Astragalus mongolicus endophytic fungus and application thereof

Abstract

The invention discloses astragalus mongolicus endophytic fungi and application thereof, and belongs to the technical field of plant disease prevention and control. The endophytic fungus is separated from a leguminous plant astragalus mongholicus living body by adopting an endophytic fungus separation and purification technology, belongs to ascomycophyta, hypocrea, cordyceps and coremia lanceolata fungi, is classified and named as Akanthomyces lecanii AMH95, and has a strain preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 41956. The endophytic fungi can significantly inhibit the growth of various plant pathogens, and the relative bacteriostasis rates are respectively 70.47%, 63.96%, 64.14%, 81.39% and 85.40%. The endophytic fungi have a good inhibition effect on various plant pathogens, and a biocontrol strain is provided for prevention and treatment of fungal diseases of medicinal plants and crops.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant disease prevention and control, in particular to a Mongolian Astragalus membranaceus endophytic fungus and its application in inhibiting the growth of plant pathogenic bacteria. BACKGROUND

[0002] Mongolian Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao is a perennial herb of Leguminosae, and its dried root is used as a medicinal material of Astragalus membranaceus, which has the effects of tonifying qi and ascending yang, consolidating exterior and stopping sweating, promoting water and reducing swelling, producing saliva and nourishing blood, removing stagnation and relieving arthralgia, removing pus and discharging abscess, and astringing and granulating flesh, and can be used for treating qi deficiency and debilitation, poor appetite and loose stool, middle qi collapse, chronic diarrhea and prolapse of the rectum, metrorrhagia and menorrhagia, exterior deficiency and spontaneous sweating, qi deficiency and edema, internal heat and polydipsia, blood deficiency and emaciation, hemiplegia, arthralgia and numbness, and ulcer and chronic sore.

[0003] Plant diseases are an important problem in current agricultural production, and traditional chemical control methods are effective, but can easily cause environmental pollution and increase the drug resistance of pathogenic bacteria. As a natural biological control resource, plant endophytic fungi have the characteristics of environmental friendliness and strong sustainability. Studies have shown that plant endophytic fungi can enhance the disease resistance of plants through various ways, including direct inhibition of pathogenic bacteria growth, ecological niche competition, and induction of systemic resistance in plants, and these characteristics make endophytic fungi an important strategy for biological control and sustainable development of traditional Chinese medicinal materials.

[0004] Endophytic fungus refers to a kind of fungus living in plant cells or plant tissues at a certain period of its life history, which does not cause obvious diseases to plant tissues. Endophytic fungi are various, distributed in different parts of different parasitic plants, can produce various metabolites, and have the effects of antifungal, antiviral, insecticidal and the like. In the long-term evolution process, endophytic fungi and plant hosts gradually form a complex symbiotic relationship, which affects plant growth and development, participates in physiological metabolism and immune defense response, and researches show that after endophytic fungi are inoculated into host plants or specific plants, the resistance of plants to pathogenic microorganisms can be enhanced, and the yield of crops can be improved. Ding Ting et al. found that the inhibition rate of the fermentation liquor of Thielavia strain DZGS08, an endophytic fungus of Eucommia ulmoides, to Rhizoctonia solani reached more than 50%. Hu Lijie et al. found that the inhibition rate of NQ8GII4, an endophytic fungus of Lycium barbarum, to Colletotrichum gloeosporioides reached 93.43%. Li Fengfu et al. found that the ethyl acetate extract of Schizophyllum commune could significantly inhibit the mycelial growth and spore germination of Fusarium oxysporum, and the inhibition rate reached 56.75% after 7 days of culture at a minimum inhibitory mass concentration of 5.00 mg / mL. In summary, endophytic fungi are important microbial resources in the field of plant prevention and control technology.

[0005] However, there is no report on the application of endophytic fungi of Mongolian milkvetch in inhibiting the growth of pathogenic bacteria. SUMMARY

[0006] The present application aims at the deficiencies of the prior art, and provides an endophytic fungus of Mongolian milkvetch and its application in inhibiting the growth of pathogenic bacteria.

