Burkholderia strain YJ-2 derived from the herbaceous plant Woodsia ilvensis, microbial inoculum, preparation method and application thereof

Abstract

The present invention discloses a Burkholderia strain YJ-2 derived from the herbaceous plant Woodsia ilvensis, a microbial agent, a preparation method and applications thereof, which are currently deposited in the China Center for Type Culture Collection, deposit number: CCTCC No: M20232448. The strain of the present invention is Burkholderia, which has a short culture time, a fast reproduction speed, and the cells and active extracts thereof after fermentation have the function of inhibiting plant pathogenic fungi. Through experimental verification, the cells and active extracts of Burkholderia strain YJ-2 have inhibitory effects on a variety of plant pathogenic fungi, especially have extremely strong inhibitory ability against Valsa mali. The present invention also provides an active extract of Burkholderia strain YJ-2 and a preparation method thereof. The active extract in the present invention is insensitive to temperature and its acid-base conditions and has extremely strong stability. The cells and active extracts of Burkholderia strain YJ-2 can be used to prepare biological agents against plant pathogenic fungi.

Description

[0001] The present invention relates to the technical field of agricultural microorganisms, and specifically to a Burkholderia strain YJ-2 derived from the herbaceous plant Woodsia ilvensis, a bacterial agent, a preparation method thereof, and an application thereof. Background Art

[0002] Plant diseases have always been important factors affecting crop yield, quality, and food safety, restricting the development of China's crop industry.

[0003] To date, chemical pesticides have always been regarded as one of the most effective methods for controlling plant diseases. However, the long-term and large-scale use of pesticides not only causes problems such as human safety and ecological system damage, but also leads to the emergence of drug resistance in the pathogens that cause plant diseases. With the increasing attention of the public to food safety and environmental protection in agricultural products, in recent years, the construction of ecological agriculture has become a new development trend. Microbiological control is considered to be one of the most economical and sustainable methods in plant disease control because of its safety for humans, the ecosystem, and the environment, and its relatively long-lasting control effect, which can provide guarantee for the sustainable development of agriculture. In addition, biocontrol microorganisms are derived from nature, are pollution-free to the environment, and are safe for non-target organisms. Using microorganisms and their metabolites to control plant diseases has very broad application prospects. Therefore, adopting the strategy of "controlling bacteria with bacteria" and screening microbial functional strains with biocontrol characteristics has very important economic and ecological value in plant disease control.

[0004] Burkholderia is an important group of biocontrol bacteria that can produce a variety of secondary metabolites with inhibitory effects on different plant diseases. These metabolites endow Burkholderia with unique biocontrol potential, and thus many strains of this genus can effectively inhibit the growth of plant pathogens. In recent years, there have been more and more reports on the biocontrol effects of Burkholderia. Many studies have confirmed that these bacteria have significant effects in the control of diseases in plants such as tomatoes and rice, but there has been no report on the biocontrol of apple tree canker. Therefore, discovering and studying the biocontrol mechanism of Burkholderia is of great significance for the development of new biological pesticides for treating plant diseases such as apple tree canker, and the biocontrol bacterial agents prepared from these bacteria also have high promotion and application value. Summary of the Invention

[0005] The first object of the present invention is to provide a Burkholderia strain YJ-2 derived from the herbaceous plant Woodsia ilvensis, whose cells have extremely strong inhibitory activity against plant pathogenic fungi.

[0006] The second object of the present invention is to provide an active extract of the above-mentioned Burkholderia strain YJ-2.

[0007] The third object of the present invention is to provide a microbial agent obtained from the above-mentioned Burkholderia strain YJ-2.

[0008] The fourth object of the present invention is to provide a method for preparing an active extract of the above-mentioned Burkholderia strain YJ-2.

[0009] The fifth object of the present invention is to provide the application of the cells of the above-mentioned Burkholderia strain YJ-2 in the biological control of phytopathogenic fungi.

[0010] The sixth object of the present invention is to provide the application of the active extract of the above-mentioned Burkholderia strain YJ-2 in the biological control of phytopathogenic fungi.

