Serratia prolinea CD3, bioinoculant and application thereof

Abstract

The present application relates to the technical field of microorganism, and particularly relates to a Serratia nematodiphila CD3, a biological bacterial agent and application thereof.The present application provides a Serratia nematodiphila CD3, and the preservation number of which is CGMCC No.31709.It is verified by experiments that the Serratia nematodiphila CD3 has a significant toxic effect on tomato root-knot nematode, and has a good application potential in the field of biological control of tomato root-knot nematode.In addition, the strain can effectively promote the growth of tomato and the improvement of fruit quality, and is a good growth-promoting bacteria.Therefore, the Serratia nematodiphila CD3 provided by the present application has potential application value in the aspects of growth-promoting application of tomato and biological control of tomato root-knot nematode.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Serratia nematodes CD3, a biological agent, and its application. Background Technology

[0002] Root-knot nematode disease is one of the most serious soil-borne diseases affecting crops. Vegetable production in most parts of China, both north and south, is affected by root-knot nematodes, with approximately 30% of greenhouse vegetables infested by the southern root-knot nematode (Meloidogyne incognita), causing severe production losses. Currently, chemical control is still the primary method for controlling root-knot nematodes. However, chemical control methods present problems such as high costs, pesticide residues, resistance, food safety concerns, and environmental pollution. Therefore, finding efficient and environmentally friendly methods for controlling root-knot nematodes remains a pressing issue.

[0003] Commonly studied nematicidal microorganisms include: *Pochonia*, *Porphyromonas*, *Hirstella*, *Arthrobotrys*, *Bacillus*, *Psedomonas*, *Streptomyces*, *Xenorhabd*, *Agrobacterium*, and *Achromobacter*. However, there are few reports on *Serratia nematodes*, which kills root-knot nematodes and promotes plant growth. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Serratia nematodes CD3, a biological agent, and its application, in order to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a strain of Serratia nematodiphila CD3, the preservation number of which is CGMCC No.31709.

[0007] This invention provides the application of the above-mentioned Serratia nematodes CD3 in the preparation of biological agents.

[0008] The present invention provides a biological agent comprising the above-mentioned Serratia nematodes CD3, the fermentation broth of the Serratia nematodes CD3, or the fermentation supernatant of the Serratia nematodes CD3.

[0009] Preferably, the method for preparing the Serratia nematodes CD3 fermentation broth includes the steps of inoculating the Serratia nematodes CD3 into a fermentation medium, carrying out fermentation culture, and obtaining the Serratia nematodes CD3 fermentation broth.

[0010] Preferably, the fermentation medium is based on LB liquid medium and further includes 5 g / L soluble starch, 15 g / L tryptone, 15 g / L yeast extract and 2.5 g / L NaCl; the pH of the fermentation medium is 7.

[0011] The fermentation culture time is 48 h or 60 h, the temperature is 28℃, and the liquid volume is 100 mL;

[0012] During inoculation, the inoculation amount of Serratia nematodes CD3 is 1-9%.

[0013] This invention provides the use of the above-mentioned Serratia nematodes CD3 or the above-mentioned biological agent in any one or more of the following:

[0014] (1) Control of root-knot nematodes;

[0015] (2) Promotes tomato growth;

[0016] (3) Improve the quality of tomato fruits;

[0017] (4) Increase tomato yield.

[0018] More preferably, the promotion of tomato growth includes promoting the increase of tomato plant height, promoting the increase of plant weight of both above-ground and underground parts, promoting root growth, promoting the increase of chlorophyll content, soluble sugar content, and proline content in tomato leaves, and reducing malondialdehyde content in tomato leaves.

[0019] More preferably, improving the quality of tomato fruit involves increasing the content of soluble protein, free amino acids, soluble sugars, ascorbic acid, titratable acid, and lycopene in the tomato fruit.

[0020] More preferably, increasing tomato yield includes increasing the number of tomatoes set, the fruit set rate, the average weight of a single fruit, and the total yield.

[0021] More preferably, the root-knot nematode is the tomato root-knot nematode.

[0022] The present invention provides a method for controlling root-knot nematodes, comprising the step of applying the above-mentioned biological agent to tomatoes.

[0023] The present invention provides a method for promoting tomato growth, comprising the step of applying the above-mentioned biological agent to tomatoes.

[0024] This invention provides a method for improving the quality of tomato fruit, including the step of applying the above-mentioned biological agent to the tomatoes.

[0025] The present invention provides a method for increasing tomato yield, comprising the step of applying the above-mentioned biological agent to tomatoes.

[0026] The present invention discloses the following technical effects:

[0027] This invention provides a strain of *Serratia nematodiphila* CD3, with the preservation number CGMCC No. 31709. This strain exhibits good toxicity against tomato root-knot nematodes, and its toxicity is further enhanced after optimization of liquid fermentation conditions. Furthermore, this strain significantly improves the morphological and physiological indicators of tomatoes. It promotes tomato plant growth, increases plant height, and enhances the dry and fresh weight of both above-ground and underground parts. The strain also increases the pigment content, soluble sugar content, and proline content in tomato leaves, while reducing malondialdehyde content. In addition, this strain significantly improves tomato fruit yield and nutritional value, specifically including fruit set number, fruit set rate, average single fruit weight, yield, soluble protein content, free amino acid content, soluble sugar content, vitamin C content, titratable acid content, and lycopene content.

