Plant growth inhibitory monomeric compounds derived from strain EY-2-4, their preparation methods and applications

Abstract

This invention discloses a plant growth inhibitory monomeric compound derived from strain EY-2-4, its preparation method, and its application. The structural formula of the monomeric compound is shown in formula (I), which is derived from the leaves of *Cephalotaxus fortunei* isolated from Songshan Lake, Dongguan. Nodulisporium sp The EY-2-4 strain was obtained through fermentation and purification of crude extract. This monomeric compound exhibits potent and concentration-dependent inhibition of tobacco seed germination and post-germination growth. The monomeric compound of this invention has a well-defined structure, specific activity, and clear mechanism of action. Its preparation process is controllable, making it a novel, highly efficient, and environmentally friendly plant growth regulator applicable to agricultural production and horticulture. It addresses the problems of unclear active ingredients, ambiguous mechanisms of action, and unstable effects in existing plant growth inhibitors, possessing significant theoretical importance and broad industrialization prospects.

Description

Technical Field

[0001] This invention relates to the field of plant growth regulators and microbial metabolites, specifically to a plant growth inhibitory monomeric compound derived from strain EY-2-4, its preparation method, and its application. Background Technology

[0002] Plant growth inhibitors, as indispensable regulators in agriculture and horticulture, play a crucial role in controlling excessive vegetative growth, optimizing plant shape, enhancing stress resistance, and regulating flowering time. With increasing environmental awareness, plant growth inhibitors that are naturally derived and environmentally compatible have become a research hotspot; however, existing products still have many shortcomings.

[0003] (1) Some synthetic inhibitors pose a high risk of environmental residue and are prone to causing ecological pollution;

[0004] (2) Most natural source inhibitors are mixed extracts with unclear core active ingredients, resulting in unstable effects and poor controllability;

[0005] (3) The mechanisms of action of most inhibitors are vague, making it difficult to achieve precise regulation of plant growth. Moreover, their specificity is insufficient and may have adverse effects on non-target organisms.

[0006] (4) No reports have been found on the isolation of single plant growth inhibitors with well-defined structures and clear mechanisms of action from the metabolites of endophytic fungi of Taxus chinensis. Summary of the Invention

[0007] In view of the above problems, the present invention provides a method derived from... Nodulisporium sp Plant growth inhibitory monomeric compounds of strain EY2-4, their preparation methods, structural identification, mechanism of action, and applications. This provides new technologies for bio-agriculture and related industries. The chemical structural formula of the monomeric compound is shown in formula (I), and it is named compound 3; the monomeric compound is derived from... Nodulisporium sp. The EY-2-4 strain (GenBank ID: PX870240) was obtained through fermentation and purification of crude extract. The strain was isolated from leaf tissue of healthy bald cypress trees in Songshan Lake, Dongguan City, Guangdong Province.

[0008] The first objective of this invention is to provide Nodulisporium sp. Application of strain EY-2-4 in the preparation of monomeric compounds, the structural formula of which is shown in formula (Ⅰ): ; Equation (Ⅰ).

[0009] A second object of the present invention is to provide a method for preparing the above-mentioned monomeric compound, comprising the following steps: from Nodulisporium spThe .EY-2-4 strain was obtained through fermentation and purification of crude extract.

[0010] Preferably, the specific steps are as follows:

[0011] 1) Fermentation by strain: Nodulisporium sp. EY-2-4 strain seed culture was inoculated into potato glucose broth medium and fermented in shake flasks at 27-29℃ and 110-130 rpm for 6-8 days to obtain fermentation broth;

[0012] 2) Preparation of crude extract: After filtration of the fermentation broth, it is extracted with ethyl acetate 3-4 times. The extracts are combined and concentrated to dryness under reduced pressure to obtain crude extract paste.

