ammonia, which the plant can use, thus promoting plant growth. This symbiotic relationship is beneficial to both the plant and the rhizobium; the plant provides the rhizobium with the carbohydrates and energy it needs, while the rhizobium provides the plant with the nitrogen it requires.

[0004] Slow-growing rhizobia colonies grow slowly; after 5 to 7 days, the colony diameter is less than 1 mm, and it takes 6 to 8 days for them to show significant growth on culture media. They do not produce acid on carbohydrate-containing media, but rather remain alkaline. These characteristics make slow-growing rhizobia valuable in agricultural production, especially in legume cultivation. Inoculating with slow-growing rhizobia can improve nitrogen fixation efficiency and reduce the use of chemical nitrogen fertilizers, which is of great significance for improving soil fertility and promoting sustainable agricultural development.

[0005] In agricultural production, the application of slow-growing rhizobia mainly focuses on improving nitrogen fixation efficiency and yield in legumes, reducing the use of chemical nitrogen fertilizers, and improving the agricultural ecological environment. Through synthetic biology techniques, scientists are also striving to obtain novel rhizobium agents suitable for peanut cultivation to further improve crop productivity and environmental sustainability. However, the main problems with the application of slow-growing rhizobia include: slow growth rate, sensitivity to environmental conditions (especially temperature and pH), narrow range of carbon and nitrogen source utilization, and weak antibiotic resistance.

[0006] Therefore, it is urgent to develop diverse application strategies for slow-growing rhizobia to ensure their effective application in different environments, taking into account different crops and soil conditions. Summary of the Invention

[0007] To address the aforementioned problems in existing technologies, the technical problem this invention aims to solve is to provide the application of *Staphylococcus aureus* G94 in the improvement of acidic soils. Another technical problem this invention aims to solve is to provide the application of *Staphylococcus aureus* G94 in promoting crop growth in acidic soils. A further technical problem this invention aims to solve is to provide the application of *Staphylococcus aureus* G94 in regulating crop nutrient accumulation in acidic soils. Yet another technical problem this invention aims to solve is to provide the application of *Staphylococcus aureus* G94 in improving crop stress resistance, for soil improvement and plant growth promotion.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] Application of slow-growing rhizobium G94 in acid soil improvement. The slow-growing rhizobium G94 is deposited at Guangdong Provincial Microbial Culture Collection Center, deposit date: August 14, 2023, accession number GDMCC.No: 63598, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] The soil improvement aims to reduce the total amount of exchangeable acid in acidic soils.

[0011] The soil improvement aims to promote the growth of H+ in acidic soils. + The content decreased.

[0012] The soil improvement aims to promote the exchange of Al in acidic soils. 3+ The content decreased.

[0013] The role of slow-growing rhizobium G94 in promoting crop growth in acidic soil. The slow-growing rhizobium G94 is deposited at Guangdong Provincial Microbial Culture Collection Center, deposit date: August 14, 2023, accession number GDMCC.No: 63598, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0014] The promotion of crop growth refers to the increase in the fresh weight and dry weight of the root system.

[0015] The role of slow-growing rhizobium G94 in regulating crop nutrient accumulation in acidic soil. The slow-growing rhizobium G94 is deposited at the Guangdong Provincial Microbial Culture Collection Center, date of deposit: August 14, 2023, accession number GDMCC.No: 63598, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0016] The regulation of crop nutrient accumulation aims to increase the total nitrogen, total carbon, and total phosphorus content in crop roots.

[0017] The regulation of crop nutrient accumulation aims to increase the total nitrogen and total phosphorus content in the aboveground parts of soybeans.

[0018] Application of slow-growing rhizobium G94 in improving crop stress resistance. The slow-growing rhizobium G94 is deposited at Guangdong Provincial Microbial Culture Collection Center, deposit date: August 14, 2023, accession number GDMCC.No: 63598, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1) Inoculation with slow-growing rhizobium G94 (AS+B) under acid stress significantly increased the pH of non-thermal soils of maize and soybean, while having no significant effect on thermal soils; it significantly reduced the total exchangeable acid in thermal soils of maize and soybean; and it increased the exchangeable H+ in both thermal and non-thermal soils of maize. + The content of [unclear] was reduced, but H2 content in soybean hot zone soils was significantly decreased. + The content of Al2O3 was significantly reduced in hot soils of maize and soybean; 3+ The content of.

