Method for enhancing biological control of bacteriophage on phytopathogen by using probiotics

Abstract

The invention discloses a method for enhancing biological control of bacteriophage on phytopathogen by utilizing probiotics. According to the invention, a carrying system composed of bacteriophages and probiotics is constructed, and the bacteriophages and the probiotics are utilized to synergistically improve the inhibition effect on pathogenic bacteria. In the carrying system, the bacteriophages can carry the probiotics to enter biological membranes and plant tissues where pathogenic bacteria are located, and the probiotics can promote movement and infection of the bacteriophages and promote increase of the number of the bacteriophages. Therefore, the carrying system can obviously reduce the number of the viable plant pathogenic bacteria and reduce the EPS content of protein, polysaccharide and eDNA generated by the plant pathogenic bacteria. The invention can be used for prevention and sustainable biological control of plant pathogens.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for enhancing the biological control of phages on plant pathogens by using probiotics. Background Art

[0002] Leaves are the main site of plant photosynthesis and are essential for the Earth's ecosystems, carbon cycles, energy conversion, and the stability and productivity of ecological and agricultural systems. However, pathogen infection and the resulting leaf diseases can damage leaf health, significantly reduce agricultural yields, and threaten food security. Chemical fungicides have been shown to be very effective in controlling plant pathogens and are widely used in modern agriculture, but their overuse has caused various environmental and health problems. These problems include soil and water quality degradation, increased resistance of plant pathogens, and potential threats to food safety from chemical residues. In addition, the non-selectivity of these chemicals can harm beneficial microorganisms, disrupt ecological balance, and reduce soil fertility. In the face of these challenges, bacteriophage biocontrol technology is emerging as a promising alternative for preventing bacterial infections in plants. Bacteriophages are viruses that specifically infect bacteria and can target and eliminate specific bacterial hosts with high precision without affecting other beneficial microorganisms or ecosystem health. This biocontrol strategy can effectively control plant diseases and has advantages in environmental sustainability. Therefore, the application of bacteriophages provides a new perspective for agricultural disease management and may play a central role in future crop protection strategies. However, pathogens sporadically colonize the surface of phylospheres and are shielded by biofilms, resulting in unstable phage biocontrol effects. In addition, resistance of plant pathogens to phages and regeneration of plant pathogens after treatment limit the scope and efficiency of phage biocontrol in agriculture. Harsh conditions on leaf surfaces, such as solar radiation, hinder the persistence of phages, which affects the prevention and sustainable biocontrol of plant pathogens. Therefore, it is important to enhance the adaptability of phages on leaf surfaces and their sustained removal effect on pathogens. Summary of the invention

[0003] The purpose of the present invention is to solve the problems existing in the prior art and provide a method for enhancing the biological control of plant pathogens by bacteriophages using probiotics.

[0004] The specific technical solutions adopted by the present invention are as follows:

[0005] In a first aspect, the present invention provides a method for enhancing the biological control of plant pathogens by bacteriophages using probiotics, comprising:

[0006] S1. For the target plant pathogen to be controlled, the first screening condition is that the target plant pathogen can be infected and replicated and multiplied with the target plant pathogen as a host, and the second screening condition is that the target phage can be adsorbed on a non-host probiotic that has an inhibitory effect on the target plant pathogen, and a target phage that satisfies both screening conditions is screened from the phage library;

[0007] S2, mixing and culturing the target bacteriophage with the non-host probiotics, so that the target bacteriophage is adsorbed on the non-host probiotics in a non-host form to form a carrying system;

[0008] S3. Adding the carrying system to the plant tissue or biofilm infected by the target plant pathogen to perform biological control on the growth of the target plant pathogen.

[0009] As a preferred embodiment of the first aspect, the target plant pathogen is screened and isolated from plant tissues with target disease symptoms by bacterial screening technology, and each candidate pathogen is subjected to a re-infection verification experiment on asymptomatic plants. The candidate pathogen that causes asymptomatic plants to show target disease symptoms in the verification experiment is used as the target plant pathogen.

