Agrobacterium plurifermans f32 with activity of promoting ectomycorrhiza formation and application thereof

Abstract

The present application relates to the technical field of microbial inoculant, and discloses Agrobacterium tumefaciens F32 with the activity of promoting ectomycorrhizal formation and application thereof. Agrobacterium tumefaciens F32 is preserved in Guangdong Microbial Culture Collection Center, the address is No. 59, Building 5, 100, Martyrs' Road, Guangzhou, the preservation time is January 9, 2026, the preservation number is GDMCC No: 67611, and the classification and naming are Agrobacterium pusense In the present application, Agrobacterium tumefaciens F32 is isolated from the fruiting body of ectomycorrhizal fungus Morchella angusticeps, has excellent solubility for insoluble organic phosphorus and inorganic phosphorus, can enhance the mycorrhizal phosphorus absorption and enrichment of Morchella angusticeps, can not only promote the mycorrhizalization of plant roots and improve the mycorrhizal infection rate, but also promote the growth of Morchella angusticeps under the conditions of separate inoculation or co-inoculation with ectomycorrhizal fungi, and has a synergistic effect with ectomycorrhizal fungi, and can be used for preparing biological agents for promoting the growth of ectomycorrhizal seedlings such as Morchella angusticeps.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, and in particular to *Pusarobacter* F32, which has the activity of promoting ectomycorrhizal formation, and its applications. Background Technology

[0002] Ectomycorrhizal fungi (EMF) are a type of soil fungi whose hyphae can invade the intercellular spaces of the root cortex of higher economic woody plants, extending to form a network-like tissue called a Hartig net. Simultaneously, they spread on the root surface to form a mantle. Through the exchange of mineral nutrients between the EMF fungi and the plant's photosynthetic products, a mutually beneficial symbiotic relationship is formed between the plant and the fungi. They have excellent growth-promoting and disease-resistant effects on plants, and as microbial fertilizers, ectomycorrhizal fungi do not cause environmental pollution, making them environmentally friendly.

[0003] As an important reproductive organ of ectomycorrhizal fungi, the fruiting body provides a stable internal microenvironment that offers suitable conditions for the survival and reproduction of specific microorganisms. These bacteria are generally considered to be endophytic bacteria within the fruiting body. From a co-evolutionary perspective, most of these endophytic bacteria possess plant growth-promoting properties and belong to the category of Plant Growth-Promoting Rhizobacteria (PGPR), a component of the rhizosphere micro-ecosystem with beneficial effects on plants. They can act independently on plants and promote plant growth and enhance stress and disease resistance through direct or indirect means. More importantly, as the basidiospores of the fruiting body mature and disperse, the PGPR within these fruiting bodies also spread, considered "natural allies" of the mycorrhizal fungi's basidiospores, exerting a synergistic effect to enhance the mycorrhizal fungi's growth-promoting and nutrient absorption effects on the host plant. Meanwhile, endophytic fungi in fruiting bodies can also leverage the fruiting bodies and hyphal networks of EMFs to overcome the limitations of the rhizosphere environment and colonize more stably within the symbiotic system of plants and fungi. Therefore, in natural ecosystems, ectomycorrhizal symbiosis is actually considered a unified system of plant roots, ectomycorrhizal fungi, and endophytic fungi in fruiting bodies. Based on this, exploring the functions of endophytic PGPR fungi in ectomycorrhizal fruiting bodies and further amplifying the comprehensive benefits of EMFs provides a novel resource and application approach for developing more efficient and stable compound microbial fertilizers. This has significant theoretical and practical value for increasing forest yields and maintaining the stability of agroforestry ecosystems.