[0013] 3) Separation and purification of monomeric compounds: The crude extract was separated by silica gel column chromatography using a gradient elution system of n-hexane:ethyl acetate at a volume ratio of 100:1 → 1:3. The fraction obtained by elution with n-hexane:ethyl acetate at a volume ratio of 2:1 and developed by TLC at a volume ratio of n-hexane:ethyl acetate at 2:1, yielding an Rf of 0.5, was collected. This fraction was then processed by silica gel column chromatography using a volume ratio of n-hexane:ethyl acetate at 20:1 → 1:3. The eluted product was then purified by Sephadex LH-20 gel chromatography with chloroform:methanol at a volume ratio of 1:3. The fraction with an Rf of 0.4 when developed by TLC at a volume ratio of n-hexane:ethyl acetate at 2:1 was collected, which is the target monomeric compound.

[0014] A third objective of this invention is to provide the application of the aforementioned monomeric compounds in regulating plant growth.

[0015] Preferably, the above-mentioned monomeric compound is used in any of the following:

[0016] 1) Application in inhibiting seed germination and / or root growth in seedlings;

[0017] 2) Regulates the phenylpropane biosynthesis pathway, auxin signal transduction pathway, and energy metabolism-related pathways;

[0018] 3) Applications in upregulating the expression of PIP2-1, a gene related to the sphingolipid signaling pathway; upregulating the expression of ITPK1-like, a gene related to the phenylpropanone metabolism pathway; and / or downregulating the expression of RGL3-like, a gene related to the auxin signaling pathway.

[0019] 4) Applications in interfering with plant hormone homeostasis and secondary metabolic flux, altering the accumulation levels of terpenoids, lipids, flavonoids, and lipid secondary metabolites, and disrupting cell wall structural integrity and redox balance.

[0020] A fourth objective of this invention is to provide the application of the aforementioned monomeric compounds in agricultural production or horticultural cultivation for controlling excessive crop growth, dwarfing plants, regulating flowering time, and / or improving plant stress resistance.

[0021] A fifth objective of this invention is to provide the application of the aforementioned monomeric compounds in the preparation of plant growth inhibitors.

[0022] Preferably, the plant is tobacco or Arabidopsis thaliana.

[0023] The sixth objective of this invention is to provide a plant growth inhibitor containing the aforementioned monomeric compound as an active ingredient.

[0024] Preferably, the plant growth inhibitor is prepared by mixing the above-mentioned monomeric compound with an agriculturally acceptable carrier or adjuvant, and the formulation is an aqueous solution, powder, emulsifiable concentrate or suspension.

[0025] Advantages of this invention:

[0026] The monomeric compounds have clearly defined structures, which solves the problems of complex components, vague active ingredients, and unstable effects of crude extracts, and provides clear molecules for precise regulation of plant growth.

[0027] The preparation process is controllable. The separation steps are optimized based on the fermentation process. The mature purification technology of "silica gel column-gel column" is adopted, which is easy to scale up and has a low production cost.

[0028] The mechanism of action is clear. Through multi-omics joint analysis and molecular experimental verification, the key pathways (phenylpropane biosynthesis pathway, auxin signal transduction pathway, etc.) and core genes involved in the regulation of the compound have been identified. PIP2-1 , ITPK1-like , RGL3-like It can achieve precise regulation of plant growth, reduce the impact on non-target organisms, and has good environmental compatibility;

[0029] It exhibits potent and specific activity, showing significant concentration-dependent growth inhibitory activity against both tobacco and Arabidopsis thaliana. It is effective even at low concentrations, making it superior to some existing natural plant growth inhibitors.

[0030] This has enriched the variety of plant growth inhibitors and provided new molecular templates and theoretical basis for the development of novel, efficient, and environmentally friendly plant growth regulators, with broad application prospects.