[0021] 2) The present invention showed that inoculation with slow-growing rhizobium G94 under acid stress conditions significantly increased the fresh weight of the aboveground parts of the plants; inoculation with slow-growing rhizobium G94 increased the fresh and dry weight of the roots of both crops under acid stress levels.

[0022] 3) Under acid stress treatment, inoculating Bradyrhizobium japonicum G94 significantly increased the TN accumulation, TC accumulation and TP content in the roots; it had no significant effect on the TN accumulation, TC accumulation and TP content in the aboveground parts; it significantly increased the TC accumulation, TP content in the aboveground parts of soybean and the TN accumulation, TC accumulation in the roots, and had no significant effect on the TN accumulation in the aboveground parts and the TC accumulation, TP content in the roots. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the scanning electron microscope image of plant ash;

[0024] Figure 2 It is the N2 adsorption - desorption isotherm diagram of plant ash;

[0025] Figure 3 It is the FT-IR diagram of plant ash;

[0026] Figure 4 It is the diagram of physical and chemical properties of paddy soil after adding plant ash;

[0027] Figure 5 It is the diagram of dynamic changes of physical and chemical properties of paddy soil after adding plant ash;

[0028] Figure 6 It is the correlation analysis diagram of physical and chemical properties of paddy soil after adding plant ash ("*" indicates 0.01 < p ≤ 0.05, "**" indicates p ≤ 0.01);

[0029] Figure 7 It is the two-dimensional analysis diagram of PC1 and PC2 of physical and chemical properties of paddy soil after adding plant ash;

[0030] Figure 8 It is the diagram of the effect of inoculating Bradyrhizobium japonicum G94 on the soil microecology of corn and soybean under acid stress (A is soil pH; B is total amount of soil exchangeable acid; C is soil exchangeable hydrogen ion; D is soil exchangeable aluminum ion);

[0031] Figure 9 It is the diagram of the effect of inoculating Bradyrhizobium japonicum G94 on the biomass of corn and soybean under acid stress;

[0032] Figure 10 It is the diagram of the effect of inoculating Bradyrhizobium japonicum G94 on the nutrient content of corn and soybean plants under acid stress. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified in detail, the technical means used are all conventional means well known to those skilled in the art.

[0034] The slow-growing rhizobium G94 used in the following examples is classified as Bradyrhizobium centrosematis and is deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 14, 2023, with accession number GDMCC.No: 63598, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0035] Example 1

[0036] 1. Physicochemical properties of wood ash

[0037] The wood ash used in this embodiment is the residue left after high-temperature combustion of eucalyptus bark in a biomass power plant. The results are shown in Table 1.

[0038] Table 1. Basic physicochemical properties of the wood ash obtained in this application.

[0039] Material Ash pH 12.2 Electrical conductivity (μS / cm) 2540 Cation exchange capacity (cmol+ / kg) 6.12 Total carbon (%) 6.74 <![CDATA[Available potassium (calculated as K2O) (%)]]> 1.47 Water-soluble calcium (%) 0.35 Water-soluble magnesium (mg / kg) 9.12 Total cadmium (mg / kg) 0.00 Effective cadmium (mg / kg) 0.00

[0040] 3. Surface morphology

[0041] The surface morphology of biochar and wood ash was recorded at different magnifications using a German Zeiss Merlin Compact scanning electron microscope, with an accelerating voltage of 5 kV.

[0042] The results are as follows Figure 1 As shown, wood ash has many small particles on its surface, but its overall structure is intact with few pores. At the same magnification, biochar is found to have smaller particle size and significantly increased pore size than wood ash, and its interior is hollow. This indicates that biochar has more adsorption sites than wood ash and can adsorb more functional groups, thereby increasing the cation exchange capacity of biochar.

[0043] 4. Specific surface area and pore structure

[0044] The specific surface area and pore size of biochar and wood ash were determined using a Micromeritics Tristar 3000 surface area and pore size analyzer. The samples were first degassed at 200℃, and then the adsorption-desorption of N2 was measured at 77K. The specific surface area and pore size of the samples were calculated using the BET isotherm model.

