Serratia marcescens and application thereof in nematode control

Abstract

The invention belongs to the technical field of microorganisms, particularly relates to serratia marcescens and application of the serratia marcescens in nematode prevention and control, and particularly discloses serratia marcescens BP1 with the taxonomic name of Serratia marcescens and the preservation number of GDMCC No: 65796. The serratia marcescens BP1 has a strong lethal effect on nematodes, can be widely applied to biological prevention and control of harmful nematodes, and has a good application prospect. Meloidogyne is treated by a BP1 crude protein extract for nematicidal rate detection and pathological microscopic observation, the nematicidal rate is 99%, the enzyme activity of the strain BP1 is 112 U / mL after 24 hours of culture, the enzyme yield of the strain BP1 is the highest and reaches 116 U / mL after 48 hours of culture, and the enzyme activity of the strain BP1 is 114 U / mL after 72 hours of fermentation.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and specifically relates to a Serratia marcescens and its application in nematode control. Background Art

[0002] With the advocacy of green and pollution-free production, biological control has broad application prospects in the field of plant disease prevention and control due to its advantages such as strong environmental compatibility and low drug resistance. Screening biocontrol bacterial resources with the function of controlling root-knot nematodes is of great significance for developing new biological pesticide products and reducing or gradually replacing chemical pesticides for the green control of crop root-knot nematode diseases.

[0003] Bacteria are widely distributed in the environment, with a large number of population categories, and the types of protease in bacterial secondary metabolites are rich. Previous studies on enzyme-producing bacteria in insect intestines, caves, and marine sediments have shown that the main protease-producing bacteria in these environments are Bacillus, Streptomyces, Pseudomonas, Microbacterium, Nocardioides, etc. Bacteria are the main resources for producing protease. The specificity of the environment leads to the diversity of enzyme-producing bacteria and the ability of different bacteria to produce a variety of protease types. By screening protease-producing bacteria from other cave habitats, plant endophytic bacteria, vegetable soil, earthworm compost, and marine sediments, the diversity of enzyme-producing bacteria in the same type of habitat, different geographical environments, and different habitat types is studied, providing certain materials for the development of enzyme-producing microbial resources. Enzyme-producing microorganisms and their metabolites have significant effects in controlling nematodes, not only directly killing nematodes, but also reducing nematode reproduction by inhibiting egg hatching and larval activity. These biological control methods provide important technical support for the sustainable development of agriculture and are expected to replace traditional chemical control methods to reduce pesticide residues and environmental pollution. Summary of the Invention

[0004] The main object of the present invention is to provide a protease-producing bacterium, Serratia marcescens BP1, to provide a new technical means for nematode control. Specifically, the present invention provides the following technical solutions: A Serratia marcescens BP1, with the taxonomic name of Serratia marcescens , and the preservation number is GDMCC No: 65796.

[0005] The fermented broth of the Serratia marcescens BP1, which is obtained by inoculating the Serratia marcescens BP1 into a PDB liquid medium for culture.

[0006] A crude protein extract, which is obtained by centrifuging and precipitating the cells from the fermented broth of the Serratia marcescens BP1, and taking the supernatant as the crude protein extract.

[0007] A protease is obtained by separating and / or purifying the bacterial liquid or the crude protein extract.

[0008] A bacterial agent contains the fermented bacterial liquid of Serratia marcescens BP1.

[0009] A nematicide contains the fermented bacterial liquid of Serratia marcescens BP1, or the crude protein extract, or the protease, or the bacterial agent.

[0010] The following applications of Serratia marcescens BP1, or the bacterial liquid, or the crude protein extract, or the protease, or the bacterial agent in any one of (1)-(5) below; (1) Killing nematodes; (2) Preparing a biological control agent for nematodes; (3) Hydrolyzing casein; (4) Degrading the collagen in the body wall of nematodes; (5) Preparing a casein or collagen degrading agent.

[0011] Further, the nematodes include Meloidogyne spp. or Caenorhabditis elegans.

[0012] The Serratia marcescens BP1 described in the present invention can be used as a biological control agent for Meloidogyne spp. in the prevention and control of Meloidogyne spp. diseases.

