Use of a strain of fungus in promoting growth of nauclea diderrichii and increasing yield of active substances

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a fungus in promoting growth of Nauclea officinalis and increasing yield of active substance in a microbial agent. The fungus has a preservation number of GDMCC No: 66543, and belongs to Cladosporium herbarum Cladosporium tenuissimum. Researches show that the fungus can well colonize in the roots of Nauclea officinalis, promote growth of the plant, and increase the yield of vinosamycin lactam in the leaves of Nauclea officinalis, thereby facilitating further improvement of availability of medicinal substance vinosamycin lactam and promoting cultivation of Nauclea officinalis.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the use of a fungus in a microbial agent that promotes the growth of *Gynostemma pentaphyllum* and increases the yield of active substances. Background Technology

[0002] *Nauclea officinalis* Pierre ex Pitard is a traditional Chinese medicine from Hainan Province. Its source plant is *Nauclea officinalis*, a key protected wild plant in Hainan Province, belonging to the genus *Nauclea* within the family Rubiaceae. *Nauclea officinalis* is mainly distributed in southern my country, including Hainan, Guangxi, and Yunnan. It prefers warm and humid environments and often grows in sheltered, damp areas near mountain peaks or half-mountains. Due to its narrow distribution range, wild populations are limited. As a traditional Li medicine, *Nauclea officinalis* has the effects of clearing heat and detoxifying, reducing swelling and relieving pain. Studies have found that its extracts have various pharmacological effects, including antitumor, antiviral, antibacterial, antimalarial, antipyretic, analgesic, anti-inflammatory, and immunomodulatory effects. *Nauclea officinalis* contains various active ingredients, among which indole alkaloids are the most representative, and isocynopsidin is considered one of the important quality markers of *Nauclea officinalis*. However, *Gnaphalium affine* is a perennial tree with a slow growth rate and low levels of many active ingredients. These factors result in a limited yield of active ingredients from *Gnaphalium affine*, which cannot meet the growing clinical demand. Therefore, it is imperative to identify the factors affecting the biosynthesis of secondary metabolites in *Gnaphalium affine* and increase the content of active ingredients in it.

[0003] Cladosporium tenuissimum is a filamentous fungus belonging to the genus Cladosporium. This fungus is a pathogenic agent for many plants, classified as a Class IV biohazard. For example, it has been identified as a pathogen in literature such as "Pathogen Identification of Pomegranate Leaf Mold in Xichang Area" (Zheng Xiaohui et al., Plant Protection, 2010, Vol. 36, No. 1, pp. 131-133). It is also a factor contributing to the spoilage of some foods, such as increasing the acid value of camellia seed kernels during food storage.

[0004] In addition, some studies have found that certain microclustery fungi possess excellent cellulase synthesis capabilities, thus making them suitable for cellulase synthesis. Other microclustery fungi have been found to have microbial control effects, such as a strain of microclustery fungi disclosed in CN201711072883.7 (CN109749938A) that can reduce the incidence of root nodule disease in Panax notoginseng; and a strain of microclustery fungi disclosed in CN201910693800.9 (CN110438011A) that can increase the synthesis of tanshinone and salvianolic acid-like substances in Salvia miltiorrhiza.

[0005] However, to date, there are still few studies or reports on methods to promote the growth of *Gnaphalium affine* plants or to promote the synthesis and yield of medicinal substances from *Gnaphalium affine*. Summary of the Invention

[0006] To address the aforementioned problems, this invention aims to provide the use of a fungus in a microbial agent that promotes the growth of *Gnaphalium affine* and increases the yield of active substances. The technical solution of this invention is as follows:

[0007] The use of a fungus in the preparation of a growth-promoting agent for *Cladosporium tenuissimum* was investigated. The fungus, classified as *Cladosporium tenuissimum*, has the accession number GDMCC No:66543, was deposited on June 18, 2025, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province. The study found that this fungus can establish a good colony-symbiotic relationship with the roots of *Cladosporium tenuissimum*, promoting the growth of the plant.

