Plant burkholderia sp. Strain BpMS90 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a plant burkholderia plantara strain BpMS90 and application thereof.The plant burkholderia plantara strain BpMS90 is obtained by separating and screening from root tissue of potentilla kleiniana, the plant burkholderia plantara strain BpMS90 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 3, 2024, and the preservation number is CGMCC NO.30848. The invention further discloses a preparation method of the plant burkholderia plantara strain BpMS90. The burkholderia plantarum BpMS90 strain is safe, non-toxic and environment-friendly, the biological agent containing the burkholderia plantarum BpMS90 is wide in antibacterial spectrum and has antibacterial activity on various plant pathogenic fungi, and the burkholderia plantarum BpMS90 strain is a broad-spectrum microbial agent / bacterial fertilizer with good development and application prospects and has broad application prospects. The method has important significance on green prevention and control of the tomato verticillium wilt disease.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a plant Burkholderia strain BpMS90 and an application thereof. Background Art

[0002] Tomato Verticillium wilt is a highly destructive soil-borne fungal disease caused mainly by Verticillium dahliae. In addition to tomatoes, Verticillium dahliae can infect more than 400 dicotyledonous plants, including some important economic and vegetable crops, posing a huge threat to agricultural production. Verticillium dahliae belongs to the subdivision Deuteromycotina, family Moniliaceae, genus Verticillium, which is widely distributed worldwide and has high genetic diversity. In the past few decades, Verticillium dahliae infecting tomatoes has been divided into physiological subspecies 1 (V. dahliarace 1) and physiological subspecies 2 (V. dahliarace 2) based on the presence or absence of the avirulent effector VdAve1. Physiological subspecies 1 has the VdAve1 avirulent effector, which can be recognized by tomato varieties containing the Ve1 resistance gene, so that physiological subspecies 1 can only infect tomato varieties lacking the Ve1 resistance gene. Race 2 does not contain VdAve1, which allows it to escape Ve-mediated resistance and is pathogenic to all tomato varieties. However, wild tomato varieties S. neorickii that are resistant to race 2 have recently been discovered and cultivated into rootstock varieties, such as Aibou and Ganbarune-Kari. Further analysis revealed that rootstock varieties that are resistant to race 2 are controlled by a single dominant locus, V2. However, the resistance controlled by V2 seems to be effective only against some strains of race 2, because several isolated strains were found to cause severe leaf yellowing and vascular browning on these varieties, and these strains were defined as race 3. Therefore, tomatoes can currently be divided into three races based on their resistance to Verticillium dahliae.

[0003] Verticillium dahliae has high genetic diversity and wide pathogenicity. It can co-evolve with its host to produce new highly pathogenic physiological strains. In addition, its microsclerotial structure is difficult to eliminate, which has long been a huge agricultural problem. Currently, the main prevention and control measures for tomato Verticillium wilt are agricultural prevention, chemical prevention and biological prevention.

[0004] Agricultural prevention and control measures mainly include useful resistant varieties, intercropping and other technical means. The rationality and diversified application of agricultural prevention and control can help provide a good growth environment for plants and reduce the adverse effects of the external environment on plant growth. However, this method also has some limitations, such as being affected by factors such as season, geography and climate, as well as slow prevention and control effects. To prevent and control tomato Verticillium wilt, agricultural prevention and control methods cannot fundamentally eliminate it, but can only reduce a certain incidence rate. Once the disease breaks out on a large scale, agricultural prevention and control methods cannot quickly control its spread.

[0005] Chemical control is a method of targeted prevention and control of crop diseases using chemical agents, drip irrigation, spraying, fumigation and other methods. Methyl bromide is considered to be the most effective chemical fumigant for controlling soil pathogens. However, due to the destructive effects of methyl bromide on the ozone layer and other environmental and health issues, the international community has gradually taken measures to restrict and eliminate its use. For a long time, people have been trying to find new chemical agents to replace methyl bromide. Common substitutes currently include chloropicrin (chloropicrin), dimethyl disulfide (DMDS), etc. The chemical control method for tomato wilt in my country is mainly to spray carbendazim and use methyl thiophanate root irrigation during the seedling stage or planting period. Chemical methods are simple, fast and effective, however, they will interfere with the environment, have adverse effects on human health, destroy aquatic ecosystems, harm pollinators and reduce the number of beneficial microorganisms in the soil, which is not conducive to sustainable development.

