Aquilaria sinensis strain r254 and application thereof in promoting growth of aesculus chinensis

Abstract

The application discloses a strain of aquilaria sinensis R254 and application of the strain in promoting growth of a Manchurian ash tree, and belongs to the technical field of microorganisms.The strain of the aquilaria sinensis R254 has a preservation number of CGMCC No.42907.The aquilaria sinensis R254 disclosed by the application has the abilities of producing indole acetic acid, producing an iron carrier and fixing nitrogen, can significantly promote synthesis of photosynthetic core pigments of leaves of the Manchurian ash tree, enhance the light energy capturing capacity of the leaves, and promote growth of the Manchurian ash tree.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to an Aquila mold R254 and its application in promoting the growth of Catalpa trees. Background Technology

[0002] Catalpa ( Catalpa bungei Catalpa (Catalpa bungei) is a precious timber and landscaping tree species unique to my country, known as the "King of Woods." It boasts excellent wood quality, beautiful tree shape, and rapid growth, making it widely used in high-end furniture manufacturing, architectural decoration, and urban greening. However, during seedling cultivation and artificial forest planting, Catalpa bungei commonly suffers from poor root development and slow growth, especially in poor soils and under adverse conditions, where seedling survival rates and growth rates fail to meet production demands. While traditional fertilization management methods can promote Catalpa bungei growth to some extent, the long-term and excessive use of chemical fertilizers not only increases production costs but may also lead to soil degradation and environmental pollution. Therefore, it is urgent to explore and vigorously develop green, efficient, and sustainable Catalpa bungei growth-promoting technologies.

[0003] Plant endophytic fungi are a class of microorganisms that are ubiquitous within plant tissues and form a mutualistic symbiotic relationship with the host plant. They promote the growth of the host plant by synthesizing plant hormones, enhancing the plant's absorption of nutrients, and increasing the accumulation of secondary metabolites. While some applications of endophytic fungi in promoting tree growth have been reported, current techniques largely focus on observing epigenetic indicators and lack a systematic and in-depth analysis of their growth-promoting molecular mechanisms using metabolomics. This is particularly relevant to the genus *Aquilaria* (…). Aquilomyces Research on fungi promoting forest tree growth is extremely limited.

[0004] Aquilomyces This genus is a new fungal genus formally established in 2015, belonging to the family Morosphaeriaceae within the phylum Ascomycota. These fungi are root endophytic and widely distributed in semi-arid regions worldwide. Existing technology regarding the genus *Aquila* (…) Aquilomyces Research on fungi promoting forest tree growth is extremely limited, and studies on the promotion of catalpa tree growth by this genus have not been reported at all. Its unique taxonomic characteristics suggest that it may contain novel mechanisms of action or active substances different from common growth-promoting fungi. In-depth research on the growth-promoting effects of *Aquilaria* on plants may not only bring new solutions to sustainable agriculture but will also greatly deepen our understanding of the complexities of the co-evolution of fungi and plants.

[0005] Therefore, providing a strain of Aquilaria rhynchophylla R254 and its application in promoting the growth of Catalpa trees is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides an Aquila rhizome R254 and its application in promoting the growth of Catalpa trees.

[0007] Strain R254 possesses multiple growth-promoting properties, including indoleacetic acid (IAA) production, siderophore production, and nitrogen fixation. It can stably colonize the roots of *Catalpa bungei*, forming characteristic structures such as dark mycelia and microsclerotia. Non-targeted metabolomics analysis revealed that strain R254 systematically reprogrammed the metabolic network of *Catalpa bungei* roots after colonization, comprehensively promoting *Catalpa bungei* growth by activating brassinolide and indoleacetic acid signaling pathways and enhancing nitrogen assimilation and amino acid metabolism. This invention provides a novel microbial resource and an efficient bio-promoting technology paradigm for addressing the problems of slow growth and poor root development in *Catalpa bungei* and other forest tree species.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A strain of Aquila mold ( Aquilomyces sp.) R254, with accession number CGMCC No. 42907, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is July 23, 2026. It is classified and named as follows: Aquilomyces sp.