[0004] Simao pine ( Pinus kesiya var. langbianensisPine (Pinus sima) is a coniferous tree species endemic to Yunnan Province. With its straight and tall trunk, it is an excellent native timber species and possesses diverse and efficient economic value: high resin yield per tree and a long resin production cycle; its bark can be used to extract tannins; and its fiber length and palm cellulose content are excellent, making it an ideal raw material for pine fiber pulp. Therefore, it occupies a pivotal position in regional forestry development. However, the growth of Pinus sima is highly dependent on mycorrhizal fungi, making it a typical ectomycorrhizal nutrient-dependent tree species. Furthermore, the low background phosphorus levels in Yunnan's acidic red soil, coupled with its easy loss, easily lead to poor growth of Pinus sima. Therefore, utilizing mycorrhizal auxiliary bacteria to promote the formation and establishment of ectomycorrhizae, thereby improving phosphorus absorption efficiency and promoting the growth of Pinus sima, is crucial. Currently, there are no reports on the screening and application of mycorrhizal auxiliary bacteria for Pinus sima. Further research can provide key theoretical support and technical directions for solving the growth limitation problem of Pinus sima, increasing its yield, and promoting the application of compound microbial fertilizers in the forestry field. Summary of the Invention

[0005] The purpose of this invention is to provide *Plasmobium pusanense* F32 with ectomycorrhizal-promoting activity and its applications. *Plasmobium pusanense* F32 is isolated from the fruiting body of *Lycoperdon perlatum*, an ectomycorrhizal fungus of *Pinus sylvestris*. It has excellent solubility for insoluble organic and inorganic phosphorus, which can enhance the absorption and accumulation of mycorrhizal phosphorus in *Pinus sylvestris*. It can not only promote mycorrhizalization of plant roots and increase mycorrhizal infection rate, but also promote the growth of *Pinus sylvestris* under conditions of inoculation alone or co-inoculation with ectomycorrhizal fungi. It also has a synergistic effect with ectomycorrhizal fungi and can be used to prepare biological agents that promote the growth of *Pinus sylvestris*.

[0006] To achieve the above objectives, this invention provides *Agrobacterium pusa* F32 with ectomycorrhizal-promoting activity. *Agrobacterium pusa* F32 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on January 9, 2026, with accession number GDMCC No: 67611, and classified as... Agrobacterium pusense The 16S rDNA sequence of Agrobacterium pusanum F32 is shown in SEQ ID NO.1.

[0007] Furthermore, when Agrobacterium pusanum-F32 uses insoluble organic phosphorus calcium phytate as the sole phosphorus source, the soluble phosphorus produced is 2.61 ± 0.21 mg / L.

[0008] Furthermore, when Agrobacterium pusanum-F32 uses insoluble inorganic phosphorus tricalcium phosphate as the sole phosphorus source, the soluble phosphorus produced is 4.32 ± 0.05 mg / L.

[0009] Furthermore, the siderophore production capacity of *Pusamobacillus* F32 was 60.74.

[0010] Furthermore, Agrobacterium pusanum-F32 produced plant growth regulator IAA at a concentration of 62 ± 2.05 mg / L.

[0011] Furthermore, *Pseudomonas pusanus* F32 secreted ACCD enzyme at a concentration of 95.98 ± 1.61 mg / L.

[0012] Furthermore, the present invention also provides the application of the above-mentioned Psammonium F32 in promoting ectomycorrhizal formation.

[0013] Furthermore, the present invention also provides the application of the above-mentioned Agrobacterium pusanum-F32 in the preparation of biological agents that promote the growth of Pinus kesiao and increase the yield of Pinus kesiao.

[0014] Furthermore, the present invention also provides a biological agent that promotes the growth of Pinus kesiao and increases the yield of Pinus kesiao, wherein the active ingredient of the biological agent is Pseudomonas aeruginosa F32.