[0028] In summary, the Serratia serratiae CD3 strain provided by this invention can not only control tomato root-knot nematode disease, but also effectively promote tomato growth and improve tomato fruit quality. This strain shows good application prospects in tomato disease control, plant growth promotion and fruit quality improvement. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 The colony morphology diagram (A) and phylogenetic tree (B) of strain CD3 are shown.

[0031] Figure 2 The effects of different carbon sources (A), nitrogen sources (B), and inorganic salt sources (C) on the nematicidal effect of Serratia marcescens CD3;

[0032] Figure 3The effects of different shake-flask fermentation treatments on the nematicidal effect of *Serratia nematodes* CD3 were investigated. Specifically, A represents the effect of different fermentation times on the nematicidal effect of *Serratia nematodes* CD3, B represents the effect of different pH values ​​on the nematicidal effect of *Serratia nematodes* CD3, C represents the effect of different temperatures on the nematicidal effect of *Serratia nematodes* CD3, D represents the effect of different liquid volumes on the nematicidal effect of *Serratia nematodes* CD3, and E represents the effect of different inoculum sizes on the nematicidal effect of *Serratia nematodes* CD3.

[0033] Figure 4 The growth phenotypes of tomato plants under different treatments were shown. The original fermentation broth was treated with Serratia nematodes CD3 fermentation broth, the 50% fermentation broth was treated with 50% Serratia nematodes CD3 fermentation broth, the 25% fermentation broth was treated with 25% Serratia nematodes CD3 fermentation broth, the culture medium was sterile LB liquid medium, and the water was sterile water.

[0034] Figure 5 The standard curve for proline content determination in tomato plants is shown, where absorbance is on the ordinate and proline concentration is on the abscissa.

[0035] Figure 6 The values ​​represent the growth indicators of tomato plants under different treatments; where A is root weight, B is plant height, C is fresh weight, and D is dry weight.

[0036] Figure 7 These are physiological indicators of tomato plants under different treatments; where A is pigment content, B is soluble sugar, C is malondialdehyde, and D is proline.

[0037] Figure 8 The values ​​represent fruit quality indicators of tomato plants under different treatments; where A represents vitamin C (ascorbic acid), B represents free amino acids, C represents soluble protein, and D represents soluble sugar.

[0038] Figure 9 The lycopene index of tomato plants under different treatments. Detailed Implementation

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0041] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0042] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0043] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0044] Example 1 Screening of strains

[0045] Thirty healthy, uniformly grown, third-instar nymphs of the forked-horn bug were selected and placed in sterile petri dishes. After starvation for 24 hours, they were frozen at -20°C for 5 minutes. After removal, the thoracic legs were cut off in a laminar flow hood, and the body surface was disinfected with 75% alcohol for 20 seconds. They were then rinsed 3-5 times in sterile water and placed in a petri dish containing sterile phosphate buffer. The complete intestine was removed under a stereomicroscope using sterile dissection tools. After rinsing with sterile water, the intestine was transferred to a 1.5 mL centrifuge tube, ground, and then diluted to 1 mL with sterile water for later use.

[0046] Take the above-mentioned stock solution and mix it at 10... -4 10 -5 10 -6 and 10 -7Prepare bacterial suspensions using serial dilutions. Spread 100 μL of the suspension evenly onto NA plates, with three replicates for each dilution. Incubate at 28°C for 2-3 days. Observe the growth of single colonies. Based on morphological characteristics such as colony color, size, shape, and texture, select single colonies and streak them onto LB plates until single colonies grow. Repeat purification 2-3 times. The colony morphology of the purified strain is shown in the image below. Figure 1 As shown in A in the diagram.

[0047] Templates were prepared using a bacterial genomic DNA extraction kit, and 16S rRNA was amplified using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID NO.1) and 1492R (5′-ACGGCTACCTTGTTACGACT-3′, SEQ ID NO.2). A 50 μL reaction system contained 45 mL of 1×TSE102 Gold Mix, 2 mL each of upstream and downstream primers, and 1 mL of DNA template. The program was as follows: pre-denaturation at 98℃ for 2 min, followed by 35 cycles of 98℃ for 10 s, 56℃ for 10 s, and 72℃ for 10 s, and then extension at 72℃ for 5 min. After passing 1.0% agarose gel electrophoresis, the PCR products were sent to Beijing Qingke Kunming Company for sequencing. The obtained 16S sequences were uploaded to NCBI for BLAST alignment, yielding other sequences with extremely high sequence similarity (high homology sequences). After downloading the high homology sequences, a phylogenetic tree was constructed using MEGA 7.0 neighbor-joining to determine the taxonomic position of the strains. The phylogenetic tree is shown below. Figure 1 As shown in B in the diagram. (By...) Figure 1 The results of A and B indicate that CD3 is Serratianematodiphila.