[0010] Furthermore, the application of Aquilae r254 in promoting the growth of Catalpa trees, the specific steps of the application method are as follows: 1) Preparation of Aquilaria rhynchophylla R254 inoculum Inoculate the R254 strain preserved on a 4 ℃ slant onto the center of a PDA solid medium plate and incubate in a constant temperature incubator at 25-28 ℃ in the dark for 10-15 days for morphological observation or molecular identification; or inoculate the R254 strain preserved on a slant into a 250 mL Erlenmeyer flask containing 50 mL of PD liquid medium and incubate at 25-28 ℃ with shaking at 180-200 r / min for 5-7 days to prepare a seed culture; inoculate the seed culture at 10% (volume ratio) into a 500 mL Erlenmeyer flask containing 100 mL of fresh PD liquid medium and incubate at 25-28 ℃ with shaking at 180-200 r / min for 7-10 days for later use.

[0011] The PDA solid culture medium consisted of 0.6% potato extract powder, 2% glucose, 2% agar, and pH 5.6; the PD liquid culture medium consisted of 0.6% potato extract powder, 2% glucose, and pH 5.6; both were autoclaved at 121 °C for 20 min.

[0012] 2) Cultivation and inoculation treatment of Catalpa trees Select catalpa trees (including but not limited to seedlings, tissue culture seedlings, or cuttings) that are in good growth condition and have a basically uniform growth. Mechanically disperse the R254 mycelial suspension prepared in step 1) to ensure the mycelium is fully dispersed, then dilute it 3-50 times with water to prepare a treatment solution. Use a root irrigation inoculation method, watering each tree with 500-1000 mL of the prepared treatment solution, ensuring the mycelial solution evenly saturates the rhizosphere soil area. Use catalpa trees watered with an equal volume and dilution of PD culture solution as a control group.

[0013] The inoculation frequency is once every 10-20 days, for a total of 3 treatments; or the treatment interval can be adjusted appropriately according to the plant growth status and temperature conditions.

[0014] Furthermore, the mechanical dispersing treatment can be carried out by using a tissue homogenizer or a homogenizer with low-speed stirring to fully disperse the mycelial balls into a uniform mycelial fragment suspension.

[0015] 3) Colonization detection After inoculation, root samples of *Catalpa bungei* were collected periodically. The roots were washed, cut into 3-5 cm segments, and fixed in FAA fixative for 24 h. The fixative was then discarded, and the roots were rinsed three times with deionized water. After clearing treatment by incubating in 10% KOH solution at 95°C for 1 h, cytoplasmic pigments were removed. After rinsing three times with deionized water, the roots were acidified with 1% HCl for 30 min, the acid solution was discarded, and the roots were rinsed three times with ddH2O. Then, 0.05% trypan blue staining solution (lactic acid:glycerol:water = 1:3:6, volume ratio) was added, and staining was performed at room temperature for at least 12 h. Excess staining was washed away with 50% lactic acid. Randomly selected root segments were mounted and examined under a microscope. The observation of the typical characteristic structures of dark-colored septate endophytic fungi—dark hyphae and microsclerotia—indicated successful colonization of strain R254 within the *Catalpa bungei* roots.

[0016] 4) Evaluation of growth-promoting effects and mechanisms After inoculation, growth indicators such as tree height, root growth, and leaf chlorophyll content of the catalpa trees in the treatment and control groups were observed and measured. Simultaneously, root samples were collected from the catalpa trees for non-targeted metabolomics analysis to reveal the metabolic regulatory mechanism by which strain R254 promotes the growth of catalpa trees.

[0017] Furthermore, the application of Aquilaria rhynchophylla R254 in the production of indoleacetic acid, siderophore production, and nitrogen fixation.

[0018] Furthermore, the application of Aquila rhamnoides R254 in increasing the chlorophyll content of Catalpa tree leaves.

[0019] Furthermore, a microbial preparation for promoting the growth of catalpa trees contains mycelium or fermentation broth of the aforementioned Aquila rhamnoides R254.

