Penicillium chrysogenum fn07 with multiple growth promoting ability and application thereof

Abstract

The application discloses a penicillium chrysogenum FN07 with multiple growth-promoting abilities and an application thereof, and belongs to the technical field of microorganisms. The classification and naming of the penicillium chrysogenum FN07 strain is Penicillium chrysogenum FN07, which has been preserved in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 20261381, a preservation date of June 18, 2026, and a preservation address of Wuhan University in Wuhan, China. The penicillium chrysogenum FN07 has multiple growth-promoting abilities such as nitrogen fixation, phosphorus dissolution, iron carrier production and indole-3-acetic acid production, can significantly improve the seedling height, ground diameter, biomass and nutrient content of limestone mountain afforestation tree species such as Tsoongiodendron odorum, Acer pentagonum and Ligustrum lucidum, and is suitable for preparing a plant growth promoter, a biological fertilizer or a soil conditioner, and has a wide application prospect in the field of limestone hilly ecological restoration and seedling cultivation.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Penicillium chrysogenum FN07 with multiple growth-promoting abilities and its applications. Background Technology

[0002] Limestone mountains refer to exposed or semi-exposed carbonate karst mountains, distributed in most provinces (municipalities and autonomous regions) of my country. The parent material of the soil in this region is weathered carbonate rocks, and the soil type is mainly coarse-textured leached brown soil. Common problems include thin soil layers, abundant exposed rocks, and sparse natural vegetation, making vegetation restoration difficult and the ecosystem relatively fragile. In recent years, tree species such as Platycladus orientalis, Ligustrum lucidum, Koelreuteria paniculata, Pistacia chinensis, Ailanthus altissima, Zelkova serrata, and Celtis sinensis have been selected and widely used in limestone mountain vegetation restoration practices. Combined with measures such as planting robust seedlings, meticulous land preparation, and forest closure for natural regeneration, ecological restoration has achieved certain results. However, limestone mountain soils have low nutrient content and poor water and fertilizer retention capacity. After artificial afforestation, seedling growth is slow and the survival rate is low. How to maintain the long-term effects of vegetation restoration and steadily improve ecological functions has become a key issue for ecological restoration in this region. Utilizing plant growth-promoting rhizobacteria (PGPR) to enhance plant adaptability to barren environments has become a research hotspot in the field of ecological restoration in recent years. Currently, resources of dedicated growth-promoting bacteria for afforestation tree species in limestone mountains are still relatively scarce, especially strains derived from the extreme environment of limestone mountains that possess multiple growth-promoting functions and are highly compatible with host tree species have not yet been reported. Summary of the Invention

[0003] To address the problem of the lack of efficient, stable, and multifunctional growth-promoting microbial resources suitable for afforestation of limestone mountainous areas in existing technologies, the primary technical problem to be solved by this invention is to provide a strain with multiple growth-promoting abilities. The secondary technical problem to be solved by this invention is to provide a microbial inoculum containing the aforementioned strain with multiple growth-promoting abilities. The final technical problem to be solved by this invention is to provide specific applications of the aforementioned strain or microbial inoculum with multiple growth-promoting abilities.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0005] A Penicillium chrysogenum FN07 with multiple growth-promoting abilities, wherein the Penicillium chrysogenum FNO7 is classified as Penicillium chrysogenum FNO7, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO: M 20261381, deposit date: June 18, 2026, and deposit address: Wuhan University, Wuhan, China.

[0006] A microbial inoculant comprising Penicillium chrysogenum FN07, which has multiple growth-promoting capabilities, and an agriculturally acceptable carrier.

[0007] In some embodiments, the microbial agent is a liquid microbial agent or a solid microbial agent.

[0008] In some embodiments, the spore concentration of Penicillium chrysogenum FN07, which has multiple growth-promoting abilities, in the liquid bacterial agent is 10. 6 -10 8 Spores / mL.

[0009] The application of Penicillium chrysogenum FN07, which has multiple growth-promoting abilities, or the microbial agent described therein, in promoting the growth of afforestation tree species in limestone mountains is characterized in that the afforestation tree species include at least one of Chinese parasol tree, five-lobed maple, and privet.

[0010] In some embodiments, the strains or microbial agents with multiple growth-promoting abilities are inoculated into the rhizosphere of the plant using a root irrigation inoculation method.

[0011] In some embodiments, the specific manifestation of promoting the growth of tree species in limestone mountain afforestation is that after inoculation with strains or microbial agents with multiple growth-promoting abilities, at least one of the following indicators of the plant seedling height, ground diameter, biomass, chlorophyll content, soluble protein content, soluble sugar content, and total nitrogen, total phosphorus, and total potassium content in the leaves is significantly higher than that of the uninoculated control.

[0012] In some embodiments, the inoculation step specifically involves inoculating once every 10 days, with each plantlet inoculated with 40 mL of liquid microbial agent each time, for a total of 3 inoculations.

[0013] The application of Penicillium chrysogenum FN07, which has multiple growth-promoting abilities, or any of the microbial agents described herein, in the preparation of plant growth promoters, bio-fertilizers, or soil conditioners.

[0014] In some embodiments, the plant growth promoter, bio-fertilizer, or soil conditioner is used for ecological restoration or seedling cultivation in limestone hilly areas.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) Unique strain origin. The strain Penicillium chrysogenum FN07 of this invention was isolated from the root system of Chinese parasol tree, maple, or privet in limestone mountains. The soil in this area is barren and the bare rock rate is high. The strain has undergone long-term natural selection and has natural adaptability and colonization ability to barren environments. It has better effect when applied to the same type of ecological restoration.

[0017] (2) The strain has diverse functions. The strain obtained by screening has all the growth-promoting abilities of nitrogen fixation, phosphorus solubilization, siderophore production and IAA production, achieving "one strain with multiple effects" and excellent overall effect.

[0018] (3) Significant growth-promoting effect. Pot experiments showed that after inoculation with Penicillium chrysogenum FN07, the height, diameter at root, aboveground biomass fresh weight, and underground biomass fresh weight of *Firmiana simplex* seedlings were significantly increased compared with the control. The aboveground biomass fresh weight increased by 137.79%, the underground biomass fresh weight by 76.26%, the aboveground biomass dry weight by 217.43%, the underground biomass dry weight by 222.93%, the SPAD value by 42.38%, and the root soluble sugar by 66.18%. The height of *Acer pentaphyllum* seedlings increased by 56.32%, and the diameter at root increased by 137.79%, the diameter at root increased by 76.26 ... The aboveground biomass fresh weight increased by 69.31%, the underground biomass dry weight increased by 69.57%, leaf soluble protein increased by 21.79%-29.93%, leaf soluble sugar increased by 27.24%, and root soluble sugar increased by 45.31%. For Ligustrum lucidum, the aboveground biomass fresh weight increased by approximately 300%, the aboveground biomass dry weight increased by 155.69%-190.24%, the SPAD value increased by 41.73%-53.78%, and root soluble sugar increased by 145.63%. All indicators were significantly better than the control.

[0019] (4) Broad application prospects. The strain of this invention can be prepared into liquid or solid inoculants, which are easy to operate and low in cost. It is suitable for ecological restoration of limestone hills and seedling cultivation, and has outstanding ecological benefits and industrial promotion value. Attached Figure Description

[0020] Figure 1 This is the experimental route map;

[0021] Figure 2 Dilution curves for the diversity of endophytic flora in the roots of different tree species; A: Endophytic fungi; B: Endophytic bacteria;

[0022] Figure 3 Diagram of OTU Venn, an endophytic fungus common to and unique to different tree species;

[0023] Figure 4 PCoA and PLS-DA diagrams of endophytic fungal OTU composition in the roots of different tree species; A: PCoA analysis of endophytic fungal community; B: PCoA analysis of endophytic bacterial community; C: PLS-DA diagram of endophytic fungal community; D: PLS-DA diagram of endophytic bacterial community.

