A methylotrophic Bacillus sp. SY-22 and its applications

Abstract

The present invention discloses a methylotrophic Bacillus SY-22 and its application. This bacterium was registered and preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on August 8, 2022, with the preservation number CGMCC No. 25501 and the preservation address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Methylotrophic Bacillus SY-22 has good effects in preventing and / or treating Anthracnose of Epimedium sagittatum, and its disease prevention effect is much higher than that of chemical pesticides such as carbendazim and prochloraz-manganese chloride complex. It is a green and pollution-free biocontrol bacterium that can reduce the use of chemical pesticides.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control, and particularly relates to a methylotrophic Bacillus SY-22 and its application. Background Art

[0002] Epimedium sagittatum (Sieb. et Zucc.) Maxim. is a perennial herb of the genus Epimedium in the family Berberidaceae, and is one of the original sources of the traditional Chinese medicine Epimedium in China. Its leaves are used as medicine, and it has the medicinal effects of tonifying kidney yang, strengthening tendons and bones, and dispelling wind and dampness. Modern pharmacological studies have shown that Epimedium has various functions such as anti-tumor, anti-inflammatory, anti-osteoporosis, beneficial to the nervous system, and protecting the cardiovascular system. Icaritin, a derivative of icariin, a flavonol compound in Epimedium, has significant anti-cancer activity. Currently, the soft capsule of icariin (acoradin, Icaritin) was approved by the National Medical Products Administration (NMPA) in January 2022 for the treatment of advanced liver cancer, and the Chinese herbal medicine Epimedium is the only source of icariin. At the same time, as a commonly used traditional Chinese medicine in clinical practice, Epimedium is widely used in the fields of medicine, health care, etc. It is the main raw material of various drugs and health products, and is also one of the traditional Chinese medicines with the greatest development potential and market potential.

[0003] In recent years, the demand for Epimedium has increased sharply and the price has been rising continuously, resulting in predatory excavation of wild Epimedium, which has caused the exhaustion of wild resources. At present, the artificial cultivation of Epimedium sagittatum has been successfully achieved, and it has become the species with the largest cultivation area among the medicinal Epimedium plants, and has also become the main source of medicinal Epimedium at home and abroad. At present, the largest Epimedium sagittatum planting base in Asia has been established in Zhumadian area, Henan Province, and it has also become the main supply place of artificially cultivated Epimedium medicinal materials in the country. With the introduction, domestication and long-term asexual reproduction of Epimedium sagittatum, various disease problems have emerged, and the more serious one is the leaf disease of Epimedium sagittatum. Since the medicinal part of Epimedium is the leaf, the occurrence of leaf diseases will seriously affect the yield and quality of Epimedium. Through disease investigation and identification, it is found that the leaf diseases of Epimedium sagittatum include various different fungal diseases, among which anthracnose is one of the main leaf diseases in the Henan production area, and the incidence rate can reach 30-40%, which has seriously affected the yield and quality of Epimedium sagittatum.

[0004] Currently, the main means of controlling plant fungal diseases is chemical pesticides. However, the extensive use of chemical pesticides can cause problems such as pesticide residues and environmental pollution. Since the medicinal part of Epimedium sagittatum is the leaves, if a large amount of chemical pesticides are used for disease control, it will cause problems such as excessive pesticide residues, resulting in unqualified products. Moreover, the Chinese Pharmacopoeia has also strictly restricted the pesticide residue problems of traditional Chinese medicines. In recent years, with the rise of ecological planting, microbial control of diseases has also become an important biological control method, and the acquisition of highly effective biocontrol bacteria is the basis for controlling plant microbial diseases. At present, there is no relevant report on biocontrol bacteria for the anthracnose of Epimedium sagittatum. In addition, through the applicant's preliminary trial, it is found that the commercial biocontrol bacteria on the market have poor effects, and the control effect is not ideal even after applying multiple pesticides. Summary of the Invention

[0005] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present invention is to provide a Bacillus methylotrophicus SY-22 and its application, which can provide a new biocontrol agent for the biological control of anthracnose of Epimedium sagittatum.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: The first object of the present invention is to provide a Bacillus methylotrophicus, which is named Bacillus methylotrophicus SY-22. It was registered and preserved in the General Microbiology Center of the China Microbial Culture Collection Management Committee on August 8, 2022, with the preservation number CGMCC No. 25501 and the preservation address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The second object of the present invention is to provide the application of the above-mentioned Bacillus methylotrophicus SY-22 in preventing and / or treating anthracnose of Epimedium sagittatum.

