A Rhodococcus qingshengii FJ5 plant that promotes tobacco growth and controls bacterial wilt and its applications

Abstract

This invention provides a method for promoting tobacco growth and preventing bacterial wilt. Rhodococcus qingshengii This invention relates to the field of microbial technology, specifically to Rhodococcus faecalis strain FJ5, with the preservation number CGMCC No. 32631. The FJ5 strain exhibits a wide adaptability range, growing well within a temperature range of 16–34°C and a pH range of 5.0–8.0. The FJ5 strain promotes early growth and enhances stress resistance in tobacco by mitigating aluminum stress, increasing the activity of tobacco defense enzymes, improving tobacco growth hormone secretion and carbon and nitrogen metabolism, while simultaneously improving acidic soil fertility and controlling tobacco bacterial wilt. The Rhodococcus faecalis FJ5 provided by this invention has significant development value for high-quality tobacco development under acidified soil conditions in rice-tobacco rotation, and is also important for improving plant stress resistance and the biological control of root and stem diseases.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain that promotes tobacco growth and prevents bacterial wilt. Rhodococcus qingshengii FJ5 and its applications. Background Technology

[0002] Acidic soils have always been a concern, with acidified soils having a pH below 5 widely distributed across multiple regions. Globally, acidic soils account for more than 35% of existing potential arable land. The causes of acidic soils can be categorized into natural and anthropogenic factors. Natural factors primarily involve the leaching of silicon and basic ions, leading to the accumulation of large amounts of iron and aluminum compounds. Anthropogenic factors include continuous cropping, long-term excessive application of synthetic fertilizers causing imbalances in soil nutrient cycling, and excessive absorption and leaching of alkaline cations, all of which are significant causes of increased soil acidity. The impact of soil acidification on crops is exacerbated by the toxicity of metals.

[0003] Soil acidification and aluminum toxicity are considered the most destructive soil conditions affecting the growth of most crops. Aluminum exists in soil as aluminosilicates, which converts into toxic soluble trivalent aluminum ions (Al₂O₃) under acidic conditions. 3+ ), Al 3+ Aluminum is a significant acidifying factor, and aluminum toxicity (aluminum stress) is one of the main limiting factors for crop growth in acidic soils. Aluminum toxicity can trigger a series of phytotoxicity syndromes. 3+ Primarily acting on plant roots, it reduces root vigor and inhibits growth; it also increases reactive oxygen species (ROS) levels, which act on the cell membrane and interact with lipid components, triggering lipid peroxidation and cytoskeleton damage, leading to reduced stress resistance and causing cell death through damage and autophagy. Previous studies have reported that under high aluminum stress, when Al... 3+ >2 cmol / kg, aluminum is toxic to plants; when Al 3+ At a concentration of 4.4 cmol / kg, soybean growth was inhibited.

[0004] Acidified soils are widely distributed in tobacco-growing areas, especially in rice-tobacco rotation soils where acidification is severe. Soil acidification is not only a decisive factor affecting soil nutrient transformation and availability, but also a significant factor influencing tobacco growth, yield, and quality. Aluminum toxicity, as a major acidifying factor, has a noteworthy impact on tobacco. Under aluminum toxicity stress, root growth and development in tobacco are inhibited, resulting in reduced plant height, stem circumference, single leaf area, root volume, and dry matter accumulation, leading to decreased photosynthesis and consequently reduced tobacco yield. Furthermore, tobacco is susceptible to pathogen infection during its growing season, leading to disease outbreaks. Plant diseases are a major factor restricting sustainable agricultural development. Root and stem diseases caused by soil-borne pathogens are characterized by high infectivity, high mortality, and difficulty in control, causing significant economic losses to agricultural production. Tobacco bacterial wilt is caused by Ralstonia pseudosolanacea (…).Ralstonia pseudosolanacearum Ralstonia solanacearum (hereinafter referred to as Bacillus spp.) is a bacterial vascular disease that is prevalent and causes serious damage worldwide. The pathogen mainly affects the roots, and after invading, it spreads within the vascular bundles, causing necrosis and rot of the vascular bundles, ultimately leading to the death of the entire plant.

[0005] Guided by the plant protection policy of "prevention first, integrated management," the control of plant root and stem diseases mainly relies on disease-resistant breeding, combined with agricultural, chemical, and biological control techniques. However, the selection of disease-resistant varieties and agricultural cultivation management suffer from long cycles and slow results. Currently, chemical control is the primary method, as chemical pesticides have a broad spectrum of fungicides, low cost, and rapid effect, making a significant contribution to crop pest and disease control. However, the long-term irrational use of chemical pesticides has led to increasingly prominent problems such as pesticide residues, food safety, and environmental pollution. Therefore, it is urgent to accelerate the change from the traditional approach of over-reliance on chemical pesticides in crops. Under the premise of stable and increased yields, it is crucial to vigorously develop low-toxicity, low-residue biological pesticide technologies and related product research and development, promote the transformation from traditional chemical control to modern biological control, reduce the input of chemical pesticides in production, and achieve sustainable development that coordinates crop yield and quality safety with agricultural ecological environmental protection. Biological control is a method that uses beneficial organisms or their secondary metabolites to inhibit the occurrence, development, or reduce the severity of harmful organisms. It can control plant diseases and pests without polluting the environment, and has advantages such as safety for humans, no residues, and high specificity. It helps protect the ecological environment and increase agricultural production, thus providing both ecological and economic benefits. Therefore, biological control has gradually become a research hotspot in the pesticide industry both domestically and internationally, and has significant development value as an alternative to chemical pesticides.

[0006] Biocontrol agents used for the control of plant root and stem diseases mainly include antagonistic bacteria, fungi and their produced antibiotics and plant-derived inducers. Among them, antagonistic bacteria are characterized by their large number, broad spectrum of inhibition, rapid growth and reproduction, and rich variety of antagonistic metabolites. Rhizosphere probiotics have been proven to promote crop nutrient absorption and alleviate abiotic stress, and are the most widely used in plant disease control. Biocontrol bacteria mainly include Bacillus and Pseudomonas. However, current microbial pesticide products suffer from relatively limited variety, a scarcity of superior strains, and the inability to stably colonize after application.

[0007] There are currently no reports on the effects of Rhodococcus on promoting plant growth, improving plant stress resistance, improving soil, or alleviating aluminum toxicity. Summary of the Invention

[0008] In view of the deficiencies in the prior art, the purpose of this invention is to provide a strain of Rhodococcus faecalis FJ5, which has a wide range of adaptability, can improve the stress resistance of plants, improve acidic soil, alleviate aluminum toxicity, promote the growth of plants such as tobacco, and at the same time improve the control effect of tobacco against bacterial wilt.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] This invention provides a strain of Rhodococcus ( Rhodococcus qingshengii The Rhodococcus FJ5 has the preservation number CGMCC No. 32631.

[0011] This invention provides a microbial inoculant, comprising Rhodococcus FJ5 as described in the above technical solution.

[0012] Preferably, the viable count of Rhodococcus FJ5 in the microbial agent is ≥1×10⁻⁶. 8 cfu / mL.

[0013] This invention provides a method for preparing the microbial inoculant described in the above-mentioned technical solution, comprising:

[0014] The Rhodococcus FJ5 was cultured in a culture medium to obtain a microbial inoculum.

[0015] Preferably, the culture temperature is 16~34℃; and the pH value of the culture medium is 5.0~8.0.

[0016] This invention provides the application of Rhodococcus FJ5 described in the above technical solution, the microbial agent described in the above technical solution, or the microbial agent prepared by the preparation method described in the above technical solution in the prevention and control of tobacco bacterial wilt.

[0017] This invention provides the application of the Rhodococcus FJ5 described in the above technical solution, the microbial agent described in the above technical solution, or the microbial agent prepared by the preparation method described in the above technical solution in promoting plant growth and / or improving plant stress resistance; the plant includes tobacco.

[0018] This invention provides the application of the Rhodococcus FJ5 described in the above technical solution, the microbial agent described in the above technical solution, or the microbial agent prepared by the preparation method described in the above technical solution in alleviating plant acid and / or aluminum stress.

[0019] This invention provides the application of the Rhodococcus FJ5 described in the above technical solution, the microbial agent described in the above technical solution, or the microbial agent prepared by the preparation method described in the above technical solution in improving soil.

[0020] This invention provides a method for promoting plant growth, improving plant stress resistance, and / or improving soil, comprising: applying the Rhodococcus FJ5 described in the above technical solution, the microbial agent described in the above technical solution, or the microbial agent prepared by the preparation method described in the above technical solution to the plant roots.

