Ampelomyces quisqualis rg231 and uses thereof

Abstract

The application discloses an Acremonium cavaraeanum RG231, the preservation unit of the Acremonium cavaraeanum RG231 strain is the General Microbiological Center of China Microorganism Culture Collection Committee, and the preservation number is CGMCC No.40817. The application also provides a method for preparing an antibacterial substance by using the Acremonium cavaraeanum RG231. The Acremonium cavaraeanum RG231 strain and / or a fermentation extract thereof provided by the application have good bacteriostatic activity on pathogenic bacteria, and can inhibit the growth of pathogenic bacteria such as Colletotrichum gloeosporioides, Escherichia coli and Micrococcus luteus.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology. More specifically, this invention relates to a strain of pyrophyllosis RG231 and its applications. Background Technology

[0002] Cinnamon (Cinnamomum cassia (L.) D. Don), the dried bark and branch bark of the cinnamon tree, a medium-sized tree in the Lauraceae family. As an important plant used for both food and medicine, cinnamon bark is not only commonly used in Western countries to make cinnamon powder for baking bread and curing meats, but also in China, where dried cinnamon bark was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), listed as a superior-grade herb. It is described as having a pungent, sweet, and hot nature, possessing the effects of tonifying the primordial yang, warming the spleen and stomach, dispelling cold accumulation, and promoting blood circulation. Cinnamon volatile oil is the main active ingredient of cinnamon. Modern pharmacological studies have shown that cinnamaldehyde, the main component of cinnamon volatile oil, has a wide range of pharmacological effects, including strong vasodilatory, anti-gastric ulcer, antibacterial, and antioxidant properties.

[0003] Anthracnose is one of the most important diseases in cinnamon cultivation, causing serious damage and posing a pressing problem in the cinnamon production chain. Currently, the control of anthracnose in cinnamon cultivation mainly relies on chemical pesticides, which easily leads to environmental pollution, pesticide residues, and the development of pesticide resistance.

[0004] In recent years, the emergence of novel coronaviruses and microbial diseases such as monkeypox has posed an unprecedented threat to human life and health. Currently, with the overuse of antibiotics, bacterial resistance is increasing dramatically, becoming one of the most serious problems threatening human survival. Although research into new antibiotics has never ceased, very few have been discovered. Therefore, broadening the channels for antibiotic discovery and accelerating the pace of antibiotic research are urgently needed.

[0005] Plant endophytic fungi are a class of fungi that can infect and colonize healthy plant tissues at a certain stage of their life cycle without causing obvious symptoms in the host plant. Since the discovery of paclitaxel-producing endophytic fungi in the yew tree, research on plant endophytic fungi has experienced explosive growth in the field of physiologically active substances. Plant endophytic fungi possess a strong ability to synthesize secondary metabolites, producing many active substances with significant applications in medicine and pesticides, which can be developed into novel drugs and novel biopesticides. The control effect of plant endophytic fungi on plant diseases is stable and they can persist in the host for a long time; therefore, plant endophytic fungi also have important significance in plant disease prevention. Summary of the Invention

[0006] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0007] One object of the present invention is to provide Acremonium cavaraeanum RG231, which can inhibit the growth of Escherichia coli, Micrococcus luteus, and Anthracnose cinnamon.

[0008] To achieve these objectives and other advantages of the present invention, a strain of Acremonium cavaraeanum RG231 is provided. The depository of this strain is the China General Microbiological Culture Collection Center, with accession number CGMCC No. 40817.

[0009] A method for preparing an antibacterial substance using *Aquilaria lycopersica* RG231 includes the following steps:

[0010] 1) The RG231 strain of *Acer buergerianum* was inoculated onto a PDA plate and cultured. Then, the colonies obtained from the culture were inoculated into sterile potato dextrose liquid medium and cultured in the dark on a shaker to obtain a fermentation mixture.

[0011] 2) The fermentation mixture is subjected to solid-liquid separation to obtain fermentation broth and mycelium;

[0012] 3) Concentrate the fermentation broth under reduced pressure, then add an equal amount of anhydrous ethanol, shake well and soak for 1-5 hours. After filtration, concentrate the filtrate under reduced pressure to a paste to obtain antibacterial substance A.