[0020] As can be seen from the above technical solution, compared with the prior art, the present invention discloses an Aquilae mold R254 and its application in promoting the growth of Catalpa trees, which has the following beneficial effects: (1) First time Aquilomyces The genus *Penicillium* is used to promote the growth of catalpa trees. Currently, the application of endophytic fungi in promoting forest tree growth mainly focuses on the genus *Penicillium*. Penicillium Trichoderma () Trichoderma A few common groups, such as ) and Aquilomyces This genus is a new genus established in 2015, possessing a unique taxonomic position. This invention is the first to apply fungus R254 of this genus to the cultivation of Catalpa trees; there are currently no other known examples of this genus, either domestically or internationally. Aquilomyces Reports indicate that fungi can promote the growth of catalpa or other trees.

[0021] (2) For the first time, strain R254 was found to have triple growth-promoting characteristics of producing IAA, producing siderophores, and fixing nitrogen. Existing growth-promoting strains usually only have single or dual growth-promoting characteristics, while strain R254 of this invention has the functions of secreting plant growth hormone IAA, producing siderophores, and fixing nitrogen. The synergistic effect of the triple growth-promoting characteristics makes its growth-promoting effect more significant and comprehensive.

[0022] (3) This study is the first to systematically reveal the metabolic regulatory network by which *Aquilaria sinensis* promotes the growth of *Catalpa bungei* using metabolomics. Existing research on growth promotion largely focuses on observing epigenetic indicators, with very limited analysis of its mechanisms of action. This invention employs non-targeted metabolomics technology to systematically analyze the metabolic reprogramming map of *Catalpa bungei* roots after R254 colonization, identifying 3,062 significantly differentially expressed metabolites. Metabolic network analysis revealed that brassinolide, as a core signaling molecule, was significantly activated, with IAA precursors and bound IAA accumulating simultaneously, synergistically promoting cell division and lateral root development. Simultaneously, the core nitrogen carrier and the key TCA cycle node α-ketoglutarate (VIP=1.59) accumulated synchronously, driving nitrogen assimilation and carbon skeleton diversion. This systematic metabolic network analysis is the first of its kind in *Aquilaria sinensis*-forest tree interaction research. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 The colony morphology of strain R254 on PDA plates.

[0025] Figure 2Phylogenetic tree of strain R254 constructed based on ITS sequence (maximum likelihood method).

[0026] Figure 3 To detect the siderogenic vector of strain R254 on CAS double-layer plates.

[0027] Figure 4 This is the IAA standard curve for strain R254.

[0028] Figure 5 To detect the colonization of strain R254 in the roots of Catalpa bungei using trypan blue staining; where A is the control and B is the treatment; scale bar is 50 µm.

[0029] Figure 6 Effects of inoculation with strain R254 on the growth of Catalpa trees (left: control, right: treatment).

[0030] Figure 7 Effects of inoculation with strain R254 on root development of Catalpa bungei (left: control, right: treatment).

[0031] Figure 8 The effect of inoculation with strain R254 on the chlorophyll content of Catalpa tree leaves. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1: Isolation, purification, and identification of strain R254 (1) Collect healthy plant root samples, wash the plant root segments with clean water, and then transfer them to a clean bench for the following operations: Soak the root segments in 10% (mass ratio) hydrogen peroxide for 8 min, gently turning them several times with sterile tweezers during this period to ensure thorough disinfection; rinse 2-3 times with sterile water, then transfer them to 6% (mass ratio) sodium hypochlorite for 15 min, and rinse 2 times with sterile water; blot the residual liquid on the surface of the root segments with filter paper. Cut the disinfected root segments into 0.5 cm long root segments with sterile scissors, inoculate them into prepared PDA plates, and incubate them upside down in the dark in a constant temperature incubator at 25 ℃. The formula of PDA plate: 0.6% potato extract powder, 2% glucose, 2% agar, pH 5.6. Sterilize at 115 ℃ for 20 min, and when cooled to 60 ℃, add sterile ampicillin (50 μg / mL) and streptomycin sulfate (100 μg / mL). The concentrations of all the components mentioned above are the final concentrations in PDA medium.

[0034] (2) When fungi are found growing at the tissue incision site, use an inoculation needle to pick them into a PDA plate without penicillin and streptomycin as soon as possible. Incubate them in the dark and inverted in a constant temperature incubator at 25 ℃ for purification culture. After transferring 3-5 times, pure colonies are obtained and inoculated onto PDA slant. Store the culture at 4 ℃.