[0045] The results are shown in Table 2. The specific surface area of ​​the wood ash was 9.22 m². 2 / g, the specific surface area of ​​biochar is 50.88m². 2 / g, significantly higher than that of wood ash. The increased specific surface area indicates that biochar contains more adsorption sites, which is beneficial for the adsorption and solidification of heavy metals. While the total pore volume of wood ash and biochar is similar, the micropore volume of wood ash is only 0.001m³. 2 / g, mostly mesoporous; while the micropore volume of biochar is 0.015m³. 2 / g, mesopore volume is 0.022m 2 / g. The micropore volume in biochar was significantly increased, a result consistent with observations made during scanning electron microscopy.

[0046] The results are as follows Figure 2 As shown, the N2 adsorption-desorption isotherm of wood ash is concave and without an inflection point. The N2 adsorption capacity increases with increasing component partial pressure, classifying it as a Type III N2 adsorption-desorption isotherm. The concave shape of the curve is due to the intermolecular attraction between adsorbate molecules exceeding the attraction between adsorbate and adsorbent, indicating significant difficulties for the adsorbent in the initial adsorption phase. However, as the adsorption process progresses, the adsorption behavior begins to exhibit a self-accelerating trend, and the formation of adsorbate layers is no longer confined to the isothermal adsorption curve. The inflection point often appears near the stage of monolayer adsorption. With further increases in relative pressure, multilayer adsorption begins to form, and upon reaching the saturated vapor pressure of the liquid, the number of adsorbate layers tends to be infinite. This situation makes it difficult to accurately determine the limiting equilibrium adsorption capacity through experimental measurements.

[0047] Table 2 Pore structure parameters of wood ash

[0048]

[0049]

[0050] 5. Surface functional groups

[0051] Various functional groups on the surface of biochar and wood ash were analyzed and determined using a Nicolet iS10 Fourier transform infrared spectroscopy (FTIR) instrument. A suitable amount of sample was ground into powder in an agate mortar. This powder was then mixed thoroughly with ground potassium bromide at a ratio of KBr:S = 20:1. The mixture was compressed into tablets using a solid tablet press for 1 minute. Using potassium bromide as the background, the spectral range was scanned from 4000 to 400 cm⁻¹. -1 Between, with a resolution of 4cm -1 Each sample was scanned 32 times.

[0052] The results are as follows Figure 3 As shown, wood ash and biochar are present at 1690-1635 cm⁻¹ -1 1125-1000cm -1 775cm -1 Similar characteristic peaks were observed in the band, indicating that wood ash may contain C=C, CH-OH, and -COO. - Functional groups, etc.

[0053] Example 2

[0054] The soil used in this example was collected from paddy fields, and its specific physicochemical properties were as follows: pH (CaCl2) 4.38, potential total acidity 15.7 cmol / kg, electrical conductivity 290 μS / cm, available potassium content 35 mg / kg, water-soluble calcium content 65.8 mg / kg, and water-soluble magnesium content 6.7 mg / kg. The wood ash used in this experiment was obtained from a biomass power plant, with a pH of 12.2, electrical conductivity of 2540 μS / cm, available potassium (calculated as K2O) content of 1.47%, water-soluble calcium content of 0.35%, and water-soluble magnesium content of 9.12 mg / kg. Four treatments were set up: 1%, 5%, 10% of soil mass, and no wood ash added. Each treatment was replicated three times, for a total of 12 pots. The soil culture experiment began on January 4, 2022. Paddy field soil and wood ash were thoroughly mixed and placed in pots. All pots were randomly placed, and water was added to each treatment every 3 days using a weighing method to maintain the soil moisture content at approximately 60% of field capacity during the experiment. The experiment was conducted in the laboratory greenhouse of Guangxi University from January to April 2022.

[0055] Soil samples of 0-10 cm were collected using a soil auger on days 7, 16, 23, 50, 58, 69, 76, and 87 after the start of the experiment. The samples were air-dried, ground, and sieved through a 2 mm sieve for determining soil pH and EC. The soil samples that passed through the 2 mm sieve were spread out, and half of the soil was taken using the "quartering method" and ground again before being sieved through a 1 mm sieve for determining the content of available potassium, water-soluble calcium, water-soluble magnesium, and potential total acidity in the soil.