[0031] The monomeric compound provided by this invention is a monomeric compound with significant plant growth inhibitory activity, which is isolated from the leaves of *Cephalotaxus fortunei* var. *mongolica* in Songshan Lake, Dongguan. Nodulisporium sp.The EY-2-4 strain (GenBank ID: PX870240) was obtained through fermentation and purification of crude extract. The chemical structure of the monomeric compound was determined by detecting its optical rotation, as shown in formula (I), and named compound 3. Bioactivity tests confirmed that this monomeric compound had a potent and concentration-dependent inhibitory effect on tobacco seed germination and post-germination growth, as well as on Arabidopsis seed germination and seedling growth. Further transcriptome-metabolome analysis and qRT-PCR verification clarified its mechanism of action as: specifically regulating the phenylpropanone biosynthesis pathway, auxin signal transduction pathway, and energy metabolism-related pathways, upregulating... PIP2-1 , ITPK1-like Gene expression downregulated RGL3-like Gene expression is disrupted, interfering with plant hormone homeostasis and secondary metabolic flux, and damaging cell wall structural integrity and redox balance, thereby exerting a growth-inhibiting effect. The monomeric compounds of this invention have well-defined structures, specific activities, and clear mechanisms of action. Their preparation process is controllable, making them suitable as novel, highly efficient, and environmentally friendly plant growth regulators for application in agricultural production and horticulture. This addresses the problems of unclear active ingredients, ambiguous mechanisms of action, and unstable effects in existing plant growth inhibitors, and has significant theoretical implications and broad industrialization prospects.

[0032] The applicant has previously isolated and identified from the leaves of Cephalotaxus fortunei in Songshan Lake, Dongguan. Nodulisporium sp The applicant also holds the EY-2-4 strain (GenBank ID: PX870240) and guarantees to make it available to the public for 20 years from the date of application. Attached Figure Description

[0033] Figure 1 This involves high-performance liquid chromatography (HPLC) for the chromatographic analysis of secondary metabolites in the crude ethyl acetate extract of the strain.

[0034] Figure 2 This is the 1H NMR spectrum (500 MHz, CD3OD) of compound 1.

[0035] Figure 3 This is the carbon NMR spectrum of compound 1 (125 MHz, CD3OD).

[0036] Figure 4 This is the 1H NMR spectrum of compound 2 (500 MHz, CD3OD).

[0037] Figure 5 This is the carbon NMR spectrum of compound 2 (125 MHz, CD3OD).

[0038] Figure 6 This is the 1H NMR spectrum of compound 3 (500 MHz, CD3OD).

[0039] Figure 7 This is the carbon NMR spectrum of compound 3 (125 MHz, CD3OD).

[0040] Figure 8 It is the chemical structural formula of a monomeric compound.

[0041] Figure 9 The figure shows that compounds 1 and 2 (100 μg / mL) had no effect on tobacco seeds and growth and development.

[0042] Figure 10 The phenotypic diagram and inhibition rate of different concentrations of monomeric compound 3 on the germination rate of tobacco seeds are shown.

[0043] Figure 11 Phenotypic diagram and inhibition rate of different concentrations of monomeric compound 3 on root length of tobacco growth and development.

[0044] Figure 12 This is a distribution map of GO functional annotations for differentially expressed genes. A represents the control group, B represents the treatment group with 50 μg / mL compound 3, and C represents the treatment group with 100 μg / mL compound 3.

[0045] Figure 13 This is a graph showing the enrichment analysis of differentially expressed genes via the KEGG pathway.

[0046] Figure 14 This is a graph showing the changes in the content of differentially metabolite categories. A represents the control group (Control), B represents the treatment group with 50 μg / mL compound 3, and C represents the treatment group with 100 μg / mL compound 3.

[0047] Figure 15 This is a schematic diagram of a gene-metabolite interaction network.

[0048] Figure 16 This is a bar chart showing the results of qRT-PCR validation of key regulatory genes. Detailed Implementation

[0049] The technical approach of this invention is as follows:

[0050] (1) Preparation, separation and purification of monomeric compounds

[0051] by Nodulisporium sp. Using strain EY-2-4 as the target strain, fermentation broth was obtained through liquid fermentation. Crude extract was prepared by ethyl acetate extraction, and a stepwise purification strategy of "silica gel column chromatography for coarse separation and gel column chromatography for fine separation" was employed to obtain high-purity monomeric compounds. The specific steps are as follows:

[0052] Fermentation by strain: Take Nodulisporium sp.The slant culture of strain EY-2-4 was inoculated into potato glucose broth medium and cultured in a shaker at 28°C and 120 rpm for 7 days to obtain the fermentation broth.