[0013] A method for preventing and controlling Meloidogyne spp. is to apply the bacterial agent or the nematicide to the crops affected by Meloidogyne spp. pests.

[0014] Further, the crops include Solanaceae, Leguminosae, Cucurbitaceae or Cruciferae crops. Specifically, the crops include cucumber, tomato, yam, tobacco, ginger, peanut, watermelon, melon, radish, banana, pepper, potato, strawberry, sugarcane, kiwifruit tree, citrus, Panax notoginseng or Astragalus membranaceus.

[0015] The technical effects achieved by the present invention: The Serratia marcescens BP1 provided by the present invention has a strong lethal effect on Meloidogyne spp. and can be widely used for the biological control of Meloidogyne spp. Through the detection of the nematode killing rate and the microscopic observation of pathology by treating Meloidogyne spp. with the crude protein extract of BP1, the nematode killing rate is 99%. The enzyme activity of strain BP1 is 112 U / mL after culturing for 24 hours, the enzyme production of strain BP1 is the highest at 48 hours, reaching 116 U / mL, and the enzyme activity is 114 U / mL after fermentation for 72 hours. Description of the Drawings

[0016] Figure 1 Schematic diagram of the process of screening protease-producing bacteria by the skim milk plate method; Figure 2Treatment of Caenorhabditis elegans (A) and Meloidogyne incognita (B) with the heated fermentation broth of bacterium BP1; Figure 3 Treatment of Meloidogyne incognita with the crude protein extract (crude enzyme solution) of bacterium BP1 and microscopic pictures; Figure 4 Determination of protease activity produced by bacterium BP1: Graphs of casein standard curve (A) and enzyme activity at different time periods (B); Figure 5 Graph of detection of collagenase activity produced by bacterium BP1; Figure 6 Graphs of propidium iodide (PI) staining of Meloidogyne incognita treated differently. BP1 (A and B), treatment with negative control PDB (C and D), treatment with positive control by heating (E and F); Figure 7 Graphs of propidium iodide (PI) staining of Caenorhabditis elegans treated differently. BP1 (A), treatment with negative control PDB treatment (B), treatment with positive control by heating (C-D) Figure 8 Graphs of propidium iodide staining of Caenorhabditis elegans at different treatment times. 24 hours (A-B) and 48 hours (C-D); Figure 9 Graphs of detection results of reactive oxygen species fluorescence of Caenorhabditis elegans treated differently. BP1 (A), OP50 (B); Figure 10 Scanning electron microscope pictures of Meloidogyne incognita at 72 hours. BP1 (A-B) and negative control (C); Figure 11 Transmission electron microscope pictures of Meloidogyne incognita. Control (A and D), 24 hours (B and E), 48 hours (C and F); Figure 12 Graphs of the colonization of BP1 in Caenorhabditis elegans. BP1 labeled with GFP (A), number of BP1 colonies in nematodes (B), and fluorescence distribution of bacteria in nematodes at 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, and 72 hours.

[0017] Note: Different letters between different treatments indicate significant differences (p < 0.05) Detailed implementation manners

[0018] The following will, in conjunction with embodiments, elaborate in detail on the implementation manners of the present invention, so as to fully understand how the present invention applies technical means to solve technical problems and achieve the realization process of technical effects and implement accordingly.

[0019] Example 1 Screening, identification and preservation of Serratia marcescens BP1 As Figure 1The present invention screened protease-producing strains of multiple bacteria isolated from different habitats by the lactose-free skim milk plate hydrolysis circle method. We used the presence or absence of a transparent circle as a standard to judge whether the strain can produce protease, and evaluated the enzyme production ability of the strain according to the difference between the hydrolysis circle and the colony radius. First, we screened the difference between the hydrolysis circle and the colony radius ≥ 0.5 cm as a standard for strong enzyme production to evaluate the enzyme production ability of the strain, and further screened strains with strong casein hydrolysis ability. The strains were screened to obtain strains with super strong protease hydrolysis activity, which were identified and named according to the 16SrRNA of the strain: Serratia marcescens BP1. The difference between the transparent circle radius and the colony radius of Serratia marcescens BP1 was 0.8 cm, showing strong protease hydrolysis activity.

