A Burkholderia plantarii strain BpMS90 and its applications

Abstract

The present invention relates to the field of microbial technology, and in particular to a Burkholderia plantarii strain BpMS90 and its application. The Burkholderia plantarii BpMS90 strain of the present application was isolated and screened from the root tissue of Potentilla kleiniana, and was deposited on June 3, 2024 at the China General Microbiological Culture Collection Center (CGMCC), with the deposit number of CGMCC NO. 30848. The Burkholderia plantarii BpMS90 strain of the present application is safe and non-toxic by itself, environmentally friendly, and the biological agent containing Burkholderia plantarii BpMS90 has a wide antibacterial spectrum and has antibacterial activity against a variety of plant pathogenic fungi. It is a broad-spectrum microbial bactericide / bacterial fertilizer with good development and application prospects, and is of great significance for the green prevention and control of tomato Verticillium wilt disease.

Description

Technical Field

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

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

[0003] Verticillium dahliae has a high degree of genetic diversity and broad pathogenicity, can co-evolve with its host to produce new highly pathogenic physiological strains, and coupled with the difficulty of eliminating its microsclerotia structure, has long been a huge problem in agriculture. At present, the prevention and control of tomato Verticillium wilt mainly include agricultural control, chemical control and biological control.

[0004] Agricultural control measures mainly include technical means such as using resistant varieties and intercropping. The rationality and diversified application of agricultural control help to 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, and the slow control effect. For the control of tomato verticillium wilt, the agricultural control method cannot fundamentally eliminate it, but only reduce the incidence to a certain extent. Once the disease breaks out on a large scale, the agricultural control method simply cannot quickly control its spread.

[0005] Chemical control is a method of specifically controlling crop diseases by using chemical agents in various ways such as drip irrigation, spraying, and fumigation. Methyl bromide is considered the most effective chemical fumigant for controlling soil pathogens. However, due to its ozone-depleting effect and other environmental and health problems, the international community has gradually taken measures to restrict and phase out its use. For a long time, people have been working hard to find new chemical reagents to replace methyl bromide. Currently, common alternatives include chloropicrin (trichloronitromethane), dimethyl disulfide (DMDS), etc. The chemical control methods for tomato verticillium wilt in China mainly involve spraying carbendazim and irrigating the roots with thiophanate-methyl during the seedling stage or planting stage. Chemical methods are simple, fast, and effective. However, they can interfere with the environment, have an adverse impact on human health, damage 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 terms of nutrition and ecological niche, induction of host plants to produce resistance substances, secretion of antimicrobial substances by microorganisms themselves, and induction of the effective role of resistant pathogens, so as to effectively control 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, thus offsetting the disease impact of pathogens on plants. Currently, the biological control strategies for plant diseases mainly involve the use of biofertilizers and antagonistic microorganisms. These antagonistic microorganisms cover various types such as bacteria, fungi, and actinomycetes.

[0007] Tomato (S. lycopersicum), as an important economic crop, is widely cultivated globally. However, the application of chemical fertilizers in tomato production is highly dependent, and it is vulnerable to various plant pathogens, including Verticillium dahliae (V. dahliae). Currently, the control of plant diseases mainly relies on chemical control. This has a serious impact on the emergence of drug-resistant pathogens, the environment, and human health. Compared with chemical control, biological control has many advantages, such as being environmentally friendly, harmless to the health of humans and livestock, having good specificity for pathogens, leaving no residues in agricultural products, having a broad antibacterial spectrum, and having diverse disease-resistant pathways. The introduction of biological control not only effectively makes up for the disadvantages 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 beneficial for further promoting the development of biological control. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide a Burkholderia plantarii strain BpMS90 and its application. The provided Burkholderia plantarii can effectively inhibit a variety of plant pathogenic fungi and can effectively control Verticillium wilt of tomatoes.

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

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

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

[0012] In one aspect of the present invention, the present invention also provides a biological agent, and the biological agent contains the above-mentioned Burkholderia plantarii BpMS90 or its fermented broth. The Burkholderia plantarii BpMS90 of the present application can be used in the production of biological agents by large-scale fermentation culture to prepare the broth. The broth of the Burkholderia plantarii BpMS90 can also be separated to obtain its metabolites, and these metabolites are believed to have the same control effect as the fermented broth.