[0006] The essence of biological control mainly includes direct mechanisms such as competition between biocontrol bacteria and plant pathogens in nutrition and ecological niches, inducing host plants to produce resistance substances, secretion of antimicrobial substances by microorganisms themselves, and inducing the effective role of resistant pathogens, thereby effectively preventing and controlling plant diseases. At the same time, biological control can also play an indirect role by activating systemic plant resistance. Some biocontrol bacteria can also promote plant growth, thereby offsetting the disease effects of pathogens on plants. At present, biological control strategies for plant diseases mainly involve the use of biological fertilizers and antagonistic microorganisms. These antagonistic microorganisms cover a variety of types such as bacteria, fungi and actinomycetes.

[0007] As an important economic crop, tomato (S. lycopersicum) is widely planted around the world. However, the tomato production process is highly dependent on the application of chemical fertilizers and is susceptible to infection by a variety of plant pathogens including Verticillium dahliae (V. dahliae). Nowadays, the prevention and control of plant diseases mainly rely on chemical control. This has a serious impact on the generation of drug-resistant pathogens, the environment, and human health. Compared with chemical control, biological control has many advantages, such as environmental friendliness, harmlessness to human and animal health, good specificity to pathogens, no residue in agricultural products, broad antibacterial spectrum, and diverse disease resistance pathways. The introduction of biological control not only effectively compensates for the drawbacks of chemical control, but also provides a green and feasible solution for the sustainable development of agriculture. Based on this, exploring the growth-promoting and disease-resistant effects of biocontrol agents on plants is conducive to the further promotion and development of biological control. Summary of the invention

[0008] In view of this, the object of the present invention is to provide a plant Burkholderia strain BpMS90 and an application thereof. The provided plant Burkholderia can effectively inhibit a variety of plant pathogenic fungi and can effectively prevent and control tomato Verticillium wilt.

[0009] The present invention solves the above technical problems by the following technical means:

[0010] In one aspect of the present invention, the present invention provides a plant Burkholderia strain, which is the plant Burkholderia (Burkholderia plantarii) BpMS90 strain, which was deposited in the China General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on June 3, 2024, with a deposit number of CGMCC NO.30848.

[0011] Preferably, the nucleotide sequence of 16S rRNA of the plant Burkholderia sp. BpMS90 is as shown in SEQ ID NO.3.

[0012] In one aspect of the present invention, the present invention also provides a biological preparation, which contains the plant Burkholderia BpMS90 or its fermented bacterial liquid. The plant Burkholderia BpMS90 of the present application can be used in the production of biological preparations by preparing bacterial liquid through large-scale fermentation culture. The bacterial liquid of plant Burkholderia BpMS90 can also be separated to obtain its metabolites, and these metabolites are believed to have the same preventive and therapeutic effects as the fermented bacterial liquid.

[0013] Preferably, the biological preparation is a liquid preparation, a lyophilized powder or an immobilized preparation. The liquid preparation of the present application can be in the form of common microbial pesticides such as suspensions, liposome liquids, emulsions, etc.; lyophilized powder is also a commonly used preparation form, which can be conveniently transported and used, and can effectively protect the activity of the bacteria in a lyophilized form. Of course, the lyophilized powder can also contain lyophilization protectants and stabilizers such as trehalose, protein, etc. that are well known in the art. Immobilized preparations are microorganisms fixed in porous carriers, and porous carriers include diatomaceous earth, activated carbon, porous silica, etc. The advantage of being fixed in a porous carrier is that the vitality of the microorganism can be maintained for a long time and it is protected from external impacts.

[0014] In one aspect of the present invention, the present invention also provides a use of the plant Burkholderia strain as described above or the biological agent as described above in inhibiting plant pathogenic fungal diseases.

[0015] Preferably, the plants include tomatoes, cotton and potatoes.

[0016] Preferably, the plant pathogenic fungi include Botrytis cinerea, Phytophthora capsici, Fusarium oxysporum, Alternariabrassicae, Cercospora arachidicola, or Piricularia oryzae.

[0017] In one aspect of the present invention, a method for inhibiting plant pathogenic fungal diseases using the above-mentioned biological agent is provided, wherein the biological agent containing plant Burkholderia solani BpMS90 is applied to the test plant to inhibit plant fungal diseases.

[0018] Further preferably, the biological preparation of the present invention can also be used together with other preparations for controlling plant fungal diseases to enhance their effects.

[0019] The present application adopting the above scheme has at least the following beneficial effects:

[0020] 1. The plant Burkholderia strain BpMS90 of the present invention is isolated and screened from the root tissue of Potentilla kleiniana. The whole plant can be used as medicine, and the young stems and leaves can also be used as vegetables. It is safe, non-toxic and environmentally friendly.