[0053] Preparation of crude extract: The fermentation broth was filtered through gauze to remove mycelium. The filtrate was extracted three times with an equal volume of ethyl acetate. The extracts were combined and concentrated to dryness under reduced pressure at 45℃ and 0.08 MPa to obtain crude extract paste.

[0054] High-performance liquid chromatography (HPLC) was used to analyze the chromatograms of secondary metabolites from the crude ethyl acetate extract of the strain. The chromatographic conditions were as follows: C18 semi-preparative column, detection wavelength 254 nm, and linear gradient elution with a methanol-water system. The chromatogram showed multiple characteristic absorption peaks; after subsequent separation, purification, and structural analysis, three main characteristic peaks (retention time t) were identified. R The corresponding compounds 1-3 were 6.68 min, 7.08 min, and 7.71 min, respectively. Figure 1 ).

[0055] Compound structure identification was completed by combining systematic spectral analysis: the assignment of ¹H NMR and ¹³C NMR data is summarized in Table 1, and the relevant spectra are detailed in the supporting information diagram (). Figure 2-7 ).

[0056] (2) Structural identification of monomeric compounds

[0057] The compound structure was determined using a combination of multiple spectroscopic techniques, as detailed below:

[0058] Sample preparation: The purified compound 3 was dissolved in methanol to prepare a sample solution of appropriate concentration;

[0059] Structural confirmation: By comparing the spectral data of the compound with a database of known compounds, the chemical structure of this monomeric compound was determined as follows: ; Compound 3

[0060] (3) Verification of the bioactivity of monomeric compounds

[0061] With tobacco ( Nicotiana tabacum L. SR1) was used as a model plant to conduct bioactivity tests and clarify the growth-inhibiting effects of the compounds:

[0062] Experimental design: The concentrations of monomer compound 3 were set at 0 μg / mL (0.05% DMSO, control group), 50 μg / mL, and 100 μg / mL, with 3 biological replicates in each group;

[0063] Tobacco seed germination inhibition test: Sterilized tobacco seeds were evenly sown on MS medium, sprayed with different concentrations of compound 3 solution, and cultured at 25℃ for 7 days under 16 h light / 8 h dark conditions. Seed germination rate was counted, root length was measured, and inhibition rate was calculated.

[0064] Results: Compound 3 showed significant inhibitory activity at a concentration of 50 μg / mL, with the best effect at a concentration of 100 μg / mL: the germination rate of tobacco seeds was inhibited by 73.06% compared with the control, and the root length was significantly shortened by 51.67% (p<0.001); and the inhibitory effect showed a clear concentration dependence with increasing concentration.

[0065] (4) Analysis of the mechanism of action of monomeric compounds

[0066] The mechanism of action of the compounds was systematically elucidated using a combined transcriptome-metabolome analysis and qRT-PCR validation strategy.

[0067] Transcriptome analysis: Three treatment groups (100 μg / mL compound 3) and a control group (0.05% DMSO) were set up. Samples were collected from tobacco seedlings after treatment (3 biological replicates) and transcriptome sequencing was performed. The results showed that a total of 3687 differentially expressed genes (DEGs) were identified in the treatment groups, of which 1421 genes were upregulated and 2266 genes were downregulated. GO functional annotation and KEGG pathway enrichment analysis showed that the differentially expressed genes were mainly enriched in auxin signal transduction, phenylpropanoid metabolism, and energy metabolism-related pathways.

[0068] Metabolomics analysis: Non-targeted metabolomics analysis was performed on tobacco seedlings in the treatment group and the control group, and 238 differential metabolites were identified. Among them, the changes in flavonoid and auxin-related metabolites were the most significant, and the accumulation levels of secondary metabolites such as terpenoid alcohols, lipids, oxygenated organic compounds and terpenes were significantly altered.

[0069] Multi-omics integrated analysis: a gene-metabolite interaction network was constructed, 17 core regulatory nodes were screened, and pathway topology analysis showed that the core modules were concentrated in the flavonoid biosynthesis pathway, confirming that the phenylpropane biosynthesis pathway is a key metabolic hub for the regulation of plant growth by compounds.