[0013] 4) Soak the mycelium in anhydrous ethanol, crush it with a pulverizer, then sonicate it for 10-30 minutes, let it stand and soak for 1-5 hours, filter it to obtain the first filtrate and mycelium residue. Add anhydrous ethanol to the mycelium residue, sonicate it for 10-30 minutes, let it stand and soak for 1-5 hours, filter it to obtain the second filtrate, combine the first filtrate and the second filtrate, concentrate it under reduced pressure to a paste to obtain antibacterial substance B.

[0014] Preferably, in step 1), the temperature for incubation in a shaker is 25-30℃, the rotation speed is 100-150rpm, and the culture is carried out in the dark for 10 days.

[0015] An antimicrobial preparation comprising an organic solvent extract of the aforementioned *Acer hygroscopicum* RG231 and / or its fermentation products.

[0016] Application of a bottle-grown pyrophorus fungus RG231 in inhibiting the growth of animal and plant pathogens.

[0017] Preferably, the animal pathogens are Escherichia coli and Micrococcus luteus, and the plant pathogens are Anthracnose cinnamon.

[0018] The present invention has at least the following beneficial effects:

[0019] First, the *Acer oryzae* strain RG231 provided by this invention was deposited on September 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 40817.

[0020] Secondly, the *Acer gargarizans* strain RG231 and / or its fermentation extract provided by this invention have good antibacterial activity against pathogens, and can inhibit the growth of pathogens such as *Anthracnose cinnamon*, *Escherichia coli*, and *Micrococcus luteus*, with an inhibition rate of 64.55% against plant pathogenic fungi.

[0021] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0022] Figure 1 These are morphological images of the strain; where A is the top view; B is the bottom view; and C is the electron microscope view.

[0023] Figure 2 Phylogenetic tree constructed for strain RG231;

[0024] Figure 3 The figures are from an antibacterial experiment; where A is the top view of the treatment group; B is the bottom view of the treatment group; C is the top view of the control group; and D is the bottom view of the control group. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0026] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0027] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0028] <Isolation and Identification of Strains>

[0029] I. Isolation of *Aqueous Species* RG231

[0030] 1.1 Culture medium preparation

[0031] In a clean bench, add streptomycin and tetracycline powder to PDA medium that has been melted and is at a temperature of about 55-65°C, shake to mix thoroughly, and prepare PDA plate medium containing streptomycin and tetracycline. Let it cool and solidify before use.

[0032] 1.2 Biological Sample Collection

[0033] The bark of fresh, healthy cinnamon plants was collected from Zhaoping County, Hezhou City, Guangxi Zhuang Autonomous Region. The fresh, healthy bark was rinsed under running tap water for 10 minutes to remove surface dust. Then, it was transferred to a clean bench and immersed in a 75% ethanol aqueous solution for 30 seconds for disinfection. Next, it was immersed in a 2.5% sodium hypochlorite solution for 5 minutes and rinsed three times with sterile water (the sterile water from the last rinse was inoculated onto PDA agar plates and incubated in an incubator for 30 days. If no colonies grew, it indicated that the plant surface was thoroughly disinfected). This yielded the thoroughly disinfected bark of the cinnamon plant.

[0034] 1.3 Strains Isolation

[0035] After thoroughly sterilizing the cinnamon plant stem bark obtained in step 1.2, blot the moisture with sterile filter paper, trim the edges, and cut into 5mm × 5mm pieces. Inoculate these pieces onto PDA agar plates. Once mycelia have grown on the surface of the tissue pieces, pick the edge portions of the colonies and transfer them to fresh PDA agar. After 3–5 purifications, obtain the endophytic strain. Store the purified strain at 4°C.

[0036] II. Morphological observation of the strain

[0037] The purified strain RG231 obtained in step 1.3 was inoculated onto potato dextrose agar (PDA) and cultured in the dark at 28°C for 10 days. The strain diameter reached 24–26 mm, with uniform edges, and the colony color was white to pale yellow. Figure 1 A), the back is initially wine red, then gradually turns dark red. Figure 1 B), simultaneously producing a wine-red pigment that spreads into the culture medium; the colony's aerial hyphae are well-developed and velvety, while the vegetative hyphae are transparent, thin-walled, septate, and 2–2.5 μm wide, often with warts and branches. The conidiophores are light-colored, smooth, unbranched, and erect on the vegetative hyphae. Figure 1 C); The conidiophores are phialidating cells, 18–30 μm in length and 2.0–2.5 μm in width at the base, with constriction 1 μm from the vegetative hyphae. The conidia are smooth, colorless or pale yellow, unicellular, elliptical or fusiform, and are chained together at the apex of the conidiophore, reaching up to 100 μm in length. The conidia are 3.2–4 (–4.5) × 1.2–2 (–2.5) μm in size, and there are no chlamydospores.