[0021] 2. The plant Burkholderia BpMS90 biological agent of the present invention has a wide antibacterial spectrum and has antibacterial activity against a variety of plant pathogenic fungi. It is a broad-spectrum microbial agent / fertilizer with good development and application prospects, and is of great significance for the green prevention and control of tomato Verticillium wilt disease;

[0022] 3. The plant Burkholderia BpMS90 strain of the present invention has simple culture conditions, fast reproduction speed, and is easy to store and transport. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The morphological characteristics of BpMS90, where A and B are the single colony morphology of the strain on the LB plate; C is Gram staining; D is scanning microscopy;

[0024] Figure 2 Figure 1 is a tolerance analysis diagram of BpMS90, where A is the growth state of BpMS90 at different temperatures; B is the growth state of BpMS90 at different pH values; C is the growth state of BpMS90 at different salt contents, and different letters indicate significant differences among treatments (p < 0.05);

[0025] Figure 3 is the phylogenetic tree of the 16S rRNA sequence of the BpMS90 strain;

[0026] Figure 4 This is a plate confrontation picture of BpMS90 against six plant pathogenic fungi;

[0027] Figure 5 The figure shows the effect of BpMS90 on tomato Verticillium wilt JR2, where A is the morphology of mycelium on the JR2 plate with normal growth; B is the morphology of mycelium under scanning electron microscope; C is the cross-section morphology of mycelium under transmission electron microscope; D is the morphology of mycelium on the JR2 plate treated with BpMS90; E is the morphology of mycelium under scanning electron microscope of JR2 treated with BpMS90; F is the cross-section morphology of mycelium under transmission electron microscope of JR2 treated with BpMS90; the scale bars of A and D are 2 cm, the scale bars of B and E are 20 μm, and the scale bars of C and F are 500 nm;

[0028] Figure 6 The control effect of BpMS90 strain on tomato Verticillium wilt is shown in Figure 1. A and C are potted tomato phenotypes; B is a cross-section of a tomato stem; and D is a stem grafted back onto PDA. CK is inoculated with clean water; BpMS90-JR2 is inoculated with both BpMS90 and pathogen JR2; JR2 is inoculated with pathogen JR2 only;

[0029] Figure 7 This is the result of the biomass of the Verticillium wilt pathogen in tomato plants, where the left is the root and the right is the stem;

[0030] Figure 8The graph shows the effect of co-culture of BpMS90 and JR2 on the growth status of JR2, where AD represents JR2 cultured alone; EH represents BpMS90 and JR2 co-cultured; d represents the number of culture days, scale bar = 10 μm. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The raw materials, equipment or instruments used, if no manufacturer is specified, are all conventional products that can be obtained commercially.

[0033] The culture medium used in the following examples of the present invention is as follows:

[0034] LB medium: 5 g yeast extract, 5 g sodium chloride, 10 g tryptone, 15 g agar powder, dissolved in 1000 mL deionized water, natural pH, sterilized at 121°C for 20 min after aliquoting.

[0035] LB liquid medium: 5 g yeast extract, 5 g sodium chloride, 10 g tryptone, dissolved in 1000 mL deionized water, natural pH, sterilized at 121°C for 20 min after aliquoting.

[0036] PDA culture medium: 200g of potatoes, peel and slice, add 1000mL of deionized water, boil and keep for 25min, then filter through double-layer gauze, add 20g of glucose and 15g of agar powder to the filtrate, finally make up to 1000mL, natural pH, and sterilize at 121℃ and high temperature and high pressure for 20min after packaging.

[0037] PDB medium: 200g of potato, peel and slice, add 1000mL of deionized water, boil for 25min, filter through double gauze, add 20g of glucose to the filtrate, adjust the volume to 1000mL, set the pH to natural, and sterilize at 121℃ and high pressure for 20min after packaging.

[0038] Basic culture medium: 20 g glucose, 2 g peptone, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 12 g agar powder, 1000 mL deionized water, natural pH, sterilize at 121°C for 20 min after packaging.

[0039] Nitrogen source utilization culture medium: replace the glucose in the basal culture medium with 20 g maltose, galactose, and mannose respectively, 1000 mL of deionized water, natural pH, and sterilize at 121°C for 20 min after packaging.

[0040] Carbon source utilization culture medium: replace the peptone in the basal culture medium with 2 g of glutamic acid, L-aspartic acid, serine, and creatine respectively, 1000 mL of deionized water, natural pH, and sterilize at 121°C for 20 min after packaging.

