Application of baeospora purpurea metabolites in plant growth regulation

Abstract

The application belongs to the technical field of biology, and particularly discloses application of a metabolism product of a yellow-cap small brittlegill in plant germination and growth regulation. The metabolism product is obtained through fermentation, and can not only promote germination of rhynchostylis retusa seeds, but also regulate growth of white and arabidopsis thaliana, and presents plant hormone-like effects and plant universality. The application provides a new method and application potential for plant growth regulation, and has important ecological and economic values.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of metabolites of *Pleurotus ostreatus* in the regulation of plant seed germination and growth. Background Technology

[0002] Fungi are among the most diverse and functionally varied groups of organisms in the microbial kingdom, and their natural products occupy an important position in ecosystems and scientific research. Orchids are one of the world's most precious plant resources, possessing significant economic and ecological value. However, because orchid seeds lack endosperm and possess only undifferentiated proembryos, they are extremely difficult to germinate under natural conditions, severely restricting their population expansion and artificial cultivation. The rare and medicinal orchid, *Cremastra appendiculata*, is a typical example; its seeds are slender, lack endosperm, and are difficult to germinate under natural conditions. Research shows that most orchid seeds rely on a symbiotic relationship with specific fungi to germinate. Fungi provide nutrients and produce signaling molecules through mycorrhizae, regulating seed germination and seedling growth.

[0003] In plant growth regulation research, there exists an important class of terpenoid compounds—gibberellin (GA). As a tetracyclic diterpenoid compound, it plays an important regulatory role in plant growth and development, such as promoting seed germination, stem elongation, parthenocarpy, inhibiting maturation, lateral bud dormancy, and senescence.

[0004] The inventors of this application isolated several novel guanacastane-type diterpenes from *Psathyrella candolleana* (Fr.) AH Smith, now known as *Candolleomyces candolleanus* (Fr.) D. Wächt. & A. Melzer). These are diterpenoid compounds with a unique 5 / 7 / 6 tricyclic skeleton, significantly different from gibberellins with a pentacyclic structure. Existing research on guanacastane-type diterpenes mainly focuses on the analysis of their chemical structures and the evaluation of their pharmacological activities. Whether guanacastane-type diterpenes play a role in plant growth and development has not yet been reported, and their application potential in fungal-plant symbiotic systems remains an unknown area. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide the application of *Pleurotus ostreatus* metabolites in plant germination and growth regulation: *Pleurotus ostreatus* metabolites are prepared by fermentation; the metabolites are co-cultured with *Rhododendron simsii* seeds to verify the germination-promoting effect of *Pleurotus ostreatus* metabolites on *Rhododendron simsii* seeds; and the hormone-like effects of *Pleurotus ostreatus* metabolites are universally verified by co-culturing with *Bletilla striata* and *Arabidopsis thaliana*.

[0006] When *Agaricus bisporus* seeds were co-cultured with *Auricularia auricula-judae* seeds, the seeds only swelled to a certain extent and remained at that stage, failing to germinate completely. However, the inventors of this application discovered that its metabolites have a significant effect on promoting *Auricularia auricula-judae* seed germination, and also have growth and development regulatory effects on plants such as *Bletilla striata* and *Arabidopsis thaliana*. Through systematic screening and functional verification, this invention reveals for the first time the potential of this type of metabolite (mycelium powder, fermentation broth, ethyl acetate extract, guanacastane-type diterpenes and their derivatives) in regulating plant growth and development.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides the application of metabolites from Candolleomyces candolleanus (Fr.) D. Wächt. & A. Melzer in regulating plant growth, wherein the metabolites are prepared by fermentation.

[0008] Furthermore, the regulation of plant growth includes regulating plant seed germination, regulating plant true leaf growth, regulating plant height, and / or regulating plant stem diameter.

[0009] Furthermore, the plant is one of the following: Rhododendron, Bletilla striata, or Arabidopsis thaliana.

[0010] Furthermore, the metabolites of *Pleurotus ostreatus* are selected from one or more of the following: mycelium obtained from fermentation of *Pleurotus ostreatus*, fermentation broth, ethyl acetate extract, and monomeric compounds.