[0035] (3) Inoculate the R254 strain, which was stored in a 4 ℃ refrigerator, onto the center of a PDA solid medium plate and incubate in a 28 ℃ incubator in the dark for 15 days. Figure 1 As shown, strain R254 grew well on PDA plates, with grayish-white, felt-like colonies. The central area was densely raised with abundant hyphae, surrounded by radially growing white new hyphae that spread evenly outwards, preliminarily identifying the strain as a filamentous fungus. Molecular identification was performed targeting the ITS (Internal Transcribed Spacer) sequence. Genomic DNA was extracted from freshly cultured hyphae using a genomic DNA extraction kit. Genomic DNA integrity was assessed using 1% agarose gel electrophoresis, and DNA concentration and purity were determined using a nucleic acid concentration analyzer. Using the qualified genomic DNA as a template, PCR amplification was performed using the universal fungal ITS primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′; SEQ ID NO.1) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′; SEQ ID NO.2). The total volume of the PCR reaction system was 50 μL, including 25 μL of 2×PCR Master Mix, 2.5 μL each of forward and reverse primers, and 5 μL of template DNA, with the volume made up with sterile ddH2O. The PCR amplification program was as follows: 94 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 52 ℃ annealing for 30 s, 72 ℃ extension for 1 min, for a total of 35 cycles; and a final extension at 72 ℃ for 10 min. After passing the PCR product test by 1% agarose gel electrophoresis, the product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0036] The ITS sequence of strain R254 was determined as shown in SEQ ID NO.3.

[0037] ; SEQ ID NO.3.

[0038] The above sequences were searched for homology in the NCBI GenBank database using the BLAST program. The obtained homologous sequences were then used to perform multiple sequence alignment of the ITS sequences of the target strain and the reference strain using MEGA 12.0 software. A phylogenetic tree was constructed using the maximum likelihood (ML) method, and the reliability of each branch node was tested using the bootstrap method with 1000 repeated samplings. Figure 2 As shown, strain R254 is related to this strain and Aquilomyces The genus clusters in the same branch and has a very close phylogenetic relationship, hence the strain was named... Aquilomyces sp. R254.

[0039] Example 2: Determination of the siderophore production capacity of strain R254 The siderophore-producing ability of the strain was detected using a CAS double-layer medium. The bottom layer medium was iron-free Czapek's agar: 30.0 g sucrose, 2.0 g NaNO3, 1.0 g KH2PO4, 0.5 g KCl, 0.5 g MgSO4·7H2O, and 15.0 g agar, diluted to 1000 mL with deionized water, pH 6.8. The mixture was autoclaved at 121 °C for 20 min and then poured into plates. The top layer, the display agar medium, was prepared by mixing the CAS colorimetric solution with 1.2% low-melting-point agarose (incubated at 55 °C) at a 1:1 volume ratio and immediately covering the surface of the bottom layer medium. Preparation of CAS colorimetric solution: Dissolve 60.5 mg of Chromium Azure S in 50 mL of deionized water (solution A); dissolve 10 mg of FeCl3·6H2O in 10 mL of 10 mM HCl (solution B); dissolve 72.9 mg of hexadecyltrimethylammonium bromide (HDTMA) in 40 mL of deionized water (solution C). Slowly add solution B dropwise to solution A, then slowly add solution C, mix thoroughly, and store at 4 °C protected from light.

[0040] Using a 5 mm punch, strain R254, activated on PDA solid medium, was inoculated onto the bottom layer of iron-free Czapek's agar. The growth of the strain was observed daily. After approximately 14 days of culture, the top layer was covered with the aforementioned chromogenic agar. After 48 hours of culture, the presence of an orange-yellow transparent ring around the colony was observed. The mechanism of this colorimetric reaction is as follows: the originally bound iron-chromium azure S complex is blue-green; when the siderophore secreted by the microorganism releases the Fe... 3+ After capture, the free chromium azure S appears orange-yellow, thus forming a yellow halo around the colony. For example... Figure 3 As shown, after strain R254 was cultured on a CAS double-layer plate, a distinct orange-yellow transparent ring appeared around the colony, indicating that strain R254 can produce and secrete siderophores and has the ability to chelate iron ions in the environment. This suggests that strain R254 can improve the efficiency of iron utilization in plants by chelating iron in the soil and play a potential role in promoting plant growth.