[0041] Example 1

[0042] This example provides the isolation, screening and identification of plant Burkholderia spp. BpMS90 strain

[0043] 1. The specific steps for the isolation and screening of plant Burkholderia spp. BpMS90 are as follows:

[0044] Separation from the root tissue of Potentilla kleiniana. The root pieces of healthy plants of Potentilla kleiniana collected from Tieshan Town, Dazu District, Chongqing (105.5984°E, 29.6850°N) were washed with clean water, rinsed with sterile water 5 times on a clean bench, cut into 0.5×0.5 cm small pieces with scissors, soaked in 75% ethanol for 1 min, soaked in 5% sodium hypochlorite for 30 s, soaked in 75% ethanol for 1 min, rinsed with sterile water 5 times, and dried with sterile filter paper on the surface of the tissue and placed on an LB plate, cultured at 37°C, and observed every day for the formation of colonies. When new colonies grew on the plate, different single colonies were transferred to new culture media according to the different morphologies of the colonies, and single colonies (without other forms of colonies around) were obtained by repeated streaking purification and preserved. At the same time, take a small amount of sterile water from the last rinse and apply it to the LB culture dish as a blank control, culture it at 37℃ for 24h, and check whether the sample surface is thoroughly disinfected.

[0045] The isolated and purified endophytes were inoculated into LB liquid medium, and cultured at 37°C and 150 rpm for 24 h to obtain the strain solution. 50 μL of the diluted Verticillium dahliae suspension (1×10 7 CFU / mL) was evenly spread on the PDA medium, and then a 6mm diameter sterile punch was used to punch holes symmetrically at 3cm from the center of the culture dish, and 50μL of the strain solution to be tested was added to the holes. Each treatment was repeated 3 times, and cultured at 28℃ for 48h. The wells with only PDB medium were used as blank controls to observe whether a transparent inhibition zone appeared. When a transparent inhibition zone appeared, the screened plant Burkholderia BpMS90 was obtained.

[0046] 2. The morphological and molecular biological identification of plant Burkholderia solani BpMS90 is as follows:

[0047] (1) Morphological observation and identification

[0048] The B. plantarii BpMS90 strain was streaked on an LB agar plate and cultured overnight in a constant temperature incubator at 37°C. After the culture was completed, the morphological characteristics of the single colonies of the BpMS90 strain were observed, including shape, color, size, wetness, transparency, and edge neatness, and Gram staining and scanning electron microscopy were performed.

[0049] The Gram staining method is as follows: the selected strain BpMS90 is streaked on LB medium and grown at 37°C for 24 hours. Then a round, smooth, milky white single colony is picked for Gram staining. The bacteria are smeared, fixed, and initially stained with crystal violet stain for 60 seconds, and the stain is washed away with water. Iodine solution is added for mordant staining for 60 seconds, washed with water, decolorized with 95% ethanol, washed with water, and counterstained with diluted carbolic acid fuchsin for about 30 seconds, washed with water, excess water is absorbed by absorbent paper, dried, and observed under a microscope. Figure 1 As shown in C.

[0050] The scanning electron microscopy method is as follows: ferment the screened strain BpMS90 in LB liquid culture medium at 37°C and 135rpm for 24 hours, take 1ml of bacterial solution and centrifuge at 12000rpm for 10min, and remove the supernatant. Add 1ml of 2.5% glutaraldehyde fixative to the precipitate, shake it appropriately, and let it stand overnight. Then take out the fixed BpMS90 sample from the 2.5% glutaraldehyde and dehydrate it with 30%, 50%, 70%, 100%, and 100% ethanol gradients, each gradient for 10 minutes. After dehydration, take out the sample from 100% ethanol and put it into a supercritical dryer for critical point drying. The drying time is about 1 hour. After drying, take out the sample from the sample chamber of the critical point dryer, fix it on the sample stage with conductive tape, spray gold, and test it, such as Figure 1 As shown in D.

[0051] from Figure 1 It can be seen that the colonies of BpMS90 on LB medium are round or oval, slightly yellow, opaque, dry and wrinkled on the surface, sinking inward in the middle, with irregular edges, sticky and difficult to pick up ( Figure 1 A, 1B), Gram-negative bacteria ( Figure 1 C). Under a scanning electron microscope, BpMS90 is in the shape of a long rod with arc-shaped upper and lower ends ( Figure 1 D).

[0052] (2) Physiological and biochemical analysis

[0053] The physiological and biochemical characteristics of BpMS90 were determined by referring to the existing methods in the "Common Bacterial System Identification Manual" and the "Berger's Bacterial Identification Manual". The items tested were: starch hydrolase, gelatin liquefaction, glucose fermentation test; carbon source utilization was: maltose, galactose, mannose; nitrogen source utilization was: L-aspartic acid, glutamic acid, serine, creatine. The physiological and biochemical results are shown in Table 1.

