Broad-spectrum growth promoting microbial agent combination, bio- agent for tung tree and tea-oil tree and application thereof

Abstract

The application discloses a broad-spectrum growth promoting bacterial agent combination for oil camellia and oil tea, a biological preparation and application thereof, and relates to the field of microbial technology.The endophytic bacteria are separated from healthy oil camellia plants, the abilities of the strains in dissolving phosphorus, fixing nitrogen, producing IAA, producing iron carrier and antagonizing the pathogenic bacteria of oil tea anthracnose are determined, the onion Burkholderia and Aspergillus niger with excellent comprehensive growth promoting functions are screened to prepare a combined bacterial agent, and the combined bacterial agent is verified to have a significant growth promoting effect on the oil camellia and the oil tea through a potting experiment.The combined bacterial agent promotes the growth, nutrient absorption and root development of the oil tea, significantly improves the enzyme activities related to the carbon, phosphorus and nitrogen cycles of the soil, and realizes the overall optimization of the "plant-soil" system.The application finds a broad-spectrum mixed bacterial agent suitable for the artificial planting of the oil camellia and the oil tea in a rocky desertification area, effectively improves the adaptability of the plants in the rocky desertification habitat, and provides a new microbial technical approach for the collaborative repair of multiple tree species in the area.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically, it relates to a combination of broad-spectrum growth-promoting bacteria for tung oil and camellia oil, biological agents and their applications. Background Technology

[0002] As an ecologically and economically important tree species in rocky desertification areas, the tung tree (Vernicia fordii) possesses abundant endophytic microbial resources. These endophytic fungi have co-evolved with the host plant over a long period, potentially developing unique functions adapted to the barren habitats of rocky desertification, such as phosphorus solubilization, nitrogen fixation, and plant hormone production. They represent a potential source for discovering superior growth-promoting strains. Previous studies have shown that tung tree endophytic fungi can significantly promote the growth of the host plant in phosphorus-poor soils; however, whether the same strain can transcend plant family boundaries and exert similar growth-promoting effects on other plants remains to be systematically verified.

[0003] Camellia oleifera is an important woody oilseed tree species in southern my country and a key economic forest species promoted by the country. However, in rocky desertification areas, Camellia oleifera suffers from low survival rate, slow growth, and delayed fruiting, which seriously restricts the sustainable development of the Camellia oleifera industry in these areas.

[0004] Therefore, there is an urgent need to develop a combination of broad-spectrum growth-promoting bacteria and biological agents for the growth of tung oil trees and camellia oleifera, so as to effectively promote the growth of tung oil trees and camellia oleifera in rocky desertification and barren habitats, and improve the development of the tung oil and camellia oleifera industry in rocky desertification and barren environments. Summary of the Invention

[0005] This invention provides a broad-spectrum growth-promoting microbial agent combination for tung oil trees and camellia oleifera, the microbial agent combination including Burkholderia cepacia and Aspergillus niger, the microbial agent combination promotes the plant height growth, fresh weight and dry weight accumulation, nitrogen and phosphorus absorption and plant root development of tung oil trees and camellia oleifera.

[0006] Preferably, Burkholderia cepacia and Aspergillus niger are mixed in a mass ratio of 1:1.

[0007] Preferably, the microbial agent combination is a liquid microbial agent, a wettable powder, or a solid microbial agent.

[0008] The present invention also provides a biological agent for tung oil and camellia oil, comprising the microbial agent combination described above.

[0009] This invention also provides an application of a broad-spectrum growth-promoting fungal agent combination for tung oil and camellia oil, comprising the step of preparing the above-mentioned fungal agent combination with a concentration greater than or equal to 1×10⁻⁶. 8 A bacterial suspension of CFU / mL was applied to the roots of tung oil trees or camellia trees.

[0010] The present invention has the following beneficial effects:

[0011] 1. This invention uses healthy *Tung tree* plants from the rocky desertification area of ​​Qingping Town State-owned Forest Farm in Xiangxi as materials. Endophytic bacteria were isolated from roots, stems, and leaves using a combination of tissue surface disinfection and plate culture. Molecular identification was performed using 16S rRNA and ITS sequence sequencing. The phosphorus-solubilizing, nitrogen-fixing, IAA production, siderophore production, and antagonistic abilities against *Camellia oleifera* anthracnose pathogens of each strain were systematically measured to screen strains with excellent comprehensive growth-promoting functions. Pot experiments were conducted to verify the growth-promoting effects of different mixed strains. The results showed that the growth-promoting effect of mixed strains was significantly better than that of single strains, exhibiting a synergistic effect of "1+1>2". The mixed strains showed significant combination specificity in promoting the growth of *Tung tree*. *Burkholderia cepacia* and *Aspergillus niger* achieved multidimensional synergistic enhancement in biomass accumulation, nutrient absorption, and root development through functional complementarity and metabolic synergy, making them the optimal mixed strain combination screened in this study.

[0012] 2. This invention experimentally verified that the mixed bacterial strains have a combination-specific effect on the growth-promoting effects of Camellia oleifera on biomass, nutrient absorption, and root development. The treatment with Burkholderia cepacia and Aspergillus niger showed the best overall performance. Through functional complementarity and metabolic synergy, the combined bacterial agent of Burkholderia cepacia and Aspergillus niger not only promotes the growth, nutrient absorption, and root development of Camellia oleifera, but also significantly enhances the activity of enzymes related to soil carbon, phosphorus, and nitrogen cycles, thus achieving overall optimization of the "plant-soil" system.

[0013] 3. This invention isolates and screens superior endophytic strains of tung oil tree and selects functionally complementary strains to prepare a combined microbial agent. This not only has a good growth-promoting effect on tung oil tree but also achieves cross-host growth promotion on camellia oleifera, effectively improving the growth and development of camellia oleifera. This invention has found a broad-spectrum mixed microbial agent suitable for the artificial planting of tung oil tree and camellia oleifera in rocky desertification areas, effectively improving the adaptability of tung oil tree and camellia oleifera in rocky desertification habitats, and providing a new microbial technology approach for the synergistic restoration of multiple tree species in this region. Attached Figure Description

[0014] Figure 1 These are photographs of the isolation, culture, and colony morphology of endophytic bacteria from the tung tree in Experimental Example 1 of this invention.

[0015] Figure 2 Photographs showing the growth of tung oil trees after inoculation with the mixed microbial agent in Experiment Example 4 of this invention;

[0016] Figure 3 This is a comparative result of the accumulation of tung oil biomass after inoculation with mixed microbial agents in Experiment Example 4 of the present invention;

[0017] Figure 4 This is a statistical analysis of the effect of mixed bacterial inoculation on nitrogen, phosphorus, and potassium absorption in tung oil trees in Experiment Example 4 of this invention.