[0015] The advantages and positive effects of *Pseudomonas aeruginosa* F32 with ectomycorrhizal formation-promoting activity and its application described in this invention are as follows: The present invention provides Agrobacterium pusa F32 ( Agrobacterium pusense The strain *Pseudomonas pusa* F32, under phosphorus-deficient conditions, significantly promoted the growth of both the aboveground and underground parts of *Pinus koraiensis*, promoted the colonization of ectomycorrhizae, and promoted phosphorus absorption. This invention provides *Pseudomonas pusa* F32... Agrobacterium pusense This strain possesses several excellent plant growth-promoting properties, including the ability to dissolve poorly soluble organic and inorganic phosphorus, produce siderophores, and produce plant hormones IAA and ACC enzymes. Therefore, *Agrobacterium pusa* F32 (… Agrobacterium pusense The strain can be used to prepare special biological agents for Pinus kesiya, which have the advantages of low cost, good effect and no toxic side effects. It can help Pinus kesiya grow and develop and increase yield for a longer period of time, and provides a new technical approach for the efficient cultivation of Pinus kesiya plantations.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 The colony characteristics of *Psammonella pusa* F32 strain, an endophytic bacterium of the fruiting body of colored puffball in Example 1 of this invention, on LB plates and in shake flasks are shown, where a represents colony characteristics and b represents growth. Figure 2 In Example 4 of this invention, a single inoculation of the ectomycorrhizal fungus *Puffballia scoparia* (E+F32-) and a single inoculation of the endophytic bacterium *Pseudomonas aeruginosa* F32 (…) were performed. Agrobacterium pusense The effects of sterile seedlings inoculated with both E-F32+ and double inoculation with both (E+F32+) on the growth of Pinus sylvestris var. mongolica were investigated. The treatment of sterile seedlings inoculated with both inactivating agents was the control group without inoculation (CK). Figure 3 This invention describes the effects of single inoculation with the ectomycorrhizal fungus *Puffballia scoparia* (E+F32-), single inoculation with the endophytic bacterium *Agrobacterium praecox* F32 (E-F32+), and a double inoculation treatment (E+F32+) on the height and root length of *Pinus koraiensis* in Example 4 of this invention. The treatment of sterile seedlings double-inoculated with both inactivating agents served as the uninoculated control group (CK) (data are mean ± standard error, n=5). A represents plant height, and B represents root length. Different lowercase letters indicate significant differences (P<0.05). Figure 4 This invention describes the effects of single inoculation with the ectomycorrhizal fungus *Puffballia scoparia* (E+F32-), single inoculation with the endophytic bacterium *Agrobacterium pusanense* F32 (E-F32+), and a double inoculation treatment (E+F32+) on the chlorophyll a, chlorophyll b, and total chlorophyll content of *Pinus koraiensis* in Example 4 of this invention. The treatment of sterile seedlings double-inoculated with both inactivating agents served as the uninoculated control group (CK) (data are mean ± standard error, n=5). A represents chlorophyll a content, B represents chlorophyll b content, and C represents total chlorophyll content. Different lowercase letters indicate significant differences (P<0.05). Figure 5 This invention describes the effects of single inoculation with the ectomycorrhizal fungus *Puffballia scoparia* (E+F32-), single inoculation with the endophytic bacterium *Agrobacterium pusanense* F32 (E-F32+), and a double inoculation treatment (E+F32+) on the aboveground, underground, and total biomass of *Pinus koraiensis* in Example 4 of this invention. The treatment of sterile seedlings double-inoculated with both inactivating agents served as the uninoculated control group (CK) (data are mean ± standard error, n=5). A represents aboveground and underground biomass, and B represents total biomass. Different lowercase letters indicate significant differences (P<0.05). Figure 6 This document presents the statistical results of the colonization rate of ectomycorrhizae of *Pleurotus ostreatus* F32 inoculated onto the fruiting bodies of *Pleurotus ostreatus* in Example 4 of this invention. The control group consisted of a single inoculation with the ectomycorrhizal fungus *Pleurotus ostreatus* (E+F32-). Data are presented as mean ± standard error, n=5. Figure 7 This invention describes the effects of single inoculation with the ectomycorrhizal fungus *Puffballia scoparia* (E+F32-), single inoculation with the endophytic bacterium *Agrobacterium pusan* F32 (E-F32+), and a double inoculation treatment (E+F32+) on the aboveground and underground phosphorus concentrations and the average phosphorus concentration of *Pinus koraiensis*. The treatment of sterile seedlings double-inoculated with both inactivating agents served as the uninoculated control group (CK) (data are mean ± standard error, n=5). A represents the aboveground and underground phosphorus concentrations, and B represents the average phosphorus concentration of *Pinus koraiensis*. Different lowercase letters indicate significant differences (P<0.05). Figure 8This invention describes the effects of single inoculation with the ectomycorrhizal fungus *Pleurotus ostreatus* (E+F32-), single inoculation with the endophytic bacterium *Agrobacterium pusa* F32 (E-F32+), and double inoculation with both (E+F32+) on the aboveground and underground phosphorus content and total phosphorus content of *Pinus sapiens*. The treatment of sterile seedlings double-inoculated with both inactivating agents served as the uninoculated control group (CK) (data are mean ± standard error, n=5). A represents the aboveground and underground phosphorus content, and B represents the total phosphorus content of *Pinus sapiens*. Different lowercase letters indicate significant differences (P<0.05).

