A Panax notoginseng endophytic fungus strain SQGX-6 and its application

Abstract

The invention discloses a Panax notoginseng endophytic fungus strain SQGX‑6 and an application thereof. The strain is Chaetomium aureum SQGX‑6, which was deposited in the China Center for Type Culture Collection on May 11, 2022, with a deposit number of CCTCC M 2022601. The endophytic fungus has a strong inhibitory effect on Gibberella tritici and can inhibit the germination of Gibberella tritici spores, which is of great significance for the biological control of Gibberella tritici.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a Panax notoginseng endophytic fungus strain SQGX-6 and applications thereof. Background Art

[0002] According to statistics, about 40% of the world's population relies on wheat as their staple food. my country is the world's largest wheat producer and consumer. It is of certain significance to conduct research on wheat's resistance to pathogenic fungal diseases and ensure safe wheat production.

[0003] Wheat fusarium head blight is a worldwide disease caused by Fusarium graminearum, F. asiaticum, F. culmorum and other Fusarium species. In recent years, due to changes in climate conditions, the prevalence of wheat fusarium head blight has led to a loss of millions of hectares of wheat production in China each year. At present, the prevention and control of wheat fusarium head blight mainly relies on chemical drugs, which are prone to environmental pollution and greatly increased risks to human and animal health.

[0004] Microbial control has the advantages of being green, environmentally friendly and highly specific, making it an ideal way to control plant diseases and insect pests. Microbial control is a type of application that uses beneficial organisms and their products in nature to kill or reduce the number of harmful organisms to control the occurrence of crop and plant diseases. This application has broad development and application prospects; however, there are few reports on antagonistic bacteria that can effectively control wheat fusarium. Summary of the invention

[0005] In view of this, the present application provides a Panax notoginseng endophytic fungus strain SQGX-6 and its application for biological control of wheat Gibberella.

[0006] In order to achieve the above technical objectives, this application adopts the following technical solutions:

[0007] In the first aspect, the present application provides a Panax notoginseng endophytic fungus strain SQGX-6, which was deposited in the China Center for Type Culture Collection on May 11, 2022, with the deposit number CCTCC M 2022601.

[0008] In a second aspect, the present application provides an application of a Panax notoginseng endophytic fungus strain SQGX-6 in preventing and controlling wheat fusarium sphaeroides.

[0009] Furthermore, the Panax notoginseng endophytic fungus strain SQGX-6 was used to inhibit the germination of wheat fusarium spores.

[0010] Furthermore, the Panax notoginseng endophytic fungus strain SQGX-6 was used to inhibit the hyphae of wheat fusarium sphaeroides.

[0011] In a third aspect, the present application provides a wheat fusarium pathogen agent, the components of which include the fermentation filtrate of the Panax notoginseng endophytic fungus strain SQGX-6 described in claim 1.

[0012] Furthermore, the preparation step of the fermentation filtrate includes: inoculating the bacterial cake of the Panax notoginseng endophytic fungus SQGX-6 into the PDB culture medium, and after fermentation, filtering and removing the mycelium to obtain the fermentation filtrate.

[0013] Furthermore, the preparation step of the fermentation filtrate includes: inoculating the bacterial cake of the Panax notoginseng endophytic fungus SQGX-6 into the PDB culture medium, culturing with shaking at 28-35° C. for 7-10 days, filtering and removing the mycelium to obtain the fermentation filtrate.

[0014] In a fourth aspect, the present application provides a fungicide for wheat fusarium, the components of which include an extract of the fermentation filtrate of the Panax notoginseng endophytic fungus strain SQGX-6 described in claim 1.

[0015] Furthermore, the extract is a n-butanol extract.

[0016] Furthermore, the preparation method of the extract comprises adding an equal volume of water-saturated n-butanol to the fermentation filtrate of the Panax notoginseng endophytic fungus strain SQGX-6, mixing thoroughly and letting stand, taking the organic phase, rotary evaporating the solvent of the organic phase, dissolving it with n-butanol, and filtering to obtain the extract.

[0017] The beneficial effects of the present application are as follows: the Panax notoginseng endophytic fungi of the present scheme have an antagonistic effect on wheat Gibberella, the fermentation filtrate of the Panax notoginseng endophytic fungi and the n-butanol extract of the fermentation filtrate have a strong inhibitory effect on wheat Gibberella, can inhibit the germination of wheat Gibberella spores, and are of great significance for the biological control of wheat Gibberella; the strain culture conditions are simple, easy to preserve, easy to industrialize and have good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The growth of Panax notoginseng endophytic fungus SQGX-6 strain in PDA medium (7 days);

[0019] Figure 2 Molecular biological identification of the endophytic fungus SQGX-6 in Panax notoginseng;

[0020] Figure 3 To establish the phylogenetic tree of the endophytic fungus SQGX-6 from Panax notoginseng;

[0021] Figure 4 The endophytic fungus SQGX-6 of Panax notoginseng antagonizes wheat fusarium;

[0022] Figure 5 The antagonistic effect of Panax notoginseng endophytic fungus SQGX-6 and wheat fusarium rust pathogen;

[0023] Figure 6 is the inhibition rate of Panax notoginseng endophytic fungus SQGX-6 against wheat fusarium sphaeroides;

[0024] Figure 7 The inhibitory effect of n-butanol extract of Panax notoginseng endophytic fungus SQGX-6 on wheat fusarium sphaeroides. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] For the screening methods, buffer configurations and common culture medium recipes involved in the embodiments, reference can be made to the contents described in the seventh edition of Microbiology edited by Chen Wenli and the Molecular Cloning Experiment Guide (see J. Sambrook et al., 2002, Molecular Cloning Experiment Guide, third edition, Jin Dongyan et al. (translation), Science Press, Beijing).

