Decarboxylation of lactic acid by lactobacillus kefiri n183 and application thereof

Abstract

The application discloses a decarboxylation Lecythophora sp. N183 strain and application thereof, and belongs to the technical field of microbial inoculants. The strain is isolated from fruiting bodies of a dominant ectomycorrhizal fungus of Pinus sylvestris var. sylvestris var. and is a plant growth-promoting rhizobacteria (PGPR). The strain has multiple plant growth-promoting functions such as IAA production and phosphorus solubilization and can significantly promote the growth of Pinus sylvestris var. sylvestris var. In addition, the strain can promote the formation of mycorrhiza of Pinus sylvestris var. sylvestris var. roots, improve mycorrhiza infection, has a synergistic effect with the ectomycorrhizal fungus of Laccaria bicolor, and the double-inoculation effect is better than the single-inoculation effect, and the performance is stable and the effect is outstanding. Based on the above characteristics, the strain can be used for developing biological agents for the seedling raising of Pinus sylvestris var. sylvestris var. and other forest trees, has the advantages of low cost, good effect and environmental friendliness and the like, can durably promote the growth and development and yield formation of plants, and provides a new microbial technical solution for the efficient cultivation of Pinus sylvestris var. sylvestris var. and other artificial forests.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, and in particular to a decarboxylated Rechlerella N183 strain and its application. Background Technology

[0002] Simao pine ( Pinus kesiya var. langbianensis Pinus sapiens (also known as Chinese pine) is a native multi-purpose tree species belonging to the genus Pinus in the family Pinaceae. It is one of the fastest-growing pine species in my country, with an average annual height growth of over 1.5 meters and an average annual diameter at breast height (DBH) growth of over 2 cm. Its excellent rapid growth characteristics, coupled with superior long fiber properties and outstanding resin quality, make it a preferred species for industrial raw material forest cultivation. In recent years, the rapid development of forestry industries such as wood-based panels, paper, and chemical processing has created a huge demand for Pinus sapiens raw material forests. Pinus sapiens is a typical ectomycorrhizal dependent plant; the formation of ectomycorrhizae can significantly improve the water and mineral nutrition of Pinus sapiens, promoting seedling growth. Furthermore, the main distribution area of ​​Pinus sapiens is in the typical southern acidic red soil region of my country, where soil phosphorus deficiency is prominent, which is a major limiting factor for the rapid development of Pinus sapiens forestry as industrial raw material. To address the common problems of soil fertility decline and low biological activity in the cultivation of Pinus kesiya plantations, the application of mycorrhizal technology to optimize the rhizosphere microecology of Pinus kesiya is a key approach to overcome the bottleneck of seedling growth and achieve healthy and sustainable cultivation.

[0003] However, greenhouse-bred single ectomycorrhizal seedlings often face antagonistic effects from native field microbiota during transplantation, resulting in limited mycorrhizalization and low mycorrhizal activity. The fruiting bodies of ectomycorrhizal fungi are not only their reproductive organs but also carriers of mycorrhiza helper bacteria (MHB). Along with the maturation and dispersal of basidiospores from the fruiting bodies, a large number of mycorrhizal helper bacteria also spread to the new habitat, effectively forming a three-in-one symbiotic system of "roots-ectomycorrhizal fungi-MHB" in the new host plant. From a co-evolutionary perspective, these endophytic bacteria on the fruiting bodies are recruited by mycorrhizal fungi through their unique metabolically active substances during the development of the fruiting bodies. Most of them possess plant growth-promoting properties, especially groups with phosphorus-solubilizing functions. Furthermore, some endophytic bacteria, in addition to their own strong phosphorus-solubilizing abilities, can synergistically interact with ectomycorrhizal fungi, significantly alleviating phosphorus stress in the host plant and promoting plant growth. Therefore, exploring the functions of endophytic bacteria in the fruiting bodies of ectomycorrhizal fungi and further expanding the comprehensive benefits of mycorrhizae is of great theoretical significance and practical value for improving the growth of Pinus kesiya, promoting phosphorus absorption by plants, and enhancing the technical level of Pinus kesiya seedling cultivation and artificial forest construction. Summary of the Invention