[0020] Serratia marcescens strain BP1, taxonomic name Serratia marcescens , the deposit number is GDMCCNo:65796; it is deposited in Guangdong Provincial Microbiological Culture Collection Center, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the deposit date is January 16, 2025. The viability of the biological material was tested by the collection center on January 16, 2025, and the result was viable.

[0021] Example 2 Pathogenicity and enzyme production activity of strain BP1 on nematodes 1 Experimental materials and instruments (1) Experimental materials: Serratia marcescens BP1, nematode: Caenorhabditis elegans ( Caenorhabditis elegans ), food is Escherichia coli Escherichia Coli OP50, culture temperature 22℃; southern root-knot nematode ( Meloidogyne incognita ), the laboratory purchased diseased plant roots and then picked out egg masses to hatch.

[0022] (2) Experimental reagents Acetone (Chengdu Kelong); 2% casein: 2.00g casein was added with 10-20mL 0.5mol / L NaOH, heated in a water bath to dissolve, and diluted to 100mL with pH 8.0 Tris-HCl; 0.4mol / L trichloroacetic acid: 65.4g, 1000mL; 0.55mol / L Na2CO3: 56.65g, 1000mL; Folin-reagent method was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Gelatin screening medium (1000mL): gelatin 20g, NaCl 0.1g, peptone 5g, KH2PO4 0.5g, MgSO4·7H2O 0.2g, pH 7.2-7.5; acidic mercuric reagent: HgCl2 15g, concentrated hydrochloric acid 20g, diluted to 100mL with distilled water; plasmid pGFPuv (laboratory storage); propidium iodide (PI) (SigmaAldrich) (3) Main instruments Eppendorf large benchtop centrifuge at 4°C, 0.45 μm bacterial filter membrane, shaker (Tiancheng, TS-100B), visible spectrophotometer (Eppendorf, BioSpectrometer), high-speed centrifuge (Eppendorf, 5424R), Bio-Rad MicroPulser electroporator 2 Experimental methods 2.1 Serratia marcescens BP1 exerts a lethal effect on nematodes through extracellular secreted proteins 1. Test the nematicidal activity by heating the bacterial fermentation supernatant at high temperature. The supernatant of the 48-hour bacterial fermentation broth was treated at 80°C for 30 min, cooled, and then used to treat nematodes. The viability of the nematodes was observed under a 4-fold optical microscope, and the number of surviving and dead nematodes was recorded to calculate the nematicidal activity.

[0023] Nematode mortality rate (%) = number of dead nematodes / number of tested nematodes × 100 Corrected nematicidal rate = (treatment mortality rate - control mortality rate) / (1 - control mortality rate) * 100% 2. Extract crude protein extract from the bacterial fermentation broth to test the nematicidal activity. After culturing the bacteria for 48 hours, centrifuge at 8000 r / min at 4°C for 15 minutes, collect the cell supernatant, and filter it using a 0.45 μm filter membrane. Add three volumes of cold acetone and incubate at -20°C for 2 hours. Centrifuge for 15 minutes to remove the upper aqueous phase in the tube, and resuspend the precipitate in 1 mL of 50 mM phosphate buffer (pH 7.4). Calculate the nematicidal activity and take microscopic pictures of the nematodes after 48 hours of treatment.