[0070] qRT-PCR validation: Targeted validation of key candidate genes was performed, and the results showed: PIP2-1 Gene (sphingolipid signaling pathway) expression was significantly upregulated with increasing concentration of compound 3; ITPK1-like High expression of the gene (phenylpropanoid metabolism pathway) promotes the accumulation of phenylpropanoids; RGL3-like Downregulation of gene expression (auxin signaling pathway) leads to weakened auxin signaling;

[0071] Mechanism summary: Compound 3 specifically interferes with the biosynthesis of phenylpropane compounds, regulates auxin signal transduction and energy metabolism pathways, affects the accumulation of key secondary metabolites such as lignin and flavonoids, disrupts cell wall structural integrity and redox balance, downregulates the expression of growth-related genes, and ultimately inhibits plant seed germination and root growth and development.

[0072] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0073] Example 1:

[0074] one, Nodulisporium Isolation, purification and identification of sp. EY-2-4

[0075] The endophytic fungus strain EY-2-4 (GenBank ID: PX870240) used in this study was isolated from the leaves of *Taxus chinensis* collected in Songshan Lake, Dongguan City, Guangdong Province in October 2024. ITS sequence analysis, BLAST alignment, and homology analysis confirmed that this strain is... Nodulisporium sp., named Nodulisporium sp. EY-2-4 (hereinafter referred to as strain EY-2-4).

[0076] two, Nodulisporium Liquid fermentation of sp. EY-2-4

[0077] Liquid fermentation: The purified strain was inoculated into a 1000 mL Erlenmeyer flask containing 400 mL of potato glucose broth (composition: 200 g / L potato, 20 g / L glucose, 3 g / L K2HPO4, 1.5 g / L MgSO4·7H2O, and 10 mg / L vitamin B1, with the remainder being water, natural pH, and autoclaved at 121℃ for 20 min). The flask was incubated with shaking at 28℃ and 120 rpm for 7 days.

[0078] Obtaining the crude extract: Following the fermentation method described above... Nodulisporium Sp. EY-2-4 was fermented, and the fermentation broth (5 L in total) was collected. The mycelium was removed by filtration through 5 layers of gauze to obtain the filtrate. The filtrate was extracted three times with an equal volume of ethyl acetate (EtOAc), shaking for 30 min each time. After standing and separating the layers, the upper ethyl acetate organic phase was collected. The organic phases were combined and concentrated to dryness under reduced pressure in a rotary evaporator (45℃, 0.08 MPa) to obtain 0.35 g of crude extract.

[0079] High-performance liquid chromatography (HPLC) was used to analyze the chromatograms of secondary metabolites from the crude ethyl acetate extract of the strain. The chromatographic conditions were as follows: Aters XBridge C18 semi-preparative column (5 μm, 10 mm × 250 mm), detection wavelength 254 nm, linear gradient elution of methanol (A)-water (B): 0–2 min, 70% A; 2–9 min, 70%–85% A; 9–12 min, 85%–100% A, 2.0 mL·min. -1 Column temperature 30℃. The chromatogram showed multiple characteristic absorption peaks; after subsequent separation, purification, and structural analysis, three main characteristic peaks were identified (retention time t). R The corresponding compounds 1-3 were 6.68 min, 7.08 min, and 7.71 min, respectively. Figure 1 ).

[0080] III. Preparation of Compounds 1-3

[0081] Isolation and purification of compounds: The crude extract (0.35 g) was treated by silica gel column chromatography (CC) using a gradient elution system of n-hexane-ethyl acetate (100:1 → 1:3, v / v). Fraction Fr. 1, obtained by elution of n-hexane-ethyl acetate at a volume ratio of 5:1 and developed by TLC at a ratio of n-hexane:ethyl acetate = 2:1 v / v, yielded an Rf of 0.4-0.5; fraction Fr. 2, obtained by elution of n-hexane-ethyl acetate at a volume ratio of 2:1 and developed by TLC at a ratio of n-hexane:ethyl acetate = 2:1 v / v, yielded an Rf of 0.5; and fraction Fr. 3, obtained by elution of n-hexane-ethyl acetate at a volume ratio of 1:1 and developed by TLC at a ratio of n-hexane:ethyl acetate = 1:1 v / v, yielded an Rf of 0.6.