[0010] As a preference for the first aspect above, the plant tissue having target disease symptoms is soybean leaves showing ** symptoms.

[0011] As a preferred embodiment of the first aspect, the non-host probiotic is Bacillus subtilis (BS).

[0012] As a preferred embodiment of the first aspect, the Bacillus subtilis (BS) is Bacillus subtilis ATCC 6633.

[0013] As a preferred embodiment of the first aspect, the method for screening target phage from the phage library is:

[0014] For different phages in the phage library, respectively, they are mixed and cultured with the target plant pathogen in LB culture medium to isolate candidate phages that can infect and replicate and proliferate using the target plant pathogen as a host cell;

[0015] Each candidate phage is mixed with the non-host probiotic bacteria and cultured, and transmission electron microscopy is used to verify whether there is non-host hitchhiking adsorption behavior between each phage and the non-host strain. If so, the corresponding candidate phage is used as the target phage.

[0016] As a preferred embodiment of the first aspect, in S3, the culture solution containing the carrying system is sprayed onto plant leaves infected by the target plant pathogen, so as to perform biological control on the growth of the target plant pathogen that causes disease symptoms on the plant leaves.

[0017] As a preferred embodiment of the first aspect, the spraying treatment of the culture solution containing the carrying system is carried out at night to avoid the inhibitory effect of ultraviolet light on bacteriophages.

[0018] As a preferred embodiment of the first aspect, the phage library is obtained by extracting and separating and purifying phages from root soil of plants infected by the target plant pathogen.

[0019] In a second aspect, the present invention provides a carrying system consisting of a bacteriophage and a probiotic, wherein the carrying system is composed of a bacteriophage adsorbed on the non-host probiotic in a non-host form; the probiotic is Bacillus subtilis ATCC6633; and the bacteriophage is a bacteriophage of a plant pathogen capable of infecting soybean bacterial leaf spot.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention constructs a carrying system composed of bacteriophages and probiotics, which utilizes bacteriophages and probiotics to synergistically improve the inhibitory effect on pathogens. In the carrying system, bacteriophages can carry probiotics into the biofilm and plant tissues where pathogens are located, and probiotics can promote bacteriophage movement and infection and promote the increase in the number of bacteriophages. Therefore, the carrying system can significantly reduce the number of surviving plant pathogens and reduce the EPS content of proteins, polysaccharides and eDNA produced by plant pathogens. The present invention can be used for prevention and sustainable biological control of plant pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Comparison of the changes in the total number of pathogens over time after treating mixed pathogen biofilms with different methods.

[0023] Figure 2 Dynamic changes in chlorophyll content and number of pathogens in soybean leaves after treatment with the control group, free phages and phage-carrying system.

[0024] Figure 3 (A) Comparison of total viable cell numbers and standard biomass after 6 hours under different treatments. (B) Comparison of normalized extracellular polymer components after 6 hours under different treatments. DETAILED DESCRIPTION

[0025] In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific implementation mode of the present invention is described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The technical features in each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0026] In the description of the present invention, it is to be understood that when an element is considered to be "connected" to another element, it may be directly connected to the other element or indirectly connected, that is, there are intermediate elements. On the contrary, when an element is said to be "directly" connected to another element, there are no intermediate elements.

[0027] In the description of the present invention, it should be understood that the terms "first" and "second" are only used for the purpose of distinguishing descriptions, and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features.

[0028] In a preferred embodiment of the present invention, a method for enhancing the biological control of plant pathogens by bacteriophages using probiotics is provided, the method comprising the following steps:

[0029] S1. For the target plant pathogen to be controlled, the first screening condition is that the target plant pathogen can be infected and replicated and multiplied with the target plant pathogen as a host, and the second screening condition is that the target phage can be adsorbed on a non-host probiotic that has an inhibitory effect on the target plant pathogen, and a target phage that satisfies both screening conditions is screened from the phage library;

[0030] S2, mixing and culturing the target bacteriophage with the non-host probiotics, so that the target bacteriophage is adsorbed on the non-host probiotics in a non-host form to form a carrying system;

[0031] S3. Adding the carrying system to the plant tissue or biofilm infected by the target plant pathogen to perform biological control on the growth of the target plant pathogen.