[0018] Figure 5 This is a photograph of the root system of the tung tree after inoculation with the mixed microbial agent in Experiment Example 4 of the present invention.

[0019] Figure 6 This is a statistical analysis of the root system indicators of tung trees after inoculation with the mixed microbial agent in Experiment Example 4 of the present invention.

[0020] Figure 7 Photographs showing the growth of Camellia oleifera after inoculation with the mixed microbial agent in Experiment Example 5 of this invention;

[0021] Figure 8 This is a comparative statistical analysis of the accumulation of Camellia oleifera biomass after inoculation with mixed microbial agents in Experimental Example 5 of the present invention.

[0022] Figure 9 This is a statistical analysis of the effect of mixed bacterial inoculation on nitrogen, phosphorus, and potassium absorption in Camellia oleifera in Experimental Example 5 of this invention;

[0023] Figure 10 This is a photograph of the root system of Camellia oleifera after inoculation with the mixed microbial agent in Experimental Example 5 of the present invention;

[0024] Figure 11 This is a statistical analysis of root indicators of Camellia oleifera after inoculation with mixed microbial agents in Experimental Example 5 of the present invention;

[0025] Figure 12 This is a statistical analysis of the effect of mixed bacterial inoculation on the rhizosphere soil enzyme activity of Camellia oleifera in Experiment Example 5 of this invention. Detailed Implementation

[0026] The specific embodiments of this disclosure are described in detail below with reference to the accompanying drawings. Specific details of the implementation are set forth in the following description to facilitate understanding of the invention. Unless otherwise specified, all materials used in this embodiment are commercially available products.

[0027] The culture media used in the various experimental examples of this invention are as follows: LB and PDA media were used for the isolation, purification and culture of endophytic bacteria, with a pH of 7.0; PKO media were used to determine the phosphorus-solubilizing ability of endophytic bacteria, with a pH of 7.0; Assumption nitrogen-free medium was used to determine the nitrogen-fixing ability of endophytic bacteria, with a pH of 7.0; IAA determination medium was used to determine the IAA production ability of endophytic bacteria, with a formula of LB medium supplemented with 100 mg / L of tryptophan, with a pH of 7.0. CAS medium was used to determine the siderophore production ability of endophytic bacteria, with a pH of 7.0. For the specific formulas and preparation methods of the aforementioned culture media, please refer to the literature "Isolation and Screening of Strawberry Endophytic Bacteria and Their Functional Characteristics" (Jie Yujia et al., Zhejiang Journal of Agricultural Sciences, 2025, 37 (12): 2525-2534).

[0028] All reagents used were analytical grade or biochemical reagents, specifically as follows: The molybdenum blue method reagent was used to determine the phosphorus-solubilizing ability of endophytic bacteria, mainly including ammonium molybdate, sulfuric acid, and ascorbic acid; the Kjeldahl nitrogen determination method reagent was used to determine the nitrogen content of plant samples, including concentrated sulfuric acid, catalyst (copper sulfate-potassium sulfate mixed reagent), sodium hydroxide, boric acid, and hydrochloric acid standard solution; the IAA colorimetric method reagent was used to determine the IAA production capacity of endophytic bacteria, including Salkowski reagent (containing FeCl3 and perchloric acid). All of the above reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0029] Experimental Example 1: Isolation and Identification of Endophytic Fungi in Tung Oil Trees

[0030] The tung oil plant samples were collected from the rocky desertification area of ​​the State-owned Forest Farm in Qingping Town, Yongshun County, Xiangxi Tujia and Miao Autonomous Prefecture, Hunan Province (110°23′E, 29°03′N). This area is a typical karst rocky desertification landform, and the soil is limestone soil. The effective phosphorus content was measured to be less than 5 mg / kg, which is a typical phosphorus-deficient soil.

[0031] Isolation and culture: Bacterial isolation was performed using the dilution-spreading method. The sterilized tissue was cut into small segments of about 0.5 cm, ground, and serially diluted with sterile physiological saline. The appropriate concentration of bacterial solution was spread onto LB agar plates and incubated at 37°C for 1-2 days. Fungal isolation was performed using the cutting-inoculation method. The sterilized tung oil tissue was cut into small segments of about 0.5 cm and directly inoculated onto PDA agar plates. Each plate was inoculated with 3-5 segments and incubated at 28°C for 3-5 days.

[0032] Purification and preservation: Single colonies or edge hyphae were selected for purification culture, and the purification was repeated three times to obtain pure strains. The purified strains were then inoculated onto appropriate slant agar plates and stored at 4°C for later use.

[0033] Molecular identification: Genomic DNA was extracted from the strains. Bacteria were sequenced using 16S rRNA gene amplification, and fungi were sequenced using ITS gene amplification. The sequencing results were compared with sequences in the NCBI database to determine the taxonomic position of the strains.

[0034] All measurements were performed in triplicate. Results are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS 26.0 software, and Tukey HSD post-hoc multiple comparisons were also performed (α=0.05).

[0035] To comprehensively compare the multiple growth-promoting potentials of various strains, SPSS 26.0 software was used to standardize the five functional indicators mentioned above. The specific method is as follows: For the three quantitative indicators of phosphorus solubility, nitrogen fixation, and IAA production, the minimum and maximum values ​​of each strain were calculated, and the original values ​​(xx) were converted into standardized scores (x′x′), using the formula x′=(x-xmin) / (xmax-xmin); for siderophore production ability (qualitative), strains with siderophore production ability were assigned a value of 1, and those without were assigned a value of 0; for antagonistic ability, the antagonistic rate (%) was used as the original value for the same range standardization. The five standardized scores were summed to obtain the comprehensive growth-promoting function score for each strain; a higher score indicates a better comprehensive growth-promoting potential. During the standardization calculation, if a strain did not possess a certain function, its original value was recorded as 0.

[0036] Thirty-one endophytic microorganisms (including bacteria and fungi) were successfully isolated and purified from the root, stem, and leaf tissues of *Tung Oil Tree* in rocky desertification areas using a surface sterilization combined with plate culture method. Statistical analysis showed that the colonization of endophytic bacteria in different tissues exhibited significant tissue preference: 28 strains were isolated from the roots, accounting for 90.32% of the total, including 13 fungi and 15 bacteria; 15 strains were isolated from the stems, accounting for 48.39%, including 10 fungi and 2 bacteria (8 fungi and 7 bacteria); and 12 strains were isolated from the leaves, accounting for 38.71%. Eight strains were isolated from roots, stems, and leaves simultaneously. The distribution frequency of specific strains in each tissue is detailed in Table 1.