[0048] The strain CD3 is named Serratia nematodiphila CD3 in this invention. It was deposited on November 13, 2024 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 31709.

[0049] Example 2: The toxic effect of Serratia nematode CD3 fermentation broth on tomato root-knot nematodes.

[0050] The in vitro toxic activity of *Serratia marcescens* CD3 fermentation broth against isolated tomato root-knot nematodes was determined using the immersion method. The specific method is as follows: A single colony of *Serratia marcescens* CD3 preserved in LB liquid medium (10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl, sterilized at 121℃ for 30 min) was inoculated into 100 mL of LB liquid medium and cultured with shaking at 28℃ and 180 r / min for 48 h to obtain the original fermentation broth (effective viable count 1.23 × 10⁻⁶). 9 The original fermentation broth was then diluted with sterile water by a factor of 1 to obtain a 50% fermentation broth (effective viable count of 5.86 × 10⁻⁶ CFU / mL). 8 The fermentation broth was centrifuged (12000 r / min for 10 min) and sterilized (121℃ for 30 min) to obtain sterilized supernatant.

[0051] Tomato root-knot nematodes were collected from diseased root tissues and rhizosphere soil of naturally infected tomatoes in the field. The root and soil solutions were poured into sieves of 20 mesh, 80 mesh, and 400 mesh (from top to bottom), rinsed with a high-pressure water gun, and the supernatant was filtered. This process was repeated several times. The nematode mixture from the 400 mesh sieve was collected into a beaker, centrifuged at 5000 rpm for 5 min, and the supernatant was discarded. A 45 wt% sucrose solution was added, stirred, and centrifuged again at 5000 rpm for 2 min. The nematode suspension was poured into a 500 mesh sieve and rinsed into a beaker to prepare a nematode suspension for later use. The tomato root-knot nematodes used in this invention have been disclosed in the literature "Preliminary Study on the Inhibition of Tomato Root-Knot Nematode Disease by Bioactive Organic Fertilizer 'Hongtuyun'", with a commitment to distribute them for 20 years.

[0052] 100 μL of fermentation stock broth, 50% fermentation broth, sterile supernatant, LB liquid medium, and sterile water were taken respectively. 10 μL of tomato root-knot nematode suspension (approximately 40 nematodes) was added to each treatment in a 96-well plate, with each treatment replicated three times. After incubation at 28℃ for 24 h, the nematodes were observed under an optical microscope. Stimulation with 1 mol / L NaOH resulted in stiffness, which was considered a sign of death. Sterile water was used as a blank control. The corrected mortality rate was calculated, and the results are shown in Table 1.

[0053] Table 1. Toxicity of different treatments of Serratia nematodes CD3 against nematodes

[0054]

[0055] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences (p<0.05), and the same applies below.

[0056] As shown in Table 1, *Serratia nematode* CD3 exhibits good toxicity against tomato root-knot nematodes, and the corrected mortality rate of tomato root-knot nematodes increases with increasing concentration. After 24 h of immersion treatment, the nematicidal activity of both the fermentation broth and the sterilized supernatant is above 80%. When the fermentation broth is the one obtained after 48 h of culture, the corrected mortality rate of nematodes after 24 h of treatment is 100.00%.

[0057] Example 3 Optimization of liquid fermentation conditions for Serratia nematodes CD3

[0058] This embodiment uses the nematode mortality rate as a metric to screen out the fermentation conditions with the best nematode-killing effect.

[0059] (1) Screening of different carbon sources: Based on LB liquid medium, sucrose, glucose, soluble starch, lactose or maltose were used as carbon sources to be screened, while other components remained unchanged, to obtain LB medium containing different carbon sources. The concentration of carbon source in the medium was 5 g / L. Each treatment was repeated 3 times. After shaking culture at 28℃ and 180 r / min for 48 h, the OD of the fermentation broth of the strain was measured. 600 nm The nematicidal activity of the supernatant (obtained by centrifuging the fermentation broth of the strain at 12000 rpm for 10 min) was determined using the immersion method. The specific steps were as follows: 100 μL of supernatant or LB liquid medium and 10 μL of tomato root-knot nematode suspension (approximately 40 nematodes) were placed in a 96-well plate and incubated at 28℃ for 24 h. The nematodes were then observed under an optical microscope. Stimulation with 1 mol / L NaOH resulted in stiffness, indicating death. Each treatment was repeated three times. The OD values ​​of the fermentation broth were then analyzed. 600 nm The optimal carbon source was determined by the value and the nematode-corrected mortality rate, and the results are as follows: Figure 2 As shown in Figure A. The results showed that different carbon sources had a significant impact on the nematicidal effect of *Serratia marcescens* CD3. The medium with soluble starch as the carbon source showed the highest corrected mortality rate (93.19%) of tomato root-knot nematodes after 24 hours of treatment with its supernatant. The order of influence of other carbon sources on the corrected mortality rate was lactose > LB broth > glucose > maltose > sucrose. *Serratia marcescens* CD3 utilized polysaccharides significantly more than disaccharides and monosaccharides; therefore, soluble starch was chosen as the carbon source for subsequent experiments.

