Method for controlling ipomoea nil by sequentially using beneficial epiphyte fungi and pathogenic bacteria

Abstract

The present application relates to the technical field of invasion plant prevention and control, in particular to a method for controlling ipomoea nil by sequentially using beneficial epiphyte fungi and pathogenic bacteria, which comprises the following steps: S1, stimulating excessive immunity, spraying G.fujikuroi spore suspension with a concentration of 3×10 7 Individual leaf spraying amount is 2.5 ml, and the ipomoea nil plants are placed in an environment with a temperature of 25±5 DEG C and a relative humidity of 85±5% for 2 days; S2, immunity decline, waiting for 3 days; S3, applying pathogenic bacteria, spraying Colletotrichum gloeosporioides spore suspension with a concentration of 2×10 6 Individual leaf spraying amount is 2.5 ml, and waiting for 2 days. A new technology of using low pathogenic Colletotrichum gloeosporioides as a biological control agent for ipomoea nil is created, and a green new way of weakening the immunity of the plant without using chemical fungicides is provided. The disease resistance of the plant is weakened, and then the ipomoea nil plants with weakened immunity are infected by Colletotrichum gloeosporioides.

Description

Technical Field

[0001] This invention relates to the field of invasive plant control technology, specifically a method for controlling *Gynostemma pentaphyllum* by sequentially using beneficial epiphytic fungi and pathogenic bacteria. Background Technology

[0002] Five-clawed golden dragon is a perennial evergreen vine mainly distributed in South China. It is one of the most harmful invasive plant species in the region. This species is highly resistant to disease and rarely suffers from diseases in the natural environment. It mainly reproduces through vegetative clonal propagation via roots and stems. It is characterized by rapid growth and strong competitiveness, causing great damage to field crops and plant diversity in South China.

[0003] Currently, the control of *Gynostemma pentaphyllum* mainly employs manual removal, chemical control, and biological control methods. Manual removal requires significant manpower and resources, is inefficient, and the roots, stems, and other plant debris left behind easily regenerate in situ. Chemical removal is effective against *Gynostemma pentaphyllum*, but the application of chemical pesticides causes serious environmental pollution. *Gynostemma pentaphyllum* occasionally exhibits very mild symptoms of *Colletotrichum gloeosporioides* infection in its natural habitat. Therefore, some have proposed using this fungus for biological control of *Gynostemma pentaphyllum*. However, *Colletotrichum gloeosporioides* is a low-pathogenic pathogen of *Gynostemma pentaphyllum*, and simply applying *Colletotrichum gloeosporioides* is insufficient to cause severe disease symptoms, thus failing to achieve the goal of controlling the plant. Studies have found that the strong resistance of *Gnaphalium affine* to *Colletotrichum candida* is closely related to the symbiosis with a beneficial epiphytic fungus (*G. fajikuroi*) on the plant's leaves. Therefore, a control strategy was proposed: first, eliminate *G. fajikuroi* to weaken the plant's disease resistance, and then use *Colletotrichum candida* to infect the weakened *Gnaphalium affine* plants. However, the chemical fungicides used to eliminate this epiphytic species pose a significant risk of environmental pollution. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for controlling *Gynostemma pentaphyllum* by sequentially using beneficial epiphytic fungi and pathogenic bacteria, comprising the following steps:

[0005] S1, Stimulate excessive immunity, with a concentration of 3×10 7 A spore suspension of G. fjikuroi per ml was sprayed onto the leaves of the five-clawed golden dragon plant, with 2.5 ml sprayed onto each leaf. The five-clawed golden dragon plant was placed in an environment with a temperature of 25±5℃ and a relative humidity of 85±5% for 2 days.

[0006] S2, Immune system weakens, wait 3 days;

[0007] S3. Pathogenic bacteria are introduced, with a concentration of 2×10⁻⁶. 6Spray a suspension of *Colletotrichum candida* spores (2.5 ml / ml) onto the leaves of the *Pterocarya stenoptera* plant and wait 2 days.