[0037] Phylogenetic tree constructed based on 16S rRNA and ITS sequences showed that the 31 endophytic bacteria were clearly divided into two major branches: bacteria and fungi. The bacteria mainly clustered in the phyla Proteobacteria and Firmicutes, while the fungi were predominantly Ascomycota, encompassing multiple functional groups. Colony morphology characteristics of some representative strains are shown below. Figure 1 As shown.

[0038] Table 1. Isolation, identification, and tissue distribution of endophytic fungi in tung oil trees.

[0039]

[0040]

[0041]

[0042] Note: In the table, "++" indicates that the strain was isolated 2 or more times in the corresponding tissue, and "+" indicates that it was isolated once.

[0043] Experiment Example 2: Functional determination of endophytic fungi in tung oil trees

[0044] Table 2. Screening results of growth-promoting characteristics of endophytic strains of tung oil tree

[0045]

[0046] Note: Data in Table 2 are "mean ± standard deviation"; different lowercase letters after the data in the same column indicate that the differences are significant at the P<0.05 level after one-way ANOVA and Tukey HSD multiple comparisons. Blank spaces indicate that the strain does not have this ability.

[0047] 2.1 Determination of phosphorus solubility

[0048] As shown in Table 2, 18 out of 31 endophytic bacteria exhibited phosphate-solubilizing ability. For the qualitative (plate culture method) and quantitative (molybdenum blue method) tests of phosphate-solubilizing ability, please refer to the reference "Screening and Identification of Highly Effective Phosphate- and Potassium-Solving Fungi and Their Phosphate- and Potassium-Solving Abilities and Growth-Promoting Effects" (Zhao Minrui, Microbiology Bulletin, 2026, 53(02): 947-959). One-way ANOVA showed highly significant differences in phosphate-solubilizing ability among different strains (P<0.01). Burkholderia cepacia (RP2) exhibited the best phosphate-solubilizing ability, with an average phosphate solubility of 459.63 mg / L and a maximum of 473.5 mg / L. Multiple comparisons confirmed that its phosphate-solubilizing ability was significantly higher than all other tested strains. Aspergillus niger (SP13) and Serratia marcescens (RP3) showed the second best phosphate-solubilizing ability, with average phosphate solubilities of 410.86 mg / L and 399.1 mg / L, respectively, significantly higher than most tested strains.

[0049] 2.2 Nitrogen fixation capacity determination

[0050] For specific methods of qualitative testing (plate culture method) and quantitative testing (Kjeldahl method) of nitrogen capacity, please refer to the reference "Screening and Application Effect Analysis of Phosphorus-Solubilizing and Nitrogen-Fixing Endophytic Bacteria in Millet" (Yang Gang, Master's Thesis, Northwest A&F University, 2020).

[0051] The results showed that only 6 out of 31 endophytic bacteria possessed nitrogen-fixing capabilities (as shown in Table 2), accounting for 19.35% of the total strains. One-way ANOVA indicated highly significant differences in nitrogen-fixing capabilities among different nitrogen-fixing strains (P<0.01). *Burkholderia cepacia* (RP2) exhibited the strongest nitrogen-fixing ability, with an average nitrogen fixation amount of 53.1 mg / g dry cell weight, significantly higher than all other nitrogen-fixing strains. It is speculated that this strain may achieve nitrogen fixation by synthesizing a nitrogenase complex, which efficiently catalyzes the nitrogen reduction reaction. *Burkholderia arvense* (RP12) and *Aspergillus niger* (SP13) showed the second strongest nitrogen-fixing abilities, with average nitrogen fixation amounts of 29.93 mg / g dry cell weight and 28.53 mg / g dry cell weight, respectively.

[0052] 2.3 IAA Production Capacity Determination

[0053] For specific determination methods, please refer to the reference "Determination and Identification of IAA Production and Antibacterial Ability of Endophytic Fungus Z5 of Polygonum villosa" (Li Zhendong et al., Journal of Grassland Science, 2010, 19(02): 61-68). The results of this determination are shown in Table 2. Among the 31 endophytic fungi, 18 strains had the ability to produce IAA, accounting for 58.06% of the total number of strains, and they were present in roots, stems, and leaves. One-way ANOVA showed that there were extremely significant differences in the IAA production ability among different strains (P<0.01). Among them, Burkholderia cepacia (RP2) had the strongest IAA production ability, with an average IAA production of 97.53 mg / L and a maximum of 99.7 mg / L. After multiple comparisons, its IAA production ability was significantly higher than that of all other tested strains. Aspergillus niger (SP13) and Serratia marcescens (RP3) had the second highest IAA production capacity, with average IAA yields of 78.84 mg / L and 74.9 mg / L, respectively, which were significantly higher than most of the tested strains.

[0054] 2.4 Determination of Iron Production Capacity

[0055] Siderogenic capacity was determined using the CAS plate method. Endophytic bacteria were inoculated onto CAS agar plates and incubated at 28°C for 3 days. The presence of red or orange-yellow halos around the colonies was observed. The siderogenic capacity of 31 endophytic bacteria strains was determined using the CAS plate method. The results are shown in Table 2. Only 5 strains (RP2, RP3, SP11, SP13, and SP20) exhibited siderogenic capacity, accounting for 16.13% of the total strains.

[0056] 2.5 Determination of antagonistic ability against Fusarium oxysporum

[0057] Antagonistic ability was determined using the plate confrontation method. For details, please refer to the reference "Screening of Biocontrol Bacteria and Effective Fungicides for Dry Rot Disease of Fritillaria thunbergii" (Yan Ziyan et al., Journal of Pesticide Science, 2025, 27(03): 533-542). Using *Fusarium oxysporum*, a common pathogen of *Tungus thunbergii*, as the indicator pathogen, the antagonistic ability of 31 endophytic fungi was determined. The results are shown in Table 2. Only 5 strains showed antagonistic ability: RP2, SP4, SP13, LP15, and SP20, accounting for 16.13% of the total strains. One-way ANOVA showed significant differences in antagonistic ability among different antagonistic strains (P<0.05). Among them, *Trichoderma harzianum* (SP20) showed the strongest antagonistic ability, with an antagonistic rate of 50.00%; LP15 showed the second strongest antagonistic ability, with an antagonistic rate of 43.33%.

[0058] Example 3: Comprehensive analysis of superior growth-promoting strains and growth-promoting experiments of 6 endophytic bacteria on tung oil trees.

[0059] A standardized scoring method was used to standardize five core growth-promoting functional indicators: phosphorus solubility, nitrogen fixation, IAA production, siderophore production, and antagonism. The standardized sum of each strain was calculated, and this sum was used to rank the strains. A higher standardized sum indicates a better overall growth-promoting function, demonstrating the ability of the strain to provide growth support to the host plant from multiple dimensions.