[0021] The beneficial effects of this invention are:

[0022] This invention provides a strain of Rhodococcus faecalis FJ5, with the preservation number CGMCC No. 32631. The Rhodococcus faecalis FJ5 provided by this invention has a wide adaptability range, growing well within a temperature range of 16–34℃ and a pH range of 5.0–8.0. This Rhodococcus faecalis FJ5 promotes early growth and enhances stress resistance in tobacco by alleviating acid-aluminum stress, increasing the activity of tobacco defense enzymes, improving tobacco growth hormone secretion and carbon and nitrogen metabolism levels, while simultaneously improving acidic soil fertility and controlling tobacco bacterial wilt. The Rhodococcus faecalis FJ5 provided by this invention has significant development value for high-quality tobacco development under acidified soil conditions in rice-tobacco rotation, and is also important for improving plant stress resistance and the biological control of root and stem diseases.

[0023] Preservation Instructions

[0024] Rhodococcus faecalis FJ5, Latin scientific name: Rhodococcus qingshengii It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 14, 2024, with accession number CGMCC No. 32631. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Phylogenetic tree analysis of the core genes of strain FJ5;

[0027] Figure 2 This is a diagram showing the culture characteristics of strain FJ5 in NA medium.

[0028] Figure 3 The diagram shows the disease prevention effect of each treatment group on tobacco bacterial wilt in Example 2;

[0029] Figure 4 Figure showing the effect of Rhodococcus faecalis FJ5 on the height of tobacco seedlings;

[0030] Figure 5 Figure showing the effect of Rhodococcus faecalis FJ5 on the aboveground fresh weight of tobacco seedlings;

[0031] Figure 6 Figure showing the effect of Rhodococcus faecalis FJ5 on the fresh weight of tobacco seedling roots;

[0032] Figure 7 Photographs showing the effects of Rhodococcus faecalis FJ5 on the whole tobacco seedling, roots, and above-ground parts;

[0033] Figure 8 Figure 1 shows the effect of Rhodococcus faecalis FJ5 on IAA, JA and SA in tobacco seedlings.

[0034] Figure 9 The figure shows the effects of Rhodococcus faecalis FJ5 on SOD, CAT, PPO and ROS activities in tobacco seedlings.

[0035] Figure 10 Figure showing the effect of Rhodococcus faecalis FJ5 on the activities of nitrate reductase and sucrose synthase;

[0036] Figure 11 The graph shows the effect of Rhodococcus FJ5 on total soluble sugars.

[0037] Figure 12 The graph shows the effect of Rhodococcus FJ5 on reducing sugars;

[0038] Figure 13 The graph shows the effect of Rhodococcus FJ5 on protein content;

[0039] Figure 14 Figure showing the effect of strain FJ5 on aluminum content in acidified soil;

[0040] Figure 15 Figure showing the effect of strain FJ5 on urease activity in acidified soil;

[0041] Figure 16 Figure showing the effect of strain FJ5 on phosphatase activity in acidified soil;

[0042] Figure 17 Figure showing the effect of strain FJ5 on sucrase activity in acidified soil;

[0043] Figure 18 Figure 1 shows the effect of strain FJ5 on organic matter and Bc content in acidified soil.

[0044] Figure 19 Figure 1 shows the effect of strain FJ5 on the hydrolyzable nitrogen content in acidified soil.

[0045] Figure 20 Figure 1 shows the effect of strain FJ5 on the ammonium nitrogen content of acidified soil.

[0046] Figure 21 The figure shows the effect of strain FJ5 on the available phosphorus content in acidified soil. Detailed Implementation

[0047] This invention provides a strain of Rhodococcus ( Rhodococcus qingshengii The Rhodococcus FJ5 has the preservation number CGMCC No. 32631.

[0048] The present invention provides Rhodococcus qingshengii FJ5 was extracted from the rhizosphere soil of healthy tobacco plants in a rice-tobacco rotation field in Huangzhouba Village, Nanping City, Fujian Province. R. qingshengii FJ5 colonies grow on NA agar plates as small, round, milky-white colonies with a moist and glossy surface. R. qingshengii The core gene sequence of FJ5 is shown in SEQ ID NO.1. The present invention provides... R. qingshengii FJ5 can promote early growth and enhance stress resistance of tobacco by reducing acid-aluminum stress, increasing the activity of tobacco defense enzymes, increasing the secretion of tobacco growth hormones and carbon and nitrogen metabolism, while improving the fertility of acidic soils and preventing tobacco bacterial wilt.

[0049] This invention provides a microbial inoculant, comprising the components described in the above technical solution. R. qingshengii FJ5. As an optional embodiment of the present invention, the microbial agent contains... R. qingshengii FJ5 viable count ≥ 1×10 8 cfu / mL.

[0050] This invention provides a method for preparing the microbial inoculant described in the above technical solution, comprising: taking the... R. qingshengii FJ5 was cultured in a culture medium to obtain a microbial inoculum.

[0051] In an optional embodiment of the present invention, the culture medium includes NA medium and / or NB medium. In an optional embodiment of the present invention, the culture temperature can be 16~34℃, or 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34℃; the pH value of the culture medium can be 5.0~8.0, or 5.0, 6.0, 7.0, or 8.0. In an optional embodiment of the present invention, the culture time can be 2 days; the culture process preferably involves shaking; the shaking speed can be 120~150 rpm; the culture preferably includes dark culture. After the culture is completed, the present invention can directly use the obtained culture medium as a microbial inoculant; or the bacterial sludge in the culture medium can be separated, resuspended, and a bacterial suspension can be obtained; the bacterial suspension can then be used as a microbial inoculant.

[0052] The present invention provides the technical solution described above. R. qingshengii FJ5. The application of the microbial inoculant described in the above technical solution or the microbial inoculant prepared by the preparation method described in the above technical solution in the control of tobacco bacterial wilt. The results of the embodiments of the present invention show that... R. qingshengii FJ5 can significantly reduce the incidence of tobacco bacterial wilt, lower the incidence index of tobacco bacterial wilt, and improve the control effect of tobacco on tobacco bacterial wilt.

[0053] The present invention provides the technical solution described above. R. qingshengii FJ5. The application of the microbial inoculants described in the above technical solutions or the microbial inoculants prepared by the preparation methods described in the above technical solutions in promoting plant growth and / or improving plant stress resistance; the plant includes tobacco. As an optional embodiment of the present invention, promoting plant growth includes increasing plant height, aboveground fresh weight, and root fresh weight, or two or more of these. As an optional embodiment of the present invention, improving plant stress resistance includes activating plant defense responses, improving plant antioxidant capacity, and enhancing carbon and nitrogen metabolism levels, or two or more of these. In the present invention, activating plant defense responses includes increasing the jasmonic acid content and / or decreasing the salicylic acid content of the plant. Jasmonic acid and salicylic acid play key roles in plant defense signaling pathways. In plant immunity, the endogenous hormones salicylic acid (SA) and jasmonic acid (JA) antagonize each other, enabling plants to flexibly defend against different types of harmful organisms. In the present invention, improving plant antioxidant capacity includes increasing plant SOD activity, increasing plant CAT activity, increasing plant PPO activity, and / or decreasing plant ROS, or two or more of these. In this invention, the enhanced carbon and nitrogen metabolism includes enhancing the activity of tobacco nitrate reductase and / or sucrose reductase.

[0054] The present invention provides the technical solution described above. R. qingshengii FJ5, the application of the microbial agent described in the above technical solution or the microbial agent prepared by the preparation method described in the above technical solution in alleviating phytic acid and / or aluminum stress. The results of the embodiments of the present invention show that... R. qingshengii FJ5 can significantly increase the stress resistance of plants growing in acidic soil conditions, and at the same time, the R. qingshengii FJ5 can significantly reduce the aluminum content in the soil, thereby alleviating phytoacidity and / or aluminum stress.

[0055] The present invention provides the technical solution described above. R. qingshengii FJ5. Application of the microbial inoculants described in the above technical solutions or the microbial inoculants prepared by the preparation methods described in the above technical solutions in soil improvement. As an optional embodiment of the present invention, the soil includes acidic soil. The results of the embodiments of the present invention show that... R. qingshengii FJ5 can significantly reduce the aluminum content in the soil, increase the content of urease, phosphatase, sucrase, soil organic matter and microbial carbon, and further increase the content of nitrogen and phosphorus nutrients in the soil.

[0056] This invention provides a method for promoting plant growth, improving plant stress resistance, and / or improving soil, comprising: applying the above-described technical solution to the plant roots.R. qingshengii FJ5, the microbial agent described in the above technical solution or the microbial agent prepared by the preparation method described in the above technical solution.

[0057] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0058] The following technical solutions are described in the accompanying drawings. This indicates a significant difference (p < 0.05). This indicates that p < 0.01; This indicates that p < 0.001. p < 0.0001 indicates a highly significant difference.