[0008] A method for promoting the growth of *Gnaphalium affine*, the method comprising the following steps:

[0009] S1: The *Cladosporium filamentosa* with accession number GDMCC No:66543 was inoculated into PDA slant medium for culture and activation;

[0010] S2: Take a PDA agar block containing activated Cladosporium fasciatus, and bring the PDA agar block into contact with the roots of the *Gnaphalium affine* seedlings for co-culture, so that the Cladosporium fasciatus can colonize and symbiotically grow on the roots of the *Gnaphalium affine*.

[0011] Preferably, the culture temperature in step S1 is 20℃~37℃ and the humidity condition is 50%~70% relative humidity; the culture temperature in step S2 is 20℃~37℃ and the humidity condition is 50%~70% relative humidity.

[0012] The aforementioned *Cladosporium* species, with accession number GDMCC No:66543, was used in the preparation of a fungal agent to enhance the yield of active substances in *Gnaphalium affine*. The study found that this fungus can establish a good colony-symbiotic relationship with the roots of *Gnaphalium affine*, thereby increasing the yield of isocinolone lactones in the leaves.

[0013] A method for increasing the yield of bioactive substances in *Gynostemma pentaphyllum*, the method comprising the following steps:

[0014] S1: The *Cladosporium filamentosa* with accession number GDMCC No:66543 was inoculated into PDA slant medium for culture and activation;

[0015] S2: Take a PDA agar block containing activated Cladosporium fasciatus, and bring the PDA agar block into contact with the roots of the *Gnaphalium affine* seedlings for co-culture, so that the Cladosporium fasciatus can colonize and symbiotically grow on the roots of the *Gnaphalium affine*.

[0016] Preferably, the culture temperature in step S1 is 20℃~37℃ and the humidity condition is 50%~70% relative humidity; the culture temperature in step S2 is 20℃~37℃ and the humidity condition is 50%~70% relative humidity.

[0017] The fungus of this invention was obtained through natural selection. Its accession number is GDMCC No:66543; the accession date is June 18, 2025; the depositary institution is the Guangdong Provincial Center for Microbial Culture Collection; the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province. After classification and identification, the fungus is taxonomically named *Cladosporium tenuissimum*.

[0018] The beneficial effects of this invention are:

[0019] The plant grows slowly, and although it contains a variety of medicinal substances, its slow growth and narrow distribution limit its medicinal value.

[0020] The fungus of this invention not only promotes the growth of *Gnaphalium affine* (e.g., plant height and leaf density), but also increases the content of isocinolone lactones in the leaves, thus promoting the biosynthesis of isocinolone lactones in the leaves. This benefits both the cultivation and planting of *Gnaphalium affine* seedlings and the extraction of more medicinal substances from only a portion of the leaves without destroying the entire plant, thereby achieving a balance between plant protection and medicinal substance acquisition.

[0021] The fungi of this invention provide technical support for the rapid promotion of the cultivation of *Gnaphalium affine* seedlings and the growth of medicinal parts through soilless culture technology. Attached Figure Description

[0022] Figure 1 Phylogenetic tree of Cladosporium sp. strain with accession number GDMCC No:66543;

[0023] Figure 2 Microscopic images of the strain colonizing the roots of *Gastrodia elata* at day 0 of co-culture (scale bar: 200µm).

[0024] Figure 3 Microscopic images of the strain colonizing the roots of *Gnaphalium affine* at day 0 of co-culture (scale bar: 100µm).

[0025] Figure 4 Microscopic images of the strain colonizing the roots of *Gnaphalium affine* after 3 days of co-culture (scale bar: 200µm).

[0026] Figure 5 Microscopic images of the strain colonizing the roots of *Gnaphalium affine* after 3 days of co-culture (scale bar: 100µm).

[0027] Figure 6 Microscopic images of the strain colonizing the roots of *Gastrodia elata* after 7 days of co-culture (scale bar: 200µm).

[0028] Figure 7 Microscopic images of the strain colonizing the roots of *Gnaphalium affine* after 7 days of co-culture (scale bar: 100µm).

[0029] Figure 8 Images of the growth status of sterile *Gnaphalium affine* seedlings in the control group at different times; C-1 to C-6 are 6 parallel samples of sterile *Gnaphalium affine* seedlings in the control group.