[0011] Furthermore, the fermentation broth and mycelium are prepared by the following method: the activated strain of *Pleurotus ostreatus* is fermented in a liquid culture medium (composition: glucose 50 g / L, peptone 5 g / L, yeast extract 1.5 g / L, KH2PO4 0.5 g / L, MgSO4 0.5 g / L) at 160 rpm and incubated in the dark at 25°C for 30 days; after fermentation, the solid-liquid mixture of the fermentation system is separated by centrifugation to obtain the fermentation broth and mycelium respectively.

[0012] Furthermore, the ethyl acetate extract is an ethyl acetate extract of mycelium (obtained by concentrating the acetone extract of mycelium to remove acetone, dissolving it in water, and then extracting it with ethyl acetate) and / or an ethyl acetate extract of fermentation broth.

[0013] Furthermore, the monomeric compound is a guanacastane-type diterpene.

[0014] Furthermore, the guanacastane-type diterpenoid is a compound called guanacastanein, with the structural formula shown in Formula 1 below:

[0015] The compound, sclerotin, was prepared by the following method: the ethyl acetate extract of mycelium and the ethyl acetate extract of fermentation broth were combined and concentrated to obtain a total extract; the total extract was dissolved in methanol and then purified by normal phase column chromatography, medium-pressure liquid chromatography, normal phase column chromatography, and high-performance liquid chromatography to obtain the compound.

[0016] Furthermore, the application includes the following steps: (1) Dissolve the metabolites of *Pleurotus ostreatus* in a solvent (preferably anhydrous ethanol) to prepare a metabolite solution; (2) Mix the solution obtained in step (1) with the co-culture medium, sterilize it to obtain the metabolite culture medium, wherein the concentration of the metabolite is 0.2~5 mg / L; (3) The plant is cultured in the metabolite culture medium obtained in step (2).

[0017] Furthermore, the co-culture medium mentioned in step (2) is oat agar medium or 1 / 2 MS medium.

[0018] Furthermore, in step (2), the concentration of the metabolite in the metabolite culture medium is 1 mg / L.

[0019] This invention also provides a plant growth regulator comprising the metabolites of the *Pleurotus ostreatus* and acceptable excipients. The plant growth regulator includes liquid sprays, bio-fertilizers, microbial inoculants, etc., and can be prepared using methods well known in the art.

[0020] Compared with the prior art, the advantages and beneficial effects of the present invention are: This invention provides the application of the metabolites of *Pleurotus ostreatus* in the regulation of plant seed germination and growth. These metabolites can not only promote the germination of *Cymbidium goeringii* seeds, but also regulate the growth of *Bletilla striata* and *Arabidopsis thaliana*, exhibiting plant hormone-like effects and plant universality, showing great application potential as a novel plant hormone substance.

[0021] Experiments have shown that the metabolites of *Pleurotus ostreatus* have a germination-promoting effect on *Rhododendron simsii* seeds. The best germination-promoting effect was observed in 1 / 2 MS medium containing 1 mg / L ethyl acetate extract. After 60 days of culture on oat agar (OMA) medium containing 1 mg / L ethyl acetate extract, the seeds swelled and formed white protocorms, with a germination rate of 4.87%. After 60 days of culture on 1 / 2 MS medium containing 1 mg / L ethyl acetate extract, the seeds swelled and formed white protocorms, with a germination rate of 18.62%.

[0022] Meanwhile, the metabolites of *Pleurotus ostreatus* var. *flavovirens* regulated the growth and development of *Bletilla striata* plants, and most seeds germinated and formed green plants after 60 days. The ethyl acetate extract (1 mg / L) treatment group showed the best performance, with plant height of 2.56-5.32 mm and diameter of 0.28-0.75 mm.

[0023] Meanwhile, the metabolites of *Pleurotus ostreatus* showed a concentration-dependent plant hormone-like effect on *Arabidopsis thaliana*. After adding different concentrations of ethyl acetate extract (0 mg / L, 0.2 mg / L, 1 mg / L, 5 mg / L) to 1 / 2 MS medium, it was observed that the low concentration (0.2 mg / L-1 mg / L) treatment group promoted the growth of *Arabidopsis thaliana*, while the high concentration (5 mg / L) treatment group inhibited the growth of *Arabidopsis thaliana*, exhibiting a plant hormone-like biological effect. Attached Figure Description

[0024] To more clearly illustrate the technical solution of the present invention, the applicant will briefly introduce the drawings used in the description of the embodiments or the prior art 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.