[0013] Preferably, the biological agent is a liquid preparation, a freeze-dried powder preparation or an immobilized preparation. The liquid preparation of the present application can be in the form of common microbial pesticides such as suspension, liposome liquid, emulsion, etc.; the freeze-dried powder is also a commonly used preparation form, which can be conveniently transported and used, and the activity of the bacteria can be efficiently protected in the freeze-dried form. Of course, the freeze-dried powder preparation can also contain freeze-drying protectants and stabilizers well-known in the art such as trehalose, protein, etc. The immobilized preparation is to immobilize microorganisms in a porous carrier, and the porous carrier includes diatomite, activated carbon, porous silica, etc. The advantage of immobilizing in the porous carrier is that the vitality of the microorganisms can be maintained for a long time and protected from external impacts.

[0014] In one aspect of the present invention, the present invention also provides an application of the Burkholderia plantarii strain 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, Alternaria brassicae, Cercospora arachidicola, or Piricularia oryzae.

[0017] In one aspect of the present invention, the present invention also provides a method for inhibiting plant pathogenic fungal diseases by using the biological agent as described above, applying the biological agent containing Burkholderia plantarii BpMS90 to the test plants to inhibit plant fungal diseases.

[0018] More preferably, the biological agent of the present invention can also be used together with other preparations for controlling plant fungal diseases to enhance its effect.

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

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

[0021] 2. The biological agent of Burkholderia plantarii BpMS90 of the present invention has a wide antibacterial spectrum and has antibacterial activity against a variety of phytopathogenic fungi. It is a broad-spectrum microbial agent / bacterial 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 culture conditions of the Burkholderia plantarii BpMS90 strain of the present invention are simple, the reproduction speed is fast, and it is easy to preserve and transport. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a morphological characteristic diagram of BpMS90. Among them, A and B are the single colony morphologies of the strain on the LB plate; C is Gram staining; D is a scanning microscope;

[0024] Figure 2 It is an analysis diagram of the tolerance of BpMS90. Among them, 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. Different letters indicate significant differences between treatments (p < 0.05);

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

[0026] Figure 4 It is a plate confrontation diagram of BpMS90 against 6 kinds of phytopathogenic fungi;

[0027] Figure 5 It is the confrontation effect diagram of BpMS90 against tomato verticillium wilt JR2. Among them, A is the mycelium morphology of the normal-growing JR2 plate; B is the mycelium morphology diagram under the scanning electron microscope; C is the transverse section morphology of the mycelium under the transmission electron microscope; D is the mycelium morphology diagram of the JR2 plate treated with BpMS90; E is the mycelium morphology of the JR2 treated with BpMS90 under the scanning electron microscope; F is the transverse section morphology of the mycelium of the JR2 treated with BpMS90 under the transmission electron microscope. The scales of A and D are 2 cm, the scales of B and E are 20 μm, and the scales of C and F are 500 nm;

[0028] Figure 6 It is the control effect diagram of the BpMS90 strain against tomato verticillium wilt. Among them, A and C are the phenotypes of tomato potted plants; B is the cross-section diagram of the tomato stem; D is the stem re-inoculated on PDA. CK is inoculated with clear water; BpMS90-JR2 is inoculated with BpMS90 and the pathogen JR2 at the same time; JR2 is only inoculated with the pathogen JR2;

[0029] Figure 7 It is the result diagram of the biomass of the verticillium wilt pathogen in tomato plants. Among them, the left is the root and the right is the stem;

[0030] Figure 8Figure showing the effect of co - culturing BpMS90 and JR2 on the growth state of JR2. Among them, A - D are for JR2 cultured alone; E - H are for co - culturing BpMS90 and JR2; d represents the number of days of culture, scale bar = 10μm. Detailed implementation manners

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to 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 of 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 belong to the scope of protection of the present invention.

[0032] In the following embodiments, those not specified in detail are carried out according to conventional conditions or conditions recommended by the manufacturer. For raw materials, equipment or instruments not specified in the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0033] The culture media used in the following embodiments of the present invention are as follows:

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

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

[0036] PDA medium: 200g of potato, sliced after peeling, added with 1000 mL of deionized water, boiled for 25 min, then filtered through double - layer gauze. 20g of glucose and 15g of agar powder were added to the filtrate, and finally made up to 1000 mL, natural pH, after aliquoting, autoclaved at 121 °C for 20 min.

[0037] PDB medium: 200g of potato, sliced after peeling, added with 1000 mL of deionized water, boiled for 25 min, then filtered through double - layer gauze. 20g of glucose was added to the filtrate, and finally made up to 1000 mL, natural pH, after aliquoting, autoclaved at 121 °C for 20 min.