[0032] It should be noted that the target plant pathogen in the present invention is a plant pathogen that needs to be biocontrolled, and the specific bacterial species is not limited. The methods described in S1 to S3 above are general methods. Based on this method, a delivery system capable of controlling the target plant pathogen can be constructed for different target plant pathogens, and this delivery system can be used to biologically control the growth of the target plant pathogen. In this delivery system, phages and probiotics are used to synergistically enhance the inhibitory effect on the pathogen. Among them, phages can carry probiotics into the biofilm and plant tissues where the pathogen is located, and probiotics can promote the movement and infection of phages and increase the number of phages.

[0033] Therefore, like the target plant pathogen, the target phage and non-host probiotics are not specific bacterial species, and the specific bacterial species type needs to be screened according to the target plant pathogen. Among them, the target phage needs to meet the above two screening conditions. The first screening condition is that it can infect the target plant pathogen and replicate and proliferate with the target plant pathogen as the host, so as to cause lysis of the target plant pathogen during the replication and proliferation process, thereby having a certain inhibitory effect on the target plant pathogen. The second screening condition is that it can adsorb on non-host probiotics that have an inhibitory effect on the target plant pathogen. In this condition, the probiotics only serve as an adsorption carrier for the phage to hitchhike and migrate, but will not replicate and proliferate with the probiotics as the host to damage the probiotics. In addition, in addition to meeting the condition of being able to adsorb phages in a non-host form, the above probiotics should also meet another condition, that is, they should have an inhibitory effect on the target plant pathogen. For different target plant pathogens, there are different probiotics. Therefore, the specific probiotic species can be selected by referring to the reports on the inhibitory bacterial species of the target plant pathogen in the existing technical literature.

[0034] In the present invention, for any one type of disease symptom (referred to as the target disease symptom), the target plant pathogen, target phage, and non-host probiotics can be screened in the following manner.

[0035] In the embodiments of the present invention, the target plant pathogen can be screened and isolated from the plant tissues with the target disease symptom by bacterial screening techniques, and each candidate pathogen is subjected to a reinfection verification experiment on asymptomatic plants. The candidate pathogen that causes the asymptomatic plants to exhibit the target disease symptom in the verification experiment is used as the target plant pathogen.

[0036] Taking soybean bacterial leaf spot as an example, a series of candidate pathogens can be screened and isolated from soybean leaves infected with soybean bacterial leaf spot by bacterial screening technology. After purification, these candidate pathogens can be used for reinfection verification experiments on symptomless plants. If a candidate pathogen causes soybean leaves that originally grew normally to show soybean bacterial leaf spot in the verification experiment, this candidate pathogen can be used as a target plant pathogen. It should be noted that the target plant pathogens actually extracted may be one or more.

[0037] In addition, for any target disease symptom, non-host probiotics that can inhibit target plant pathogens can be found through literature reports or actual experiments. Taking soybean bacterial leaf spot as an example, the corresponding non-host probiotic can be Bacillus subtilis (BS). In an embodiment of the present invention, the Bacillus subtilis (BS) can be commercially available Bacillus subtilis, with a product catalog number of ATCC 6633.

[0038] In addition, for any target disease symptom, the method for screening the target phage from the phage library is:

[0039] First, for different phages in the phage library, they are mixed with the corresponding target plant pathogens in LB medium to isolate candidate phages that can infect and replicate with the target plant pathogens as host cells;

[0040] Then, each candidate phage was mixed with a non-host probiotic capable of inhibiting the target plant pathogen, and transmission electron microscopy was used to verify whether there was non-host hitchhiking adsorption behavior between each phage and the non-host strain. If so, the corresponding candidate phage was used as the target phage.