[0054] Table 1: Physiological and biochemical analysis of BpMS90

[0055]

[0056] In Table 1, "+" indicates a positive reaction, and "-" indicates a negative reaction. The data in Table 1 show that the results were positive in the starch hydrolyzing enzyme, gelatin liquefaction, and glucose fermentation tests. Carbon and nitrogen source utilization analysis found that BpMS90 can utilize maltose, galactose, mannose, glutamic acid, and serine, but cannot utilize L-aspartic acid and creatine.

[0057] (3) Molecular biological identification

[0058] The 16S rRNA gene of BpMS90 was amplified and sequenced. The primers were: 27F (5'-AGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-TACGACTTAACCCCAATCGC-3', SEQ ID NO.2). The PCR reaction system (20 μL) was: DNA template 2.0 μL, 27F primer 2.0 μL, 1492R primer 2.0 μL, Premix Taq enzyme 10.0 μL, RNase-free ultrapure water 4.0 μL;

[0059] PCR amplification reaction program: preheat at 95°C for 5 min, denature at 95°C for 45 s, anneal at 55°C for 45 s, extend at 72°C for 45 s, amplify for 30 cycles, and finally extend at 72°C for 5 min. The PCR product was detected by 1% agarose gel electrophoresis and sent to BGI for sequencing. The sequencing results were aligned by Blast. After sequencing, the sequences were submitted to the GenBank database for homology analysis. Subsequently, the phylogenetic tree was constructed using MEGA6.0 software, as shown in Figure 3 shown.

[0060] 16S sequence

[0061]

[0062] exist Figure 3 In the present study, the 16S rRNA of the BpMS90 strain was sequenced, and the 16S rRNA sequencing results of the BpMS90 strain were subjected to Blast comparison. The comparison results showed that the similarity between the BpMS90 strain and the Burkholderia plantarii GCF-001411805.1 strain was 99.804%, and the phylogenetic tree analysis was in the same branch. At the same time, the morphological characteristics of the strain BpMS90 were consistent with those of the members of the genus Burkholderia. Based on morphological observation, physiological and biochemical analysis and phylogenetic tree analysis, the classification and naming of BpMS90 were determined as: Burkholderia plantarii. The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 3, 2024, with the deposit number CGMCC NO.30848, and the deposit address is Beijing, China.

[0063] 3. Analysis of BpMS90 strain tolerance

[0064] ① Growth activity at different temperatures: Take BpMS90 seed solution and inoculate it into LB liquid medium at 2% inoculation volume. Then place it in a shaker at 16℃, 28℃, 37℃, and 42℃, shake and culture at 150r / min for 24h, and measure the OD of the bacterial solution at different temperatures with a spectrophotometer. 600 Each treatment was repeated 3 times in the experiment. Figure 2 As shown in A.

[0065] ② Salt tolerance: BpMS90 was inoculated into 100 mL of LB culture medium containing 1%, 3%, 5%, 7%, and 9% (W / V) sodium chloride. After culturing at 37°C and 150 r / min for 24 hours, the OD of the bacterial solution at different salt concentrations was measured using a spectrophotometer. 600 Each treatment was repeated 3 times in the experiment. Figure 2 As shown in B.

[0066] ③ Acid and alkali resistance: BpMS90 was inoculated into LB medium with a pH of 3 to 10, and cultured at 37°C and 150 r / min for 24 h. The OD of the bacterial solution at different pH values ​​was measured using a spectrophotometer. 600 Each treatment was repeated 3 times in the experiment. Figure 2 As shown in C.

[0067] according to Figure 2 A shows that in the temperature range of 16℃ to 42℃, the concentration of BpMS90 bacteria is lower than the initial OD 600The values ​​showed different degrees of growth; in the temperature range of 26℃ to 37℃, the growth condition of BpMS90 was particularly ideal, but the growth state was inhibited at 42℃.

[0068] according to Figure 2 B shows that the BpMS90 strain grows well in an environment of pH 5 to 8, especially at pH 8, where it grows best. However, at pH 9, the growth of the strain is significantly inhibited.

[0069] according to Figure 2 C shows that with the increase of salt concentration, the growth state of BpMS90 presents an opposite trend; when the salt content is 7%, the growth state of the bacteria begins to be inhibited.