[0030] Figure 9 Images show the growth status of *Gnaphalium affine* seedlings in the co-culture group at different times. Images 1-1 to 1-6 are six parallel samples of *Gnaphalium affine* seedlings in the co-culture group.

[0031] Figure 10 This is the standard curve of the HPLC method in Example 3;

[0032] Figure 11 The chromatogram of isovinctin lactam reference standard obtained by HPLC method in Example 3;

[0033] Figure 12 The chromatogram of the *Gnaphalium affine* test solution obtained by the HPLC method in Example 3 is shown below.

[0034] Figure 13 This is a chromatogram of isovinctin lactam detection in the leaves of sterile *Gnaphalium affine* seedlings in the control group of Example 3 on day 0.

[0035] Figure 14 This is a chromatogram of isovinctin lactone detection in the leaves of sterile *Gnaphalium affine* seedlings in the control group of Example 3 on day 20.

[0036] Figure 15 The chromatogram of isovinca lactone detection in the leaves of *Gnaphalium affine* seedlings on day 0 of the co-culture group of the strains in Example 3;

[0037] Figure 16 The chromatogram shows the detection of isovincin lactam in the leaves of *Gnaphalium affine* seedlings on day 20 of the co-culture group of the strains in Example 3. Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. 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. Unless otherwise specified, the reagents used in the following embodiments of the present invention are all commercially available and commonly used reagents. The method for cultivating sterile seedlings of *Gnaphalium affine* in the following embodiments is a commonly used method disclosed in the prior art, which can be found in CN201510721255.1 (An induction culture medium for sterile seedlings of *Gnaphalium affine* and a method for rapid virus-free propagation of *Gnaphalium affine*). The soilless culture medium is WPM medium.

[0039] Example 1: Activation of Cladosporium strains

[0040] The *Cladosporium* strain with accession number GDMCC No:66543 is a fungus isolated and purified from leaves of *Gnaphalium affine* and preserved in PDA slant medium. This fungus exhibits strong environmental adaptability and can grow rapidly under conditions of 20℃~37℃ and relative humidity of 50%~70%.

[0041] Using an inoculation loop, pick up the mycelia and inoculate them into PDA medium. Incubate at 28°C and 66% relative humidity for 4-5 days, then reactivate for later use. (The viable count of *Cladosporium microphyllum* with accession number GDMCC No: 66543 is approximately 10). 8 CFU / g).

[0042] The following is the isolation and identification information of Cladosporium microsporidium with accession number GDMCC No:66543:

[0043] 1. Sample collection

[0044] Fresh, healthy leaves of *Gnaphalium affine* were collected as samples from the Haikou campus of the Chinese Academy of Tropical Agricultural Sciences. Leaves without obvious pests or diseases and in good growth condition were selected as *Gnaphalium affine* leaf samples. The selected plants were identified as *Gnaphalium affine* by the School of Pharmacy of Hainan Medical University before sampling. The samples were placed in sealed bags, labeled, and transported to the laboratory. The leaf samples were used for the isolation and purification of endophytic bacteria from *Gnaphalium affine*.

[0045] 2. Disinfection of the leaf surface of *Gnaphalium affine*

[0046] To ensure effective surface disinfection, fresh, healthy, and sterile leaves of *Gnaphalium affine* were selected as the experimental material. After rinsing with running water for 10 minutes, the leaves were rinsed with sterile water, and excess water was absorbed with sterile filter paper. Under sterile conditions, the leaves were disinfected in 75% alcohol for 1 minute, then transferred to a 2% sodium hypochlorite solution for 5 minutes. The leaves were rinsed 4-5 times with sterile water. The sterile water from the final rinse was then spread onto PDA and LB agar media. The results showed no microbial growth in the media, indicating thorough disinfection of the leaf surface.

[0047] 3. Isolation of endophytic fungi from leaves of *Gnaphalium affine*

[0048] For the isolation of endophytic fungi, the tissue isolation method was used: the surface-sterilized leaves were cut into 1cm pieces with incisions on all four sides. 2 Place the leaves into PDA medium in cubes of various sizes and label them. Incubate the medium at 28°C. After 4-5 days, observe whether colonies have grown from the leaf cuts on the medium. Once visible colonies have grown from the leaf edges in the medium, pick a small amount of mycelium and inoculate it onto the PDA medium. Repeat this process several times until the colonies are uniform. Store the resulting pure colony of the endophytic fungus on a slant.