[0018] Instructions for the Preservation of Biological Materials Agrobacterium pusanum-F32 ( Agrobacterium pusense This sample is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on January 9, 2026. The accession number is GDMCCNo:67611, and the classification name is... Agrobacterium pusense . Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0022] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0023] The culture medium formulation (per liter) used in the examples is as follows: LB medium: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride.

[0024] NRBIP liquid medium: glucose 10 g, calcium phytate 5 g, tricalcium phosphate 5 g, magnesium chloride 2.35 g, magnesium sulfate 0.25 g, potassium chloride 0.2 g, ammonium sulfate 0.1 g, pH=7.

[0025] PVK medium: glucose 10 g, calcium phytate 5 g, tricalcium phosphate 5 g, ammonium sulfate 0.5 g, sodium chloride 0.3 g, potassium chloride 0.3 g, ferric sulfate heptahydrate 0.03 g, manganese sulfate 0.023 g.

[0026] MKB liquid culture medium: 5 g casein amino acids, 15 ml glycerol, 2.5 g magnesium sulfate heptahydrate, 2.5 g dipotassium hydrogen phosphate, adjust pH to 7.2.

[0027] PDA medium: 200 g potato, 20 g glucose, 15 g agar.

[0028] MMN culture medium: glucose 15 g, calcium chloride 0.05 g, magnesium sulfate 0.15 g, sodium chloride 0.5 g, ferric chloride (1%) 1.2 ml, potassium dihydrogen phosphate 0.5 g, vitamin B 0.1 mg, diammonium hydrogen phosphate 0.25 g, pH=5.5.

[0029] Example 1 Agrobacterium pusanum-F32 ( Agrobacterium pusense Separation of ) Agrobacterium pusanum-F32 ( Agrobacterium pusense The fruiting bodies of the ectomycorrhizal fungus *Calvatia spp.*, collected from under *Pinus simaoensis* forests in Pu'er City, Yunnan Province, were isolated. The specific steps were as follows: Freshly collected fruiting bodies were soaked in 10% NaClO for 5 min, then in 75% alcohol for 5 min for surface disinfection. After rinsing three times with sterile water, the surface of the fruiting bodies was removed with a sterile scalpel. 0.5 g of the internal tissue was placed in a 2 mL sterile centrifuge tube, physically disrupted, and an appropriate amount of sterile water was added to prepare an endophytic bacterial suspension. After centrifugation, the supernatant was serially diluted and plated onto LB agar plates. The plates were incubated at 37℃ in the dark for 2-3 days. Single colonies with uniform growth and suitable density were selected and inoculated into sterile LB liquid agar plates, then incubated at 37℃ in a shaker for 24 h. After incubation, 1 mL of the bacterial suspension was used for molecular identification. The remaining suspension was added to sterile glycerol and stored at -80℃ for later use.