[0025] Figure 1 The images show the co-culture of *Pleurotus ostreatus* metabolites and *Cymbidium goeringii* seeds on OMA and 1 / 2 MS media (60 days) in Examples 1 and 2. Figure 2 The growth and development diagrams of Bletilla striata seeds obtained by co-culturing in Example 3 (30 days and 60 days); Figure 3 This is a diagram showing the co-culture of *Pleurotus ostreatus* metabolites and *Arabidopsis thaliana* seeds on 1 / 2 MS medium (7 days) in Example 4. Detailed Implementation

[0026] The applicant below combines Figures 1-3The technical solutions in the embodiments of the present invention are clearly and completely described, and the present invention is described in detail. These embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection claimed by the present invention.

[0027] The metabolites of *Pleurotus eryngii* used in the following examples were obtained by fermentation; this invention provides a metabolite of *Pleurotus eryngii* that promotes seed germination and regulates plant growth and development; through co-culture experiments with *Rhododendron simsii* seeds, it was found that the metabolite of *Pleurotus eryngii* can promote the germination of *Rhododendron simsii* seeds, while regulating the growth and development of plants such as *Bletilla striata* and *Arabidopsis thaliana*.

[0028] The inventors of this application, through systematic isolation and functional verification, obtained a series of metabolites from *Agaricus flavomarginata*, including mycelial powder, fermentation broth, ethyl acetate extract, and guanacastane-type diterpenes and their derivatives. Studies have found that these metabolites exhibit significant promoting effects on plant growth and development, demonstrating great application potential. The fermentation broth can be formulated into a liquid spray for spraying plant leaves to enhance plant resistance; the mycelial powder can be formulated into a bio-fertilizer to improve the soil microbial environment; and the guanacastane-type diterpenes and their derivatives can be formulated into biostimulants, stress-resistant agents, and microbial inoculants to improve plant quality. In the pharmaceutical field, there are reports of using this type of diterpenes and their derivatives as analgesics. In summary, the metabolites of *Agaricus flavomarginata* show promising development prospects.

[0029] Example 1: Co-culture experiment of metabolites of *Pleurotus ostreatus* and seeds of *Rhododendron simsii* in basal medium 1-oat agar. The specific steps are as follows: 1. Materials and Methods 1.1 Ethyl acetate extract In this embodiment, the metabolites of *Candolleomyces candolleanus* (Fr.) D. Wächt. & A. Melzer (strain preserved at the School of Pharmacy, South-Central University for Nationalities, Wuhan, Hubei Province) were prepared by fermentation. The preparation method is as follows: The preserved *Candolleomyces candolleanus* was inoculated onto potato dextrose agar (PDA) medium and cultured at 28°C for 7 days. Then, the mycelial blocks were inoculated onto a second medium for fermentation at 160 rpm in the dark at 25°C for 30 days. The second medium used distilled water as a solvent and consisted of: 50 g / L glucose, 5 g / L peptone, 1.5 g / L yeast extract, 0.5 g / L KH₂PO₄, and 0.5 g / L MgSO₄. The fermentation broth and mycelium were separated by centrifugation. The fermentation broth was concentrated under reduced pressure and extracted five times with ethyl acetate. The ethyl acetate layers were combined and concentrated under reduced pressure to obtain the fermentation extract. The mycelium was extracted five times with acetone, each time for 12 hours. The five acetone extracts were combined, concentrated under reduced pressure to remove the acetone, dissolved in water, and extracted five times with ethyl acetate. The ethyl acetate layers were combined and concentrated under reduced pressure to obtain the mycelial extract. The fermentation broth and mycelial extract were combined to obtain the total extract, i.e., the metabolite.

[0030] 1.2 Culture medium Weigh 1 mg of the metabolite and dissolve it in 1 mL of anhydrous ethanol to prepare a 1 mg / mL (1000 mg / L) stock solution. The stock solution is clear and pale yellow. Take 0 mL, 0.02 mL, 0.1 mL, and 0.5 mL of the stock solution respectively and add them to 100 mL of the following basal culture medium 1 to prepare extract media with concentrations of 0 mg / L, 0.2 mg / L, 1 mg / L, and 5 mg / L. Sterilize at 121 °C for 20 min, then remove and shake well. Pour the culture medium, which has been cooled to about 40 °C, into sterile petri dishes and allow it to solidify before storing for later use.

[0031] Basic culture medium 1 - Oat agar medium (OMA) is prepared as follows: Weigh 3 g of oat flour (Solarbio, Cat#FA0280), add an appropriate amount of water, boil for 30 min, then add 15 g of agar powder, stir and mix well, cool slightly, and then add water to make up to 1000 mL.

