Methods of using probiotics to enhance the biocontrol of plant pathogens by bacteriophages

Abstract

This invention discloses a method for enhancing the biocontrol of plant pathogens by bacteriophages using probiotics. The invention constructs a transport system composed of bacteriophages and probiotics, which synergistically enhances the inhibitory effect on pathogens. In this transport system, bacteriophages can carry probiotics into the biofilm and plant tissues where pathogens reside, while the probiotics promote bacteriophage motility and infection, and increase the number of bacteriophages. Therefore, this transport system can significantly reduce the number of surviving plant pathogens and decrease the EPS content of proteins, polysaccharides, and eDNA produced by plant pathogens. This invention can be used for the prevention and sustainable biocontrol of plant pathogens.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for enhancing the biological control of plant pathogens by bacteriophages using probiotics. Background Technology

[0002] Leaves are the primary site of photosynthesis in plants, crucial for the stability and productivity of Earth's ecosystems, carbon cycles, energy conversion, and ecological and agricultural systems. However, pathogen infection and resulting leaf diseases can damage leaf health, significantly reduce agricultural yields, and threaten food security. Currently, chemical fungicides have proven highly 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 degradation, increased resistance to plant pathogens, and the potential threat of chemical residues to food safety. Furthermore, the non-selectivity of these chemicals can harm beneficial microorganisms, disrupt ecological balance, and reduce soil fertility. In response to these challenges, bacteriophage biocontrol technology is emerging as a promising alternative for preventing plant bacterial infections. Bacteriophages are viruses that specifically infect bacteria, enabling them to precisely target and eliminate specific bacterial hosts without affecting other beneficial microorganisms or ecosystem health. This biocontrol strategy can effectively control plant diseases and offers advantages in terms of environmental sustainability. Therefore, the application of bacteriophages provides a new perspective on agricultural disease management and may play a central role in future crop protection strategies. However, pathogens sporadically colonize the surface of leaf blades and are shielded by biofilms, leading to inconsistent biocontrol efficacy of bacteriophages. Furthermore, the development of resistance to bacteriophages by plant pathogens and the regeneration of pathogens after treatment limit the scope and efficiency of bacteriophage biocontrol in agriculture. Harsh conditions on leaf surfaces, such as solar radiation, hinder the persistence of bacteriophages, affecting the prevention of plant pathogens and sustainable biocontrol. Therefore, enhancing the adaptability of bacteriophages to leaf surfaces and their sustained removal of pathogens is crucial. Summary of the Invention

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

[0004] The specific technical solution adopted in this invention is as follows:

[0005] In a first aspect, the present invention provides a method for enhancing the biocontrol 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 infect the target plant pathogen and replicate and proliferate using the target plant pathogen as a host, and the second screening condition is that the target plant pathogen can be adsorbed onto non-host probiotics that have an inhibitory effect on the target plant pathogen. Target phages that meet both screening conditions are screened from the phage library.

[0007] S2. The target phage is mixed and cultured with the non-host probiotics, so that the target phage is adsorbed onto the non-host probiotics in a non-host form to form a transport system;

[0008] S3. The transport system is added to the plant tissue or biofilm infected by the target plant pathogen to biologically control 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 symptoms of the target disease using bacterial screening technology, and each candidate pathogen is subjected to a reinfection verification experiment on asymptomatic plants. The candidate pathogen that causes asymptomatic plants to exhibit symptoms of the target disease in the verification experiment is selected as the target plant pathogen.

[0010] As a preferred embodiment of the first aspect above, the plant tissue exhibiting the symptoms of the target disease is a soybean leaf displaying the symptoms.

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

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

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

[0014] Different phages in the phage library were mixed with the target plant pathogen in LB medium to isolate candidate phages that could infect and replicate using the target plant pathogen as a host cell.

[0015] Each candidate phage was co-cultured with the non-host probiotic, and the presence of non-host free-riding adsorption behavior between each phage and the non-host strain was verified by transmission electron microscopy. If such behavior was found, the corresponding candidate phage was selected as the target phage.

[0016] As a preferred embodiment of the first aspect above, in step S3, the culture solution containing the transport system is sprayed onto the leaves of plants infected by the target plant pathogen to biologically control the growth of the target plant pathogen causing disease symptoms on the plant leaves.