[0011] The first technical solution adopted by the present invention is to provide Burkholderia strain YJ-2, which is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC No: M20232448, and the deposit date: December 04, 2023.

[0012] The second technical solution adopted by the present invention is: a microbial agent for biological control, the active ingredient of the microbial agent is mainly the cells, active extract or fermentation broth of Burkholderia strain YJ-2, and the microbial agent can be in the form of particles, powders, tablets in solid shape or liquids, pastes, gels, creams in fluid form. The microbial agent can be mainly used for the control of phytopathogenic fungal diseases such as apple tree canker.

[0013] The third technical solution adopted by the present invention is: a method for preparing an active extract of Burkholderia strain YJ-2, which specifically includes the following preparation steps:

[0014] Step 1, preparation of the seed liquid;

[0015] Inoculate Burkholderia strain YJ-2 into the seed medium, and culture it at 37°C under the oscillation condition of 180 r / min to 200 r / min for 1 d to 2 d to obtain the seed liquid;

[0016] Step 2, fermentation culture;

[0017] The seed liquid obtained in Step 1 is inoculated into the fermentation medium, and the inoculation amount is 5% to 7%, and it is cultured at 37°C under the oscillation condition of 180 r / min to 200 r / min for 3 d to 5 d to obtain the fermentation broth;

[0018] Step 3, preparation of the active extract;

[0019] Centrifuge the fermentation broth obtained in Step 2 to collect the fermentation supernatant; add 2 volumes of acetone and assist the extraction with ultrasonic waves. Repeat this three times, combine the acetone extracts, and concentrate and evaporate to dryness using a rotary evaporator to obtain the supernatant extract.

[0020] The characteristics of the third technical solution adopted in the present invention also lie in that

[0021] The seed medium (PIA) in Step 1 is composed of the following substances in the following weight ratios: peptone 17.0 g / L - 22.0 g / L, magnesium chloride 1.0 g / L - 1.5 g / L, potassium sulfate 9.0 g / L - 11.0 g / L, glycerol 3% - 5%, triclosan 20.0 mg / L - 25.0 mg / L, pH 7.2 - 7.3, and the solvent is water.

[0022] The fermentation medium (YSP) in Step 2 is composed of the following substances in the following weight ratios: carbon source 9.0 g / L - 12.0 g / L; nitrogen source 15.0 g / L - 20.0 g / L, pH 7.1 - 7.3, and the solvent is water.

[0023] In Step 2, the carbon source is any one of sucrose, glycerol, rice flour, soybean cake powder, starch, peanut cake powder, cottonseed cake powder, corn steep liquor or cellulose; the nitrogen source is any two of yeast extract, peptone, and beef extract.

[0024] The fourth technical solution adopted in the present invention is that the cells of Burkholderia sp. strain YJ-2 can be used for the preparation of a biological agent against plant pathogenic fungi and its application in the control of plant fungal diseases.

[0025] The fifth technical solution adopted in the present invention is that the active extract and fermentation broth of Burkholderia sp. strain YJ-2 can be used for the preparation of a biological agent against plant pathogenic fungi and its application in the control of plant fungal diseases.

[0026] Beneficial effects

[0027] The present invention provides Burkholderia sp. strain YJ-2, a bacterial agent, and a preparation method and application thereof derived from the herbaceous plant Woodsia ilvensis. It has the following beneficial effects:

[0028] The present invention provides a Burkholderia strain YJ-2 bacterial agent derived from the herbaceous plant Woodsia ilvensis, a preparation method thereof, and applications. The present invention provides a Burkholderia strain YJ-2 that has an inhibitory effect on plant pathogenic fungi, especially a strong inhibitory effect on Valsa mali Miyabe et Yamada. The present invention also provides a method for fermenting and producing an antibacterial active extract using this strain. At the same time, the present invention also provides a bacterial agent for preventing and controlling plant pathogenic fungal diseases by using the cells, active extracts, and fermentation broth of Burkholderia strain YJ-2, thereby providing a new prevention and control means for plant fungal diseases. The advantages of Burkholderia strain YJ-2 of the present invention are as follows:

[0029] 1. It has a strong inhibitory effect on 5 kinds of plant pathogenic fungi;

[0030] 2. It grows and reproduces quickly, has a short culture cycle, and can be used for preparing bacterial agents after 3 days of fermentation;

[0031] 3. The active substances of Burkholderia strain YJ-2 have extremely strong stability to temperature, acids, and alkalis, which is convenient for subsequent separation, purification, application, and storage;

[0032] 4. Burkholderia strain YJ-2 has the functions of promoting plant growth, nitrogen fixation, and producing siderophores;

[0033] 5. The cells of Burkholderia strain YJ-2 and its active extracts have a strong prevention and control effect on plant fungal diseases such as apple tree canker;

[0034] 6. The biological bacterial agent prepared from the fermentation broth of Burkholderia strain YJ-2 has a strong prevention and control effect on plant fungal diseases such as apple tree canker.

[0035] In summary, Burkholderia strain YJ-2 is an ideal initial material strain for research, transformation, and application. It has great potential for industrial production in the fields of inhibiting plant pathogenic fungi, producing biological pesticides and biological bacterial fertilizers, and has a broad range of industrial and agricultural applications and market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a diagram of the Gram staining result observed under an optical microscope for Burkholderia strain YJ-2 of the present invention;

[0037] Figure 2It is the morphological result diagram of the Burkholderia strain YJ-2 of the present invention observed under a biological electron microscope;

[0038] Figure 3 It is the colony photo of the Burkholderia strain YJ-2 of the present invention on the screening solid medium (PIA);

[0039] Figure 4 It is the colony photo of the Burkholderia strain YJ-2 of the present invention on the tryptone soya broth solid medium (TSB);

[0040] Figure 5 It is the colony photo of the Burkholderia strain YJ-2 of the present invention on the fermentation solid medium (YSP);

[0041] Figure 6 It is the biological characteristic result diagram of the Burkholderia strain YJ-2 of the present invention;

[0042] Figure 7 It is the phylogenetic tree constructed based on the 16S rDNA sequence of the Burkholderia strain YJ-2 of the present invention;

[0043] Figure 8 It is the effect diagram of the active extracts of the bacterial cells and fermentation broth of the Burkholderia strain YJ-2 of the present invention in inhibiting plant pathogenic fungi;

[0044] Figure 9 A-B is the effect diagram of the preventive effect of the active extracts of the bacterial cells and fermentation broth of the Burkholderia strain YJ-2 of the present invention on the detached apple tree branches infected with Valsa mali;

[0045] Figure 10 It is the control effect diagram of the bacterial agent of the Burkholderia strain YJ-2 of the present invention on the potted apple saplings infected with Valsa mali. Detailed implementation manners

[0046] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0047] Examples:

[0048] An embodiment of the present invention provides a Burkholderia strain YJ-2 derived from the herbaceous plant Woodsia ilvensis, a bacterial agent, a preparation method and an application thereof. The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0049] The screening and isolation process of the Burkholderia strain YJ-2 in the present invention is as follows:

[0050] The Burkholderia strain YJ-2 was isolated from the rhizosphere soil of Woodsia ilvensis in a certain area; 10 g of the collected rhizosphere soil of Woodsia ilvensis was aseptically weighed and placed in 90 mL of sterilized physiological saline, and cultured with shaking at 37 °C and 180 r / min for 3 h, then diluted, and the diluted culture solution was evenly spread on a screening plate, and then inverted and cultured in a biochemical incubator at 37 °C for 2-5 d. After the colonies grew out, observe the colony morphology, color and size on the plate, pick a single colony and isolate it after three streakings, and inoculate the obtained pure strain onto a slant screening medium, which is the Burkholderia strain YJ-2; the screening medium is composed of the following substances in weight ratio: glycerol 4%, peptone 20 g / L, magnesium chloride 1.4 g / L, potassium sulfate 10 g / L, triclosan 25 mg / L, agar powder 15.0 g / L, pH 7.2, and the solvent is water.