[0060] As shown in Table 3 of the comprehensive scoring results, the standardized total of the 31 endophytic bacteria showed significant differences, and the top 7 strains had excellent overall performance: Burkholderia cepacia RP2 > Aspergillus niger SP13 > Burkholderia arborescens RP12 > Serratia marcescens RP3 > Trichoderma longibranchs LP15 > Trichoderma harzianum SP20 > Bacillus aquaticus SP4.

[0061] Table 3 Standardized scores and rankings of the comprehensive growth-promoting function of endophytic strains

[0062]

[0063]

[0064] The top-ranked bacteria were selected for the growth-promoting experiment on tung oil trees. Since both *Burkholderia cepacia* and *Burkholderia arborescens* belong to the *Burkholderia* genus, previous experiments showed that the former's functions were superior to the latter in various aspects. Therefore, after excluding *Burkholderia arborescens*, *Burkholderia cepacia* (RP2), *Aspergillus niger* (SP13), *Serratia marcescens* (RP3), *Trichoderma longifolia* (LP15), *Trichoderma harzianum* (SP20), and *Bacillus aquaticus* (SP4) were selected as treatment groups. A blank control group (CK) without inoculation was used. Tung oil tree seedlings were collected from the rocky desertification area of ​​the Qingping Town State-owned Forest Farm in Yongshun County, Xiangxi Tujia and Miao Autonomous Prefecture, Hunan Province. Sowing and seedling cultivation were carried out in mid-to-late March of the following year. Seedlings were cultivated in the plantation of Central South University of Forestry and Technology (112.995°E, 28.132°N). Approximately 60 days after sowing, robust, uniformly growing seedlings free from diseases and pests were selected as test materials. After centrifugation of 200ml of bacterial culture, it was resuspended in sterile water to prepare a concentration of 10. 8A bacterial suspension of CFU / mL was applied to each treatment group, with 50 ml per pot applied each time. The control group was applied with the same amount of sterile water. Applications were repeated every 7 days for a total of 4 applications. Each treatment was replicated in triplicate, with one potted plant per replicate. Each pot contained 2 kg of sterilized mixed substrate. The potted experiments were conducted in a greenhouse. During cultivation, the plants were watered regularly to maintain soil moisture at 60%-70% of field capacity. Various indicators were measured after 60 days of cultivation. The results showed that the optimal nitrogen content in *Tung Oil Tree* was achieved by the *Burkholderia cepacia* (RP2) treatment group (20.03±0.29 g / kg) and the second optimal phosphorus content was achieved by the RP2 treatment group (4.13±0.06 g / kg) and the second optimal phosphorus content was achieved by the SP13 treatment group (3.14±0.09 g / kg).

[0065] Experiment Example 4: Growth-promoting effect of mixed endophytic bacteria on tung oil trees

[0066] This experiment used Burkholderia cepacia RP2 (phosphorus-solubilizing and nitrogen-fixing dominant) as the core strain, and constructed mixed combinations with Aspergillus niger SP13 (root-expansion dominant), Serratia marcescens RP3 (phosphorus-potassium synergistic enhancement), and Trichoderma harzianum SP20 (which previously showed good antagonistic ability against Fusarium oxysporum in plate confrontation experiments). An SP20+LP15 fungal combination was also included. The two strains were mixed at a 1:1 mass ratio and inoculated. After centrifugation of 200 ml of the mixed bacterial solution, the suspension was resuspended in sterile water to prepare a concentration of 10. 8 A bacterial suspension of CFU / mL was used to irrigate each pot of *Tung Oil Tree* seedlings in each treatment group with 50 ml of the suspension, while the control group was irrigated with the same amount of sterile water. Irrigation was performed every 7 days for a total of 4 times. Biomass accumulation, nitrogen and phosphorus nutrient absorption, and root development were systematically measured. The following combinations were prepared by mixing the centrifuged samples at a 1:1 mass ratio: *Burkholderia cepacia*: *Aspergillus niger* (RP2+SP13), *Burkholderia cepacia*: *Serratia marcescens* (RP2+RP3), *Burkholderia cepacia*: *Trichoderma harzianum* (RP2+SP20), and *Trichoderma longifolia*: *Trichoderma harzianum* (SP20+LP15). Pot experiments were conducted in a greenhouse, and the cultivation method for the *Tung Oil Tree* seedlings was the same as in Experiment 3.

[0067] Biomass index determination: plant height, fresh and dry weight, chlorophyll content (SPAD method), root index, diameter at breast height (mm): nitrogen content determination (Kjeldahl method), phosphorus content determination (molybdenum blue method), potassium content determination (flame photometry), etc. For specific operating methods, please refer to the above references.

[0068] Colonization Validation Experiment: Construction of RP2-gfp Engineered Bacteria: Competent cells of Burkholderia (RP2) were prepared using the CaCl2 method. RP2 strains were inoculated into LB liquid medium and cultured until OD600 = 0.6-0.8. The cells were collected, resuspended in a suitable amount of pre-chilled 0.1 mol / L CaCl2 solution (containing 15% glycerol), aliquoted, and stored at -80℃ for later use. 100 μL of competent cells were taken, and 10 μL of plasmid vector pPROBE-NT was added. After mixing, the cells were incubated on ice for 30 min, heat-shocked in a 42℃ water bath for 90 s, and then transferred to an ice bath for cooling for 2 min. 900 μL of LB liquid medium was added, and the cells were cultured with shaking for 1 h to allow the strain to recover growth and express the resistance gene. The transformed bacterial culture was spread on LB solid medium containing kanamycin resistance. A single colony with good growth was picked and inoculated into LB liquid medium. Genomic DNA of the strain was extracted and PCR amplification was performed using gfp gene-specific primers. The PCR product was detected by electrophoresis. The strain that showed the expected band size was the positive engineered bacteria and named RP2-gfp.

[0069] Colonization observation and detection: To rapidly detect the colonization ability of the strain, 60-day-old tung oil seedlings with robust growth, uniform growth, a height of 15-20cm, well-developed root systems, and no diseases or pests were selected as experimental materials. The RP2-gfp engineered bacteria were inoculated into LB liquid medium and cultured until OD600 = 0.8, then diluted to a concentration of 1×10⁻⁶. 8 A bacterial suspension of CFU / mL was prepared. Tung tree seedlings were immersed in the bacterial suspension for 30 min, then transplanted into a sterilized substrate (vermiculite: peat moss = 1:1) for cultivation. A blank control group (roots immersed in an equal volume of sterile saline) was set up. Each treatment group had 10 replicates. Cultivation conditions were: temperature 25-28℃, light duration 12 h / d, relative humidity 60%-70%, and regular watering to maintain substrate moisture at 60% of field capacity.