[0008] The third object of the present invention is to provide a biological agent, which includes the above-mentioned Bacillus methylotrophicus SY-22. This biological agent can be the fermentation broth of Bacillus methylotrophicus SY-22, or directly make the bacterial cells of Bacillus methylotrophicus SY-22 into solid preparations such as suspension agents or powder agents, or make a preparation with Bacillus methylotrophicus SY-22 as the sole active ingredient or the main active ingredient in combination with excipients.

[0009] The present invention has the following beneficial effects:

[0010] The present invention isolates endophytes from the leaves of Epimedium sagittatum, and screens an endophytic bacterium with a strong antagonistic effect against the pathogen causing anthracnose disease of Epimedium sagittatum. Through morphological and 16S rDNA molecular biological identification, it is determined to be Bacillus methylotrophicus, and it is named Bacillus methylotrophicus SY-22. The Bacillus methylotrophicus SY-22 is used in the field control experiment and also achieves an ideal disease control effect. Moreover, the disease prevention effect is much higher than that of chemical pesticides such as carbendazim and prochloraz-manganese chloride complex. It is a green and pollution-free biocontrol bacterium, which can reduce the use of chemical pesticides. At the same time, since the antagonistic bacterium is isolated from healthy Epimedium sagittatum plants, the antagonistic bacterium is very friendly to the environment, meets the requirements of the country for ecological agricultural planting, and has a very broad application prospect in the field of ecological control of diseases of the Chinese medicinal material Epimedium. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Figure 6 shows the colony morphology of Bacillus methylotrophicus SY-22 on LB medium.

[0012] Figure 2 Figure 10 shows the plate inhibition effect of Bacillus methylotrophicus SY-22 on the anthracnose pathogen causing anthracnose of Epimedium sagittatum. Among them Figure 2 -A is the control of anthracnose pathogen, Figure 2 -B is the plate confrontation between the antagonistic bacterium SY-22 and the anthracnose pathogen.

[0013] Figure 3 Figure 18 shows the phylogenetic tree of Bacillus methylotrophicus SY-22.

[0014] Figure 4 Figure 22 shows the control effect diagram of Bacillus methylotrophicus SY-22 on anthracnose of Epimedium sagittatum in the field experiment. Among them Figure 4 -A is the control of healthy Epimedium sagittatum, Figure 4 -B is the Epimedium sagittatum infected by anthracnose pathogen and showing disease symptoms, Figure 4 -C is the Epimedium sagittatum after spraying the antagonistic bacterium SY-22 to control anthracnose. DETAILED DESCRIPTION OF THE INVENTION

[0015] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are followed. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0016] Example 1: Isolation, purification and identification of strains

[0017] I. Isolation of endophytic antagonistic bacteria

[0018] Collection of Epimedium sagittatum plant samples: In 2021, healthy Epimedium sagittatum plants were collected from the GAP planting base of Epimedium sagittatum in Pingyu County, Zhumadian City, Henan Province for the isolation of endophytic bacteria.

[0019] Take fresh and healthy Epimedium sagittatum plants, wash the surface soil with clean water, soak the leaves successively with 75% ethanol, 0.1% mercuric chloride solution, and 75% ethanol in a super clean bench, then wash with sterile water 5 - 7 times, place on sterile absorbent paper to dry. Spread the water from the last wash on an LB medium and culture at 37°C to detect whether the surface disinfection is complete. Use a sterile scalpel to cut the Epimedium sagittatum leaves into leaf tissues about 0.2 - 0.3 cm in length and width, transfer the leaf tissues to an LB medium, and culture at 28°C. After colonies grow around the leaves, pick the colonies for streak plating. After single colonies grow on the streaked area, pick different single colonies separately for culture. After obtaining pure cultures, store them for later use.

[0020] Composition of the LB medium used above: Yeast extract 5 g / L, Tryptone 10 g / L, Sodium chloride (NaCl) 5 g / L, pH 7.0.

[0021] II. Screening of antagonistic strains by the plate confrontation method

[0022] Use a punch to take a 5 - mm - diameter inoculum of the anthracnose pathogen of Epimedium sagittatum (Colletotrichum fructicola) and inoculate it onto a PDA medium. Then use a pipette to aspirate 50 μL of the bacterial solution and inoculate it 3 cm away from the fungal block of the anthracnose pathogen of Epimedium sagittatum. Then place it in an incubator at 28°C for 3 days to observe the antagonistic effect. Measure the diameter of the pathogen colony and calculate the inhibition rate to determine the antagonistic effect of the strain. Select the strains with obvious antagonistic effects for identification and storage for later use.