[0008] The preferred method for the isolation and purification of G. fujikuroi is as follows:

[0009] Collect vigorous growth of *Gynostemma pentaphyllum* plants. Select bright green, disease-free leaves from the stems of the plants. Place 8 leaves in a 250mL Erlenmeyer flask, add 75mL of sterile water, seal the mouth of the flask with sealing film, and place the flask on a shaker at 170rpm for 60min. In a clean bench, use a sterile pipette tip to draw 200μL of the suspension and inoculate it onto sterile PDA medium. Spread the suspension evenly, invert all the plates, and incubate them in a 26℃ incubator in the dark for 7 days. Pick out each colony on the PDA medium and transfer it to a new sterile PDA medium for purification culture for 2-3 generations.

[0010] The preferred method for isolating and purifying *Colletotrichum gloeosporioides* is as follows:

[0011] Collect leaves of *Gynostemma pentaphyllum* that have already shown symptoms of infection with *Colletotrichum gloeosporioides*. Use a sterile blade to cut leaf spots from the surface of the *Gynostemma pentaphyllum* and transfer them to sterile PDA medium. Incubate in a 26°C incubator in the dark for 7 days. Pick out each colony on the PDA medium and transfer it to a new sterile PDA medium for purification culture for 2-3 generations.

[0012] Preferably, the PDA culture medium is prepared by using 200g potato, 20g glucose, 15g agar, 0.1g streptomycin sulfate, and 1000mL deionized water.

[0013] The preferred method for preparing the fungal spore suspension is as follows:

[0014] The purified colonies were transferred from PDA medium to sterile PDB medium, which was prepared by mixing 200g of potato, 20g of glucose, and 1000mL of deionized water. The culture was then placed in a dark incubator at 26°C for 24 hours. The fungal culture was filtered through three layers of sterile gauze to obtain a spore suspension stock solution. Both stock solutions were transferred to 50mL centrifuge tubes and centrifuged at 4000rpm for 10 minutes to allow the spores to settle at the bottom of the centrifuge tube. The PDB layer on top of the centrifuge tube was discarded, and sterile water was added to the centrifuge tube. The centrifuge tube was then shaken to obtain a fungal spore suspension.

[0015] The present invention has the following beneficial effects:

[0016] A new technology has been developed to use the low-pathogenicity Golgiformis glomeratus as a biological control agent for Gynostemma pentaphyllum, providing a green approach to weaken the plant's immunity without the need for chemical fungicides. By using the beneficial epiphytic fungus (G. fjikuroi) of Gynostemma pentaphyllum, the plant's over-immunity can be stimulated and subsequently weakened in a short period of time, avoiding the environmental pollution caused by directly eliminating the beneficial epiphytic fungus (G. fjikuroi) with chemical fungicides.

[0017] This method uses low-pathogenicity Colloidal anthracnose bacteria to cause serious diseases in *Gynostemma pentaphyllum*, making this low-pathogenicity strain meet the requirements for biological control of *Gynostemma pentaphyllum*, thus solving the problem of a severe shortage of natural high-pathogenicity biological control strains for *Gynostemma pentaphyllum*.

[0018] In this method, both beneficial epiphytic fungi and pathogenic fungi can be isolated from the leaves of the five-clawed golden dragon. The isolation method is simple, and the equipment required for preparing the spore suspension of each fungal species is simple, with high yield and low cost. Attached Figure Description

[0019] Figure 1 This is a flowchart of a method for controlling *Gynostemma pentaphyllum* by sequentially using beneficial epiphytic fungi and pathogenic bacteria, according to the present invention.

[0020] Figure 2 This diagram illustrates the effects of beneficial epiphytic fungi applied for 2 and 5 days on the H2O2 content, chitinase activity, β-1,3-glucanase activity, and PAL activity of *Gynostemma pentaphyllum*.

[0021] Figure 3 This is a schematic diagram illustrating the effects of the beneficial epiphytic fungi of the present invention on the antioxidant enzyme activity and MDA content of *Gynostemma pentaphyllum* after 2 and 5 days of application.