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

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

[0019] Secondly, the present invention provides a transport system composed of bacteriophages and probiotics, wherein the bacteriophages adsorb onto the non-host probiotics in a non-host form to form the transport system; the probiotics are Bacillus subtilis ATCC6633; and the bacteriophages are bacteriophages of plant pathogens capable of infecting soybean bacterial leaf spot disease.

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

[0021] This invention constructs a transport system composed of bacteriophages and probiotics, which synergistically enhances the inhibitory effect against pathogens. In this system, bacteriophages can carry probiotics into the biofilm and plant tissues where pathogens reside, while the probiotics promote bacteriophage motility and infection, and increase bacteriophage numbers. Therefore, this transport system can significantly reduce the number of surviving plant pathogens and decrease the EPS (extracellular protein, polysaccharide, and eDNA) content produced by plant pathogens. This invention can be used for the prevention and sustainable biocontrol of plant pathogens. Attached Figure Description

[0022] Figure 1 This study compares the changes in the total number of pathogens over time after treating mixed pathogen biofilms using different methods.

[0023] Figure 2 The dynamic changes in chlorophyll content and pathogen count in soybean leaves after treatment with control group, free bacteriophage, and bacteriophage transport system.

[0024] Figure 3 (A) Comparison of total surviving cell count and standard biomass after 6 hours under different treatments. (B) Comparison of normalized extracellular polymeric components after 6 hours under different treatments. Detailed Implementation

[0025] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the present invention can be combined accordingly without mutual conflict.

[0026] In the description of this invention, it should be understood that when an element is considered to be "connected" to another element, it can be a direct connection to the other element or an indirect connection, i.e., there is an intermediate element. Conversely, when an element is said to be "directly" connected to another element, there is no intermediate element.

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

[0028] In a preferred embodiment of the present invention, a method for enhancing the biocontrol 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 infect the target plant pathogen and replicate and proliferate using the target plant pathogen as a host, and the second screening condition is that the target plant pathogen can be adsorbed onto non-host probiotics that have an inhibitory effect on the target plant pathogen. Target phages that meet both screening conditions are screened from the phage library.

[0030] S2. The target phage is mixed and cultured with the non-host probiotics, so that the target phage is adsorbed onto the non-host probiotics in a non-host form to form a transport system;

[0031] S3. The transport system is added to the plant tissue or biofilm infected by the target plant pathogen to biologically control the growth of the target plant pathogen.

[0032] It should be noted that the target plant pathogens in this invention are plant pathogens requiring biocontrol, and are not limited to specific species. The methods described in S1 to S3 above are general methods. Based on this method, a transport system capable of controlling different target plant pathogens can be constructed, and this transport system can be used to biocontrol the growth of the target plant pathogens. In this transport system, bacteriophages and probiotics are used to synergistically enhance the inhibitory effect on pathogens. Bacteriophages can carry probiotics into the biofilm and plant tissue where the pathogens reside, while probiotics can promote bacteriophage motility and infection, and increase the number of bacteriophages.

[0033] Therefore, similar to the target plant pathogen, the target bacteriophage and non-host probiotics are not specific species; the specific species need to be screened against the target plant pathogen. Specifically, the target bacteriophage must meet the two screening criteria mentioned above. The first criterion is its ability to infect the target plant pathogen and replicate and proliferate using the pathogen as a host, causing lysis of the pathogen during replication and thus inhibiting its growth. The second criterion is its ability to adsorb onto non-host probiotics that inhibit the target plant pathogen. In this case, the probiotic merely acts as a carrier for the bacteriophage's migration, without replicating and proliferating on the probiotic and thus destroying it. In addition to being able to adsorb bacteriophages in a non-host form, the probiotic must also meet another condition: it should have an inhibitory effect on the target plant pathogen. Different probiotics exist for different target plant pathogens. Therefore, the specific types of probiotics can be selected by referring to the reports on inhibitory strains of target plant pathogens in existing technical literature.

[0034] In this invention, for any disease symptom (referred to as the target disease symptom), the target plant pathogen, target bacteriophage, and non-host probiotic can be screened in the following manner.

