Serratia marcescens serving as biological control bacterium and application of serratia marcescens in preparation of soybean sclerotinia rot control agent

Abstract

The invention discloses serratia marcescens serving as a biological control bacterium and application of the serratia marcescens in preparation of a soybean sclerotinia rot control agent, and belongs to the technical field of agricultural microbiology. The strain is preserved in the China Center for Type Culture Collection on November 29, 2024, the preservation number is CCTCC M 20242667, the strain is classified and named as Serratia marcescens G4, the bacterial colony is red, the surface is thick, smooth and moist, the bacterial colony is slightly glossy and opaque, and the strain can be used for preparing a soybean sclerotiniose control agent. The G4 strain can inhibit the growth of sclerotinia sclerotiorum and change the form of hyphae. Experiments prove that the serratia marcescens G4 separated from soybean rhizosphere soil is an effective biocontrol strain for preventing and treating the soybean sclerotiniose, and compared with the conventional chemical synthesis pesticide for prevention and treatment, the serratia marcescens G4 has the advantages of high efficiency, low toxicity, no environmental pollution, difficulty in generating drug resistance and the like, and has a wide application prospect and a good application value.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microbiology, and particularly relates to Serratia marcescens as a biological control bacterium and application thereof in preparing a soybean sclerotinia disease control agent. Background Art

[0002] Soybean sclerotinia is also known as white rot and white hair disease. The pathogen is soybean sclerotinia [Sclerotinia sclerotiorum (Lib.) deBary], which belongs to the phylum Ascomycota. It was first discovered in 1837. It has a wide distribution range and a wide variety of host species. It can infect more than 400 plant species in 41 families, including Leguminosae, Asteraceae, and Apiaceae, in addition to Gramineae.

[0003] For a long time, the main method for preventing and controlling soybean sclerotinia disease has been to apply chemical pesticides. However, the long-term use of chemical pesticides will cause problems such as drug resistance, pesticide residues, environmental pollution, inhibition of soybean nodulation and nitrogen fixation. Therefore, in order to prevent and control soybean sclerotinia disease, it is urgent to find a new environmentally friendly and low-toxic method. Compared with chemical pesticides, microbial pesticides have the advantages of strong selectivity, no pollution, not easy to produce drug resistance, no damage to the ecological environment, and effective prevention and control of plant diseases. At present, biocontrol bacteria have become a research hotspot.

[0004] Biological control has received more extensive attention and developed rapidly. More than 40 kinds of microorganisms have been studied to control the occurrence of sclerotinia disease. Fungi such as C. minitans, Clonostachys rosea and Fusarium oxysporum have good antagonistic effects on soybean sclerotinia disease. The biological control strains for the prevention and control of soybean sclerotinia disease are mainly Bacillus sp. and Trichoderma sp. Therefore, it is urgent to explore and isolate more beneficial microorganisms that are widely present in the soil and grow rapidly to prevent and control soybean sclerotinia disease (Sclerotinia sclerotiorum).

[0005] Serratia marcescens belongs to the genus Serratia of the family Enterobacteriaceae. It is a Gram-negative bacterium that is widely found in air, water, soil, various plants, and the digestive tract of animals. It can colonize in the rhizosphere, plant bodies, and insects of plants, and has a certain biological control effect on plant pathogens and weeds. Therefore, biological control Serratia and its preparations have broad application prospects in the green prevention and control of plant diseases.

[0006] At present, there are few reports on Serratia marcescens used for preventing and controlling soybean sclerotinia sclerotiorum. Therefore, the present invention provides a greater theoretical and practical basis for finding new strains for preventing and controlling soybean sclerotinia sclerotiorum. Summary of the invention

[0007] The purpose of the present invention is to provide a Serratia marcescens as a biological control bacterium and its application in preparing a soybean sclerotinia (Sclerotinia sclerotiorum) control agent.

[0008] In order to achieve the above object, the present invention adopts the following technical measures:

[0009] The Serratia marcescens described in the present invention is isolated from the rhizosphere soil of soybean fields in the teaching and research base of Jilin Agricultural University in Changchun City, Jilin Province. The strain has been deposited in the China Center for Type Culture Collection, with a deposit date of November 29, 2024, a deposit number of CCTCC M 20242667, and a classification name of Serratia marcescens G4. The address of the depository unit is Wuhan University, Wuhan City, Hubei Province, China (430072). Colony characteristics: The colony is red, with a sticky and smooth surface, moist, slightly shiny, and opaque ( Figure 1 ).

[0010] The Serratia marcescens provided by the present invention can effectively inhibit the growth of important pathogens on soybeans, and the inhibition effect is good, avoiding the potential adverse effects of chemical control on the environment and crop safety. Compared with the current use of chemical synthetic pesticides for control, it has the advantages of high efficiency, low toxicity, no environmental pollution, and not easy to produce drug resistance, and has broad application prospects and good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Schematic diagram of the G4 strain colony characteristics (LB, 30°C, 16h);

[0012] Figure 2 This is the scanning electron micrograph of the G4 strain (LB, 30°C, 16h);

[0013] Figure 3 is the phylogenetic tree of G4 strain (based on 16S rDNA sequence);