[0022] Figure 4 This is a schematic diagram illustrating the effect of sequentially combining beneficial epiphytic fungi and pathogenic bacteria on the symptoms of anthrax caused by *Colletotrichum gloeosporioides*.

[0023] Figure 5 This is a schematic diagram illustrating the effect of sequentially combining beneficial epiphytic fungi and pathogenic bacteria on the leaf disease rate of *Gynostemma pentaphyllum*. Detailed Implementation

[0024] The principles and steps of the present invention are described below. The embodiments given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] In one specific implementation, such as Figure 1-5 As shown, a method for controlling *Gynostemma pentaphyllum* by sequentially using beneficial epiphytic fungi and pathogenic bacteria includes the following steps:

[0027] S1, Stimulate excessive immunity, with a concentration of 3×10 7 A spore suspension of G. fjikuroi was sprayed onto the leaves of the five-clawed golden dragon plant, with 2.5 ml sprayed onto each leaf. The five-clawed golden dragon plant was then placed in an environment with a temperature of 25±5℃ and a relative humidity of 85±5% for 2 days.

[0028] S2, Immune system weakens, wait 3 days.

[0029] S3. Pathogenic bacteria are introduced, with a concentration of 2×10⁻⁶. 6 Spray a suspension of *Colletotrichum candida* spores (2.5 ml / ml) onto the leaves of the *Pterocarya stenoptera* plant and wait 2 days.

[0030] In this embodiment, the method for isolating and purifying the beneficial epiphytic fungus G. fajikuroi is as follows:

[0031] Collect vigorous *Gnaphalium affine* plant populations, selecting bright green, disease-free leaves from the stems and preserving them in sealed plastic bags. Place eight leaves in a 250mL Erlenmeyer flask, add 75mL of sterile water, and seal the flask opening with sealing film. Place the flask on a shaker at 170rpm for 60min. In a laminar flow hood, use a sterile pipette tip to aspirate 200μL of the suspension and inoculate it onto sterile PDA medium, spreading it evenly. The PDA medium was prepared by mixing 200g potato, 20g glucose, 15g agar, 0.1g streptomycin sulfate, and 1000mL deionized water. Invert all plates and incubate at 26℃ in the dark for 7 days. Pick out each colony from the PDA medium and transfer it to fresh sterile PDA medium for purification culture. After 2-3 generations, purified strains of various leaf-epithelial fungi, including *Gnaphalium affine*, can be obtained.

[0032] Identification of *G. fajikuroi* was performed using a combination of fungal morphology and ITS sequence alignment. First, based on the colony shape, color, and the shape and size of conidia and microconidia described in *Fusarium Taxonomy* (Leslie & Summerell, 2006), preliminary identification and screening of *G. fajikuroi* from the isolated purified foliar fungi strains were conducted. Then, the ITS sequences of the screened strains were sequenced and compared with NCBI BLAST. A 100% similarity was found between the ITS sequence and the *G. fajikuroi* species (MN704851.1) reported by NCBI, thus confirming the species as *G. fajikuroi*.

[0033] In this embodiment, the method for isolating and purifying *Colletotrichum gloeosporioides* is as follows:

[0034] Leaves of *Gynostemma pentaphyllum* exhibiting symptoms of *Colletotrichum candelilla* infection were collected. Leaf spots were cut from the surface of the *Gynostemma pentaphyllum* using a sterile blade and transferred to sterile PDA medium. The PDA medium was prepared by mixing 200g potato, 20g glucose, 15g agar, 0.1g streptomycin sulfate, and 1000mL deionized water and incubated at 26℃ in the dark for 7 days. Each colony on the PDA medium was picked out and transferred to fresh sterile PDA medium for purification. Purified strains of *Colletotrichum candelilla* were obtained after 2-3 generations.