[0051] The Burkholderia strain YJ-2 isolated in the present invention is preserved in the National Collection of Type Cultures, and the preservation date is December 4, 2023.

[0052] The characteristics of the Burkholderia strain YJ-2 in the present invention are as follows:

[0053] (1) Morphological characteristics:

[0054] The Burkholderia strain YJ-2 was identified as a Gram-negative bacterium by Gram staining (such as Figure 1 ). The cells of the strain were observed to be short rod-shaped and non-flagellated under an electron scanning microscope (such as Figure 2 ). The colonies of the strain on PIA, TSB, and YSP media were small and non-spreading, 1-2 mm in diameter, round, milky white, with a rough and opaque surface and a neatly concave edge (such as Figure 3 , 4 , 5).

[0055] (2) Biological characteristics:

[0056] Burkholderia strain YJ-2 has the ability to produce indole acetic acid, with a yield of approximately 8.75 mg / L( Figure 6 A). The CAS plate was used to test the ability of strain YJ-2 to produce siderophores. The results showed an obvious orange ring on the CAS plate, indicating that strain YJ-2 can produce siderophores( Figure 6 B). A nitrogen-free medium was used to test the nitrogen fixation ability of strain YJ-2. The results showed that YJ-2 is a strain with nitrogen fixation ability( Figure 6 C).

[0057] (3) 16S rDNA sequence identification:

[0058] The primers used for amplifying bacterial 16S rDNA were respectively:

[0059] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0060] 1492R: 5'-GGTTACCTTGTTACGACTT-3';

[0061] The PCR amplification reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 95°C for 50 s, 55°C for 50 s, 72°C for 90 s, for 31 cycles; complete extension at 72°C for 10 min; after the PCR amplification was completed, the PCR product was analyzed by electrophoresis using 0.8% agarose gel; the PCR product of about 1500 bp after purification and recovery was ligated to the vector pMD19-T and then transformed into competent Escherichia coli TG1 cells; positive transformants were picked and sent to a certain Sangon Biotech Co., Ltd. for determination; the obtained 16S rDNA sequence, the sequence is shown in SEQ ID NO: 1, this sequence was subjected to sequence similarity search and alignment on EzTaxon (http: / / eztaxon-e.ezbiocloud.net), and it was found that this strain has a very high similarity with Burkholderia strains, indicating that this strain belongs to Burkholderia strains. Using BioEdit and MEGA7.0 software, a phylogenetic tree was constructed by the neighbor-joining method, and it was found that this strain has a similarity of 99.72% with Burkholderia stagnalis LMG28156 T ), as Figure 7 shown, so this strain was named Burkholderia strain YJ-2.

[0062] A microbial agent for biological control, the active ingredient of which is mainly the cells, active extracts or fermentation broth of Burkholderia strain YJ-2. It can be in the form of granules, powders, tablets or other solid forms, or in the form of liquids, pastes, gels, creams or other fluid forms. The microbial agent can be used for the control of plant fungal diseases.

[0063] The preparation method of the above-mentioned microbial agent is specifically carried out according to the following steps:

[0064] The fermented fermentation broth, the cells collected by centrifugation or the active extracts extracted are respectively mixed with one or more of the following auxiliary materials: water, vaseline, liquid paraffin, peat, animal manure, straws of various crops, pine shells, rice straw, peanut skins, etc. to prepare a microbial agent. According to the usage situation, the microbial agent can be in the form of granules, powders, tablets or other solid forms, or in the form of liquids, pastes, gels, creams or other fluid forms for the control of plant fungal diseases.

[0065] The preparation method of the active extract of the present invention is specifically prepared according to the following steps:

[0066] Step 1, preparation of the seed liquid;

[0067] Inoculate Burkholderia strain YJ-2 into the seed medium and culture it at 37°C under the oscillation condition of 180 r / min - 200 r / min for 1 d - 2 d to obtain the seed liquid;

[0068] The seed medium is composed of the following substances in weight ratio: peptone 17.0 g / L - 22.0 g / L, magnesium chloride 1.0 g / L - 1.5 g / L, potassium sulfate 9.0 g / L - 11.0 g / L, glycerol 3% - 5%, triclosan 20.0 mg / L - 25.0 mg / L, pH 7.2 - 7.3, and the solvent is water.