[0024] Figure 5 A graph showing the relative abundance of root endophytic bacteria at the phylum and genus levels for different tree species;

[0025] Figure 6-1 Comparison charts showing differences among endophytic fungal groups at the genus level in the root systems of different tree species: A: Comparison chart of differences among endophytic fungal groups; B: Comparison chart of differences among endophytic bacterial groups.

[0026] Figure 6-2LDA bar charts for endophytic fungi in the roots of different tree species: C: LDA bar chart for endophytic fungi; D: LDA bar chart for endophytic bacteria;

[0027] Figure 7 Bar chart for the functional classification of FUNGuild, an endophytic fungus in the roots of different tree species;

[0028] Figure 8 Functional classification of COG in the roots of different tree species;

[0029] Figure 9 Morphological characteristics of endophytic fungi on PDA solid medium;

[0030] Figure 10 Morphological characteristics of endophytic bacteria on LB solid medium;

[0031] Figure 11 These are some representative strains with nitrogen-fixing capabilities;

[0032] Figure 12 Statistics on the phosphorus solubility of some strains; A: Solubility of organic phosphorus; B: Solubility of inorganic phosphorus;

[0033] Figure 13 This shows the growth of some strains on potassium-solubilizing medium;

[0034] Figure 14 This is a statistical analysis of the siderophore production capacity of some strains; Note: The upper right corner of the image shows the effect of some siderophore-producing positive strains, and the middle is the colony. You can observe an orange-yellow or purplish-red halo around the colony.

[0035] Figure 15 This image shows the statistical analysis of the indoleacetic acid (IAA) production capacity of some strains. Note: The upper right corner of the image shows the colorimetric reaction results: CK is the negative control (uninoculated), 100 μg·mL -1 IAA serves as a positive control, while the others represent the colorimetric effects of some IAA-producing positive strains; the higher the IAA content in the supernatant, the deeper the pink color.

[0036] Figure 16 Phylogenetic tree of three endophytic fungi based on ITS sequences; Note: The values ​​on the branches of the phylogenetic tree represent bootstrap values, the numbers in parentheses are GenBank accession numbers, and the scale bar represents the number of nucleotide substitutions.

[0037] Figure 17 Phylogenetic tree of two endophytic bacteria based on 16S rRNA sequences;

[0038] Figure 18 To investigate the effects of inoculation with different bacterial strains on the growth of three seedlings: A: Firmiana simplex; B: Acer pentaphyllum; C: Ligustrum lucidum;

[0039] Figure 19 The effect of inoculation with different bacterial strains on seedling height (A) and ground diameter (B) of three seedlings; Note: Different lowercase letters indicate significant differences between different treatments (P < 0.05).

[0040] Figure 20 To investigate the effect of inoculation with different bacterial strains on the SPAD values ​​of three seedlings;

[0041] Figure 21 To investigate the effect of inoculation with different bacterial strains on the soluble protein content of three seedlings; A: leaf; B: root;

[0042] Figure 22 To investigate the effect of inoculation with different bacterial strains on the soluble sugar content of three seedlings; A: leaf; B: root. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0044] The Penicillium chrysogenum FNO7 in the following examples is classified as Penicillium chrysogenum FNO7 and has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20261381, deposit date: June 18, 2026, and address: Wuhan University, Wuhan, China.

[0045] Example 1

[0046] 1. Materials and Methods

[0047] 1.1 Overview of the Experimental Site

[0048] The experimental area is located in Zhaotuan State-owned Forest Farm, Maoshan Town, Tongshan District, Xuzhou City, Jiangsu Province (117°24'49''-117°25'13'' E, 34°13'7''-34°13'14'' N). The area has a temperate monsoon climate with four distinct seasons: hot and rainy summers and cold and dry winters. The average annual temperature is 14.3℃, the average annual precipitation is 869 mm, the average annual sunshine duration is 2366 h, and the frost-free period is approximately 210 days. The study area features typical limestone hilly terrain, with soils mainly composed of coarse-grained brown soil and leached brown soil developed from limestone parent material. Common problems include high bare rock ratio, shallow soil layer, and low nutrient content. The main tree species are Chinese arborvitae plantations. Since 2007, relying on the "Xuzhou City's Second Advance into Barren Mountains Project", artificial forests of privet, five-lobed maple, trident maple (Acer buergerianum), chinaberry (Melia azedarach), ailanthus, Quercus acutissima, hackberry, and zelkova have been planted to restore the limestone mountains ecologically. Auxiliary tree species such as Chinese parasol tree, white elm (Ulmus pumila) and silk cottonwood (Euonymus bungeanus) are also scattered to restore the limestone mountains ecologically.

[0049] 1.2 Test Materials

[0050] In September 2024, root samples were randomly selected from 15 healthy plants in three plantations (Ligustrum lucidum, Acer pentaphyllum, and Firmiana simplex, 21, 22, and 22 years old, respectively) for sampling. Masks and sterile gloves were worn during sampling, and tools (scissors and shovels) were disinfected with 75% ethanol before use. After removing surface vegetation and other debris, roots were dug to a depth of 10-20 cm at a distance of approximately 50 cm from the base of the trunk. Roots approximately 10 cm long were cut, and surface soil was carefully removed. Roots from three plants of the same species were combined to form one composite sample, considered a biological replicate. Five replicates were obtained for each species, for a total of 15 root samples. Immediately after collection, samples were placed in sterile sampling bags. In the laboratory, all collected samples were aliquoted: one sample was stored at -80°C for DNA extraction, and another was used for endophytic bacteria isolation.

[0051] 1.3 Test Methods

[0052] 1.3.1 Endophytic DNA extraction, amplification, and sequencing

[0053] (1) Disinfection of plant tissue surfaces

[0054] First, the root samples were repeatedly washed with tap water to remove surface soil. Then, the roots were cut into 3-4 cm sections using sterilized scissors. Surface sterilization was performed entirely in a clean bench, following a specific procedure: rinsing with sterile water for 30 seconds, washing with 75% ethanol for 1 minute, washing with 2% sodium hypochlorite solution for 3 minutes, washing again with 75% sterile ethanol for 1 minute, and rinsing with sterile water 2-3 times. After sterilization, all root sections were blotted dry with sterile filter paper. To verify the effectiveness of surface sterilization, 200 µL of the final rinse solution was evenly spread on PDA and LB agar plates and incubated at 28°C for 3-7 days. If no colonies appeared on the agar plates, it indicated that microorganisms on the surface of the plant material had been eliminated, and the material could be used for subsequent endophytic bacteria analysis.

[0055] (2) Extraction of total DNA

[0056] The root samples, after the epidermis was removed, were ground into powder under liquid nitrogen conditions, transferred to sterile centrifuge tubes, and stored at -80°C for total DNA extraction. (According to EZNA) ® Total genomic DNA was extracted from microorganisms using the DNA kit (Omega Bio-tek, USA). The quality of the extracted genomic DNA was assessed using 1% agarose gel electrophoresis, and the purity and concentration of the DNA were determined using a micro-volume UV spectrophotometer (NanoDrop 2000, USA).

[0057] (3) PCR amplification and sequencing

[0058] Using the extracted DNA as a template, the ITS1 region of the fungal ITS gene was amplified using the universal primers ITS1F / ITS2R. ] The V5-V7 region of the bacterial 16S gene was amplified using primers 799F / 1193R. Specific primer information is shown in Table 1.

[0059] Table 1 Sequencing Primer Information

[0060]

[0061] PCR reactions were performed using Pro Taq DNA polymerase, with a 20 μL reaction system. Endophytic fungal PCR amplification conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 35 cycles; stable extension at 72℃ for 10 min. Endophytic bacterial PCR amplification conditions: 95℃ pre-denaturation for 3 min; 27 primer cycles in the first round; 13 primer cycles in the second round (95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s); stable extension at 72℃ for 10 min; finally, storage at 10℃ (PCR instrument: T100 Thermal Cycler PCR, USA).