[0014] Figure 4The graph shows the inhibitory effect of different concentrations of G4 strain on the colony diameter (Figure A) and hyphae growth (Figure B) of soybean sclerotinia sclerotiorum (PDA, 25°C, 7d); the horizontal axis is the concentration of G4 bacterial solution in PDA medium (cfu / mL), and the vertical axis is the colony diameter (mm) of soybean sclerotinia sclerotiorum, the dry weight of hyphae (mg) and the inhibition rate (%) of different concentrations of biocontrol strain G4 on soybean sclerotinia sclerotiorum;

[0015] Figure 5 The following are photos of the effect of G4 strain on the mycelial morphology of soybean sclerotinia sclerotiorum (PDA, 25°C, 7d); Figure A shows the mycelial morphology of soybean sclerotinia sclerotiorum pathogen under normal growth; Figure B shows the mycelial morphology of soybean sclerotinia sclerotiorum pathogen under the condition of G4 strain concentration of 5.0×10 5 The morphology of mycelial growth of soybean sclerotinia (Sclerotiniasclerotiorum) at cfu / mL;

[0016] Figure 6 Figure 2 is the inhibitory effect curve of G4 strain on soybean Sclerotinia sclerotiorum at different temperatures (Figure A) and pH (Figure B) (PDA, 25℃, 7d); the horizontal axis is different temperatures (℃) and pH, and the vertical axis is the OD of biocontrol bacteria G4 at different temperatures and pH. 600nm Values ​​and inhibition rate (%) of biocontrol bacteria G4 against soybean sclerotinia sclerotiorum at different temperatures and pH values;

[0017] Figure 7 The following are the inhibition curves of soluble protein (Figure A), reducing sugar content (Figure B), and malondialdehyde content (Figure C) in the mycelium of soybean sclerotinia rot after being treated with different concentrations of G4 strain; the horizontal axis is the concentration of G4 bacterial solution in PDB medium (cfu / mL), and the vertical axis is the soluble protein content (mg / mL), reducing sugar content (mg / mL), and malondialdehyde content (mmol / g);

[0018] Figure 8Figure 3 is the effect curve of G4 strain on the activity of cell wall degrading enzymes of soybean sclerotinia sclerotinia; the horizontal axis represents the time points of different sampling, the vertical axis of Figure A represents the cellulase (Cx) content (U / mL), and the vertical axis of Figure B represents the β-glucosidase (β-G) content (U / mL); the vertical axis of Figure C represents the polygalacturonase (PG) content (U / mL), and the vertical axis of Figure D represents the polymethylgalacturonase (PMG) content (U / mL).

[0019] Fig. 9 It is a curve diagram of the effect of G4 strain on the defense enzyme activity and different enzyme contents of soybean leaves; the horizontal axis is the time point of different sampling, the vertical axis of Figure A is the content of superoxide dismutase (SOD), the vertical axis of Figure B is the content of polyphenol oxidase (PPO), the vertical axis of Figure C is the content of catalase (CAT), the vertical axis of Figure D is the content of peroxidase (POD), the vertical axis of Figure E is the content of malondialdehyde (MDA), and the vertical axis of Figure F is the content of phenylalanine ammonia lyase (PAL). DETAILED DESCRIPTION

[0020] In order to better explain the present invention, the main contents of the present invention are further explained below in conjunction with specific examples, but the contents of the present invention are not limited to the following examples. The technical solutions involved in the embodiments of the present invention, if not otherwise specified, are all conventional solutions in the art; the reagents or materials, if not otherwise specified, are all derived from commercial channels.

[0021] Example 1: Isolation, Identification and Biological Characteristic Analysis of Serratia marcescens G4 I. Isolation of the strain and analysis of its biological characteristics

[0022] Soybean rhizosphere soil samples were collected from the teaching and research base of Jilin Agricultural University. 10 g of the soil sample was placed in a triangular flask containing 90 mL of sterile water, and then placed on a shaker at 30 °C and 155 rpm for 30 min to fully mix it. After the obtained sample was allowed to stand for 10 min, 1 mL of the supernatant was drawn and diluted to 10 with sterile water. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7Concentration gradient suspension; 100 μL of each concentration gradient suspension was aspirated and dropped onto the center of the NA solid culture medium, spread evenly with a spreader, and each gradient was repeated 3 times. The plate was inverted and cultured in a 30°C constant temperature incubator for 2 days. The separation and purification method is as follows: the bacterial colonies with different morphologies obtained in the above experiment were picked and streaked onto the LB solid culture medium, and the colonies were cultured in a 30°C constant temperature incubator for 2 days. The above operation was repeated until a single colony with the same morphology appeared on the LB solid culture medium. A single colony was picked with an inoculation loop and inoculated into the LB liquid culture medium. The culture was cultured at 30°C and 155 rpm for 12 hours to obtain the strain fermentation broth, and the concentration of the strain fermentation broth was adjusted to 1×10 9 cfu / m.

[0023] The specific method of the confrontation experiment is as follows: take 0.5 mL of the above concentration of 1×10 9 cfu / m strain fermentation broth was added to PDA medium (temperature was 40-45℃), and the mixed medium was obtained by thorough mixing; then 10mL of the obtained mixed medium was poured into a culture dish with a diameter of 90mm, and after the mixed medium solidified, a 5mm diameter Sclerotinia sclerotiorum pathogenic bacteria cake was inoculated in the center of the mixed medium as the experimental group, and a Sclerotinia sclerotiorum pathogenic bacteria cake was inoculated in a PDA medium without the fermentation broth of the strain as the control group, and each treatment was repeated three times. The experimental group and the control group were incubated in an incubator at 30℃ for 7 days, and the colony diameter was measured with a vernier caliper using the cross method to calculate the inhibition rate. Inhibition rate (%) = [(control group colony diameter - experimental group colony diameter) / control group colony diameter] × 100%. Finally, a strain with the highest inhibition rate against soybean sclerotinia sclerotiorum was screened out, named Serratia marcescens G4 (G4 strain), and stored in a -80°C refrigerator.