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

[0039] Nitrogen source utilization medium: Replace glucose in the basal medium with 20 g of maltose, galactose, and mannose respectively, add 1000 mL of deionized water, adjust to natural pH, dispense, and autoclave at 121 °C for 20 min.

[0040] Carbon source utilization medium: Replace peptone in the basal medium with 2 g of glutamic acid, L-aspartic acid, serine, and creatine respectively, add 1000 mL of deionized water, adjust to natural pH, dispense, and autoclave at 121 °C for 20 min.

[0041] Example 1

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

[0043] I. The specific steps for the isolation and screening of Burkholderia plantarii BpMS90 are as follows:

[0044] Isolate from the root tissue of Potentilla kleiniana. Wash the root tubers of healthy Potentilla kleiniana plants collected from Tieshan Town, Dazu District, Chongqing City (105.5984° E, 29.6850° N) with clean water, then rinse 5 times with sterile water in a laminar flow hood, cut into 0.5×0.5 cm small pieces with scissors, soak in 75% ethanol for 1 min, soak in 5% sodium hypochlorite for 30 s, soak in 75% ethanol for 1 min, rinse 5 times with sterile water, dry the surface moisture of the tissue with sterile filter paper, and place on an LB plate. Incubate at 37 °C and observe daily for colony formation. When new colonies grow on the plate, transfer different single colonies into a new medium according to their different morphologies, repeatedly streak and purify to obtain single colonies (no other morphological colonies around), and preserve them. At the same time, take a small amount of the sterile water from the last rinse and spread it on an LB petri dish as a blank control, incubate at 37 °C for 24 h, and check whether the surface disinfection of the sample is complete.

[0045] Inoculate the isolated and purified endophytes into LB liquid medium respectively, incubate on a shaker at 37 °C and 150 rpm for 24 h to obtain the test strain bacterial solutions; take 50 μL of the diluted Verticillium dahliae suspension (1×10 7 CFU / mL) and spread it evenly on PDA medium. Then, use a sterile punch with a diameter of 6 mm to make symmetric holes 3 cm away from the center of the petri dish, add 50 μL of the test strain bacterial solution into the holes, repeat each treatment 3 times, incubate at 28 °C for 48 h, and use the holes with only PDB medium as the blank control to observe whether a clear inhibition zone appears. When a clear inhibition zone appears, the screened Burkholderia plantarii BpMS90 is obtained.

[0046] II. The morphological and molecular biological identification of Burkholderia plantarii BpMS90 is as follows:

[0047] (1) Morphological observation and identification

[0048] Inoculate the B. plantarii BpMS90 strain by streaking on an LB agar plate and incubate overnight in a constant temperature incubator at 37°C. After incubation, observe the morphological characteristics of the single colonies of the BpMS90 strain, including shape, color, size, humidity, transparency, and the regularity of the edges, and perform Gram staining and scanning electron microscopy observation.

[0049] The method of Gram staining is as follows: Streak the screened strain BpMS90 on an LB medium plate and grow at 37°C for 24 hours. Then pick round, smooth, milky white single colonies for Gram staining. Make a bacterial smear, fix it, add crystal violet stain solution for primary staining for 60 seconds, and rinse off the stain with water. Add iodine solution for mordanting for 60 seconds, wash with water, decolorize with 95% ethanol, wash with water, add diluted carbol fuchsin for counterstaining for about 30 seconds, wash with water, blot off the excess water with absorbent paper, dry, and observe under a microscope, as Figure 1 shown in C.

[0050] The method of scanning electron microscopy is as follows: Ferment the screened strain BpMS90 in an LB liquid medium at 37°C and 135 rpm for 24 hours. Take 1 ml of the bacterial liquid and centrifuge at 12000 rpm for 10 minutes to remove the supernatant. Add 1 ml of 2.5% glutaraldehyde fixative to the precipitate, shake gently, and let stand overnight. Then take out the fixed BpMS90 sample from 2.5% glutaraldehyde, perform gradient dehydration with 30%, 50%, 70%, 100%, 100% ethanol, 10 minutes for each gradient. After dehydration, take out the sample from 100% ethanol, 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, sputter gold, and test, as Figure 1 shown in D.