[0035] In embodiments of the present invention, the target plant pathogen can be screened and isolated from plant tissues with symptoms of the target disease using bacterial screening technology. Each candidate pathogen is then subjected to a reinfection verification experiment on asymptomatic plants. The candidate pathogen that causes asymptomatic plants to exhibit symptoms of the target disease in the verification experiment is selected 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 the disease using bacterial screening technology. These candidate pathogens, after purification, can be used in reinfection verification experiments on asymptomatic plants. If a candidate pathogen causes normally growing soybean leaves to exhibit soybean bacterial leaf spot in the verification experiment, then this candidate pathogen can be used as the target plant pathogen. It should be noted that the actual extracted target plant pathogen may be one or more species.

[0037] Furthermore, for any target disease symptom, non-host probiotics capable of inhibiting the target plant pathogen can be identified 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 the embodiments of this invention, the Bacillus subtilis (BS) can be commercially available Bacillus subtilis, with product catalog number ATCC 6633.

[0038] Furthermore, for any target disease symptom, the method for screening target phages from the phage library is as follows:

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

[0040] Then, each candidate phage was co-cultured with a non-host probiotic that could inhibit the target plant pathogen, and the presence of non-host free-riding adsorption behavior between each phage and the non-host strain was verified by transmission electron microscopy. If such behavior was found, the corresponding candidate phage was selected as the target phage.

[0041] It should be noted that, theoretically, the more phage types included in the aforementioned phage library, the better, as it would allow for the selection of more target phages. However, an excessively large phage library increases the workload and reduces efficiency during screening. In the embodiments of this invention, considering that the root soil of plants infected with the target plant pathogen generally contains target phages that meet the screening criteria, root soil samples can be collected from infected plants, and the phages contained therein can be extracted. The extracted mixed phages can then be separated and purified to obtain a series of pure phages, forming the aforementioned phage library. This allows for verification of whether each phage meets the two aforementioned screening criteria. This screening method can narrow down the phage library and quickly and accurately screen for target phages.

[0042] It should be noted that the final selected target phage is not necessarily just one type. Due to the wide variety of phages in nature, it is generally possible to ultimately select a series of target phages that meet the aforementioned two selection criteria. These target phages can be used individually or in combination as the final target phages for constructing the delivery system.

[0043] It should be noted that if there are multiple target plant pathogens causing the symptoms of the target disease, when screening candidate phages by co-culturing them with the target plant pathogens in LB medium, it is necessary to screen for candidate phages that can simultaneously infect and replicate using all target plant pathogens as host cells. At the same time, the corresponding probiotics should also be able to inhibit all target plant pathogens causing the symptoms of the target disease, so as to ensure that the final transport system can inhibit all target plant pathogens.

[0044] Furthermore, in this invention, the method of adding the final transport system during application depends on the target plant pathogen. Plant tissues or biofilms can both be considered as targets. Taking plant leaves as an example, in practical applications, the transport system can be prepared in liquid form. When using it, a culture solution containing a large amount of the transport system is sprayed onto the leaves of plants infected with the target plant pathogen, thus biologically controlling the growth of the pathogen causing disease symptoms on the plant leaves. This spraying process can be carried out periodically, and it is recommended to spray at night to avoid the inhibitory effect of ultraviolet light on bacteriophages.

[0045] The following examples, using soybean bacterial leaf spot as an example, demonstrate how to construct a transport system composed of bacteriophages and probiotics for this type of diseased plant. In the examples described below, the transport system consists of bacteriophages adsorbing onto non-host probiotics in a non-host form; the probiotic is Bacillus subtilis ATCC 6633; and the bacteriophage is a plant pathogen capable of infecting soybean bacterial leaf spot, screened from the soil where the diseased plant is located. However, it should be noted that this example is merely illustrative, and the present invention is not limited to using the specific bacteriophages and probiotics extracted in this example.

[0046] Example

[0047] The first step is to screen and obtain pathogenic bacteria and probiotics from soybean bulbs.