[0004] The purpose of this invention is to provide a decarboxylating Rechlerellae strain N183 and its applications. This strain possesses multiple growth-promoting functions, including phosphorus solubilization, siderophore production, and the production of IAA and ACC enzymes. Furthermore, it can synergistically interact with the dominant mycorrhizal fungus *Lycoperdon perlatum* of *Pinus yew*, enhancing mycorrhizalization of the *Pinus yew* root system and improving phosphorus nutrient absorption efficiency. This invention provides a highly efficient and beneficial microbial resource, which can not only facilitate the efficient cultivation of robust mycorrhizalized seedlings of *Pinus yew*, but also provide key materials for the development of fertilizers specifically for *Pinus yew* plantations, thus having a positive impact on the sustainable development of *Pinus yew* plantations.

[0005] To achieve the above objectives, the present invention provides a decarboxylating Rechler bacteria strain N183, with the Latin name... Leclercia adecarboxylata The 16S rDNA sequence, as shown in SEQ ID NO.1, was deposited on February 4, 2026, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCCNo:67812.

[0006] The application of the decarboxylated Rechlerella N183 strain as described above in the preparation of microbial inoculants.

[0007] Preferably, the microbial agent has the ability to solubilize phosphorus, produce siderophores, and generate IAA and ACC enzymes.

[0008] Preferably, the microbial agent uses Decarboxylated Rechlerella N183 strain as the active ingredient or as a single component.

[0009] The application of the decarboxylated Rechler bacteria strain N183 as described above in the cultivation of Pinus simonii seedlings.

[0010] Preferably, the decarboxylated Rechler bacteria N183 strain is applied alone.

[0011] Preferably, the decarboxylated Lycium barbarum strain N183 is applied together with the culture medium of colored puffball mycelium.

[0012] Preferably, the method for preparing the colored puffball mycelial culture medium is as follows: inoculate the colored puffball mycelium into liquid PDA medium and culture at 28°C and 180 rpm for 15-20 days to obtain the mycelial culture.