[0032] 1.3 Plant materials The rhododendron seed solution used in this embodiment was prepared by surface sterilization of mature rhododendron pods that had not yet cracked: the pods were surface sterilized with 75% (volume percentage, the same below, not repeated) ethanol for 30 seconds, and rinsed three times with distilled water; then surface sterilized with 0.1% mercuric chloride for 5 minutes, and rinsed three times with distilled water; then surface sterilized with 2% sodium hypochlorite for 1 minute, and rinsed five times with sterile water. The sterilized pods were then opened to release the seeds, which were stored in a 0.1% sterile water agar suspension for later use.

[0033] 1.4 Co-culture Open the fully prepared extract culture medium in step 1.2 on a clean bench. Use a sterile dropper to draw 2 mL of the sterilized Rhododendron seed solution obtained in step 1.3 and evenly disperse it on the surface of the culture medium. Divide the medium into three dishes for each concentration, seal the dishes with sealing film, and incubate them in an artificial climate chamber.

[0034] 1.5 Seed germination Seed morphological changes were observed regularly under a stereomicroscope and photographed. After 60 days of culture, it was found that the seeds in the blank group (OMA group, 0 mg / L extract medium) did not germinate, while the seeds in the ethyl acetate extract treatment group (OMA+B-1 group, 1 mg / L extract medium) showed the best germination, with seeds swelling and forming white protocorms (see...). Figure 1 The germination rate was calculated to be 4.87%, and no germination was observed in the Rhododendron seeds treated with 0.2 mg / L and 5 mg / L extract culture media.

[0035] Example 2: Co-culture experiment of *Pleurotus ostreatus* metabolites and *Rhododendron simsii* seeds in basal medium 2-1 / 2 MS medium. The specific steps are as follows: 1. Materials and Methods 1.1 Ethyl acetate extract Same as Example 1.

[0036] 1.2 Culture medium Weigh 1 mg of the metabolite and dissolve it in 1 mL of anhydrous ethanol to prepare a 1000 mg / L stock solution. Take 0 mL, 0.02 mL, 0.1 mL, and 0.5 mL of the stock solution respectively, add them to 100 mL of the following basal culture medium 2 to prepare extract media with concentrations of 0 mg / L, 0.2 mg / L, 1 mg / L, and 5 mg / L. Sterilize at 121 °C for 20 min, then remove and shake well. Pour the culture medium, which has been cooled to about 40 °C, into sterile petri dishes and store it for later use after solidification.

[0037] The basal medium 2-1 / 2 MS medium is prepared as follows: Weigh 1.24 g of 1 / 2 MS (Hangzhou Best Biotechnology Co., Ltd., BS 3004), 10 g of sucrose, 3.75 g of activated carbon, and 3.75 g of agar, add water to 480 mL, and adjust the pH to 7.10 with NaOH solid particles.

[0038] 1.3 Plant materials Same as Example 1.

[0039] 1.4 Co-culture Same as Example 1.

[0040] 1.5 Seed germination Seed morphological changes were observed regularly under a stereomicroscope, and photographs were taken for recording. After 60 days of cultivation, it was observed that the Rhododendron seeds swelled and formed white protocorms (see...). Figure 1 ), calculate its germination rate on 1 / 2 MS medium.

[0041] The germination rate of Rhododendron seeds in the 1 / 2 MS group (0 mg / L extract medium) was 9.45%.

[0042] The seed germination rate of Rhododendron simsii in the 1 / 2 MS+B-0.2 group (0.2 mg / L extract medium) was 16.75%.

[0043] The seed germination rate of Rhododendron simsii in the 1 / 2 MS+B-1 group (1 mg / L extract medium) was 18.62%.

[0044] The seed germination rate of Rhododendron simsii in the 1 / 2 MS+B-5 group (5 mg / L extract medium) was 14.37%.

[0045] The results showed that, compared with the control group (1 / 2 MS group), the addition of different concentrations of *Pleurotus eryngii* metabolites (ethyl acetate extract) to the 1 / 2 MS medium significantly improved the germination rate of *Rhododendron simsii* seeds. Among them, the treatment with 1 mg / L of *Pleurotus eryngii* metabolites (ethyl acetate extract) resulted in the highest germination rate of *Rhododendron simsii* seeds.