[0024] The NA solid culture medium is: 30.00 g beef extract, 50.00 g peptone, 30.00 g sodium chloride, 20.00 g agar powder, and 1000 mL distilled water;

[0025] The LB solid medium is: 10.00 g of tryptone, 5.00 g of yeast extract powder, 10.00 g of sodium chloride, 20.00 g of agar powder, and 1000 mL of distilled water;

[0026] The LB liquid culture medium is: 10.00 g of tryptone, 5.00 g of yeast extract powder, 10.00 g of sodium chloride, and 1000 mL of distilled water;

[0027] The PDA culture medium comprises: 20.00 g of glucose, 20.00 g of agar powder, 200.00 g of potato and 1000 mL of distilled water.

[0028] The strain activation, cultivation and fermentation methods are as follows:

[0029] (1) Strain activation: Use a sterile inoculation needle to pick colonies from the G4 strain slant stored in a -80 refrigerator, inoculate them on LB solid medium, and culture them in a 30°C incubator for 16 h;

[0030] (2) Strain culture: The G4 strain cultured for 16 h in step (1) was transferred to fresh LB solid medium and cultured in a 30° C. incubator for 16 h;

[0031] (3) Strain fermentation: The G4 strain in step (2) was inoculated into LB liquid medium using an inoculation loop and cultured at 30°C and 155 rpm until the logarithmic phase (OD 600 =0.6), prepare G4 seed solution. Pipette 0.5mL of G4 seed solution into a flask containing 100mL LB liquid medium, shake culture at 155rpm and 30℃ for 18h, centrifuge at 7000rpm for 5min, and adjust the concentration of the centrifuged bacteria to 1×10 with sterile water. 9 cfu / mL, and G4 fermentation broth was prepared.

[0032] 2. Morphological characteristics of strains

[0033] (1) Colony characteristics

[0034] The G4 strain stored in a -80 refrigerator was inoculated on LB solid medium and cultured in a 30°C constant temperature incubator for 16 hours. The colonies were red, sticky, smooth, moist, slightly shiny, and opaque ( Figure 1 ).

[0035] (2) Scanning electron microscopy results

[0036] The strain was observed under electron microscope to have a rough surface and was a rod-shaped bacterium ( Figure 2 ).

[0037] 3. Analysis of Physiological and Biochemical Characteristics

[0038] The physiological and biochemical characteristics of the G4 strain were determined with reference to Microbiology Experiments (Shen Ping, Fan Xiurong, Li Guangwu, Microbiology Experiments (3rd edition); Beijing: Higher Education Press, 1999) and Manual of Identification of Common Bacterial Systems (Dong Xiuzhu, Cai Miaoying, Manual of Identification of Common Bacterial Systems; Beijing: Science Press, 2011).

[0039] The test results showed that the strain was negative for Gram and methyl red reactions, but positive for VP reaction, starch hydrolysis test, glycolysis test (fructose, sucrose, maltose, glucose), citric acid utilization test, catalase test, and indole reaction, indicating that the strain can use citrate to hydrolyze fructose, sucrose, maltose, glucose, and produce indole.

[0040] 4. Identification of the taxonomic attributes of strain G4

[0041] The whole genomic DNA was extracted using the TIANamp bacterial DNA kit (Tiangen Biotechnology Co., Ltd., Beijing, China). The 16S rDNA gene was amplified by universal primer polymerase chain reaction (PCR) using the primers in the kit: 27F (5'-AGAGTTGATCCTGCTCAG-3'), 1492R (5'-GTTACCTTTACGACTT-3'). 50 μL amplification system: Premixtaq 25 μL, template (G4 strain DNA) 500 ng, 1492R and 27F 10 μL each, and sterile water was used to make up to 50 μL. The PCR cycle parameters were: 95 °C preheating for 5 min, 95 °C denaturation for 30 s, 56 °C annealing for 30 s, 72 °C extension for 1.5 min, 72 °C extension for 10 min, after purification by 1% agarose gel electrophoresis, EasyPure gel purification was used. Then sequencing was performed by Sangon Biotechnology Co., Ltd. (Shanghai, China). The PCR product was purified and recovered using the V-gene nucleic acid purification kit (TIANGEN), and the DNA was eluted into a collection tube and refrigerated at 4°C. The sequencing was completed by Shanghai Bioengineering Engineering Co., Ltd. The sequencing results were compared with the 16S rDNA sequence in Genbank using the Blast software. The 16S rDNA sequence of the G4 strain was obtained. The 16S rDNA sequence of the strain is shown in SEQ ID NO.1 and has been submitted to the GenBank database. The phylogenetic tree of the biological control bacteria was constructed, and G4 was determined to be a genus of Serratia marcescens and named Serratia marcescens G4 ( Figure 3 ).

[0042] The strain has been deposited in China Center for Type Culture Collection with the deposit number of CCTCC M 20242667 and the classification name of Serratia marcescens G4; address: Wuhan University, Wuhan, China; postal code: 430072, telephone: (027) 68754052. In the present invention, Serratia marcescens G4 is abbreviated as G4 strain.