[0023] Formula for calculating the plate inhibition rate: Inhibition rate (%) = (Diameter of the control pathogen fungal colony - Diameter of the pathogen fungal colony co - cultured with the antibacterial agent) / Diameter of the control pathogen fungal colony × 100%

[0024] Result: The plate inhibition rate of the antagonistic bacteria against the anthracnose pathogen of Epimedium sagittatum for 3 days was 87%, as Figure 2 shown.

[0025] III. Identification of strains

[0026] The above - screened antagonistic bacteria were identified as follows:

[0027] 1. Morphological identification

[0028] The surface of the colonies of the selected antagonistic bacteria is smooth and opaque, white or slightly yellowish( Figure 1 ), and after Gram staining, it is observed as Gram-positive bacteria containing spores under an optical microscope, and the shape is bacillus.

[0029] 2. Molecular identification

[0030] The genomic DNA of the antagonistic bacteria was extracted and amplified using 16S rDNA universal primers (27f: 5’-AGAGTTTGATCCTGGCTCAG-3’ and 1492R: 5’-TACGACTTAACCCCAATCGC-3’). The PCR products were sent to a sequencing company for sequencing. The sequencing results were input into the GenBank database for comparison. The comparison results showed that the strain had the highest similarity with Bacillus methylotrophicus in bacterial classification (see its phylogenetic tree in Figure 3 ). It was identified as Bacillus methylotrophicus and named Bacillus methylotrophicus SY-22. This strain was deposited at the China General Microbiological Culture Collection Center (abbreviation: CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101) on August 8, 2022, and the deposit number was CGMCC No. 25501.

[0031] Example 2: Field biocontrol effect of the strain

[0032] The verification of the field biocontrol effect of Bacillus methylotrophicus SY-22 against anthracnose of Epimedium sagittatum is as follows:

[0033] In the GAP planting base of Epimedium sagittatum in Pingyu County, Zhumadian City, Henan Province, 5 experimental plots were selected from healthy Epimedium sagittatum, namely the blank control plot (healthy Epimedium sagittatum), chemical control plot 1 (sprayed with the pesticide carbendazim), chemical control plot 2 (sprayed with the pesticide prochloraz manganese chloride), experimental plot 1 (the plot inoculated with the spores of Colletotrichum fructicola, the pathogen of anthracnose of Epimedium sagittatum), and experimental plot 2 (the plot sprayed with the bacterial solution of Bacillus methylotrophicus SY-22 at a concentration of 1*10 6 cfu / ml after inoculation with anthracnose bacteria, and about 3-5 ml of the bacterial solution was evenly sprayed on each leaf, and sprayed once). All the Epimedium sagittatum in the 5 experimental plots were stabbed with sterilized toothpicks on the leaves, and the operation was carried out according to the above experimental group design. The disease occurrence of the leaves of Epimedium sagittatum was recorded every day after 3 days of inoculation. The results showed that Bacillus methylotrophicus SY-22 had a good control effect on anthracnose of Epimedium sagittatum, and the field control efficiency could reach 54.15%( Figure 4), which is much higher than that of the control agent 1, carbendazim (control efficiency: 17.16%), and the control agent 2, prochloraz-manganese chloride complex (control efficiency: 33.38%).

[0034] Field control efficiency (%) = (incidence rate in the control area - incidence rate in the control area) / incidence rate in the control area × 100%, where the incidence rate (%) = number of diseased seedling leaves / number of inspected leaves × 100%.

[0035] In summary, a strain of antagonistic bacterium SY-22 (the strain preservation number is: CGMCC No. 25501) screened by the present invention has a strong antagonistic effect on the pathogenic bacteria causing the anthracnose disease of Epimedium sagittatum, and the field control experiment has also achieved an ideal disease control effect. Moreover, the disease prevention effect is much higher than that of the chemical pesticides carbendazim and prochloraz-manganese chloride complex. It is a green and pollution-free biological control bacterium, which can reduce the use of chemical pesticides. At the same time, since the antagonistic bacterium is isolated from healthy Epimedium sagittatum plants, the antagonistic bacterium is very friendly to the environment, meets the requirements of the country for ecological agricultural planting, and has a very broad application prospect in the field of ecological control of diseases of the Chinese medicinal material Epimedium.

[0036] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of Bacillus methylotrophicus ( Bacillus methylotrophicus ) SY-22 in controlling Colletotrichum gloeosporioides on Epimedium sagittatum in the field; Among them, Methylotrophic Bacillus SY-22 was deposited at the General Microbiological Center of the China National Committee for Culture Collection of Microorganisms on August 8, 2022, with the deposit number CGMCC No. 25501 and the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; The pathogen causing anthracnose of Epimedium sagittatum is Colletotrichum fructicola Colletotrichum fructicola .

Images