[0046] Example 3: The regulatory effect of *Pleurotus eryngii* metabolites on the growth and development of *Bletilla striata*, the specific steps of which are as follows: 1. Materials and Methods 1.1 Ethyl acetate extract Same as Example 1.

[0047] 1.2 Culture medium Same as Example 2.

[0048] 1.3 Plant materials The Bletilla striata seed solution used in this embodiment was prepared by surface sterilization of mature Bletilla striata pods that had not been cracked: surface sterilization with 75% ethanol for 30 s, followed by rinsing three times with distilled water; then surface sterilization with 0.1% mercuric chloride for 5 min, followed by rinsing three times with distilled water; then surface sterilization with 2% sodium hypochlorite for 1 min, followed by rinsing five times with sterile water. The sterilized pods were then opened to release the seeds, which were then stored in a 0.1% sterile water agar suspension for later use.

[0049] 1.4 Co-culture Open the fully prepared extract culture medium in step 1.2 on a clean bench. Use a sterile dropper to draw 2 mL of the sterilized Bletilla striata seed solution obtained in step 1.3 and evenly disperse it on the surface of the culture medium. Divide the medium into three dishes for each concentration, seal the dishes with sealing film, and incubate them in an artificial climate chamber.

[0050] 1.5 Seed germination Seed morphological changes were observed regularly under a stereomicroscope and photographed for record-keeping. After 30 days of culture, it was observed that the seeds of *Bletilla striata* in the culture dish swelled and formed green protocorms (see...). Figure 2 After 60 days of cultivation, observation revealed that most of the Bletilla striata seeds had germinated. The ethyl acetate extract treatment group (1 / 2 MS + B-1 group, 1 mg / L extract medium) showed the best results, and green plants had formed (see...). Figure 2 (1 / 2 MS+B-1 group already had cotyledons), and their plant health status was recorded.

[0051] 1 / 2 MS group (0 mg / L extract medium): plant height 3.32-5.67 mm, diameter 0.28-0.45 mm.

[0052] 1 / 2 MS+B-1 group (1 mg / L extract medium): plant height 2.56-5.32 mm, diameter 0.28-0.75 mm.

[0053] The results showed that, compared with the control group (1 / 2 MS group), treatment with 1 mg / L of the metabolite (ethyl acetate extract) of *Pleurotus ostreatus* in 1 / 2 MS medium resulted in more robust growth of *Bletilla striata* plants and a better promoting effect.

[0054] Example 4: The regulatory effect of *Pleurotus eryngii* metabolites on the growth and development of *Arabidopsis thaliana*, the specific steps of which are as follows: 1. Materials and Methods 1.1 Ethyl acetate extract Same as Example 1.

[0055] 1.2 Culture medium Same as Example 2.

[0056] 1.3 Plant materials The Arabidopsis seeds used in this embodiment are of the Col-0 type. The Arabidopsis seed disinfection steps are as follows: Take an appropriate amount of preserved wild-type seeds and pour them into a 1.5 mL centrifuge tube. Add sterile water, vortex several times, and centrifuge at high speed for 15 seconds. Discard the supernatant and floating seeds. Add 1 mL of 70% ethanol to the centrifuge tube, vortex for 10 seconds, and carefully remove the supernatant with a pipette. Add 1 mL of sterile deionized water to the centrifuge tube, vortex, and aspirate. Repeat twice. Add 1 mL of 3% sodium hypochlorite solution to the centrifuge tube, vortex vigorously for 10 minutes, and carefully remove the supernatant with a pipette. Add 1 mL of sterile water to the centrifuge tube to obtain the fully sterilized Arabidopsis seed solution.

[0057] 1.4 Co-culture Open the fully prepared extract culture medium from step 1.2 on a clean bench. Pour the sterilized Arabidopsis seed solution obtained in step 1.3 into a petri dish containing sterile filter paper. After the water has slightly dried, use small tweezers to sow Arabidopsis seeds into the extract culture medium. Use three dishes for each concentration and seal the petri dishes with sealing film. Place the sealed petri dishes in a refrigerator at 4 ℃ for 3 days for vernalization treatment to break the seed dormancy period. After 3 days, transfer the fully vernalized petri dishes to an artificial climate chamber for further cultivation.

[0058] 1.5 Seed germination Seed morphological changes were observed regularly under a stereomicroscope, and photographs were taken for recording. After 3-4 days of cultivation, Arabidopsis seeds in the petri dish were observed to germinate; after 7 days of cultivation, seedlings were observed to have formed and had grown two true leaves (see...). Figure 3 ). Statistical analysis of the plant's health status.