[0059] Example 1 Rhodococcus qingshengii Isolation, identification and culture

[0060] 1. R. qingshengii Separation and identification

[0061] In June 2024, rhizosphere soil (pH=5.16) from healthy tobacco plants in a rice-tobacco rotation field in Huangzhouba Village, Nanping City, Fujian Province was collected. One gram of soil was weighed and mixed with 9 mL of sterile water to prepare a solution with a concentration of 10. -1 Soil leachate was then prepared into 10 by a sterile water gradient dilution. -4 Soil leachate was used to extract 200 μL of solution, which was then placed on an NA (broth nutrient solid medium, autoclaved at 121°C for 20 min) plate using a sterile pipette tip. The solution was then evenly spread onto the plate surface using a sterile spreader. After incubation in the dark at 28°C for 2 days, a dominant single colony was picked and streaked onto an NA plate for purification, yielding strain FJ5. The strain was identified as Rhodococcus (Rhodococcus) by PCR amplification of the conserved 16S gene region and GenBank Blast sequencing. Rhodococcus qingshengii The 16S rDNA gene sequence of strain FJ5 is shown in SEQ ID NO.1, specifically as follows:

[0062]

[0063] Based on the FJ5 whole genome data (NCBI Genbank accession number SUB15557392), average nucleotide identity (ANI) analysis was used to analyze FJ5 and... Rhodococcus qingshengii JCM 15477 (genome number GCF_023221595.1) has an ANI value of 98.7422%, which is higher than... Rhodococcus erythropolis 95.5575% of NBRC 15567 (genome number GCF_001552595.1); based on the standard of ANI value ≥95%-96% to determine that they are the same species, FJ5 can be clearly identified as... Rhodococcus qingshengii They are more closely related by blood.

[0064] Core gene phylogenetic tree analysis: Based on the strain's chromosome sequence, a core gene phylogenetic tree was constructed using ubcg software. The results showed that this strain is related to... Rhodococcus qingshengii JCM 15477 is located in the same high-support branch, confirming a close evolutionary relationship between the two at the core gene level. The phylogenetic tree analysis of the core genes of strain FJ5 is shown below. Figure 1 As shown. The results confirmed that strain FJ5 is... Rhodococcus qingshengii .

[0065] R. qingshengii FJ5 training

[0066] The original bacterial strain, cryopreserved at -80℃ in 30% glycerol, was activated by streaking on NA medium plates. After incubation at 32℃ in the dark for 36 hours, and the formation of microbial growth on the plates, the appropriate temperature and pH conditions were determined. The steps are as follows: The bacterial growth of strain FJ5 on NA medium plates was washed and diluted with an appropriate amount of sterile water. The bacterial suspension was read using a spectrophotometer, and the OD was adjusted accordingly. 600 The value is 0.1, reserved.

[0067] (1) Determination of suitable growth temperature

[0068] Different temperature treatments were performed at 4, 10, 16, 22, 28, 34, 40, 44, and 50 °C; 500 µL of OD was collected. 600 Bacterial suspensions with a nutrient solution concentration of 0.1 were placed in pre-sterilized NB (broth nutrient solution) centrifuge tubes (5 mL), with each treatment repeated three times. The bacterial suspensions from different treatments were placed in shaking incubators at different temperatures and incubated overnight (12 h) at 120 rpm in the dark. The OD values ​​of the bacterial suspensions from different treatments were measured using a spectrophotometer. 600 value.

[0069] (2) Determination of suitable pH for growth

[0070] The culture medium was prepared using concentrated acid and concentrated alkali titration. Six different pH treatments were included in the experiment: 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0. NB medium was prepared first, and then the pH was measured using test strips (pH 7.2). Titration was performed using either acid or alkali solutions, and the pH was confirmed using test strips until the desired pH value was reached. The bacterial suspension was inoculated into the culture media at different pH values ​​and placed in an incubator at 28°C, 120 rpm, and in the dark with shaking overnight (12 h). The OD values ​​of the bacterial suspensions under different treatments were measured using a spectrophotometer. 600 value.

[0071] Test results

[0072] FJ5 colonies grow on NA agar plates as small, round, milky-white colonies with a moist and glossy surface. Figure 1 The spectrophotometer reads the OD values ​​of FJ5 bacterial suspensions under different temperature and pH conditions. 600 The optimal growth temperature for FJ5 was found to be 28℃ (OD). 600 =0.55), suitable growth range is 22℃-34℃ (OD 600 =0.42~0.47); FJ5 basically stops growing when the temperature is above 40℃ or below 10℃. Meanwhile, the optimal pH for FJ5 growth is 5.0 and 6.0, with a growth pH range of 5.0~8.0. FJ5 basically stops growing at pH=4.0 and pH=9.0.

[0073] Example 2 R. qingshengii The effect of FJ5 on the prevention and control of tobacco bacterial wilt

[0074] R. qingshengii Preparation of FJ5 bacterial suspension: After streaking FJ5 strain on nutrient broth (NA) plates for 36 h, single colonies were picked using a sterile inoculation loop and inoculated into NB (NA without agar) medium. The culture was incubated at 28℃ and 150 rpm in the dark for 2 days with shaking. The bacterial pellet was then centrifuged at 8000 rpm for 5 min and diluted with an appropriate amount of sterile water to prepare a 10% concentration. 8 CFU / mL bacterial suspension, for later use.

[0075] R. qingshengii The disease prevention test of FJ5 against tobacco bacterial wilt was conducted under greenhouse conditions.

[0076] The tested tobacco variety was K326 (growing period of 45 days) seedlings.

[0077] The experiment included three treatments: treatment 1 was FJ5 bacterial suspension, treatment 2 was 40% thiamethoxam zinc suspension (1000-fold dilution) for the chemical control of bacterial wilt, and treatment 3 was a blank control.

[0078] The root drenching inoculation method was used. 10 mL of FJ5 bacterial suspension and 1000-fold diluted 40% thiamethoxam zinc suspension were applied to the root zone soil of tobacco seedlings. An equal volume of sterile water served as a blank control. Each treatment consisted of 15 seedlings, with 3 replicates.

[0079] Five days after inoculation, take 10 mL (concentration approximately 10). 8 A suspension of Ralstonia solanacearum (cfu / mL) was applied to the roots of tobacco seedlings and inoculated. All tested seedlings were then cultured in a greenhouse at 30℃ and 80% relative humidity. Disease incidence was observed every 3 days. When the disease incidence rate (ratio of diseased plants to total plants) in the control seedlings exceeded 0.6, the severity of disease was classified according to the "Classification and Investigation Methods of Tobacco Diseases and Pests" (GB / T23222), and the disease index was calculated. R. qingshengii The effect of FJ5 on the prevention and control of tobacco bacterial wilt.

[0080] The statistical methods for disease incidence are as follows:

[0081] a) Incidence rate (%) = (Number of infected plants / Total number of plants surveyed) × 100;

[0082] b) Disease index = [∑(number of diseased plants at each level × disease level value)] / (total number of plants surveyed × highest level value) × 100;

[0083] c) Disease prevention effect (%) = [1 - (disease index after treatment in the treatment area × disease index before treatment in the control area) / (disease index before treatment in the treatment area × disease index after treatment in the control area)] × 100, where the disease index represents the disease severity index.

[0084] On day 10 after inoculation with Ralstonia solanacearum, the morbidity rate, disease index, and relative efficacy of each treatment group were as follows: Figure 3 As shown in the figure, under greenhouse pot cultivation conditions, FJ5 bacterial suspension showed significant control effects against tobacco bacterial wilt, delaying the onset and reducing the severity of the disease. Compared with the control group, seedlings treated with FJ5 bacterial suspension showed a 4-day delay in disease development. On day 10 after inoculation with Ralstonia solanacearum, the control group exhibited relatively severe disease, with an incidence rate of 66.67% and a disease index of 34.57. FJ5-treated seedlings showed relatively milder disease, with an incidence rate of 61.11% and a disease index of 6.79, resulting in a control efficacy of 80.36%. In contrast, the control group treated with 40% thiamethoxam zinc suspension (1000-fold dilution) showed an incidence rate of 55.56% and a disease index of 7.41, with a control efficacy of 78.57%. Therefore, the FJ5 bacterial suspension treatment was comparable to the chemical treatment in disease control. In conclusion, FJ5 bacterial suspension showed ideal control effects against tobacco bacterial wilt in pot cultivation and has significant application potential in the green control of tobacco bacterial wilt.

[0085] Example 3 R. qingshengiiFJ5 promotes early tobacco growth in acidic soils and activates the host plant's defense response.

[0086] R. qingshengii Preparation of FJ5 bacterial suspension: After activating and culturing FJ5 strain on NA medium plates for 2 days, the bacterial growth on the plates was mixed with a small amount of sterile water, and 1% of the bacterial suspension was inoculated into NB medium. The mixture was then incubated in the dark at 28°C and 150 rpm with shaking for 2 days. The bacterial pellet was then centrifuged at 8000 rpm for 5 minutes, and diluted with an appropriate amount of sterile water to prepare a suspension with a concentration of 10%. 8 CFU / mL bacterial suspension, for later use.