[0069] Step 2, fermentation culture;

[0070] Inoculate the seed liquid obtained in Step 1 into the fermentation medium, with an inoculation amount of 5% - 7%, and culture it at 37°C under the oscillation condition of 180 r / min - 200 r / min for 3 d - 5 d to obtain the fermentation broth;

[0071] The fermentation medium is composed of the following substances in weight ratio: carbon source 9.0 g / L - 12.0 g / L; nitrogen source 15.0 g / L - 20.0 g / L, pH 7.1 - 7.3, and the solvent is water.

[0072] The carbon source is any one of sucrose, glycerol, rice flour, soybean cake powder, starch, peanut cake powder, cottonseed cake powder, corn steep liquor or cellulose; the nitrogen source is any two of yeast extract, peptone and beef extract.

[0073] Step 3, Preparation of the active extract;

[0074] Collect the supernatant by centrifuging the fermentation broth obtained in Step 2 to obtain the fermentation supernatant; add 2 volumes of acetone and perform ultrasonic-assisted extraction three times. Combine the acetone extracts, and after concentrating and evaporating to dryness using a rotary evaporator, the supernatant concentrate is obtained, which is the active extract.

[0075] Example 1

[0076] The method for preparing the active extract of the Burkholderia strain YJ-2 specifically includes the following steps:

[0077] Step 1, Preparation of the seed liquid;

[0078] Inoculate the Burkholderia strain YJ-2 into the seed medium and culture it at 37 °C with shaking at 200 r / min for 2 days to obtain the seed liquid.

[0079] Among them, the seed medium is composed of the following substances in weight ratio: peptone 17 g / L, magnesium chloride 1 g / L, potassium sulfate 9 g / L, glycerol 3%, triclosan 20 mg / L, pH 7.2, and the solvent is water.

[0080] Step 2, Fermentation culture;

[0081] Inoculate the seed liquid obtained in Step 1 into the fermentation medium with an inoculation amount of 5%, and culture it at 37 °C with shaking at 200 r / min for 5 days to obtain the fermentation broth.

[0082] Among them, the fermentation medium is composed of the following substances in weight ratio: peptone 9 g / L, yeast extract 3 g / L, sucrose 15 g / L, pH 7.1, and the solvent is water.

[0083] Step 3, Preparation of the active extract;

[0084] Collect the supernatant by centrifuging the fermentation broth obtained in Step 2 to obtain the fermentation supernatant; add 2 volumes of acetone and perform ultrasonic-assisted extraction three times. Combine the acetone extracts, and after concentrating and evaporating to dryness using a rotary evaporator, the supernatant concentrate is obtained, which is the active extract;

[0085] Example 2

[0086] The method for preparing the active extract of the Burkholderia strain YJ-2 specifically includes the following steps:

[0087] Step 1, Preparation of the seed liquid;

[0088] Inoculate Burkholderia strain YJ-2 into the seed medium and culture it at 37°C under shaking conditions at 180 r / min for 1 day to obtain a seed solution.

[0089] Among them, the seed medium is composed of the following substances in weight ratio: peptone 20 g / L, magnesium chloride 1.4 g / L, potassium sulfate 10 g / L, glycerol 4%, triclosan 2.5 mg / L, pH 7.3, and the solvent is water.

[0090] Step 2, fermentation culture:

[0091] Inoculate the seed solution obtained in Step 1 into the fermentation medium at an inoculation amount of 7%, and culture it at 37°C under shaking conditions at 180 r / min for 3 days to obtain a fermentation broth.

[0092] Among them, the fermentation medium is composed of the following substances in weight ratio: rice flour 10 g / L, yeast extract 5.0 g / L, sucrose 20 g / L, pH 7.3, and the solvent is water.