[0059] 1 / 2 MS group (0 mg / L extract medium): plant height 1.49-2.89 mm.

[0060] 1 / 2 MS+B-0.2 group (0.2 mg / L extract medium): plant height 1.55-3.39 mm.

[0061] 1 / 2 MS+B-1 group (1 mg / L extract medium): plant height 3.66-4.13 mm.

[0062] 1 / 2 MS+B-5 group (5 mg / L extract medium): plant height 0.45-0.80 mm.

[0063] The results showed that treatment with low concentrations (0.2 mg / L-1 mg / L) of ethyl acetate extract promoted the growth and development of Arabidopsis thaliana, while treatment with high concentrations (5 mg / L) of ethyl acetate extract inhibited the growth and development of Arabidopsis thaliana, exhibiting the plant hormone-like effects of the extract.

[0064] Example 5: The acquisition and effects of monomeric compounds from the metabolites of *Pleurotus ostreatus*, the specific steps of which are as follows: 1. Materials and Methods 1.1 Preparation of Monomer Compounds The metabolite (monomer compound 1) of *Pleurotus ostreatus* used in this embodiment was obtained by chromatographic separation of the total extract obtained by fermentation and ethyl acetate extraction in Example 1. The separation method is as follows: The total extract obtained in section 1.1 of Example 1 was dissolved in methanol and mixed with 80-100 mesh normal phase silica gel. The mixture was then subjected to normal phase column chromatography in a petroleum ether / acetone system (petroleum ether:acetone = 1:0, 20:1, 1:1, 0:1, v / v). Similar components were detected by TLC (developing solvent was dichloromethane:acetone = 5:1, v / v) to obtain 5 components (labeled as AE in the order of elution). Fragment B (45 g, eluted from petroleum ether-acetone at a volume ratio of 1:1) was eluted by medium-pressure liquid chromatography gradient elution (methanol:water = 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, v / v), and similar components were analyzed by TLC (developing solvent: dichloromethane:acetone = 5:1, v / v). The fractions were then divided into 10 subfractions (B1~B1) according to the elution order. 10 Subfraction B9 (1.6 g, eluted from methanol-water at a volume ratio of 40:60) was eluted by gradient elution on a normal-phase column (dichloromethane / ethyl acetate: 20:1, 15:1, 10:1, 5:1, 2:1, 0:1, v / v), and similar components were analyzed by TLC (developing solvent: petroleum ether: acetone = 3:1, v / v). Subfraction B9 was divided into six subfractions according to the elution order (B9, 1.6 g ... 9-1 ~B 9-6 ), B 9-5 The fraction eluted from dichloromethane to ethyl acetate at a volume ratio of 2:1 was further purified by Agilent 1260 high-performance liquid chromatography (Zorbox SB-C18 semi-preparative column, 5 μm, 9.4 mm × 150 mm; acetonitrile-0.01% formic acid aqueous solution = 43:57, v / v; flow rate 4 mL / min; detection wavelength 220 nm) to obtain monomer compound 1 (59.5 mg, retention time = 15.5 min). Its structure was determined by NMR and high-resolution mass spectrometry. The structural characterization results are as follows: The physicochemical data of monomer compound 1 are as follows: Colorless oily substance; specific rotation: + 165.2 (c 0.053, MeOH); UV(MeOH) λ max(log ε) 205 (3.79), 235 (3.73), 280 (4.05) nm; 1 H NMR (600 MHz) and 13 C10 NMR (150MHz) data (CD3OD), see Table 1; High-resolution mass spectrometry (HRESIMS) m / z 415.20920 [M+Na] + , (calcd.for C 22 H 32 O6Na + , 415.20966).

[0065] The structural formula of monomer compound 1 is shown in Formula 1 below, and the NMR data are shown in Table 1 below:

[0066] Table 1: NMR data of monomeric compound 1

[0067] 1.2 Culture medium The monomeric compound 1 described above was named sclerotin. 1 mg of this compound was weighed and dissolved in 1 mL of anhydrous ethanol to prepare a 1 mg / mL (1000 mg / L) stock solution. 0 mL, 0.02 mL, 0.1 mL, and 0.5 mL of the stock solution were added to 100 mL of the following basal culture medium 2 to prepare compound culture media with concentrations of 0 mg / L, 0.2 mg / L, 1 mg / L, and 5 mg / L, respectively. The media were sterilized at 121°C for 20 min, then removed and shaken well. The culture media, cooled to approximately 40°C, was poured into sterile petri dishes and allowed to solidify before storage for later use.