[0049] 4. Morphological identification

[0050] Macroscopic morphology of colonies: When the strain was cultured in PDA medium at a constant temperature of 28℃, the colonies grew in a fluffy, flat manner; after maturity, the colonies were generally olive gray-brown to smoky brown, with a darker color in the center and lighter gray and sparse hyphae on the outer edge, and irregular wavy edges; the back of the medium was dark brown with no obvious exudate.

[0051] Colony micromorphology: The hyphae of this fungus are septate and branched, light yellowish-brown; conidiophores are erect, septate, and sympodially branched at the apex; conidia are small, oval, mostly clustered, and occasionally in short chains. Preliminary morphological identification identifies it as Cladosporium tenuissimum.

[0052] 5. Fungal DNA extraction and PCR amplification

[0053] The purified fungi were inoculated into PDA liquid medium and cultured at 30°C and 150 rpm for 48 h on a shaker. The cells were collected by centrifugation at 5000 rpm for 5 min, and the supernatant was discarded. Liquid nitrogen was added to the fungal sample, and the sample was ground. Fungal DNA was extracted from the ground sample using a universal DNA extraction kit. The extracted DNA was then subjected to PCR amplification. The primers used for fungal amplification were:

[0054] ITS4(5′-TCCTCCGCTTATTGATATGC-3′) / ITS1-F(5′-CTTGGTCATTTAGAGGAAGTAA-3′), PCR reaction system (50 μL): 2×Accurate Taq Master Mix: 25 μL, forward and reverse primers: 1 μL each, template DNA: 1 μL, RNase-free water: 22 μL. PCR reaction conditions:

[0055] Table 1 PCR reaction conditions

[0056] After PCR, the PCR products were detected by 1% agarose gel electrophoresis, and the qualified PCR products were sent to Guangzhou BGI Biotechnology Co., Ltd. for molecular identification. Sequencing results were compared using BLAST on the NCBI website to obtain the molecular identification results of the endophytic bacteria.

[0057] DNA was extracted and PCR amplified from the isolated and purified endophytic fungi, and electrophoresis was performed on a 1% agarose gel. The agarose gel electrophoresis results of the fungal PCR products showed that there were obvious bands in the range of 500~750bp, indicating that the PCR was successful and the target fragment could be recovered and sequenced.

[0058] The qualified PCR products were sent to Guangzhou BGI Biotechnology Co., Ltd. for molecular identification. Sequencing results were compared using BLAST on the NCBI website to obtain the molecular identification results of the endophytic fungus. Its ITS nucleotide sequence is shown in SEQ ID NO.1. The sequenced gene sequence was compared using BLAST on the NCBI website, confirming that the isolated endophytic fungus had 100% similarity to *Cladosporium tenuissimum*.

[0059] 6. Phylogenetic tree

[0060] like Figure 1 As shown, to further clarify the species-level taxonomic position of strain D1, a phylogenetic tree was constructed using the type strain sequence (NR_119855) of Cladosporium tenuissimum and sequences of closely related species. The results showed that strain D1 clustered with all reference sequences (including the type strain) of C. tenuissimum into the same monophyletic branch, with a self-development support rate of 93%, and was clearly separated from the closely related species C. salinae. Figure 1In the table: A phylogenetic tree of strain D1 and closely related species of the genus Rachicladosporium, constructed based on the target sequence, was built using the maximum likelihood (ML) method with 1000 bootstrap replicates; Rachicladosporium americanum is an outgroup; node numbers represent expansion support. All reference sequences were downloaded from the NCBI GenBank database; sequence information, strain IDs, and accession numbers are detailed in Table 2.

[0061]

[0062] Example 2: Co-culture of Cladosporium microsporidium strain with aseptic seedlings of *Gnaphalium affine*

[0063] This embodiment is a follow-up experiment to Embodiment 1.