[0070] 4. Determination of the broad-spectrum antibacterial activity of BpMS90 strain

[0071] The plant pathogens to be tested are shown in Table 2:

[0072] Table 2 Pathogenic bacteria strains tested

[0073]

[0074] Activate on PDA solid medium and place in a constant temperature incubator at 26℃ for 5-7 days. After cultivation, use a puncher to take a 0.5cm diameter bacterial cake from the edge of the pathogen hyphae and inoculate it in the center of the new PDA solid medium. Inoculate the BpMS90 strain 2.5cm from the left and right ends of the bacterial block (the control group is only inoculated with pathogens), and culture at 26℃. Repeat the treatment 3 times. After 7 days of cultivation, calculate the antibacterial rate. The calculation method of the antibacterial rate is as follows:

[0075]

[0076] The results of confrontation culture between BpMS90 strain and 6 tested pathogens are as follows Figure 4 Its antibacterial effect on 6 tested pathogens is shown in Table 3:

[0077] Table 3 Antibacterial activity of BpMS90 against six plant pathogenic fungi

[0078]

[0079] As shown in Table 3, the growth of pathogens was significantly inhibited around the single colony of BpMS90; the inhibition rate of BpMS90 strain against the six tested pathogens was more than 50%; among them, the inhibition effect on Fusarium oxysporum was the best, with an inhibition rate of 84.59%, followed by the inhibition rate on Botrytis cinerea at 75.22%; this shows that BpMS90 has a strong potential for biological control.

[0080] Example 2

[0081] This example mainly analyzes the control effect of BpMS90 on tomato Verticillium wilt.

[0082] 1. Effect of BpMS90 on Verticillium wilt JR2

[0083] ① The tomato Verticillium wilt pathogen JR2 was activated on the PDA solid culture medium and cultured in a constant temperature incubator at 26°C for 5 to 7 days. After cultivation, a 0.5 cm diameter bacterial cake was taken from the edge of the pathogen hyphae using a puncher and inoculated in the center of a new PDA solid culture medium. The BpMS90 strain was inoculated 2.5 cm from both ends of the bacterial block (the control group was only inoculated with the pathogen JR2) and cultured at 26°C for 7 days.

[0084] The inhibition rate of BpMS90 against JR2 was calculated as follows:

[0085]

[0086] The plate confrontation method was used to analyze the antagonistic ability of BpMS90 against tomato Verticillium wilt pathogen JR2. After 7 days of cultivation, according to Figure 5 A and 5D show that the BpMS90 colony periphery significantly inhibited the growth of JR2, with an inhibition rate of 58.6%.

[0087] ②Scanning electron microscope observation of confrontation effect

[0088] Sampling and fixation: Cut 8mm at the edge of the inhibited pathogen 3 The control group took the normal growth of pathogenic bacteria 8mm 3 The bacterial blocks were quickly placed in centrifuge tubes with 2.5% glutaraldehyde solution and fixed at 4°C overnight. The remaining were washed, replaced, and observed by scanning electron microscopy according to the method of Golding et al. (Golding CG, Lamboo LL, Beniac DR, et al. The scanning electron microscope in microbiology and diagnosis of infectious disease. Scientific Reports, 2016, 6 (1): 1-8.). The results are as follows Figure 5 B and 5E.

[0089] By scanning the inhibited area of ​​JR2, the control group JR2 mycelium structure was complete, the mycelium was plump, uniform in thickness, firm, without wrinkles or depressions, and the cell wall structure was complete. The mycelium of JR2 treated with BpMS90 was obviously twisted, uneven in thickness, wrinkled, broken, and shrunken. The outer layer of the mycelium was broken and the surface was dissolved, indicating that the activity of JR2 mycelium was significantly inhibited.

[0090] ③ Transmission electron microscopy observation of confrontation effect

[0091] Sampling and fixation: Cut 8mm at the edge of the inhibited pathogen 3 The control group took the normal growth of pathogenic bacteria 8mm 3 The bacterial blocks were fixed, dehydrated, infiltrated, embedded, sliced, and stained according to the method of Liu et al. (Liu Z, Fan C, Xiao J, et al. Metabolomic and transcriptome analysis of the inhibitory effects of Bacillus subtilis strain Z-14 against Fusarium oxysporum causing Vascular wilt diseases in cucumber. Journal of Agricultural and Food Chemistry, 2023, 71(5): 2644-2657.) and observed by transmission electron microscopy. The results are as follows Figure 5 C and 5F.

[0092] Transmission electron microscopy results showed that the internal structure of JR2 cells was changed after treatment with strain BpMS90; the cell thickness and cell wall thickness of the control hyphae were uniform, the morphology was normal, the organelle structure was relatively complete and evenly distributed in the cytoplasm; the JR2 cells treated with BpMS90 were deformed, the thickness changed, the cell membrane deformed, the cell wall thickness changed, and obvious ruptures occurred. BpMS90 had a significant inhibitory effect on the growth of JR2 cells.