[0035] The *Colletotrichum gloeosporioides* strain was identified using a combination of fungal ITS sequencing, NCBI BLAST alignment, and morphological characteristics. First, the purified fungal strain was preliminarily identified based on the colony shape, color, and the shape and size of large and small conidia described by Rabha et al. (2006). Then, the strain's ITS sequence was sequenced and aligned using NCBI BLAST. The ITS sequence showed 100% similarity to *Colletotrichum gloeosporioides* (MN704853.1) reported by NCBI, confirming the strain as *Colletotrichum gloeosporioides*.

[0036] Furthermore, the method for preparing the fungal spore suspension is as follows:

[0037] Two-week-old colonies of *G. fajikuroi* and *Colletotrichum gloeosporioides* were transferred from PDA medium to sterile PDB medium, prepared with 200g potato, 20g glucose, and 1000mL deionized water. The cultures were then incubated at 26°C in a dark incubator for 24 hours. The fungal culture was filtered through three layers of sterile gauze to remove mycelia, and the resulting filtrate served as the stock solutions of spore suspensions for both *G. fajikuroi* and *Colletotrichum gloeosporioides*. These stock solutions were transferred to 50mL centrifuge tubes and centrifuged at 4000rpm for 10 minutes to allow the spores to settle at the bottom of the tubes. Discard the PDB layer at the top of the centrifuge tube, add 10 mL of sterile water, and shake vigorously to resuspend the spores in the sterile water. Take 1 mL of the stock solution of both *G. fajikuroi* and *Colletotrichum gloeosporioides* spore suspensions and add it to the counting cells of a hemocytometer. Count the spores under a standard optical microscope and calculate their concentrations. Adjust the concentration of the *G. fajikuroi* spore suspension to 3 × 10⁻⁶ with sterile water. 7 The concentration of the *Colletotrichum candida* spore suspension was adjusted to 2 × 10⁶ spores / ml. 6 The concentration of fungal spores per ml should be prepared and used immediately before use. If storage at 4°C is required, it should not exceed 6 hours.

[0038] In this embodiment, in step S1, to determine whether the plant has achieved excessive immunity, a portion of the *Gynostemma pentaphyllum* plant can be taken, and its leaves can be used as material. The determination is made by analyzing plant immunity-related physiological indicators and membrane lipid peroxidation indicators. These indicators include H2O2 content, chitinase activity, β-1,3-glucanase activity, phenylalanine ammonia-lyase (PAL) activity, superoxide dismutase (SOD) activity, peroxidase (POD) activity, catalase (CAT) activity, and malondialdehyde content. Similarly, in step S2, it can be tested whether the *Gynostemma pentaphyllum*'s immunity has weakened.

[0039] Example 1:

[0040] Activating excessive immunity: Fifty-eight leafy stem segments, each 20cm long and 3mm in diameter, from healthy *Aglaonema edulis* plants were collected from the wild. Each segment retained two leaves before being inserted into Hoagland nutrient solution. The 58 *Aglaonema edulis* plants were divided into two groups of 29 plants each. Then, a solution with a concentration of 3×10⁻⁶ was used. 7A diluted suspension of *G. f. f. f. f. spores (2.5 mL / ml) was sprayed onto the upper and lower surfaces of the leaves of one group of 29 *G. f. f. plants, serving as the treatment group. The remaining 29 *G. f. plants were left untreated as a control group. Finally, both groups of *G. f. f. plants were placed in separate incubators with different light and humidity levels. The incubators were maintained at 25±5℃, 85±5% relative humidity, a 14h / 10h light / dark cycle, and a light intensity of 200 μmol·m⁻¹. -2 ·s -1 After 2 days of cultivation, 13 plants were randomly harvested from each group, and their leaves were used to detect plant immune-related physiological indicators and membrane peroxidation physiological indicators. Four plants from the same group were used to detect H2O2 content, chitinase activity, and β-1,3-glucanase activity; another four plants from the same group were used to detect phenylalanine ammonia-lyase (PAL) activity; and the remaining five plants from the same group were used to analyze superoxide dismutase (SOD) activity, peroxidase (POD) activity, catalase (CAT) activity, and malondialdehyde (MDA) content. Both groups of plants were tested.