[0062] PCR products were recovered using 2% agarose gel electrophoresis. The recovered products were purified using a DNA gel recovery and purification kit (PCR Clean-Up Kit, China Yuhua), and quantified using Qubit 4.0 (Thermo Fisher Scientific, USA). High-throughput sequencing was performed on the Illumina Nextseq 2000 platform, outsourced to Shanghai Meiji Biopharmaceutical Technology Co., Ltd.

[0063] 1.3.2 Isolation, purification and preservation of plant endophytic bacteria

[0064] (1) Isolation of endophytic fungi

[0065] Blot the sterilized plant tissue dry on sterile filter paper. In a laminar flow hood, use a sterile scalpel to remove the epidermis around the roots, then longitudinally cut the root segments in half. Use sterile forceps to pick up small sample pieces and place them on the surface of PDA solid culture medium containing chloramphenicol. Press the small pieces gently with the forceps to ensure full contact between the cut surface and the culture medium. Inoculate four small root samples of the same type into each culture medium, with five replicates per sample. Label the inoculated material with the name and date. Seal the culture medium with sealing film and incubate upright in a 28°C incubator for 3-7 days.

[0066] (2) Isolation of endophytic bacteria

[0067] Under aseptic conditions, the epidermis of the sterilized plant material was removed using a sterile scalpel, and the central portion was cut into pieces approximately 1 cm in size. 3 Small pieces. Weigh 1 g of material and place it in a flame-sterilized mortar. Add 9 mL of sterile water and grind thoroughly to form a homogenate. Then, pack the homogenate into tubes. Pipette 1 mL of the supernatant into 9 mL of sterile water and mix well. Dilute sequentially to obtain 10. -1 10 -2 10 -3 10 -4 10-5 The concentration gradient was calculated, and 100 μL of the supernatant for each concentration was evenly spread on LB medium and incubated upside down in a dark chamber at 28°C for 2-5 days.

[0068] (3) Purification of endophytic bacteria

[0069] During the cultivation process, continuously observe the plant tissue cut surfaces for mycelial growth. Once mycelia appear on the PDA medium, promptly use a sterile inoculation loop to pick out different morphologies of mycelia and transfer them to new PDA plates, which are then placed in a 28°C incubator for further cultivation. After the newly inoculated mycelia have grown further and formed stable colonies, endophytic fungi are purified using the tip mycelial picking method. Through repeated purification processes, single colonies are eventually obtained. Subsequently, the surface morphology of the colonies is observed, and samples are numbered and recorded until no new morphologies of fungi appear.

[0070] On LB medium, bacteria were initially distinguished based on colony color, morphological characteristics, and growth time differences, and isolated using the streak plate method. Different strains were then transferred to fresh LB medium and cultured at 28°C. The cultures were purified repeatedly until a single strain was obtained.

[0071] (4) Short-term preservation methods

[0072] The endophytic bacteria, after undergoing several purification processes, were numbered and inoculated onto corresponding slant agar plates, and cultured at 28°C. Once colony growth stabilized, their morphology was observed to ensure consistency with that of the plate culture, and that there was no contamination. The strains that met the requirements were then transferred to a 4°C refrigerator for storage.

[0073] (5) Long-term preservation method

[0074] Dissolve 50 mL of glycerol reagent in 100 mL of distilled water, mix thoroughly, and autoclave at 121 °C for 20 min. Cool to room temperature and store. Obtain endophytic fungal mycelium using a punch, place the mycelium in cryovials containing 50% glycerol, and store at -80 °C.

[0075] The bacterial strain was inoculated into liquid culture medium and incubated at 37°C for 2-3 days to obtain a large amount of bacterial suspension in the logarithmic phase. The bacterial suspension was then thoroughly mixed with sterile 50% glycerol at a 1:1 ratio in cryovials and stored at -80°C.

[0076] 1.3.3 Screening of growth-promoting bacteria

[0077] (1) Nitrogen-fixing bacteria

[0078] The activated test strains were inoculated onto Ashby medium, with each strain in triplicate. The medium was incubated at 28°C for three consecutive subcultures, and the growth of the strains was observed. If the strains could still grow normally on the medium, it indicated that the strains had potential nitrogen-fixing capabilities.

[0079] (2) Phosphate-solubilizing bacteria

[0080] Activated fungal mycelia were directly inoculated onto inorganic and organic phosphorus agar plates. Activated bacteria were then agglutinated onto the same agar plates using sterile toothpicks for further incubation. Each strain was cultured in triplicate, with fungi incubated at 28°C for 5 days and bacteria at 28°C for 3 days. After incubation, the formation of a transparent phosphate-solubilizing zone on the agar plate was observed. The diameter of the phosphate-solubilizing zone (HD) was measured, and the ratio of the two was calculated to preliminarily screen strains with phosphate-solubilizing capabilities.

[0081] (3) Potassium-solubilizing and growth-promoting bacteria

[0082] The activated strains were inoculated onto potassium-solubilizing solid medium, with each strain in triplicate, and incubated at 28°C for several days. The appearance of the clear zone was observed, and the ratio of the clear zone diameter (HD) to the colony diameter (CD) was measured and calculated to preliminarily screen for strains with potassium-solubilizing capabilities.

[0083] (4) Siderogenic bacteria

[0084] The siderophore-producing ability of the strains was detected using the CAS plate assay. Activated strains were inoculated onto CAS agar medium, with three replicates for each strain, and cultured at 28°C in the dark for 5 days. After incubation, the color change of the medium around the colonies was observed. When an orange-yellow or purplish-red halo appeared around the colonies, the halo diameter (HD) and colony diameter (CD) were measured, and their ratio was calculated to preliminarily screen strains with siderophore-producing ability.

[0085] (5) Indole-3-acetic acid (IAA) producing bacteria

[0086] The Salkowski colorimetric method was used to screen for strains capable of producing the auxin indoleacetic acid (IAA). The test strains were inoculated with a solution containing L-tryptophan (100 mg·L⁻¹). -1 In LB medium or PDB liquid medium, at 28°C and 180 r·min -1 Under dark conditions, bacteria were cultured for 2 days and fungi for 7 days, with three replicates for each strain. After culture, 1 mL of bacterial culture was incubated at 10000 r·min⁻¹. -1Centrifuge for 10 min, take 100 µL of the supernatant and mix thoroughly with an equal volume of Salkowski colorimetric solution. Incubate at room temperature in the dark for 30 min. Preliminary assessment of the strain's IAA secretion ability is based on the color change. Use sterile culture medium as a negative control and IAA standard solution (100 μg / mL) as the reagent. -1 As a positive control, those whose color matches the standard solution are considered positive (indicating that the strain can secrete IAA), and those whose color does not match are considered negative.

[0087] Use 10, 20, 30, 40, 50 mg·L -1 The IAA standard solution was mixed with an equal volume of Salkowski colorimetric solution and reacted in the dark for 30 min. Sterile water was used as a control for zeroing, and the reaction was performed using a Spark ELISA reader at a wavelength of 530 nm. ® The absorbance of each solution was measured using a spectrophotometer (Switzerland). The absorbance (OD) was plotted on the x-axis as IAA concentration and the corresponding absorbance on the y-axis as OD. 530 Plot the IAA standard curve with the vertical axis as the ordinate. The screened IAA-producing strains were cultured under the same conditions, and the absorbance at 530 nm was measured using the method described above. The results were then substituted into the standard curve to calculate the IAA yield of the strains.

[0088] 1.3.4 Molecular biological identification of growth-promoting strains

[0089] Under aseptic conditions, a suitable amount of mycelium was scraped from endophytic fungal colonies cultured on PDA plates for 7 days, and genomic DNA was extracted from the growth-promoting strains according to the instructions of the fungal genomic DNA extraction kit. DNA was extracted from endophytic bacteria using a bacterial genomic DNA extraction kit (TSP701-50, Qingke Biotechnology).