[0064] Take sterile seedlings of *Cladosporium glomeratum* with the same culture time, and use a sterile punch to take a uniform fungal agar block (about 0.8 mm in diameter) with hyphae from the activated *Cladosporium glomeratum* culture of Example 1. Place the block about 1 cm away from the root-stem junction of the sterile seedling and co-culture it in a soilless environment under the conditions of 12 h / d light duration (light intensity 90 lx), 25°C and 66% relative humidity. This is the co-culture group.

[0065] The sterile seedlings in the blank control group were cultured in soilless conditions under the same conditions, with a blank PDA medium agar block (about 0.8 mm in diameter) without mycelium placed about 1 cm from the root-stem junction.

[0066] Each of the above treatments was performed in 6 replicates, and all operations were performed under aseptic conditions.

[0067] Example 3: Effects of Cladosporium fasciatus on the growth and medicinal substance synthesis of *Gnaphalium affine* seedlings

[0068] This example is a follow-up experiment to Example 2.

[0069] 1. Observation on the colonization of Cladosporium filamentosa on the roots of *Gnaphalium affine*

[0070] Roots of *Cladosporium spp.* co-cultured for 0, 3, and 7 days were collected and placed in sterile centrifuge tubes. FAA fixative was added for fixation, followed by rinsing with sterile water. The root samples were then soaked in 100 g / L KOH solution and incubated at 90°C for 60 min. After rinsing with sterile water, 30% H₂O₂ was added for 5 min. The samples were rinsed with sterile water and drained. Lactic acid was added for 5 min, and after discarding the lactic acid, the samples were stained with trypan blue for 5 min. The stained root samples were then destained overnight in a lactic acid-glycerol mixture (lactic acid:glycerol = 1:1). The destained samples were observed under an optical microscope.

[0071] The results are as follows Figure 2-7 As shown, *Cladosporium filamentosa* colonizes rapidly on the roots of *Gnaphalium affine* seedlings. After 3 days of co-culture, the mycelium initially invades the roots; after 7 days of co-culture, a large number of mycelia accumulate on the roots of *Gnaphalium affine*.

[0072] 2. The promoting effect of Cladosporium filamentosa on the growth of *Gnaphalium affine* seedlings

[0073] The growth of the *Gnaphalium affine* seedlings was photographed and compared regularly during the cultivation process. The results are as follows: Figure 8 and Figure 9 As shown in the figure. Under the same culture conditions and culture period, compared with the control group, the *Gnaphalium affine* seedlings in the co-culture group showed significantly enhanced growth. After 20 days of co-culture, the plant height of the *Gnaphalium affine* seedlings in the co-culture group increased significantly more than that in the control group (p<0.05). The plant height in the control group increased from the initial value of (4.42 ± 0.41) cm to (6.27 ± 0.85) cm, a net increase of (1.85 ± 0.94) cm; the plant height in the co-culture group increased significantly from the initial value of (5.86 ± 0.58) cm to (10.69 ± 1.94) cm, a net increase of (4.83 ± 2.02) cm.

[0074] 3. Effects of Cladosporium fasciatus on the synthesis of medicinal substances in *Gnaphalium affine* seedlings

[0075] The following method for determining the content of isocinolone lactone in *Gnaphalium affine* seedlings is an improvement upon the method described in the literature (see: Zhang Junqing, Mao Caini, Yang Weili, et al. Study on the content of isocinolone lactone in different parts of *Gnaphalium affine* [J]. Journal of Pharmaceutical Analysis, 2008, 28(10):4). The main differences between this literature and the following HPLC method are the column length and the mobile phase volume ratio.

[0076] Preparation of the test solution: Dry the leaves of *Gnaphalium affine* seedlings to constant weight, then take equal weights of leaves from each sample. Use 65% ethanol as the extraction solvent, with a material ratio of 1:100 (g:mL), and extract ultrasonically for 40 min. After extraction, filter and collect the filtrate as the extract. Take 1 mL of the extract and dilute it to 10 mL with acetonitrile:0.1% phosphoric acid solution (25:75). Filter the diluted solution through a 0.22 μm microporous membrane to obtain the test solution for later use.

[0077] Preparation method of reference solution: Accurately weigh 1.6 mg of isocynoplasmin lactam standard, dissolve it in acetonitrile: 0.1% phosphoric acid water (25:75) solvent, and make up to 10 mL to obtain a stock solution of isocynoplasmin lactam reference solution with a concentration of 160 μg / mL for later use.