[0041] In the natural environment, beneficial epiphytic fungi have formed a stable symbiotic relationship with the leaves of *Gynostemma pentaphyllum*, enhancing its immunity and disease resistance. In this example, the *Gynostemma pentaphyllum* plants in the control group were collected from the natural environment without any spraying treatment; therefore, the immunity level of the control group plants due to the epiphytic fungal symbiosis can be considered as natural immunity. Figure 2 It was observed that after 2 days of application of *G. fajikuroi* spore suspension, the H2O2 content, chitinase activity, β-1,3-glucanase activity, and PAL activity in the treatment group were significantly higher than those in the control group. This result indicates that 2 days of application of *G. fajikuroi* spore suspension can induce an excessive immune response in the *G. fajikuroi* plant. Figure 3 It is evident that the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and malondialdehyde (MDA) in the control group were not significantly different from those in the control group. This indicates that applying G. fujikuroi spore suspension for 2 days did not affect the antioxidant capacity of the G. fujikuroi plant or cause membrane lipid peroxidation damage to the plant cells.

[0042] Weakened Immunity: Unharvested *Gynostemma pentaphyllum* plants from both the control and treatment groups were further cultured. The incubator temperature was 25±5℃, relative humidity was 85±5%, light / dark cycle was 14h / 10h, and light intensity was 200μmol·m–2·s⁻¹. After 3 days of further culture, 13 plants were randomly harvested from each group. Plant immune-related physiological indicators and membrane peroxidation physiological indicators were detected in their leaves. Four plants from each group were used for H₂O₂ content, chitinase activity, and β-1,3-glucanase activity; another four plants from each group were used for phenylalanine ammonia-lyase (PAL) activity; and the remaining five plants from each group were used for superoxide dismutase (SOD) activity, peroxidase (POD) activity, catalase (CAT) activity, and malondialdehyde (MDA) content. The detection was performed on both groups of plants.

[0043] Depend on Figure 2 , Figure 3 It is evident that, compared to the treatment group after 2 days of application of *G. fajakuroi* spore suspension, the activities of superoxide dismutase (SOD), peroxidase (POD), chitinase, and β-1,3-glucanase were significantly decreased after 5 days of application of *G. fajakuroi* spore suspension. Furthermore, compared to the control group, the activities of chitinase, β-1,3-glucanase, PAL, SOD, POD, and CAT in the treatment group were all significantly lower. These results indicate that the immunity of the over-immunized *G. fajakuroi* plants significantly decreased automatically after 3 days of culture. Notably, even after 5 days of application of *G. fajakuroi* spore suspension, the H2O2 in the treatment group remained significantly higher than that in the control group. This prolonged excessive accumulation of H2O2 can easily lead to membrane lipid peroxidation damage in plant cells. Figure 3 It is evident that the activities of SOD, POD, and CAT in the treated plants were significantly lower than those in the control group, but their MDA content was significantly higher. These results further confirm that the antioxidant capacity of the over-immunized *Gynostemma pentaphyllum* plants was significantly weakened after 3 days of culture, rendering them unable to clear the excessively accumulated H2O2, thus resulting in severe cell membrane lipid peroxidation damage. Therefore, the immunity of the over-immunized *Gynostemma pentaphyllum* plants showed a significant decline after 3 days of continued culture.

[0044] Pathogenic bacteria: Dilute the concentration to 2×10 6 A suspension of *Colletotrichum candida* spores at a concentration of 2.5 mL / ml was sprayed onto the upper and lower surfaces of leaves from unharvested plants in both the control and treatment groups. The plants were then placed in their respective incubators for further cultivation at a temperature of 25±5℃, a relative humidity of 85±5%, a light / dark cycle of 14h / 10h, and a light intensity of 200 μmol·m⁻¹. –2 ·s-1 Two days later, all plants in the treatment and control groups were harvested, with three biological replicates in each group. The number of diseased leaves per plant in both the control and treatment groups was counted, and the disease rate per plant was calculated. The appearance of yellow and black spots of varying sizes, as well as rotten patches on the leaves, was used as the criteria for identifying diseased leaves.