[0007] In a first aspect, the present application provides an endophytic fungus of Mongolian milkvetch, which belongs to Ascomycota, Xylariomycetes, Cordycipitaceae and Akanthomyces lecanii AMH95. The endophytic fungus of Mongolian milkvetch is obtained by using endophytic fungus separation and purification technology from the living body of the perennial herbaceous plant Mongolian milkvetch, and is identified as Akanthomyces lecanii by microbiological taxonomy. The strain has been preserved, the strain preservation number is CGMCC No.41956, the preservation date is May 26, 2025, the preservation unit is China General Microbiological Culture Collection Center (CGMCC), the address is No.3, Beichen West Road, Chaoyang District, Beijing, and the postcode is 100101.

[0008] The solid culture characteristics of the endophytic fungus are as follows:

[0009] The growth rate is moderate, the colony is white and slightly thick, the middle part is slightly raised, the reverse center of the colony can be seen yellowish bacterial ring, the surface is dense and soft, the edge mycelium is thin and the edge mycelium is thin, and the edge mycelium is thin. Figure 1 .

[0010] The morphological characteristics of the endophytic fungus under an optical microscope are as follows: the mycelium is colorless and transparent, thin and thin-walled, smooth surface, and spores are clearly visible around the mycelium. Figure 2 .

[0011] The ITS molecular sequence of the endophytic fungus is shown as SEQ ID NO. 1. The sequencing result is subjected to sequence (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) on the NCBI website, and it is found that the sequence homology of 3 reference strains Akanthomyces lecanii (MW165534), Akanthomyces lecanii (OP437999) and Akanthomyces lecanii (MH312007) of Akanthomyces lecanii is 99.03%, 99.34% and 99.34% respectively. MEGA7.0 is used for multiple sequence alignment, and the reference sequence of lodophanus taxi is used as an outgroup, and the neighbor-joining method (NJ) is used for analysis, and the alignment is repeated 1000 times. The ITS agarose gel electrophoresis map and phylogenetic tree of the endophytic fungus are shown in Figure 3 、 Figure 4 .

[0012] The second aspect of the present application provides the application of the above-mentioned endophytic fungus in inhibiting the growth of plant pathogenic fungi.

[0013] Specifically, the AMH95 fungus is inoculated into the PDA culture medium, and is co-cultured with the pathogenic fungi Fusarium oxysporum, Bipolaris sorokiniana, Rhizoctonia solani, Colletotrichum gloeosporioides and Fusarium solani, so that the growth of the pathogenic fungi can be significantly inhibited.

[0014] The above-mentioned application is mainly realized through the following technical solutions:

[0015] (1) Take an AMH95 cryovial and, under sterile conditions, pick a small amount of mycelium with an inoculating needle and inoculate it onto a sterilized PDA medium. The medium is formulated as follows: 200 g potato, 20 g glucose, 15 g agar, 1000 mL distilled water, pH natural, incubate in the dark at 26°C and 65% humidity for 5 days. Once the mycelium has fully grown on the plate, it is ready for use.

[0016] (2) In a clean bench, use a marker to mark a dot at the center of the bottom of the culture medium, and then mark a dot every 120° at a distance of 2.25 cm from the center on a line passing through the center radius. Under sterile conditions, use a sterilized punch to punch three AMH95 bacterial cakes at the edge of the colony, and use tweezers to inoculate the bacterial cakes into the three marked points on the culture medium.

[0017] (3) Use a sterilized punch to punch out the bacterial cakes of the pathogens Fusarium oxysporum, Bipolaris sorokiniana, Rhizoctonia solani, Colletotrichum gloeosporioides, and Fusarium solani at the edge of the colony and inoculate them into the center of the culture medium. The experiment was repeated three times.

[0018] (4) All culture media were sealed and placed in a 26°C mold incubator with 65% humidity for 5-7 days in the dark. The morphology and diameter of the pathogens in each group were observed and the inhibition rate was calculated. The results showed that AMH95 significantly inhibited the growth of the hyphae of the above pathogens ( Figure 5 、 Figure 6 ).