[0043] Example 2: Inhibitory effect of strain G4 on colony diameter and mycelial growth of soybean sclerotinia (Sclerotinia sclerotiorum)

[0044] A single colony of the G4 strain was picked up with an inoculation loop and inoculated into 100 mL of LB liquid medium. The culture was continued at 30°C and 155 rpm until the logarithmic phase (OD 600nm =0.6), prepare G4 seed solution. Pipette 0.5mL seed solution into 100mL LB liquid medium, culture at 30℃, 155rpm for 16h, centrifuge, discard the supernatant, wash the bacteria with sterile water 3 times to obtain bacterial solution, and adjust the concentration of the bacterial solution to 1×10 5 cfu / mL, 1×10 6 cfu / mL, 1×10 7 cfu / mL, 1×10 8 cfu / mL, 1×10 9 cfu / mL for later use. Prepare a PDA medium containing G4 bacterial solution by adding 1mL of bacterial solution to 100mL of PDA medium. A well-grown 5mm soybean sclerotinia (Sclerotinia sclerotiorum) pathogen cake was inoculated in the center of the PDA medium containing G4 bacterial solution as the experimental group, and a medium without G4 bacterial solution but only inoculated with pathogen cake was used as the control group; cultured at 25°C for 7 days, the colony diameter was measured, and then the hyphae were scraped off with a sterile blade, dried in an oven at 60°C for 6h and weighed, and the hyphae weight of the control group and the experimental group were measured. Calculate the inhibition rate, repeat the treatment 3 times, and the experiment was performed twice.

[0045] like Figure 4 As shown in the figure, the inhibitory effect of G4 strain on the colony growth and mycelial dry weight of soybean sclerotinia sclerotiorum increased with the increase of strain concentration. 5 cfu / mL, the inhibition rate of soybean sclerotinia colony diameter and mycelium dry weight was the highest, which were 92.43% and 92% respectively. Inhibition rate (%) = [(mycelium dry weight of control group - mycelium dry weight of experimental group) / mycelium dry weight of control group] × 100%.

[0046] Example 3: Effect of strain G4 on mycelial morphology of soybean sclerotinia (Sclerotinia sclerotiorum)

[0047] A single colony of the G4 strain was picked up with an inoculation loop and inoculated into 100 mL of LB liquid medium. The culture was continued at 30°C and 155 rpm until the logarithmic phase (OD 600nm=0.6), prepare G4 seed solution. Pipette 0.5mL seed solution into 100mL LB liquid medium, culture at 30℃, 155rpm for 16h, centrifuge, discard the supernatant, wash the bacteria with sterile water 3 times to obtain bacterial solution, and adjust the concentration of the bacterial solution to 1×10 9 cfu / mL for later use. Prepare a PDA medium containing G4 bacterial solution according to the ratio of adding 1mL bacterial solution to 100mL PDA medium. A well-grown 5mm soybean sclerotinia (Sclerotinia sclerotiorum) pathogen cake was inoculated in the center of the PDA medium containing the G4 strain as the experimental group, and a medium without G4 bacterial solution but only inoculated with pathogen cake was used as the control group; after culturing at 25°C for 7 days, the colony was scraped off with a slide and placed under an optical microscope for observation to observe the changes in hyphae morphology. The treatment was repeated three times and the experiment was performed twice.

[0048] The morphological changes of soybean sclerotinia mycelium after treatment with G4 strain are shown in Figure 2. Figure 5 In the control group, the hyphae of soybean sclerotinia in normal growth showed low density, long hyphae, and extended in all directions ( Figure 5 A). The concentration is 5.0×10 5 After treatment with the cfu / mLG4 strain, the hyphae were severely swollen and deformed; the cell wall was thickened, and some hyphae were twisted and broken, resulting in the leakage of intracellular protoplasts ( Figure 5 B). Therefore, the G4 strain can effectively inhibit the growth of soybean sclerotinia. The G4 strain has an inhibitory effect on the mycelial morphology and can effectively reduce the activity of soybean sclerotinia.

[0049] Example 4: Inhibitory effect of strain G4 on soybean sclerotinia sclerotiorum at different temperatures and pH values

[0050] A single colony of the G4 strain was picked up with an inoculation loop and inoculated into 100 mL of LB liquid medium. The culture was cultured overnight for 16 h and the concentration of the bacterial solution was adjusted to 1.0 × 10 9 cfu / mL, 1 mL of bacterial suspension was taken and added into 100 mL of LB liquid medium (pH 7), and then cultured at 20°C, 25°C, 30°C, 35°C, 40°C, pH 5, 6, 7, 8, 9, and 155 rpm for 12 h to obtain G4 bacterial suspension, and the OD of the bacterial suspension was measured by UV spectrophotometer 600 nm The growth of biological control bacteria was determined by quantifying the value of the value; a 5 mm soybean sclerotinia (Sclerotinia sclerotiorum) cake was inoculated in the center of the PDA medium, and 5 μL of the G4 bacterial suspension (OD 600=0.6) as the experimental group, and PDA medium without G4 bacterial solution as the control group. The culture dishes were cultured in a constant temperature incubator at 25°C for 7 days, and the colony diameters of the control group and the experimental group were measured. The antibacterial effect was observed and the inhibition rate of G4 strain on soybean sclerotinia (Sclerotinia sclerotiorum) was calculated. Each treatment was repeated three times.