[0024] 2.2 Determination of protease activity of Serratia marcescens BP1 Draw the casein standard curve according to Li Jiancheng's method, and use the Folin-phenol reagent method to determine the crude enzyme activity of the fermentation broth. The bacteria were cultured in PDB medium at 37 °C and 180 rpm / min for 24 hours, 48 hours, and 72 hours. Then, 2 mL of the fermentation broth was taken, centrifuged at 8000 r / min, and the supernatant was taken as the crude protein extract (abbreviated as "crude enzyme solution"). 2.5 mL of 2% casein solution preheated at 40 °C for 10 min was added to 1 mL of the supernatant, and then incubated in a water bath at 40 °C for 10 min. 2.5 mL of 0.4 mol / L trichloroacetic acid (TCA) was added to terminate the reaction, and incubated at 40 °C for 15 min. After the reaction, it was filtered through filter paper. 1 mL of the supernatant was taken, 2.5 mL of 0.55 mol / L Na2CO3 solution and 0.5 mL of Folin-phenol reagent were added, and incubated at 40 °C for 10 min. The absorbance at 660 nm was measured. The inactivated crude protein extract was used as the negative control, and water was used as the blank control. The meaning of one unit of enzyme activity U is: at 40 °C, the amount of enzyme required to release 1 µg of tyrosine per minute when hydrolyzing casein per milliliter of liquid enzyme is 1 unit (U). The activity of each mL of protease = (OD 660 -b) × f / (k × t); t is the time of the enzymatic reaction; f: is the dilution factor of the enzyme 2.3 Screening for the collagenase activity of Serratia marcescens BP1 Use gelatin plates to detect the collagenase activity of bacteria. An appropriate amount of bacterial fermentation broth was inoculated on the gelatin screening plate and cultured at 37 °C for 3 - 5 days. Acidic mercury reagent was added dropwise around the colonies, and the formation of clear zones was observed.

[0025] 2.4 Observation of nematode cell damage by propidium iodide staining Propidium iodide (PI) dye can stain damaged cells and emit fluorescence under a fluorescence microscope. Propidium iodide was used to stain nematodes to observe cell damage in nematodes. After BP1 was cultured in PDB medium for 48 hours, 300 µL of bacterial liquid was aspirated and spread on NGM (Nematode Growth Medium) plates, and then taken out and placed at room temperature after overnight culture. Synchronized Caenorhabditis elegans at the L4 stage was washed from the Escherichia coli OP50 plate using M9 buffer; for Meloidogyne incognita, it could be directly centrifuged and enriched and treated in the bacterial fermentation supernatant for 24 hours and 48 hours. The bacterial plates were replicated three times, at least 30 nematodes (10 nematodes / plate) were selected from the bacterial plates, and washed twice in 20 µL of M9 buffer. The nematodes were transferred to the wells of a 96-well microtiter plate containing 10 µM propidium iodide PI in M9 buffer and incubated in the dark at 25 °C for 3 hours. Subsequently, the nematodes were rinsed with M9 buffer and observed using a fluorescence microscope. When cells were damaged and the integrity of their cell membranes was lost, resulting in the dye being observed in the cells near the nematode intestinal cavity, cell necrosis in Caenorhabditis elegans was confirmed. PI staining of heat-treated nematodes and nematodes fed on Escherichia coli OP50 were used as positive and negative controls, respectively.

[0026] 2.5 ROS Reactive Oxygen Species Detection To detect whether bacterial infection can trigger the production of reactive oxygen species in nematodes by reactive oxygen species detection, reactive oxygen species are not only one of the most primitive natural defense mechanisms for nematodes to resist many invading microorganisms, but also one of the damage mechanisms caused by the invasion of pathogenic bacteria into nematodes. Caenorhabditis elegans treated on the BP1 bacterial plate for 24 hours was incubated with 5 mM CM-H2DCFDA (Full Chinese name: 5-(and6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate) at room temperature for 3 hours, and then transferred to a 2% agarose plate and observed for ROS production under blue light excitation of a fluorescence microscope.

[0027] 2.6 Scanning and Transmission Electron Microscopy Observation of the Damage of the Body Surface and Internal Tissues of Meloidogyne incognita 1. BP1 bacterial liquid was contacted with nematodes (2000 J2s) for 48 hours, and after centrifugation, the nematodes were washed 3 times in Tris-HCl buffer (pH 7.0) and fixed in 4% glutaraldehyde for 2 hours. Then, they were dehydrated stepwise in ethanol (10, 30, 50, 70, 95, and 100%) and dehydrated 3 times in 100% ethanol, 15 minutes each time. The nematode materials were dried overnight at the critical point and sputter-coated with gold. The nematodes were observed using a scanning electron microscope (SEM).

[0028] 2. To study the tissue damage in Meloidogyne incognita during infection, we used transmission electron microscopy (TEM) to detect the intestinal structure of the nematodes. The nematodes were treated in the same way as in the nematicide test. After collecting the nematodes, they were fixed with 2.5% glutaraldehyde and sent to the Kunming Institute of Zoology, Chinese Academy of Sciences for dehydration, embedding, and section preparation.