[0041] Example 3: Determination of IAA production capacity of strain R254 A standard curve for IAA was established using the Salkowski colorimetric method. IAA standard solutions with concentrations of 10, 20, 30, 40, and 50 mg / L were prepared. 5 mL of each solution was mixed thoroughly with an equal volume of Salkowski colorimetric solution, and the mixture was allowed to react in the dark for 30 min. The absorbance was then measured at 530 nm. The Salkowski colorimetric solution was prepared by mixing 1 mL of 0.5 M FeCl3 with 50 mL of 7.9 M H₂SO₄. Figure 4 As shown, a standard curve was plotted with IAA concentration on the x-axis and absorbance at 530 nm on the y-axis, showing a good linear relationship (R0). 2>0.99).

[0042] The IAA secretion capacity of the strain was determined using PD liquid medium supplemented with L-tryptophan. Fresh mycelial growths (5 mm in diameter) were inoculated into 100 mL of PD liquid medium supplemented with 100 mg / L L-tryptophan and cultured in the dark with shaking at 28 ℃ and 180 r / min for 7 days. The fermentation broth was centrifuged at 8000 r / min for 10 min, and the supernatant was collected. The supernatant was thoroughly mixed with an equal volume of Salkowski chromogenic solution and incubated in the dark for 30 min. The absorbance was measured at 530 nm. Calculations using a standard curve showed that the IAA content in the fermentation broth under 100 mg / L tryptophan supplementation was approximately 21.69 mg / L, demonstrating a good IAA secretion capacity among previously reported filamentous fungi.

[0043] Example 4: Determination of nitrogen fixation capacity of strain R254 The nitrogen-fixing capacity of the strain was evaluated using nitrogen-free Ashby solid medium. The nitrogen-free Ashby solid medium formulation was as follows: mannitol 10.0 g, K₂HPO₄ 0.5 g, MgSO₄·7H₂O 0.2 g, NaCl 0.2 g, CaSO₄·2H₂O 0.1 g, CaCO₃ 1.0 g, agar 18.0 g, diluted to 1000 mL with distilled water, pH at rest, and autoclaved at 121 °C for 20 min.

[0044] Vigorous, fresh mycelia were selected from PDA medium and streaked onto nitrogen-free Ashby solid medium. The culture was repeated 2-3 times, and the growth of the strain was observed. The results showed that strain R254 could grow continuously and stably on nitrogen-free Ashby medium, indicating its ability to utilize atmospheric nitrogen and possessing a certain nitrogen-fixing potential, thus providing additional nitrogen nutrition for the catalpa tree.

[0045] Example 5: Colonization detection of strain R254 in the roots of Catalpa trees Catalpa tree tissue culture transplant seedlings (approximately 22-25 cm in height) with similar growth were selected, and treatment and control groups were established. R254 bacterial suspension (concentration 1×10⁻⁶) cultured in PD liquid medium was then used. 7 The mycelium was mechanically dispersed (CFU / mL) to ensure full dispersion, and then diluted 10 times with water to prepare a treatment solution. Each pot was watered with 800 mL of the treatment solution each time. The control group was watered with the same amount of water. Treatment was carried out every 10 days for a total of 3 treatments.

[0046] Catalpa root samples were collected 60 days after the last treatment. Root colonization observation was performed using a modified trypan blue staining method: The collected roots were washed with clean water, cut into 3-5 cm long segments, fixed in FAA fixative for 24 h, then the fixative was discarded. The roots were rinsed three times with deionized water, and then placed in a 10% KOH solution at 95 ℃ for 1 h for clearing treatment. After rinsing three times with deionized water, 1% HCl was added for acidification for 30 min, the acid solution was discarded, and the roots were rinsed three times with ddH2O. 0.05% trypan blue staining solution (solvent: lactic acid: glycerol: water = 1:3:6) was added and stained at room temperature for more than 12 h. Excess staining solution was washed away with 50% lactic acid. Root segments were randomly selected, pressed into slides, and mounted for microscopic examination to observe the adhesion of the bacterial strain to the root surface and the internal infection.

