CN115820424A - Strain and application thereof - Google Patents

Abstract

The invention discloses a strain and application thereof, wherein the strain is Colletotrichumaeschynomenes SF-1, and the strain is deposited in the following parts: china general microbiological culture Collection center; the preservation date is as follows: 21/4/2022; the preservation number is: CGMCC No.40133. The invention has the beneficial effects that: 1. the invention adopts separated and purified endophytes under the aseptic condition, screens antagonistic strains through a plate confrontation test, identifies the morphology and molecular biology, and optimizes the strain fermentation condition through a single-factor test. 2. The prevention and control effect of the microbial inoculum and the various applications are verified through a pot experiment.

Description

Strain and application thereof

Technical Field

The invention relates to the technical field of strains, in particular to a Colletotrichum aeschynomenes strain and application thereof.

Background

Root rot is one of the most serious root diseases which harm plants of panax, and mainly infects the roots of the plants to cause the roots to soften, rot and wilting, thereby causing large-area plant death and seriously affecting the healthy development of panax industry. With the remarkable improvement of the awareness of people on the protection of the ecological environment, the safe, pollution-free, low-cost and efficient prevention and control of the root rot is one of the conditions for ensuring the safety and effectiveness of the traditional Chinese medicinal materials. Therefore, screening of biocontrol strains with high efficiency against pathogenic bacteria and development of biocontrol microbial inoculum are hot spots of the current prevention and treatment research on root rot of panax plants.

The endophyte refers to bacteria, fungi or actinomycetes which exist in healthy plant tissues or organs at a certain stage or whole stage of life history and form a reciprocal symbiotic relationship with a host. The endophyte not only can provide required nutrient substances for plants and promote the growth of the plants, but also participates in the defense function of the plants and enhances the stress resistance and disease resistance of the plants. Therefore, screening based on endophyte antagonistic bacteria and research and development of biocontrol microbial inoculum are effective methods and ways for preventing and treating root rot.

The application screens out biocontrol bacteria for resisting root rot from endophytes separated from panax notoginseng, and verifies the effect of resisting panax notoginseng root rot under a pot experiment through fermentation culture, thereby laying a foundation for developing a specific microbial agent for panax notoginseng plants.

Disclosure of Invention

The invention aims to provide a strain and application thereof, wherein panax notoginseng endophytes are separated and identified, the panax notoginseng endophytes are screened through a plate confrontation test, antagonistic strains are identified through morphology and molecular biology, strain fermentation conditions are optimized through a single-factor test, and the control effect of a microbial inoculum is verified through a pot experiment.

In order to solve the technical problems, the invention adopts the following technical scheme:

the invention relates to a strain, which is Colletotrichum aeschynomenes SF-1 and is deposited in the following places: china general microbiological culture Collection center; the preservation date is as follows: 21/4/2022; the preservation number is: CGMCC No.40133.

The strain is applied to inhibiting Fusarium oxysporum.

The application of the strain in reducing root rot of pseudo-ginseng seedlings is disclosed.

The application of the strain in producing IAA.

The strain of the invention is used for producing GA ₃ The use of (1).

The application of the strain in promoting the growth of pseudo-ginseng is disclosed.

The application of the strain in promoting the height of the pseudo-ginseng seedling is provided.

The application of the strain in promoting the leaf length of pseudo-ginseng seedlings is disclosed.

The application of the strain in promoting the fresh weight of pseudo-ginseng seedlings is disclosed.

Compared with the prior art, the invention has at least one of the following beneficial effects: 1. the invention adopts separated and purified endophytes under the aseptic condition, screens antagonistic strains through a plate confrontation test, identifies the morphology and molecular biology, and optimizes the strain fermentation condition through a single-factor test. 2. The prevention and control effect of the microbial inoculum and the various applications are verified through a pot experiment.

Drawings

FIG. 1 is a morphological diagram (left) and a microscopic view (right) of the strain SF-1 of the present invention.

FIG. 2 is a graph showing the plate confrontation test in the present invention.

FIG. 3 is a phylogenetic tree diagram of ITS sequence homology of the strain SF-1 of the present invention.