[0019] The present invention has the advantage that the endophytic fungus Akanthomyces lecanii AMH95 of Astragalus membranaceus can effectively inhibit the growth of plant pathogens Fusarium oxysporum, Bipolaris sorokiniana, Rhizoctonia solani, Colletotrichum gloeosporioides, and Fusarium solani, with inhibition rates of 70.47%, 63.96%, 64.14%, 81.39%, and 85.40%, respectively. The endophytic fungus of Astragalus membranaceus has broad application prospects in the field of biological control of plant fungal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments;

[0021] Figure 1:The colony morphology of Akanthomyces lecanii AMH95 on PDA solid medium;

[0022] Figure 2 :Mycelial and spore morphology of Akanthomyces lecanii AMH95 under optical microscope;

[0023] Figure 3 :Gel electrophoresis of the Akanthomyces lecanii AMH95 sequence amplified based on ITS5 and ITS4 primers;

[0024] Figure 4 : Phylogenetic tree of Akanthomyces lecanii AMH95 based on ITS sequences (NJ method);

[0025] Figure 5 : Co-culture of Akanthomyces lecanii AMH95 and pathogens; (T1 is the pathogen group, from left to right are the colony morphology of pathogens Fusarium oxysporum, Bipolaris sorokiniana, Rhizoctonia solani, Colletotrichum gloeosporioides and Fusarium solani; T2 is the positive bacteria treatment group, from left to right are Trichoderma harzianum Colony morphology of co-culture of AMH95 with Fusarium oxysporum, Helminthosporium spp., Rhizoctonia solani, Colletotrichum gloeosporioides, and Fusarium solani; T3 is the AMH95 treatment group, from left to right, colony morphology of co-culture of AMH95 with Fusarium oxysporum, Helminthosporium spp., Rhizoctonia solani, Colletotrichum gloeosporioides, and Fusarium solani);

[0026] Figure 6 : The results of the inhibitory effect of Akanthomyces lecanii AMH95 on the growth of pathogens; (A: Fusarium oxysporum group; B: Helminthomyces solani group; C: Rhizoctonia solani group; D: Colletotrichum gloeosporioides group; E: Fusarium solani group; F: AMH95 inhibition rate). DETAILED DESCRIPTION

[0027] The endophytic fungus of the present invention is separated from the living body of healthy fresh plants of Mongolian Astragalus in Hohhot City, Inner Mongolia Autonomous Region.

[0028] Example 1:

[0029] The endophytic fungus was isolated by the following steps: fresh Astragalus mongolica plants were rinsed 3-4 times with tap water to remove any soil from the plant surface, and then the surface moisture was blotted dry with sterile filter paper. A three-step disinfection method was used: soaking in 75 vol% ethanol for 1 minute, rinsing with 2.5 vol% sodium hypochlorite in a 3-minute root: stem: leaf pattern (3 minutes: 1 minute: 30 seconds), rinsing again with 75 vol% ethanol for 30 seconds, and rinsing with sterile water five times until the surface ethanol was completely rinsed. The roots, stems, and leaves were cut into small pieces (0.5 cm × 0.5 cm × 0.5 cm) using a sterile scalpel and directly inoculated onto PDA culture medium (PDA medium formulation: 200 g potato, 20 g glucose, 1000 mL distilled water, 15 g agar, natural pH). The culture dish was sealed with parafilm and incubated in a 26°C incubator for 5 days. The growth of the endophytic fungus colonies was observed. Once the endophytic fungus had fully grown, it was purified using an inoculation needle until a single colony was formed. Classification is based on the color, morphology and other characteristics of hyphae and colonies. Figure 1 As shown, it was cultured in the dark at 26℃ on potato dextrose agar (PDA) medium. The growth rate was medium. The colonies were white, slightly thick, slightly raised in the middle, and a light yellow circle was visible in the center of the reverse side of the colony. The hyphae were fuzzy or cotton-like, with a dense and soft surface. The hyphae at the edge were thin and velvety. Figure 1 The morphological characteristics under the optical microscope are: the mycelium is colorless and transparent, thin and thin-walled, with a smooth surface, and the spores around the mycelium are clearly visible. Figure 2 .