[0051] like Figure 6 As shown in the figure, with the increase of temperature, the inhibitory effect of G4 strain on soybean Sclerotinia sclerotiorum first increased and then decreased; at 20-40℃, the inhibition rate of G4 strain on soybean Sclerotinia sclerotiorum was 77.73%-92.10% ( Figure 6 A). The inhibition rate was the highest at 30°C (92.10%). In the pH range of 5 to 9, the inhibition rate was between 50.66% and 82.32%, and the inhibition rate was the highest at pH 7 (82.32%). Therefore, the G4 strain had the highest inhibitory activity against soybean sclerotinia at pH 7 and 30°C. Inhibition rate (%) = [(control group colony diameter - experimental group colony diameter) / control group colony diameter] × 100%.

[0052] Example 5: Inhibitory effect of strain G4 on metabolites of soybean sclerotinia

[0053] Use a 6mm puncher to punch out bacterial cakes on the soybean sclerotinia plates with the same growth trend. Place the bacterial cakes in a conical flask containing 100 mL of PDB medium and shake culture at 28°C and 160 rpm for 4 days. Further, different concentrations of G4 strain (1 mL, 5.0×10 1 ~5.0×10 5 cfu / mL) as the laboratory, and the control group used sterile water (1mL) instead of G4 strain. After 24 hours of culture, the mycelium was collected. The mycelium was wiped with sterile filter paper to remove moisture and stored in a -20°C refrigerator. The mycelium was placed in a vacuum freeze dryer and dried for 48 hours, and crushed into powder with a tissue grinder. The mycelium powder (1g) and Tris-HCl buffer (4mL) were mixed and centrifuged at 15000rpm for 20min. The supernatant was collected and stored at -20°C for the determination of soluble protein, reducing sugar, and malondialdehyde content.

[0054] The reducing sugar content was determined by the DNS method. Take 0.1 mL of the supernatant of the mycelial extract into a test tube, add 2 mL of DNS solution, mix well, boil in a boiling water bath for 5 min, then quickly cool, add 1 mL of Tris-HCl buffer, and measure the absorbance of each treatment at a wavelength of 540 nm, using Tris-HCl buffer as the zero point control, and repeat 3 times for each treatment.

[0055] Method for determining the soluble protein content. Take 0.1 mL of the supernatant from each treatment, add 5 mL of Coomassie Brilliant Blue G-250 solution, mix well and keep for 5 minutes. Then measure the absorbance of each treatment at a wavelength of 595 nm. Tris-HCl buffer plus Coomassie Brilliant Blue G250 solution was used as the zero control, and each treatment was repeated 3 times.

[0056] Thiobarbituric acid method was used to determine the content of malondialdehyde. Take 2 mL of supernatant and mix with 2 mL of 0.5% thiobarbituric acid solution, put the test tube into boiling water for 10 minutes, and centrifuge at 5000r / min for 10 minutes after cooling. Take 0.5% thiobarbituric acid solution as group zero, and measure the absorbance at wavelengths of 532nm, 600nm and 450nm. Each treatment was repeated 3 times.

[0057] Depend on Figure 7 As shown in Figure 2, after 24 h of treatment with strain G4, the reducing sugar content in the mycelium of soybean sclerotinia was significantly lower than that in the control group ( Figure 7 A). The concentration of G4 bacterial solution was 5.0×10 1 ~5.0×10 5 cfu / mL, the reducing sugar content was 7.88, 4.82, 4.07, 6.31, and 5.21 mg / mL, respectively, all lower than the control group (13.5 mg / mL). The soluble protein content in soybean sclerotinia showed a significant downward trend ( Figure 7 C) The concentration of G4 bacterial solution was 5.0×10 1 ~5.0×10 5 cfu / mL, the soluble protein content was reduced by 25.64%, 27.96%, 36.25%, 42.86% and 36.76% compared with the control (7.63 mg / mL), respectively. Therefore, the G4 strain can inhibit protein synthesis and promote protein hydrolysis in the body, resulting in abnormal protein content in soybean sclerotinia, thereby disrupting the physiological metabolic process and inhibiting the normal growth of soybean sclerotinia. After treatment with the G4 strain, the MDA level of soybean sclerotinia increased significantly ( Figure 7 B), increased by 91.84%, 86.47%, 81.01%, 78.95% and 19.51% respectively.

[0058] Example 6: Effect of strain G4 on the activity of cell wall degrading enzymes in soybean sclerotinia sclerotiorum

[0059] At the three-leaf stage, healthy soybean leaves were collected, washed with 70% alcohol for 1 min, washed with sterile water 3 times, and dried with sterile gauze. There were 4 treatment groups: 1) G4 strain group (sprayed with 20 mL OD 600=0.6 of G4 bacterial suspension), 2) control group (sprayed with 20 mL of sterile water, i.e., Control group), 3) soybean sclerotinia group (each leaf was inoculated with 1 bacterial cake), 4) soybean sclerotinia + G4 strain group (each leaf was sprayed with 20 mL of G4 bacterial suspension before inoculating 1 bacterial cake). Each treatment included 20 leaves. In addition, leaves were taken on the 1st, 2nd, 3rd, 4th and 5th day after treatment and ground in a mortar at 4°C. Buffer solution (20 mmol L -1 Tris-HCl buffer and 1 mol L - 1 NaCl, pH 7.4) and leaf powder were incubated at 4°C for 10 minutes. Further, the mixture was centrifuged at 4°C and 12,000 rpm, and the supernatant contained crude enzyme solution. The activities of cell wall degrading enzymes such as cellulase (Cx), β-glucosidase (β-G), polygalacturonase (PG) and polymethylgalacturonase (PMG) were measured using an ultraviolet spectrophotometer.