[0078] The content of isovincinolactam in each sample was determined by high-performance liquid chromatography (HPLC). The HPLC detection conditions were as follows:

[0079] The chromatographic column was a Phenomenology Luna C18(2) 100Å (150×4.6mm, 5μm) column; the mobile phase was acetonitrile and 0.1% phosphoric acid aqueous solution, eluted isocratically at a ratio of 25:75; the working wavelength was 226nm, the column temperature was 30℃, the flow rate was 1.0mL / min, and the injection volume was 10μL. The formula for calculating the content of isocynoplasmin in the sample is as follows:

[0080]

[0081]

[0082] Where X is the concentration of isovincinolactam in the test solution, in μg / mL; D is the dilution factor of the extract (10 times); V is the total volume of the extract, 100 mL; W is the constant weight sample mass of the leaves of the *Gnaphalium affine* seedling (g); ÷1000 is used to convert μg to mg; y is the peak area (mAU*min).

[0083] (1) Standard curve and linear range: 0.25 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, and 2.5 mL of vinblastine lactone reference solution stock solution with a concentration of 160 μg / mL were taken and diluted to 5 mL with acetonitrile:0.1% phosphoric acid water (25:75). Standard curves were plotted using the injection concentrations and peak areas of the six gradient concentrations, and regression equations were obtained. The concentration of the reference solution was plotted on the x-axis, and the peak area was plotted on the y-axis. By plotting the standard curve of the vinblastine lactone content, the standard curve equation of this detection method was obtained as y = 0.7035x - 0.1561, with a correlation coefficient of 0.9997 and a linear range of 8 μg / mL to 80 μg / mL. Figure 10 This is the standard curve for this HPLC method.

[0084] (2) Precision: The reference solution (the reference solution stock solution diluted to a concentration of 8 μg / mL) was injected 6 times consecutively and measured under the aforementioned chromatographic conditions. The peak areas obtained were 5.1299, 5.1059, 5.1099, 5.1235, 5.1353, and 5.0624, respectively. The calculated RSD was 0.52%, indicating that the method has good precision.

[0085] (3) Stability: After the test solution was prepared, it was injected at 0, 2, 4, 8, 12 and 24 h and measured under the above chromatographic conditions. The peak areas obtained were 33.6916, 33.9239, 33.8658, 33.9725, 34.2009 and 33.3169 respectively. The peak area RSD was 0.89%, which indicates that the test solution has good stability within 24 h under the conditions of room temperature and protection from light.

[0086] (4) Repeatability: Six samples of *Gnaphalium affine* leaves from the same batch of treated plants (all from *Gnaphalium affine* seedlings in the co-culture group of the strains; the co-culture time between sterile seedlings and the strains was 20 days) were accurately weighed. Six test solution samples were prepared in parallel according to the aforementioned extraction and test solution preparation methods and injected for analysis. The content of isocinolone lactam was calculated, and the relative standard deviation of the content was also calculated. The peak areas obtained were 32.0045, 29.7906, 30.5710, 31.5040, 29.1506, and 31.3257, respectively. The refolding test showed that the RSD of this invention was 2.71%, indicating good repeatability of the method.

[0087] Figure 11 , 12 The figures are HPLC chromatograms of the reference standard (diluted to 80 μg / mL, with a retention time of 12.178 min for isocinolone lactam) and the test solution (from a sample that underwent a stability study for 24 h, with a retention time of 12.230 min for isocinolone lactam) under the HPLC chromatographic conditions described above.

[0088] Quantitative detection results of isovinctin lactam in the sample:

[0089] The quantitative detection results of isovincin lactone in the leaves of *Gnaphalium affine* seedlings in the co-culture group of strains 0 and 20 and the leaves of sterile *Gnaphalium affine* seedlings in the control group (the sampling positions of the leaves were similar in height and the sampling positions were comparable) are shown in Tables 3 and 4, respectively. Figure 13 , 14 The corresponding HPLC chromatograms are the HPLC chromatograms of the detection of isovinctin lactam content in the leaves of sterile seedlings of *Gnaphalium affine* on days 0 and 20 of the control group. Figure 15 , 16 The corresponding HPLC chromatograms are those for the detection of isovincin lactam content in the leaves of *Gnaphalium affine* seedlings on days 0 and 20 of the co-culture group of the strains.