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

[0052] (2) Physiological and biochemical analysis

[0053] Referring to the methods in the "Common Bacterial System Identification Manual" and the "Bergey's Manual of Determinative Bacteriology", the physiological and biochemical characteristics of BpMS90 were determined. The items tested were: starch hydrolase, gelatin liquefaction, glucose fermentation test; for carbon source utilization: maltose, galactose, mannose; for nitrogen source utilization: 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 starch hydrolase, gelatin liquefaction, and glucose fermentation tests are all positive. Analysis of carbon source and nitrogen source utilization found that BpMS90 can utilize maltose, galactose, mannose, glutamic acid, and serine, but cannot utilize L-aspartic acid and creatine.

[0057] (3) Molecular Biology 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 procedure: After preheating at 95°C for 5 min, denaturation at 95°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 45 s, amplified for 30 cycles, and finally terminal extension at 72°C for 5 min. The PCR product was detected by 1% agarose gel electrophoresis and then sent to BGI for sequencing. After the sequencing results were compared by Blast, the sequence was submitted to the GenBank database for homology analysis after sequencing. Subsequently, a phylogenetic tree was constructed using MEGA6.0 software, as Figure 3 shown.

[0060] 16S sequence

[0061]

[0062] In Figure 3 it, by sequencing the 16S rRNA of strain BpMS90 and performing a Blast alignment on the 16S rRNA sequencing results of strain BpMS90, the alignment results showed that the similarity between strain BpMS90 and strain Burkholderia plantarii GCF-001411805.1 reached 99.804%, and phylogenetic tree analysis showed that they were in the same branch. At the same time, the morphological characteristics of strain BpMS90 were consistent with those of members of the genus Burkholderia. Through comprehensive morphological observation, physiological and biochemical analysis, and construction of phylogenetic tree analysis, the taxonomic naming of BpMS90 was determined as: Burkholderia plantarii. This strain was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on June 3, 2024, with the deposit number CGMCC NO.30848 and the deposit address in Beijing, China.

[0063] III. Tolerance analysis of strain BpMS90

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

[0065] ② Salt tolerance: Inoculate BpMS90 into 100 mL of LB culture solution containing 1%, 3%, 5%, 7%, and 9% (W / V) sodium chloride respectively. After shake-culturing at 37°C and 150 r / min for 24 h, use a spectrophotometer to measure the OD 600 value of the bacterial solution at different salt concentrations. Each treatment in the experiment was repeated 3 times, as Figure 2 shown in B.

[0066] ③ Acid and alkali tolerance: Inoculate BpMS90 into LB medium with a pH of 3 - 10 respectively. After shake-culturing at 37°C and 150 r / min for 24 h, use a spectrophotometer to measure the OD 600 value of the bacterial solution at different pH values. Each treatment in the experiment was repeated 3 times, as Figure 2 shown in C.

[0067] According to Figure 2 A, in the temperature range of 16°C to 42°C, the cell concentration of BpMS90 compared to the initial OD 600The values showed varying degrees of growth; in the temperature range of 26°C to 37°C, the growth of BpMS90 was particularly ideal, but the growth state was inhibited at 42°C.

[0068] According to Figure 2 B, in the environment with pH 5 - 8, the strain BpMS90 grew well; especially when the pH was 8, its growth was in the best state; but when the pH was 9, the growth of the strain was significantly inhibited.

[0069] According to Figure 2 C, with the increase of salt concentration, the growth state of BpMS90 showed an opposite trend; when the salt content was 7%, the growth state of the bacteria began to be inhibited.

[0070] IV. Determination of the broad-spectrum antibacterial property of BpMS90 strain

[0071] The test plant pathogens, as shown in Table 2:

[0072] Table 2 Test pathogen strains

[0073]

[0074] Activate on the PDA solid medium and place it in a constant temperature incubator at 26°C for 5 - 7 days. After culturing, use a punch to take a 0.5 cm diameter bacterial cake at the edge of the pathogen mycelium and inoculate it at the center of a new PDA solid medium. Inoculate the BpMS90 strain 2.5 cm from both ends of the bacterial block (the control group only inoculates the pathogen), culture at a constant temperature of 26°C, and repeat the treatment 3 times. After culturing for 7 days, calculate the inhibition rate, and the calculation method of the inhibition rate is as follows:

[0075]

[0076] The confrontation culture results of the BpMS90 strain and 6 test pathogens are as Figure 4 shown, and its antibacterial effects on 6 test pathogens are shown in Table 3:

[0077] Table 3 Antibacterial activity of BpMS90 against 6 plant pathogenic fungi

[0078]

[0079] According to Table 3, it can be seen that around the single colony of BpMS90, the growth of the pathogen was significantly inhibited; the inhibition rates of the BpMS90 strain against 6 test pathogens all reached over 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 being 75.22%; this indicates that BpMS90 has strong potential for biological control.