[0060] The determination method of cellulase (Cx) is to take 0.2mL of crude enzyme solution, add 1mL of substrate (1% carboxyhydroxycellulose solution) and 1.8mL of buffer solution (50mmol / L disodium hydrogen phosphate-citric acid buffer solution), mix well, and place in a 50℃ constant temperature water bath for 1h, then add 1.5mL of DNS reagent to terminate the reaction, shake well, and place in a boiling water bath for 10min. Cool and dilute to 25mL with distilled water. The activity of the enzyme is measured at a wavelength of 540nm, and the enzyme solution treated with boiling water bath is used as a control. The enzyme activity unit is 1mL of enzyme solution at 50℃ catalyzing the substrate to produce 1μg of glucose per minute as one enzyme activity unit (U), expressed as U / mL.

[0061] The determination method of β-glucosidase (β-G) is to take 0.2mL of crude enzyme solution, add 1mL of substrate (1% salicin solution) and 1.8mL of buffer solution (50mmol / L acetic acid-sodium acetate buffer solution), mix well, and place in a 50℃ constant temperature water bath for 1h, then add 1.5mL of DNS reagent to terminate the reaction, shake well, and place in a boiling water bath for 10min. After cooling, distilled water is used to make the volume 25mL. The activity of the enzyme is measured at a wavelength of 540nm, and the enzyme solution after adding the boiling water bath is used as the control. The enzyme activity unit is 1mL of enzyme solution at 50℃ catalyzing the substrate to produce 1μg of glucose per minute as one enzyme activity unit (U), expressed as U / mL.

[0062] The determination method of polygalacturonase (PG) is to take 0.2mL crude enzyme solution, add 1mL substrate (0.1% polygalacturonic acid solution) and 1.8mL buffer solution (50mmol / L disodium hydrogen phosphate-citric acid buffer solution), mix well, and place in a 50℃ constant temperature water bath for 1h, then add 2.5mL DNS reagent to terminate the reaction, shake well, and place in a boiling water bath for 10min. After cooling, distilled water is used to make the volume 25mL. The activity of the enzyme is measured at a wavelength of 540nm, and the enzyme solution treated with boiling water is used as a control. The enzyme activity unit is 1mL enzyme solution at 50℃ catalyzes the production of 1μg galacturonic acid per minute, which is one enzyme activity unit (U), expressed as U / mL.

[0063] The determination method of polymethylgalacturonase (PMG) is to take 0.2mL of crude enzyme solution, add 1mL of substrate (0.1% pectin solution) and 1.8mL of buffer solution (50mmol / L disodium hydrogen phosphate-citric acid buffer solution), shake well, and place in a 50℃ constant temperature water bath for 1h, then add 2.5mL of DNS reagent to terminate the reaction, and then place in a boiling water bath for 10min. After cooling, distilled water is used to make the volume 25mL. The activity of the enzyme is measured at a wavelength of 540nm, and the enzyme solution after adding the boiling water bath is used as the control. The enzyme activity unit is 1mL of enzyme solution at 50℃ catalyzes the production of 1μg of galacturonic acid per minute, which is one enzyme activity unit (U), expressed as U / mL.

[0064] Depend on Figure 8 As shown, the G4 strain effectively reduced the activities of cell wall degrading enzymes of soybean sclerotinia. Specifically, it reduced the activities of Cx, PG, β-G, and PMG. The activities of these enzymes did not change significantly over time in the control group. In the soybean sclerotinia and soybean sclerotinia+G4 groups, the activities of these enzymes increased over time.

[0065] Example 7: Determination of the activity of defense-related enzymes and malondialdehyde (MDA) content in soybean leaves by strain G4

[0066] There are 4 treatments in the experiment: 1) control (irrigating the roots with 20 mL of sterile water), 2) G4 (irrigating the roots with OD 600 =0.6 G4 suspension 20mL watering roots), 3) soybean sclerotinia (inoculate 1 sclerotinia cake per leaf), 4) soybean sclerotinia + G4 (spray 20mL of G4 suspension before inoculating soybean sclerotinia cake). When soybean plants were in the 3-leaf stage, the roots were sprayed with G4 suspension (OD 600nm=0.6; 20 mL) or sterile water. After 48 h, soybean sclerotinia was inoculated on soybean leaves. Soybean seedling leaves were collected on the 1st, 2nd, 3rd, 4th and 5th day after treatment. Peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and malondialdehyde (MDA) were determined by spectrophotometer.

[0067] Malondialdehyde (MDA) determination method: Weigh 1.00g soybean leaves, add 2mL of 10% trichloroacetic acid, grind into a homogenate at 4℃, then add 8mL of trichloroacetic acid for further grinding, centrifuge at 4000rpm for 10min at 4℃, and the supernatant is the MDA crude extract. Take 2mL of the crude extract, add 2mL of 0.6% thiobarbituric acid solution, and add 2mL of distilled water as a control. The mixed solution reacts in a boiling water bath for 15min, and after cooling, take the supernatant and measure the absorbance at wavelengths of 450nm, 532nm and 600nm respectively. The MDA content is expressed in units of μmol / g·FW.