[0045] Depend on Figure 4 As can be seen, the leaves of the control group plants remained healthy, with no yellow or black spots or rotten patches appearing on the leaves. Figure 5 It is evident that the disease incidence rate per plant in the control group was 0.0 ± 0.0%. These results further demonstrate that *Colletotrichum gloeosporioides* has extremely low pathogenicity against *Gynostemma pentaphyllum*, and that simply spraying *Colletotrichum gloeosporioides* is unlikely to cause disease in *Gynostemma pentaphyllum*. However, from Figure 4 As can be seen, the plants in the treatment group exhibited obvious symptoms of collodion anthracnose, with yellow and black spots and rotten patches of varying sizes appearing on the leaves. Figure 5 It can be seen that the disease rate of individual plants in the treatment group was 66.7±16.7%. These results indicate that the application of the beneficial epiphytic fungus G. fajikuroi can weaken the immunity of Gynostemma pentaphyllum and thus aggravate the symptoms of Gynostemma pentaphyllum anthracnose, thereby achieving the purpose of biological control.

[0046] The optimal ambient temperature for applying the spore suspension is 25±5℃ and the relative humidity is 85±5%, which is consistent with the environmental conditions in most areas and can be widely used in most regions.

[0047] Figure 2 and Figure 3 In the table, different uppercase letters indicate significant differences between the treatment group and the control group after 2 days of application of the diluted beneficial epiphytic fungal spore suspension (P < 0.05), different lowercase letters indicate significant differences between the treatment group and the control group after 5 days of application of the diluted beneficial epiphytic fungal spore suspension (P < 0.05), and * and ** indicate significant (P < 0.05) and highly significant (P < 0.01) differences between the same group after 2 days and 5 days of application of the diluted beneficial epiphytic fungal spore suspension, respectively.

[0048] The method described in this embodiment, which uses beneficial epiphytic fungi and pathogenic bacteria in sequence to control *Gynostemma pentaphyllum*, firstly stimulates an excessive immune response in the *Gynostemma pentaphyllum* plant through the application of beneficial epiphytic fungi. Then, the excessively immune *Gynostemma pentaphyllum* plant undergoes further culture to reduce its immunity, thus laying a crucial foundation for the low-pathogenicity *Colletotrichum gloeosporioides* to infect the *Gynostemma pentaphyllum* plant and aggravate its disease symptoms.

[0049] In summary, this method presents a novel approach using the low-pathogenicity *Colletotrichum gloeosporioides* as a biocontrol agent for *Gnaphalium affine*, a green method that weakens the plant's immunity without the need for chemical fungicides. By utilizing the beneficial epiphytic fungus *Gnaphalium affine* (*G. fajikuroi*), the method can quickly induce excessive immunity in *Gnaphalium affine* and subsequently reduce its immunity, avoiding the environmental pollution caused by directly eliminating this beneficial epiphytic fungus (*G. fajikuroi*) with chemical fungicides. This method effectively inflicts severe disease on *Gnaphalium affine* using the low-pathogenicity *Colletotrichum gloeosporioides*, making this low-pathogenicity fungus suitable for biocontrol and solving the problem of a severe shortage of naturally occurring high-pathogenicity biocontrol fungi for *Gnaphalium affine*. Both the beneficial epiphytic fungus and the pathogenic fungus can be isolated from the leaves of *Gnaphalium affine* using simple isolation methods. The equipment required for preparing the spore suspensions of each fungus is simple, high-yield, and low-cost.

[0050] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Those skilled in the art can readily implement the present invention based on the accompanying drawings and the description above. However, any modifications, alterations, or variations made by those skilled in the art without departing from the scope of the present invention, using the disclosed technical content, are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, or variations made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the present invention.