FIG. 4 is a graph showing the growth of the strain SF-1 (Colletotrichum aeschynomenes) according to the present invention under various factors. ( Note: different lower case letters indicate significance of up to a 0.05 level of difference. )

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.

The invention relates to a strain, which is Colletotrichum aeschynomenes SF-1 and is deposited in the following places: china general microbiological culture Collection center; the preservation date is as follows: 21/4/2022; the preservation number is: CGMCC No.40133.

The strain is applied to inhibiting Fusarium oxysporum.

The application of the strain in reducing root rot of pseudo-ginseng seedlings is disclosed.

The application of the strain in producing IAA.

The strain of the invention is used for producing GA ₃ The use of (1).

The application of the strain in promoting the growth of pseudo-ginseng is disclosed.

The application of the strain in promoting the height of the pseudo-ginseng seedling is provided.

The application of the strain in promoting the leaf length of pseudo-ginseng seedlings is disclosed.

The application of the strain in promoting the fresh weight of pseudo-ginseng seedlings is disclosed.

Example 1

The endophyte is separated from root, stem and leaf of the Yunnan pseudo-ginseng sample. The root rot pathogen Fusarium oxysporum was isolated from the laboratory and the strain was stored. The pseudo-ginseng seeds to be tested were provided by department of herbaceous medicine, stachys sieboldii, inc.

Reagent consumables: 200g of potato, 20g of glucose (national drug group chemical reagent Co., ltd., lot number 20180327) and 13g of agar (national drug group chemical reagent Co., ltd., lot number 20180722).

Spin Kit for Soil Kit (MPBIO, lot 20200731), fungus universal primer (beijing optima biotechnology limited, lot 20200824), and gossypol lactate cotton blue staining solution (fuzhou feijing biotechnology limited, lot 20200526).

The test instrument: DL-CJ-1 NP-II type sterile operating platform (Beijing Toyobo Harr instruments manufacturing Co., ltd.), FR-2L type trinocular fluorescence microscope (Beijing century Kexin scientific instruments Co., ltd.), 2720 type polymerase chain reaction apparatus (Applied United states)

Company), DYY-8C type electrophoresis apparatus (Beijing Heishi Instrument Co., ltd.), DH420 type electric heating constant temperature incubator (Beijing Kogyi Yongxing instruments Co., ltd.).

1. Endophyte isolation

The endophytic fungi are cultured by a pure culture method. The separation and purification method comprises the following steps: cleaning fresh and healthy root, stem and leaf of Panax notoginseng with running water, sterilizing the surface, cutting the tissue into blocks with a sterilized scalpel, cutting into 0.5cm pieces, inoculating into PDA culture medium, culturing at 25 deg.C, picking different bacterial colonies into PDA culture medium, and gradually separating and purifying to obtain purified single bacterial strain.

2. Screening of antagonistic bacteria against root rot

Screening antagonistic strains of pathogenic fusarium oxysporum by adopting a plate confronting method, taking fusarium oxysporum cakes out by using a puncher with the diameter of 1.00cm, transferring the fusarium oxysporum cakes to a new PDA culture medium, simultaneously culturing endophytic fungi at 4 points 2.50cm away from the center of the PDA solid culture medium, and screening the strains with antagonistic action. PDA plates inoculated with only blocks of Fusarium oxysporum hyphae served as controls. Culturing in 28 deg.C incubator for 5 days, repeating each strain for 3 times, measuring the colony diameter of Fusarium oxysporum in the culture dish, and calculating the antibacterial rate. Bacteriostasis rate = (control colony diameter-treated colony diameter)/(control colony diameter-cake diameter) × 100%.

3. Identification of antagonistic functional bacteria

Hypha and spore with good growth condition are picked and prepared into temporary tablets, the properties such as hypha morphology, spore shape, spore septum and septum quantity are observed under a microscope, and the morphology identification is carried out according to the manual for fungus identification.

Total DNA of endophytic fungi is extracted by a Spin Kit for Soil (MPBIO) Kit, and ITS2 rRNA gene fragments are amplified by using general fungal primers ITS1/ITS 2. The sequencing results were aligned to sequences in the NCBI database, homology alignment analysis was performed using the Blast online tool, and phylogenetic trees were constructed using MEGA7.0 software (fig. 3).