[0013] Therefore, the present invention provides a decarboxylating Rechler bacteria strain ( Leclercia adecarboxylata The N183 strain and its applications, and its specific technical effects are as follows: (1) The endophytic bacteria of the ectomycorrhizal fungi fruiting body provided by the present invention is decarboxylating Leavenweg N183 ( Leclercia adecarboxylataThis strain possesses a variety of excellent plant growth-promoting properties, including the ability to dissolve insoluble organic and inorganic phosphorus, produce siderophores, produce the plant hormone IAA, and produce ACC enzymes. (2) The endophytic bacteria of the ectomycorrhizal fungi fruiting bodies provided by this invention are decarboxylating Leavenweg ( Leclercia adecarboxylata The N183 strain can significantly promote the growth of both the aboveground and underground parts of Pinus kesiao under phosphorus-deficient conditions, promote the colonization of ectomycorrhizae, and promote phosphorus absorption. (3) The endophytic bacteria of the ectomycorrhizal fungi fruiting bodies provided by this invention are decarboxylating Leavenweg ( Leclercia adecarboxylata The N183 strain can be used to prepare microbial agents that promote the growth of Pinus sylvestris. It has the advantages of low cost, good effect and no toxic side effects, and can help Pinus sylvestris grow and develop and increase yield for a longer period of time, providing a new technical approach for the efficient cultivation of Pinus sylvestris plantations. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 The endophytic bacteria *Decarboxylylereus* of the fruiting bodies of colored puffballs in Example 1 of this invention are... Leclercia adecarboxylata Colony characteristics of strain N183 on LB plates and growth in shake flasks; where a represents the colony characteristics on LB plates and b represents the growth in shake flasks. Figure 2 In Example 3 of this invention, the ectomycorrhizal fungus *Lycoperdon perlatum* (Pt+N183-) and the endophytic bacterium *Decarboxylella oleracea* (decarboxylated *Lycium barbarum*) were inoculated alone. Leclercia adecarboxylata The effects of Pt-N183 (Pt-N183+) and its double inoculation treatment (Pt+N183+) on the growth of Pinus sylvestris; where CK was sterile seedlings inoculated with the two inactivated fungicides; Figure 3 In Example 3 of this invention, the ectomycorrhizal fungus *Lycoperdon perlatum* (Pt+N183-) and the endophytic bacterium *Decarboxylella oleracea* (decarboxylated *Lycium barbarum*) were inoculated alone. Leclercia adecarboxylata The effects of Pt-N183 (Pt-N183+) and its double inoculation treatment (Pt+N183+) on the height and root length of Pinus sylvestris var. mongolica; where a represents plant height, b represents root length, and CK represents the treatment of sterile seedlings inoculated with the two inactivating agents; different lowercase letters in the bar chart indicate significant differences. P<0.05; data are mean ± standard error, n=5; Figure 4 In Example 3 of this invention, the ectomycorrhizal fungus *Lycoperdon perlatum* (Pt+N183-) and the endophytic bacterium *Decarboxylella oleracea* (decarboxylated *Lycium barbarum*) were inoculated alone. Leclercia adecarboxylata The effects of Pt-N183 (Pt-N183+) and its double inoculation treatment (Pt+N183+) on the chlorophyll a, chlorophyll b, and total chlorophyll content of Pinus sylvestris var. simonii; where a represents chlorophyll a content, b represents chlorophyll b content, and c represents total chlorophyll content; CK represents the treatment of sterile seedlings inoculated with the two inactivating agents; different lowercase letters in the bar chart indicate significant differences. P <0.05; data are mean ± standard error, n=5; Figure 5 In Example 3 of this invention, the ectomycorrhizal fungus *Lycoperdon perlatum* (Pt+N183-) and the endophytic bacterium *Decarboxylella oleracea* (decarboxylated *Lycium barbarum*) were inoculated alone. Leclercia adecarboxylata The effects of N183 (E-N183+) and its double inoculation treatment (Pt+N183+) on the fresh weight of the aboveground parts, underground parts, and whole plant of *Pinus sylvestris*; where a represents the fresh weight of the aboveground parts, b represents the fresh weight of the underground parts, c represents the fresh weight of the whole plant, and CK represents the treatment of sterile seedlings inoculated with the two inactivating agents; different lowercase letters in the bar chart indicate significant differences. P <0.05; data are mean ± standard error, n=5; Figure 6 In Example 3 of this invention, the endophytic bacteria *Decarboxylylereus* of the fruiting bodies of colored puffballs were inoculated. Leclercia adecarboxylata The effect of N183 on the colonization rate of ectomycorrhizae of Pinus simaoensis is statistically analyzed, where the data are mean ± standard error, n=5; Figure 7 In Example 3 of this invention, the ectomycorrhizal fungus *Lycoperdon perlatum* (Pt+N183-) and the endophytic bacterium *Decarboxylella oleracea* (decarboxylated *Lycium barbarum*) were inoculated alone. Leclercia adecarboxylata The effects of Pt-N183 (Pt-N183+) and its dual inoculation treatment (Pt+N183+) on the aboveground, underground, and average P concentrations of Pinus kesiao. Where a represents the aboveground P concentration, b represents the underground P concentration, c represents the average P concentration, and CK represents the treatment of sterile seedlings inoculated with both inactivating agents. Different lowercase letters in the bar chart indicate significant differences. P <0.05; data are mean ± standard error, n=5; Figure 8 In Example 3 of this invention, the ectomycorrhizal fungus *Lycoperdon perlatum* (Pt+N183-) and the endophytic bacterium *Decarboxylella oleracea* (decarboxylated *Lycium barbarum*) were inoculated alone. Leclercia adecarboxylataThe effects of Pt-N183 (Pt-N183+) and its dual inoculation treatment (Pt+N183+) on the P content in the aboveground parts, underground parts, and total P content of Pinus kesiao. Where a represents the P content in the aboveground parts, b represents the P content in the underground parts, c represents the total P content, and CK represents the treatment of sterile seedlings inoculated with both inactivating agents. Different lowercase letters in the bar chart indicate significant differences. P <0.05; data are mean ± standard error, n=5. Detailed Implementation

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

[0017] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0018] The instruments, equipment, reagents and materials used in the embodiments were all obtained through commercial means; the methods and steps not described in detail are all conventional techniques in the field.

[0019] The culture medium formulation (per liter) used in the examples is as follows: LB medium: 5g yeast extract, 10g tryptone, 10g sodium chloride.