[0060] (2) Screening of nitrogen sources: Based on LB liquid medium containing 5 g / L soluble starch, yeast extract, tryptone, peptone, potassium nitrate, and ammonium chloride were used as nitrogen sources to be screened, while other components remained unchanged, to obtain LB medium containing different nitrogen sources. The concentration of nitrogen source in this medium was 10 g / L. Each treatment was repeated 3 times. After shaking culture at 28℃ and 180 r / min for 48 h, the OD of the fermentation broth of the strain was measured. 600 nm The nematicidal activity of the supernatant (obtained by centrifuging the fermentation broth of the strain at 12000 rpm for 10 min) was determined using the immersion method, with the specific steps being the same as in step (1). The results are as follows: Figure 2 As shown in B. The results showed that, when using different nitrogen sources as culture medium components, the corrected mortality rate of *Serratia nematode* CD3 supernatant against tomato root-knot nematodes, from highest to lowest, was: tryptone, yeast extract, peptone, ammonium chloride, and potassium nitrate. Among these, in the culture media using tryptone and yeast extract as nitrogen sources, the toxicity of *Serratia nematode* CD3 supernatant against tomato root-knot nematodes was significantly higher than that of other nitrogen source media (P<0.05). *Serratia nematode* CD3 utilized organic nitrogen sources more efficiently than inorganic nitrogen sources. Therefore, tryptone and yeast extract were selected as nitrogen sources for subsequent experiments.

[0061] (3) Screening of inorganic salts: LB liquid medium containing 5 g / L soluble starch, 10 g / L tryptone and 10 g / L yeast extract was used. Sodium chloride, potassium sulfate, dipotassium hydrogen phosphate and disodium hydrogen phosphate were used as inorganic salts to be screened, while other components remained unchanged. LB medium containing different inorganic salts was obtained. The concentration of inorganic salts in this medium was 5 g / L. Each treatment was repeated 3 times. After shaking culture at 28℃ and 180 r / min for 48 h, the OD of the fermentation broth was measured. 600 nm The nematicidal activity of the supernatant (obtained by centrifuging the fermentation broth of the strain at 12000 rpm for 10 min) was determined using the immersion method, with the specific steps being the same as in step (1). The results are as follows: Figure 2 As shown in C. The results showed that different inorganic salt culture media had a significant effect on the killing effect of *Serratia nematode* CD3 on tomato root-knot nematodes (P<0.05). When sodium chloride was used as a culture medium component, the corrected mortality rate of tomato root-knot nematodes reached the highest level, which was significantly higher than that of other inorganic salt treatments. Therefore, sodium chloride was selected as the inorganic salt in the culture medium for component ratio optimization.

[0062] (4) Orthogonal optimization of fermentation medium: Single-factor screening of the fermentation medium revealed that the optimal carbon source was soluble starch, the optimal nitrogen source was tryptone and yeast extract, and the optimal inorganic salt was NaCl. The four factors obtained from the screening were then arranged according to L9(3) 4The factors and levels of the orthogonal experiment were designed to compare the corrected mortality rate of tomato root-knot nematodes under different component ratios, and to determine the optimal component ratio of the fermentation medium. The factors and levels of the orthogonal optimization experiment of the fermentation medium components of Serratia nematodes CD3 are shown in Table 2.

[0063] Table 2. Factors and levels of orthogonal optimization experiment for the components of Serratia nematodes CD3 fermentation medium.

[0064]

[0065] Range analysis results (Table 3) showed that tryptone had the greatest impact on the activity of *Serratia marcescens* CD3 supernatant in killing tomato root-knot nematodes, followed by yeast extract, while soluble starch had a smaller impact. The optimal ratio was A2B3C3D1. Therefore, the optimal fermentation medium for *Serratia marcescens* CD3 was determined to be LB liquid medium as the basal medium, also containing 5 g / L soluble starch, 15 g / L tryptone, 15 g / L yeast extract, and 2.5 g / L NaCl.

[0066] Table 3. Range evaluation results of orthogonal experiments for optimizing culture medium

[0067]

[0068] Note: (1) Lowercase letters in the table indicate that the difference is significant at the 0.05 level; (2) K1, K2 and K3 represent the average values ​​of the levels of each factor.