[0087] Soil tested: Soil was collected from a rice-tobacco rotation field in Jianyang, Nanping, Fujian Province. The soil was acidic (pH=5.16). Tobacco seedlings of variety K326 with a growth period of 30 days were transplanted into pots (9 cm in diameter) containing the acidic soil. After 5 days of recovery, 10 mL of FJ5 bacterial suspension (concentration 1×10⁻⁶) was collected. 8 (cfu / mL) was inoculated into the rhizosphere soil of the tested tobacco seedlings via root drenching. An equal volume of sterile water was used as a control. Each treatment consisted of 10 seedlings, with three replicates. The inoculated seedlings were then cultured in an artificial climate chamber at 28℃ and 70% relative humidity, under routine management. Twenty days after inoculation, growth-related indicators of the seedlings in each treatment were investigated.

[0088] I. Investigate the plant height, above-ground fresh weight, and fresh root weight of each treatment of tested tobacco seedlings.

[0089] II. Endogenous Hormones: The detection steps for auxin indoleacetic acid (IAA), jasmonic acid (JA), and salicylic acid (SA) are as follows:

[0090] Plant endogenous hormones were extracted from the samples using acetonitrile as the extraction solvent and the QuEChERS purification method. The plant endogenous hormones IAA, JA, and SA were simultaneously determined by ultra-high performance liquid chromatography (UPLC, Qsight LX 50, PerkinElmer, USA) coupled with triple quadrupole mass spectrometry (QQQ, Qsight420, PerkinElmer, USA). Internal standards were added to the extract to correct the detection results.

[0091] 1. Materials and Reagents

[0092] 1.1 Reference standards (IAA, JA and SA) and deuterated reference standards (D-IAA, D-JA and D-SA) were purchased from Shanghai Zhenzhun Biotechnology Co., Ltd.

[0093] The packing materials (Carbon-GCB, 120-400 mesh; HC-C18 SPE, 40-63μm) were all purchased from Shanghai Anpu Experimental Technology Co., Ltd.

[0094] Methanol and acetonitrile for chromatography were purchased from Merck, Germany. Formic acid for chromatography was purchased from Thermo Fisher Scientific, USA. Unless otherwise specified, all laboratory water was ultrapure water (Watsons).

[0095] 1.2 Instruments and Equipment

[0096] Benchtop high-speed centrifuge (TG-16G, Hunan Kaida Scientific Instruments Co., Ltd., China); 0.0001 g electronic analytical balance (MA55 / A, Mettler Toledo Instruments Shanghai Co., Ltd., Switzerland); 0.0001 g electronic balance (LE204E / 02, Mettler Toledo Instruments Shanghai Co., Ltd., Switzerland); Ultra-high performance liquid chromatography-tandem mass spectrometry (Qsight LX 50, Qsight 420, PerkinElmer, USA); Water bath nitrogen evaporator (CM-24, Beijing Chengmeng Weiye Technology Co., Ltd., China); Nitrogen generator (CM-60L, Beijing Chengmeng Weiye Technology Co., Ltd., China); Vortex mixer (XH-C, Jintan District Baita Xinbao Instrument Factory, China); Ultrasonic cleaner (YM-100S, Shenzhen Fangao Microelectronics Co., Ltd., China).

[0097] 1.3 Preparation of Standard Solutions

[0098] 1.3.1 Internal Standard Working Solution

[0099] (1) Internal standard stock solution (100 μg / mL): Weigh 1 mg (accurate to 0.0001 g) into a 10 mL volumetric flask, dilute to the mark with chromatographic methanol, and sonicate to mix.

[0100] (2) Internal standard working solution (100 ng / mL): Pipette 5 μL of the required internal standard stock solution (100 μg / mL) into a 5 mL volumetric flask and prepare an internal standard working solution with a mass concentration of 100 ng / mL using chromatographic methanol.

[0101] 1.3.2 Standard working solution

[0102] (1) External standard stock solution (1 mg / mL): Weigh 10 mg (accurate to 0.0001 g) into a 10 mL volumetric flask, dilute to the mark with chromatographic methanol, and sonicate.

[0103] (2) Standard working solutions for the calibration curve: Hormone mixed standard working solutions with concentrations of 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL were prepared using ultrapure water. The concentration of the hormone internal standard solution was 20 ng / mL for all solutions. A standard curve was plotted with the ratio of the measured characteristic ion mass chromatographic peak area of ​​the external standard to the internal standard as the ordinate and the corresponding standard solution concentration as the abscissa. The regression equation and correlation coefficient were then calculated.

[0104] 1.4 Mobile Phase

[0105] Inorganic phase (A: water-0.02% formic acid): 0.2 mL of chromatographic formic acid was diluted to 1 L with ultrapure water and sonicated. Organic phase (B: methanol): 100% chromatographic methanol.

[0106] 2. Detection Method

[0107] 2.1 Sample Pretreatment

[0108] (1) Grind the plant sample in liquid nitrogen until it is pulverized, accurately weigh the sample (accurate to 0.0001 g) into a test tube, add 10 times the volume of chromatographic acetonitrile, and add 80 μL of internal standard stock solution;

[0109] (2) Extract by ultrasonication at 4℃ for 30 min, centrifuge at 4℃, 5,000 rpm for 10 min, and collect the supernatant;

[0110] (3) Add 5 times the volume of chromatographic acetonitrile to the precipitate and repeat the above extraction steps.

[0111] (4) Combine the supernatants obtained from the two centrifugations, add 35 mg C18 and 20 mg GCB, shake for 30 s, centrifuge at 5,000 rpm for 10 min at 4℃, and take the supernatant;

[0112] (5) Dry the supernatant with nitrogen, redissolve it with 200 μL methanol and 200 μL ultrapure water, centrifuge at 12,000 rpm for 5 min at 4℃, take the supernatant, filter it through a 0.11 μm organic phase filter membrane, and place it in a -20℃ refrigerator for instrument testing.

[0113] 2.2 Chromatographic conditions

[0114] Chromatographic column: Poroshell 120 EC-C18 reversed-phase column (2.1 × 100 mm, 2.7 μm); guard column: Poroshell 120 EC-C18 (2.1 × 5 mm, 2.7 μm); chromatographic conditions: column temperature: 40℃; flow rate: 0.3 mL / min; injection volume: 10 µL; mobile phase: A:B = (water - 0.02% formic acid): (chromatographic methanol). The gradient elution program is shown in Table 1.

[0115] Table 1 Gradient elution program

[0116]

[0117] 2.3 Mass Spectrometry Conditions

[0118] Ionization mode: Electrospray ionization source (ESI), multi-stage reaction monitoring (MRM), positive and negative ion switching scan.

[0119] Backflush drying gas: 120 mL / min;

[0120] Mass spectrometer interface heating (HSID Temperature): 300℃;

[0121] Nebulizer Gas: 240 mL / min;

[0122] Ionization voltage (ElectroSpray V): +5500 V / -5000 V;

[0123] Ion source temperature: 300℃.

[0124] The multi-stage reaction monitoring parameters are shown in Table 2.

[0125] Table 2 Multistage reaction monitoring parameters

[0126]

[0127] Note: Mark The ions are quantitative.

[0128] 2.4 Experimental Data and Analysis

[0129] Calculation formula:

[0130] ;

[0131] In the formula: c: concentration of the target compound in the solution, ng / mL; V: extraction volume, mL; m: sample mass, g; ω: content of the target compound per gram of sample (ng / g).

[0132] 3. Experimental Results

[0133] (1) The effects of the two treatment groups on the growth indicators of tobacco seedlings are as follows: Figures 4 - 7 As shown. Figure 7 The left side of the image shows the FJ5 treatment group, and the right side shows the control group.

[0134] Depend on Figures 4 - 7 It was found that under acidic soil conditions, the application of strain FJ5 could significantly promote the growth of tobacco seedlings, including plant height, aboveground part and root fresh weight. The treatment increased by 39.39%, 21.62% and 53.84% respectively compared with the control, and the differences between the treatments were significant (p<0.01), especially the root biomass accumulation.

[0135] (2) The results of endogenous hormone detection in the two treatment groups are as follows: Figure 8 As shown.

[0136] As shown in Figure 8, under acidic soil conditions, the application of FJ5 bacterial suspension altered the secretion of endogenous hormones in tobacco seedlings to varying degrees. The content of the growth hormone IAA increased slightly, by 2.54% compared to the control treatment, with a significant difference between treatments. Compared to the control treatment, the contents of jasmonic acid and salicylic acid showed extremely significant increases and decreases, respectively (p<0.001). JA and SA play key roles in plant defense signaling pathways. In plant immunity, the endogenous hormones salicylic acid (SA) and jasmonic acid (JA) antagonize each other, enabling plants to flexibly defend against different types of harmful organisms. The results of this study indicate... R. qingshengii FJ5 stimulates the host's defense response through an antagonistic pattern of extremely significant increases in JA and extremely significant decreases in SA, enhancing the tobacco's ability to resist biotic or abiotic stresses, and has important application potential in promoting the green and high-quality development of tobacco leaves.