[0029] 2.7 Detection of the colonization ability of strain BP1 in nematodes (1) GFP labeling of Serratia marcescens BP1 a. The overnight-grown bacterial culture was used as the seed culture and inoculated into 50 mL of LB liquid medium at an addition amount of 1%. The culture was incubated at 37 °C and 200 rpm / min until the OD 600 reached 0.6, and then the competent cells were prepared.

[0030] b. The bacterial culture was ice-bathed for 20 min and centrifuged at 6000 rpm for 15 min. The supernatant was removed, and the cells were resuspended in ice-cold 10% glycerol and centrifuged. This was repeated three times. Then the cell pellet was suspended in an appropriate amount of 10% glycerol.

[0031] c. 50 μL of competent cells and 5 μL of pGFPuv plasmid were mixed and incubated on ice for 1 min.

[0032] d. The mixture was added to a 1 mm electroporation cuvette and gently mixed. The electroporation voltage was set to 2.5 KV, and the electroporation time was 4.9 s. Immediately, 1 mL of LB liquid medium was added to resuscitate the cells.

[0033] e. The mixture was resuscitated at 37 °C and 180 rpm / min for 2 h. An appropriate amount of the resuscitated mixture was taken and spread on an LB plate containing 100 μg / mL kanamycin and incubated overnight.

[0034] (2) Detection of CFU of Serratia marcescens BP1 in nematodes The experimental method for detecting the number of bacteria colonized in nematodes was carried out as follows: a. The overnight-grown NGM plates with bacteria were cooled to room temperature, and no less than 100 L4 Caenorhabditis elegans were inoculated on each plate. After being treated for 6 h, 24 h, 36 h, 48 h, and 72 h respectively, the nematodes were washed off the plates, crawled on the NA plates for 2 - 3 h to remove the excess bacteria, and 20 - 30 nematodes were picked and placed in a 1.5 mL centrifuge tube.

[0035] b. The nematodes treated for 24 h, 48 h, and 78 h were washed off and then washed 3 times in sterile M9 solution. Then they were incubated in sterile M9 solution containing 10 mM levamisole, ampicillin (100 μg / mL), and kanamycin (200 μg / mL) for 1 - 3 h. After centrifuging at 4000 rpm for 1 min, the precipitate was collected.

[0036] c. Add sterile M9 solution to the centrifuge tube to wash the nematodes by centrifugation, and repeat three times to remove excess antibiotics.

[0037] d. Add 100 μL of sterile M9 solution to the centrifuge tube, and then grind it thoroughly with an electric grinding rod.

[0038] e. After dilution, pipette different concentrations of the ground solution and spread it on NA plates. After overnight incubation at 37 °C, count the colonies on the plates, and finally calculate the number of colonies in each nematode. CFU / perworm = (number of colonies on the plate * dilution factor * 100 μL) / (volume used for spreading * number of nematodes) f. Take fluorescence microscopy pictures of the nematodes treated with bacteria for 6 hours, 24 hours, 36 hours, 48 hours, and 72 hours to explore the accumulation results of bacteria in the nematode intestine at different time periods.

[0039] 3 Experimental results 3.1 The biocontrol enzyme-producing bacterium BP1 of nematodes has a lethal effect on nematodes through extracellular secreted proteins 1. Conduct direct contact killing experiments on Meloidogyne incognita J2 and Caenorhabditis elegans with the fermentation supernatant of strain BP1 and the filtered fermentation supernatant after heat treatment at 80 °C respectively. The effects are as Figure 2 shown. The experimental results show that the nematicidal activity of the filtered fermentation supernatant after heat treatment on nematodes is significantly reduced, indicating that the nematicidal substance of strain BP1 may be extracellular proteins or protein metabolites secreted by bacteria.