[0020] NBRIP liquid medium: glucose 10.0g, calcium phytate 5g, calcium phosphate 5.0g, magnesium chloride 2.35g, magnesium sulfate 0.25g, potassium chloride 0.2g, ammonium sulfate 0.1g, pH=7.

[0021] PVK medium: glucose 10g, calcium phytate 5g, tricalcium phosphate 5g, ammonium sulfate 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, ferric sulfate heptahydrate 0.03g, manganese sulfate 0.023g.

[0022] MKB liquid culture medium: 5g casein amino acids, 15mL glycerol, 2.5g magnesium sulfate heptahydrate, 2.5g dipotassium hydrogen phosphate, adjust pH to 7.2.

[0023] PDA medium / plate: 200g potato, 20g glucose, 15g agar; PDA liquid medium without agar.

[0024] MMN culture medium: glucose 15g, calcium chloride 0.05g, magnesium sulfate 0.15g, sodium chloride 0.5g, ferric chloride (1%) 1.2mL, potassium dihydrogen phosphate 0.5g, vitamin B 0.1mg, diammonium hydrogen phosphate 0.25g, pH=5.5.

[0025] CAS quantitative detection solution: Solution A: Chromium azurite S solution (60.5 mg Chromium azurite S dissolved in 50 mL dd H2O), Fe 3+ Solution (0.0273 g ferric chloride hexahydrate, 1 mL 1 mol / L HCl, dissolved in 99 mL dd H₂O), take 10 mL Fe 3+ Mix the solution with the chromium azurite S solution; Solution B: CTAB solution (72.9 mg CTAB dissolved in 40 mL dd H2O); Pour solution A into solution B and mix well.

[0026] Salkowski colorimetric solution: 1.5 mL of 0.5 mol / L FeCl3 solution, 30 mL of concentrated sulfuric acid (H2SO4), and 50 mL of distilled water.

[0027] Example 1 Decarboxylated Lechlerbe ( Leclercia adecarboxylata Separation of N183.

[0028] Decarboxylated Lechlerbe ( Leclercia adecarboxylata N183 was isolated from the fruiting bodies of the ectomycorrhizal fungus *Calvatia spp.* collected from under *Pinus simaoensis* forests in Jinggu County, Pu'er City, Yunnan Province. The specific steps were as follows: Freshly collected fruiting bodies were soaked in 10% NaClO for 5 minutes, then in 75% alcohol for 5 minutes for surface disinfection. After rinsing three times with sterile water, the surface of the fruiting bodies was removed with a sterile scalpel. 0.5g of the internal tissue was placed in a 2mL sterile centrifuge tube, physically disrupted, and an appropriate amount of sterile water was added to prepare an endophytic 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 hours. Part of the culture was used for subsequent experiments, and the remainder was preserved in sterile glycerol at -80℃ for later use.

[0029] Images of colonies on LB plates, as shown Figure 1 As shown in a, the endophytic decarboxylating Leavenweed (Ectomycorrhizal fungi) fruiting bodies of the ectomycorrhizal fungi Leclercia adecarboxylata N183 colonies are moist, milky white to pale yellow, and round, with neat and clear edges, a slightly raised center, and a diameter of approximately 1-2 mm. See [link to shake flask culture] for growth information. Figure 1 b. The 16S rDNA sequence of *Lechlerae decarboxylates* N183 is shown in SEQ ID NO.1.

[0030] SEQ ID NO.1: Example 2 Investigating decarboxylating Leavenbacterium ( Leclercia adecarboxylata N183 evaluates the growth-promoting properties of plants, as detailed below: 1) Phosphorus solubilization capacity.

[0031] Take 0.5 mL of decarboxylated Leavenella N183 bacterial suspension (OD) 600 =0.8), inoculated into 250mL Erlenmeyer flasks containing 100mL NBRIP liquid medium, which uses tricalcium phosphate (inorganic phosphorus source) or calcium phytate (organic phosphorus source) as the sole phosphorus source, and is prepared by adding 5g of the corresponding phosphorus source per liter of medium. Incubated continuously at 37℃ and 180rpm in a constant temperature shaker for 3 days, with 5 biological replicates for each treatment, 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 3000rpm for 5min, and the supernatant was collected. The OD was measured using a microplate reader with a molybdenum-antimony colorimetric method. 700 The value is used to calculate the soluble phosphorus concentration.