[0027] The various other experimental operations involved in the present invention are all routine techniques in the art. For the parts not specifically described herein, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of the present invention.

[0028] Example 1

[0029] Fresh Panax notoginseng required for this experiment was purchased from Tuojia Village, Yanshan County, Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province. The roots of Panax notoginseng were first rinsed with tap water and washed. The surface was disinfected with 75% anhydrous ethanol for 1 min on a clean bench, then soaked in 3% sodium hypochlorite for 3 min, rinsed with sterile water 5 times, placed on dust-free filter paper, and then cut the roots of Panax notoginseng into small pieces of 0.5 cm in length, width and height with a scalpel, and transferred to a PDA plate, 4 pieces were placed on each plate, and cultured at 28°C. After 7 days, the edge of the colony was picked with a sterile pick and inoculated on a new PDA medium to purify the strain. Repeat three times to obtain the Panax notoginseng endophytic fungus strain SQGX-6 of the present application.

[0030] The morphological and molecular biological identification of the endophytic fungus SQGX-6 of Panax notoginseng was carried out:

[0031] The biological characteristics of the Panax notoginseng endophytic fungus SQGX-6 strain are as follows: the colonies on the PDA medium are red and green, with neat edges, the mycelium is felt-like and round, the inner circle on the back of the colony is dark green, and the outer circle is light red. After 7 days of cultivation, the PDA medium turns red ( Figure 1 ), Figure 1The left picture shows the front of SQGX-6, and the right picture shows the back of SQGX-6.

[0032] At the same time, the gene sequence of the SQGX-6 strain was isolated and obtained (see sequence 1 and sequence 2), and the ITS sequence of the Panax notoginseng endophytic fungus SQGX-6 was amplified, ITS1: 5'-TCC GTA GGT GAA CCT GCG G-3' (see sequence 3); ITS4: 5'-TCC TCCGCT TAT TGA TAT GC-3' (see sequence 4), and the size of the target fragment was 580 bp, which was consistent with the expectation. The electrophoresis results are shown in Figure 2 The phylogenetic tree of SQGX-6 and highly similar strains was constructed using MEGA7.0 software, and the homology with Arcopilus aureus was 100% ( Figure 3 Therefore, SQGX-6 was preliminarily identified as Arcopilus aureus, which belongs to the phylum ascomycetes, class Sordariomycetes, order Sordariales, family Chaetomiaceae, and genus Arcopilus.

[0033] Example 2

[0034] A fermentation filtrate of a Panax notoginseng endophytic fungus SQGX-6 strain, and a preparation method thereof is as follows:

[0035] (1) Activation of strains: After taking out the strain SQGX-6 from the -80°C refrigerator, inoculate it onto the PDA solid culture medium slant and culture it at 28°C for 5 days. The preparation method of the PDA culture medium is to peel 200g of potatoes and cut them into shreds, add 1000ml of double distilled water and boil for 30 minutes, then filter it with a 300-mesh filter, add 15g of agar and 20g of glucose to the obtained filtrate, add double distilled water to make up to 1000ml, and sterilize it at 121°C for 30 minutes;

[0036] (2) Liquid seed culture: First, prepare PDB liquid seed PDB medium. The preparation method is as follows: peel 200 g of potatoes and cut them into shreds, add 1000 ml of double distilled water, boil for 30 minutes, and filter with a 200-mesh filter. Add 20 g of glucose to the filtrate, add double distilled water to make the volume 1000 ml, put 200 ml of liquid seed medium into each 500 ml Erlenmeyer flask, and sterilize at 121° C. for 30 minutes.

[0037] (3) Fermentation culture: The SQGX-6 on the PDA plate was punched with a 6 mm hole puncher and inoculated into a conical flask containing 200 ml of liquid culture medium. The shaking speed was 170 rpm and the temperature was 28° C. The culture was carried out for 7 days to obtain SQGX-6 fermentation liquid; the mycelium was removed by filtering with a 0.22 μm filter to obtain the antibacterial fermentation liquid of Panax notoginseng endophytic fungus (SQGX-6);

[0038] Fermentation culture characteristics: On the first day of culture, the bacteria are a small number of red spheres; on the third day of culture, the number of small red mycelial balls increases; on the fifth day of culture, the red mycelial balls continue to increase and become larger in size, and the fermentation liquid is light red; on the seventh day of culture, a large number of red mycelial balls appear and become larger in size, and the fermentation liquid is dark.