[0090] Using extracted genomic DNA as a template, PCR amplification was performed using universal primers for fungi ITS1 and ITS4, and universal primers for bacteria 27F and 1492R, respectively (GeneAmp). ® PCR System 9700 (USA). Primer information is shown in Table 2, and the PCR reaction system is shown in Table 3. Fungal amplification conditions were: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s; 55℃ annealing for 30 s; 72℃ extension for 1 min, for a total of 30 cycles; after each cycle, a final extension at 72℃ for 10 min was performed, with three replicates. Bacterial amplification conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, with the remaining cycle parameters consistent with those for fungi. The amplified products were detected by 1% agarose gel electrophoresis. After confirming the detection of the target band, the products were sent to Nanjing Qingke Biotechnology Co., Ltd. for Sanger sequencing analysis.

[0091] Table 2 Primer information used for strain identification

[0092]

[0093] Table 3 PCR amplification system

[0094]

[0095] 1.3.5 Preparation of bacterial suspension

[0096] The selected growth-promoting fungi were activated, and the fungal cakes were inoculated into 50 mL of PDB liquid medium and incubated at 28℃ and 180 r·min. -1 Under shaking conditions, the culture medium was incubated for 5 days. After incubation, the culture medium was filtered through sterile gauze, and the spore suspension was collected and cultured at 3000 r·min. -1 Centrifuge for 5 min under the specified conditions. Resuspend the spore pellet in sterile water and adjust the spore concentration to 10. 6 -10 8 Spores / mL, stored at 4℃ for short-term use.

[0097] The selected growth-promoting bacteria were activated, and single colonies were picked and inoculated into LB liquid medium. The culture was then incubated at 28°C and 180 rpm. -1 The culture was incubated under shaking conditions for 3 days. After the incubation period, the temperature was increased to 6000 r·min. -1 Centrifuge for 10 min under the specified conditions, discard the supernatant to obtain bacterial precipitate, and resuspend in an appropriate amount of sterile water, washing twice. Then add sterile water and adjust the bacterial suspension concentration to OD at 600 nm. 600 ≈ 1, store at 4℃ for short-term use. Add sterile water to dilute to OD before inoculation. 600 Use after preparing a bacterial solution of 0.4-0.5.

[0098] 1.3.6 Functional verification of growth-promoting strains

[0099] The growth-promoting effect of the strains was verified using a pot experiment. One-year-old seedlings of *Firmiana simplex*, *Acer pentaphyllum*, and *Ligustrum lucidum*, all with good growth, free from pests and diseases, and exhibiting relatively uniform growth, were selected as test plants. An organic cultivation substrate, sterilized at high temperature and free of insect eggs and weed seeds, was used as the culture medium. The selected growth-promoting strains were inoculated into the rhizosphere of the plants using a root drenching inoculation method. A control group without inoculation and irrigated with an equal volume of sterile water served as a blank control. Each treatment group and control group had three replicates.

[0100] The roots of the seedlings were repeatedly rinsed with tap water until the rinse solution was clear and free of turbidity. The roots were then immersed in 75% ethanol for surface sterilization for 5 minutes, followed by repeated rinsing with sterile water to remove residual ethanol. Finally, the surface moisture was blotted dry with sterile paper. The treated plants were planted in pots containing sterilized substrate and cultured for 10 days before inoculation. The bacterial suspension was evenly poured along the root zone using a syringe, with 40 mL applied every 10 days for a total of 3 inoculations. Throughout the experimental period, regular watering, weeding, and pest and disease control were carried out to ensure normal seedling growth. Subsequent indicators were measured 50 days after inoculation.

[0101] 1.3.7 Measurement Indicators and Methods

[0102] (1) Plant growth indicators

[0103] Seedling height was measured using a folding tape measure, and diameter at ground level was measured using vernier calipers. After the experiment, destructive sampling of seedlings was performed to determine biomass. For each treatment of different tree species, three plants were randomly selected, and the plants were divided into above-ground and below-ground parts using scissors. The fresh weight of each part was measured. The parts were marked, dried in an oven to constant weight, and then weighed.

[0104] (2) Physiological and biochemical indicators of plants

[0105] Chlorophyll content was measured using a chlorophyll meter. Three healthy leaves from the upper, middle, and lower parts of the plant were selected from each treatment group and the control group for measurement.

[0106] The soluble protein content in plant roots and leaves was determined using the Coomassie brilliant blue staining method.

[0107] The soluble sugar content of plant roots and samples was determined using the anthrone colorimetric method.

[0108] (3) Plant nutrient content

[0109] Leaves from three plants were randomly selected from different treatment groups of each tree species as samples, with each plant considered as a replicate. The samples were dried at 65℃ to constant weight to prepare dry samples, which were then pulverized using a grinder and passed through a 100-mesh sieve to obtain uniform powder for later use.

[0110] Samples were pretreated using a sulfuric acid-hydrogen peroxide digestion method. The contents of total nitrogen (TN), total phosphorus (TP), and total potassium (TK) in leaves were determined using the Kjeldahl method, the molybdenum-antimony colorimetric method, and the flame photometry method, respectively. A blank experiment was conducted, with all reagent dosages and procedures identical to those used for the sample determination, except that no sample was added.

[0111] 1.4 Data Processing and Analysis

[0112] The raw paired-end sequences were quality controlled using FASTP software (Version 0.19.6), and sequence assembly and filtering were performed using FLASH software (Version 1.2.7) to obtain high-quality data. Based on the effective sequences, Operational Taxonomic Unit (OTU) clustering was performed using UPARSE software (Version 2.2) at a 97% similarity level, while chimeras were removed. To reduce the impact of sequencing depth differences on the analysis results, flattening was performed using the minimum sample sequencing volume as the standard.

[0113] The RDP classifier Bayesian algorithm was used to annotate the representative OTU sequences, and community species composition was statistically analyzed at the phylum and genus levels. Comparative classification of endophytic fungi and bacteria was performed using the UNITE (Release 8.0) and SILVA (Release 138) databases (confidence level 0.7), respectively. Functional prediction of endophytic fungi was performed using FUNGuild (Version 1.0), and functional prediction of endophytic bacteria was performed using PICRUSt2 (Version 2.2.0) software combined with the KEGG (Kyoto Encyclopedia of Genes and Genomes) and COG (Clusters of Orthologous Groups) databases.

[0114] The Alpha diversity index of the endophytic microbiota was calculated using Mothur software (Version 1.30.2). ] The indices included the Ace index, Chao index, Shannon index, and Simpson index. One-way ANOVA was performed using SPSS software to examine the significance of differences among different tree species.

[0115] Data analysis and visualization were performed using R (Version 3.3.1), including dilution curves, Venn plots of endophytic bacteria at the OUT level, and bar charts of relative abundance of the community. Principal Coordinates Analysis (PCoA) was performed based on the Bray-Curtis distance algorithm, and the differences in community structure were analyzed by combining the Adonis test and Partial Least Squares Discriminant Analysis (PLS-DA).

[0116] Linear discriminant analysis (LDA) in LEfSe software was used to identify significantly different bacterial communities at the genus level among different groups (LDA > 3.5, P < 0.05) and to assess their impact on community structure.

[0117] ContigExpress software was used to perform sequence assembly and quality control on the Sanger sequencing results. The obtained sequences were aligned to the NCBI database using blastn (Version 2.13) to obtain accession numbers and species annotation information for similar sequences. Reference sequences with high similarity to the target sequence were downloaded, and a phylogenetic tree was constructed using MEGA 6.0 software with neighbor-joining and 1000 bootstrap replicates to determine the phylogenetic position of the strains.

[0118] SPSS software was used to perform statistical analysis on the growth-promoting ability screening and pot experiment data, and significance tests were conducted; Prism 8.2 software was used to draw relevant bar charts.