[0041] It should be noted that the more types of phages the above-mentioned phage library theoretically contains, the better, and the more target phages that can be screened. However, a phage library that is too large will cause a greater workload for screening and a lower efficiency. In an embodiment of the present invention, considering that the root soil of plants infected by the target plant pathogens generally contains target phages that meet the screening conditions, it is possible to collect the root soil of plants infected by the target plant pathogens, extract the phages contained therein, and then separate and purify the extracted mixed phages to obtain a series of pure phages as the aforementioned phage library, and verify whether each phage meets the aforementioned two screening conditions. This screening method can narrow the scope of the phage library and quickly and accurately screen the target phages.

[0042] It should be noted that the target phages screened out in the end are not necessarily just one. Since there are many types of phages in nature, generally speaking, a series of target phages that meet the above two screening conditions can be screened out in the end, and these target phages can be used alone or in combination as the target phages for constructing the delivery system in the end.

[0043] It should be noted that if there are multiple target plant pathogens that cause the target disease symptoms, then when the phage is mixed with the target plant pathogen in LB medium to screen the candidate phage, it is necessary to screen out the candidate phage that can simultaneously infect and replicate all the target plant pathogens as host cells. At the same time, the corresponding probiotics should also be able to inhibit all target plant pathogens of the target disease symptoms to ensure that the final carrying system can inhibit all target plant pathogens.

[0044] In addition, in the present invention, the method of adding the finally constructed carrying system during application needs to be determined according to the corresponding control target. Plant tissues or biofilms can be used as control targets. Taking plant leaves as an example, in actual application, the carrying system can be formulated into a liquid form. When used, the culture solution containing a large amount of the carrying system is sprayed onto the plant leaves infected by the target plant pathogens, and the growth of the target plant pathogens that cause disease symptoms on the plant leaves can be biologically controlled. The spraying process can be carried out regularly, and it is recommended to spray at night to avoid the inhibitory effect of ultraviolet light on bacteriophages.

[0045] Taking soybean bacterial leaf spot as an example, an example is given to illustrate how to construct a transport system composed of bacteriophages and probiotics for such diseased plants. In the following example, a transport system is formed by bacteriophages adsorbed on non-host probiotics in a non-host form; the probiotics are Bacillus subtilis ATCC 6633; and the bacteriophages are phages of plant pathogens that can infect soybean bacterial leaf spot selected from the soil where the diseased plants are located. However, it should be noted that this example is only an example, and the present invention is not limited to the use of the bacteriophages and probiotics specifically extracted in this example.

[0046] Example

[0047] The first step is to screen and obtain soybean phytopathogens and probiotics

[0048] (1) In this example, bacterial plant pathogens were isolated from soybean leaves with obvious symptoms of soybean bacterial leaf spot in soybean fields of Hailun Farm, Harbin, China. Each sample was collected in a sterile glass stopper bottle and stored in an ice pack. The leaf samples were cut into small pieces of 5 mm × 5 mm and sterilized with 0.1% mercuric chloride for 2 minutes. The crushed leaf tissue was immersed in an inoculation loop and underlined on King's B medium plates supplemented with 50 mg / mL spectinomycin and cultured for 1 to 2 days. After 16S rRNA sequencing, 117 plant pathogens were identified, belonging to Pseudomonas agglomerans (PA) and Pseudomonas syringae (PS), with accession numbers PP998474 and PP998476, respectively. Morphological observations of PA and PS were performed using a standard scanning electron microscope (SEM, FEIQUANTA 200), and strains were selected based on their morphological uniqueness.