[0093] Step 3, preparation of the active extract;

[0094] Collect the supernatant from the fermentation broth obtained in Step 2 by centrifugation to obtain the fermentation supernatant; add 2 volumes of acetone and perform ultrasonic-assisted extraction. Repeat three times, combine the acetone extracts, and after concentration and evaporation to dryness using a rotary evaporator, obtain a supernatant concentrate, which is the active extract.

[0095] The antagonistic effect of Burkholderia strain YJ-2 cells and the active extract of the fermentation broth against phytopathogenic fungi was tested using the plate control method:

[0096] Collect the supernatant from the fermentation broth obtained in Step 2 of Example 2 by centrifugation to obtain the fermentation supernatant; add 2 volumes of acetone and perform ultrasonic-assisted extraction. Repeat three times, combine the acetone extracts, and after concentration and evaporation to dryness using a rotary evaporator, obtain a supernatant concentrate, which is the active extract. Mark on the back of the PDA plate using the symmetry method, and inoculate inverted different fungal disks (d = 5 mm) at the center position. The fungi are: Valsa mali Miyabe et Yamada, Fusarium graminearum, Bipolaris sorokinianum, Exserohilum turcicum, and Alternaria solani Sorauer. There are 5 PDA plates in each group.

[0097] The Burkholderia strain YJ-2 was inoculated on the four sides of the test fungal discs using the symmetry method, and the culture medium inoculated only with the test fungal discs was used as the blank control for the plate confrontation experiment. The active extract of the fermentation broth of the Burkholderia strain YJ-2 was dissolved in methanol, and sterilized punchers (d = 5 mm) were used to make a hole symmetrically at 25 mm on the four sides of the test fungal discs, and the active extract of the fermentation broth was added. The culture medium inoculated only with the test fungal discs was used as the blank control for the plate confrontation experiment. The results are as Figure 8 shown, Figure 8 in which the test fungi 1 is Valsa mali, 2 is Bipolaris sorokiniana, 3 is Setosphaeria turcica, 4 is Fusarium graminearum, and 5 is Alternaria solani. A is the confrontation experiment between the cells of Burkholderia strain YJ-2 and the test fungi; B is the confrontation experiment between the active extract of the fermentation broth of Burkholderia strain YJ-2 and the test fungi; C is the control group of the test fungi. It can be Figure 8 seen that the Burkholderia strain YJ-2 has a significant inhibitory effect on the five tested phytopathogenic fungi, whether it is the cells or the active extract of the fermentation broth.

[0098] The stability test of the active extract of the Burkholderia strain YJ-2 was carried out using the plate control method:

[0099] Prepare PDA plates, make marks on the back of the PDA plates using the symmetry method, inoculate the inverted test fungal discs (d = 5 mm) at the center of the plates, and punch holes at 25 mm on both sides of the discs using sterilized punchers (d = 5 mm). The active extract of the Burkholderia strain YJ-2 was dissolved in methanol. Using Valsa mali as the indicator fungus, the active extract was treated at 50, 60, 70, 80, 90, and 100 °C for 30 min, and the active extract was adjusted to different pH values of 2, 4, 6, 8, 10, and 12 using 1 mol / L HCl and 1 mol / L NaOH. 1 mol / L HCl and 1 mol / L NaOH were used as controls, and 200 μL of the sample was added to each well. Each treatment had 3 replicates for the stability analysis of the active extract.

[0100] As shown in Table 1, after the active extract of Burkholderia strain YJ-2 was treated at different temperatures, its activity still remained, and there was no significant change in the activity level. As shown in Table 2, among different pH values, only the antagonistic effects of the active extracts at pH 2 and 4 decreased, and there were no differences in the others. The test results indicate that the active extract of Burkholderia strain YJ-2 has extremely strong stability against temperature and acid-base properties.