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

[0049] (2) Reinfection verification experiment. A typical soybean (variety: SUINONG-14) growing in the black soil region of Northeast China was selected as the model plant. Soybean seeds were surface sterilized and incubated at 28±2℃ for 2 days before being selected for cultivation. After culturing in a water bath at 30℃ for 1-2 hours, soybeans with uniform germination and growth were selected and transplanted into flower pots using a mixture of black soil and vermiculite (1:1), with 6 soybean plants planted in each pot. All plants were exposed to light at 22℃ for 16 hours, and the relative humidity was maintained at 60%-70%. The pathogens PA and PS were cultured to the exponential growth phase and enriched by centrifugation at 14000g and 4℃ for 10 min. Subsequently, the enriched bacteria were resuspended in sterile 10mM MgCl2 solution as inoculum. Then, the diluted plant pathogen PA and PS solutions selected in the previous step were mixed at a 1:1 ratio to obtain the final inoculum pathogenic solution. The concentration was 106 CFU / mL using a sterile syringe. Approximately 0.5 mL of bacterial suspension was evenly sprayed onto both sides of the needle-like leaves at the petiole junction (trifoliate stage). The sprayed plants were then immediately covered with a transparent plastic bag for at least one day. Each plant was cultured in a plant growth chamber for 30 days under long-term observation at approximately 25°C. The development of soybean bacterial leaf spot symptoms was monitored daily for all plants. Bacteria were re-isolated from selected plants with soybean bacterial leaf spot to confirm that the symptoms were caused by the inoculum. Two single colonies (strains PA and PS) were ultimately screened, purified multiple times, and stored in an equal volume of 30% glycerol medium at -80°C. The collected plant pathogen strains were cultured at 30°C and 150 rpm for 24 hours. 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. The polymerase chain reaction (PCR) amplification was then performed under the following conditions: 96℃ for 1 minute, followed by 25 cycles of 96℃ for 10 seconds, 55℃ for 5 seconds, and 60℃ for 4 minutes, with a final extension at 72℃ for 5 minutes. The 16S rRNA sequences of these two plant pathogens were then compared with the GenBank database using an online BLAST program and stored separately in GenBank. Thus, through reinfection verification experiments, it was confirmed that pathogens PA and PS are the causative agents of bacterial leaf spot disease in soybean.

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

[0051] The second step is to screen for phages that meet the criteria.

[0052] (1) Bacteriophages were isolated from soybean rhizosphere soil in a soybean field at Hailun Farm, Harbin, China, from which bacterial plant pathogens were isolated. 10 g of soil sample was extracted using 10% beef extract buffer and incubated overnight in a shaker at 10 °C. The soil sample was vortexed and sonicated, followed by centrifugation and filtration to remove particles larger than 0.22 μm. The phage extract in the filtrate was then further concentrated by precipitation with polyethylene glycol 8000 (PEG 8000) 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 the initial phage stock solution. To remove any contaminants, phages were further isolated from the initial phage stock solution. Each phage underwent three purification cycles, and the phage titer was determined per milliliter of PFU using triplicate double-layer plaques. Different phages obtained through isolation and purification were added to a phage library for subsequent screening.

[0053] (2) Cultivation of phage-pathogen mixtures. For each purified phage in the phage library, plant pathogenic strains PA and PS were used as hosts sequentially and cultured in LB medium for 24 h to determine whether they could replicate and proliferate using both pathogenic strains simultaneously. If so, the final culture mixture was centrifuged at 10000g for 10 min at 4°C, and residual cells were removed through a 0.22μm filter. The resulting mixture was stored as candidate phages at 4°C for further analysis. In this embodiment, this step yielded multiple candidate phages.

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

[0055] In this embodiment, two candidate bacteriophages, denoted as phage HBP1 and phage HBP2, were screened and validated to exhibit both hitch-riding behavior with non-host Bacillus subtilis strains and the ability to use pathogens PA and PS as host bacteria. These two phages, in their exponential growth phase, were enriched in PS strains overnight in King's B medium. The purified phages were then stored in SM buffer at 4°C. Phage titers were quantified using the double-layer plaque method and expressed as plaque-forming units (PFUs).

[0056] The third step involves using a transport system composed of bacteriophages and probiotics to treat pathogenic biofilms and characterize the effects.

[0057] (1) The selected bacteriophages HBP1 and HBP2 were mixed with the probiotic strain BS in PBS buffer to allow the bacteriophages to adsorb onto Bacillus subtilis in a non-host form, forming a transport system. Negative staining with 1% (w / v) uranyl acetate was performed, and the electrophoretic system between the bacteriophages and bacteria was verified by transmission electron microscopy. A migration assay was then performed on a double-layer plate to verify the construction of the transport system. A bacterial lawn was formed using pathogenic strains, treated with the following settings: (i) a control group using PBS buffer (labeled BT1), (ii) a bacteriophage group (labeled BT2, bacteriophage concentration 10⁶ PFU / mL), (iii) a probiotic group using strain BS (labeled BT3, probiotic concentration 10⁶ PFU / mL), and (iv) a bacteriophage group using the Bacillus subtilis-phage system (BS-phage system, labeled BT4). Each group underwent at least three parallel trials, with the infected area measured at 6 hours, 12 hours, and 24 hours after incubation.