[0030] On LB agar plates, endophytic *Pseudomonas spp.* F32 of ectomycorrhizal fungi fruiting bodies ( Agrobacterium pusense The colonies are smooth and moist, milky white to pale yellow, translucent to opaque, and round. The edges of the colonies are neat and clear, the center is slightly raised, and the diameter is about 1-2 mm (e.g., Figure 1(a) The bacterial culture in the logarithmic growth phase was mixed with lysis buffer at a volume ratio of 1:2 (5 μL bacterial culture: 10 μL lysis buffer), boiled for 5 minutes, and briefly centrifuged. 2 μL of the mixture was used as a PCR template. PCR amplification was performed using universal bacterial primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-TACGGCTACCTTGTTACGACTT-3') and the MHiper high-speed mix enzyme. Samples that successfully amplified via electrophoresis were sent to the Kunming branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing. After correcting the successfully sequenced sequences, SeqMan clustering (99%) was used to select representative sequences for sequence alignment and taxonomic annotation using 16S-based IDs in the EzBioCloud database. The 16S rRNA sequence SEQ ID NO.1 is shown below (5'-3'):

[0031] Example 2 Agrobacterium pusanum-F32 ( Agrobacterium pusense The plant growth-promoting functional characteristics of ) are investigated as follows: 1. Evaluation of phosphorus solubility: Take 0.5 mL of bacterial suspension (OD) 600 =0.8), inoculated into 250 mL Erlenmeyer flasks containing 100 mL NRBIP liquid medium. This medium uses tricalcium phosphate (inorganic phosphorus source) or calcium phytate (organic phosphorus source) as the sole phosphorus source, prepared at 5 g of the corresponding phosphorus source per liter of medium. Incubated continuously for 3 days at 37℃ and 180 rpm in a shaker, with 5 biological replicates per group, and a blank control group without inoculation. Immediately after incubation, the pH of the fermentation broth was measured using a pH meter; simultaneously, the fermentation broth was centrifuged at 3000 rpm for 5 min, and the supernatant was collected. The OD was measured using a microplate reader via the molybdenum-antimony colorimetric method. 700 The value is used to calculate the soluble phosphorus concentration.

[0032] 2. Evaluation of iron-producing carriers: Take 0.5 mL of bacterial culture (OD) 600 =0.8), inoculated into 50 mL of sterile MKB liquid medium, and cultured at 37℃ and 180 rpm for 3 days, with 3 replicates per group. An uninoculated MKB liquid medium was also included as a blank control. The OD of the fermentation broth was measured. 600 The fermentation broth was centrifuged at 5000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm sterile filter. 1 mL of the filtrate was mixed with an equal volume of 1 mL of CAS quantitative detection solution, and the reaction was allowed to proceed at room temperature for 1 h before the OD value was measured. 630 The siderophore unit (%) is calculated using the formula: = 100 × (Ar - As) / Ar (where As is the OD of the inoculated group). 630 Value, Ar is the OD value of the blank control group. 630 The siderophore-producing capacity of this bacterium is expressed as the ratio of siderophore units to bacterial OD. 600 The ratio of .

[0033] 3. IAA production capacity assessment: Take 1 mL of the bacterial culture (OD) 600 =0.8), inoculated into 100 mL of sterile LB liquid medium (containing 100 mg / L L-tryptophan), and cultured at 37℃ and 180 rpm for 2 days, with 3 replicates per group, and uninoculated LB medium as a blank control. After culture, the fermentation broth was centrifuged at 5000 rpm for 10 min, and the supernatant was mixed with an equal volume of Salkowski colorimetric solution for color development in the dark for 30 min. The absorbance at 530 nm was measured, and the IAA yield of the bacterium was calculated based on the IAA standard curve.

[0034] 4. Evaluation of ACC enzyme production capacity: Take 1 mL of bacterial culture (OD) 600 =0.8), inoculated into 250 mL Erlenmeyer flasks containing 100 mL of sterile MMN liquid medium, with 3 replicates per group, and an uninoculated group as a control. Cultured at 37 ℃ and 180 rpm for 2 days. After culture, the fermentation broth was centrifuged at 5000 rpm for 10 min, and 1.5 mL of the supernatant was collected. The OD was measured using an ACCD kit (LMAIBio, LM-ACCD-Ot) according to the ELISA kit instructions. 450 The ACC deaminase activity was calculated using the value and the standard curve.