[0049] (2) Conduct a reinfection verification experiment. A typical soybean (variety: SUINONG-14) grown in the black soil region of Northeast China was selected as a model plant. The soybean seeds were surface sterilized, incubated at 28±2℃ for 2 days, and then selected for cultivation. After 1-2 hours of water bath culture at 30℃, soybeans with consistent growth after germination were selected and transplanted into pots with a mixture of black soil and vermiculite (1:1). During the planting process, 6 soybean plants were planted in each pot. All plants were illuminated for 16 hours at 22℃, and the relative humidity was maintained at 60%-70%. The pathogens PA and PS were cultured to the exponential growth phase, respectively, and enriched by centrifugation at 14000g and 4℃ for 10 minutes. Subsequently, the enriched bacteria were resuspended in a sterile 10mM MgCl2 solution to prepare the inoculum. Then the diluted plant pathogen PA and PS solutions screened in the previous step were mixed in a 1:1 ratio to obtain the final inoculum pathogenic solution. A sterile syringe was used to inject 106CFU / mL of the concentration. About 0.5 mL of the bacterial suspension was evenly sprayed on both sides of the needle-shaped leaves at the junction of the petiole (triplet stage). Then the sprayed plants were immediately covered with a transparent plastic bag for at least one day. Each plant was cultured in a plant growth chamber for 30 days and observed for a long time under environmental conditions of about 25°C. The development of soybean bacterial leaf spot symptoms in all plants was monitored daily. Bacteria were re-isolated from selected plants with soybean bacterial leaf spot to confirm that the symptoms were caused by the inoculum. Finally, two single colonies (strains PA and PS) were screened out, and after multiple separations and purifications, they were preserved in an equal volume of 30% glycerol medium and stored at -80°C. The collected plant pathogenic strains were cultured at 30°C and 150rpm for 24h, and 2-3 mL of bacterial culture with OD600 = 0.8-1.0 was taken, and genomic DNA was extracted using the Ezup column bacterial genomic DNA isolation kit. Then the polymerase chain reaction (PCR) for amplification was performed. The PCR conditions were as follows: 96°C for 1 min, followed by 25 cycles of 96°C for 10 s, 55°C for 5 s, and 60°C for 4 min, and finally extended at 72°C for 5 min. Then, the 16S rRNA sequences of the two plant pathogens were compared with the GenBank database by the online BLAST program and deposited in GenBank, respectively. Thus, after the reinfection verification experiment, it was proved that the pathogens PA and PS were the causative bacteria of soybean bacterial leaf spot.

[0050] (3) Bacillus subtilis (BS) (ATCC 6633) was selected as a probiotic strain due to its motility and potential to control plant pathogens. The Bacillus subtilis strain was obtained from the BeNa culture collection. This strain can form clear bacterial circles on the bacterial lawns of pathogens PA and PS. BS was added to LB medium and cultured as the basis for subsequent experiments.

[0051] The second step is to screen the phages that meet the requirements

[0052] (1) Phages were isolated from soybean rhizosphere soil of soybean fields in Hailun Farm, Harbin, China, from which bacterial plant pathogens were isolated. 10 g of soil samples were extracted using 10% beef extract buffer and incubated overnight in a constant temperature shaker at 10°C. After vortexing and sonication of the soil samples, particles larger than 0.22 μm were removed using centrifugation and filtration. The phage extract in the filtrate was then further concentrated by polyethylene glycol 8000 (PEG 8000) precipitation and resuspended in SM buffer (50 mmol / L Tris-HCl [pH 7.5], 0.1 mol / L NaCl, 8 mmol / L MgSO4, 0.01% gelatin) to obtain an initial phage stock solution. To remove any contaminants, phages were further isolated from the initial phage stock solution, three purification cycles were performed for each phage, and the phage titer was determined by triplicate double-layer plaques in PFU per milliliter. The different phages isolated and purified are added to the phage library for subsequent screening.

[0053] (2) Cultivate the mixture of phage and pathogens. For each purified phage in the phage library, use the plant pathogenic strains PA and PS as hosts in turn, and culture them in LB medium for 24 hours to determine whether they can replicate and proliferate with these two pathogenic strains as hosts at the same time. If possible, centrifuge the final cultured mixture at 10000g for 10 minutes at 4°C, remove residual cells through a 0.22μm filter, and store it at 4°C as a candidate phage for further analysis. In this embodiment, this step obtained a variety of candidate phages.