[0047] Experimental results showed that after trypan blue staining, a large number of slender, tubular, dark-colored hyphae were distributed throughout the root tissue of the Catalpa tree in the R254 treatment group, and a large number of short rod-shaped micro-nuclei were observed in some cortical cells in a string-like arrangement or scattered aggregation. Figure 5 B), this is a typical characteristic structure of root endophytic fungi colonizing plant roots; however, no corresponding hyphae and microsclerotia structures were observed in the root samples of the uninoculated control group. Figure 5 A). The above results indicate that strain R254 was able to successfully colonize the root cortical cells of Catalpa bungei and form a stable fungal-plant symbiotic relationship.

[0048] Example 6: Effects of strain R254 on the growth and photosynthetic pigment content of Catalpa bungei. The treatment of catalpa trees was the same as described in Example 5. Sixty days after the final treatment, the phenotypes of the treated and control groups were observed. It was found that the catalpa trees in the treatment group inoculated with strain R254 showed more vigorous growth. The plant height in the control group was 50.03 ± 2.90 cm, while that in the treatment group was 67.87 ± 4.40 cm, showing a significant increase in plant height and a darker green leaf color. Figure 6 Root length measurements showed that the average root length of the control group of *Catalpa bungei* trees was 53.62 ± 5.03 cm, while the average root length of the *Catalpa bungei* trees treated with strain R254 was 75.48 ± 6.86 cm. Compared with the control group, the average root length of the *Catalpa bungei* trees treated with strain R254 increased by approximately 40.8%. Simultaneously, root morphology observation revealed that, compared with the control group, the root system of the strain R254-treated group was more developed, with a significant increase in root biomass and the number of lateral roots. Figure 7 The above results indicate that strain R254 has a significant growth-promoting effect on both the aboveground and underground parts of the catalpa tree.

[0049] Weigh approximately 0.1 g of fresh catalpa leaves, cut them into small pieces, and place them in a test tube. Add 10 mL of acetone-ethanol mixture (V / V = 1:1) and extract in the dark until the leaves turn completely white. Using the extract as a blank control, measure the absorbance of the extract at wavelengths of 645 nm and 663 nm using a spectrophotometer. Calculate the chlorophyll a and chlorophyll b contents using the following formula: Chlorophyll a content (mg / g) = (12.71 × A) 663 2.59×A 645 )×V / (1000×m; Chlorophyll b content (mg / g) = (22.88 × A) 645 4.67×A 663 )×V / (1000×m; Where V is the total volume of the extract (mL) and m is the fresh weight of the sample (g).

[0050] The results showed that the chlorophyll a and chlorophyll b contents of the leaves of the control group were 0.237±0.020 mg / g and 0.084±0.007 mg / g, respectively; while in the inoculated group, these contents increased to 0.432±0.016 mg / g and 0.146±0.005 mg / g, respectively. Compared with the control group, the chlorophyll a and chlorophyll b contents of the treated group increased by 82.3% and 73.8%, respectively, showing a significant increase. Figure 8 ).

[0051] The above results indicate that the colonization of strain R254 in the root system of Catalpa not only promotes root growth to ensure sufficient absorption of water and nutrients, but also significantly promotes the synthesis of photosynthetic core pigments in Catalpa leaves, enhances the light-harvesting capacity of leaves, and provides a sufficient energy basis for the growth of Catalpa.

[0052] Example 7: Metabolomics analysis of the effect of strain R254 on the growth of Catalpa trees Young root tip samples from catalpa trees were collected from both the treatment and control groups, flash-frozen in liquid nitrogen, and then ground into powder. Store at 80 ℃. After weighing the sample, add acetonitrile:methanol (1:1) extraction solution, vortex to mix, and then extract using low-temperature ultrasonication. Proteins were allowed to precipitate at 20 ℃ and centrifuged at 12000 rpm for 15 min at 4 ℃. The supernatant was then collected for analysis.