[0119] 2 Results and Analysis

[0120] 2.1 Analysis of the composition and function of endophytic microbial communities in tree root systems

[0121] 2.1.1 Endophytic bacteria sequencing results

[0122] High-throughput sequencing results of endophytic bacteria from the roots of *Firmiana simplex*, *Acer pentaphyllum*, and *Ligustrum lucidum* (Table 4) showed that the effective sequences for endophytic fungi totaled 865,801, with a total base count of 222,200,220 bp and an average sequence length of 258 bp; while the effective sequences for bacteria totaled 769,940, with a total base count of 290,747,840 bp and an average sequence length of 377 bp. After clustering and annotation based on 97% sequence similarity, a total of 990 fungal OTUs and 2,769 bacterial OTUs were obtained. The dilution curves for each sample tended to flatten after reaching a certain sequencing depth, indicating that the current sequencing depth can effectively reflect the true diversity of endophytic bacteria in the samples and can be used for subsequent analysis. Figure 2 ).

[0123] Table 4. Statistical information on endophytic bacteria sequences

[0124]

[0125] Venn diagram analysis shows that ( Figure 3The endophytic fungal communities of the three tree species contained a total of 82 OTUs, accounting for only 8.28% of the total OTUs. Each tree species had a high proportion of endemic OTUs, with *Firmiana simplex*, *Acer pentaphyllum*, and *Ligustrum lucidum* having 219 (22.12%), 267 (26.97%), and 253 (25.56%), respectively. The bacterial communities of the three tree species contained a high number of out-of-cell (OUT) OTUs, totaling 591 (21.34%), indicating a high degree of commonality among different tree species. The endemic OTUs for each tree species were 428 (15.46%) for *Firmiana simplex*, 533 (19.25%) for *Acer pentaphyllum*, and 515 (18.60%) for *Ligustrum lucidum*. In terms of the total number of OTUs, endophytic fungi had 391, 469, and 463 OTUs detected in *Firmiana simplex*, *Acer pentaphyllum*, and *Ligustrum lucidum*, respectively, while bacteria had 1388, 1677, and 1588 OTUs, respectively, all significantly higher than the fungal communities. Pairwise comparisons revealed that the number of OTUs (79 fungi and 333 bacteria) was highest between Acer pentaphyllum and Ligustrum lucidum, indicating a high degree of similarity in the community structure between these two tree species.

[0126] 2.1.2 Endophytic community diversity analysis

[0127] The results of α-diversity analysis of root endophytic fungi (Table 5) showed that the richness indices (Ace and Chao indices) of endophytic fungi and bacteria did not differ significantly among different tree species (P > 0.05), while the diversity indices (Shannon and Simpson indices) showed significant differences (P < 0.05). The Shannon index of endophytic fungi in the roots of *Acer pentaphyllum* was significantly higher than that of *Firmiana simplex*, while the Simpson index was the opposite (P < 0.05), indicating higher fungal diversity. The Shannon index of endophytic bacteria in the roots of *Ligustrum lucidum* was significantly higher than that of *Acer pentaphyllum*, while the Simpson index was the opposite (P < 0.05), indicating higher bacterial diversity.

[0128] Table 5 Alpha diversity index of root endophytic bacteria in different tree species

[0129]

[0130] Note: Different lowercase letters indicate significant differences among samples of different tree species (P < 0.05).

[0131] PCoA analysis showed that the first two principal axes (PC1 and PC2) of the fungal community explained 21.39% and 15.30% of the variation, respectively, with a cumulative contribution of 36.69%. Figure 4 A); the first two principal axes of the bacterial community explained 36.52% and 24.65% of the variation, respectively, contributing a total of 61.17% ( Figure 4B). Combined with the Adonis test results, it was found that tree species significantly affected the differences in endophytic fungal and bacterial community structure (P < 0.05), with the bacterial community being more strongly affected by tree species differences (fungi R² = 0.2925, P = 0.002; bacteria R² = 0.4797, P = 0.001). The PLS-DA results further indicated that endophytic fungi from different tree species were relatively independently distributed across different dimensions, exhibiting a clear trend of segregation. Figure 4 C, D).

[0132] 2.1.3 Analysis of Endophytic Community Composition

[0133] A total of 10 fungal phyla and 350 fungal genera, as well as 32 bacterial phyla and 668 bacterial genera, were detected in root samples from the three tree species. To compare the dominant taxa and their relative abundance characteristics of the root endophytic microbial communities of different tree species, the top 5 most abundant microbial taxa at the phylum level and the top 15 most abundant microbial taxa at the genus level were selected to create community abundance bar charts. Figure 5 The remaining low-abundance taxa were merged into "Others".

[0134] At the phylum level, endophytic fungi were predominantly Basidiomycota (62.58%) and Ascomycota (30.98%), with an average combined share exceeding 90%. Other phyla had lower abundances, with only Glomeromycota exceeding 1% in Ligustrum lucidum. Endophytic bacteria were dominated by Pseudomonadota, Actinomycetota, and Bacillota, with an average combined share of 97.43%. Pseudomonadota were more abundant in *Firmiana simplex* and *Acer pentaphyllum*, while Bacillota were more abundant in *Acer pentaphyllum* and Ligustrum lucidum.

[0135] At the genus level, "Others" genera accounted for a large proportion in each sample, indicating a complex root microbial community structure with a large number of low-abundance genera. The common endophytic fungi among the three tree species were mainly *Naganishia*, *Porotheleum*, *Filobasidium*, and *Aspergillus*; the common endophytic bacteria were mainly *Enterobacter*, *Pseudomonas*, *Bradyrhizobium*, and *Actinophytocola*. The fungal community of *Firmiana simplex* was relatively balanced, with endemic genera such as *Marasmius* and *Lysurus*, while the bacterial community showed a high proportion of *Escherichia-Shigella*. Among the endophytic fungi in the roots of *Acer pentaphyllum*, a few dominant genera were abundant, such as *Leptoxyphium*, *Enterobacter*, and *Pseudomonas*. In contrast, the root microbial community of *Ligustrum lucidum* was more complex and diverse. The endemic *Trechispora* fungus was not detected in either *Firmiana simplex* or *Acer pentaphyllum*. Dominant bacterial genera included *Niallia*, *Paenibacillus*, and *Mycobacterium*.

[0136] 2.1.4 Analysis of Endophytic Microbiota Marker Species

[0137] At the genus level, the Kruskal-Wallis rank-sum test was performed on the top 15 root endophytic fungi with the highest sum of abundance among the three tree species. Figure 6-1 A, B), and used LEfSe analysis to screen out microbial groups with LDA scores > 3.5 ( Figure 6-2 (C, D). The results showed that the endophytic fungal communities of the three tree species showed significant differences in genera including *Chaetothyriales*, *Ustilago maydis*, *Trogia*, *Dominikia*, *Cladophialophora*, and *Scytalidium*. Among them, *Chaetothyriales* (LDA = 5.29) and *Ustilago maydis* (LDA = 4.50) were significantly enriched in *Acer pentaphyllum*, with relative abundances of 30.04% and 4.68%, respectively; *Chaetothyriales_gen_Incertae_sedis* (LDA = 4.63, 6.39%) and *Dominikia* (LDA = 4.43, 2.14%) were enriched in *Ligustrum lucidum*; and *Cladophialophora* (LDA = 4.03, 1.53%) was enriched in *Firmiana simplex*.

[0138] Significantly different genera in the endophytic bacterial community included *Escherichia coli*-*Shigella*, *Pseudomonas*, *Rhizobium*, and *Actinomadura*. Among these, *Escherichia coli*-*Shigella* (LDA = 4.94, 21.15%), *Rhizobium* (LDA = 4.57, 9.35%), *Actinophytocola* (LDA = 4.70, 9.54%), and *Actinomadura* (LDA = 4.60, 7.64%) were significantly enriched in *Firmiana simplex*; *Pseudomonas* (LDA = 4.92, 17.3%) was significantly enriched in *Acer pentaphyllum*; and *Nereidum* (LDA = 4.83, 9.73%) and *Bacillus* (LDA = 4.61, 5.95%) were significantly enriched in *Ligustrum lucidum*.