[0056] 1. Adding wood ash improves the physical and chemical properties of paddy field soil.

[0057] The results are as follows Figure 4 As shown, different application rates of wood ash had significant effects on soil physicochemical properties. When wood ash was applied to paddy soil at concentrations of 1%, 5%, and 10%, the soil pH value was significantly increased (p<0.05). Compared with the control, the pH value of paddy soil increased by 0.62, 1.84, and 2.04 units, respectively. Figure 4 A). Applying wood ash can significantly reduce the potential total acidity of the soil. Figure 4 B) Compared with the control, the application of 1%, 5%, and 10% wood ash in paddy fields reduced the potential total acidity of the soil by 35.9%, 79.0%, and 92.1%, respectively (p<0.05). In this study, the application of 1% wood ash to paddy field soil did not cause significant differences in soil electrical conductivity, available potassium, water-soluble calcium, and water-soluble magnesium; however, when the wood ash addition reached 5% and 10%, the soil electrical conductivity, available potassium, water-soluble magnesium (excluding the case with 5% wood ash addition), and water-soluble calcium content significantly increased (p<0.05). Figure 4 CF).

[0058] 2. Dynamic changes in the physical and chemical properties of paddy field soil after the addition of wood ash

[0059] The results are as follows Figure 5 As shown, in acidic paddy soil, the application of wood ash in the early stage (0-7 days) significantly increased soil pH, EC, available potassium, and water-soluble calcium and magnesium content, while reducing the soil's potential total acidity. With increasing wood ash application, soil pH significantly increased, reaching 6.8 when the application rate reached 10%. In the middle stage (7-69 days), soil pH tended to stabilize, while potential total acidity, EC, and water-soluble calcium content gradually decreased, and available potassium content initially increased and then decreased. In the later stage (after 69 days), soil pH and available potassium remained relatively stable, but potential total acidity and EC showed an increasing trend. Therefore, in paddy soil, the application of wood ash in the early stage (first 7 days) can significantly reduce soil acidity and increase the number of basic ions. However, due to the soil's buffering effect, this effect gradually diminished, and after 69 days, a low wood ash application rate (1%) had no significant effect on increasing soil basic ions.

[0060] 3. Correlation and principal component analysis of soil physicochemical properties after the addition of wood ash

[0061] Correlation analysis was performed on six soil physicochemical properties of paddy field soil, and the results are as follows: Figure 6 As shown, there were highly significant correlations between soil pH and the contents of potential total acidity, electrical conductivity, available potassium, water-soluble calcium, and water-soluble magnesium in paddy fields. Specifically, pH was negatively correlated with potential total acidity (p<0.01) and strongly positively correlated with electrical conductivity, available potassium, water-soluble calcium, and water-soluble magnesium (p<0.01). Potential total acidity in paddy fields was also highly significantly negatively correlated with soil electrical conductivity, available potassium, water-soluble calcium, and water-soluble magnesium (p<0.01).

[0062] The results are as follows Figure 7 As shown, the first principal component (PC1) has the highest contribution rate, explaining 88.2% of the total variance, while the second principal component (PC2) contributes 6.0%. Therefore, PC1 can represent most of the screening information. In PC1, indicators such as EC, available potassium, and water-soluble calcium have a relatively high proportion; therefore, we named this principal component the "basic ion factor." To more clearly represent the effect of treatment on the "basic ion factor," we performed a two-dimensional principal component analysis (PCA) with PC1 and PC2 as the x and y axes and plotted the results. Figure 3-7 In this graph, an increase in the horizontal axis represents a greater gain in the "basic ion factor" of the treatment. In this experiment, with the increase of wood ash application, the horizontal axis of the corresponding treatment in the two-dimensional PCA analysis graph also increased. This indicates that wood ash can increase the content of basic ions in the soil, thereby increasing the soil pH.

[0063] Example 3

[0064] The soil used in this example was strongly acidic agricultural soil with a pH of 4.3. The specific nutrient contents were: total carbon 8.93 g / kg; total nitrogen 0.95 g / kg; available phosphorus 12.16 g / kg; and available potassium 76.54 mg / kg. The soil was sieved through a 2 mm sieve and sterilized before the experiment.

