Enterobacter strain and application thereof

Abstract

The invention provides an Enterobacter sp (Enterobacter sp) strain and an application thereof, and relates to the technical field of biocontrol microorganisms. The strain is preserved in the China Center for Type Culture Collection on November 1, 2019, and the preservation number is CCTCC NO: M 2019830. The strain has a good control effect on plant nematodes and has a remarkable growth promoting effect on plants. In addition, the method is low in cost, environment-friendly and wide in application prospect in the field of rice planting.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms and biological control, and more particularly to an Enterobacter strain for preventing and controlling soil-borne plant diseases and promoting plant growth, and an application thereof. Background Art

[0002] Plant parasitic nematodes are highly destructive soil-borne plant diseases in global agricultural production, causing enormous economic losses annually. The root-knot nematode (Meloidogyne graminicola) primarily parasitizes rice, causing spiral or hook-shaped root knots to form at the root tips, severely impacting root development. This disease is widespread in rice ecosystems across tropical and subtropical regions and has gradually spread from southern my country to northern China in recent years.

[0003] Currently, the main method for controlling soil-borne plant diseases in agricultural production relies on traditional chemical control. However, long-term use can easily lead to drug resistance and cause environmental safety issues. Therefore, the search for efficient, safe, and pollution-free green biological control methods has become an urgent need.

[0004] Biological control shows great potential for sustainable agricultural plant nematode control, and rhizosphere microorganisms are considered effective biocontrol agents. Screening beneficial microbial strains and utilizing their metabolites for biocontrol offers significant advantages over traditional control methods, both in terms of efficiency and environmental friendliness, contributing to the development of healthy and sustainable agricultural ecosystems. Providing new biocontrol strains for soilborne diseases and developing novel, more effective, and safer microbial agents to control soilborne plant diseases and improve seedling growth and stress resistance are essential for ensuring the production safety of rice and other food crops, as well as agricultural security and sustainable development. Summary of the Invention

[0005] Based on this, in order to address the technical problem that the above-mentioned chemical control methods have a negative impact on the ecological environment and human health, this application provides a biocontrol bacterium that is friendly to the ecological environment and human health and can prevent and control soil-borne diseases and promote plant growth.

[0006] The present invention solves the above-mentioned technical problems in the following ways:

[0007] The present invention provides a strain for preventing and controlling soil-borne plant disease nematodes and promoting plant growth. The strain was identified as Enterobacter sp and named CG-7. It was deposited in the China Center for Type Culture Collection on November 1, 2019, with the deposit number CCTCC NO: M2019830.

[0008] The present invention also provides a microbial agent containing the Enterobacter strain CG-7.

[0009] In some embodiments, the microbial agent includes the Enterobacter strain or a fermentation culture of the Enterobacter strain.

[0010] In some embodiments, the fermentation culture includes fermentation supernatant and bacterial cells.

[0011] The present invention also provides a method for preparing the microbial agent according to any of the above embodiments, the method comprising the following steps:

[0012] Slant seed culture: Streak Enterobacter strain CG-7 onto a slant medium to obtain slant seeds.

[0013] Liquid seed culture: Pick a single activated colony from the slant and inoculate it into the seed culture medium to obtain the seed culture liquid;

[0014] Fermentation culture: inoculate the seed culture solution into the fermentation medium at an inoculation rate of 1%-5%, and shake culture to obtain the fermentation culture;

[0015] Preparation of fermentation broth: Collect the fermentation broth from the fermentation culture and prepare it into microbial liquid or solid inoculum.

[0016] In one embodiment, the conditions for the slant seed culture are 30°C and the culture time is 24 hours; the conditions for the liquid seed culture are 180 r / min, 30°C, and the culture time is 24 hours; the conditions for the fermentation culture are 30°C, a rotation speed of 180 r / min, and a fermentation time of 48 hours; the slant culture medium, seed culture medium, and fermentation medium are all LB medium.

[0017] The present invention also provides the use of the Enterobacter strain or the microbial agent in preventing and controlling soil-borne plant nematodes.

[0018] In one embodiment, the active ingredient of the product includes at least one of the Enterobacter strain CG-7 and its fermentation culture (including fermentation supernatant and bacteria).

[0019] The present invention also provides the use of the Enterobacter strain CG-7 or the microbial agent in preparing a product for promoting plant growth and stress resistance.

[0020] In one embodiment, the active ingredient of the product includes at least one of the Enterobacter strain CG-7 and its fermentation culture (including fermentation supernatant and bacteria).

[0021] The present invention also provides the use of the Enterobacter strain CG-7 or the microbial agent in the preparation of microbial pesticides and biofertilizers.

[0022] The present invention also provides a biofertilizer containing at least one of the Enterobacter strain CG-7 and a fermentation culture of the Enterobacter strain.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. This invention provides a novel Enterobacter strain CG-7, which significantly controls plant nematodes, laying the foundation for the development of biological control for soil-borne diseases. Furthermore, treatment of seedlings with the fermentation broth of this strain significantly and consistently improves plant growth and stress resistance.