[0053] The supernatant was analyzed using ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry (UHPLC-MS / MS) for metabolite detection. The UHPLC system used a BEH C18 column (100 mm × 2.1 mm, 1.7 μm) for separation. Mobile phase A was 2% acetonitrile-water (containing 0.1% formic acid), and mobile phase B was acetonitrile (containing 0.1% formic acid). The flow rate was 0.40 mL / min, the column temperature was 40 ℃, and the injection volume was 2 μL. Mass spectrometry used an electrospray ionization (ESI) source with alternating positive and negative ion scanning, and the mass range was m / z 70–1050. Raw data were processed using Progenesis QI software for peak extraction, alignment, and normalization. Metabolite identification was performed using the MJDBPM, HMDB, and KEGG databases. The MS mass error was <10 ppm. Differential metabolites were screened using the OPLS-DA model, with a VIP>1 and p<0.05 standard, and pathway enrichment analysis was performed using KEGG.

[0054] The results showed that a total of 14,342 metabolites were detected, and 3,062 significantly differentially expressed metabolites were screened out, of which 1,847 were upregulated and 1,215 were downregulated. The key differentially expressed metabolite changes are as follows: Regarding nitrogen metabolism and amino acid turnover, the core nitrogen carriers L-glutamate (VIP=1.54), L-glutamine, and L-asparagine were all significantly upregulated; α-ketoglutarate (VIP=1.59), the crossover node between the TCA cycle and nitrogen assimilation, was significantly accumulated, indicating an effective diversion of the carbon skeleton to nitrogen assimilation; and the osmotic regulator L-proline (VIP=1.26) was upregulated, enhancing the root tip's resistance to stress in the microenvironment of microbial colonization.

[0055] In terms of plant hormone metabolism, brassinolide has a VIP as high as 3.58, making it a core signaling molecule that promotes cell elongation and division. At the same time, the IAA precursors L-tryptophan, indole-3-butyric acid (IBA), and indole-3-acetylinositol are upregulated simultaneously, indicating that the root system flexibly regulates the level of active IAA by increasing the bound IAA pool, thus synergistically promoting lateral root development and elongation.

[0056] The above results indicate that strain R254 promotes the overall growth of Catalpa trees by synergistically regulating the metabolic network of Catalpa root through multiple pathways, including activating the plant hormone brassinolide signaling and auxin metabolic precursor supply, and enhancing nitrogen assimilation and amino acid metabolism.

[0057] The *Aquilaria* R254 strain and its application method provided by this invention can be widely used in the cultivation of *Catalpa bungei* seedlings, the establishment of artificial forests, and urban greening. The fermentation process of strain R254 is simple and inexpensive, and the resulting *Catalpa bungei* growth promoter is easy to store, transport, and use. Its cultivation is easily scaled up, enabling large-scale application. This invention provides important technical support for the green and sustainable development of the *Catalpa bungei* industry.

[0058] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Aquila mold ( Aquilomyces sp.) R254, characterized in that, Its accession number is CGMCCNo.42907.

2. The application of Aquilaria rhynchophylla R254 as described in claim 1 in promoting the growth of Catalpa trees.

3. The application according to claim 2, characterized in that, The mycelial suspension of Aquilaria rhynchophylla R254 was obtained by fermentation culture, mechanically dispersed and diluted, and then inoculated into the rhizosphere of Catalpa trees by root irrigation.

4. The application according to claim 3, characterized in that, The fermentation culture was carried out using PD liquid medium and cultured at 25-28℃ and 180-200 r / min for 5-10 days.

5. The application according to claim 3, characterized in that, The root irrigation inoculation method involves watering each plant with 500-1000 mL of diluted mycelial suspension, once every 10-20 days, for a total of 2-3 treatments.

6. The use of Aquilaria rhynchophylla R254 as described in claim 1 in the production of indoleacetic acid, the production of siderophores, and nitrogen fixation.

7. The application of Aquilaria rhynchophylla R254 as described in claim 1 in increasing the chlorophyll content of Catalpa tree leaves.

8. A microbial preparation for promoting the growth of Catalpa trees, characterized in that, Mycelium or fermentation broth containing the Aquila rhizome R254 as described in claim 1.

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