Application of extract of entophytic fungus in hibiscus yangii in preparing medicine for resisting plant fungal disease

Abstract

The application discloses application of an extract of an endophytic fungus of Broussonetia papyrifera in preparation of a medicine for resisting plant fungal diseases, wherein the endophytic fungus of Broussonetia papyrifera is an endophytic fungus (Colletotrichum sp.) J1-20 with a preservation number of CCTCC NO: M20231761, a fermentation strain, a fermentation culture obtained through liquid fermentation, mycelia and a fermentation liquor, the mycelia are extracted by a mixed solvent composed of ethyl acetate, methanol and acetic acid, and the extract is obtained by concentration of the extract. The applicant finds in experiments that the endophytic fungus of Broussonetia papyrifera or the mycelium extract has inhibiting effects on plant diseases caused by Colletotrichum gloeosporioides, Colletotrichum gloeosporioides, Colletotrichum gloeosporioides, Fusarium oxysporum, Pseudomonas syringae and Alternaria alternata, and is expected to be used in preparation of corresponding plant disease medicines.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of an extract of endophytic fungi from *Hibiscus syriacus* in the preparation of drugs for treating plant fungal diseases. Background Technology

[0002] Mangroves are a group of salt-tolerant plant species that grow in tropical and subtropical intertidal estuaries, possessing a unique ecosystem. They can be divided into mangrove and semi-mangrove plants. Endophytic fungi are an important component of the plant micro-ecosystem and are widely present in healthy plant tissues. Mangrove endophytic fungi can tolerate various stresses and exhibit outstanding biological activity by producing a large number of metabolites with special biological functions.

[0003] *Thespesia populnea* is a semi-mangrove medicinal plant belonging to the genus *Thespesia* in the family Malvaceae. It possesses various pharmacological activities, including antibacterial, antioxidant, antidiabetic, antihyperlipidemic, antitumor, and hepatoprotective effects. However, research on its endophytic fungi and active substances is currently limited. Xiao Shenglan et al. screened eight endophytic fungi from *Thespesia populnea*, one of which showed strong antibacterial activity against *Escherichia coli* (Xiao Shenglan, Lei Xiaoling, She Zhigang, et al. Isolation and identification of eight mangrove endophytic fungi in Shankou, Guangxi and screening of antibacterial strains [J]. Journal of Tropical Crops, 2011, 32(12):2259-2263.). Lin Aiyu et al. screened 12 endophytic fungi from *Pinus sylvestris*, two of which showed strong antibacterial activity against *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans* (Lin Aiyu, Xing Xiaoke, Guo Shunxing, et al. Isolation and antibacterial activity of endophytic fungi from four medicinal semi-mangrove plants [J]. Chinese Pharmaceutical Journal, 2006, 41(12): 892-894.). Furthermore, invention patents CN114452309A and CN114404460A disclose the application of fermentation extract of *Penicillium sp. YX-002* (GDMCC NO: 62179), an endophytic fungus from *Pinus sylvestris*, as an active ingredient in the preparation of anti-inflammatory drugs and drugs that scavenge DPPH free radicals. However, there are currently no reports of endophytic fungi screened from *Pinus sylvestris* exhibiting antibacterial activity against plant wilt and plant diseases caused by *Alternaria*. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide the application of mycelial extracts of endophytic fungi of *Hibiscus syriacus* in the preparation of drugs against plant fungal diseases.

[0005] The technical solution of this invention is: the application of endophytic fungi of *Hibiscus syriacus* or mycelial extracts of endophytic fungi of *Hibiscus syriacus* in the preparation of drugs against plant fungal diseases, wherein,

[0006] The endophytic fungus described is Colletotrichum sp. J1-20, which was deposited on September 20, 2023, at the China Center for Type Culture Collection (CCTCC, address: No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, within the campus of Wuhan University), with accession number CCTCC NO: M 20231761.