[0040] 2. To further confirm that the biocontrol enzyme-producing target strain BP1 of nematodes has a lethal effect on nematodes through extracellular secreted proteins, conduct nematicidal rate detection and pathological microscopic observation on root-knot nematodes treated with the crude protein extract (crude enzyme solution) of BP1. As Figure 3 shown, the nematicidal rate at 48 hours is 99%, which is higher than the lethality of treating nematodes with the fermentation broth of strain BP1. Moreover, the morphological and structural pathological characteristics of root-knot nematodes treated with the crude protein extract of BP1 are damage to the body wall of nematodes, intestinal vacuoles, and degradation of the nematode body, which are similar to the pathological characteristics shown by treating nematodes with the fermentation broth of strain BP1. These data fully illustrate that the biocontrol enzyme-producing target strain BP1 of nematodes has a lethal effect on nematodes through extracellular secreted proteins.

[0041] 3.2 Analysis of enzyme-producing characteristics of Serratia marcescens BP1 1. Determination of protease activity of the biocontrol enzyme-producing bacterium BP1 of nematodes In this study, crude protein extracts were extracted at three time periods of 24 hours, 48 hours, and 72 hours during the cultivation of strain BP1. Calculate the activity of protease per mL according to the casein standard curve = (OD 660(-0.02)×f / (0.0028×10) was used to determine the enzyme activity of the crude protein extract fermented by strain BP1. The results are as Figure 4 shown. The enzyme activity of strain BP1 was 112 U / mL after 24 hours of cultivation, and the enzyme production of strain BP1 reached the highest level of 116 U / mL after 48 hours, and the enzyme activity was 114 U / mL after 72 hours of fermentation.

[0042] 3.3 Determination of the collagenase activity of the nematode biocontrol enzyme-producing bacterium BP1 The collagenase activity of strain BP1 was detected by the hydrolysis zone method on a gelatin screening medium plate. The plate inoculated with bacteria was taken out after three days of incubation, and acidic mercury reagent was added dropwise around the colonies. The test results showed that obvious hydrolysis zones were produced around the colonies of strain BP1 on the gelatin plate, and the hydrolysis zone effect is as Figure 5 shown, indicating that bacterium BP1 has the ability to degrade collagen.

[0043] 3.4 The nematode biocontrol enzyme-producing bacterium BP1 exerts nematicidal effects by destroying the nematode body wall and colonizing the intestine 3.4.1 Observation of nematode cell damage by propidium iodide (PI) staining To analyze the tissue localization of the entry pathway of the toxic protein secreted by the nematode biocontrol enzyme-producing target strain BP1 bacteria into nematodes, nematodes were treated with the fluorescent dye propidium iodide, and a fluorescence signal tracing experiment of PI was carried out. Root-knot nematodes and Caenorhabditis elegans treated for 48 hours were stained with PI and observed under a fluorescence microscope. The experimental results are as Figure 6 (root-knot nematode) and Figure 7 (Caenorhabditis elegans) shown. Compared with the negative control, the nematodes treated on the bacterial plate showed strong fluorescence throughout the body. The experiment also showed ( Figure 8 ) that as the treatment time extended, fluorescence first appeared in the pharynx of the nematodes treated with bacterium BP1 for 6 hours, and then the whole body of the nematodes showed fluorescence, suggesting that the bacterium first caused damage to the pharynx of the nematodes and might enter the nematode intestinal cavity through the damage formed by the toxic protein, colonize in the cavity and spread throughout the body, causing systemic damage.

[0044] 3.4.2 ROS reactive oxygen species detection After 24 hours of infection of nematodes by bacterium BP1, the nematodes were treated with a staining agent, and the fluorescence generation in the nematodes was observed under a fluorescence microscope. The results showed ( Figure 9 ) that compared with the control treated with OP50, the reactive oxygen species in the Caenorhabditis elegans treated with BP1 increased significantly. The accumulation of high levels of ROS can cause serious damage to various organelles by changing DNA, proteins, and lipids.