[0065] This study used in-situ enzyme profiling to analyze three enzymes and defined soil zones in the experiment. Areas exceeding 25% of the grayscale value in the longitudinal vertical image (including the root system) of the entire planting box were defined as enzyme activity hotspots, while areas below this level were considered non-hotspots. The specific steps are as follows:

[0066] In situ enzyme spectrometry was used to directly study the distribution of enzyme activity at the root-soil interface of maize and soybean plants. The distribution of rhizosphere enzyme activity was observed using a saturated filter membrane (20cm × 20cm) pre-soaked with 4-methyl umbelliferone (MUF). The principle is that the substrate fluoresces upon hydrolysis by a specific enzyme, and the fluorescence intensity released after enzyme hydrolysis under ultraviolet light is proportional to the enzyme activity. The transparent root box was opened from one side, and the substrate-soaked saturated membrane was placed tightly against the root-soil interface to allow for sufficient reaction between the substrate and enzyme. A layer of aluminum foil was placed over the membrane to create a dark environment, and the transparent root box was replaced. After 1 hour of enzymatic reaction, the membrane was carefully peeled off from the soil surface, and adhering soil particles were gently removed with tweezers and a small brush. The membrane was then placed in a dark chamber, and images were taken using a camera under an ultraviolet lamp with an excitation wavelength of 355nm and an emission wavelength of 460nm. Differences in grayscale values ​​observed in the images, combined with variations in fluorescence intensity, were used to distinguish between hot and non-hot regions.

[0067] The biomass ash used in this experiment was sourced from Beihai Hepu Anong Co., Ltd., with a pH of 11.78.

[0068] The crops tested in this study were soybeans, variety Guixia 7, and corn, variety Zhengdan 958.

[0069] The slow-growing rhizobium G94 tested in this study, classified as Bradyrhizobium centrosematis, is deposited at the Guangdong Provincial Microbial Culture Collection Center on August 14, 2023, with accession number GDMCC.No.: 63598, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0070] Each root box was filled with 2.5 kg of soil, and ventilation and drainage pipes were installed on both sides. The soil was irrigated with sterile water using a weighing method until the field water holding capacity was approximately 70% ± 5%. The boxes were then placed in a walk-in incubator for two weeks of pre-cultivation. Before germination, the seeds were sterilized by soaking them in 75% alcohol for 30 seconds and then in a 1% sodium hypochlorite solution for 10 minutes. After rinsing with sterile water, the seeds were placed in sterilized containers covered with gauze, and sprayed with sterile water to keep the gauze moist. The containers were then placed in a 28℃ incubator for 48 hours to promote germination. Seeds with similar germination rates were selected and sown about 1 cm deep along the root window. The transparent side of the root box was then placed face down, and the transparent side was sealed with aluminum foil to maintain a dark environment. The boxes were tilted at a 45° angle to encourage root growth along the transparent root window. Soil moisture content was controlled at 70% ± 5% of field capacity using a weighing method. A walk-in incubator was used to simulate the growing environment, with daytime temperature of 28℃ and nighttime temperature of 22℃, daytime light intensity of 600 μmol, and humidity of 65%. No fertilization was applied to any treatment, and all treatments followed the same management practices. Destructive sampling was performed after 30 days to measure relevant indicators.

[0071] The experiment included a blank control group (NS), acid stress (AS), acid stress inoculated with slow-growing rhizobium G94 (AS+B), and no stress inoculated with slow-growing rhizobium G94 (NS+B). Each treatment was replicated three times, with an inoculation amount of 2% (w / w) (i.e., 2 mL of bacterial solution per 100 g of air-dried soil). The blank control group (no stress soil) was prepared by adding 2% biomass wood ash to the acid stress soil to adjust the pH of the strongly acidic soil to 6.7.

[0072] 1. Slow-growing rhizobium G94 improves soil microecology.