[0137] Example 4 R. qingshengii FJ5 enhances the antioxidant capacity of tobacco and improves carbon and nitrogen metabolism.

[0138] The pretreatment is the same as in Example 3, specifically:

[0139] R. qingshengii The method for preparing FJ5 bacterial suspension is the same as in Example 3.

[0140] Soil tested: Soil was collected from a rice-tobacco rotation field in Jianyang, Nanping, Fujian Province. The soil was acidic (pH=5.16). Tobacco seedlings of variety K326 with a growth period of 30 days were transplanted into pots (9 cm in diameter) containing the acidic soil. After 5 days of recovery, 10 mL of FJ5 bacterial suspension (concentration 1×10⁻⁶) was collected. 8The inoculation solution (cfu / mL) was applied to the rhizosphere soil of the tested tobacco seedlings via root irrigation. An equal volume of sterile water was used as a control. Each treatment consisted of 10 seedlings, with three replicates. The inoculated tobacco seedlings were then cultured in an artificial climate chamber at 28°C and 70% relative humidity, under routine management.

[0141] 1. Twenty days after inoculation, root tissue samples were collected from tobacco seedlings of each treatment. The activities of three antioxidant enzymes—superoxide dismutase (SOD), catalase (CAT), and polyphenol oxidase (PPO)—and the content of reactive oxygen species in the roots were determined using a kit method. Leaves (upper part) of FJ5 and CK treatments were collected, and changes in carbon and nitrogen metabolism were determined using a kit method. Each treatment was replicated three times.

[0142] The specific measurement method is as follows:

[0143] 1. Activities of tobacco defense enzymes SOD, CAT, and PPO

[0144] The assays were performed using SOD kits, CAT kits, and PPO kits (Beijing Solarbio Science & Technology Co., Ltd.), with the procedures following the kit instructions.

[0145] 2. Determination of Reactive Oxygen Species (ROS) Content

[0146] The ROS assays were performed using a reagent kit (Beijing Solarbio Science & Technology Co., Ltd.), and the procedures were performed in accordance with the kit's instruction manual.

[0147] 3. Experimental Results

[0148] The effects of strain FJ5 on the antioxidant capacity of tobacco plants are shown in Table 3. Figure 9 As shown.

[0149] Table 3. Effects of FJ5 strain on the antioxidant capacity of tobacco plants

[0150]

[0151] R. qingshengii The activities of antioxidant enzymes, including SOD, CAT, and PPO enzymes, in the roots of tobacco seedlings changed after FJ5 treatment. The results are shown in Table 3 and 4. Figure 9As shown, SOD activity in tobacco roots was significantly increased to 189.09 U / g (average of three replicates), a 24.89% increase compared to the control (CK) treatment, with highly significant differences between treatments. CAT activity in tobacco roots was also significantly increased to 335.34 U / g (average of three replicates), a 204.17% increase compared to the CK treatment. PPO activity in tobacco roots was significantly enhanced to 112.49 U / g (average of three replicates), a 19.77% increase compared to the CK treatment. Plant root SOD and CAT enzymes are the main enzyme systems for eliminating reactive oxygen species (ROS), participating in two aspects of plant activity: firstly, normal plant growth and morphogenesis, playing a role in plant growth and development; and secondly, related to plant stress and disease resistance, serving as important protective enzymes in plant protective enzyme systems and crucial parameters for evaluating plant stress resistance. These ROS scavenging mechanisms mediated by antioxidant enzymes are the first line of defense for plants against stress, directly reflecting the impact of stress on plants. Effective antioxidant capacity is essential to mitigate the negative impact of stress on plant metabolism and growth. These results indicate that FJ5 ​​inoculation can increase the activity of SOD and CAT enzymes in tobacco roots and reduce ROS content, effectively scavenging excess reactive oxygen species and reducing ROS damage to the host plant, thus significantly improving the host plant's disease resistance. Polyphenol oxidase (PPO) plays an important role in plant secondary metabolism, its main function being pigment production and the biosynthesis of various phenolic compounds, including flavonoids and lignin. This study shows that FJ5 ​​inoculation can increase PPO activity in tobacco plants, stimulate the production of phenolic compounds in soybeans, thereby increasing the substrate level for PPO activity, exerting a defensive effect, and ultimately improving the stress resistance of tobacco seedlings.

[0152] two, R. qingshengii FJ5 enhances carbon and nitrogen metabolism in tobacco.

[0153] 1. Assay of sucrose synthase (SS) activity

[0154] Twenty days after inoculation, the test material consisted of upper tobacco leaves treated with FJ5 and CK. The leaves were homogenized on ice at a ratio of leaf weight (g): extract volume (mL) of 1:(5-10) (approximately 0.1g tissue was added to 1 mL of extract). The mixture was then centrifuged at 8000g, 4℃ for 10 min, and the supernatant was collected and placed on ice for testing. The assay was performed using a kit method (Beijing Solarbio Biotechnology Co., Ltd.). Sucrose synthase (SS) activity was calculated.

[0155] Unit definition: One unit of enzyme activity is defined as the amount of sucrose produced per gram of tissue per minute.

[0156] SS activity (U / g mass) = (C standard tube × V1 × ΔA measured ÷ ΔA standard) ÷ (W × V1 ÷ V2) ÷ T;

[0157] Where: C standard tube: standard tube concentration, 500µg / mL; V1: sample volume added to the reaction system, 0.01mL; V2: extraction liquid volume added, 1mL; W: sample fresh weight, g; T: reaction time, 10 min.

[0158] 2. Nitrate reductase (NR) activity assay

[0159] Sample pretreatment was the same as described in section 1 above, and the NR kit was used for determination.

[0160] Based on sample quality: Definition of enzyme activity unit: The amount of 1 μmol NADH consumed per gram of sample per hour is defined as one NR activity unit.

[0161] NR activity (U / g mass) = [ΔA×V total ÷ (ε×d)×106] ÷ (W ÷ V extract × V sample) ÷ T;

[0162] Where: Vreaction total: volume of the reaction system, 2 × 102 -4 L; Vsample: Sample volume aspirated, 0.012 mL; Vextract: Extraction volume added, 1 mL; T: Reaction time, 0.5 h; ε: Molar extinction coefficient of NADH, 6220 L / mol / cm; d: Cuvette path length, 0.6 cm; W: Sample mass, g; 10 6 Unit conversion factor, 1 mol = 10 6 μmol.

[0163] 3. Determination of total soluble sugar, reducing sugar and soluble protein content

[0164] (1) Total soluble sugars

[0165] The anthrone method was used for determination. Preparation of the standard curve: Add 0, 0.2, 0.4, 0.8, 1.2, and 1.6 mL of 100 μg / mL sucrose solution to test tubes, respectively, and add water to a final volume of 2 mL. Add 0.5 mL of anthrone-ethyl acetate reagent and 5 mL of concentrated sulfuric acid to each tube, respectively. After thorough shaking, place the tubes in a boiling water bath for 1 min, and allow them to cool naturally to room temperature. Using a blank as a control, measure the absorbance at a wavelength of 630 nm. Plot the standard curve with absorbance on the x-axis and sugar content on the y-axis, and derive the standard linear equation.

[0166] Extraction of soluble sugars from the sample. Weigh 0.2000 g of the sample into a 15 mL centrifuge tube, add 10 mL of distilled water, extract in a boiling water bath for 30 min, remove and cool, centrifuge, transfer the supernatant to a 25 mL volumetric flask, add another 10 mL of distilled water to the residue, extract in a boiling water bath for 20 min, remove and cool, transfer all to a 25 mL volumetric flask, rinse the centrifuge tube and residue repeatedly, dilute to the mark, filter, and the filtrate is ready for analysis.

[0167] Colorimetric determination. Pipette 2 mL of the sample dilution into a 20 mL graduated test tube. Following the same steps as preparing the standard curve, add anthrone-ethyl acetate reagent and concentrated sulfuric acid solution in sequence, develop the color, and measure the absorbance. Calculate the amount of sugar (μg) using the standard linear equation, and then calculate the sugar content in the test sample.

[0168] Result calculation:

[0169] Total soluble sugar content (mg / g) = ;

[0170] In the formula: C: sugar content of the sample in the colorimetric tube obtained from the standard curve, in μg; V t : Total volume of sample extraction, mL; V: Volume of liquid taken for determination, mL; D: Dilution factor; 0.001: Convert μg to mg; m: Sample weight, g.