[0069] (5) Screening of optimal fermentation time: After inoculating Serratia nematodes CD3 into the optimal fermentation medium obtained above, the culture was carried out at 28℃. The inoculation amount was 1%, and the volume of the fermentation liquid in a 250mL fermentation bottle was 100mL. During the fermentation period of 0-96h, the OD of the fermentation liquid was measured once every 12h. 600 nm The nematode mortality rate was determined by centrifuging the fermentation broth of the strain at 12000 rpm for 10 min using the immersion method, which was used to collect samples from different time periods until the end of 96 h. Specifically, 100 μL of supernatant and 10 μL of tomato root-knot nematode suspension (approximately 40 nematodes) were placed in a 96-well plate and incubated at 28℃ for 24 h. The nematodes were then observed under an optical microscope. Stimulation with 1 mol / L NaOH resulted in stiffness, indicating death. Each treatment was repeated three times. The OD value of the fermentation broth was then used as the nematode mortality rate. 600 nm Using the value and corrected mortality rate of nematodes as indicators, the optimal fermentation time was determined, and the results are as follows: Figure 3As shown in Figure A. The results showed that the nematode-killing effect of the supernatant of *Serratia nematodes* CD3 obtained at different fermentation times differed significantly (P<0.05). The nematode mortality rate was significantly lower at 12 h than at other times. The corrected mortality rates of tomato root-knot nematodes were highest at 48 h and 60 h, at 91.84% and 92.26%, respectively, with no significant difference between the two. Therefore, 48 h and 60 h can be selected as the optimal fermentation times for *Serratia nematodes* CD3.

[0070] (6) Screening of optimal pH: After adjusting the pH of the optimal fermentation medium obtained above to 5, 6, 7 and 8, Serratia marcescens CD3 was inoculated and cultured at 28℃ for 48 h. The inoculation amount was 1%, and the volume of the fermentation liquid in a 250 mL fermentation bottle was 100 mL. The OD of the fermentation liquid obtained from the optimal fermentation medium at different pH was measured once. 600 nm The nematode corrected mortality rate was determined by immersion method in the supernatant obtained from samples taken at different time periods (the supernatant was obtained by centrifuging the fermentation broth of the strain; the centrifugation conditions were: 12000 rpm for 10 min). The specific steps were the same as in step (5), and the results were as follows. Figure 3 As shown in B in the figure. The results showed that the toxic effect of Serratia nematodes CD3 supernatant on tomato root-knot nematodes varied significantly under different initial pH conditions (P<0.05), but the overall mortality rate of tomato root-knot nematodes was higher than 80%. When the initial pH was between 6 and 7, there was no significant difference in the mortality rate of tomato root-knot nematodes. When the initial pH was 7, the mortality rate of tomato root-knot nematodes reached its maximum of 93.27%. When the initial pH exceeded 7, the mortality rate of nematodes gradually decreased. Therefore, pH 7 was selected as the optimal initial pH value for the fermentation culture of Serratia nematodes CD3.

[0071] (7) Screening of optimal fermentation temperature: The pH of the optimal fermentation medium obtained above was adjusted to 7, and Serratia nematodes CD3 was inoculated and cultured for 48 h. The inoculation amount was 1%, and the volume of the fermentation liquid in a 250 mL fermentation bottle was 100 mL. The fermentation temperature was 22, 25, 28, 31 or 34 °C. The OD of the fermentation liquid obtained at different fermentation temperatures was measured once. 600 nm The nematode corrected mortality rate was determined by immersion method in the supernatant obtained from samples taken at different time periods (the supernatant was obtained by centrifuging the fermentation broth of the strain; the centrifugation conditions were: 12000 rpm for 10 min). The specific steps were the same as in step (5), and the results were as follows. Figure 3 As shown in C. The results showed that there was no significant difference in the mortality rate of tomato root-knot nematodes at temperatures ranging from 25 to 31℃, with the highest mortality rate (93.98%) observed at 28℃. 28℃ was selected as the optimal fermentation temperature for *Serratia nematodes* CD3.

[0072] (8) Screening of optimal liquid volume: The pH of the optimal fermentation medium obtained above was adjusted to 7, and Serratia nematodes CD3 was inoculated and cultured at 28℃ for 48 h. The inoculation amount was 1%. The liquid volume of the 250 mL fermentation bottle was set to 50, 100, 150 or 200 mL. The OD of the fermentation liquid obtained with different bottle volumes was measured once. 600 nm The nematode corrected mortality rate was determined by immersion method in the supernatant obtained from samples taken at different time periods (the supernatant was obtained by centrifuging the fermentation broth of the strain; the centrifugation conditions were: 12000 rpm for 10 min). The specific steps were the same as in step (5), and the results were as follows. Figure 3 As shown in D in the figure. The results showed that there was no significant difference in the mortality rate of tomato root-knot nematodes when the liquid volume was 50-100 mL. Among them, the mortality rate of tomato root-knot nematodes was the highest at 100 mL, which was 99.10%. 100 mL was selected as the optimal liquid volume.