[0068] The basal medium 2-1 / 2 MS medium is prepared as follows: Weigh 1.24 g of 1 / 2 MS (Hangzhou Best Biotechnology Co., Ltd., BS 3004), 10 g of sucrose, 3.75 g of activated carbon, and 3.75 g of agar, add water to 480 mL, and adjust the pH to 7.10 with NaOH solid particles.

[0069] 1.3 Plant materials The plant material used in this embodiment is azalea seeds, and the treatment method is the same as in Embodiment 1.

[0070] 1.4 Co-culture Same as Example 1.

[0071] 1.5 Seed germination Seed morphological changes were observed regularly under a stereomicroscope. After 60 days of culture, germination of Rhododendron seeds was observed. The germination rates of Rhododendron seeds in media containing 0 mg / L, 0.2 mg / L, 1 mg / L, and 5 mg / L of sclerotin were 9.45%, 12.67%, 9.97%, and 7.45%, respectively. With increasing concentration, the germination-promoting effect of sclerotin first increased and then decreased.

[0072] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0073] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any transformations or substitutions that can be understood by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of the present invention.

Claims

1. Application of metabolites of Candolleomyces candolleanus (Fr.) D. Wächt. & A. Melzer in regulating plant growth.

2. The application according to claim 1, characterized in that, The regulation of plant growth includes regulating plant seed germination, regulating plant true leaf growth, regulating plant height, and / or regulating plant stem diameter.

3. The application according to claim 2, characterized in that, The plant in question is one of the following: Rhododendron, Bletilla striata, or Arabidopsis thaliana.

4. The application according to claim 3, characterized in that, The application includes the following steps: (1) Dissolve the metabolites of *Pleurotus ostreatus* in a solvent to prepare a metabolite solution; (2) Mix the solution obtained in step (1) with the co-culture medium, sterilize it to obtain the metabolite culture medium, wherein the concentration of the metabolite is 0.2~5 mg / L; (3) The plant is cultured in the metabolite culture medium obtained in step (2).

5. The application according to claim 4, characterized in that, In step (2), the concentration of the metabolites in the culture medium is 1 mg / L.

6. The application according to any one of claims 1-5, characterized in that, The metabolites of *Pleurotus ostreatus* are selected from one or more of the following: mycelium obtained from fermentation of *Pleurotus ostreatus*, fermentation broth, ethyl acetate extract, and monomeric compounds.

7. The application according to claim 6, characterized in that, The fermentation broth and mycelium are prepared using the following methods: The activated mycelium of *Pleurotus ostreatus* was fermented in liquid culture medium at a speed of 160 rpm and incubated in the dark at 25°C for 30 days. After fermentation, the solid-liquid mixture of the fermentation system was separated by centrifugation to obtain the fermentation broth and mycelium, respectively. The liquid culture medium consists of: 50 g / L glucose, 5 g / L peptone, 1.5 g / L yeast extract, 0.5 g / L KH2PO4, and 0.5 g / L MgSO4.

8. The application according to claim 7, characterized in that, The ethyl acetate extract is an ethyl acetate extract of mycelium and / or an ethyl acetate extract of fermentation broth; The ethyl acetate extract of the mycelium is prepared by the following method: after concentrating the acetone extract of the mycelium to remove acetone, water is added to dissolve it, followed by extraction with ethyl acetate. The ethyl acetate layer is collected to obtain the ethyl acetate extract of the mycelium.

9. The application according to claim 8, characterized in that, The monomeric compound is a guanacastane-type diterpene, with the structural formula shown in Formula 1 below: ； The monomeric compound was prepared by the following method: the ethyl acetate extract of mycelium and the ethyl acetate extract of fermentation broth were combined and concentrated to obtain a total extract; the total extract was dissolved in methanol and then purified by normal phase column chromatography, medium pressure liquid chromatography, normal phase column chromatography and high performance liquid chromatography in sequence to obtain the monomeric compound.

10. A plant growth regulator, characterized in that, Includes metabolites and acceptable adjuvants from Candolleomyces candolleanus (Fr.) D. Wächt. & A. Melzer.

Images