[0073] The results are as follows Figure 8 As shown, inoculation with the slow-growing rhizobium G94 (AS+B) under acid stress significantly increased the pH of non-thermal soils of maize and soybean by 2.73% and 2.27%, respectively, while having no significant effect on soils in thermal zones. Under no stress, inoculation significantly decreased the pH of maize soils in thermal zones, but had no significant effect on soybean soils in thermal zones. Under acid stress, inoculation with G94 significantly reduced the total exchangeable acid in maize and soybean soils in thermal zones by 10.72% and 11.1%, respectively, showing a decreasing trend in non-thermal soils, but not reaching a significant level. Under no stress, inoculation with G94 had no significant effect on the total exchangeable acid in maize and soybean soils in both thermal and non-thermal zones. Under acid stress, inoculation with G94 increased the exchangeable pH in maize soils in both thermal and non-thermal zones. + The content of [unclear] was reduced, but H2 content in soybean hot zone soils was significantly decreased. + The content of [unspecified substance] decreased by 14.28%; under no-stress treatment, inoculation with G94 showed no significant difference in exchangeable Al content in maize and soybean soils in both hot and non-hot zones. Under acid stress treatment, inoculation with G94 significantly reduced the exchangeable Al content in hot zone maize and soybean soils.3+ The contents decreased by 13.9% and 9.62% respectively, and had no significant effect on the non-heat zone soil. Under the non-stress treatment, inoculation with G94 had no significant effect on the exchangeable Al in the heat zone and non-heat zone soils of both crops. 3+ There was no significant effect.

[0074] 2. Bradyrhizobium japonicum G94 promotes crop growth under acid stress

[0075] As shown in Table 3, through three-factor variance analysis, it can be seen that the three factors of crop type, whether to inoculate Bradyrhizobium japonicum G94, and the presence or absence of acid stress, as well as some of their interactions, had significant effects on the fresh weight and dry weight of the above-ground parts and roots; among them, the three factors and their interactions all had significant effects on the dry and fresh weights of the roots.

[0076] Table 3 Effect test of the interaction of treatments on the biomass of different crops

[0077]

[0078]

[0079] Note: * indicates 0.01 < p < 0.05, ** indicates 0.001 < p < 0.01, *** indicates p < 0.001, NS indicates p > 0.05.

[0080] The results are as Figure 9As shown, in both maize and soybean, acid stress treatment significantly reduced aboveground fresh weight compared to the no-stress treatment. Furthermore, inoculation with *S. truncatula* G94 significantly increased aboveground fresh weight under both acid stress and no-stress conditions compared to the corresponding uninoculated treatment. Specifically, for maize, acid stress significantly reduced aboveground fresh weight by 136.8% compared to the no-stress treatment, while inoculation significantly increased by 101.3% under acid stress and by 27.9% under no-stress conditions. For soybean, acid stress significantly reduced aboveground fresh weight by 67.9% compared to the no-stress treatment, while inoculation significantly increased by 31.5% under acid stress and by 12.02% under no-stress conditions. Similarly, aboveground dry weight significantly increased under both acid stress and no-stress conditions compared to the uninoculated treatment. In maize, a fibrous-rooted crop, acid stress significantly reduced aboveground dry weight by 100.22% compared to no stress treatment, and the inoculation rate increased significantly by 74.38% under acid stress, compared to 21.84% under no stress treatment. In soybean, a taprooted crop, acid stress significantly reduced aboveground dry weight by 70.34% compared to no stress treatment, and the inoculation rate increased significantly by 55.3% under acid stress, compared to 11.73% under no stress treatment. Notably, inoculation with *Syntrophus stenoptera* G94 under acid stress had a greater positive impact on aboveground dry and fresh weight than inoculation under no stress treatment, and maize showed a greater stress response than soybean. This indicates that the strain plays a greater role under stress than under non-stress conditions, and inoculation increased the stress resistance of both crops, with maize showing a greater response than soybean.

[0081] In the case of not inoculating with Bradyrhizobium, acid stress treatment significantly reduced the fresh and dry weights of the roots of both crops compared with non-stress treatment, while inoculating with Bradyrhizobium G94 increased the fresh and dry weights of the roots under both acid stress and non-stress conditions. Specifically, for maize, compared with non-stress treatment, acid stress treatment significantly reduced the fresh and dry weights of the roots by 102.75% and 113.15% respectively, while inoculating with Bradyrhizobium G94 under acid stress treatment significantly increased the fresh and dry weights of the roots by 99.2% and 122.9% respectively, basically restoring to the non-stress level. Inoculating with bacteria under non-stress treatment significantly increased the fresh and dry weights of the roots by 14.63% and 11.37% respectively; for soybean, compared with non-stress treatment, acid stress treatment significantly reduced the fresh and dry weights of the roots by 71.03 and 32.39% respectively, while inoculating with Bradyrhizobium G94 under acid stress treatment significantly increased the fresh and dry weights of the roots by 39.37% and 26.32% respectively. Inoculating with bacteria under non-stress treatment significantly increased the fresh and dry weights of the roots by 23.2% and 4.28% respectively; similar to the dry and fresh weights of the above-ground parts, the positive effects of inoculating with Bradyrhizobium G94 on the dry and fresh weights of the roots of the two different types of crops under acid stress were greater than those of inoculating with bacteria under non-stress treatment.