[0054] (3) When the Bacillus subtilis cultured in LB medium is in the stable growth phase, the suspension is diluted to OD600 = 0.1, and 1 mL of the Bacillus subtilis suspension is added to 45 mL of the phage stock solution of each candidate phage, and incubated separately in a shaker (30°C, 130 rpm) to promote the adhesion of the candidate phage to the Bacillus subtilis cells. Then, the mixture of Bacillus subtilis cells and candidate phages is collected and centrifuged at 4000g for 10 min at 4°C. Transmission electron microscopy (TEM) is used to verify the hitchhiking behavior between each candidate phage and the non-host strain. 5 μL of each phage suspension with a concentration of 109 PFU / mL is loaded onto a carbon-coated copper grid for 10 minutes and stained with 1% (w / v) uranyl acetate for 1 minute. The stained samples are washed twice with water and air-dried for at least 30 minutes. Subsequently, the samples are observed by transmission electron microscopy to verify the hitchhiking behavior between each candidate phage and the non-host strain Bacillus subtilis.

[0055] In this example, two candidate phages that can not only exhibit hitchhiking behavior with the non-host strain Bacillus subtilis but also use the pathogenic bacteria PA and PS as host bacteria were screened and verified, denoted as phage HBP1 and phage HBP2. In King's B medium, these two phages in the exponential growth phase were enriched in the PS strain overnight, and the purified phages were separately stored in SM buffer at 4 °C. The phage titer was quantified using the double-layer plaque method and expressed as plaque-forming units (PFU).

[0056] Step 3: Treat the pathogenic biofilm with the delivery system composed of phages and probiotics and characterize the effect

[0057] (1) Mix the screened phages HBP1 and HBP2 with the probiotic strain BS in PBS buffer so that the phages adsorb on Bacillus subtilis in a non-host form to form a delivery system. Negative staining was performed with 1% (w / v) uranyl acetate, and then transmission electron microscopy imaging was used to verify the electrophoresis system between the phages and bacteria. Subsequently, a migration test was carried out on a double-layer plate to verify the construction of the delivery system. A bacterial lawn was formed using the pathogenic strain and treated with the following settings: (i) a control group using PBS buffer (labeled BT1), (ii) a phage group (labeled BT2, phage concentration of 106 PFU / mL), (iii) a probiotic group using strain BS (labeled BT3, probiotic concentration of 106 PFU / mL), (iv) a phage group using the Bacillus subtilis-phage phage system (BS-phage system, labeled BT4). At least 3 parallel tests were performed for each group, and the infected area was measured after 6 hours, 12 hours, and 24 hours of incubation respectively.

[0058] (2) Establish a mixed-species biofilm of the plant pathogens PA and PS in a 96-well plate and then treat it with the above different groups. Each biofilm was cultured in 8 parallel cultures for 6 hours.

[0059] (3) Using d-glucose, bovine serum and calf thymus DNA as standards, the contents of polysaccharides, proteins and eDNA were determined by phenol-sulfuric acid method, Coomassie brilliant blue spectrophotometry and diphenylamine reagent method, respectively. The total cell count of pathogens was determined using King's B agar plates containing 50 mg / mL spectinomycin and 50 mg / mL ampicillin, and the number of phages was counted using the double-layer plate method. The biofilm structure and cell count of each treatment were observed using confocal laser scanning microscopy (CLSM), using 488 nm laser line and 560 nm laser line. Living cells emitted green light under the excitation of 488 nm laser line, and dead bacteria emitted red light under the excitation of 560 nm laser line. The biofilms in 96-well plates were stained with SYTO 9 and propidium iodide (PI) of the LIVE / DEAD BacLight kit (ApexBio Technology). The structure of the biofilm was then detected using Nikon A1-Rsi CLSM and the number of dead and living cells was calculated using Image J (1.8.0) software.

