Ginseng endophyte with high yield of pyrroloquinoline quinone and application of ginseng endophyte

Abstract

The ginseng endophyte for high yield of PQQ is characterized in that the classification name of the ginseng endophyte is Methylorbum sp. W7N-70, the ginseng endophyte is preserved in the China Center for Type Culture Collection (CCTCC), the preservation address is No. 299, eight road, Wuchang District, Wuhan City, Hubei Province, the preservation number is CCTCCM20241885, and the preservation date is September 2, 2024. The obtained ginseng endophyte W7N-70 has the capacity of producing PQQ, the PQQ synthesis amount detected in supernate of the ginseng endophyte W7N-70 reaches 75.81 mg / L, the ginseng endophyte W7N-70 can be effectively applied to fermentation engineering for synthesizing the PQQ, in addition, the strain can produce antibacterial metabolites and can effectively inhibit growth of various pathogenic bacteria, and the strain has a good application prospect in the aspects of promoting growth of plants, resisting bacteria and synthesizing the PQQ. The effect is excellent.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a ginseng endophyte with high yield of pyrroloquinoline quinone and application thereof. Background Art

[0002] In nature, the relationship between plants and microorganisms is close and complex. Microorganisms exist on the surface and in the body of almost all plant tissues, and the two have co-evolved to form a relatively stable relationship. Endophytes refer to microorganisms that live in various tissues and organs of healthy plants at certain stages or all stages of their life history, and their presence does not cause any diseases. They include multiple microbial groups such as endophytic fungi, endophytic bacteria and endophytic actinomycetes. In plants, the bioactive metabolites produced by endophytes can promote plant growth, inhibit plant pathogens, and improve plant tolerance to biotic and abiotic stresses. In addition, some bioactive metabolites synthesized by endophytes can also be used as raw materials for medicines, cosmetics and foods.

[0003] Ginseng( Panax ginseng CA Meyer) is a perennial herbaceous plant of the genus Panax in the Araliaceae family, mainly distributed in Asia and North America. As a traditional precious Chinese herbal medicine, it has a long history of medicinal use and is widely used in medicines, functional foods, dietary supplements and cosmetics. Due to overexploitation in recent years, wild ginseng resources have become increasingly scarce, and cultivated ginseng has gradually become the mainstream of the market. However, compared with other herbal medicinal plants, ginseng has a longer growth cycle, generally taking 4-6 years to harvest, and has more stringent environmental requirements. It is easily affected by multiple factors such as temperature, soil, light, precipitation, and pests and diseases. As a result, ginseng cultivation has complex cultivation technology, long production cycle, uncontrollable quality, and unstable yield, which greatly restricts the development of the ginseng industry.

[0004] Pyrroloquinoline quinone (PQQ) is a reddish brown, light-degradable, heat-stable, water-soluble orthoquinone compound. It is the third coenzyme of oxidoreductase after pyridine nucleotides and riboflavin. PQQ has multiple physiological functions, such as participating in quinone protease and respiratory chain electron transfer, regulating gene expression and protein function, regulating signal transduction and DNA function repair, and regulating the level of free radicals in the body. In addition, as a plant growth regulator, PQQ can improve the physical and chemical properties of soil, enhance plant stress resistance, and promote plant growth and development, making it an important development prospect in the food industry, light industry, agriculture, and medicine. The synthesis methods of PQQ mainly include chemical synthesis and microbial fermentation. Compared with chemical synthesis, microbial fermentation has the advantages of simple operation, low cost, and easy subsequent treatment, and has become the main production method of PQQ. Therefore, screening and obtaining ginseng endophytes with the ability to synthesize PQQ is of great significance for obtaining new PQQ production strains and the development of ginseng microbial agents. Summary of the invention

[0005] The present invention aims to provide a ginseng endophyte with high PQQ production. The endophyte has high PQQ production and has an inhibitory effect on various pathogenic bacteria of ginseng. The endophyte effectively improves the antibacterial ability of ginseng and the growth quality of ginseng, and promotes the development of the ginseng industry.

[0006] Another object of the present invention is to provide an application of the ginseng endophyte.

[0007] The purpose of the present invention is achieved through the following technical solutions: A high-yield PQQ ginseng endophyte, characterized in that: the ginseng endophyte is classified as Methyl Rhodobacterium ( Methylorubrum sp.)W7N-70, deposited in China Center for Type Culture Collection (CCTCC), the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the deposit number is CCTCCM20241885, and the deposit date is September 2, 2024.