[0073] (9) Screening of optimal inoculum size: The pH of the optimal fermentation medium obtained above was adjusted to 7, and Serratia nematodes CD3 was inoculated and cultured at 28°C for 48 h. The volume of the fermentation liquid in a 250 mL fermentation bottle was 100 mL. The inoculum size was 1%, 3%, 5%, 7% or 9%. The OD of the fermentation liquid obtained with different inoculum sizes was measured once. 600 nm The nematode corrected mortality rate was determined by immersion method in the supernatant obtained from samples taken at different time periods (the supernatant was obtained by centrifuging the fermentation broth of the strain; the centrifugation conditions were: 12000 rpm for 10 min). The specific steps were the same as in step (5), and the results were as follows. Figure 3 As shown in E in the figure. The results showed that different inoculation amounts had no significant effect on the efficacy of Serratia nematode CD3 against tomato root-knot nematodes. However, the mortality rate of tomato root-knot nematodes was the highest at a 5% inoculation amount, reaching 100%. Therefore, 5% was selected as the optimal inoculation amount.

[0074] Example 4: Preparation of CD3 fermentation broth of Serratia nematodes

[0075] Serratia nematodes CD3 was inoculated into a fermentation medium (LB liquid medium as the basal medium, also containing 5 g / L soluble starch, 15 g / L tryptone, 15 g / L yeast extract, and 2.5 g / L NaCl, pH 7) for fermentation at 28℃ for 48 h. The volume of the fermentation solution in a 250 mL fermentation flask was 100 mL, and the inoculum size was 5%, yielding the Serratia nematodes CD3 fermentation stock broth (effective viable count 1.73 × 10⁻⁶). 9 (cfu / mL).

[0076] Example 5: Preparation of 50% Serratia nematodes CD3 fermentation broth and 25% Serratia nematodes CD3 fermentation broth

[0077] The *Serratia nematodes* CD3 fermentation broth prepared in Example 4 was diluted (using sterile water as the diluent) by a factor of 1 to obtain a 50% *Serratia nematodes* CD3 fermentation broth (effective viable count of 8.63 × 10⁻⁶). 8 The concentration of cfu / mL was diluted 4 times to obtain a 25% Serratia nematode CD3 fermentation broth (effective viable count of 3.69 × 10⁻⁶). 8 (cfu / mL).

[0078] Example 6: Tomato growth-promoting effect of Serratia nematodes CD3 fermentation broth

[0079] Preliminary preparation: Tomato seeds were surface-sterilized with 2% NaClO for 5 min, rinsed 3 times with sterile water, and then placed in petri dishes lined with moist filter paper. They were cultured at 28℃ for 3-4 days. After germination, the seeds were transplanted into plastic pots filled with autoclaved soil (vermiculite:soil = 1:5 (v / v)). These pots were placed in a greenhouse at 28℃-35℃, with 14 h / d of light and approximately 73% relative humidity, and watered regularly. Once the tomato plants reached a height of about 10 cm, they were transplanted into culture pots for further testing.

[0080] Experimental Design: Five treatments were established, including Serratia nematodes CD3 fermentation stock broth (prepared in Example 4), 50% Serratia nematodes CD3 fermentation broth (prepared in Example 5), 25% Serratia nematodes CD3 fermentation broth (prepared in Example 5), sterile LB liquid medium, and sterile water. Each treatment had eight replicates. Each pot was inoculated with 30 mL of Serratia nematodes CD3 fermentation stock broth, 50% Serratia nematodes CD3 fermentation broth, 25% Serratia nematodes CD3 fermentation broth, sterile LB liquid medium, or sterile water, for a total of four inoculations, with an interval of 7 days between adjacent inoculations. After 30 days of treatment, the morphological indicators (root weight, plant height, fresh weight, dry weight) and physiological indicators (pigment content, soluble sugar content, malondialdehyde content, proline content) of tomatoes in each treatment were measured.

[0081] The specific measurement method is as follows:

[0082] (1) Determination of pigment content: Accurately weigh 0.1 g of fresh sample into a 25 mL graduated tube, add 10 mL of 95% ethanol solution, and extract overnight in the dark until the tissue is completely bleached. Then dilute the extract to 15 mL with 95% ethanol solution, take 1 mL of chloroplast pigment extract, add 3 mL of 95% ethanol for dilution, and measure the absorbance at wavelengths of 665 nm, 649 nm and 470 nm, with 95% ethanol as a blank control.

[0083] The calculation formula is as follows:

[0084] Ca=13.95OD 665 -6.88OD 649 ;

[0085] Cb=24.96OD 649 -7.32OD 665 ;

[0086] Cxc = (1000OD) 470 -2.05Ca-114.8Cb) / 245;

[0087] Pigment content (mg / g·FW) = (pigment concentration (mg / L) × extraction liquid volume (L) × dilution factor) / sample fresh weight (g);

[0088] Where Ca, Cb, and Cxc are the total concentrations of chlorophyll a, chlorophyll b, and carotenoids, respectively, in mg / L.