[0093] 2. Potted experiment on prevention and control of tomato Verticillium wilt

[0094] 1. Mix the BpMS90 spores with sterilized soil 7 days in advance to make the soil contain 1×10 7cfu / g concentration of BpMS90 strains as the soil treatment group, and the sterile water treatment as the soil control group. MoneyMaker susceptible tomato seeds were sterilized and evenly sown on the pre-prepared 1 / 2MS solid medium. After germination, they were transferred to the pre-inoculated BpMS90 medium. Seven days after transplanting, the tomato seedlings of the treatment group and the control group were carefully pulled up and immersed in a medium containing 1×10 6 cfu / mL of JR2 pathogen spore solution for 30 minutes.

[0095] In addition, some seedlings in the control group were soaked in sterile water for 30 minutes to eliminate the influence of root injury on tomato growth. After JR2 and sterile water soaking, they were transplanted back to the original pot. The biological control group was recorded as BpMS90-JR2, the pathogen treatment group was recorded as JR2, and the group without any treatment was recorded as CK (such as Figure 6 A and 6C), each treatment was parallel to 24 small pots. The water used for plant growth must be sterilized by high pressure. After 21 days of cultivation in the smart greenhouse, the disease incidence, disease index and biological control effect were detected according to the method of Chen et al. (Chen Y, Gao X, Chen Y, et al. Inhibitory efficacy of endophytic Bacillus subtilis EDR4 against sclerotinia sclerotiorum on rapeseed. Biological Control, 2014, 78: 67-76.), and the biomass was measured (as shown in Table 4), with 24 plants treated each time.

[0096] 2. Experiment on the browning of vascular bundles of tomato plants and fungal recovery: The root-stem junction of tomato plants was cut off and cut into two small pieces with a blade. The browning of vascular bundles of plants in the JR2-BpMS90, JR2, and CK groups was observed and photographed (e.g. Figure 6 B); the experimental and control stems were cut into small sections of about 3 cm, sterilized in 5% sodium hypochlorite and 0.05% Tween solution for 7 min, 70% ethanol for 5 min, washed twice with distilled water, 5 min each time, and placed on PDA plates to re-isolate the fungi. The plates were incubated at 26°C for 7 days, and the fungi were observed to see if they could grow and photographed (such as Figure 6 D), each treatment was repeated three times.

[0097] Based on the above experiments, a pot experiment was conducted to analyze the effect of BpMS90 strain on tomato Verticillium wilt caused by JR2 ( Figure 6A and 6C). Compared with the blank control (CK), JR2 infection had a significant effect on tomato growth, manifested as slow growth, short plants, and yellowing and falling of cotyledons.

[0098] After inoculation with BpMS90 for biological control, tomato Verticillium wilt caused by JR2 was alleviated to a great extent, as shown in Table 4:

[0099] Table 4 Disease-resistant biomass of tomato seedlings inoculated with BpMS90

[0100]

[0101]

[0102] Note: “-” represents no disease; different lowercase letters indicate statistically significant differences among treatments (least significant difference test, P < 0.05);

[0103] According to Table 4, the blank control group decreased by 38.8%, 44.2%, 49.1%, 29.7% and 21.4% in plant height, stem width, root length, fresh weight and dry weight respectively compared with the control group JR2 inoculated group; while after being treated with BpMS90 strain, they only decreased by 16.9%, 28.8%, 32.3%, 15.6% and 7.1%. In terms of morbidity, the BpMS90-JR2 treatment group decreased from 78.3% to 35.7% compared with the JR2 treatment group. The disease index decreased from 48.53 to 19.26, and the biological control effect reached 60.9%. This shows that BpMS90 can produce a good biological control effect on tomato Verticillium wilt caused by JR2.

[0104] In order to further observe the browning of tomato stems in each treatment group, the stems were cut longitudinally with a scalpel. Figure 6 As shown in B, the stems of the JR2-inoculated group showed obvious browning, while the BpMS90-JR2 group did not show obvious browning, indicating that the presence of BpMS90 can significantly slow down the infection of JR2 hyphae on the stems. In addition, after the surface of each stem was disinfected, it was cut into blocks and cultured on PDA medium for 7 days to observe whether JR2 hyphae grew in the block tomato stems; Figure 6 As shown in D, obvious JR2 mycelium appeared around the stem tubers of the JR2 group, while the colony morphology of the stem tubers of the BpMS90-JR2 group was consistent with that of BpMS90, and no JR2 mycelium appeared; this further demonstrates that BpMS90 can significantly prevent and control the infection of JR2 to tomatoes.