[0032] 2) Iron-producing carrier.

[0033] Take 0.5 mL of decarboxylated Leavenella N183 bacterial suspension (OD) 600 =0.8), inoculated into 50 mL of sterile MKB liquid medium, and cultured at 37°C 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 Value; Centrifuge the fermentation broth at 5000 rpm for 10 min, collect the supernatant and filter it through a 0.22 μm sterile filter. Mix 1 mL of the filtrate with an equal volume of 1 mL of CAS quantitative detection solution, react at room temperature for 1 h, and then measure the OD value. 630 The siderophore unit is calculated using the formula: Siderophore unit (%) = 100 × (Ar - As) / Ar (where As is the OD of the inoculated group). 630 The value, Ar, represents the OD value of the blank control. 630 (Value). The siderophore-producing capacity of this bacterium is expressed as the ratio of siderophore units to bacterial OD. 600 The ratio of .

[0034] 3) IAA production capacity.

[0035] Take 1 mL of decarboxylated Rechler bacteria N183 suspension (OD) 600=0.8) was inoculated into 100 mL of sterile LB liquid medium (containing 100 mg / L L-tryptophan) and cultured at 37 °C and 180 rpm for 2 days, with 3 replicates per group. An uninoculated LB liquid medium was also included as a blank control. After culture, the fermentation broth was centrifuged at 5000 rpm for 10 min. The supernatant was mixed with an equal volume of Salkowski colorimetric solution and developed in the dark for 30 min. The absorbance at 530 nm was measured, and the IAA yield of the strain was calculated using the IAA standard curve.

[0036] 4) Evaluation of ACC deaminase production capacity.

[0037] Take 1 mL of bacterial culture (OD) 600 =0.8), inoculated into 250mL Erlenmeyer flasks containing 100mL of sterile MMN liquid medium, with 3 replicates per group, and an uninoculated group as a control. Incubated at 37℃ and 180rpm for 2 days. After incubation, the fermentation broth was centrifuged at 5000rpm for 10min, and 1.5mL of the supernatant was collected. The OD was measured using an ACCD kit (LMAI Bio, LM-ACCD-Ot) following the ELISA kit instructions. 450 The ACC deaminase activity was calculated using the value and the standard curve.

[0038] Test results decarboxylated Lechlerella ( Leclercia adecarboxylata The results of the growth-promoting function test of N183 are as follows: Decarboxylated Leavenler bacteria N183 exhibits excellent plant growth-promoting properties and has a highly efficient ability to dissolve insoluble phosphorus. When insoluble organic phosphorus calcium phytate is used as the sole phosphorus source, the soluble phosphorus produced is 4.52±0.12 mg / L; when insoluble inorganic phosphorus tricalcium phosphate is used as the sole phosphorus source, the soluble phosphorus produced is 4.11±0.07 mg / L. In addition, decarboxylated Leavenler bacteria N183 has an extremely strong ability to produce siderophores. With a SU content of 59.34% and an As / Ar ratio of 0.372, it has an iron-carrier capacity of 48.77, which helps plants absorb iron and alleviate iron deficiency stress. It can synthesize the plant growth hormone IAA at a concentration of 69.14±2.69 mg / L and secrete ACC enzyme (103.88±2.00 mg / L) to alleviate plant stress. Through synergistic effects of multiple mechanisms, it can significantly promote the growth of plants such as Pinus sylvestris, and has important application value in the field of plant growth-promoting biological agents.

[0039] Example 3 Decarboxylated Lechlerbe ( Leclercia adecarboxylata The specific steps regarding the growth-promoting effect of N183 on Pinus kesiya are as follows: 1) Preparation of mycorrhizal and non-mycorrhizal seedlings of Pinus simonii.

[0040] After surface disinfection of the seeds of Pinus kesao-yomi by 75% alcohol, they are washed three times with sterile water. Then, in a seedling tray filled with sterile vermiculite, the seeds are evenly spread on the surface of the vermiculite, covered with an appropriate amount of sterile vermiculite, thoroughly watered with sterile tap water, and placed in a greenhouse for germination and cultivation for 15-20 days to obtain Pinus kesao-yomi seedlings.