[0080] Example 2

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

[0082] I. Confrontation effect of BpMS90 on Verticillium dahliae JR2 of tomatoes

[0083] ① Activate the tested Verticillium dahliae pathogen JR2 on PDA solid medium, place it in a constant temperature incubator at 26 °C for 5 - 7 days. After culturing, use a punch to cut a 0.5 cm diameter fungal plug at the edge of the pathogen mycelium and inoculate it in the center of a new PDA solid medium. Inoculate the BpMS90 strain 2.5 cm from both ends of the fungal block (the control group only inoculates the pathogen JR2), and culture at a constant temperature of 26 °C for 7 days.

[0084] Calculate the inhibition rate of BpMS90 on JR2. The calculation method of the inhibition rate is as follows:

[0085]

[0086] Adopt the plate confrontation method to analyze the antagonistic ability of BpMS90 against the Verticillium dahliae pathogen JR2 of tomatoes. After culturing for 7 days, according to Figure 5 As shown in A and 5D, the growth of JR2 was significantly inhibited around the BpMS90 colony, and the inhibition rate was 58.6%.

[0087] ② Observe the confrontation effect by scanning electron microscopy

[0088] Sampling and fixation: Cut an 8 mm fungal block at the edge of the inhibited pathogen, and cut an 8 mm fungal block of the normally growing pathogen in the control group. Quickly place them into centrifuge tubes containing 2.5% glutaraldehyde solution respectively, and fix overnight at 4 °C. The remaining cleaning, replacement, and observation are carried out 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 shown in 3 as shown in B and 5E. 3 Figure 5

[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 7The BpMS90 strain at a concentration of cfu / g was used as the soil treatment group, and the sterile water treatment was used as the soil control group. The susceptible tomato seeds of MoneyMaker were disinfected, and the disinfected seeds were evenly sown on the pre-prepared 1 / 2MS solid medium. After germination, they were transferred to the pre-inoculated BpMS90. Seven days after transplantation, the tomato seedlings in the treatment group and the control group were carefully uprooted and immersed in the JR2 pathogen spore solution containing 1×10 6 cfu / mL for 30 min.

[0095] In addition, a part of the seedlings in the control group were immersed in sterile water for 30 min to exclude the influence of root injury on the growth of tomatoes. After soaking in JR2 and sterile water, they were transplanted back into the original pots. The biocontrol group was denoted as BpMS90-JR2, the pathogen treatment group was denoted as JR2, and the untreated group was denoted as CK (such as Figure 6 A and 6C), and each treatment had 24 small pots in parallel. The water used for plant growth needed to be autoclaved. After being placed in the intelligent greenhouse for 21 days, the disease incidence, disease index, and biocontrol 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 various biomass were measured (as shown in Table 4), with 24 plants treated each time.

[0096] 2. Tomato plant vascular browning and fungal recovery experiment: Cut off the rhizome junction of the tomato plant, and longitudinally cut it into two small pieces with a blade. Observe the vascular browning of the plants in the JR2-BpMS90, JR2, and CK groups and take pictures for record (as Figure 6 shown in B); Cut the experimental group and control stems into sections about 3 cm long, sterilize them in 5% sodium hypochlorite and 0.05% Tween solution for 7 min, sterilize them in 70% ethanol for 5 min, wash them twice with distilled water for 5 min each time, and place them on PDA plates to re-isolate the fungi. Incubate the plates at 26 °C for 7 days, observe whether the fungi can grow and take pictures for record (as Figure 6 shown in D), and each treatment was repeated three times.

[0097] According to the above experiments, the effect of the BpMS90 strain on tomato verticillium wilt caused by JR2 was analyzed through pot experiments ( Figure 6A and 6C). Compared with the blank control (CK), the infection of JR2 had a significant impact on the growth of tomatoes; it was manifested as slow growth, short plants, and accompanied by yellowing and shedding of cotyledons.

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

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

[0100]

[0101]

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

[0103] According to Table 4, compared with the control group JR2 inoculation group, the blank control group decreased by 38.8%, 44.2%, 49.1%, 29.7%, and 21.4% respectively in plant height, stem width, root length, fresh weight, and dry weight; after treatment with the BpMS90 strain, they only decreased by 16.9%, 28.8%, 32.3%, 15.6%, and 7.1%. In terms of the incidence rate, 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%. It shows that BpMS90 can have a good biological control effect on the Verticillium wilt of tomatoes caused by JR2.