[0038] III. Identification of Strains

[0039] The ITS of strain RG231 was amplified and sequenced. The 18S rDNA sequence of the strain (SEQ ID No. 1) was compared with the relevant DNA sequence in GenBank using BLAST. Multiple sequence homology analysis was performed using MEGA 11.0 software, and a phylogenetic tree was constructed. Figure 2 ITS sequence alignment revealed that the sequence of strain RG231 shared over 99% similarity with the corresponding sequence of *Acremonium cavaraeanum* (accession number QQ429446) in the NCBI database. In the phylogenetic tree, strain RG231 clustered with *Acremonium cavaraeanum* (accession number QQ429446). Based on colony morphology and 18S rDNA sequence analysis, strain RG231 was identified as *Acremonium cavaraeanum*. This *Acremonium cavaraeanum* strain RG231 was deposited on September 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 40817.

[0040] <Preparation of Antibacterial Substances>

[0041] A method for preparing antibacterial substances using *Aquilaria lycopersica* RG231 includes the following steps:

[0042] 1) The *Aquilaria lingua* RG231 strain was inoculated onto a PDA plate medium and cultured. The *Aquilaria lingua* RG231 colonies were punched with a puncher to obtain mycelial cakes with a diameter of 6 mm. Three mycelial cakes were picked and inoculated into a 500 mL Erlenmeyer flask containing 300 mL of sterile potato dextrose liquid medium. The mixture was cultured in the dark at 28 °C and 130 rpm for 10 days in a shaker to obtain a fermentation mixture.

[0043] 2) Filter the fermentation mixture to obtain fermentation broth and mycelium;

[0044] 3) The fermentation broth was concentrated under reduced pressure at 45℃, and then an equal amount of anhydrous ethanol was added. After shaking, it was soaked for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to a paste to obtain antibacterial substance A. It was weighed and stored in a -20℃ refrigerator for later use.

[0045] 4) Soak the mycelium in anhydrous ethanol (enough to soak the mycelium), crush it with a pulverizer, then sonicate for 20 minutes, let it stand and soak for 2 hours, filter to obtain the first filtrate and mycelium residue. Add anhydrous ethanol to the mycelium residue, sonicate for 20 minutes, let it stand and soak for 2 hours, filter to obtain the second filtrate. Combine the first and second filtrates, concentrate under reduced pressure to a paste to obtain antibacterial substance B.

[0046] <Antibacterial Efficacy Test>

[0047] I. Tests on inhibiting plant pathogenic fungi

[0048] The plate confrontation method was used: a 6mm mycelial cake of the plant pathogenic fungus *Anthracnose cinnamon* was inoculated in the center of a PDA plate. A 6mm mycelial cake of *Acer cinnamon* RG231 was inoculated 25mm away using a cross-hatching method. A control group was inoculated with a 6mm PDA cake using the same cross-hatching method at a distance of 25mm from the pathogenic mycelial cake. The plates were then incubated at 28℃ for 7 days before observation. Each group had three replicates. The presence of an inhibition zone between the colonies in the plate confrontation was used to determine whether there was an inhibitory effect. If an inhibition zone existed, a straight line was drawn between the center points of the two colonies on the treatment plate, and the growth radius of the pathogenic fungus colony in the treatment group was measured with a ruler (see Table 1), denoted as r1. Figure 3 A, Figure 3 B), the growth radius of the pathogenic fungus in the control group was measured (see Table 1) and recorded as r0( Figure 3 C, 3D). Calculate the antibacterial rate using the following formula.

[0049] Antibacterial rate (%) = (r0-r1) / (r0-r j )*100;

[0050] r0 is the growth radius of colonies when only pathogenic bacteria are inoculated;

[0051] r1 is the growth radius of the pathogenic bacteria in the confrontation culture;

[0052] r j The radius of the mushroom cake.

[0053] The inhibition rate was calculated to be 64.55%.

[0054] Table 1 Results of antibacterial tests of RG231 against plant pathogens

[0055]

[0056] II. Antibacterial test of antimicrobial substances

[0057] 1. Prepare active test samples

[0058] The antibacterial substances A and B prepared above were dissolved in dimethyl sulfoxide (DMSO) to prepare a test sample solution of 10 mg / ml.