[0171] (2) Reducing sugar content

[0172] The 3,5-dinitrosalicylic acid colorimetric method was used. Preparation of the standard curve: 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of 1 mg / mL glucose solution were added to test tubes, and the volume was brought up to 2 mL with distilled water. 1.5 mL of DNS reagent was added, and the mixture was shaken well. The tubes were then incubated in a boiling water bath for 5 min, rapidly cooled to room temperature, and brought to a final volume of 25 mL with distilled water. The tubes were zeroed using a blank tube, and the absorbance was measured at 540 nm. A standard curve was plotted with absorbance on the x-axis and sugar content on the y-axis, and the regression equation was obtained.

[0173] Extraction of reducing sugars. Weigh 0.2000g of sample into a 15mL test tube, add 10mL of distilled water, and extract in a 50℃ water bath for 20min to leach out the reducing sugars. Centrifuge or filter, and filter the extract into a 25mL volumetric flask. Rinse the test tube and residue repeatedly with 10mL of distilled water into the volumetric flask, and dilute to the mark with distilled water. Mix well, filter, and the filtrate is the reducing sugar test solution.

[0174] Colorimetric determination. Pipette 1 mL of sample solution into a 25 mL graduated test tube, add water to make up to 2 mL, add 1.5 mL of DNS reagent, and perform the remaining operations as in the preparation of the standard curve. Measure the absorbance of each tube.

[0175] The formula for calculating the result is as follows:

[0176] Reducing sugar content (mg / g) = ;

[0177] C: Sugar content of the sample in the colorimetric tube, obtained from the standard curve, in mg; V t : Total volume of sample extraction, mL; V: Volume of liquid taken for determination, mL; D: Dilution factor; m: Sample weight, g.

[0178] (3) Protein content

[0179] The protein content was determined using a BCA protein assay kit. Sample extraction: Approximately 0.1 g of leaf tissue from different treatments was weighed and added to 1 mL of extraction buffer (enzyme extraction buffer, distilled water, or physiological saline could be used). The mixture was homogenized on ice, centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was collected. The microplate reader was preheated for 30 min, and the wavelength was adjusted to 562 nm. The reaction mixture was preheated in a 60°C water bath for 30 min. See Table 4 for details.

[0180] Table 4 Experimental Procedures

[0181]

[0182] Results were calculated based on the fresh weight of the sample.

[0183] Protein content (mg / g fresh weight) = y ÷ (V1 ÷ V × W) × D;

[0184] In the formula: y: content in the sample solution obtained from the standard curve; V1: sample volume added to the reaction system, mL; V: volume of extract added, mL; W: sample mass, g; D: dilution factor.

[0185] 4. Experimental Results

[0186] R. qingshengii The effects of FJ5 on nitrate reductase and sucrase activities, such as Figure 10 As shown. R. qingshengii The effects of FJ5 on the content of soluble total sugar, reducing sugar and soluble protein in tobacco are as follows: Figures 11 - 13 As shown.

[0187] Depend on Figures 10 - 13 As shown: Inoculation R. qingshengii After FJ5 treatment, the activities of nitrate reductase and sucrose reductase in tobacco were enhanced; the activities of NR and SS were 0.35 U / g fresh weight and 98.12 U / g fresh weight, respectively, while the activities of the two enzymes in the control group were 0.27 U / g fresh weight and 77.47 U / g fresh weight, respectively, with growth rates of 28.79% and 21.05%. At the same time, the total soluble sugar content after FJ5 treatment increased to 3.53 mg / g, an increase of 6.88% compared with the control group, and the reducing sugar content increased significantly compared with the control group, reaching 1.58 mg / g, with a growth rate of 16.28%; while the protein content decreased slightly by 7.86% compared with the control group.

[0188] Carbon (C) and nitrogen (N) are essential elements for plant growth and development. As the structural and functional framework of organic compounds, they drive key biological processes such as photosynthesis, carbohydrate metabolism, and nitrogen assimilation. Carbon metabolism fixes CO2 through photosynthesis, producing soluble sugars such as glucose, sucrose, and fructose. These not only provide energy for plant growth and development as respiratory substrates but also, as metabolic intermediates, are resynthesized into other substances through metabolic pathways, such as cellular structural components, storage substances, and the carbon skeletons of amino acids and proteins, providing energy and structural materials for plants. Nitrogen metabolism involves the absorption of nitrates (NO3). - ) and ammonium salts (NH4) + Sugars synthesize amino acids, proteins, and other substances, driving cell division and organ development, thereby enhancing plant resistance to stress. Sugars not only provide energy for plant growth and development as respiratory substrates, but also, as metabolic intermediates, are resynthesized into other substances through metabolic pathways, such as structural components of cells, storage substances, and the carbon skeletons of amino acids and proteins. R. qingshengii FJ5 enhances the activity of nitrate reductase and sucrose synthase in tobacco, promotes the synthesis of soluble sugars and increases the content of reducing sugars, improves the osmotic regulation capacity of leaf tissue cells, alleviates damage to membrane system integrity under acidic soil stress, and enhances the leaves' defense stress resistance to cope with adverse environments. Simultaneously, it promotes carbon assimilation and nitrogen absorption, accelerates nutrient accumulation, increases the nutrient requirements of tobacco plants, promotes healthier tobacco growth, and enhances stress resistance to cope with adverse environments.

[0189] Example 5 R. qingshengii FJ5 improves the activity and nutrients of acidified soil.

[0190] The pretreatment is the same as in Example 3, specifically: R. qingshengii The method for preparing FJ5 bacterial suspension is the same as in Example 3.

[0191] Soil tested: Soil was collected from a rice-tobacco rotation field in Jianyang, Nanping, Fujian Province. The soil was acidic (pH=5.16). Tobacco seedlings of variety K326 with a growth period of 30 days were transplanted into pots (9 cm in diameter) containing the acidic soil. After 5 days of recovery, 10 mL of FJ5 bacterial suspension (concentration 1×10⁻⁶) was collected. 8 The inoculation solution (cfu / mL) was applied to the rhizosphere soil of the tested tobacco seedlings via root irrigation. An equal volume of sterile water was used as a control. Each treatment consisted of 10 seedlings, with three replicates. The inoculated tobacco seedlings were then cultured in an artificial climate chamber at 28°C and 70% relative humidity, under routine management.

[0192] Twenty days after inoculation, rhizosphere soil samples were collected from tobacco seedlings in each treatment, and the concentration of aluminum ions, urease, catalase, sucrase, phosphatase activities, and the content of organic nitrogen, available phosphorus, available potassium, and organic carbon nutrients in the rhizosphere soil were measured.

[0193] 1. Determination of the effect of strain FJ5 on aluminum ion content in acidified soils of rice-tobacco rotation.

[0194] Aluminum was extracted from the rhizosphere soil of tobacco plants under different treatments using four chemical extraction liquids: KCl (1 mol / L), NH4Ac (1 mol / L, pH 4.8), HCl (1 mol / L), and NaOH (0.5 mol / L). Aluminum ions adsorbed by soil colloids were extracted using 1 mol / L KCl; acid-soluble inorganic aluminum was extracted using 1 mol / L HCl; the aluminum extracted using neutral salts was mainly trivalent aluminum (Al), also known as exchangeable aluminum. The aluminum ion concentration in the filtrate, i.e., the aluminum content, was further determined by colorimetry (Ma Huichang, Comparison of Active Aluminum Species in Coniferous Forest Soils of Different Regions, *Environmental Chemistry*). The results showed the effect of strain FJ5 on the aluminum content of acidified soil as follows: Figure 14 As shown. Figure 14 The results showed that in acidic soil with a pH of 5.16, the aluminum content in the rhizosphere soil of tobacco plants decreased by 10.59% after inoculation with FJ5 compared with the control (CK) treatment, and the difference between the treatments was significant (p<0.05). This indicates that FJ15 can reduce the aluminum content in acidic soil, thereby alleviating the damage of soil aluminum stress to the root system of tobacco plants, and has great application value for the healthy growth of tobacco.

[0195] 2. FJ5 enhances the activity of enzymes in acidifying soils during rice-tobacco rotation.

[0196] 2.1 Urease activity

[0197] The indophenol colorimetric method was used. Standard curve preparation: Take 1, 3, 5, 7, 9, 11, and 13 mL of nitrogen working solution into 50 mL colorimetric tubes, respectively, and prepare a blank control tube. Add distilled water to a final volume of 20 mL. Then add 4.0 mL of sodium phenolate solution and 3 mL of sodium hypochlorite solution, shaking constantly after each addition. After 20 minutes, allow the solution to develop color and dilute to volume. Measure the color at 578 nm using a spectrophotometer within 1 hour. Plot a standard curve based on the optical density versus solution concentration.