[0008] The strain W7N-70 belongs to Gram-negative bacteria, is light pink in color, and deepens in color after being cultured for a period of time. The colonies are small and round, shiny, easy to pick, and opaque.

[0009] The 16S rDNA sequence was sequenced, and the sequencing results were subjected to Blast comparison analysis in NCBI, and sequence similarity analysis was performed using DNAman. Then, a phylogenetic tree was constructed using MEGA, and strain W7N-70 was identified as Methylobacterium ( Methylorubrum sp.).

[0010] Furthermore, after enrichment culture, the strain W7N-70 screened out had the ability to produce pyrroloquinoline quinone (PQQ), and the PQQ content detected in its supernatant reached 75.81 mg / L.

[0011] Furthermore, the strain W7N-70 also produces metabolites with antibacterial activity against ginseng rust pathogen ( Cylindrocarpon destructans )、Ginseng Botrytis cinerea ( Botrytis cinerea )、Ginseng Anthracnose ( Colletotrichum panacicola )、Rhizoma ginseng blight ( Rhizoctonia solani ) Alternaria panax ) and Sclerotinia ginseng ( Sclerotinia libertiana ) has an inhibitory effect on the growth of

[0012] In view of the ability of strain W7N-70 to produce PQQ and inhibit the growth of pathogens, the strain can be used to promote plant growth.

[0013] The application of the above-mentioned ginseng endophyte W7N-70 in promoting plant growth is specifically to promote the growth of ginseng.

[0014] Furthermore, the above-mentioned ginseng endophyte W7N-70 is used in the fermentation project of synthesizing PQQ.

[0015] In addition, the above-mentioned ginseng endophyte W7N-70 is used to inhibit the growth of pathogenic bacteria.

[0016] Furthermore, the ginseng endophyte W7N-70 inhibits the growth of at least one pathogen of ginseng rust, ginseng gray mold, ginseng anthracnose, ginseng damping-off, ginseng black spot and ginseng sclerotinia.

[0017] The present invention has the following technical effects: The ginseng endophyte W7N-70 obtained in the present invention belongs to the genus Methylorubrum ( Methylorubrum sp.), presumably Methylobacterium zasneri ( Methylobacterium zatmanii ), which has the ability to produce PQQ, and the PQQ synthesis amount detected in its supernatant reached 75.81 mg / L, which can be effectively applied to the fermentation project of synthesizing PQQ. In addition, the strain can produce antibacterial metabolites, which can effectively inhibit the growth of various pathogens. The strain is applied to promote plant growth and antibacterial, as well as synthesize PQQ, with excellent effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Non-enzymatic detection of PQQ production of strain W7N-70 corresponding to the standard curve.

[0019] Figure 2: Colony morphological observation results of strain W7N-70.

[0020] Figure 3 : Agarose gel electrophoresis of PCR amplification products of 16S rDNA sequence of strain W7N-70.

[0021] Figure 4 : Phylogenetic tree of strain W7N-70 constructed based on 16S rDNA sequence.

[0022] Figure 5 :The antibacterial effect of strain W7N-70 on 6 ginseng pathogens.

[0023] Figure 6 :The antibacterial effect of the supernatant of the culture fluid of strain W7N-70 on 6 ginseng pathogens. DETAILED DESCRIPTION

[0024] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0025] Example 1 Isolation and purification of strains 1. Sample Collection In October 2023, a total of 21 healthy ginseng plants were collected in Weishahe Town and Naozhi Town, Linjiang City, Jilin Province, China for the isolation of ginseng endophytes. After collection, the stems, leaves and other tissues were removed, only the main root was retained, the soil adhering to the surface was washed off with running water, the water was absorbed by absorbent paper, and the self-sealing bags were packaged and stored at 4℃ for later use. The information of the collected samples is shown in Table 1.

[0026] Table 1:

[0027] 2. Isolation and purification of endophytes Endophytes were isolated by tissue grinding: the ginseng roots that had been surface disinfected were cut into small pieces, placed in a sterile mortar, ground with an appropriate amount of physiological saline, and the ground tissue was placed on a culture medium. The culture medium used was potato dextrose agar (PDA), beef extract peptone (NA) and Gao's medium No. 1.