[0104] To further observe the browning of the tomato stems in each treatment group, the stems were longitudinally cut with a scalpel. As Figure 6 shown in B, obvious browning occurred in the stems of the JR2 inoculation group, while no obvious browning occurred in the BpMS90-JR2 group; it shows that the presence of BpMS90 can significantly slow down the infection of JR2 hyphae on the stems. In addition, after disinfecting the outer surface of each stem, it was cut transversely into blocks and cultured on PDA medium for 7 days to observe whether there was growth of JR2 mycelium on the blocky tomato stems; as Figure 6 shown in D, obvious JR2 mycelium appeared around the stem blocks of the JR2 group, while the colony morphology of the stem blocks in the BpMS90-JR2 group was consistent with that of BpMS90, and no JR2 mycelium appeared; it further shows that BpMS90 can significantly prevent the infection of JR2 on tomatoes.

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

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

[0107] PCR reaction procedure: pre-denaturation at 94°C for 30 s; denaturation at 94°C for 5 s, annealing at 60°C for 30 s, for a total of 30 cycles; amplification and analysis were performed on a quantitative PCR instrument (CFX96) from BIO-RAD. The results are as Figure 7 shown.

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

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

[0110] The preserved BpMS90 strain was activated by streaking on an LB plate, 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 approximately 1×10 5 cfu / mL with fresh PD liquid medium. After the JR2 strain was inoculated on a PDA solid medium and cultured at 26°C for 5 - 7 d, six 0.5-cm-diameter fungal blocks were taken with a punch and placed into 100 mL of PD liquid medium, and cultured with shaking at 28°C for 48 h. The concentration of JR2 was adjusted to 1×10 7 cfu / mL with fresh PD liquid medium. The JR2 bacterial solution with adjusted concentration was mixed with the BpMS90 bacterial solution in a 9:1 ratio for co-culture as the treatment group (BpMS90 + JR2), and the JR2 bacterial solution was mixed with sterile water in a 9:1 ratio as the control group (JR2). They were co-incubated with shaking at 28°C for a period of time (2 - 5 days).

[0111] Take the fermentation broth of the above for 2 - 5 days and observe the growth status of JR2 and BpMS90 under co-culture under a microscope. The results are as Figure 8 shown. From Figure 8It can be seen that as Figure 8 shown in A-8D, the strain JR2 grew significantly on the second day after cultivation, and by the fifth day, JR2 had multiplied abundantly on the culture medium. As Figure 8 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 during the period from 2 to 5 days, JR2 could hardly produce conidia. This indicates that the presence of BpMS90 has an obvious inhibitory effect on the growth of JR2.

[0112] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention. The technologies, shapes, and structures not described in detail in the present invention are all well-known technologies.

Claims

1. A Burkholderia plantarii strain, characterized in that, The strain is Burkholderia plantarii BpMS90, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (CGMCC) on June 3, 2024, with the deposit number of CGMCC NO. 30848.

2. The Burkholderia plantarii according to claim 1, wherein The nucleotide sequence of the 16S rRNA of the Burkholderia plantarii BpMS90 is shown as SEQ ID NO.

3.

3. A biological preparation, characterized in that, The biological agent contains the Burkholderia plantarii BpMS90 described in claim 1 or its fermentation broth.

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

5. Use of the Burkholderia plantarii strain according to claim 1 or 2 or the biological agent according to claim 3 or 4 in inhibiting plant pathogenic fungal diseases, wherein the plant pathogenic fungi are Botrytis cinerea, Phytophthora capsici, Fusarium oxysporum, Alternaria brassicae, Cercospora arachidicola, Piricularia oryzae or Verticillium dahliae JR2.

6. The application according to claim 5, wherein The plants include tomatoes, cotton, and potatoes.

7. A method for inhibiting plant pathogenic fungal diseases by using the biological agent according to claim 3 or 4, characterized in that, Applying the biological agent to the test plants to inhibit plant fungal diseases, wherein the plant pathogenic fungi are Botrytis cinerea, Phytophthora capsici, Fusarium oxysporum, Alternaria brassicae, Cercospora arachidicola, Piricularia oryzae or Verticillium dahliae JR2.

Images