[0090] Table 3 Peak area and content of 6 parallel samples from the control group

[0091] Table 4. Peak area and content of 6 parallel samples from the co-culture group

[0092] The biosynthesis of isocinolone acetonide in the leaves of both groups of *Gnaphalium affine* seedlings showed a dynamic change with continuous accumulation. Experimental data showed that during the culture period (0-20 days), the average isocinolone acetonide content in the leaves of the control group increased from 23.6969 mg / g to 29.5853 mg / g, an increase of 24.85%. In contrast, the accumulation effect in the co-culture group was more significant. In this group, the average isocinolone acetonide content increased from 25.5207 mg / g to 40.4285 mg / g during the same culture period, an increase of 58.41%. The growth rate of isocinolone acetonide content in the co-culture group was significantly higher than that in the control group (p<0.05). This result indicates that co-culture of the strains has a significant promoting effect on the biosynthesis of isocinolone acetonide in the leaves of *Gnaphalium affine* seedlings.

[0093] The above results indicate that *Cladosporium filamentosa* with accession number GDMCC No:66543 has a significant promoting effect on the growth of *Gnaphalium affine* seedlings and a significant increasing effect on the content of isocinolone lactone in the leaves.

Claims

1. The use of a fungus in the preparation of a fungal agent to promote the growth of *Gnaphalium affine*, characterized in that, The fungus is *Cladosporium*, classified and named... Cladosporium tenuissimum The accession number is GDMCC No:66543, the accession date is June 18, 2025, the depositary institution is Guangdong Provincial Center for Microbial Culture Collection, and the depositary address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

2. A method for promoting the growth of *Gnaphalium affine*, characterized in that, The method includes the following steps: S1: The *Cladosporium filamentosa* with accession number GDMCC No:66543 was inoculated into PDA slant medium for culture and activation; S2: Take a PDA agar block containing activated Cladosporium fasciatus, and bring the PDA agar block into contact with the roots of the 'Gynostemma pentaphyllum' seedlings for co-culture, so that the Cladosporium fasciatus can colonize the roots of the 'Gynostemma pentaphyllum'.

3. The method for promoting the growth of *Gnaphalium affine* according to claim 2, characterized in that, The culture temperature in step S1 is 20℃~37℃; the humidity condition is 50%~70% relative humidity; the culture temperature in step S2 is 20℃~37℃, and the humidity condition is 50%~70% relative humidity.

4. The use of a fungus in the preparation of a fungal agent that enhances the yield of active substances in *Gynostemma pentaphyllum*, characterized in that, The fungus is *Cladosporium*, classified and named... Cladosporium tenuissimum The accession number is GDMCC No:66543, the accession date is June 18, 2025, the depositary institution is Guangdong Provincial Center for Microbial Culture Collection, and the depositary address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.

5. The use according to claim 4, characterized in that, The active substance in the *Cinnamomum camphora* is isovinctin lactam.

6. A method for increasing the yield of active substances in *Gynostemma pentaphyllum*, characterized in that, The method includes the following steps: S1: The *Cladosporium filamentosa* with accession number GDMCC No:66543 was inoculated into PDA slant medium for culture and activation; S2: Take a PDA slant agar block containing activated Cladosporium fasciatum, and bring the PDA agar block into contact with the roots of the 'Gynostemma pentaphyllum' seedlings for co-culture, so that the Cladosporium fasciatum colonizes the roots of the 'Gynostemma pentaphyllum'.

7. The method for increasing the yield of active substances in *Gynostemma pentaphyllum* according to claim 6, characterized in that, The culture temperature in step S1 is 20℃~37℃; the humidity condition is 50%~70% relative humidity; the culture temperature in step S2 is 20℃~37℃, and the humidity condition is 50%~70% relative humidity.

8. A method for increasing the yield of active substances in *Gynostemma pentaphyllum* according to claim 6 or 7, characterized in that, The active substance in the *Cinnamomum camphora* is isovinctin lactam.

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