[0028] After the colonies were observed on the culture medium, the colonies were picked up with an inoculation loop and streaked three times on the corresponding culture medium according to their morphological characteristics to purify the endophytes, and they were named and labeled and stored in a -80℃ refrigerator for a long time. A total of 206 ginseng endophytes were isolated, of which 97 were isolated from NA medium, 64 from PDA medium, and 45 from Gao's No. 1 medium.

[0029] Example 2 Screening and identification of endophytic bacteria in ginseng 1. Screening of ginseng endophytes with PQQ synthesis ability The 206 endophytic bacteria isolated and purified in Example 1 were inoculated into enrichment medium, cultured at 28°C and 170rpm with shaking for 5-6 days, and the growth of the bacteria was observed. The enrichment medium components are as follows: CH3OH 15 mL, (NH4)2SO4 4g, KH2PO4 1.4 g, Na2HPO4 5 g, MgSO4·7H2O 1 g, CaCl2·2H2O 0.075 g, FeSO4·7H2O 0.05 g, water 1 L, pH 6.8.

[0030] The PQQ synthesis capacity of ginseng endophytes was initially screened by non-enzymatic method: 100 mg / L, 80 mg / L, 60 mg / L, 40 mg / L, 20 mg / L, 10 mg / L and 5 mg / L PQQ standard solutions were prepared for the construction of standard curves. 240 μL of reaction system was added to a 96-well plate, including 48 μL of bacterial supernatant, 32 μL of borate buffer, and 160 μL of NBT-potassium glycine solution. The reaction was incubated at 37 °C in the dark for 20 min, and the OD was detected by microplate reader. 530 The absorbance value under the conditions was 0.0077x + 0.1726, and the uninoculated culture medium was used as a blank control. Each treatment was repeated 3 times. A strain with the ability to synthesize PQQ was screened from 206 ginseng endophytes, and the strain was recorded as W7N-70 (from a 7-year-old forest ginseng sample from Weishahe Town). The results are shown in Figure 1. The regression equation of the standard curve is y = 0.0077x + 0.1726, R 2 = 0.9954, with a good linear relationship. A higher PQQ content of 75.81 mg / L was detected in the supernatant of the culture solution of strain W7N-70. The strain W7N-70 can synthesize PQQ by fermentation in fermentation engineering, and PQQ can be used as a reagent to promote plant growth in the process of plant growth.

[0031] 2. Strain Identification The colony morphology of strain W7N-70 cultured on plate medium is as followsFigure 2 As shown, strain W7N-70 is a Gram-negative bacterium with a light pink color. The color deepens after a period of culture. The colonies are small and round. The colonies are shiny and easy to pick. The colonies are opaque and not tightly bound to the culture medium.

[0032] DNA of strain W7N-70 was extracted using the Bio-Tech Ezup column-type bacterial genomic DNA extraction kit. The extracted genomic DNA was used as a template and the universal primers for bacterial species identification were used to perform PCR amplification of the 16S rDNA sequence.

[0033] The obtained PCR products were subjected to 1% agarose gel electrophoresis. Figure 3 As shown, the bands are clear and bright. The bands of the appropriate size were cut out and recovered using the Sangon SanPrep column DNA gel recovery kit. The gel recovery products were sent to Sangon Biotechnology Co., Ltd. for sequencing.

[0034] The sequencing results were analyzed by Blast in NCBI, and the sequences with high homology were downloaded. DNAman was used for sequence similarity analysis and MEGA was used to construct a phylogenetic tree. The phylogenetic tree result is shown in Figure 4. The identification results showed that strain W7N-70 belonged to the genus Methylobacterium ( Methylorubrum sp.), and further speculated to be Methylobacterium zasneri ( Methylobacterium zatmanii ).

[0035] The above-mentioned strain W7N-70 is deposited in the China Center for Type Culture Collection (CCTCC), the storage address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the storage number is CCTCCM20241885, and the storage date is September 2, 2024.

[0036] Example 3 Inhibitory effect of strain W7N-70 on ginseng pathogens The pathogens of ginseng tested include: Ginseng rust pathogen ( Cylindrocarpon destructans )、Ginseng Botrytis cinerea ( Botrytis cinerea )、Ginseng Anthracnose ( Colletotrichum panacicola )、Rhizoma ginseng blight ( Rhizoctonia solani ) Alternaria panax ) and Sclerotinia ginseng ( Sclerotinia libertiana The above ginseng pathogens were provided by the College of Plant Protection, Jilin Agricultural University.