[0105] 3. Analysis of biomass of Verticillium wilt pathogen in tomato plants

[0106] The JR2 pathogen DNA in the total DNA extracted from tomato roots and stems was quantified by PCR using OLG primers (F: 5′-CAGCGAAACGCGATATGTAG-3′, SEQ ID NO.4; R: 5′-GGCTTGTAGGGGGTTTAGA-3′, SEQ ID NO.5) and internal reference primers (UBI-F: 5′-GCCGACTACAACATCCAGAAGG-3′, SEQ ID NO.6; UBI-R: 5′-TGCAACACAGCGAGCTTAACC-3′, SEQID NO.7).

[0107] PCR reaction procedure: 94℃ pre-denaturation for 30s; 94℃ denaturation for 5s, 60℃ annealing for 30s, a total of 30 cycles; amplification analysis on BIO-RAD quantitative PCR instrument (CFX96), the results are as follows Figure 7 shown.

[0108] Depend on Figure 7 It can be seen that compared with the tomato experimental group inoculated with JR2 only, the root biomass in the BpMS90-JR2 group decreased by 6.32 times, and the biomass of fungal pathogens in its stems decreased more significantly, by 343 times, further confirming that the BpMS90 strain has a good preventive and control effect on tomato Verticillium wilt caused by JR2.

[0109] 3. Co-culture of BpMS90 and JR2

[0110] The preserved BpMS90 strain was activated by streaking culture on LB plates, and then a single colony of the activated BpMS90 strain was inoculated into 100 mL of PD liquid medium and cultured with shaking at 37 °C for 24 h. The concentration of the bacterial suspension was adjusted to about 1 × 10 5 cfu / mL. After inoculating JR2 strain on PDA solid medium at 26°C for 5-7 days, six bacterial blocks with a diameter of 0.5 cm were taken out with a cork puncher and placed in 100 mL PD liquid medium. The culture was shaken at 28°C for 48 hours, and the concentration of JR2 was adjusted to 1×10 7 cfu / mL. The JR2 bacterial solution after adjusting the concentration was mixed with the BpMS90 bacterial solution at a ratio of 9:1 for co-culture as the treatment group (BpMS90+JR2), and the JR2 bacterial solution was mixed with sterile water at a ratio of 9:1 as the control group (JR2). The mixture was incubated at 28°C for a period of time (2 to 5 days).

[0111] Take the fermentation liquid from 2 to 5 days and observe the growth of JR2 and BpMS90 under the microscope. Figure 8 As shown. Figure 8It can be seen that Figure 8 As shown in A-8D, JR2 strains grew significantly on the second day after culture, and on the fifth day, JR2 multiplied in large numbers on the culture medium. Figure 8 As shown in E-8H, the growth of JR2 co-cultured with BpMS90 was significantly inhibited on the second day, the spore morphology was also severely deformed, and JR2 could hardly produce conidia during the period of 2-5 days, indicating that the presence of BpMS90 had a significant inhibitory effect on the growth of JR2.

[0112] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all known technologies.

Claims

1. A plant Burkholderia strain, characterized in that The strain is Burkholderia plantari BpMS90 strain, which was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on June 3, 2024, with the deposit number CGMCC NO.30848.

2. The plant Burkholderia according to claim 1, characterized in that The nucleotide sequence of 16SrRNA of the plant Burkholderia sp. BpMS90 is shown in SEQ ID NO.

3.

3. A biological agent, characterized in that: The biological preparation contains the Burkholderia plantarum BpMS90 or its fermented bacterial liquid as described in claim 1.

4. The biological agent according to claim 3, characterized in that The biological preparation is a liquid preparation, a lyophilized powder or an immobilized preparation.

5. Use of the plant Burkholderia strain according to claim 1 or 2 or the biological preparation according to claim 3 or 4 in inhibiting plant pathogenic fungal diseases.

6. The use according to claim 5, characterized in that: The plants include tomatoes, cotton, and potatoes.

7. The use according to claim 5, characterized in that: The plant pathogenic fungi include Botrytis cinerea, Phytophthora capsici, Fusarium oxysporum, Alternaria brassicae, Cercospora arachidicola, or Piricularia oryzae.

8. A method for inhibiting plant pathogenic fungal diseases using the biological agent according to claim 3 or 4, characterized in that: The biological agent is applied to the test plants to suppress plant fungal diseases.

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