[0030] A small amount of mycelium from cryopreserved AMH95 strain was sterilely picked with an inoculating needle and activated in fresh PDA medium. The culture was then incubated in the dark at 26°C until mature for ITS sequence amplification and molecular identification. DNA was extracted using DNA iso reagent, and the ITS sequence was amplified by PCR using primers ITS4 (5'-GGAAGTAAAAGTCGTAAGG-3', SEQ ID NO. 2) and ITS5 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO. 3). PCR reaction parameters were as follows: 1. Initial denaturation at 95°C for 3 minutes; 2. Denaturation at 94°C for 40 seconds; 3. Annealing at 52°C for 50 seconds; 4. Extension at 72°C for 1 minute; 5. Repeat 2-4 cycles 35 times; 6. Extension at 72°C for 10 minutes. PCR products, after agarose gel electrophoresis, were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequence determination. The sequence obtained by PCR product was used as the target sequence, and the homologous sequence was searched in the GenBank database of NCBI. The reference sequence most similar to the morphological sequence was downloaded, and the phylogenetic analysis was performed using the neighbor-joining method (NJ) to determine the phylogenetic status of the strain to be identified. Figure 3 、 4 The endophytic fungal strain of the present invention has a sequence homology of more than 99% with three reference strains of Akanthomyces lecanii, namely Akanthomyces lecanii (MW165534), Akanthomyces lecanii (OP437999), and Akanthomyces lecanii (MH312007). It belongs to the phylum Ascomycota, order Hypocreales, family Cordyceps, genus Akanthomyces, and is classified as Akanthomyces lecanii AMH95. It was deposited in the China General Microbiological Culture Collection Center (CGMCC) on May 26, 2025, and the culture deposit number is CGMCC No. 41956.

[0031] Example 2:

[0032] A small amount of mycelium was aseptically extracted from a frozen tube of AMH95 using an inoculating needle and inoculated into a sterile PDA medium (PDA medium formulation: 200 g potato, 20 g glucose, 15 g agar, 1000 mL distilled water, natural pH). The culture was incubated in the dark at 26°C and 65% humidity for 5 days. In a laminar flow hood, a dot was made at the center of the bottom of the medium using a marker, and dots were made at 120° intervals at 2.25 cm from the center along a line passing through the center radius. Aseptically, three AMH95 cakes were punched from the edge of the colony using a sterile borer. These cakes were then inoculated into the three dotted spots on the medium using tweezers. The experiment was repeated three times. Simultaneously, sterile microparticles of Fusarium oxysporum, Bipolarissorokiniana, Rhizoctonia solani, Colletotrichum gloeosporioides, and Fusarium solani were punched from the edge of the colony using a sterile borer and inoculated at a dot in the center of the culture medium. The experiment was repeated three times. Trichoderma harzianum was used as a positive control and treated in the same manner as AMH95. The inoculated culture medium was sealed and placed in a mold incubator at 26°C, 65% humidity, and incubated in the dark for 5-7 days. The morphology and diameter of the pathogens in each group were observed, and the inhibition rate was calculated as (control pathogen growth radius - treatment pathogen growth radius) / control pathogen growth radius × 100%. The results showed that AMH95 could significantly inhibit the growth of Fusarium oxysporum, Bipolarissorokiniana, Rhizoctonia solani, Colletotrichum gloeosporioides, and Fusarium solani, with relative inhibition rates of 70.47%, 63.96%, 64.14%, 81.39%, and 85.40%, respectively. Among them, the inhibitory effect of AMH95 on Colletotrichum gloeosporioides and Fusarium solani was stronger than that on the positive bacteria Trichoderma harzianum ( Figure 5 、 Figure 6 ).

[0033] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. An endophytic fungus of Astragalus mongolica, characterized in that The endophytic fungus of Mongolian Astragalus belongs to the Ascomycota, Hypocreales, Cordyceps family, and Akanthomyces genus, and is classified and named Akanthomyces lecanii AMH95. It was deposited in the China General Microorganism Culture Collection Center on May 26, 2025, and the culture collection number is CGMCC No. 41956.

2. A use of the endophytic fungus of Astragalus mongolica according to claim 1, characterized in that: Used to inhibit the growth of plant pathogens.

3. The use according to claim 2, characterized in that The plant pathogenic bacteria are one or more of Fusarium oxysporum, Bipolaris sorokiniana, Rhizoctonia solani, Colletotrichum gloeosporioides and Fusarium solani.

4. The use according to claim 2, characterized in that The application specifically involves co-culturing the endophytic fungus with plant pathogens.

5. The use according to claim 4, characterized in that The co-cultivation conditions were as follows: culture in a mold incubator at 26°C, 65% humidity, and darkness.

6. The use according to claim 4, characterized in that The co-cultivation medium was potato dextrose agar (PDA).

7. The use according to claim 6, characterized in that The formula of potato dextrose agar (PDA) medium is as follows: 200 g potato, 20 g glucose, 15 g agar, 1000 mL distilled water, natural pH.

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