[0037] Test steps: (1) The plate confrontation method was used to study the antibacterial effect of strain W7N-70 on ginseng pathogens. Specifically, six ginseng pathogens were inoculated in the center of the PDA solid culture medium and placed in a 26°C incubator for 5 days. Then, a 5 mm punch was used to punch out the bacterial cake and inoculated it on the center point of the new PDA culture medium. The strain W7N-70 was inoculated at four points 2.5 cm away from the center point at the same time. Only the pathogen was inoculated as a control and placed in a 26°C incubator for culture. The relevant data were observed and recorded every 12 hours. The colony diameters of the experimental group and the control group were determined by the cross method to calculate the antibacterial rate. The antibacterial rate calculation formula is as follows:

[0038] Where L1 is the colony diameter of the control group, and L2 is the colony diameter of the experimental group.

[0039] The results are as follows Figure 5 As shown in the table (A is W7N-70 and rust pathogen; B is W7N-70 and anthracnose pathogen; C is W7N-70 and solani pathogen; D is W7N-70 and black spot pathogen; E is W7N-70 and sclerotinia pathogen; F is W7N-70 and gray mold pathogen), strain W7N-70 can effectively inhibit the growth of 6 ginseng pathogens, preventing their colonies from expanding outward and producing obvious inhibition zones. The calculation results of the inhibition rate are shown in Table 2.

[0040] Table 2: Inhibition rate of strain W7N-70 against ginseng pathogens

[0041] The strain W7N-70 had the best antibacterial effect against anthracnose pathogens, followed by R. solani, with inhibition rates of 56.3% and 42.4%, respectively.

[0042] (2) The growth rate method was used to study the antibacterial effect of the supernatant of the culture medium of strain W7N-70 on ginseng pathogens: Specifically, strain W7N-70 was inoculated into the liquid culture medium and the OD value of the culture medium was 600 When the value is 0.6-0.8, centrifuge at 10,000 rpm for 10 min, take the supernatant into a 500 mL beaker, heat in a water bath and concentrate to 50 mL, then sterilize at 115°C for 30 min, then mix the culture supernatant with PDA culture medium in a ratio of 1:10 and set aside. Use a 5 mm puncher to punch out the ginseng pathogen cake to be tested, and inoculate it into the center of the culture medium containing the culture supernatant, and culture at 26°C. Observe the growth of ginseng pathogen mycelium every 12 h, and use the same volume of sterile water instead of the concentrate in the control group. The results are as follows Figure 6As shown in the figure (A is rust pathogen; B is anthracnose pathogen; C is damping-off pathogen; D is black spot pathogen; E is sclerotinia pathogen; F is gray mold pathogen), obvious inhibition zones were formed between the culture supernatant of strain W7N-70 and the six pathogens, indicating that the supernatant of strain W7N-70 has a good inhibitory effect on ginseng pathogens. The inhibition rate was calculated and the results are shown in Table 3.

[0043] Table 3: Inhibition rate of strain W7N-70 culture supernatant against ginseng pathogens

[0044] As can be seen from the above table, the culture supernatant of strain W7N-70 also has a good antibacterial effect on ginseng pathogens, among which the inhibition effect on Rhizoctonia solani is better, with an inhibition rate of 32.1%.

[0045] The above tests show that strain W7N-70 and its culture supernatant have good antibacterial effects on a variety of ginseng pathogens, which further illustrates that ginseng endophyte W7N-70 produces metabolites with broad-spectrum antibacterial activity, which can be applied to fermentation engineering to synthesize metabolites to inhibit pathogens.

Claims

1. A ginseng endophyte with high PQQ production, characterized by: The ginseng endophyte is classified as Methyl Rhodobacterium ( Methylorubrum sp.)W7N-70, deposited in China Center for Type Culture Collection (CCTCC), the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the deposit number is CCTCCM20241885, and the deposit date is September 2, 2024.

2. Use of the ginseng endophyte W7N-70 as claimed in claim 1 in promoting plant growth.

3. Use of the ginseng endophyte W7N-70 as claimed in claim 1 in the fermentation project of synthesizing PQQ.

4. The use of ginseng endophyte W7N-70 as claimed in claim 1, characterized in that: Application in inhibiting the growth of pathogenic bacteria.

5. The use according to claim 4, characterized in that: Specifically, the method inhibits the growth of at least one pathogen selected from the group consisting of ginseng rust pathogen, ginseng gray mold pathogen, ginseng anthracnose pathogen, ginseng damping-off pathogen, ginseng black spot pathogen and ginseng sclerotinia pathogen.

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