[0007] The mycelial extract of the endophytic fungus Colletotrichum sp. mentioned above was obtained by using Colletotrichum sp. J1-20 as the fermentation strain, obtaining the fermentation culture through liquid fermentation, separating the mycelium and fermentation broth, extracting the mycelium with a mixed solvent composed of ethyl acetate, methanol and acetic acid, and concentrating the extract.

[0008] The plant fungal diseases mentioned include at least one of the following: plant anthracnose, plant wilt, and diseases caused by Alternaria.

[0009] In the above technical solution, the endophytic fungus *Colletotrichum sp.* J1-20 is screened from *Colletotrichum sp.*. The liquid fermentation involves placing *Colletotrichum sp.* J1-20 in PD liquid medium and culturing it in a shaker or static environment at 25–30°C to obtain the fermentation culture. When using shaker culture, the preferred rotation speed is 150–200 r / min, and the preferred culture time is 7–10 days. When using static culture, the preferred culture time is 20–30 days. The PD liquid medium used in the liquid fermentation is a conventional choice in the prior art, and its composition can be: 180–220 g of potato, 18–22 g of glucose, and 1000 mL of 50% artificial seawater; a more preferred composition is 200 g of potato, 20 g of glucose, and 1000 mL of 50% artificial seawater. The formula for the artificial seawater (salinity 3.34%) is as follows: per liter of water, it contains 26.726g of sodium chloride (NaCl), 2.26g of magnesium chloride (MgCl2), 3.248g of magnesium sulfate (MgSO4), 1.153g of calcium chloride (CaCl2), 0.198g of sodium bicarbonate (NaHCO3), 0.721g of potassium chloride (KCl), 0.058g of sodium bromide (NaBr), 0.058g of boric acid (H3BO3), 0.0024g of sodium silicate (Na2SiO3), 0.0015g of sodium silicate (Na2Si4O9), 0.002g of phosphoric acid (H3PO4), 0.013g of aluminum hexachloride (Al2Cl6), 0.002g of ammonia (NH3), and 0.0013g of lithium nitrate (LiNO3).

[0010] In the above technical solution, the volume ratio of ethyl acetate, methanol and acetic acid in the mixed solvent is preferably 70-80:15-20:4-8, and more preferably 75-80:15-18:5-8.

[0011] In the above technical solution, the plant fungal diseases specifically include at least one of the following: persimmon anthracnose, walnut anthracnose, litchi anthracnose, banana wilt, cucumber leaf blight, and plant diseases caused by Alternaria.

[0012] The present invention also includes a drug for treating plant fungal diseases, which contains a mycelial extract of Colletotrichum sp. J1-20 or Colletotrichum sp. J1-20.

[0013] The applicant discovered in experiments that the endophytic fungus Colletotrichum sp. J1-20 or its mycelial extract has a significant inhibitory effect on plant diseases caused by *Colletotrichum persimmon*, *Colletotrichum walnut*, *Colletotrichum litchi*, *Fusarium wilt* of banana, *Fusarium solani* of cucumber, and *Alternaria alternata*. It is expected to be used to prepare drugs against *Colletotrichum persimmon*, *Colletotrichum walnut*, *Colletotrichum litchi*, *Fusarium wilt* of banana, *Fusarium solani* of cucumber, and plant diseases caused by *Alternaria alternata*. Attached Figure Description

[0014] The endophytic fungus Colletotrichum sp. J1-20 involved in this invention was deposited on September 20, 2023, at the China Center for Type Culture Collection (CCTCC, address: No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, within Wuhan University), with accession number CCTCC NO: M20231761.

[0015] Figure 1 The images show the morphology of the endophytic fungus strain J1-20 of *Colletotrichum sp.* described in this invention on a culture medium, wherein (a) is a colony photograph on PDA medium; (b) is a mycelial photograph of the strain under an optical microscope (magnification 10×40); and (c) is a mycelial photograph of the strain under an optical microscope (magnification 10×40).

[0016] Figure 2 This is the ITS sequence phylogenetic tree of Colletotrichum sp. J1-20, the endophytic fungus of Populus tomentosa described in this invention.