[0041] Colored puffball mycelia were inoculated onto PDA plates and incubated upside down in the dark at 28°C for approximately 15 days. After a large number of mycelia had grown, three 5mm diameter mycelial blocks were taken using a punch and inoculated into 250mL Erlenmeyer flasks containing 150mL of PDA liquid medium. The flasks were then incubated at 28°C and 180rpm on a shaker for 15 days. The mycelial cells were then removed using a vacuum pump and transferred to sterile 5mL EP tubes. Sterile water was added for tissue homogenization, and the resulting solution was washed to obtain the mycelial suspension. The prepared mycelial suspension was divided into two portions: one portion was used as an active ectomycorrhizal inoculant; the other portion was autoclaved at 121°C for 20 minutes to serve as an inactivated ectomycorrhizal inoculant. Two inoculants were inoculated into the root system of Pinus kesina seedlings with uniform growth. The seedlings were placed in a greenhouse and cultured under natural light for about 30 days to obtain mycorrhizal seedlings (Pinus kesina seedlings inoculated with active ectomycorrhizal inoculants, denoted as E+) and non-mycorrhizal seedlings (Pinus kesina seedlings inoculated with inactivated ectomycorrhizal inoculants, denoted as E-).

[0042] 2) Preparation of inoculum.

[0043] A single colony of *Decarboxylated Rechlerellae* strain N183 was picked and inoculated into a 250 mL Erlenmeyer flask containing 150 mL of LB medium. The flask was incubated at 37 °C and 180 rpm for 2 days, followed by centrifugation at 5000 rpm for 10 min. The precipitated bacterial cells were then mixed with sterile water and the concentration was adjusted to OD0.05. 600 = 0.8, and a bacterial suspension was prepared. The bacterial suspension was divided into two equal parts. One part was left untreated and was designated as the live bacterial agent (N183+). The other part was sterilized by autoclaving (121℃, 20min) and designated as the inactivated bacterial agent (N183-), which served as the inoculum for the uninoculated control group.

[0044] The method for preparing colored puffball fungal inoculant is as shown in 1), which is a suspension of colored puffball fungal inoculant.

[0045] 3) Design and cultivation of the experimental and control groups.

[0046] Using the prepared colored puffball fungal inoculant, decarboxylated Chainleckella N183, and their inactivation inoculants (121℃, 20min) as microbial inoculants, the non-mycorrhizal seedlings prepared in step 1) were treated with double inoculation of the two inactivated inoculants (CK) and single inoculation of N183 (Pt-N183+), respectively. The mycorrhizal seedlings prepared in step 1) were treated with single inoculation of colored puffball fungal inoculant (Pt+N183-) and double inoculation of the two active inoculants (Pt+N183+). Therefore, a total of four treatment groups were obtained.

[0047] The culture medium was a mixture of humus, sterile vermiculite, and Pinus sylvestris forest soil in a ratio of 1:15:15 (w / w), with 3‰ of insoluble inorganic tricalcium phosphate and organic calcium phytate added during the process. The culture medium was sterilized at 121℃ for 2 hours, with a one-day interval, and the sterilization process was repeated 3 times before use.

[0048] During planting, the roots were inoculated with an equal amount of microbial agent using the root-dipping method, with 5 replicates per group. The plants were then placed in a greenhouse under natural light and cultivated. Uniform irrigation management was carried out during the cultivation period to keep the substrate moist. Various indicators were measured after 120 days of cultivation.

[0049] 4) Detection indicators.

[0050] By measuring plant height, root length, biomass, phosphorus use efficiency, chlorophyll content, and other indicators in different treatment groups, this study explored the growth-promoting effect of N183 on Pinus simonii, as well as its functional role in the establishment and functional enhancement of ectomycorrhizae in Pinus simonii.

[0051] 5) Test results.