[0059] 2. Antibacterial test

[0060] 2.1 Antimicrobial test of bacterial cells

[0061] Antimicrobial screening was performed using the bacterial cake method. Activated pathogens (Escherichia coli, Micrococcus luteus) were inoculated into a liquid beef extract peptone medium at approximately 40°C (i.e., when it is not hot to the touch, at which point it is liquid). The medium consisted of accurately weighed 10.0 g peptone, 15.0 g agar powder, 5.0 g beef extract, and 5.0 g sodium chloride, dissolved in 1000 mL deionized water, and the pH was adjusted to 7.2–7.4. The medium was then sterilized at 121°C for 20 min in a high-temperature, high-pressure autoclave. The resulting culture had a bacterial concentration of approximately 1 × 10⁻⁶. -6 cfu·ml -1 ~1×10 -7 cfu·ml -1 The bacterial beef extract peptone medium was promptly poured into sterile petri dishes. After the medium cooled and solidified into plates, sterile perforators with a diameter of 6 mm were used to make evenly spaced wells on the plates after heating with an alcohol lamp. The agar blocks were then removed to prepare the experimental strain plates. Agar blocks of the same size were punched from the activated *Acer oryzae* RG231 culture medium on PDA plates and transferred into the pre-punched wells of the experimental strain plates. 20 μL of 10 mg / ml streptomycin sulfate and tetracycline were used as positive controls, respectively, while the PDA agar blocks served as blank controls. The experiment was repeated three times. After incubation at 37℃ for 24 h, the diameter of the inhibition zone and antibacterial activity were measured (see Table 2).

[0062] 2.2 Antibacterial test of antibacterial substances

[0063] Antibacterial experiments were conducted using the perforation method. For example, in 2.1, pathogenic bacteria plates were prepared. After removing the agar block, 20 μL of the test sample solution was injected into the wells. 20 μL of streptomycin sulfate (10 mg / ml) and tetracycline were used as positive controls, and 20 μL of DMSO was injected as a blank control. The experiment was repeated three times. After incubation at 37℃ for 24 h, the diameter of the inhibition zone and antibacterial activity could be measured (see Table 2).

[0064] Table 2 Results of antibacterial tests against Escherichia coli and Micrococcus luteus

[0065]

[0066] As shown in Table 2, the *Acer buergerianum* RG231 and its fermentation extract of the present invention have good antibacterial activity and can significantly inhibit the growth of pathogenic bacteria *Escherichia coli* and *Micrococcus luteus*.

[0067] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. Acremonium cavaraeanum RG231, characterized in that, The culture vessel containing the RG231 strain of Acetobacter oryzae is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 40817.

2. A method for preparing antibacterial substances using *Aqueous Chlorella vulgaris* RG231, characterized in that, Includes the following steps: 1) The RG231 strain of *Acer buergerianum* as described in claim 1 is inoculated onto a PDA plate medium and cultured. Then, the colonies obtained from the culture are inoculated into a sterile potato dextrose liquid medium and cultured in the dark on a shaker to obtain a fermentation mixture. 2) The fermentation mixture is subjected to solid-liquid separation to obtain fermentation broth and mycelium; 3) Concentrate the fermentation broth under reduced pressure, then add an equal amount of anhydrous ethanol, shake well and soak for 1-5 hours. After filtration, concentrate the filtrate under reduced pressure to a paste to obtain antibacterial substance A. 4) Soak the mycelium in anhydrous ethanol, crush it with a pulverizer, then sonicate it for 10-30 minutes, let it stand and soak for 1-5 hours, filter it to obtain the first filtrate and mycelium residue. Add anhydrous ethanol to the mycelium residue, sonicate it for 10-30 minutes, let it stand and soak for 1-5 hours, filter it to obtain the second filtrate, combine the first filtrate and the second filtrate, concentrate it under reduced pressure to a paste to obtain antibacterial substance B.

3. The method for preparing antibacterial substances using *Aqueous Chlorella vulgaris* RG231 according to claim 2, characterized in that, Step 1) Incubate in a shaker at a temperature of 25-30℃ and a rotation speed of 100-150 rpm for 10 days in the dark.

4. An antibacterial agent, characterized in that, It includes the genus Acetobacter RG231 as described in claim 1.

5. The application of the jarophyte RG231 as described in claim 1 in inhibiting the growth of Escherichia coli, Micrococcus luteus, and Anthracnose cinnamon.

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