[0101] Table 1. Stability of Active Extract at Different Temperatures

[0102]

[0103] Note: +++ indicates strong activity, - indicates no antibacterial activity

[0104] Table 2. Stability of Active Extract at Different pH Values

[0105]

[0106]

[0107] Note: ++ indicates relatively strong activity, +++ indicates strong activity, - indicates no antibacterial activity

[0108] Determination of the control effect of the cells and active extract of Burkholderia strain YJ-2 against apple tree canker

[0109] Refer to the scald inoculation method to determine the control effect on detached branches. Collect several perennial 'Fuji' apple branches (d = 15 mm - 20 mm), cut them into 25 cm long segments, first rinse the branches with tap water, then disinfect them with 75% alcohol for 8 min, and finally rinse them 3 times with sterile water. Seal both ends with liquid paraffin. Scald the branches with a red-hot iron nail head (d = 5 mm), and apply the cells of Burkholderia strain YJ-2 or its original active extract, as well as 2, 5, 10, 20, 50, and 100-fold diluted solutions, to the scalded areas. After drying, inoculate a mycelial cake of apple tree canker that has been cultured for 7 days (d = 5 mm). There is 1 inoculation point on each branch, and each treatment group is repeated with 5 branches. Use the application of sterile water as the control. Incubate in the dark at 25°C with humidity maintained, measure the lesion size after 7 days, calculate the lesion area, and calculate the disease control effect. The calculation formula is:

[0110] Disease control effect = (control lesion area - treated lesion area) / control lesion area × 100%

[0111] As Figure 9, as shown in Table 3 and Table 4, among which the cells of Burkholderia strain YJ-2 shown in Table 3 can effectively inhibit the expansion of lesions, and the disease prevention effect reaches 91.13%; as shown in Table 4, the active extract of Burkholderia strain YJ-2 can also effectively inhibit the expansion of lesions. Among them, the original solution, 2-fold and 5-fold dilutions of the active extract have very good preventive effects on apple tree canker on detached branches, and the disease prevention effects reach 94.81%, 94.38% and 93.16% respectively. After that, with the increase of the dilution multiple, the preventive effect gradually decreases.

[0112] Table 3. Disease prevention effect of the cells of YJ-2 strain on apple tree canker on detached branches

[0113]

[0114] Table 4. Disease prevention effect of the active extract of YJ-2 strain on apple tree canker on detached branches

[0115]

[0116] Control experiment on the prevention and treatment of apple tree canker after infection of potted apple saplings with the pathogen of apple tree canker by the agent of Burkholderia strain YJ-2:

[0117] Agent preparation: Weigh 330 - 340 g of bone glue, 3 - 4 g of CaCO3, 500 - 600 ml of fermentation broth, 160 - 170 ml of glycerol, and 330 - 340 ml of H2O. After mixing, stir well to dissolve it to obtain a matrix. Replace the water in the matrix with the fermentation broth of strain YJ-2 to prepare the agent. Store it at room temperature for later use. Refer to the scald inoculation method to determine the prevention and treatment effect of strain YJ-2 on apple tree canker after infection of potted apple saplings. Conduct experiments on 3-year-old dwarf "Fuji" apple trees in pots after they grow new green leaves.

[0118] Preparation of apple tree canker pathogen discs: The apple tree canker pathogen cultured on PDA solid medium for 5 days was made into discs (d = 5 mm) for standby. The branches of apple tree seedlings were scalded with a red-hot iron nail head (d = 5 mm), and the apple tree canker pathogen discs (d = 5 mm) cultured for 5 days were inoculated at the scalded parts. The discs were fixed with plastic wrap. Three inoculation points were set for each seedling. The apple tree seedlings were entirely covered with a transparent plastic bag (60 cm × 90 cm) and cultured at room temperature under natural light, waiting for the apple tree canker pathogen to infect the apple tree. During this period, water was sprayed on the apple tree every day to fully moisturize the sealed environment. Five days after the inoculation wound of the apple tree canker pathogen, the infection was successful, and typical canker disease spots were formed. Next, the epidermis of the infected part was scraped off with a blade, and the infected apple trees were divided into three groups. The first group was not treated (blank control group), the second group was smeared with the matrix at the infected part (matrix group), and the third group was smeared with the ointment bactericide at the infected part. After smearing, they were cultured at room temperature under natural light and smeared once every 10 days. After 30 days, the control effect was observed and recorded.