[0070] Lateral root samples were collected from tung oil seedlings on day 14 post-inoculation. The sample preparation steps were as follows: A lateral root segment approximately 1 cm long was taken, and the surface matrix was gently rinsed with sterile water. The segment was then fixed in 4% paraformaldehyde for 2 hours, followed by washing three times with PBS buffer for 5 minutes each time. The fixed root segment was embedded in OCT embedding medium, flash-frozen in liquid nitrogen, and then sectioned to a thickness of 20 μm using a cryostat. The sections were then mounted on glass slides. The slides were stained with DAPI (4′,6-diamidinyl-2-phenylindole, 1 μg / mL) for 10 minutes, rinsed three times with PBS, and then mounted.

[0071] Observation was performed using a laser scanning confocal microscope. The excitation wavelengths were set to DAPI 405nm and GFP 488nm; the emission wavelengths were set to DAPI 460nm and GFP 507nm. Images were observed and captured to analyze the colonization of RP2-gfp engineered bacteria in the root surface, root hair zone, and cortical tissue.

[0072] SPSS 26.0 software was used for statistical analysis. One-way ANOVA was used for comparisons among multiple groups. Post-hoc multiple comparisons were performed using both LSD and Duncan's new multiple range method. The significance level was set at α=0.05. Different lowercase letters in the figure indicate significant differences between treatments (P<0.05), while the same letter indicates no significant differences.

[0073] 4.1 Effects of different mixed bacterial strains on tung oil biomass

[0074] The effects of different mixed bacterial strains on the biomass accumulation of *Tung Oil Tree* were analyzed by measuring plant height, fresh weight, dry weight, relative chlorophyll content (SPAD value), and diameter at root. The results are as follows: Figure 2 , Figure 3 As shown.

[0075] Plant height varied significantly among treatments, with the RP2+SP13 treatment group having the highest plant height (40.33±0.48cm), which was significantly higher than the CK (33.36±2.60cm).

[0076] Fresh weight varied significantly among the treatments. The RP2+SP13 (57.00±1.53g) and RP2+SP20 (53.67±2.91g) treatments had the highest fresh weights, with no significant difference between the two groups, and both were significantly higher than the other treatments. The CK group had the lowest fresh weight.

[0077] The dry weights differed significantly among the treatments. The RP2+SP13 (15.93±0.98g), RP2+SP20 (15.77±0.58g), and RP2+RP3 (13.90±1.65g) treatments had the highest dry weights, with no significant difference among the three groups, and all were significantly higher than the CK (8.83±0.41g).

[0078] The chlorophyll SPAD value was highest in the RP2+SP20 treatment group (33.34±2.04), while the SPAD values ​​of the other treatment groups ranged from 25.82 to 29.23, but there was no significant difference compared with RP2+SP20 and CK. There was no significant difference in ground diameter among the treatments, with values ​​ranging from 9.8 to 11.8 mm.

[0079] In summary, the mixed strains RP2+SP13 and RP2+SP20 were significantly better than the control in promoting the accumulation of fresh and dry weight of tung oil trees. RP2+SP13 showed the best performance in increasing plant height, while there was no significant difference in diameter at ground level among the treatments.

[0080] 4.2 Effects of different mixed bacterial strains on nitrogen and phosphorus nutrition in tung oil trees

[0081] The effects of different mixed bacterial strains on nitrogen and phosphorus nutrient uptake in tung oil trees were analyzed by measuring the nitrogen (N) and phosphorus (P) contents of the aboveground and underground parts of the tung oil tree, as well as the N / P ratio of the aboveground and underground parts. The results are as follows: Figure 4 As shown.

[0082] The nitrogen content in the aboveground parts showed highly significant differences among treatments, with RP2+SP13 (42.33±0.59 g / kg) and RP2+SP20 (39.60±1.76 g / kg) having the highest levels, and these two treatments showed no significant difference but were significantly higher than other treatments; the control (CK) had the lowest levels. The nitrogen content in the underground parts also showed highly significant differences among treatments, with RP2+SP13 (44.00±0.61 g / kg) and RP2+SP20 (41.27±1.75 g / kg) having the highest levels, and these two treatments showed no significant difference; the control (CK) had the lowest levels.

[0083] The phosphorus content in the aboveground parts showed highly significant differences among treatments. Both RP2+SP13 (6.63±0.09 g / kg) and RP2+SP20 (6.20±0.26 g / kg) were significantly higher than other treatments, with RP2+SP13 being significantly higher than RP2+SP20; the control (CK) had the lowest phosphorus content. There were no significant differences in phosphorus content in the underground parts among treatments. Notably, the phosphorus content in both the aboveground and underground parts of the RP2+SP13 combination was more than 1.5 times higher than that of the single RP2 and SP13 treatments.

[0084] There were no significant differences in the nitrogen-to-phosphorus ratio among the treatments in the aboveground parts. The differences in the nitrogen-to-phosphorus ratio among the treatments in the underground parts were extremely significant, with RP2+SP13 (6.27±0.09) having the highest ratio, significantly higher than other treatments; RP2+SP20 (5.87±0.23) was the second highest; and CK had the lowest ratio.

[0085] In summary, the mixed strains RP2+SP13 and RP2+SP20 showed outstanding performance in promoting nitrogen and phosphorus absorption in tung oil trees, significantly increasing nitrogen content in both aboveground and belowground parts, as well as phosphorus content in the aboveground parts, and significantly increasing the nitrogen-to-phosphorus ratio in the belowground parts. RP2+RP3 and LP15+SP20 showed moderate effects, while the control (CK) showed the worst results. There were no significant differences in phosphorus content in the belowground parts and the nitrogen-to-phosphorus ratio in the aboveground parts among the treatments.

[0086] 4.3 Effects of different mixed bacterial strains on tung oil tree root indicators

[0087] The effects of different mixed bacterial strains on the root development of *Vernicia fordii* were analyzed by measuring root length, root surface area, number of root tips, average root diameter, and root volume. The results are as follows: Figure 5 and Figure 6 As shown.

[0088] The root lengths differed significantly among the treatments. The RP2+SP13 treatment group had the longest root length (936.59±13.72cm), which was significantly higher than all other treatments. The RP2+RP3 treatment group (602.75±23.30cm) was the second longest, which was significantly higher than the RP2+SP20 treatment (544.98±11.65cm), CK treatment (483.85±7.44cm), and LP15+SP20 treatment (344.17±16.70cm).

[0089] The root surface area differed significantly among the treatments. The RP2+SP13 treatment group had the largest root surface area (287.07±16.06 cm²), which was significantly higher than other treatments. The RP2+RP3 treatment group (249.23±17.25 cm²) was significantly higher than the CK treatment (182.48±17.29 cm²), but the RP2+SP20 treatment group (213.51±23.77 cm²) was not significantly different from the CK treatment.