[0068] C(MDA)=6.45×(A532-A600)-0.56×A450 (1)

[0069]

[0070] Superoxide dismutase (SOD) assay method: Weigh 1.00 g of soybean leaves in a mortar, add 2 mL of 50 mmol / L phosphate buffer solution containing 1% polyvinyl pyrrolidone (PVP) precooled at 4°C, grind into a homogenate under an ice bath, centrifuge at 12000 rpm at 4°C for 15 min, and the supernatant is the crude enzyme extract. Add reagents 0.3 mL of methionine (L-Met), 0.3 mL of nitro tetrazolium chloride (750 μmol / L NBT), 0.3 mL of disodium ethylenediaminetetraacetate (100 μmol / LEDTA-2Na), 0.5 mL of distilled water and 0.1 mL of enzyme solution. After adding the above reagents, mix them thoroughly and react for 20 to 30 minutes at 25°C to 35°C and 4000Lx light intensity. After the reaction is completed, cover with a black cloth to terminate the reaction. Measure the absorbance (A) at a wavelength of 560nm. Use the shading as the blank control. Replace the enzyme solution with PB solution to carry out the above reaction treatment group solution and adjust to zero. SOD activity is expressed in units of Ug-1 / min. Calculation is as shown in formula (4).

[0071]

[0072] The meanings of the above formulas are as follows:

[0073] A0: absorbance of blank control group (A); AS: absorbance of sample group (A); VT: total volume of crude enzyme solution (mL); VS: volume of crude enzyme solution used in the assay (mL); FW: fresh weight of leaves (g).

[0074] Peroxidase (POD) assay method: Weigh 1.00 g of soybean leaves in a mortar, add 2 mL of 0.2 mol / L PB and grind into a homogenate, centrifuge at 12000 rpm and 4°C for 15 min, and the supernatant is the crude enzyme solution. Take 0.3 mL of the crude enzyme solution, add 3 mL of the reaction solution (200 mL of 0.2 mol / L PB, add 0.076 mL of guaiacol while heating and stirring, and add 0.112 mL of 3% H2O2 after cooling), adjust to zero with 0.2 mol / L PB, measure the absorbance (A) at a wavelength of 570 nm, read once every 30 seconds, and at a wavelength of 470 nm, every 0.01 change in absorbance is one enzyme activity unit U. The peroxidase activity is expressed as U g-1min-1, and is calculated as shown in formula (5).

[0075]

[0076] The meanings of the above formulas are as follows:

[0077] ΔA470: absorbance change of enzyme activity at 470 nm wavelength during reaction time; Vt: total volume of crude enzyme solution (mL); Vs: volume of enzyme solution during measurement (mL); FW: fresh weight of leaves (g); t: reaction time 30 s.

[0078] Catalase (CAT) assay method: 1.00 g soybean leaves were placed in a mortar, 2 mL 0.15 mol / L PB was added, and the mixture was ground into a homogenate, and centrifuged at 12000 rpm at 4°C for 15 min. The supernatant was the crude enzyme solution. 0.1 mL of the crude enzyme solution was added to 3 mL of the reaction solution (200 mL 0.15 mol / L PB was added to 0.3902 mL 30% H2O2), and the mixture was adjusted to zero with 0.15 mol / L PB. The absorbance (A) was measured at a wavelength of 240 nm, and the reading was taken every 30 s. Each change of 0.01 in the absorbance at a wavelength of 240 nm was an enzyme activity unit U. The catalase activity was expressed as U g-1min-1, and the calculation was as shown in formula (6).

[0079]

[0080] In the formula, ΔA240 represents: the absorbance change of enzyme activity at a wavelength of 240 nm during the reaction time. The meanings of the other formulas are the same as those in formula 6.

[0081] Polyphenol oxidase (PPO) assay method: Weigh 1.0g soybean leaves, add 2mL 0.1mol / L PB, grind into homogenate, centrifuge at 12000rpm at 4℃ for 15min, and the supernatant is the crude enzyme solution. Take 2mL of crude enzyme solution and preheat it in a 25℃ constant temperature water bath for 5min, add 1mL of catechol, 0.1mL enzyme solution and 3.9mL PB solution, react in a 37℃ constant temperature water bath for 10min, immediately add 2mL 20% trichloroacetic acid to terminate the reaction after the reaction, and measure the absorbance (A) at a wavelength of 525nm after appropriate dilution. Each change of 0.01 in absorbance at a wavelength of 525nm is one enzyme activity unit U, and the polyphenol oxidase activity is expressed as U g -1 min -1 The enzyme solution treated in boiling water bath was used as blank control and the calculation was as shown in formula (7).

[0082]

[0083] The meanings of each item in the formula are expressed as follows: ΔA525: absorbance change of enzyme activity at a wavelength of 525 nm during the reaction time; FW: fresh weight of soybean leaves (g); t: reaction time (min); D: dilution multiple.