[0035] Experimental results: against Agrobacterium pusanum-F32 ( Agrobacterium pusense The results of the growth-promoting function test of F32 are as follows: F32 has excellent plant growth-promoting function characteristics and has the ability to efficiently dissolve insoluble phosphorus. When insoluble organic phosphorus calcium phytate is the only phosphorus source, the soluble phosphorus produced is 2.61±0.21 mg / L, and when insoluble inorganic phosphorus tricalcium phosphate is the only phosphorus source, the soluble phosphorus produced is 4.32±0.05 mg / L. In addition, F32 has a very strong siderophore production capacity, SU=74.3%, As / Ar=0.236, and siderophore production capacity of 60.74, which can help plants absorb iron and alleviate iron deficiency stress. It can synthesize plant growth hormone IAA at a concentration of 62±2.05 mg / L and can also secrete ACCD enzyme (95.98±1.61 mg / L) to alleviate plant stress. Through multi-mechanism synergistic action, it can significantly promote the growth of plants such as Pinus kesiao, and has important application value in the field of plant growth-promoting biological agents.

[0036] Example 3 Agrobacterium pusanum-F32 ( Agrobacterium pusense Chemotactic properties of mycorrhizal Pinus koraiensis root exudates: 1) Preparation of mycorrhizal and non-mycorrhizal pine seedlings: The method for raising seedlings of Pinus simonii is as follows: After disinfecting the surface of Pinus simonii seeds with 75% alcohol for 10 minutes, wash them three times with sterile water. Then, in a seedling tray filled with sterile vermiculite, spread the seeds evenly on the surface of the vermiculite, cover them with an appropriate amount of sterile vermiculite, water them thoroughly with sterile tap water, and place them in a greenhouse for 15-20 days.

[0037] Preparation of mycorrhizal seedlings: Colored puffball fungus was inoculated onto PDA plates and cultured upside down in the dark at 28 ℃ for 10-15 days. After a large number of hyphae had grown, an appropriate amount of hyphae was inoculated into a 250 mL Erlenmeyer flask containing 150 mL of PDA liquid medium and cultured at 28 ℃ and 180 rpm for 10-15 days. After a large number of hyphae had grown, the mycelium was removed using a vacuum pump and transferred to a sterile 5 mL EP tube. A suitable amount of sterile water was added for tissue grinding, and after washing, a fungal suspension was obtained. This fungal suspension was divided into two equal portions: one portion was used as an active ectomycorrhizal inoculum (E+); the other portion was sterilized by autoclaving at 121 ℃ for 20 min and used as an inactivated ectomycorrhizal inoculum (E-). Both inoculums were inoculated onto the root systems of pine seedlings with uniform growth, and cultured for 20-30 days to obtain mycorrhizal seedlings (E+) and non-mycorrhizal seedlings (E-).

[0038] 2) Chemotactic properties of strain F32 on root exudates of Pinus sylvestris: Mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings were collected after 90 days of greenhouse cultivation. After washing the roots with sterile pure water, the two types of Pinus sylvestris seedlings were placed in sterile plastic cups containing 250 mL of sterile pure water (6 seedlings per cup). The cups were covered with aluminum foil to block light and placed in a light incubator for 48 hours to obtain root exudates from the mycorrhizal and non-mycorrhizal seedlings. The exudates were then filtered through a 0.22 μm sterile filter for sterilization and set aside for later use.

[0039] Take 1 mL of the filtered and sterilized root exudate into a sterile centrifuge tube (2 mL), then add 200 μL of sterile LB medium and mix well to form the culture medium. The treatment containing mycorrhizal seedling root exudate is the experimental group, and the control group is the treatment containing 1 mL of non-mycorrhizal seedling root exudate. Then take 300 μL (OD) of the cultured bacterial solution. 600 Inoculate with 0.8 g of the bacteria and culture in a shaker for 2-3 days, then measure the bacterial OD value. 600 Values. Each treatment group was set up with 3 replicates, and significance was observed using an independent samples t-test. The effect of F32 on the growth of mycorrhizal seedling root exudates was calculated using the formula: [(OD value of mycorrhizal seedling group)]. 600 Value - OD of non-mycorrhizal seedlings 600 (value) / OD of non-mycorrhizal seedling group 600 The results showed that F32 exhibited a highly significant difference in the chemotactic properties of root exudates from mycorrhizal Pinus sylvestris seedlings compared to non-mycorrhizal seedlings. p <0.01, t-test).