[0090] The number of root tips varied significantly among the treatments. The RP2+SP13 treatment group had the most root tips (529±16.09), which was significantly higher than other treatments. The RP2+RP3 group (441.67±5.21) and the RP2+SP20 group (456.67±15.17) had the next most root tips, with no significant difference between the two groups. However, the number of root tips in the RP2+SP13 group was significantly higher than that in the CK group (223.33±30.66) and the LP15+SP20 group (247±25.00).

[0091] There was no significant difference in average root diameter among the treatments, with values ​​ranging from 0.72 to 0.99, indicating that different mixed strains had limited effect on the root thickness of tung trees.

[0092] The root capacity varied significantly among the treatments. The RP2+SP13 treatment group had the highest root capacity (4.69±0.22 cm³), which was significantly higher than the other treatments. The RP2+RP3 (3.71±0.18 cm³) and RP2+SP20 (3.61±0.20 cm³) treatments were next, with no significant difference between them, but both were significantly higher than the CK (2.33±0.16 cm³) and LP15+SP20 (2.50±0.12 cm³).

[0093] In summary, the effects of different mixed bacterial strains on the root development of *Vernicia fordii* exhibit combination-specificity. RP2+SP13 showed the best performance in root length, root surface area, root tip number, and root volume, significantly promoting root expansion and increasing root volume. RP2+RP3 and RP2+SP20 performed well in some root indicators, but still lagged behind RP2+SP13. LP15+SP20 and the control group (CK) performed poorly in all indicators. There was no significant difference in average root diameter among the treatments.

[0094] Based on the results of this experiment, we can conclude that:

[0095] (1) The concentration of the single bacterial suspension in Experiment 3 was the same as the concentration of the mixed bacterial suspension in Experiment 4, both being 1×10⁻⁶. 8 The concentration of CFU / mL, i.e., the corresponding single-strain concentration in the mixed bacteria in Experiment 4, was halved compared to Experiment 3. Comparing the results of Experiment 3 and Experiment 4, it can be seen that the nitrogen content in both the aboveground and underground parts of the RP2+SP13 treatment was more than twice that of the single-strain treatments of RP2 and SP13. This indicates that the growth-promoting effect of the mixed strains is significantly better than that of the single strains, and the mixed strains still exhibit a synergistic effect of "1+1>2" even with the single-strain concentration halved. The fresh weight of tung oil in the RP2+SP13 (Burkholderia cepacia + Aspergillus niger) treatment group increased by 106.0% compared to the control; the nitrogen and phosphorus contents in the aboveground parts increased by 53.8% and 53.1%, respectively. These results indicate that RP2 (phosphorus-solubilizing and nitrogen-fixing dominant type) and SP13 (root expansion dominant type) achieved a dual enhancement of "nutrient supply + absorption capacity" through functional complementarity. Their synergistic effect is not a simple functional additive effect, but rather a positive feedback loop based on metabolic interactions between strains.

[0096] (2) The growth-promoting effect of mixed strains exhibits significant combination specificity, and functional complementarity and niche differentiation are key prerequisites for synergistic effects. Burkholderia cepacia RP2 and Aspergillus niger SP13 belong to bacteria and fungi, respectively, with obvious niche differentiation. Functionally, they focus on nutrient transformation and root expansion, respectively, and their complementarity is strong. Although RP2 and RP3 have some functional complementarity, they may have similar rhizosphere niche requirements (both prefer root surface colonization), leading to resource competition and weakening the synergistic effect. SP20 and LP15 belong to the Trichoderma genus, with redundant functions and overlapping niches, and failed to produce synergistic effects. The above patterns suggest that the construction of efficient mixed microbial communities should take into account both functional complementarity and niche differentiation, and avoid the excessive accumulation of strains with similar functions. The growth-promoting effect of mixed strains on tung oil trees exhibits significant combination specificity. RP2+SP13 (Burkholderia cepacia + Aspergillus niger) achieves multidimensional synergistic enhancement in biomass accumulation, nutrient absorption, and root development through functional complementarity and metabolic synergy.

[0097] Experimental Example 5: The growth-promoting effect of mixed endophytic bacteria on Camellia oleifera

[0098] This experiment aims to clarify: (1) whether excellent endophytic strains derived from tung oil trees can achieve cross-host growth promotion on camellia oleifera; (2) whether the growth promotion effect of mixed strain combinations on camellia oleifera continues its synergistic advantage on tung oil trees; and (3) to screen broad-spectrum mixed bacterial agents suitable for artificial planting of camellia oleifera in rocky desertification areas.

[0099] The camellia oleifera seedlings used in this experiment were collected from the State-owned Forest Farm in Qingping Town, Yongshun County, Xiangxi Tujia and Miao Autonomous Prefecture, Hunan Province. Sowing and seedling cultivation took place from late March to early April of the following year, and the seedlings were managed conventionally in a greenhouse. When the seedlings reached mid-June (approximately 75–80 days after sowing), robust, uniformly growing seedlings free from pests and diseases were selected as test materials. These seedlings underwent root irrigation inoculation with mixed bacterial strains, using the same method as in Experiment 4. After 60 days of cultivation (late August to early September), various indicators were measured.

[0100] The tested mixed strains, potting substrate, and testing instruments and equipment were the same as in Experiment 4. Soil enzyme activity was measured using a Solarbio kit purchased from Beijing Solarbio Technology Co., Ltd.

[0101] 5.1 Effects of different mixed microorganisms on the biomass of Camellia oleifera

[0102] The effects of different mixed bacterial strains on biomass accumulation in *Camellia oleifera* were analyzed by measuring plant height, fresh weight, dry weight, relative chlorophyll content (SPAD value), and diameter at root. The results are as follows: Figure 7 As shown.

[0103] Plant height varied significantly among treatments. The RP2+SP13 treatment group had the highest plant height (32.03±0.68cm), which was not significantly different from the RP2+SP20 treatment group (32.53±0.90cm). Both treatments were significantly taller than the other treatments.

[0104] Fresh weight varied significantly among the treatments. The RP2+SP13 treatment group had the highest fresh weight (19.67±0.82g), followed by the RP2+SP20 treatment group (16.80±0.62g), which was significantly higher than all other treatment groups except RP2+SP13.

[0105] The dry weight varied significantly among the treatments, with the RP2+SP13 treatment group having the highest dry weight (6.63±0.32g) and the RP2+SP20 treatment group (5.00±0.47g) having the second highest, both significantly higher than the other treatment groups except RP2+SP13.

[0106] The relative chlorophyll content (SPAD value) varied among the treatments. The CK treatment group had the highest SPAD value (47.3467±4.89137), followed by the LP15+SP20 treatment group (45.5467±7.22776), with the RP2+SP20 treatment group (38.7067±2.41047) and the RP2+SP13 treatment group (37.7267±6.79953) in the middle, and the RP2+RP3 treatment group had the lowest value (29.6567±1.76972).