[0039] Example 3

[0040] The invention discloses an extract of a fermentation filtrate of an endophytic fungus of Panax notoginseng. The preparation method of the extract comprises the following steps: inoculating a bacterial cake of an endophytic fungus of Panax notoginseng into a sterile PDB liquid culture medium, placing the culture medium in a shaking incubator, shaking and culturing the culture medium at 28°C and 170 rpm for 7 days, filtering the culture medium through a 0.22 μm filter to obtain a sterile fermentation filtrate; adding an equal volume of water-saturated n-butanol to the sterile fermentation filtrate, mixing the mixture thoroughly, and then standing the mixture for 30 minutes; collecting an upper organic phase and a lower aqueous phase by a separatory funnel, and evaporating the organic phase by a rotary evaporator at 50°C; dissolving the precipitate of the extraction phase by using n-butanol, filtering the mixture, and obtaining a n-butanol extract of the fermentation filtrate.

[0041] Test Example 1

[0042] The antagonistic effect of Panax notoginseng endophytic fungus SQGX-6 on wheat gibberella was evaluated by the following methods:

[0043] Confrontation culture: The inhibitory effect of Panax notoginseng endophytic fungus SQGX-6 on wheat fusarium fusarium was determined by confrontation culture in a dish. A 6mm diameter Panax notoginseng endophytic fungus SQGX-6 cake was inoculated at one end 2cm away from the center of the PDA plate, and a 6mm diameter wheat fusarium cake was inoculated at the other end as the control group 1. The plates were sealed with sealing film and cultured in a 28℃ incubator. The plates inoculated with only Panax notoginseng endophytic fungus SQGX-6 and wheat fusarium fusarium were used as the control group, and each treatment was repeated 3 times. Compared with the control group 1, SQGX-6 can significantly inhibit the growth of wheat fusarium fusarium after six days of confrontation with it ( Figure 4 and Figure 5 ). The growth of the mycelium of wheat fusarium on the plate was observed. Compared with the control phase 1, the mycelium of the pathogen became thinner and unevenly distributed. It can be seen that the antibacterial active substances contained in the endophytic fungus SQGX-6 of Panax notoginseng can significantly inhibit the growth of wheat fusarium.

[0044] After the control mycelium fills the dish, measure the control radius (Rc) and the radius (Rp) of the wheat fusarium colony extending toward the Panax notoginseng endophytic fungus SQGX-6, and calculate the inhibition rate. Figure 6 As shown, with the passage of time, the inhibition rate of SQGX-6 against wheat fusarium sphaeroides gradually increased from the second day, reached a peak on the 3rd to 4th day, and then tended to rise steadily. The inhibition rate was 70% on the 6th day.

[0045] Antibacterial rate (%) = [(Rc-Rp) / (Rc-3)] × 100

[0046] Test Example 2

[0047] Evaluation of the antibacterial effect of n-butanol extract of Panax notoginseng endophytic fungus SQGX-6 on Gibberella tritici

[0048] Preparation of the fermentation liquid of Panax notoginseng endophytic fungus SQGX-6: The bacterial cake of SQGX-6 (6 mm in diameter) was placed in a conical flask containing 200 ml of PDB medium and cultured at 28°C and 170 rpm for 7 days to obtain the fermentation liquid. The mycelium and spores of the fermentation liquid were first filtered out with a suction pump, and then sterilized by filtering twice with a 0.22 μm microporous filter membrane to obtain the culture filtrate, which was stored at 4°C for later use.

[0049] The inhibitory effect of the culture solution of Panax notoginseng endophytic fungus SQGX-6 on the mycelial growth of wheat fusarium: the wheat fusarium cake (6mm) was inverted and pasted on the PDA plate, and 20ul of SQGX-6 n-butanol extract was dripped vertically on the cake. The control group 2 without the culture solution was used as the control group 2. Each group was treated 3 times and sealed in a 28℃ incubator for 5 days. The results showed that the colony diameter of wheat fusarium on the plate with n-butanol aqueous phase in the control group 2 exceeded 2cm, and the mycelium was dense, while the mycelium on the plate with n-butanol extract was basically not grown, which shows that the extract can significantly inhibit the mycelial growth of wheat fusarium ( Figure 7 ), Figure 7 .1 Left: SQGX-6 n-butanol water phase; Figure 7 .2 The left is the n-butanol extraction phase of SQGX-6; the right is the wheat fusarium.

[0050] The above are only preferred specific implementations of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A Panax notoginseng endophytic fungus strain SQGX-6, characterized in that: It was deposited in the China Center for Type Culture Collection on May 11, 2022, with the accession number CCTCCM2022601.

2. An application of the Panax notoginseng endophytic fungus strain SQGX-6 as claimed in claim 1 in controlling wheat fusarium sphaeroides.

3. The use according to claim 2, characterized in that: The endophytic fungus strain SQGX-6 from Panax notoginseng was used to inhibit the germination of spores of wheat fusarium.

4. The use according to claim 2, characterized in that: The endophytic fungus strain SQGX-6 from Panax notoginseng was used to inhibit the hyphae of wheat fusarium sphaeroides.

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