[0082] Fr. 1 (0.1 g) was initially separated by Sephadex LH-20 gel chromatography (eluent: chloroform / methanol, 1:3, v / v), and further purified by reversed-phase semi-preparative high-performance liquid chromatography using a YMC pack ODS-A / AQ column with elution conditions of acetonitrile / water, 20:30, v / v, 2 mL / min, yielding compound 1 (0.9 mg, t). R = 30.0 min), TLC thin-layer chromatography was performed with n-hexane:ethyl acetate = 2:1 (v / v) and Rf = 0.5-0.6.

[0083] Fr. 2 (0.13 g) was separated into two subfractions, Fr. 2.1 (i.e., compound 3, developed by TLC with hexane:ethyl acetate = 2:1, v / v, Rf = 0.4) and Fr. 2.2 (developed by TLC with hexane:ethyl acetate = 2:1, v / v, Rf = 0.5-0.6).

[0084] Subfraction Fr. 2.2 (0.07 g) was purified by Sephadex LH-20 and semi-preparative HPLC using a YMC pack ODS-A / AQ column under the following elution conditions (acetonitrile / water, 20:30, v / v, 2 mL / min) and a detection wavelength of 254 nm to give compound 2 (2.0 mg, t R = 45.0 min).

[0085] IV. Structural Identification of Compound 3

[0086] In this invention, 1 H NMR, 13 C NMR, HMBC and other nuclear magnetic resonance spectra were all measured using a Bruker Advance-500 nuclear magnetic resonance spectrometer, with tetramethylsilane (TMS) as an internal standard.

[0087] like Figure 2-7 As shown, Figure 2 It is compound 1 1 H-NMR spectrum; Figure 3 It is compound 1 13 C-NMR spectrum; Figure 4 It is compound 2. 1 H-NMR spectrum; Figure 5 It is compound 2. 13 C-NMR spectrum; Figure 6 It is compound 3. 1 H-NMR spectrum; Figure 7 It is compound 3. 13 C-NMR spectroscopy; combined analysis using nuclear magnetic resonance (¹H-NMR, ¹³C-NMR) and mass spectrometry, along with comparison with compound databases, determined the chemical structures of the compounds. The chemical structural formulas of monomer compounds 1-3 are as follows: Figure 8 As shown in equation (II), the attribution of ¹H NMR and ¹³C NMR data is summarized in Table 1.

[0088] Compound 3: Molecular formula C 10 H 10 O5, a white powder. (Through...)13 C NMR data (Table 1), with a total of 10 carbon signals: δ C 201.5 (C-1) shows the presence of a conjugated ketone carbonyl group; 6 aromatic carbon signals: δ C 167.1 (C-8) and 148.5 (C-10) are highly deshielded quaternary carbons, suggesting the presence of oxygen-containing groups, δ C 110.4 (C-9) and 166.4 (C-6) are quaternary carbons, δ C 108.4 (C-5) and 102.5 (C-7) are two aromatic carbons with two hydrogen atoms; there are three aliphatic carbon signals: δ C 73.7 (C-4) and 71.8 (C-3) are hydroxymethyl groups, δ C 44.3 (C-2) is a methylene group. Compound 3 is largely consistent with the data reported in the literature, and is identified as 4-hydroxyscytalone.

[0089] ; Formula (II)