[0040] Example 4 Agrobacterium pusanum-F32 ( Agrobacterium pusense The specific steps for promoting the growth of Pinus kesiya are as follows: 1) Test materials: The prepared Pinus sylvestris (Sima) as described above Pinus kesiya var. langbianensis Mycorrhizal and non-mycorrhizal seedlings serve as host plants, with ectomycorrhizal fungi such as Puffball (Calamus odoratum) Pisolithus tinctorius ) and the endophytic bacteria F32 on the fruiting bodies of colored puffballs ( Agrobacterium pusense The culture medium is a mixture of humus, sterile vermiculite, and Pinus sylvestris forest soil (ratio 1:15:15, w / w), with 3‰ of insoluble inorganic tricalcium phosphate and organic calcium phytate mixed in. The culture medium is sterilized at 121℃ for 2 hours, with a one-day interval, and the sterilization is repeated 3 times.

[0041] 2) Inoculum preparation: Single colonies of F32 were picked and inoculated into a 250 mL Erlenmeyer flask containing 150 mL of LB medium and incubated at 37 ℃ and 180 rpm for 2 days. The bacterial fermentation broth was then centrifuged at 5000 rpm for 10 min, and the precipitated bacterial cells were mixed with sterile water to prepare a bacterial suspension. Half of this suspension was used as the active bacterial agent (F32+); the other half of the F32 bacterial suspension was sterilized by autoclaving (121 ℃, 20 min) and used as the inoculum for the uninoculated control group. The colored puffball fungal inoculum was prepared as described above.

[0042] 3) Design and culture of the experimental and control groups: Using *Lycoperdon perlatum* fungus and its endophytic bacterium F32, along with their inactivated inoculum (121℃, 20 min), as microbial inoculum agents, four treatment groups were obtained: double inoculation (CK) of two inactivated agents on non-mycorrhizal seedlings, single inoculation (E-F32+ and E+F32-) of two inoculum agents, and double inoculation (E+F32+) of two live inoculum agents. At planting, equal amounts of inoculum agents were inoculated onto the roots using a root-dipping method. Each group was planted in five replicates. Water was applied every 2-3 days with 100 mL of water. The seedlings were cultured under greenhouse conditions for 120 days before harvesting.

[0043] 4) Detection indicators: By measuring the plant height, root length, biomass, phosphorus use efficiency, and chlorophyll content of different treatment groups of *Pinus sapiens*, this study explored the growth-promoting effect of F32 on *Pinus sapiens*, as well as its functional role in the establishment and functional enhancement of ectomycorrhizae in *Pinus sapiens*.

[0044] 5) Test results: The growth and fruiting of Pinus simonii are as follows Figure 2As shown: Compared with the uninoculated control group (CK), both the single-inoculation treatment group and the double-inoculation combination treatment group significantly promoted the growth and development of *Pinus kesiao*, with the double-inoculation treatment of endophytic bacteria F32 and *Lycoperdon perlatum* having the most significant growth-promoting effect. Further analysis of the growth indicators of *Pinus kesiao* showed that its plant height and root length (… Figure 3 ), chlorophyll content ( Figure 4 ) and biomass ( Figure 5 Physiological and growth indicators, such as those of *Pinus kesiao*, showed significant improvements compared to the control group. Treatments with single inoculation of the endophytic bacteria F32 or *Lycoperdon perlatum* significantly increased the total biomass of *Pinus kesiao*. The total biomass of the double inoculation group (F32 and *Lycoperdon perlatum*) was significantly higher than that of the control group and each single inoculation group. Simultaneously, inoculation with the endophytic bacteria F32 increased the ectomycorrhizal colonization rate of *Pinus kesiao* by 3.76%. Figure 6 There is a significant and positive synergistic effect between endophytic bacteria F32 and the colory bean puffball fungus, and the combined inoculation of the two has the most significant effect on promoting the growth of Pinus sylvestris.