[0058] (2) A mixed biofilm of plant pathogens PA and PS was established in a 96-well plate and then treated with the different groups mentioned above. 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 the 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 bacteriophages was counted using the double-layer plate method. The biofilm structure and cell count for each treatment were observed using a confocal laser scanning microscope (CLSM). Live cells emitted green light under 488 nm laser excitation, while dead bacteria emitted red light under 560 nm laser excitation. Biofilms in 96-well plates were stained with SYTO 9 and propidium iodide (PI) from the LIVE / DEAD BacLight kit (ApexBio Technology). The structure of the biofilm was then detected using a Nikon A1-Rsi CLSM, and the number of dead and live cells was calculated using Image J (1.8.0) software.

[0060] (4) Treatment of pathogen-infected leaves using a transport system composed of bacteriophages and probiotics. The superiority and synergistic mechanism of the bacteriophage infection system composed of BS-bacteriophages were verified through infection experiments within soybean leaves. Treatment experiments were conducted using soybean leaves and inoculated with pathogenic fluid, as used in the reinfection experiment in the first aspect. After the appearance of disease symptoms on each leaf, three treatments were applied: (i) a sterile water control treatment (labeled PT1); (ii) bacteriophage treatment (i.e., bacteriophages HBP1 and HBP2, 106 PFU / mL, MOI = 1, labeled PT2); and (iii) treatment using the BS-bacteriophage transport system prepared by adsorbing bacteriophages onto strain BS (labeled PT3). Experimental soybean plants were cultured in an artificial climate incubator, with each treatment sprayed continuously at night for 1, 4, 7, and 20 days to avoid the inhibitory effect of ultraviolet light on the bacteriophages. The wet spray volume was consistently maintained at 5 mL, and the plants were cultured in an artificial climate incubator. All experiments were independently replicated 6 times. Fresh leaves were collected on days 0, 10, 20, and 30 after treatment using the perforation (leaf disc) method, and bacterial and bacteriophage counts were determined using the plate method and double-layer plate method. Secondly, as previously reported, chlorophyll was extracted from soybean leaves on days 0, 10, 20, and 30, and its content was estimated using the classical Arnon equation. Thirdly, parallel leaf samples were collected on day 10 and flash-frozen in liquid nitrogen (stored at -80°C) for further subtranscriptomics and metabolomics analysis. To assess the long-term inhibitory effect of the transport system on the growth of soybean plant pathogens, mixed pathogenic suspensions of appropriate concentrations and quantities were uniformly sprayed and infiltrated on both sides of the treated leaves (PT2 and PT3 treatment groups). Leaf samples were collected after pathogen infection, and the total pathogen count was determined.

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

[0062] Figure 1 This study compares the changes in total pathogen count over time after treating mixed pathogen biofilms using different methods. Figure 1 It can be seen that the probiotic BS and phage mixture have similar inhibitory effects on pathogens, but the inhibitory effect is not very obvious, and the number of pathogens tends to increase in the later stage. The BS-phage delivery system has a better inhibitory effect on pathogens, and the number of pathogens continues to decrease within 5 days, which is consistently better than the two treatments alone, and the total number of pathogens decreases significantly.

[0063] Figure 2 This study demonstrates the dynamic changes in chlorophyll content and pathogen count in soybean leaves after treatment with control group A1, free bacteriophage A2, and bacteriophage transport system A3. The total pathogen count in the control group continuously increased to 1.36 × 10⁻⁶ over 30 days. 7 CFU / g leaf. Plant pathogen counts in the phage group alone decreased significantly to 0.92 × 10⁻⁶ within 10 days. 7 CFU / g, then remained at 0.80×10 for the next few days. 7 The CFU / g level fluctuated around [value missing]. For the phage-probiotic group, the plant pathogen count significantly decreased to 0.53 × 10 [value missing] after 10 and 30 days, respectively. 7 CFU / g and 0.18×10 7 The CFU / g (p<0.05, statistically significant) indicates that the transport system effectively controls the pathogens. After biocontrol treatment, the chlorophyll levels in A2 and A3 were 10.25 mg / g and 12.35 mg / g, respectively, higher than the chlorophyll content in the control group A1. Because probiotics promote phage motility and infection, the phage count in A3 increased faster than in A2. The final phage count in A3 was 1.2 times that of PT2, demonstrating the promoting effect of probiotics on phage increase.