[0017] Figure 3These are photographs of the antifungal activity experiments of the endophytic fungus *Colletotrichum sp.* J1-20 described in this invention. In the photographs, A shows the inhibition of *Anthracnose spp.* by the strain; B shows the inhibition of *Anthracnose spp.* by the strain; C shows the inhibition of *Anthracnose spp.* by the strain; D shows the inhibition of *Fusarium wiltii* var. *banana* (FOC1); E shows the inhibition of *Fusarium wiltii* var. *banana* (FOC4); F shows the inhibition of *Alternaria cucumeroides* var. *cucumeroides* by the strain; and G shows the inhibition of *Alternaria spp.* var. *cucumeroides* by the strain. Detailed Implementation

[0018] To better explain the technical solution of the present invention, the present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0019] The PDA culture medium used in the following examples consisted of: 200g potato, 20g glucose, 20g agar, and 1000mL of 50% artificial seawater. The artificial seawater (salinity 3.34%) was formulated as follows: per liter of water, it contained 26.726g sodium chloride, 2.26g magnesium chloride, 3.248g magnesium sulfate, 1.153g calcium chloride, 0.198g sodium bicarbonate, 0.721g potassium chloride, 0.058g sodium bromide, 0.058g boric acid, 0.0024g sodium silicate, 0.0015g sodium silicate, 0.002g phosphate, 0.013g aluminum hexachloride, 0.002g ammonia, and 0.0013g lithium nitrate.

[0020] The PD liquid culture medium used in the following examples consists of: 200g potato, 20g glucose, and 1000mL of 50% artificial seawater; wherein the formulation of the artificial seawater (salinity 3.34%) is as described above.

[0021] Example 1: Isolation and identification of Colletotrichum sp. J1-20, an endophytic fungus of Populus tomentosa.

[0022] 1. Isolation of fungi

[0023] The stems, leaves, and bark of *Hibiscus syriacus* treated with 5v / v% hypochlorous acid and 75v / v% ethanol were cut into 5mm × 5mm (length × width) pieces using sterile scissors. These pieces were then placed on PDA agar plates and incubated upside down at 25–30℃ for 10–15 days. The growth of the tissues on the agar plates was observed periodically to obtain well-grown strains. When mycelia appeared at the cut surfaces of the plant tissues, the edges of the well-grown mycelia were selected and transferred to purification agar plates (PDA agar, formulation as described above) for further cultivation. After multiple separation and purification processes, the strain was obtained.

[0024] 2. Fungal culture and morphological observation

[0025] Culture characteristics observation: The bacterial strain was inoculated onto PDA medium and incubated at 26℃ to obtain colonies. The color of the initial hyphae, changes in colony color, surface characteristics, and presence or absence of pigment production were recorded.

[0026] Slide preparation and microscopic examination: Sporulation organs and hyphae of each strain were selected to prepare water-sealed slides, stained with cotton blue solution, observed under a microscope, and recorded the hyphal characteristics, branching, spore and sporulation organ structure, and photographed.

[0027] The results are as follows Figure 1 As shown: The fungus grows well on PDA medium. The colonies are dark green, with stiff, felt-like aerial hyphae. The hyphae at the colony edge and within the substrate are yellowish-white. The colonies grow slowly, adhere tightly to the medium, and are difficult to pick up. Small black spherical structures are present on the colony surface. No spore production was observed. The hyphae have septa, and the tips of the hyphae are swollen into spherical shapes.

[0028] 3. Extraction of total fungal DNA

[0029] The isolated endophytic fungi were inoculated into PD liquid medium and cultured in shake flasks at 28°C for 8 days. The mycelium was then collected by filtration. The mycelium underwent two freeze-thaw cycles in liquid nitrogen, was ground with a sterile glass rod, and the fungal genome was extracted using the "Fungal Genomic DNA Mini-Extraction Kit" from USA Omega Bio-Tek.