[0107] The diameter at ground level varied significantly among the treatments. The RP2+SP13 (6.1±0.2082 mm) and RP2+SP20 (6.1±0.1528 mm) treatments had the highest diameter at ground level, with the two treatments having the same value. The RP2+RP3 treatment (5.133±0.4372 mm) was the second highest. The LP15+SP20 treatment (4.767±0.1333 mm) and CK (3.933±0.2906 mm) had the lowest diameter at ground level.

[0108] In summary, the mixed strains RP2+SP13 and RP2+SP20 were superior to the control and other mixed combinations in promoting the accumulation of plant height, fresh weight, and dry weight in Camellia oleifera, with RP2+SP13 showing particularly outstanding performance in fresh weight and dry weight. The RP2+RP3 and LP15+SP20 treatment groups showed little difference from the control in most biomass indicators. Chlorophyll content was higher in the CK and LP15+SP20 treatment groups, while ground diameter was optimal in the RP2+SP13 and RP2+SP20 treatment groups.

[0109] 5.2 Effects of different mixed strains on nitrogen and phosphorus nutrition in Camellia oleifera

[0110] The effects of different mixed bacterial strains on nitrogen and phosphorus nutrient uptake in Camellia oleifera were analyzed by measuring the nitrogen (N) and phosphorus (P) contents of the aboveground and underground parts of the Camellia oleifera plantation, as well as the N / P ratio between the aboveground and underground parts. The results are as follows: Figure 8 and Figure 9 As shown.

[0111] The nitrogen content in the aboveground parts varied significantly among the treatments. The RP2+SP13 treatment group had the highest aboveground nitrogen content (29.40±0.59 g / kg), which was not significantly different from the RP2+SP20 treatment group (27.60±0.81 g / kg). Both treatments were significantly higher than the other treatments. Similarly, the nitrogen content in the belowground parts varied significantly among the treatments. The RP2+SP13 treatment group had the highest belowground nitrogen content (18.83±0.38 g / kg), which was not significantly different from the RP2+SP20 treatment group (17.60±0.62 g / kg). Both treatments were significantly higher than the other treatments.

[0112] The phosphorus content in the aboveground parts varied significantly among the treatments. The RP2+SP13 treatment group had the highest aboveground phosphorus content (5.43±0.15 g / kg), which was significantly higher than all other treatments. The RP2+SP20 treatment group (5.10±0.12 g / kg) was the second highest, which was not significantly different from the RP2+RP3 treatment group (4.67±0.09 g / kg), but was significantly lower than RP2+SP13. The RP2+RP3 treatment group was significantly higher than LP15+SP20 (3.93±0.33 g / kg) and CK (3.33±0.09 g / kg). The phosphorus content in the underground parts varied significantly among the treatments. The RP2+SP13 treatment group had the highest underground phosphorus content (3.97±0.12 g / kg), which was significantly higher than other treatments. The RP2+SP20 treatment group (3.67±0.09 g / kg) and the RP2+RP3 treatment group (3.27±0.09 g / kg) had no significant difference, but the RP2+SP13 treatment group had a significantly lower phosphorus content. The RP2+RP3 treatment group had a significantly higher phosphorus content than the LP15+SP20 treatment group (2.70±0.25 g / kg) and the control group (2.23±0.09 g / kg).

[0113] The nitrogen-to-phosphorus ratio in the aboveground parts differed significantly among the treatments, with the highest ratio (5.75±0.05) in the control group, significantly higher than the other treatment groups; there were no significant differences among the other treatment groups. The nitrogen-to-phosphorus ratio in the underground parts did not differ significantly among the treatments (P>0.05).

[0114] In summary, the mixed strain RP2+SP13 showed outstanding performance in promoting nitrogen and phosphorus absorption in Camellia oleifera, with significantly higher nitrogen and phosphorus contents in both aboveground and belowground parts compared to most treatments. RP2+SP20 was second best, with some indicators showing no significant difference compared to RP2+SP13. RP2+RP3 showed moderate performance, while LP15+SP20 and the control (CK) showed poor performance. Regarding the nitrogen-to-phosphorus ratio, the control (CK) had the highest aboveground nitrogen-to-phosphorus ratio, while there was no significant difference in the belowground nitrogen-to-phosphorus ratio among the treatments.

[0115] 5.3 Effects of different mixed bacterial strains on root indicators of Camellia oleifera

[0116] The effects of different mixed bacterial strains on the root development of *Camellia oleifera* were analyzed by measuring root length, root surface area, number of root tips, average root diameter, and root volume. The results are as follows: Figure 10 and Figure 11 As shown.

[0117] The root lengths differed significantly among the treatments. The RP2+SP13 treatment group had the longest root length (574.15±64.88 mm), which was significantly higher than all other treatments. The RP2+RP3 treatment group was the second longest, with a root length of (475.77±29.89 mm), which was significantly higher than RP2+SP20, SP20+LP15 and CK. The root length of the RP2+SP20 treatment group was (350.54±35.75 mm), which was significantly higher than SP20+LP15 and CK.

[0118] The root surface area differed significantly among the treatments. The RP2+SP13 treatment group had the largest root surface area, with an average of (266.85±23.29 cm²), which was significantly higher than the other treatments. There were no significant differences among the other four treatment groups, namely RP2+RP3 (177.53±13.07 cm²), RP2+SP20 (153.91±8.03 cm²), SP20+LP15 (171.23±11.50 cm²), and CK (167.71±23.40 cm²).

[0119] The number of root tips varied significantly among the treatments. The RP2+SP13 treatment group had the highest number of root tips, with an average of (377.33±44.28), which was significantly higher than the other treatments. The RP2+RP3 treatment group was the second highest, with an average of (283.00±29.10), which was significantly higher than RP2+SP20, SP20+LP15 and CK.

[0120] The root capacity varied significantly among the treatments. The RP2+SP13 (4.35±0.36 cm³) and RP2+RP3 (3.90±0.32 cm³) treatments had the highest root capacity, with no significant difference between the two, and were significantly higher than other treatments. The RP2+SP20 treatment (3.26±0.37 cm³) was significantly higher than that of SP20+LP15 and CK.

[0121] There was no significant difference in average root diameter among the treatments.

[0122] In summary, the effects of different mixed bacterial strains on the root development of Camellia oleifera exhibit combination-specificity. The RP2+SP13 treatment showed the best performance in increasing root length, root surface area, root tip number, and root volume, significantly promoting root expansion and increasing root volume. RP2+RP3 showed good performance in increasing root length, root tip number, and root volume. RP2+SP20 showed moderate performance in increasing root length and root volume. SP20+LP15 and the control group (CK) performed poorly in all indicators, with no significant growth-promoting effect. The average root diameter did not differ significantly among the treatments.