[0139] 2.1.5 Prediction of Endophytic Microbial Ecological Functions

[0140] The results of fungal community function prediction indicate that ( Figure 7 The endophytic fungi in the three tree species mainly consist of three basic trophic types and three cross-trophic types, including saprophytic, symbiotic, pathotrophic, patho-saprophytic, patho-saprophytic-symbiotic, and saprophytic-pathological-symbiotic trophic types. Excluding unidentified groups, 13 ecological function subclasses were identified, with saprophytic having the highest proportion (31.11%-53.39%). Wood-saprophytic fungi had the highest relative abundance in Ligustrum lucidum (24.03%), but less than 2% in the other tree species. Undefined saprophytic fungi were dominant in all tree species, with an average abundance of 19.79%. Leaf saprophytic-wood saprophytic fungi were only found in Acer pentaphyllum and Ligustrum lucidum, with an average abundance of 7.50%. Litter saprophytic-soil saprophytic-wood saprophytic fungi had an average abundance of 19.72% in Firmiana simplex, significantly higher than in Acer pentaphyllum and Ligustrum lucidum.

[0141] The functional annotation results from the KEGG database (Table 6) show that the root endophytic bacteria of the three tree species contain a total of six types of biological metabolic pathways in the primary functional layer, among which metabolic pathways are the main functional categories, accounting for 74.59%-78.67%. COG functional classification shows ( Figure 8 The functional composition of endophytic bacteria in the roots of the three tree species was generally similar. Aside from classifications of unknown functions, their main functional types were concentrated in substance transport and metabolism, such as amino acid transport and metabolism (E, 10.54%-10.88%), carbohydrate transport and metabolism (G, 6.18%-7.19%), and inorganic ion transport and metabolism (P, 6.64%-7.51%). Simultaneously, functions related to genetic information processing and cell structure also accounted for a significant proportion, such as transcription (K, 6.03%-7.13%) and translation, ribosome structure and biosynthesis (J, 5.43%-5.70%).

[0142] Table 6. Functional percentage of different samples in the first-level functional layer of KEGG

[0143]

[0144] 2.2 Isolation of root endophytic bacteria

[0145] In this study, 23 endophytic fungi were isolated from the roots of three tree species using PDA medium, and 26 endophytic bacteria were isolated using LB medium, for a total of 49 endophytic fungi. Among them, 16, 13, and 20 endophytic bacteria were obtained from *Firmiana simplex*, *Acer pentaphyllum*, and *Ligustrum lucidum*, respectively. All strains were purified to obtain stable single colonies. Figure 9 and Figure 10 The images show the colony morphology of the isolated endophytic fungi and endophytic bacteria, along with their corresponding strain numbers (F indicates fungi, B indicates bacteria; the second letter indicates the source: Q for *Firmiana simplex*, W for *Acer pentaphyllum*, N for *Ligustrum lucidum*; the numbers indicate the order of strain isolation).

[0146] Fungal colonies are predominantly white, green, or brown, with regular or wavy edges. Some strains exhibit vigorous hyphae growth, resulting in a fluffy or cottony surface, such as FW02 and FW05. Other strains have dense colonies with relatively smooth surfaces, such as FQ01 and FQ04. Still others show concentric rings between the center and edge of the colony, such as FQ04 and FW03. Bacterial colonies are predominantly white or pale yellow, with some strains displaying pale pink or yellow colonies, such as BQ06 and BN06. Colony shapes are mostly round or nearly round, with smooth or viscous surfaces. Most strains have relatively regular edges, while a few have slightly wavy edges. The growth rates of different endophytic fungi and bacteria vary significantly under plate culture conditions; most strains grow rapidly, while some grow relatively slowly.

[0147] 2.3 Growth-promoting ability of endophytic fungi in tree root systems

[0148] 2.3.1 Nitrogen fixation capacity

[0149] All isolated endophytic bacteria were inoculated onto Ashby's solid medium and cultured. The results showed that ( Figure 11 Under conditions lacking exogenous inorganic nitrogen, 33 endophytic bacteria strains (67.35% of the total isolated strains) were able to grow normally, preliminarily indicating that these strains have a certain nitrogen-fixing potential. Further observation revealed that 10 endophytic fungi and 3 endophytic bacteria formed distinct clear zones on the culture plates. The growth morphology of different strains on the plates varied; some strains exhibited vigorous colony growth and relatively obvious clear zones, such as FQ01 and FN07; while others had smaller or less obvious clear zones.

[0150] 2.3.2 Phosphorus solubility

[0151] All strains were inoculated into inorganic phosphorus and organic phosphorus media, respectively. After incubation, observation revealed that some strains exhibited a clear halo around their colonies. A total of 13 endophytic bacteria were found to have the ability to dissolve organic phosphorus, and 11 endophytic bacteria were found to have the ability to dissolve inorganic phosphorus.

[0152] In the determination of organophosphorus solubility ( Figure 12 A) The ratio of the transparent zone diameter to the colony diameter (HD / CD) of each strain ranged from 1.09 to 4.27. Among them, strain BW05 had the highest HD / CD value, which was significantly higher than other strains (P < 0.05), showing the strongest organophosphate solubility. Strains FQ05, BQ06 and BQ04 had the next highest organophosphate solubility, while the HD / CD values ​​of the remaining strains were relatively low.

[0153] In the determination of inorganic phosphorus solubility ( Figure 12 (B) The HD / CD values ​​of each strain ranged from 1.08 to 4.80. Strain BW05 also exhibited the strongest phosphorus solubility, with its HD / CD value significantly higher than other strains (P < 0.05). The phosphorus solubility of the remaining strains was relatively weak. There were significant differences in phosphorus solubility among different strains, while strain BW05 showed high phosphorus solubility potential in both organic and inorganic phosphorus solubility tests.

[0154] Note: The top right corner of the bar chart shows the phosphate solubility effect of some strains, and the center shows the colonies, with a clear zone visible around each colony. Different lowercase letters indicate significant differences between different strains (P < 0.05). The same applies below.

[0155] 2.3.3 Potassium solubilization capacity

[0156] All isolated strains were inoculated onto potassium-solubilizing solid medium. After cultivation, observation revealed that no obvious transparent hydrolysis zone formed around any strain, indicating that none of the isolated strains exhibited significant potassium-solubilizing activity under the experimental conditions. All strains could grow normally on potassium-solubilizing medium, but differences existed in colony size, edge morphology, and hyphal development. Figure 13 ).

[0157] 2.3.4 Iron production capacity

[0158] All isolated strains were inoculated onto CAS solid medium, and observations were made after the culture period. Figure 14Some strains exhibited distinct orange-yellow or purplish-red halos around their colonies, and a total of 13 endophytic bacteria were detected to possess siderophore-producing capabilities. The ratio of the siderophore diameter to the colony diameter (HD / CD) ranged from 1.49 to 4.39, with strain FQ04 showing the highest HD / CD value, significantly higher than other strains (P < 0.05), demonstrating the strongest siderophore-producing ability. Strains FN03 and FW03 also showed strong siderophore-producing capabilities, with relatively clear halos. The ratios of strains BQ05, BQ04, FW05, and FN09 were relatively low, with no significant differences between groups.

[0159] 2.3.5 IAA Production Capability

[0160] All isolated strains were inoculated into a culture medium containing L-tryptophan and cultured with shaking. The supernatant of some strains turned pink, and a total of 25 endophytic bacteria were found to have the ability to produce IAA. Figure 15 The concentration range of IAA produced by each strain was 7.37-35.90 μg·mL. -1 Among them, strain BQ04 exhibited the strongest IAA production capacity, significantly higher than other strains (P < 0.05). Strains FQ05, FN05, BN01, and FN06 also showed strong IAA production capacity, with concentrations around 18 μg·mL⁻¹. -1 Of the above, strain BQ07 has the weakest IAA production capacity.