[0082] 3. Bradyrhizobium G94 Promotes Nutrient Accumulation in Crops under Acid Stress

[0083] As shown in Table 4, through three-factor variance analysis, it can be seen that acid stress, whether inoculated with bacteria, different crop types and their interaction have significant effects on the total nitrogen, total carbon and total phosphorus in the above-ground parts and roots of plants.

[0084] Table 4 Effect Test of Interaction of Treatments on Nutrients of Different Crop Plants

[0085]

[0086] Note: * indicates 0.01 < p < 0.05, ** indicates 0.001 < p < 0.01, *** indicates p < 0.001, NS indicates p > 0.05.

[0087] The results are as Figure 10As shown, under acid stress treatment, compared with the no-stress treatment, the accumulation of TN, TC, and TP in maize aboveground parts and roots all showed a decreasing trend, decreasing by 26.29%, 36.19%, and 44.69%, and 30.02%, 41.05%, and 181.81%, respectively. The decrease in TC accumulation in both aboveground parts and roots was significantly greater than that in TN accumulation. Inoculation with *S. truncatula* G94 significantly increased the accumulation of TN, TC, and TP in roots under both acid stress and no-stress treatments. However, inoculation only had a significant effect on the accumulation of TN, TC, and TP in aboveground parts under no-stress treatment, while it had no significant effect under acid stress treatment. Under acid stress treatment, inoculation increased root TN, TC accumulation, and TP content by 95.4%, 115.83%, and 171.71%, respectively. Under no stress treatment, inoculation increased these values ​​by 49.57%, 51.17%, and 15.42%, respectively. The positive effects of inoculation under acid stress were greater than those under no stress, regardless of TN, TC accumulation, or TP content. Under no stress treatment, inoculation increased aboveground TN and TC accumulation by 63.31% and 55.43%, respectively, but decreased TP content. Compared to no stress treatment, acid stress treatment reduced TC accumulation and TP content in soybean aboveground parts and roots, but had no significant effect on TN accumulation. Inoculation with slow-growing rhizobium G94 significantly increased the accumulation of TC and TP in the aboveground parts of soybean and the accumulation of TN and TC in the roots, by 29.2%, 211.21%, 21.64%, and 24.38%, respectively, under acid stress treatment. In contrast, inoculation under no stress treatment only significantly increased the accumulation of TC and TP in the aboveground parts and the accumulation of TN in the roots, by 20.37%, 142.42%, and 28.28%, respectively, without significantly affecting the accumulation of TN in the aboveground parts and the accumulation of TC and TP in the roots.

[0088] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. Application of slow-growing rhizobium G94 in the improvement of acidic soil. The slow-growing rhizobium G94 is deposited at the Guangdong Provincial Microbial Culture Collection Center, date of deposit: August 14, 2023, accession number GDMCC.No: 63598, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The improvement is to promote the growth of H+ in acidic soil. + Decreased content and exchangeable Al 3+ The content is reduced, and the specific application process is as follows: 2.5 kg of soil was placed in the root box, and 2 mL of slow-growing rhizobium G94 bacterial solution was added to every 100 g of soil. Aeration and drainage pipes were installed on both sides of the root box. The soil was irrigated with sterile water using the weighing method until the field water holding capacity was 70% ± 5%. The root box was then placed in a walk-in incubator for two weeks of pre-cultivation. Soybean seeds with the same degree of germination were selected and sown at a depth of 1 cm along the root window. The transparent side of the root box was then placed downwards, and the transparent side was sealed with aluminum foil to maintain a dark environment. The root box was tilted at 45° to allow the soybean roots to grow along the transparent root window. The soil moisture content was controlled at 70% ± 5% of the field water holding capacity using the weighing method. The walk-in incubator simulated the growth environment with a daytime temperature of 28°C and a nighttime temperature of 22°C, a daytime light intensity of 600 μmol, and a humidity of 65%. No fertilizer was applied. Soil was improved after 30 days of growth. in, The acidic soil is a strongly acidic agricultural soil with a pH of 4.