[0045] 3.4.3 Scanning and transmission electron microscopy of root-knot nematodes to detect the damage of the nematode body wall and intestine by BP1 treatment The root-knot nematodes after 72 hours of treatment were observed by scanning electron microscopy for damage to the nematode body surface. The results showed that for the nematodes treated with the bacterial solution ( Figure 10 A and Figure 10 B), there were gaps on the body surface, accompanied by severe shrinkage, indicating that the integrity of the nematode body surface was damaged; while for the negative control treated with PDB ( Figure 10 C), the nematode body shape was perfect, the surface was smooth, the lateral stripes were clear, and it showed a curved state after being fixed with glutaraldehyde. These results indicate that there are substances in the BP1 bacterial solution that can degrade the nematode body surface. To observe the damage inside the nematodes, the treated nematodes were photographed by transmission electron microscopy ( Figure 11 ), and the results showed that the tissues inside the nematodes treated for 24 hours and 48 hours changed. As the treatment time extended, the degree of tissue damage in the nematodes increased. Compared with the control, for the nematodes treated for 24 hours, the intestinal cavity of the nematodes expanded, structures such as the gonads gradually disappeared, the tissues inside the nematodes were decomposed to form cavities, the body wall was damaged and separated from the tissues, leaving only some difficult-to-degrade membrane structures.

[0046] 3.4.3 Detection of the colonization ability of the nematode biocontrol enzyme-producing strain BP1 in nematodes The BP1 strain ( Figure 12 A) successfully labeled with GFP was co-cultured with nematodes on a plate for 6 hours, 24 hours, 36 hours, 48 hours, and 72 hours, and then the colonization amount of bacteria in the nematode intestine was detected ( Figure 12 B). As the co-culture time extended, the number of bacterial colonies isolated from the nematodes increased, and the fluorescence intensity and range of bacteria in the nematodes became stronger and stronger, indicating that the colonization amount of bacteria in the nematode intestine increased with time. Compared with the nematodes cultured for 6h and 12h, the whole body of the nematodes cultured for 24h, 36h, 48h, and 72h was covered with fluorescence, and the bacteria might be able to damage the nematode intestine and spread throughout the nematode body.

[0047] In summary, the Serratia marcescens BP1 provided by the present invention has a strong lethal effect on nematodes.

[0048] The above description shows and describes several preferred embodiments of the present invention. However, as mentioned above, it should be understood that the present invention is not limited to the form disclosed herein, should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications, and can be changed within the scope of the present invention's concept through the above teachings or the technology or knowledge in the relevant field. And any changes and modifications made by those skilled in the art without departing from the spirit and scope of the present invention shall fall within the protection scope of the appended claims of the present invention.

Claims

1. A Serratia marcescens BP1, characterized in that The taxonomic name is Serratia marcescens , the deposit number is GDMCC No:65796.

2. A fermentation liquid of Serratia marcescens BP1, characterized in that: The Serratia marcescens BP1 according to claim 1 is inoculated into a PDB liquid culture medium for culturing to obtain the fermentation broth.

3. A crude protein extract, characterized in that The crude protein extract is prepared by centrifuging the fermented bacterial broth of Serratia marcescens BP1 as claimed in claim 2 to precipitate the bacterial bodies, and taking the supernatant as the crude protein extract.

4. A protease, characterized in that The protease is obtained by separating and / or purifying the bacterial solution according to claim 2 or the crude protein extract according to claim 3.

5. A bacterial agent, characterized in that A fermentation bacterial liquid containing the Serratia marcescens BP1 according to claim 2.

6. A nematicide, characterized in that: The nematicide contains the fermentation broth of Serratia marcescens BP1 according to claim 2, or the crude protein extract according to claim 3, or the protease according to claim 4, or the bacterial agent according to claim 5.

7. Use of the Serratia marcescens BP1 according to claim 1, or the bacterial solution according to claim 2, or the crude protein extract according to claim 3, or the protease according to claim 4, or the bacterial agent according to claim 5 as described in any of the following (1) to (5); (1) Kill nematodes; (2) Preparation of nematode biological control agents; (3) Hydrolyzed casein; (4) Degradation of collagen in the nematode body wall; (5) Preparing casein or collagen degradation agents.

8. The use according to claim 7, characterized in that: The nematode includes root-knot nematodes or Caenorhabditis elegans.

9. A method for controlling root-knot nematodes, characterized in that: The fungicide according to claim 5 or the nematicide according to claim 6 is applied to crops infested with root-knot nematodes.

10. The control method according to claim 9, characterized in that: The crops include crops from the Solanaceae, Leguminosae, Cucurbitaceae or Cruciferae families.

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