4. Screening suitable fermentation conditions of antagonistic strains

The single factor test examines the influence of the culture time, the shaking flask rotation speed, the temperature and the initial pH on the strain fermentation. Viable count was counted by plate dilution and spreading method, which was referred to "microbiology experiment" and was repeated 3 times per group.

5. Effect verification of biocontrol microbial inoculum

And (3) filling the sterilized humus soil into a seedling raising tray made of a plastic material, and screening pseudo-ginseng seeds with consistent germination and disinfected surfaces and sowing the pseudo-ginseng seeds in the seedling raising tray. And (4) pouring sterile water after sowing, and performing illumination treatment for 8 hours every day to ensure normal seedling emergence and growth of the pseudo-ginseng. And (5) sowing for 60 days, and keeping the seedling group which grows healthily and has consistent size for testing after the panax notoginseng seedlings emerge. The experiment was set up with 3 treatments. The treatment was as follows: (1) a negative control group; (2) a positive control group; (3) and (4) inoculating biocontrol bacteria and pathogenic bacteria (inoculating the pathogenic bacteria 2 weeks after biocontrol bacteria inoculation). Inoculating Notoginseng radix by root irrigation method, wherein the concentration of pathogenic bacteria and biocontrol bacteria is 1 × 10 ⁸ cfu/mL, inoculum size50 mu L of the solution is beaten to the root of the panax notoginseng plant along the rhizome by a pipette and 10mL of sterile water is poured into each group. After inoculation for 28 days, the growth and incidence of pseudo-ginseng seedlings under different treatments are counted, and disease index grading is carried out]. Each group was biologically replicated with 36 pseudo-ginseng seedlings.

Incidence = number of diseased plants/total number of plants × 100%

Disease index = (disease plant tree x number of representative series)/(total plant number sum x highest representative series value) × 100

The control effect = (control disease index-treatment disease index)/control disease index x 100%

6. Results

Strain morphology and molecular characterization

The growth of endophyte SF-1 is slow during culture, primary white hyphae grow after two days of culture at 28 ℃, the hyphae are thin and flocculent, the bacterial colony is round, and after 5 days of culture, the single hyphae form a gray-white round bacterial colony with regular edges on a culture medium and the center is gray-black (figure 1). Under a microscope, the large conidia of the endophyte SF-1 are in a sickle shape or a long column shape; the conidiophores are slender. The strain is compared with the morphological characteristics of the fungus listed in the manual of fungus identification, and is preliminarily identified as Colletotrichum (Colletotrichum).

The ITS rRNA gene sequence of the strain SF-1 is as follows:

after the ITS sequence comparison, the sequence similarity of the strain SF-1 and a Colletotrichum aeschynomenes ICMP 17673 (accession number NR _ 166015.1) is 99.22 percent, and the strain SF-1 is identified as the Colletotrichum aeschynomenes by combining the morphological characteristics of thalli in the same branch of a phylogenetic tree.

Antagonistic action against root rot

Through a plate confrontation test, the bacterial strain SF-1 (Colletotrichum aeschynomenes) is found to have strong bacteriostatic activity on fusarium oxysporum with the diameter of 1.40cm and the bacteriostatic rate of 73.33 percent (figure 2 and table 1).

TABLE 1 bacteriostatic percentage of the strain SF-1 (Colletotrichum aeschynomenes) (%)

Growth promoting effect

IAA and GA for strain SF-1 (Colletotrichum aeschynomenes) ₃ The content determination (Table 2) shows that the IAA yield of the strain SF-1 (Colletotrichum aeschynomenes) fermentation liquor is 40.25 mg.L ^-1 ，GA ₃ The yield of (a) was 26.38 mg.L ^-1 。

TABLE 2 IAA and GA production by fermentation broth of strain SF-1 (Colletotrichum aeschynomenes) ₃ In an amount of