[0161] 2.3.6 Comparison of Endophytic Bacterial Growth-Promoting Ability

[0162] The nitrogen fixation, phosphorus solubilization, potassium solubilization, siderophore production, and IAA production capabilities of all isolated strains were compared. The results (Table 7) showed significant differences among all strains in various growth-promoting indicators. However, no clear zone was observed on the potassium-solubilizing medium, indicating that all strains had weak or no potassium-solubilizing ability (-). If a strain had the ability to solubilize organic or inorganic phosphorus, it was considered to have phosphorus solubilization ability (+). Overall, most strains had 1-3 growth-promoting abilities, such as strains FQ01, FW01, and BQ05; 6 strains did not have the above growth-promoting abilities, such as strains FQ02, FW04, and BQ01.

[0163] Through comprehensive screening using multiple indicators, five growth-promoting endophytic bacteria with nitrogen fixation, phosphorus solubilization, siderophore production, and IAA production capabilities were finally obtained, namely strains FQ05, FN06, FN07, BQ04, and BN02. To clarify their taxonomic status, further molecular biological identification was carried out on these strains.

[0164] Table 7. Characteristics of the growth-promoting ability of root endophytic bacteria

[0165]

[0166]

[0167] Note: "+" indicates positive, and "-" indicates negative.

[0168] 2.4 Phylogenetic analysis of growth-promoting bacteria

[0169] ITS gene amplification and sequencing were performed on three endophytic fungi, yielding effective sequences of approximately 700 bp in length. BLAST alignment of the sequences with the NCBI database revealed (Table 8 and...) Figure 16 The sequences of strains FQ05, FN06, and FN07 showed high sequence identity of 99.64%, 99.66%, and 99.83% with those of Penicillium citrinum (OP237245.1), Penicillium oxalicum (OL687558.1), and Penicillium chrysogenum (JN561259.1), respectively. In the phylogenetic tree, the above three fungal strains clustered into independent branches with their corresponding reference strains with 100% support.

[0170] Two endophytic bacteria strains underwent 16S rRNA gene amplification and sequencing, yielding effective sequences of 1500 bp in length. BLAST alignment of the sequences with the NCBI database revealed the results (Table 8 and...). Figure 17 The sequences of strains BQ04 and BN02 are 100.00% and 99.93% identical to those of Bacillus cereus (MN074540.1) and Pseudomonas chlororaphis (CP050510.1), respectively. On the phylogenetic tree, the two bacteria and their corresponding reference strains cluster into independent branches with 99% and 100% support, respectively.

[0171] Based on the comprehensive molecular identification results, strain FQ05 was identified as *Penicillium citrinum*, strain FN06 as *Penicillium oxalicum*, and strain FN07 as *Penicillium chrysogenum*; strain BQ04 was identified as *Bacillus cereus*, and strain BN02 was identified as *Pseudomonas chlororaphis*.

[0172] Table 8. Sequence Comparison Results

[0173]

[0174] 2.5 Effects of inoculation with growth-promoting bacteria on seedling growth physiology

[0175] 2.5.1 Effects of bacterial strain inoculation on seedling height and ground diameter

[0176] Inoculation with different bacterial strains significantly promoted the growth of seedlings of *Firmiana simplex*, *Acer pentaphyllum*, and *Ligustrum lucidum*. Figure 18 The seedling height of the three seedlings was highest with inoculation treatments of FN06, FN07, and BN02, respectively, significantly increasing by 43.88%, 56.32%, and 81.96% compared to the control (CK). Analysis of variance results showed that ( Figure 19 The seedling heights of *Firmiana simplex* and *Acer pentaphyllum* seedlings treated with growth-promoting bacteria were significantly higher than those of the control (P < 0.05). The seedling heights of *Ligustrum lucidum* seedlings treated with BN02, FQ05, FN06, and FN07 were significantly higher than those of the control, but there was no significant difference between the BQ04 treatment and the control (CK). Overall, BN02 and FN07 inoculations showed the best effect on promoting seedling height in all three seedling types.

[0177] The ground diameter (DBD) of *Firmiana simplex*, *Acer pentaphyllum*, and *Ligustrum lucidum* seedlings was highest with inoculation treatments of FN06, BN02, and FQ05, respectively, significantly increasing by 35.12%, 37.93%, and 37.78% compared to the control. Analysis of variance showed that the DBD of *Firmiana simplex* and *Ligustrum lucidum* seedlings inoculated with growth-promoting bacteria was significantly higher than the control, while there was no significant difference in DBD between the *Acer pentaphyllum* seedlings inoculated with FQ05 and the control. In summary, inoculation with BN02 and FN06 had the best promoting effect on the DBD of the three seedlings.

[0178] 2.5.2 Effects of bacterial strain inoculation on seedling biomass

[0179] Biomass measurements (Table 9) showed that inoculation with growth-promoting bacteria had varying degrees of impact on the aboveground and belowground biomass accumulation of *Firmiana simplex*, *Acer pentaphyllum*, and *Ligustrum lucidum*. Compared with the control (CK) treatment, inoculation with FQ05, FN06, and FN07 significantly increased the fresh weight of *Firmiana simplex* (P < 0.05), with aboveground biomass increasing by 125.25%–137.79% and belowground biomass increasing by 65.50%–77.26%. Inoculation with growth-promoting bacteria had no significant effect on the aboveground fresh weight of *Acer pentaphyllum*, but after FN06 and FN07 treatments, the belowground fresh weight increased significantly by 54.96% and 69.31% compared to the CK treatment, respectively. Inoculation with all five growth-promoting bacteria significantly increased the aboveground fresh weight of *Ligustrum lucidum*, increasing it by 92.72%–389.19% compared to the CK treatment; however, only inoculation with FQ05 significantly increased the belowground fresh weight, increasing it by 134.38% compared to the CK treatment.

[0180] Table 9. Effects of inoculated strains on the biomass of the three seedlings.

[0181]

[0182] Compared with the control (CK) treatment, inoculation with different bacterial strains significantly increased the dry weight of *Firmiana simplex* (P < 0.05), with aboveground biomass increasing by 132.51%–217.43% and underground biomass increasing by 71.48%–222.93%. Inoculation with growth-promoting bacteria had no significant effect on the aboveground dry weight of *Acer pentaphyllum*, but inoculation with FN06 and FN07 significantly increased the underground dry weight by 51.06% and 69.57% compared to the CK treatment, respectively. Inoculation with BN02, FQ05, FN06, and FN07 significantly increased the aboveground dry weight of *Ligustrum lucidum*, increasing it by 155.69%–190.24% compared to the CK treatment; however, only inoculation with FQ05 significantly increased the underground dry weight, increasing it by 82.64% compared to the CK treatment. Overall, strains FN07 and FN06 showed relatively stable promoting effects on the biomass of all three seedlings, significantly increasing the aboveground and underground biomass of *Firmiana simplex*, promoting the accumulation of underground parts in *Acer pentaphyllum* seedlings, and promoting the accumulation of aboveground biomass in *Ligustrum lucidum* seedlings.

[0183] 2.5.3 Effect of bacterial strain inoculation on seedling SPAD value

[0184] Compared with the control (CK) treatment, inoculation with different strains significantly increased the SPAD values ​​of the leaves of all three seedlings (P < 0.05). Analysis of variance results showed that ( Figure 20 For *Firmiana simplex* seedlings, inoculation with strains FQ05, FN06, and FN07 showed the best performance, increasing chlorophyll content by 45.53%, 47.10%, and 42.38% respectively compared to the control (CK) treatment, while the increases at inoculations with BQ04 and BN02 were relatively smaller. For *Acer pentaphyllum* and *Ligustrum lucidum* seedlings, inoculation with BQ04 significantly increased SPAD values, increasing by 45.85% and 57.11% respectively compared to the CK treatment. Furthermore, the SPAD values ​​of *Acer pentaphyllum* and *Ligustrum lucidum* leaves inoculated with BN02, FQ05, FN06, and FN07 increased by 15.85%-31.23% and 41.73%-53.78% respectively compared to the CK treatment. Overall, different strains can promote chlorophyll accumulation in seedlings to some extent, with a more significant promoting effect on SPAD values ​​in *Ligustrum lucidum* leaves.