[0045] The concentrations of phosphorus (P) in the aboveground and underground parts of Pinus sylvestris, as well as the average phosphorus concentration of the plant (P), were studied. Figure 7 ) and phosphorus accumulation in the aboveground and underground parts, total phosphorus accumulation ( Figure 8 The results of the assay showed that, compared with the uninoculated control group (CK), both single-inoculation and double-inoculation combinations significantly increased the soluble phosphorus concentration and accumulation in the aboveground and underground parts of *Pinus koraiensis*. Particularly in the two core indicators of average phosphorus concentration and total phosphorus accumulation, the double-inoculation group showed particularly outstanding performance: its average phosphorus concentration increased by 224%, 75.2%, and 20.1% compared with the CK control and the two single-inoculation groups (E-F32+ and E+F32-), respectively; and its total phosphorus accumulation increased by 1487.9%, 482.1%, and 50.8% compared with the CK, E-F32+, and E+F32- groups, respectively. The changes in the above phosphorus nutrition indicators were consistent with the growth indicators, showing an overall gradient difference of "double-inoculation group > single-inoculation group > CK group". Endophytic *Pseudomonas aeruginosa* F32 (… Agrobacterium pusense Inoculation treatment can not only significantly promote the growth of Pinus sylvestris and increase the colonization rate of ectomycorrhizae, but also effectively enhance its ability to absorb insoluble phosphorus. In particular, the combined inoculation of F32 and ectomycorrhizal fungi has a better comprehensive effect on phosphorus nutrient absorption and growth promotion than the single inoculation treatment and the control group.

[0046] Therefore, the *Agrobacterium pusa* F32, which promotes ectomycorrhizal formation in this invention, exhibits excellent solubility for insoluble organic and inorganic phosphorus, significantly enhancing the mycorrhizal phosphorus absorption and accumulation in *Pinus kesiya*. It not only promotes mycorrhizalization of plant roots and increases mycorrhizal infection rate, but also significantly promotes the growth of both above-ground and underground parts of *Pinus kesiya* under both single inoculation and double inoculation with ectomycorrhizae. Furthermore, its synergistic effect is superior to that of single *Agrobacterium pusa* or *Lycoperdon perlatum* strains, demonstrating stable and prominent effects. It can be used to prepare biological agents for economically valuable seedlings such as *Pinus kesiya*, offering advantages such as low cost, high efficacy, and no toxic side effects. It can provide a more sustained boost to the growth, development, and yield of host plants, offering a new technical approach for the efficient cultivation of *Pinus kesiya* plantations.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of *Agrobacterium pusa* F32, which has ectomycorrhizal formation-promoting activity, in the preparation of biological agents that promote the growth and increase the yield of *Pinus kesiya*, characterized in that: The *Pseudomonas aeruginosa* F32 was isolated from the fruiting body of *Lycoperdon perlatum*, an ectomycorrhizal fungus of *Pinus kesiya*. Co-inoculation of *Pseudomonas aeruginosa* F32 with *Lycoperdon perlatum* promotes the colonization rate of ectomycorrhizae and the absorption of phosphorus by ectomycorrhizae, thereby promoting the growth of *Pinus kesiya* and increasing its yield. Agrobacterium pusa F32 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on January 9, 2026, with accession number GDMCC No:67611. It is classified as follows: Agrobacterium pusense The 16S rDNA sequence of Agrobacterium pusanum-F32 is shown in SEQ ID NO.1; When Agrobacterium pusanum-F32 uses insoluble organic phosphorus calcium phytate as the sole phosphorus source, the soluble phosphorus produced is 2.61 ± 0.21 mg / L. When Agrobacterium pusanum-F32 uses insoluble inorganic phosphorus tricalcium phosphate as the sole phosphorus source, the soluble phosphorus produced is 4.32 ± 0.05 mg / L. The siderogenic capacity of Agrobacterium pusanum-F32 was 60.

74. Agrobacterium pusa F32 produces plant growth regulator IAA at a concentration of 62±2.05 mg / L; Pseudomonas aeruginosa F32 secretes ACCD enzyme at a concentration of 95.98±1.61 mg / L.

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