3. The specific nutrient contents are: total carbon 8.93 g / kg; total nitrogen 0.95 g / kg; available phosphorus 12.16 g / kg; available potassium 76.54 mg / kg.

2. Application of *Staphylococcus aureus* G94 in regulating crop nutrient accumulation in acidic soils. *Staphylococcus aureus* G94 is deposited at the Guangdong Provincial Microbial Culture Collection Center, date of deposit: August 14, 2023, accession number GDMCC.No: 63598, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The application includes promoting H+ accumulation in acidic soils. + Decreased content and exchangeable Al 3+ The content decreases, and the regulation of crop nutrient accumulation is to promote the increase of total nitrogen and total phosphorus content in the aboveground parts of soybeans. The specific application process is as follows: 2.5 kg of soil was placed in the root box, and 2 mL of slow-growing rhizobium G94 bacterial solution was added to every 100 g of soil. Aeration and drainage pipes were installed on both sides of the root box. The soil was irrigated with sterile water using a weighing method until the field water holding capacity reached 70% ± 5%. The root box was then placed in a walk-in incubator for two weeks of pre-cultivation. Soybean seeds with similar germination levels were sown 1 cm deep along the root window surface. The transparent side of the root box was then placed downwards, and the transparent side was sealed with aluminum foil to maintain a dark environment. The root box was tilted at 45° to allow soybean roots to grow along the transparent root window surface. The soil moisture content was controlled at 70% ± 5% of field water holding capacity using a weighing method. The walk-in incubator simulated the growth environment with a daytime temperature of 28°C and a nighttime temperature of 22°C, a daytime light intensity of 600 μmol, and a humidity of 65%. No fertilizer was applied. After 30 days of growth, nutrient accumulation was regulated to promote an increase in total nitrogen and total phosphorus content in the soybean aboveground parts. in, The acidic soil is a strongly acidic agricultural soil with a pH of 4.

3. The specific nutrient contents are: total carbon 8.93 g / kg; total nitrogen 0.95 g / kg; available phosphorus 12.16 g / kg; available potassium 76.54 mg / kg.

3. Application of slow-growing rhizobium G94 in improving crop stress resistance. The slow-growing rhizobium G94 is deposited at the Guangdong Provincial Microbial Culture Collection Center, date of deposit: August 14, 2023, accession number GDMCC.No: 63598, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The application includes promoting H+ in acidic soils. + Decreased content and exchangeable Al 3+ The content is reduced, and the specific application process is as follows: 2.5 kg of soil was placed in the root box, and 2 mL of slow-growing rhizobium G94 bacterial solution was added to every 100 g of soil. Aeration and drainage pipes were installed on both sides of the root box. The soil was irrigated with sterile water using a weighing method until the field water holding capacity was 70% ± 5%. The root box was then placed in a walk-in incubator for two weeks of pre-cultivation. Soybean seeds with the same degree of germination were selected and sown at a depth of 1 cm along the root window. The transparent side of the root box was then placed downwards, and the transparent side was sealed with aluminum foil to maintain a dark environment. The root box was tilted at 45° to allow the soybean roots to grow along the transparent root window. The soil moisture content was controlled at 70% ± 5% of the field water holding capacity using a weighing method. The walk-in incubator simulated the growth environment with a daytime temperature of 28°C and a nighttime temperature of 22°C, a daytime light intensity of 600 μmol, and a humidity of 65%. No fertilizer was applied. The crop was allowed to grow for 30 days to improve its stress resistance. in, The acidic soil is a strongly acidic agricultural soil with a pH of 4.

3. The specific nutrient contents are: total carbon 8.93 g / kg; total nitrogen 0.95 g / kg; available phosphorus 12.16 g / kg; available potassium 76.54 mg / kg.

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