[0030] 4. Molecular identification of ITS in fungi

[0031] The spacer sequence (containing the ITS1, 5.8S, and ITS2 regions) of fungal rDNA was amplified using primers ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO: 1) and ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO: 2). The PCR reaction conditions were: denaturation at 94℃ for 5 min, followed by 35 cycles of amplification at 94℃ for 30 s → 55℃ for 40 s → 72℃ for 1.0 min, and a final extension at 72℃ for 10 min. The purified PCR product was directly sent to Genewiz Biotechnology Co., Ltd. for sequencing using primer ITS1. The obtained ITS sequencing sequence is shown below:

[0032] (SEQ ID NO: 3).

[0033] The obtained ITS sequencing sequences were analyzed for similarity in GenBank using Blasten. The strain with the highest similarity was Colletotrichum aotearoa (NR_120136), with a similarity of 100%.

[0034] Phylogenetic analysis of the strain revealed that the isolated strain was phylogenetically aligned with Colletotrichum gloeosporioides, Colletotrichum aotearoa, etc., on the same branch. The resulting ITS sequence phylogenetic tree is shown below. Figure 2 As shown.

[0035] Based on the combined morphological and molecular biological identification results, this strain can be identified as a fungus of the genus Colletotrichum. Molecular biological identification results indicate that its phylogenetic relationship is closest to that of Colletotrichum gloeosporioides. We tentatively name this strain Colletotrichum sp.J1-20.

[0036] Example 2: Extraction of active substances from Colletotrichum sp. J1-20, an endophytic fungus of Populus tomentosa.

[0037] 1. Seed culture

[0038] Select fungal hyphae and inoculate them onto PDA medium for slant culture, and culture at 28℃ for 5–7 days.

[0039] 2. Fermentation culture

[0040] The fungi cultured on slant were transferred to PD liquid medium and cultured in a shaker at 140 r / min for 7–10 days at 25–30°C (or static culture for 20–30 days) to obtain the fermentation culture.

[0041] 3. Filtering

[0042] The above fermentation culture was filtered through four layers of gauze, and the mycelium and fermentation broth were collected separately.

[0043] 4. Preparation of mycelial extract

[0044] The collected mycelium was rinsed with water to remove the fermentation broth and dried. Then it was extracted with a mixed solvent consisting of ethyl acetate, methanol and acetic acid (volume ratio of 80:15:5) at a temperature of 15-20℃ for 3-4 days. After filtration, the extract was placed in a rotary evaporator and concentrated to a small volume at 55℃ and a speed of 50 r / min. The solvent was then evaporated in a water bath at 70℃ to obtain the mycelial extract of Colletotrichum sp. J1-20 (hereinafter referred to as mycelial extract).

[0045] Example 3: Antibacterial activity of Colletotrichum sp. J1-20, an endophytic fungus of Populus tomentosa, and its mycelial extract.

[0046] 1. Preparation of indicator bacteria

[0047] Indicator fungi *Colletotrichum gloeosporioides*, *Gloeosporium fructigenum*, *Colletrichum litchi Trag.*, *Fusarium oxysporum*, *Alternaria cucumerina*, and *Alternaria burnsii* were inoculated onto PDA agar plates and cultured to half-fullness. These bacteria and fungi were all preserved in our laboratory. Each indicator fungus was inoculated onto two plates.

[0048] 2. Detection of antifungal activity

[0049] The antifungal activity of Colletotrichum sp. J1-20 and its mycelial extracts was screened using the plate confrontation method.

[0050] 2.1 Antifungal activity of endophytic fungi in *Hibiscus syriacus*

[0051] The colony radius (Rc) of the indicator bacteria cultured alone and the directional radius (Rp) of the opposing cultures were determined using the cross-multiplication method, and the inhibition rate was calculated to determine the inhibitory effect of the endophytic fungus *Colletotrichum sp.* J1-20 on the indicator bacteria. Inhibition rate = (Rc - Rp) / Rc × 100%. Indicator bacteria: Observe the inhibition results, such as... Figure 3 As shown in Table 1 below. A blank control was prepared using the mixed solvent (ethyl acetate:methanol:acetic acid = 80:15:5, volume ratio). The experiment was repeated three times.