[0052] The growth and fruiting of Pinus simonii are as follows Figure 2 As 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 *Lycium barbarum* N183 and *Lycoperdon perlatum* having the most significant growth-promoting effect. Further analysis of the growth indicators of *Pinus kesiao* revealed 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 koraiensis*, showed significant improvements compared to the control group. Treatments with single inoculation with either *Decarboxylated Rechlerella asiatica* N183 or *Lycoperdon perlatum* significantly increased the total biomass (fresh weight of the whole plant) of *Pinus koraiensis*. Figure 5 c); while the total biomass of the double-inoculation group of decarboxylated Leavenler N183 and colored bean puffball fungus was significantly higher than that of the control group and each single-inoculation group (c); Figure 5 c); meanwhile, inoculation with decarboxylated Rechlerella N183 increased the colonization rate of ectomycorrhizal mycorrhizae of Pinus kesiaoensis by 6.8% ( Figure 6A significant and positive synergistic effect of decarboxylated Lechlerella N183 and Puffball fungus was observed, with the combined inoculation of the two showing the most significant growth-promoting effect on Pinus sylvestris.

[0053] The phosphorus concentrations in the aboveground and underground parts of Pinus sylvestris and the average phosphorus concentration of the plant were compared. Figure 7 ) and phosphorus accumulation in aboveground and underground parts, and total phosphorus accumulation in the whole plant ( Figure 8 The results showed that, compared with the uninoculated control group (CK), all inoculation treatment groups (single inoculation and double inoculation combinations) significantly increased the concentration and accumulation of soluble phosphorus 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 performed exceptionally well: its average phosphorus concentration increased by 309.49%, 134.75%, and 51.81% compared to the CK control and the two single inoculation groups (Pt-N183+ and Pt+N183-), respectively; and its total phosphorus accumulation was 29.80 times, 10.25 times, and 2.83 times that of the CK, Pt-N183+, and Pt+N183- groups, respectively. The changes in these phosphorus nutrition indicators followed the same pattern as the growth indicators, showing an overall gradient of "double inoculation group > single inoculation group > CK group".

[0054] In summary, the endophytic decarboxylating Leavenweed (a type of ectomycorrhizal fungus) fruiting bodies of this fungus... Leclercia adecarboxylata N183 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 N183 and ectomycorrhizal fungi has a better comprehensive effect on phosphorus nutrient absorption and growth promotion than single inoculation treatment and control group.

[0055] Therefore, the endophytic bacteria *Decarboxyl-Lyclerobacterium* of the fruiting bodies of colored puffballs provided by this invention (…) Leclercia adecarboxylata The N183 strain belongs to the plant growth-promoting bacteria (PGPR) family. It has excellent solubility for insoluble organic and inorganic phosphorus, and can significantly enhance the mycorrhizal phosphorus absorption and accumulation of Pinus kesjayensis. It can not only promote mycorrhizalization of plant roots and increase mycorrhizal infection rate, but also significantly promote the growth of Pinus kesjayensis aboveground and underground parts under the conditions of single inoculation or co-inoculation with ectomycorrhizae. Moreover, the promoting effect of double inoculation is better than that of single inoculation with decarboxylating Leavenweed or colored puffball strain, showing a synergistic effect and stable and prominent effect. It can be used to prepare biological agents for the cultivation of Pinus kesjayensis seedlings. It has the advantages of low cost, good effect and no toxic side effects. It can more persistently help the growth and development of host plants and increase yield, and provides a new technical approach for the efficient cultivation of Pinus kesjayensis plantations.

[0056] 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. A strain of decarboxylated Rechler bacteria ( Leclercia adecarboxylata The application of strain N183 in the preparation of microbial inoculants is characterized by: The 16S rDNA sequence, as shown in SEQ ID NO.1, was deposited on February 4, 2026, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:67812.

2. The decarboxylating Leavenbacterium as described in claim 1 ( Leclercia adecarboxylata The application of strain N183 in the cultivation of Pinus simonii seedlings is characterized by: The decarboxylated Rechler bacteria ( Leclercia adecarboxylata The N183 strain was applied together with the culture medium of colored puffball mycelium.

3. The decarboxylating Leavenella vulgaris according to claim 2 ( Leclercia adecarboxylata The application of strain N183 in the cultivation of Pinus simonii seedlings is characterized by, The preparation method of the colored puffball mycelial culture medium is as follows: inoculate the colored puffball mycelium into liquid PDA medium and culture it at 28℃ and 180rpm for 15-20 days to obtain mycelial culture.

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