[0089] (2) Determination of soluble sugars and malondialdehyde (MDA): Weigh 0.5 g of each sample leaf, repeat 3 times for each sample. Cut the leaves into small pieces, put them in a mortar, add 1 mL of 10% trichloroacetic acid (TCA) and a small amount of quartz sand, grind into a homogenate, add 4 mL of TCA and grind further. Transfer the homogenate to a centrifuge tube and centrifuge at 4000 r / min for 10 min. Take 2 mL of the supernatant (add 2 mL of distilled water to the blank tube), add 2 mL of 0.6% thiobarbituric acid (TBA) solution, mix well, boil in a boiling water bath for 15 min, cool and centrifuge once more, take the supernatant, and use a Tµ-1901 UV-Vis spectrophotometer to measure the extinction at wavelengths of 450 nm, 532 nm and 600 nm respectively. Calculate the concentrations of soluble sugars and malondialdehyde (MDA) according to the formula.

[0090] The calculation formula is as follows:

[0091] C1=11.71D 450 ;

[0092] C2 = 6.45 (D) 532 -D 600 -0.56D 450 ;

[0093] MDA content (μmol / g) = MDA concentration (μmol / L) × extraction liquid volume (L) / fresh weight of plant tissue;

[0094] Soluble sugar content (μmol / g) = soluble sugar concentration (μmol / L) × extract volume (L) / fresh weight of plant tissue;

[0095] In the formula: C1 is the concentration of soluble sugar (μmol / L), C2 is the concentration of malondialdehyde (MDA) (μmol / L), and D... 450 D 532 D 600 The values ​​are extinction values ​​at wavelengths of 450, 532, and 600 nm (non-specific absorption), respectively, and MDA is malondialdehyde.

[0096] (3) Determination of proline content: Preparation of extract: 0.2 g fresh leaves + 5 mL 3% sulfosalicylic acid (3 g sulfosalicylic acid dissolved in 100 mL distilled water) were ground, boiled in a water bath for 10 min, cooled and the supernatant was taken and diluted to 5 mL with 3% sulfosalicylic acid. Determination: 2 mL of extract was taken, and 2 mL glacial acetic acid and 3 mL acidic ninhydrin were added. The test tube was stoppered, boiled in a water bath for 40 min, cooled and 5 mL toluene solution was added, shaken for 30 s, and allowed to stand for 2 h. The solution was separated into layers. The upper layer was taken and the absorbance at 520 nm was measured with toluene as a blank. Plotting a standard curve: Using a standard solution (25 mg proline, diluted to 250 mL with distilled water), take six test tubes numbered 1-6 and add 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mL of proline standard solution, respectively. Then, add 9.9, 9.8, 9.7, 9.6, 9.5, and 9.4 mL of distilled water accordingly, bringing the final proline concentration in each tube to 1, 2, 3, 4, and 5 μg, respectively. Measure the absorbance at 520 nm using the same steps as above. Plot a standard curve with absorbance on the ordinate and proline concentration on the abscissa.

[0097] The calculation formula is as follows:

[0098] Proline content (mg / g) = (C*V / a)*1000 / W;

[0099] Where C is the proline concentration in μg / mL, such as Figure 5 As shown; V is the total extract volume in mL; a is the measured extract volume in mL; W is the fresh weight of the leaves in g.

[0100] The effects of Serratia nematode CD3 on tomato growth are shown in Table 4. Figure 4 , Figure 6 and Figure 7 As shown in the figure. The results showed that *Serratia marcescens* CD3 improved the morphological indicators of tomato plants, significantly increasing root weight, plant height, fresh weight, and dry weight (P < 0.05), thereby significantly enhancing the growth vigor of tomatoes. Furthermore, *Serratia marcescens* CD3 significantly increased the pigment and proline content in tomato leaves, while decreasing the content of soluble sugars and malondialdehyde, indicating that it enhances the accumulation of nutrients and stress resistance in tomatoes by increasing photosynthetic rate, reducing membrane lipid peroxidation, and regulating osmotic balance.

[0101] Table 4 Effects of different treatments on tomato growth indicators

[0102]

[0103] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences (p<0.05).

[0104] Example 7: Application of Serratia nematodes CD3 fermentation broth in improving tomato fruit quality

[0105] Preliminary preparation: Tomato seeds were surface-sterilized with 2% NaClO for 5 min, rinsed 3 times with sterile water, and then placed in petri dishes lined with moist filter paper. They were cultured at 28℃ for 3-4 days. After germination, the seeds were transplanted into seedling trays containing substrate soil (vermiculite:soil = 1:5 (v / v)). The trays were placed in a greenhouse at 28℃-35℃, with 14 h / d of light and approximately 73% relative humidity, and watered regularly. Once the tomato plants reached a height of about 10 cm, they were transplanted into culture pots for further testing.

[0106] Experimental Design: Five treatments were established, including Serratia nematodes CD3 fermentation stock solution (prepared in Example 4), 50% Serratia nematodes CD3 fermentation broth (prepared in Example 5), 25% Serratia nematodes CD3 fermentation broth (prepared in Example 5), sterile LB liquid medium, and sterile water. Each treatment had eight replicates. Each pot was inoculated with 100 mL of either Serratia nematodes CD3 fermentation stock solution, 50% Serratia nematodes CD3 fermentation broth, 25% Serratia nematodes CD3 fermentation broth, sterile LB liquid medium, or sterile water. A total of five inoculations were performed, with a 7-day interval between inoculations. After the tomato fruits turned red, the fruit set rate for each treatment was recorded. Ten fruits were selected to calculate the average weight of a single fruit. Fruits at the same maturity stage were selected, and the quality indicators of tomatoes in each treatment group were measured.