[0052] Table 1: Antifungal activity of Colletotrichum sp. J1-20, an endophytic fungus of Hibiscus syriacus.

[0053]

[0054] Note: Ⅰ: Mixed solvent; Ⅱ: Colletotrichum sp. J1-20, an endophytic fungus of Populus tomentosa.

[0055] 2.2 Antifungal activity of mycelial extracts from endophytic fungi of *Hibiscus syriacus*

[0056] The mycelial extracts prepared in Example 2 were weighed and diluted with sterile water to concentrations of 400 μg / ml, 200 μg / ml, 100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, and 6.25 μg / ml, respectively. The antifungal activity of the test solutions was detected using a perforation method. Using a 5 mm diameter perforator, holes were punched at the edge of colonies of indicator fungi cultured for 2–4 days. 40 μL of the test solution was then placed into each hole and incubated at 26°C for 2 days. The antifungal effect was then observed. The mixed solvent used in the extraction (ethyl acetate:methanol:acetic acid = 80:15:5, volume ratio) was used as a blank control. The experiment was repeated three times. The results are shown in Table 2 below.

[0057] Table 2: Antifungal activity of mycelial extract (100 μg / ml) of Colletotrichum sp. J1-20 endophytic fungus.

[0058]

[0059] Note 1: Ⅰ: Mixed solvent; Ⅱ: Mycelial extract.

[0060] Note 2: +: has antibacterial activity; -: has no antibacterial activity.

Claims

1. The application of mycelial extracts of endophytic fungi of *Hibiscus syriacus* or *Hibiscus syriacus* in the preparation of drugs against plant fungal diseases, among which, The endophytic fungi of *Hibiscus syriacus* are endophytic fungi of *Hibiscus syriacus* ( Colletotrichum sp.)J1-20, its accession number is CCTCC NO: M 20231761; The mycelial extract of the endophytic fungus of Hibiscus syriacus is derived from the endophytic fungus of Hibiscus syriacus ( Colletotrichum sp.)J1-20 was used as a fermentation strain. Fermentation culture was obtained through liquid fermentation. Mycelium and fermentation broth were separated. Mycelium was extracted with a mixed solvent composed of ethyl acetate, methanol and acetic acid. The extract was concentrated. The volume ratio of ethyl acetate, methanol and acetic acid in the mixed solvent was 70~80:15~20:4~8. The plant fungal disease is Alternaria cucumeroides ( Alternaria cucumerina ) or Alternaria alternata ( Alternaria burnsii Diseases caused by ) 2. The application according to claim 1, characterized in that, The liquid fermentation is carried out in PD liquid medium at 25~30℃ using either a shaker culture or a static culture.

3. The application according to claim 2, characterized in that, The shaking incubation period is 7-10 days, and the static incubation period is 20-30 days.

4. A drug for treating plant fungal diseases, comprising an endophytic fungus of *Hibiscus syriacus* or a mycelial extract of an endophytic fungus of *Hibiscus syriacus*, wherein the endophytic fungus of *Hibiscus syriacus* is an endophytic fungus of *Hibiscus syriacus* (…). Colletotrichum sp.)J1-20, its accession number is CCTCC NO: M 20231761; The mycelial extract of the endophytic fungus of Hibiscus syriacus is derived from the endophytic fungus of Hibiscus syriacus ( Colletotrichum sp.)J1-20 was used as a fermentation strain. Fermentation culture was obtained through liquid fermentation. Mycelium and fermentation broth were separated. Mycelium was extracted with a mixed solvent composed of ethyl acetate, methanol and acetic acid. The extract was concentrated. The volume ratio of ethyl acetate, methanol and acetic acid in the mixed solvent was 70~80:15~20:4~8. The plant fungal disease is Alternaria cucumeroides ( Alternaria cucumerina ) or Alternaria alternata ( Alternaria burnsii Diseases caused by )

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