Conditions of fermentation

The viable count of the microbial inoculum C.aeschynomenes increases with the increase of the culture time (24-72 h), and when the culture time is longer than 72h, the viable count increases slowly, so that 72h is the optimal fermentation time (figure 4A). The viable count of the microbial inoculum increases (24-26 ℃) along with the rise of the temperature, when the temperature is higher than 26 ℃, the viable count of the C.aeschynomenes microbial inoculum tends to be stable, and then the number of the viable counts is reduced, and 26 ℃ is selected as the optimal culture temperature (figure 4B). The viable count of the microbial inoculum SF-1 (Colletotrichum aeschynomenes) increases (0-120 r.min) along with the increase of the rotating speed ^-1 ) When the rotating speed is more than 120 r.min ^-1 Then, the viable count tends to be stable, so 120 r.min is selected ^-1 The optimum shaking flask fermentation speed was set (FIG. 4C). The viable count of the microbial inoculum increases and then decreases with the increase of the pH, and when the pH is 6.5, the viable count of the C.aeschynomenes reaches the maximum value, so that the optimum initial fermentation pH is selected as 6.5 (fig. 4D). The results show that the suitable initial fermentation conditions for c.aeschynomenes are: 72h,26 ℃,120 r.min ^-1 ，pH 6.5。

The growth curve of the strain SF-1 (Colletotrichum aeschynomenes) under the condition of each factor is as follows:

pot culture test 1

After the C.aeschynomenes biocontrol microbial inoculum is inoculated to 28d, compared with a positive control group (a germ group), the microbial inoculum remarkably reduces the morbidity (64.07%) and the disease index (56.31%) of root rot of panax notoginseng seedlings (Table 3); compared with a negative control group, the plant height (41.01%), the leaf length (47.09%) and the fresh weight (47.06%) of the pseudo-ginseng seedlings are remarkably promoted.

TABLE 3 Effect of bacterial agents on the prevention and control of root rot and growth of Panax notoginseng

The data show that: the endophyte separated in the invention is identified as Colletotrichum aeschynomenes, and has the functions of inhibiting fusarium oxysporum and promoting plant growth. The fermentation conditions of the strain are as follows: 72h,26 ℃,120 r.min ^-1 pH6.5, the colony diameter is 1.40cm, the bacteriostasis rate is 73.08 percent, and the yield of IAA and GA3 is 40.25mg/L and 26.38mg/L respectively. The pot experiment result shows that after the C.aeschynomenes biocontrol microbial inoculum is inoculated to 28d, the incidence rate (64.07%) and the disease index (56.31%) of root rot of the pseudo-ginseng seedlings are remarkably reduced, and the plant height (41.01%), the leaf length (47.09%) and the fresh weight (47.06%) of the pseudo-ginseng seedlings are remarkably promoted. The endophyte Colletotrichum aeschynomenes can inhibit the fusarium oxysporum of the root rot and promote the growth of plants, provides a new optional strain for preventing and treating the root rot of medicinal plants, and simultaneously provides a material for developing microbial agents of the medicinal plants.

Although the invention has been described herein with reference to a number of illustrative embodiments thereof, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More specifically, various variations and modifications are possible in the component parts and/or arrangements of the subject combination arrangement within the scope of the disclosure, the drawings and the appended claims. In addition to variations and modifications in the component parts and/or arrangements, other uses will also be apparent to those skilled in the art.

Claims (9)

1. A strain of bacteria characterized by: the strain is Colletotrichum aeschynomenes SF-1, and the strain is deposited in the following places: china general microbiological culture Collection center; the preservation date is as follows: 21/4/2022; the preservation number is: CGMCC No.40133.

2. Use of a strain according to claim 1 for inhibiting Fusarium oxysporum.

3. Use of the strain of claim 1 for reducing root rot of pseudo-ginseng seedlings.

4. Use of a strain according to claim 1 for the production of IAA.

5. A method for producing GA using the strain of claim 1 ₃ The use of (1).

6. Use of the strain of claim 1 for promoting the growth of panax notoginseng.

7. The application of the strain of claim 1 in promoting the height of panax notoginseng seedlings.

8. Use of the strain of claim 1 for promoting leaf length of pseudo-ginseng seedlings.

9. Use of the strain of claim 1 for promoting the fresh weight of pseudo-ginseng seedlings.

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