[0107] The quality indicators and testing methods are as follows:

[0108] (1) Determination of soluble protein, amino acids, soluble sugars and ascorbic acid (vitamin C) in fruit: The soluble protein content in fruit was determined using the Coomassie Brilliant Blue assay kit (catalog number: G0417F); the free amino acid content was determined using the amino acid assay kit (catalog number: G0415F); the soluble sugar content in fruit was determined using the soluble sugar assay kit (catalog number: G0501F); and the ascorbic acid content was determined using the reduced ascorbic acid assay kit (catalog number: G0201F). All kits were purchased from Suzhou Grees Biotechnology Co., Ltd.

[0109] (2) Determination of lycopene: After homogenizing the tomato sample, accurately weigh 3 g, add 2 mL of anhydrous ethanol and stir well; centrifuge the mixture at 3000 r / min for 10 min, discard the supernatant, and extract the residue with 20 mL of acetone-petroleum ether (1:1) in the dark for 2 h, stirring once every 20 min. After filtration, transfer the filtrate to a separatory funnel, wash 3 times with deionized water and discard the acetone layer, and determine the total volume of the upper organic phase; accurately transfer 1 mL of the extract into a 10 mL brown volumetric flask, dilute to volume with petroleum ether, and use petroleum ether as a reference to measure the absorbance at 502 nm with a 1 cm cuvette.

[0110] The effects of Serratia nematode CD3 on tomato fruit yield and quality are shown in Tables 5-6 and 6. Figures 8-9 As shown in the figure. The results showed that after treatment with *Serratia nematodes* CD3, the total number of fruits set, fruit set rate, average single fruit weight, and yield of tomatoes were all increased. Among them, the *Serratia nematodes* CD3 fermentation broth had the best effect, with a yield increase of 33.55% compared to the control. Simultaneously, *Serratia nematodes* CD3 also significantly improved the quality of tomato fruits. After root irrigation with *Serratia nematodes* CD3 fermentation broth, the nutrients in the tomato fruits were all increased. Among them, the *Serratia nematodes* CD3 fermentation broth had the best effect; the effect gradually weakened as the bacterial concentration decreased, except for soluble sugar content. Moreover, all three treatments with *Serratia nematodes* CD3 fermentation broth significantly increased the vitamin C content and free amino acid content in tomatoes (P < 0.05).

[0111] Table 5. Effects of different treatments on tomato yield and composition.

[0112]

[0113] Table 6 Effects of different treatments on tomato fruit quality

[0114]

[0115] Note: The data in the table are mean ± standard error. Different lowercase letters after the data in the same column indicate significant differences (p<0.05).

[0116] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Serratia nematodiphila CD3, characterized in that, The preservation number of the *Serratia nematodes* CD3 is CGMCC No. 31709.

2. The application of Serratia nematodes CD3 as described in claim 1 in the preparation of biological agents.

3. A biological agent, characterized in that, The biological agent includes the *Serratia nematodes* CD3 as described in claim 1, the fermentation broth of the *Serratia nematodes* CD3, or the fermentation supernatant of the *Serratia nematodes* CD3.

4. The biological agent according to claim 3, characterized in that, The method for preparing the fermentation broth of *Serratia nematodes* CD3 includes the steps of inoculating *Serratia nematodes* CD3 into a fermentation medium, carrying out fermentation culture, and obtaining the fermentation broth of *Serratia nematodes* CD3.

5. The biological agent according to claim 4, characterized in that, The fermentation medium is based on LB liquid medium and also includes 5 g / L soluble starch, 15 g / L tryptone, 15 g / L yeast extract and 2.5 g / L NaCl; the pH of the fermentation medium is 7. The fermentation culture time is 48 h or 60 h, the temperature is 28℃, and the liquid volume is 100 mL; During inoculation, the inoculation amount of Serratia nematodes CD3 is 1-9%.

6. The use of Serratia nematodes CD3 as described in claim 1 or the biological agent as described in any one of claims 3-5 in any one or more of the following: (1) Control of root-knot nematodes; (2) Promotes tomato growth; (3) Improve the quality of tomato fruits; (4) Increase tomato yield.

7. A method for controlling root-knot nematodes, characterized in that, The step includes applying the biological agent described in any one of claims 3-5 to tomatoes.

8. A method for promoting tomato growth, characterized in that, The step includes applying the biological agent described in any one of claims 3-5 to tomatoes.

9. A method for improving the quality of tomato fruit, characterized in that, The step includes applying the biological agent described in any one of claims 3-5 to tomatoes.

10. A method for increasing tomato yield, characterized in that, The step includes applying the biological agent described in any one of claims 3-5 to tomatoes.

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