[0185] 2.5.4 Effect of bacterial strain inoculation on the soluble protein content of seedlings

[0186] Inoculation with different bacterial strains increased the soluble protein content in the leaves and roots of the three seedlings, with the promoting effect on leaves generally being greater than that on roots. Figure 21In leaves, inoculation with strain FN06 showed no significant difference in soluble protein content between *Firmiana simplex* and the control (CK) treatment, while the other inoculation treatments significantly increased it by 8.82%–16.01% compared to CK. Inoculation with growth-promoting bacteria significantly increased the soluble protein content of *Acer pentaphyllum* leaves by 21.79%–29.93% compared to CK, and significantly increased it by 19.22%–41.26% in *Ligustrum lucidum* leaves. In roots, the soluble protein content of *Firmiana simplex* treated with FQ05, FN06, and FN07 was significantly higher than CK, increasing by 11.49%–15.09%; the soluble protein content of *Acer pentaphyllum* treated with BQ04, BN02, FN06, and FN07 was significantly higher by 7.22%–11.26%; and the soluble protein content of *Ligustrum lucidum* treated with FN06 and FN07 was significantly higher than CK, increasing by 12.84%–13.57%.

[0187] 2.5.5 Effect of bacterial strain inoculation on soluble sugar content in seedlings

[0188] Inoculation with different bacterial strains increased the soluble sugar content of all three seedlings. Figure 22 In the leaves, the soluble sugar content of *Firmiana simplex* was highest in the BQ04 treatment, significantly higher than other treatments (P < 0.05); the FN06 treatment was second highest, increasing by 52.12% compared to the control (CK). The soluble sugar content of *Acer pentaphyllum* leaves was significantly higher in the FN07 and BQ04 treatments than in the control (CK), increasing by 27.24% and 27.80%, respectively; the other treatments showed no significant difference from the control. The soluble sugar content of *Ligustrum lucidum* leaves was highest in the BN02 and BQ04 treatments, reaching 281.50 mg·g⁻¹. -1 and 262.91 mg·g -1 The results were significantly higher than those of other treatments.

[0189] In the root system, the soluble sugar content of *Firmiana simplex* was highest in the FN07 treatment, followed by the FN06 treatment, which significantly increased by 66.18% and 53.95% compared to the control (CK), respectively. The soluble sugar content of roots in all inoculation treatments of *Acer pentaphyllum* was significantly higher than that of the control (CK) (P < 0.05), with the FN07, FN06, and FQ05 treatments increasing by 45.31%, 42.04%, and 36.40% compared to the control, respectively. The soluble sugar content of *Ligustrum lucidum* roots was highest in the BN02 treatment, followed by the FN07 and BQ04 treatments, which increased by 190.04%, 145.63%, and 145.12% compared to the control, respectively. Overall, comparing different tree species and organs, the promoting effect of growth-promoting bacteria on the accumulation of soluble sugars in seedling roots was stronger than that in leaves, with the most significant increase observed in the soluble sugar content of *Ligustrum lucidum* roots.

[0190] 2.5.6 Effects of bacterial strain inoculation on nutrient content in seedling leaves

[0191] After inoculation with BQ04, BN02, FQ05, and FN06, the total nitrogen content in the leaves of *Firmiana simplex* was significantly higher than that in the control (P < 0.05), with the highest total nitrogen content observed in the FQ05 treatment (37.86 g·kg⁻¹). -1 (Table 10). In both Acer pentaphyllum and Ligustrum lucidum, all inoculation treatments significantly increased the total nitrogen content in leaves, with the BQ04 treatment showing the best effect, reaching 46.00 g·kg⁻¹. -1 and 32.06 g·kg -1 Compared with the CK treatment, it improved by 40.46% and 16.75%.

[0192] In *Firmiana simplex* and *Acer pentaphyllum*, strain BQ04 promoted phosphorus uptake by leaves, with total phosphorus content reaching 6.50 g·kg⁻¹. -1 and 7.32 g·kg -1 The total phosphorus content of privet leaves increased significantly by 64.14% and 189.33% compared to the control (CK). In contrast, the changes in total phosphorus content in privet leaves after inoculation with growth-promoting bacteria were relatively small, with only the BN02 and FQ05 treatments showing significant increases compared to the control (CK).

[0193] Compared with the control (CK), all inoculation treatments significantly increased the total potassium content in the leaves of the three seedling species. In *Firmiana simplex*, there were significant differences in total potassium content among the treatments, with BQ04 and FN06 treatments showing the best effects, increasing by 48.89% and 44.69% respectively compared to the CK. In *Acer pentaphyllum*, all five treatments significantly increased the total potassium content in the leaves, with strain BQ04 showing the best effect, increasing by 98.95%. In *Ligustrum lucidum*, there were significant differences in total potassium content among the treatments, with BN02 and BQ04 treatments showing the best effects, increasing by 35.06% and 30.29% respectively compared to the CK.

[0194] Overall, the total nitrogen, total phosphorus, and total potassium contents of leaves treated with the inoculated strains showed a significant upward trend compared with the control (CK) treatment. Among them, strains BQ04 and FQ05 effectively promoted the absorption and accumulation of nutrients by seedlings.

[0195] Table 10 Effects of inoculation with different bacterial strains on the total nitrogen, total phosphorus, and total potassium content in the leaves of three seedlings.

[0196]

[0197] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A Penicillium chrysogenum FN07 with multiple growth-promoting abilities, characterized in that, The aforementioned Penicillium chrysogenum FNO7, classified as Penicillium chrysogenum FNO7, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20261381, deposit date June 18, 2026, and address at Wuhan University, Wuhan, China.

2. A microbial inoculant, characterized in that, It includes Penicillium chrysogenum FN07 with multiple growth-promoting capabilities as described in claim 1, and an agriculturally acceptable carrier.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent can be a liquid bacterial agent or a solid bacterial agent.

4. The microbial agent according to claim 3, characterized in that, The spore concentration of Penicillium chrysogenum FN07, which has multiple growth-promoting abilities, in the liquid bacterial agent is 10. 6 -10 8 Spores / mL.

5. The application of Penicillium chrysogenum FN07 with multiple growth-promoting abilities as described in claim 1, or the microbial inoculant as described in any one of claims 2-4, in promoting the growth of tree species in limestone mountain afforestation, characterized in that, The afforestation tree species include at least one of Chinese parasol tree, five-lobed maple, and privet.

6. The application according to claim 5, characterized in that, The strains or microbial agents with multiple growth-promoting abilities were inoculated into the rhizosphere of the plant using the root irrigation inoculation method.

7. The application according to claim 5, characterized in that, The specific manifestations of promoting the growth of tree species in limestone mountain afforestation are as follows: after inoculation with strains or microbial agents with multiple growth-promoting abilities, the seedling height, ground diameter, biomass, chlorophyll content, soluble protein content, soluble sugar content, and leaf total nitrogen, total phosphorus, and total potassium content of the plants are significantly higher than those of the uninoculated control.

8. The application according to claim 5, characterized in that, The specific inoculation steps are as follows: inoculate once every 10 days, each time inoculating each plant with 40 mL of liquid microbial agent, for a total of 3 inoculations.

9. The use of Penicillium chrysogenum FN07 with multiple growth-promoting abilities as described in claim 1 or the microbial agent as described in any one of claims 2-4 in the preparation of plant growth promoters, bio-fertilizers or soil conditioners.

10. The application according to claim 9, characterized in that, The plant growth promoters, bio-fertilizers, or soil conditioners are used for ecological restoration or seedling cultivation in limestone hilly areas.

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