[0123] 5.4 Effects of different mixed strains on soil enzyme activity

[0124] The effects of different mixed bacterial strains on soil biochemical processes were analyzed by measuring the activities of phytase, β-glucosidase, acid phosphatase, sucrase, acid protease, and urease in potted soil. The results are as follows: Figure 12 As shown.

[0125] Urease activity varied significantly among the treatments. The RP2+SP13 treatment group had the highest urease activity (0.83367±0.039641U / g), followed by the RP2+RP3 treatment group (0.59533±0.179393U / g). The RP2+SP20 (0.56500±0.026764U / g) and LP15+SP20 (0.51467±0.009262U / g) were in the middle, while the control group (CK) had the lowest activity (0.43333±0.016576U / g).

[0126] The activities of acidic proteases varied significantly among the treatments. The RP2+SP13 treatment group had the highest activity (0.05133±0.0020 U / g), followed by the RP2+SP20 treatment group (0.04133±0.0009 U / g). The activities of RP2+RP3 (0.03600±0.0012 U / g), LP15+SP20 (0.03233±0.0009 U / g), and CK (0.03200±0.0006 U / g) decreased in that order.

[0127] Sucrase activity varied significantly among treatments. The RP2+SP13 treatment group had the highest activity (5.4567±0.16476U / g), which was significantly higher than other treatments. The RP2+RP3 (2.8733±0.04667U / g) and LP15+SP20 (2.9533±0.05548U / g) treatments were next, with no significant difference between them. The RP2+SP20 (2.4833±0.03756U / g) treatment was next. The CK treatment had the lowest activity (1.7133±0.03333U / g).

[0128] β-glucosidase activity varied significantly among treatments. The RP2+SP13 treatment group had the highest activity (4.8233±0.1713 U / g), which was significantly higher than other treatments. The RP2+SP20 group (3.1233±0.04333 U / g) was the second highest. There was no significant difference among RP2+RP3 (2.6933±0.12454 U / g), LP15+SP20 (2.7333±0.12129 U / g), and CK (2.6400±0.10583 U / g), and all of them were significantly lower than RP2+SP13.

[0129] Phytase activity varied significantly among the treatments. The RP2+RP3 treatment group had the highest activity (3.6300±0.11136U / g), which was significantly higher than all other treatments. The RP2+SP13 treatment group was the second highest (2.7767±0.08413U / g), which was significantly higher than RP2+SP20, LP15+SP20 and CK. The RP2+SP20 treatment group (2.0800±0.01732U / g) was significantly higher than LP15+SP20 (1.7267±0.05364U / g) and CK (1.5233±0.05783U / g).

[0130] Acid phosphatase activity varied significantly among treatments. The RP2+SP13 treatment group had the highest activity (25321.66667±1290.04 U / g), which was significantly higher than other treatments. The RP2+RP3 (17465.66667±917.96 U / g) and RP2+SP20 (15873.33333±188.36 U / g) treatment groups were next, with no significant difference between the two groups. The RP2+RP3 and RP2+SP20 (15360.33333±257.12 U / g) treatment groups were also not significantly different from the LP15+SP20 (15360.33333±257.12 U / g) treatment groups, but all of them were significantly higher than the CK (12001.536±260.32 U / g).

[0131] In summary, different mixed bacterial strains significantly affected soil enzyme activities, exhibiting strain specificity. The RP2+SP13 treatment significantly increased the activities of urease, acidic protease, sucrase, β-glucosidase, and acid phosphatase, indicating that this combination can effectively promote the activities of enzymes related to soil carbon, nitrogen, and phosphorus cycles. RP2+RP3 showed good performance in phytase and acid phosphatase. RP2+SP20 showed moderate levels in some enzyme activities. LP15+SP20 and CK showed low levels in most enzyme activities.

[0132] This experiment used the optimal mixed bacterial strain combination screened from the *Tung Oil Tree* study as the test material and *Camellia oleifera* seedlings as the research object. The biomass accumulation, nitrogen and phosphorus nutrient absorption, root development, and six soil enzyme activities of *Camellia oleifera* under different mixed bacterial strain treatments were systematically measured. Compared with the results of the *Tung Oil Tree* study in Example 4, the mixed bacterial strains showed a similar functional complementarity pattern in their growth-promoting effect on *Camellia oleifera*, but also exhibited host-specific response characteristics. The main conclusions are: the mixed bacterial strains have combination-specific effects on the growth-promoting effects of *Camellia oleifera* biomass, nutrient absorption, and root development, with the RP2+SP13 treatment (*Burkholderia cepacia* + *Aspergillus niger*) showing the best overall performance. The mixed bacterial strains exhibit significant combination-specific effects on the growth-promoting effect of *Camellia oleifera*. *Burkholderia cepacia* and *Aspergillus niger*, through functional complementarity and metabolic synergy, significantly enhanced the activity of enzymes related to soil carbon, phosphorus, and nitrogen cycles while promoting the growth, nutrient absorption, and root development of *Camellia oleifera*, achieving overall optimization of the "plant-soil" system. This is the optimal mixed bacterial combination screened in this study. Excellent endophytic fungi derived from tung oil trees can promote cross-host growth in camellia oleifera, but different hosts show different response amplitudes to the same strain combination, suggesting that targeted verification is needed when applying it across tree species.

Claims

1. A broad-spectrum growth-promoting bacterial agent combination for tung oil trees and camellia oleifera, characterized in that, The microbial agent combination includes Burkholderia cepacia and Aspergillus niger, which promotes the plant height growth, fresh weight and dry weight accumulation, nitrogen and phosphorus absorption and plant root development of tung oil trees and camellia oleifera.

2. The broad-spectrum growth-promoting bacterial agent combination for tung oil and camellia oil according to claim 1, characterized in that, Burkholderia cepacia and Aspergillus niger were mixed at a mass ratio of 1:

1.

3. The broad-spectrum growth-promoting bacterial agent combination for tung oil and camellia oil according to claim 1, characterized in that, The bacterial agent combination is a liquid bacterial agent, a wettable powder, or a solid bacterial agent.

4. A biological agent for tung oil and camellia oil, characterized in that, Includes the microbial agent combination as described in any one of claims 1-3.

5. The application of a broad-spectrum growth-promoting bacterial agent combination for tung oil trees and camellia oil trees as described in any one of claims 1-3, characterized in that, The step includes preparing the bacterial agent combination to a concentration greater than or equal to 1×10⁻⁶. 8 A bacterial suspension of CFU / mL was applied to the roots of tung oil trees or camellia trees.

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