[0090] Example 2: Bioactivity testing of monomeric compounds

[0091] Tobacco Seed Germination Inhibition Test: Test Materials: Tobacco seeds ( Nicotiana tabacum L. SR1 The seeds were sterilized with 75% ethanol for 30 seconds, rinsed three times with sterile water, and air-dried for later use. Treatment settings: monomeric compounds 1-3 were at concentrations of 0 μg / mL (CK, 0.05% DMSO, v / v), 50 μg / mL (0.05% DMSO, v / v), and 100 μg / mL (0.05% DMSO, v / v), with three replicates per group and 100 seeds per replicate. Experimental method: Seeds were evenly sown on MS solid medium, sprayed with different concentrations of monomeric compound solutions, and cultured for 7 days in a 25℃, 16-h light / 8-h dark artificial climate chamber. Results: Compounds 1 and 2 (100 μg / mL) had no effect on tobacco seeds or growth and development. Figure 9 The seed germination rate of the CK group was 95.7%, while the germination rates of the 50 μg / mL and 100 μg / mL monomeric compound 3 treatment groups were 68.2% and 24.6%, respectively, with inhibition rates of 28.7% and 73.06%, respectively. Figure 10 The root length of the CK group was 2.4 cm, while the root lengths of the 50 μg / mL and 100 μg / mL treatment groups were 1.5 cm and 1.1 cm, respectively. Figure 11 The inhibition rates were 34.4% and 51.67% (p<0.001), respectively, showing a significant concentration-dependent inhibitory effect, confirming that monomer compound 3 has a significant inhibitory effect on tobacco growth.

[0092] Example 3: Mechanism of Action of Monomer Compound 3

[0093] Transcriptome analysis: Sample preparation: Tobacco seedlings from the 100 μg / mL monomer compound 3 treatment group and the CK group, cultured for 7 days as described in Example 3, were used. Three biological replicates were performed for each group, and the samples were flash-frozen in liquid nitrogen and stored. Sequencing and analysis: Total RNA was extracted, and transcriptome sequencing was performed using the Illumina NovaSeq 6000 platform. Differentially expressed genes (DEGs) were screened, and GO functional annotation and KEGG pathway enrichment analysis were conducted. Results: A total of 3687 DEGs were identified, of which 1421 were upregulated and 2266 were downregulated. The differentially expressed genes were significantly enriched in pathways related to auxin signal transduction, phenylpropanoid metabolism, and energy metabolism. Figure 12-13 ).

[0094] Metabolomics Analysis: Sample Processing: Same as transcriptomics analysis; Detection and Analysis: Untargeted metabolomics analysis was performed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) to identify differentially expressed metabolites and annotate their categories; Results: A total of 238 differentially expressed metabolites were identified, among which flavonoids and auxin-related metabolites showed the most significant changes, and the accumulation levels of terpenoids, lipids, oxygenated organic compounds, and terpenoid secondary metabolites were significantly altered. Figure 14 ).

[0095] Multi-omics integrated analysis: A gene-metabolite interaction network was constructed, and 17 core regulatory nodes were screened. Pathway topology analysis showed that the core modules were concentrated in the flavonoid biosynthesis pathway, confirming that the phenylpropane biosynthesis pathway is a key metabolic hub for the regulation of plant growth by monomeric compound 3. Figure 15 ).

[0096] qRT-PCR validation: Candidate genes: selected PIP2-1 , ITPK1-like , RGL3-likeAs candidate genes; Experimental methods: Total RNA was extracted from tobacco seedlings (7 days after culture in Example 3) treated with different concentrations of monomeric compounds (NEBMonarch Total RNA Miniprep Kit), and cDNA was synthesized by reverse transcription (PrimeScript RT Reagent Kit (Vazyme, China)). qRT-PCR was then performed. The primer sequences used for qPCR are shown in Table 2. Real-time quantitative reverse transcription PCR (qRT-PCR) was performed on a CFX96 real-time quantitative PCR instrument (Bio-Rad, USA). The reaction was performed using the TaKaRa SYBR Premix Ex Taq II kit (Japan). The qRT-PCR reaction program was: 95℃ pre-denaturation for 30 s; 40 amplification cycles (95℃ 5 s, 60℃ 30 s). Melting curve analysis was performed after the reaction to confirm amplification specificity. Results ( Figure 16 PIP2-1 gene expression was significantly upregulated with increasing concentration of monomer compound 3, with the expression level in the 100 μg / mL treatment group being 35 times that in the CK group; the expression level of ITPK1-like gene in the 100 μg / mL treatment group was 42 times that in the CK group; and the expression level of RGL3-like gene in the 100 μg / mL treatment group was 0.28 times that in the CK group, consistent with the transcriptome results.