Claims

1. A method for controlling *Gynostemma pentaphyllum* by sequentially using beneficial epiphytic fungi and pathogenic bacteria, characterized in that: Includes the following steps: S1, Stimulate excessive immunity, with a concentration of 3×10 7 A spore suspension of *G. fujikuroi* was sprayed onto the leaves of *Gymnocalycium mihanovichii* plants, with 2.5 ml sprayed onto each leaf. The plants were then placed in an environment with a temperature of 25±5 ℃ and a relative humidity of 85±5% for 2 days. The *G. fujikuroi* spore suspension was isolated, purified, and prepared from bright green, disease-free leaves of the *Gymnocalycium mihanovichii* plants. S2, Immune system weakens, wait 3 days; S3. Pathogenic bacteria are introduced, with a concentration of 2×10⁻⁶. 6 A suspension of *Colletotrichum candida* spores per ml was sprayed onto the leaves of the *Gynostemma pentaphyllum* plant, with 2.5 ml sprayed onto each leaf. The treatment was allowed to proceed for 2 days. The *Colletotrichum candida* spore suspension was isolated, purified, and prepared from diseased leaves of *Gynostemma pentaphyllum* already infected with *Colletotrichum candida*.

2. The method for controlling *Gynostemma pentaphyllum* by sequentially using beneficial epiphytic fungi and pathogenic bacteria according to claim 1, characterized in that: The methods for isolating and purifying G. fujikuroi are as follows: Collect vigorous growth of *Gynostemma pentaphyllum* plants. Select bright green, disease-free leaves from the plant stems. Place 8 leaves in a 250 mL Erlenmeyer flask, add 75 mL of sterile water, seal the mouth of the flask with sealing film, and place the flask on a shaker at 170 rpm for 60 min. In a clean bench, use a sterile pipette tip to draw 200 μL of the suspension and inoculate it onto sterile PDA medium. Spread the suspension evenly, invert all plates, and incubate them in a 26℃ incubator in the dark for 7 days. Pick out each colony from the PDA medium and transfer it to a new sterile PDA medium for purification culture for 2-3 generations.

3. The method for controlling *Gynostemma pentaphyllum* by sequentially using beneficial epiphytic fungi and pathogenic bacteria according to claim 1, characterized in that: The methods for isolating and purifying *Colletotrichum gloeosporioides* are as follows: Collect diseased leaves of *Gynostemma pentaphyllum* infected with *Colletotrichum gloeosporioides*. Use a sterile blade to cut leaf spots from the surface of the *Gynostemma pentaphyllum* and transfer them to sterile PDA medium. Incubate in a 26°C incubator in the dark for 7 days. Pick out each colony on the PDA medium and transfer it to a new sterile PDA medium for purification culture for 2-3 generations.

4. A method for controlling *Gynostemma pentaphyllum* by sequentially using beneficial epiphytic fungi and pathogenic bacteria according to claim 2 or 3, characterized in that: PDA medium is prepared by using 200 g potato, 20 g glucose, 15 g agar, 0.1 g streptomycin sulfate, and 1000 mL deionized water.

5. A method for controlling *Gynostemma pentaphyllum* by sequentially using beneficial epiphytic fungi and pathogenic bacteria according to claim 2 or 3, characterized in that: The method for preparing fungal spore suspension is as follows: The purified *G. fujikuroi* and *Colletotrichum gloeosporioides* colonies were transferred from PDA medium to sterile PDB medium, which was prepared by mixing 200 g of potato, 20 g of glucose, and 1000 mL of deionized water. The medium was then incubated at 26°C in a dark incubator for 24 h. The fungal culture was filtered through three layers of sterile gauze to obtain the spore suspension stock solution. The resulting filtrate was the spore suspension stock solution for both *G. fujikuroi* and *Colletotrichum gloeosporioides*. Both stock solutions were transferred to 50 mL centrifuge tubes and centrifuged at 4000 rpm for 10 min to allow the spores to settle at the bottom of the tube. The upper layer of PDB was discarded, and sterile water was added to the centrifuge tube. The tubes were then shaken to obtain the fungal spore suspension.

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