[0084] L-phenylalanine ammonia lyase (PAL) assay method: Weigh 1.0 g of soybean leaves in a mortar, add 0.5 mol / l boric acid buffer extract and grind into a homogenate, centrifuge at 15000 rpm and 4°C for 20 min, and the supernatant is the crude enzyme solution. Take 0.2 mL of the above enzyme solution, add 3.8 mL of 0.5 mol / l boric acid buffer solution and 1 mL of 0.02 mol / L L-phenylalanine ammonia lyase, react in a 40°C constant temperature water bath for 60 min, immediately add 1 mL of 5 mol / L HCl solution to terminate the reaction, and measure the absorbance (A) at a wavelength of 290 nm. Each change of 0.01 in absorbance at a wavelength of 290 nm is one enzyme activity unit U. The phenylalanine ammonia lyase activity is expressed as U g-1min-1, calculated as formula (8), and an equal volume of enzyme solution is used as the blank control group (Control group).

[0085]

[0086] The meanings of each item in the formula are expressed as follows: ΔA290: the absorbance change of enzyme activity at a wavelength of 290 nm during the reaction time; VT: the total volume of crude enzyme solution (mL); FW: the fresh weight of soybean leaves (g); t: reaction time (min); VL: the volume of enzyme solution during measurement (mL).

[0087] Depend on Fig. 9As shown, the soybean sclerotinia disease + G4 group showed the highest defense-related enzyme activity. The activity of the G4 group was lower than that of the soybean sclerotinia disease + G4 group, but higher than that of the soybean sclerotinia disease group. The MDA level of the soybean sclerotinia disease + G4 group was lower than that of the soybean sclerotinia disease group. These results indicate that the G4 strain can improve the disease resistance of soybean and protect soybean from infection by soybean sclerotinia disease.

[0088] Example 8: Pot control effect of G4 strain fermentation liquid on soybean sclerotinia (Sclerotinia sclerotiorum) Preparation of G4 strain fermentation liquid: After the G4 strain stored in a -80°C refrigerator was thawed on ice, it was inoculated into LB liquid culture medium, cultured overnight for 12 hours, and then inoculated into LB solid culture medium, and activated and cultured at 30°C for 24 hours. The strain colony was picked up with an inoculation loop and inoculated into 100mL (250mL triangular flask) of LB culture liquid, and cultured at 30°C and 155rpm for 18 hours to prepare G4 strain fermentation liquid, and the final concentration of G4 strain fermentation liquid was adjusted to 1×10 9 cfu / mL.

[0089] Pot treatment: Jiyu 47 soybean seeds were treated with 0.5% sodium hypochlorite solution for 1 min, and then rinsed thoroughly with sterile distilled water. The seeds were sandwiched between wet filter papers and germinated at 28°C for 4 days. Further, each seedling was planted in a plastic container 9 cm high and 10 cm in diameter, containing 0.8 kg of sterilized soil mixed with vermiculite in a ratio of 1:5 (v / v). Grow under conditions of a temperature of 25-30°C, a photoperiod of 14h light / 10h dark, and a relative humidity of 70%. There were 4 treatment groups in total: 1) G4 strain group (sprayed with 20mL OD 600 =0.6 of G4 bacterial suspension), 2) control group (sprayed with 20 mL of sterile water), 3) soybean sclerotinia group (each leaf was inoculated with 1 bacterial cake), 4) soybean sclerotinia + G4 strain group (each leaf was sprayed with 20 mL of G4 bacterial suspension before inoculating 1 bacterial cake). The disease situation was investigated on the 30th day after treatment, and the disease situation was investigated according to the soybean sclerotinia grading standard, the total number of plants and the number of diseased plants were recorded, and the disease index and control effect were calculated.

[0090] Grading Standard:

[0091] Level 0: no symptoms;

[0092] Level 1: Mild disease, slightly yellow leaves, and reddish-brown spots less than 1 cm in size appear at the base of the stems;

[0093] Level 2: The leaves turn yellow, and obvious reddish-brown spots of about 1-3 cm appear at the base of the stem;

[0094] Level 3: The leaves turn yellow and white spots appear at the base of the stems that are larger than 3 cm in diameter.

[0095] Table 1: Experimental data on the control effect of G4 strain on soybean sclerotinia

[0096] Treatments Disease severity index(%) Disease reduction (%) Soybean Sclerotinia 74.54±1.51 —— Soybean Sclerotinia + G4 34.62±0.52 53.56 G4 5.81±0.05 92.20 Comparison 32.51±0.25 ——

[0097] Disease index = [∑(number of diseased plants at each level × disease level) / (total number of plants surveyed × highest disease level)] × 100%

[0098] Control effect = [(control disease index - treatment disease index) / control disease index] × 100%

[0099] In order to evaluate the biocontrol effect of the G4 strain, a potted plant experiment was conducted. The disease index was determined. The disease index after the sclerotinia disease treatment was 74.54%, and the disease index after the sclerotinia disease + G4 treatment was 34.62%. The control effect of the soybean sclerotinia disease + G4 group was 53.56%, and the control effect of the G4 group was 92.20% (Table 1), which effectively inhibited the occurrence of sclerotinia disease. Therefore, the Serratia marcescens G4 prepared by the present invention can be used to prepare a soybean sclerotinia disease (Sclerotiniasclerotiorum) control agent.

Claims

1. A Serratia marcescens as a biological control bacterium, which has been deposited in the China Center for Type Culture Collection on November 29, 2024, with a deposit number of CCTCC M 20242667 and a classification name of Serratia marcescens G4.

2. Use of the Serratia marcescens as a biological control bacterium according to claim 1 in the preparation of a soybean sclerotinia (Sclerotinia sclerotiorum) control agent.

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