[0097] Mechanistic conclusion: Monomer compound 3 specifically regulates the phenylpropane biosynthesis pathway, auxin signal transduction pathway, and energy metabolism-related pathways, upregulates the expression of PIP2-1 and ITPK1-like genes, downregulates the expression of RGL3-like genes, interferes with plant hormone homeostasis and secondary metabolic flux, and disrupts cell wall structural integrity and redox balance, thereby inhibiting plant seed germination and root growth and development.

[0098] Table 1. 1H and 1C NMR spectra of compounds 1–3 in deuterated methanol .

[0099] Table 2 Primer sequences used for qPCR .

Claims

1. Nodulisporium The application of strain sp. EY-2-4 in the preparation of monomeric compounds, characterized in that, The structural formula of the monomer compound is shown in formula (Ⅰ): ； Equation (Ⅰ).

2. The method for preparing the monomeric compound according to claim 1, characterized in that, Includes the following steps: Depend on Nodulisporium sp The .EY-2-4 strain was obtained through fermentation and purification of crude extract.

3. The preparation method according to claim 2, characterized in that, The specific steps are as follows: 1) Fermentation by strain: Nodulisporium sp. The EY-2-4 strain was inoculated into potato glucose broth medium and cultured in a constant temperature shaker at 28°C and 120 rpm for 7 days to obtain the fermentation broth. 2) Obtaining crude extract: After filtering the fermentation broth through 5 layers of gauze, the filtrate was extracted with an equal volume of ethyl acetate 3-4 times. The ethyl acetate layers were combined and concentrated to dryness under reduced pressure to obtain crude extract paste. 3) Separation and purification of monomeric compounds: The crude extract was separated by silica gel column chromatography using a gradient elution system of n-hexane:ethyl acetate at a volume ratio of 100:1 → 1:

3. The fraction obtained by elution with n-hexane:ethyl acetate at a volume ratio of 2:1 and developed by TLC at a volume ratio of n-hexane:ethyl acetate at 2:1, yielding an Rf of 0.5, was collected. This fraction was then processed by silica gel column chromatography using a volume ratio of n-hexane:ethyl acetate at 20:1 → 1:

3. The eluted product was then purified by Sephadex LH-20 gel chromatography with chloroform:methanol at a volume ratio of 1:

3. The fraction with an Rf of 0.4 when developed by TLC at a volume ratio of n-hexane:ethyl acetate at 2:1 was collected, which is the target monomeric compound.

4. Application of monomeric compounds in regulating plant growth; The structural formula of the monomeric compound is as follows: ； Equation (Ⅰ).

5. The application according to claim 4, characterized in that, For the use of monomeric compounds in any of the following: 1) Application in inhibiting seed germination and / or seedling root growth; 2) Regulates the phenylpropane biosynthesis pathway, auxin signal transduction pathway, and energy metabolism-related pathways; 3) Applications in upregulating the expression of PIP2-1, a gene related to the sphingolipid signaling pathway; upregulating the expression of ITPK1-like, a gene related to the phenylpropanone metabolism pathway; and / or downregulating the expression of RGL3-like, a gene related to the auxin signaling pathway. 4) Applications in interfering with plant hormone homeostasis and secondary metabolic flux, altering the accumulation levels of terpenoids, lipids, flavonoids, and lipid secondary metabolites, and disrupting cell wall structural integrity and redox balance.

6. The application of monomeric compounds in agricultural production or horticultural cultivation, characterized in that, Used to control excessive crop growth, dwarf plants, regulate flowering time, and / or improve plant stress resistance; The structural formula of the monomeric compound is as follows: ； Equation (Ⅰ).

7. Application of monomeric compounds in the preparation of plant growth inhibitors; The structural formula of the monomeric compound is as follows: ； Equation (Ⅰ).

8. The application according to any one of claims 4-7, characterized in that, The plant in question is either tobacco or Arabidopsis thaliana.

9. A plant growth inhibitor, characterized in that, The active ingredient is a monomeric compound; The structural formula of the monomeric compound is as follows: ； Equation (Ⅰ).

10. The plant growth inhibitor according to claim 9, characterized in that, The plant growth inhibitor is prepared by mixing the monomeric compound described in claim 1 with an agriculturally acceptable carrier or adjuvant, and is formulated as an aqueous solution, powder, emulsifiable concentrate or suspension.

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