[0198] Weigh 5.00g of soil sample and place it in a 50mL Erlenmeyer flask. Add 1.0mL of toluene. After 15 minutes, add 10mL of 10% urea solution and 20mL of pH 6.7 citrate buffer. Shake well and incubate at 37℃ for 24 hours. After incubation, remove the sample and centrifuge at 6000rpm for 10 minutes. Take 3.0mL of supernatant (2.0mL for fresh soil samples; 0.5mL for air-dried soil and soil samples stored for one month) into a 50mL graduated test tube, and then perform colorimetric determination according to the standard curve plotting method.

[0199] Results calculation: Urease activity was expressed as the number of milligrams of NH3-N in 1g of soil after 24h.

[0200] NH3-N (mg) = a × b;

[0201] In the formula, a - the NH3-N concentration obtained from the standard curve, mg / mL; b - the coefficient for conversion to 1g of soil.

[0202] Urease activity test results as follows Figure 15 As shown, by Figure 15 The results show that strain FJ5 can significantly increase urease activity in acidic soil. The urease activity after FJ5 bacterial treatment was 0.3069 mg NH3-N / g / 24 h, while the control treatment showed 0.2146 mg NH3-N / g / 24 h, representing a growth rate of 42.96%. Soil urease is the only enzyme catalyzing the hydrolysis of urea, and its activity is positively correlated with soil fertility, namely, the content of organic matter, total nitrogen, and available phosphorus. Soil urease activity is often used to indicate soil nitrogen status. Therefore, the beneficial bacteria strain FJ5 increases urease activity in acidic soil, thereby promoting nitrogen release and increasing nitrogen content and fertility in acidic soil.

[0203] 2.2 Soil acid phosphatase activity

[0204] Construction of standard curve: Take 0, 1, 2, 3, 4, and 5 mL of standard solution, add 1 mL of 2 M CaCl2, 4 mL of 0.2 M NaOH and 4 mL of PBS buffer, and make up to 50 mL with deionized water. Measure the 405 nm OD value colorimetrically and construct the standard curve.

[0205] Acid phosphatase activity assay: Take 1.0 g of soil, add 4 mL of buffer solution, and add 1 mL of 100 mM sodium p-nitrophenyl phosphate solution. Incubate at 30℃ for 30 min. Add 1 mL of 2 M CaCl2 and 4 mL of 0.2 M NaOH to stop the reaction, add 90 mL of deionized water, and measure the OD value of the filtered filtrate at 405 nm. Calculate the phosphatase activity according to the standard curve.

[0206] Soil acid phosphatase activity test results as follows Figure 16 As shown.

[0207] Figure 16 The results showed that strain FJ5 significantly increased acid phosphatase activity in acidic soil. The acid phosphatase activity in the soil treated with strain FJ5 was 2.047 mg P / g / h, while the activity in the control group was 1.741 mg P / g / h, representing a growth rate of 17.57%. Acid phosphatase promotes the dissolution of insoluble phosphorus in soil and is one of the most important influencing factors on plant growth in medium- and low-fertility soils. Therefore, strain FJ5 can improve the phosphorus-solubilizing capacity of acidic soil, thereby promoting tobacco seedling growth.

[0208] 2.3 Sucrase activity in acidic soil

[0209] The activity of sucrase is expressed by measuring the amount of reducing sugar.

[0210] (1) Draw the standard curve

[0211] Pipette 0, 25, 50, 75, 100, 125, 150, and 200 µL of glucose standard solution into test tubes, respectively. Add distilled water to a final volume of 250 µL. Add 0.75 mL of DNS reagent and mix well. Incubate in a boiling water bath for 5 min, then remove and cool to room temperature in a cold water bath. Make up to 25 mL. Zero the volume with a blank tube and measure the absorbance at 508 nm. Plot a standard curve with absorbance on the ordinate and glucose concentration on the abscissa.

[0212] (2) Assay of sucrase activity

[0213] Weigh 2.0 g of soil sample and place it in a 50 mL Erlenmeyer flask. Add 15 mL of 8% sucrose solution, 5 mL of pH 5.5 phosphate buffer, and 5 drops of toluene. Shake well and incubate at 37℃ for 24 h. Filter quickly. Transfer 0.1 mL of the filtrate to a test tube, bring the volume to 0.25 mL with water, add 0.75 mL of DNS reagent, heat in boiling water for 5 min, and then cool under running tap water for 3 min. Make up to 25 mL. Measure the colorimetric value at 508 nm using a microplate reader. A soil-free control is required for the entire experiment.

[0214] (3) Sucrase activity is expressed as the number of milligrams of glucose produced per gram of dry soil over 24 hours.

[0215] Sucrase activity = (a sample - a soil-free - a matrix-free) × n / m;

[0216] a_sample, a_soil-free, and a_inorganic matter represent the number of milligrams of glucose obtained from the standard curve, respectively; n: fractional fraction; m: soil weight.

[0217] Experimental results are as follows Figure 1 As shown, strain FJ5 increased the activity of sucrase in the rhizosphere of tobacco plants in acidic soil to 16.61 mg / g, while the activity in the treatment group was 13.07 mg / g, representing a growth rate of 27.17%. Soil sucrase, also known as invertase, catalyzes the breakdown of sucrose in the soil into glucose and fructose, which are the direct energy source for plant growth. Higher sucrase activity indicates faster decomposition and transformation of soil organic matter, smoother nutrient cycling, and higher soil fertility. Therefore, FJ5 improved the fertility of the rhizosphere soil of tobacco plants under acidic soil conditions, which is beneficial to tobacco plant growth.

[0218] 3. Effects of FJ15 on nutrient content in acidified soils from rice-tobacco rotation

[0219] 3.1 Effects of FJ15 on organic matter and microbial carbon (Bc) content in acidic soils

[0220] The organic matter content was determined using the potassium dichromate-sulfuric acid oxidation method. The main steps were as follows: Accurately weigh 0.50 g of each treated soil sample (sieved and air-dried) into a hard glass test tube, and add 10.00 mL of 0.4 mol / L potassium dichromate-sulfuric acid solution. Heat the test tube in an oil bath at 185℃-190℃. When the solution begins to boil, continue heating for 5 min ± 0.5 min, then remove and cool. Transfer the contents of the test tube to a 100 mL Erlenmeyer flask, and rinse the test tube with water to ensure the total volume of the solution in the flask is controlled at 50 mL. Add 3 drops of o-phenanthroline indicator, and titrate the remaining K₂Cr₂O₇ with ferrous sulfate standard solution.

[0221] Result Calculation

[0222]

[0223] In the formula:

[0224] OM: Mass fraction of soil organic matter, in grams per kilogram (g / Kg); V0: Volume of ferrous sulfate standard solution consumed in the blank test, in milliliters (mL); V: Volume of ferrous sulfate standard solution consumed in the sample determination, in milliliters (mL); c: Concentration of ferrous sulfate standard solution, in mol per liter (mol / L); 3: Molar mass of 1 / 4 carbon atom (g / mol); 1.724: Coefficient for converting organic carbon to organic matter; 1.10: Oxidation correction coefficient; m: Mass of the sample, in grams (g); 1000: Conversion to content per kilogram.

[0225] Bc content was determined using the chloroform fumigation extraction method. 5.00 g of fresh soil samples from different treatments were weighed into 100 mL beakers and placed in a desiccator containing 50 mL of NaOH solution and 50 mL of ethanol-free chloroform. A small amount of petroleum jelly was added for sealing, and the mixture was vacuum-evacuated until the chloroform boiled and maintained at that temperature for at least 2 minutes. The desiccator valve was closed, and the samples were placed in the dark at 25°C for 24 hours. The valve was then opened, and the soil samples were removed until the chloroform odor was no longer detectable. An equal amount of soil was not fumigated as a control. After fumigation, all soil samples were transferred to 250 mL Erlenmeyer flasks, and 25 mL of potassium sulfate solution was added. The samples were extracted on a shaker for 30 minutes and then filtered. The control treatment was performed using the same potassium sulfate solution extraction method. The Bc content of the extract was directly determined using a carbon and nitrogen analyzer.

[0226] The formula for calculating microbial biomass (Bc) is: ω(C) = Ec × 2.2

[0227] Where: ω(C): mass fraction of microbial biomass carbon, mg / kg; Ec: difference between organic carbon content in fumigated soil sample and organic carbon content in unfumigated soil sample, mg / kg; 2.2: correction coefficient.

[0228] Experimental results are as follows ​ As shown, strain FJ5 increased the organic matter content in the rhizosphere of tobacco plants in acidic soil. The organic matter content in the treatment group treated with this strain was 118.59 g / kg, while the organic matter content in the control group was only 93.42 g / kg, representing a growth rate of 26.95%. Strain FJ5 also increased the microbial carbon content in the rhizosphere of tobacco plants in acidic soil. The microbial carbon content in the treatment group treated with this strain was 1919.80 mg / kg, while the microbial carbon content in the control group was only 1576.27 mg / kg, representing a growth rate of 21.79%. Soil organic matter is a core indicator for measuring soil fertility. Organic matter contains abundant carbon, nitrogen, phosphorus, and other nutrients. Through microbial decomposition, these elements are converted into inorganic salts (such as ammonium nitrogen, nitrate nitrogen, and phosphate ions), which can be absorbed and utilized by plant roots. This is a continuous and stable nutrient supply process. Meanwhile, soil microbial carbon is the most active and core component of organic matter, and its content is a key indicator for measuring soil health. Therefore, strain FJ5 improves soil fertility and health by increasing soil organic matter and microbial carbon content, thereby promoting tobacco plant growth and increasing tobacco leaf yield.