[0060] (4) Treating pathogen-infected leaves with a delivery system composed of bacteriophages and probiotics. The superiority of the bacteriophage infection system composed of bs-phages and its synergistic mechanism were verified by infection experiments in soybean leaf layers. The soybean leaves used in the reinfection experiment in the first aspect and the inoculated pathogenic liquid were used for treatment experiments. After the disease symptoms appeared on each leaf, it was divided into three treatments: (i) sterile water control treatment (marked as PT1); (ii) bacteriophage treatment (i.e., bacteriophage HBP1 and HBP2, 106PFU / mL, MOI=1, marked as PT2); (iii) BS-phage delivery system treatment prepared by adsorbing bacteriophages to strain BS (marked as PT3). The experimental soybean plants were cultured in an artificial climate incubator, and each treatment was sprayed continuously at night for 1, 4, 7, and 20 days to avoid the inhibitory effect of ultraviolet light on bacteriophages. The wet spray volume was stabilized at 5mL and cultured in an artificial climate incubator. All experiments were repeated 6 times independently. Fresh leaves were collected by punching (leaf disc) method at 0, 10, 20, and 30 days after treatment, and bacterial and bacteriophage counts were determined by plate method and double plate method. Secondly, chlorophyll of soybean leaves at 0, 10, 20, and 30 days was extracted and its content was estimated according to the classical Arnon equation as reported previously. Thirdly, parallel leaf samples were collected on day 10 and snap-frozen in liquid nitrogen (-80°C storage) for further metatranscriptomic and metabolomic analysis. To evaluate the long-term inhibitory effect of the carrier system on the growth of soybean plant pathogens, the mixed pathogenic suspensions of corresponding concentrations and quantities were evenly sprayed on both sides of the treated leaves (PT2 and PT3 treatment groups) and infiltrated. Leaf samples were taken after infection with pathogens to determine the total number of pathogens.

[0061] The relevant experimental results of this embodiment are shown below.

[0062] Figure 1 The comparison of the total number of pathogens over time after different methods were used to treat mixed pathogen biofilms was shown. Figure 1 It can be seen that the inhibitory effect of the probiotic BS and the phage mixture on pathogens is similar, but the inhibitory effect is not obvious, and the number of pathogens tends to increase in the later period. The BS-phage carrier system has a better inhibitory effect on pathogens, and the pathogens continue to decrease within 5 days, and it has always been better than the two treatments alone, and the total number of pathogens has decreased significantly.

[0063] Figure 2 The dynamic changes of chlorophyll content and number of pathogens in soybean leaves after treatment with control group A1, free phage A2 and phage carrier system A3 were shown. The total number of pathogens in the control group continued to increase to 1.36×10 within 30 days. 7 CFU / g leaf. The plant pathogen count of the phage group alone decreased significantly to 0.92×10 within 10 days. 7 CFU / g, and in the following days, it was 0.80×10 7 For the phage-probiotic group, the plant pathogen counts were significantly reduced to 0.53×10 after 10 and 30 days, respectively. 7 CFU / g and 0.18×10 7 CFU / g (p<0.05, statistically significant), indicating that the carrying system had a good effect on the treatment of pathogens. After biological control treatment, the chlorophyll levels of A2 and A3 were 10.25 mg / g and 12.35 mg / g, respectively, which were higher than the chlorophyll content of the control group A1. Since probiotics promote phage movement and infection, the phage count in A3 increased faster than that in A2. The final phage count in A3 was 1.2 times that of PT2, showing the promotion effect of probiotics on the increase of phage count.

[0064] Figure 3The cell survival and extracellular polymer components after 6 hours under different treatments are shown, where (A) is the comparison of the total number of surviving cells and the standard biomass after 6 hours under different treatments, and (B) is the comparison of the normalized extracellular polymer components after 6 hours under different treatments. It can be seen that after treatment with strain BS, phage and phage-BS delivery system, the total pathogenic biofilm biomass decreased by 30%, 43% and 58%, respectively (A). In addition, after treatment with the phage-BS delivery system, the number of surviving pathogenic bacteria decreased significantly, which was 3.9 times, 7.5 times and 11.2 times lower than that of strain BS, phage cocktail and control group, respectively (p<0.05) (A). Compared with the untreated control group, the phage-BS delivery system significantly reduced the EPS content of protein, polysaccharide and eDNA (p<0.05), which was reduced by 45%, 47% and 61%, respectively (B), indicating that the phage-BS delivery system has the potential to control biofilm.