[0064] Figure 3The study presents cell survival and extracellular polymeric substance (EPS) composition after 6 hours under different treatments. (A) compares the total number of surviving cells with standard biomass after 6 hours under different treatments, and (B) compares the normalized EPS composition after 6 hours under different treatments. It is evident that treatment with strain BS, bacteriophage, and the bacteriophage-BS transport system reduced the total pathogenic biofilm biomass by 30%, 43%, and 58%, respectively (A). Furthermore, treatment with the bacteriophage-BS transport system significantly reduced the number of surviving pathogenic bacteria, which was 3.9 times, 7.5 times, and 11.2 times lower than that of strain BS, the bacteriophage cocktail, and the control group, respectively (p<0.05) (A). Compared to the untreated control group, the bacteriophage-BS transport system significantly reduced the EPS content of protein, polysaccharides, and eDNA (p<0.05), by 45%, 47%, and 61%, respectively (B), indicating that the bacteriophage-BS transport system has the potential to control biofilms.

[0065] The embodiments described above are merely some preferred implementations of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A method for enhancing the biocontrol 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 phage can infect the target plant pathogen and replicate and proliferate using the target plant pathogen as a host, and the second screening condition is that the phage can adsorb onto non-host probiotics that have an inhibitory effect on the target plant pathogen. Target phages that meet both screening conditions are screened from the phage library. In the second screening condition, the non-host probiotics are merely adsorption carriers for the phage to migrate by hitchhiking, but the phages do not replicate and proliferate using the non-host probiotics as a host to destroy the non-host probiotics. S2. The target phage is mixed and cultured with the non-host probiotics, so that the target phage is adsorbed onto the non-host probiotics in a non-host form to form a transport system; S3. Spray the culture solution containing the transport system onto the leaves of plants infected by the target plant pathogen to biologically control the growth of the target plant pathogen that causes disease symptoms on the plant leaves. The target plant pathogen is selected and isolated from plant tissues with symptoms of the target blight using bacterial screening technology. Each candidate pathogen is then tested for reinfection on asymptomatic plants. The candidate pathogen that causes asymptomatic plants to exhibit symptoms of the target blight in the test is selected as the target plant pathogen. The plant tissue exhibiting the symptoms of the target disease is a soybean leaf showing bacterial leaf spot of soybean; The non-host probiotic is Bacillus subtilis ATCC 6633.

2. The method for enhancing the biocontrol of plant pathogens by bacteriophages using probiotics as described in claim 1, characterized in that, The method for screening target bacteriophages from the bacteriophage library is as follows: Different phages in the phage library were mixed with the target plant pathogen in LB medium to isolate candidate phages that could infect and replicate using the target plant pathogen as a host cell. Each candidate phage was co-cultured with the non-host probiotic, and the presence of non-host free-riding adsorption behavior between each phage and the non-host strain was verified by transmission electron microscopy. If such behavior was found, the corresponding candidate phage was selected as the target phage.

3. The method for enhancing the biocontrol of plant pathogens by bacteriophages using probiotics as described in claim 1, characterized in that, The culture medium spraying treatment containing the transport system is carried out at night to avoid the inhibitory effect of ultraviolet light on the bacteriophages.

4. The method for enhancing the biocontrol of plant pathogens by bacteriophages using probiotics as described in claim 1, characterized in that, The phage library is obtained by extracting and purifying phages from the root soil of plants infected with the target plant pathogen.

5. A transport system composed of bacteriophages and probiotics, characterized in that, A transport system is formed by bacteriophages adsorbing onto the non-host probiotics in a non-host form; the probiotics are Bacillus subtilis ATCC 6633; and the bacteriophages are bacteriophages of plant pathogens capable of infecting soybean bacterial leaf spot.

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