[0229] 3.2 FJ5 bacteria increase the nitrogen and phosphorus nutrient content in acidic soils

[0230] (1) Soil hydrolyzable nitrogen content was determined by sodium hydroxide alkaline hydrolysis method.

[0231] Weigh 2.0 g of sieved and air-dried soil sample and spread it evenly in the outer chamber of a diffusion dish. Add 1.0 g of zinc-ferrous sulfate to the soil sample in the outer chamber, and prepare a reagent blank as a control. Add 3 mL of 20 g / L boric acid indicator solution to the inner chamber of the diffusion dish. Apply alkaline adhesive, cover with frosted glass, and rotate several times to ensure the frosted glass adheres completely to the edge of the diffusion dish. Open the slit between the frosted glass and the diffusion dish, add 10 mL of 1.8 mol / L sodium hydroxide solution to the outer chamber of the diffusion dish, and then tightly cover with frosted glass. Incubate at a constant temperature of 40℃ for 24 h. Titrate the amount of ammonia absorbed in the boric acid in the inner chamber with 0.01 mol / L hydrochloric acid standard solution; the endpoint is reached when the color changes from blue-purple to red. Perform reagent blank determination simultaneously with sample determination.

[0232] The hydrolyzable nitrogen content of soil samples is calculated using the following formula:

[0233] ;

[0234] In the formula: W N: Hydrolyzable nitrogen content, in mg / kg; V0: Volume of acid standard titration solution consumed in blank test, in milliliters (mL); V: Volume of acid standard titration solution consumed in sample determination, in milliliters (mL); C: Concentration of hydrochloric acid standard solution, in mol / L; 14: Molar mass of N, in mg / mmol; m: Mass of sample, in g.

[0235] (2) The ammonium nitrogen content in the soil was determined by the indophenol blue colorimetric method. 7.0 g of fresh soil sample was weighed and placed in a 100 mL Erlenmeyer flask. 35 mL of potassium chloride solution was added, and the mixture was shaken at 180 r / min for 1 h. The sample was then filtered and stored in a refrigerator for later use. 5 mL of soil leachate was taken and placed in a 25 mL volumetric flask. 2.5 mL of phenol solution and 2.5 mL of sodium hypochlorite alkaline solution were added. The mixture was allowed to stand at room temperature (around 20 °C) for 1 h. 0.5 mL of masking agent was added, and the volume was adjusted to the mark with water. The absorbance was measured at a wavelength of 625 nm.

[0236] Standard curve determination: Pipette 0.0, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of standard solution into 25 mL volumetric flasks, respectively, and bring the volume to 5 mL with potassium chloride solution. Perform colorimetric determination using the same standard determination procedure.

[0237] Soil ammonium nitrogen content (mg / kg) = ρ × V × ts / m;

[0238] In the formula: P: ammonia nitrogen concentration of the colorimetric solution (mg / L); V: volume of the colorimetric solution (mL); ts: fractionation factor; m: sample mass (g).

[0239] (3) The available phosphorus content in acidic soil was determined by Bray 1 method.

[0240] Weigh 2.5 g of sieved and air-dried soil samples from different treatments into a 150 mL Erlenmeyer flask, add 25 mL of hydrochloric acid-ammonium fluoride extractant, shake at 25℃ and 180 r / min for 30 min, and centrifuge the filtrate for later use.

[0241] Construction of the standard curve: Pipette 0 mL, 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL of phosphorus standard solution into 25 mL volumetric flasks, respectively. Add 5 mL of ammonium fluoride-hydrochloric acid extraction agent and 5 mL of boric acid solution, shake well, then add 2 drops of dinitrophenol indicator. Adjust the solution to just turn slightly yellow with sulfuric acid solution or ammonia solution. Add 2.5 mL of molybdenum antimony colorimetric reagent, dilute to the mark with water, shake well, and let stand at room temperature for 30 min. Then, perform colorimetric determination at a wavelength of 700 nm, zeroing the instrument with the zero point of the standard solution, and construct the standard curve.

[0242] Pipette an appropriate amount of sample solution into a 25 mL volumetric flask, add 5 mL of boric acid solution, shake well, then add 2 drops of dinitrophenol indicator. Adjust the solution to a slightly yellow color with sulfuric acid solution or ammonia solution. Add 2.5 mL of molybdenum antimony colorimetric reagent, dilute to volume with water, and let stand at room temperature for 30 min. Then, zero the instrument at a wavelength of 700 nm using the zero point of the standard solution and perform colorimetric determination. Simultaneously, determine the blank solution.

[0243] Results calculated: Available phosphorus content in soil (mg / kg) =

[0244] Where: ρ: mass concentration of P obtained from the working curve (mg / L); V: volume for color development (mL); ts: fractionation factor; m: sample mass (g); 10 3 : Convert mL to L; 1000: Convert to P content per kg.

[0245] The results of the detection of hydrolyzable nitrogen, ammonium nitrogen and available phosphorus content in the rhizosphere soil of tobacco plants are as follows: ​ As shown, after adding FJ5 bacteria, the contents of hydrolyzable nitrogen, ammonium nitrogen, and available phosphorus in the rhizosphere soil of tobacco plants were 342.34 mg / kg, 3.85 mg / kg, and 181.58 mg / kg, respectively; while in the CK treatment, the contents of hydrolyzable nitrogen, ammonium nitrogen, and available phosphorus were 268.56 mg / kg, 3.29 mg / kg, and 129.32 mg / kg, respectively; the growth rates were 27.47%, 16.94%, and 40.41%, respectively. Soil hydrolyzable nitrogen, ammonium nitrogen, and available phosphorus are all key indicators of available nutrients for evaluating soil fertility. Soil hydrolyzable nitrogen (also known as alkaline available nitrogen) represents the nitrogen source in the soil that can be mineralized and absorbed and utilized by plants in the near term, reflecting the soil's nitrogen supply capacity and its recent nitrogen supply intensity. Soil ammonium nitrogen is a form of inorganic nitrogen in the soil and an important intermediate product in the nitrogen cycle; it is one of the inorganic nitrogen sources that plants can directly absorb. Available phosphorus refers to the general term for phosphorus forms in the soil that can be absorbed and utilized by plants in the current season. Phosphorus is easily fixed by the soil (with calcium). 2+ Fe 3+ Al 3+ (The phosphorus content can combine to form insoluble compounds), but a high total phosphorus content does not necessarily mean sufficient phosphorus for plants. Available phosphorus is a key indicator of phosphorus fertility; phosphorus deficiency severely inhibits seedling growth and root development. The results of this experiment show that the use of FJ5 increases the content of hydrolyzable nitrogen, ammonium nitrogen, and available phosphorus in acidified soils to varying degrees, especially effectively increasing the content of available phosphorus in the soil and promoting the development of tobacco roots and aboveground parts.

[0246] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Rhodococcus ( Rhodococcus qingshengii The preservation number of Rhodococcus FJ5 is CGMCC No. 32631.

2. A microbial inoculant, characterized in that, Includes the Rhodococcus FJ5 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The viable count of Rhodococcus FJ5 in the microbial agent is ≥1×10⁻⁶. 8 cfu / mL.

4. A method for preparing the microbial inoculant according to claim 2 or 3, characterized in that, include: The Rhodococcus FJ5 was cultured in a culture medium to obtain a microbial inoculum.

5. The preparation method according to claim 4, characterized in that, The culture temperature is 16~34℃; the pH value of the culture medium is 5.0~8.

0.

6. The application of the Rhodococcus FJ5 of claim 1 or the microbial agent of claim 2 or 3 in the prevention and control of tobacco bacterial wilt.

7. The application of the Rhodococcus FJ5 of claim 1 or the microbial agent of claim 2 or 3 in promoting tobacco growth and / or improving tobacco's resistance to stress under acidic soil conditions.

8. The application of the Rhodococcus FJ5 of claim 1 or the microbial agent of claim 2 or 3 in reducing aluminum stress in tobacco in acidic soil.

9. The application of the Rhodococcus FJ5 of claim 1 or the microbial agent of claim 2 or 3 in improving acidic soil.

10. A method for promoting tobacco growth and / or improving acidic rhizosphere soil in tobacco plants, characterized in that, include: Apply the Rhodococcus FJ5 of claim 1 or the microbial agent of claim 2 or 3 to the tobacco root system.

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