[0065] The above-described embodiments are only some preferred implementations of the present invention, but are not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A method for enhancing the biological control of plant pathogens by bacteriophages using probiotics, characterized in that: include: S1. For the target plant pathogen to be controlled, the first screening condition is that the target plant pathogen can be infected and replicated and multiplied with the target plant pathogen as a host, and the second screening condition is that the target phage can be adsorbed on a non-host probiotic that has an inhibitory effect on the target plant pathogen, and a target phage that satisfies both screening conditions is screened from the phage library; S2, mixing and culturing the target bacteriophage with the non-host probiotics, so that the target bacteriophage is adsorbed on the non-host probiotics in a non-host form to form a carrying system; S3. Adding the carrying system to the plant tissue or biofilm infected by the target plant pathogen to perform biological control on the growth of the target plant pathogen.

2. The method for enhancing the biological control of plant pathogens by bacteriophages using probiotics as claimed in claim 1, characterized in that: The target plant pathogens are screened and separated from candidate pathogens by bacterial screening technology from plant tissues with target disease symptoms, and each candidate pathogen is subjected to a reinfection verification experiment on asymptomatic plants. The candidate pathogens that cause asymptomatic plants to exhibit target disease symptoms in the verification experiment are used as target plant pathogens.

3. The method for enhancing the biological control of plant pathogens by bacteriophages using probiotics as claimed in claim 2, characterized in that: The plant tissue with target disease symptoms is soybean leaves showing soybean bacterial leaf spot.

4. The method for enhancing the biological control of plant pathogens by bacteriophages using probiotics as claimed in claim 3, characterized in that: The non-host probiotic is Bacillus subtilis (BS).

5. The method for enhancing the biological control of plant pathogens by bacteriophages using probiotics as claimed in claim 4, characterized in that: The Bacillus subtilis (BS) is Bacillus subtilis ATCC 6633.

6. The method of using probiotics to enhance the biological control of phages against plant pathogens as claimed in claim 1, characterized in that: The method for screening target phage from the phage library is: For different phages in the phage library, respectively, they are mixed and cultured with the target plant pathogen in LB culture medium to isolate candidate phages that can infect and replicate and proliferate using the target plant pathogen as a host cell; Each candidate phage is mixed with the non-host probiotic bacteria and cultured, and transmission electron microscopy is used to verify whether there is non-host hitchhiking adsorption behavior between each phage and the non-host strain. If so, the corresponding candidate phage is used as the target phage.

7. The method of using probiotics to enhance the biological control of phages against plant pathogens as claimed in claim 1, characterized in that: In S3, the culture solution containing the carrying system is sprayed onto the plant leaves infected by the target plant pathogens to biologically control the growth of the target plant pathogens that cause disease symptoms on the plant leaves.

8. The method of using probiotics to enhance the biological control of phages against plant pathogens as claimed in claim 1, characterized in that: The culture solution spraying treatment containing the carrying system is carried out at night to avoid the inhibitory effect of ultraviolet light on bacteriophages.

9. The method of using probiotics to enhance the biological control of phages against plant pathogens as claimed in claim 1, characterized in that: The phage library is obtained by extracting phages from the root soil of plants infected by the target plant pathogens and separating and purifying them.

10. A carrying system composed of bacteriophage and probiotics, characterized in that: The carrying system is composed of bacteriophages adsorbed on the non-host probiotics in a non-host form; the probiotics are Bacillus subtilis ATCC 6633; and the bacteriophages are phages of plant pathogens capable of infecting soybean bacterial leaf spot.

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