[0119] The results are as Figure 10 shown in Table 5. For the apple tree seedlings infected with the apple tree canker pathogen, after 30 days of treatment, it was shown that the survival rate of the apple tree seedlings in Group T3 smeared with the bactericide made from strain YJ-2 was as high as 83.33%, while the survival rates of the apple tree seedlings in Group T2 smeared with the matrix and Group T1 not smeared were only 23.33% respectively. It indicates that the bactericide made from strain YJ-2 has a significant control effect on apple tree canker.

[0120] Table 5 Control effect of bactericide on apple tree canker after 30 days of treatment

[0121]

[0122] The above experimental results show that the cells of Burkholderia strain YJ-2 and its active extracts can be used to prepare biocontrol bactericides for controlling plant fungal diseases such as apple tree canker.

[0123] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. The Burkholderia strain YJ-2 derived from the herbaceous plant Woodsia ilvensis is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC No: M20232448, and the deposit date: December 4, 2023.

2. An active extract, characterized in that, The active extract is extracted from the supernatant of the fermentation broth of the Burkholderia strain YJ-2 described in Claim 1. The specific preparation method of the active extract includes the following preparation steps: Step 1, preparation of the seed liquid; Inoculate the Burkholderia strain YJ-2 into the seed medium and culture it at 37°C under the oscillation condition of 180 r / min to 200 r / min for 1 d to 2 d to obtain the seed liquid; Step 2, fermentation culture; Inoculate the seed liquid obtained in Step 1 into the fermentation medium, with the inoculation amount of 5% to 7%, and culture it at 37°C under the oscillation condition of 180 r / min to 200 r / min for 3 d to 5 d to obtain the fermentation broth; Step 3, preparation of the active extract; Collect the fermentation supernatant from the fermentation broth obtained in Step 2 by centrifugation; add 2 volumes of acetone and extract it with ultrasonic assistance, repeat three times, combine the acetone extracts, and concentrate and evaporate to dryness using a rotary evaporator to obtain the supernatant concentrate, which is the active extract.

3. A biological agent for biological control, characterized in that, The active ingredient of the biological agent is the cell body of the Burkholderia strain YJ-2 described in Claim 1 or the active extract and fermentation broth described in Claim 2.

4. A bacterial agent for biological control, characterized in that, The active ingredient of the bacterial agent is the cell body of the Burkholderia strain YJ-2 described in Claim 1 or the active extract and fermentation broth described in Claim 2. The bacterial agent can be in the form of granules, powders, tablets in solid form or liquids, pastes, gels, creams in fluid form.

5. An active extract according to claim 2, wherein The fermentation medium is composed of the following substances in weight ratio: carbon source 9.0 g / L to 12.0 g / L, nitrogen source 15.0 g / L to 20.0 g / L, pH 7.2 to 7.3, and the solvent is water.

6. An active extract according to claim 5, characterized in that, The carbon source is any one of sucrose, glycerol, rice flour, soybean cake powder, starch, peanut cake powder, cottonseed cake powder, corn steep liquor or cellulose; the nitrogen source is any two of yeast extract, peptone, and beef extract.

7. The cell body of the Burkholderia strain YJ-2 described in Claim 1 can be used for preparing a biological agent against plant pathogenic fungi and its application in the prevention and control of plant fungal diseases. The plant pathogenic fungi include Valsa mali Miyabe et Yamada, Fusarium graminearum, Bipolaris sorokinianum, Exserohilum turcicum, and Alternaria solani Sorauer.

8. The active extract and fermentation broth described in claim 2 can be used for preparing a biological agent against phytopathogenic fungi and for application in the prevention and control of plant fungal diseases, and the phytopathogenic fungi include Valsa mali Miyabe et Yamada causing apple tree canker, Fusarium graminearum, Bipolaris sorokiniana causing wheat root rot, Exserohilum turcicum causing northern leaf blight of maize, and Alternaria solani Sorauer causing early blight of tomato.

Images