[0025] 2. The present invention provides a microbial agent containing the Enterobacter strain CG-7, as well as its preparation and use methods. This microbial agent can be used to prepare products related to controlling soil-borne plant nematodes and promoting plant growth, and is friendly to the ecological environment and human health. Both the preparation and use methods are very simple and convenient, and the production process does not use organic solvents, thereby reducing the degree of environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the colony morphology of the strain CG-7 obtained in Example 1;

[0027] Figure 2 This is the phylogenetic tree of strain CG-7 obtained in Example 1. DETAILED DESCRIPTION

[0028] The following description sets forth numerous specific details to facilitate a thorough understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific implementations disclosed below.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0030] An Enterobacter sp strain, named CG-7, was deposited in the China Center for Type Culture Collection on November 1, 2019, with the accession number CCTCCNO: M2019830.

[0031] In order to facilitate a further understanding of the present invention, the technical solution of the present invention is now described in detail in conjunction with preferred embodiments.

[0032] Example 1 Isolation and identification of Enterobacter strain CG-7

[0033] (1) Separation and purification

[0034] LB agar medium: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 20 g agar, 1000 mL deionized water, pH 7.4, sterilized at 121°C for 30 min.

[0035] The inventors collected rice rhizosphere soil samples from Changsha, Hunan Province, and after natural air drying, weighed 10 g of the sample and dissolved it in 90 mL of sterile water to prepare a soil suspension; then the sample was diluted 10 times with sterile water, and 10 -4 and 10 -5 100 μL of the soil suspension was evenly spread on an LB medium plate; the plate was placed in an incubator and cultured at 30°C for 36-48 hours, and different colonies were picked according to their morphology, size and color, and purified on new LB medium plates until pure colonies were obtained and numbered for preservation.

[0036] (2) Strain identification

[0037] After Gram staining of pure bacterial colonies, the strains were streaked, isolated, cultured, and observed by Gram staining using an optical microscope. Physiological and biochemical identification was performed with reference to the Bergey Manual of Bacterial Identification and the Manual of Identification of Common Bacterial Systems.

[0038] like Figure 1 The surface of the colonies on the culture medium is smooth, opaque, slightly yellow, and the bacteria are short rod-shaped. The Gram reaction is negative. Physiological and biochemical characteristics: positive in the catalase test, nitrate reduction test, starch hydrolysis test, and casein hydrolysis test.

[0039] (3) 16S rDNA sequence identification and phylogenetic tree comparison

[0040] DNA was extracted using a kit, and 16S rDNA was amplified by PCR using universal primers (8F 5-AGAGTTTGATCCTGCGTCAG-3' and 1492R 5'-GGTTACCTTGTTACGACTT-3') synthesized by Shanghai Sangon Biotechnology Co., Ltd. The amplified product was purified and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.

[0041] Amplification reaction system: 10× Buffer (Mg 2+ ) 2.5 μL, dNTPs 1 μL, primers 0.5 μL each, Enterobacter bacterial DNA 0.5 μL, Taq DNA polymerase 0.2 μL, add deionized water to 25 μL.

[0042] PCR reaction conditions: 94°C for 4 min, 94°C for 45 s, 55°C for 40 s, and 72°C for 60 s; 30 cycles; finally, repair and extension at 72°C for 10 min, and termination at 4°C.

[0043] The sequencing results were compared with the 16S rDNA sequences in the GenBank database through online analysis. The gene sequences of typical strains with high sequence similarity were selected as reference objects, and the neighbor-joining (NJ) method of Mega 7.0 was used to construct the phylogenetic tree. Figure 2 As shown, phylogenetic analysis was performed.

[0044] Based on the results of 16S rDNA sequence analysis and comprehensive consideration of the strain's colony morphology, physiological and biochemical characteristics, this strain was identified as an Enterobacter sp strain and numbered CG-7.

[0045] Example 2 Preparation of microbial agent

[0046] LB liquid medium: 5 g yeast extract, 10 g peptone, 10 g sodium chloride, 1000 mL deionized water, pH 7.4, sterilized at 121°C for 30 min.

[0047] (1) Slant seed culture: Enterobacter sp. strain CG-7 (hereinafter referred to as strain CG-7) was aseptically streaked onto a slant of LB agar medium and cultured at 30°C for 24 h to obtain slant seeds;

[0048] (2) Liquid seed culture: Pick a single activated colony from the slant and inoculate it into LB liquid medium for cultivation at 180 rpm, 30°C, and for 24 h to obtain seed culture solution;

[0049] (3) Fermentation: The seed culture solution was inoculated into LB liquid medium at a 5% inoculum volume, at 30°C, at a rotation speed of 180 r / min, and the fermentation time was 48 h;

[0050] (4) Preparation of fermentation broth: Collect the fermentation broth from the fermentation culture and prepare it into microbial liquid or solid inoculum.

[0051] Example 3 Detection of the activity of strain CG-7 in killing plant nematodes

[0052] 1. Preparation of fermentation supernatant: strain CG-7 was fermented according to the method of Example 2. The fermentation culture was collected after 48 hours of fermentation. The number of viable bacteria was 1×10 8cfu / ml. The fermentation broth was centrifuged at 10,000 rpm / min for 10 min, and the supernatant was filtered through a bacterial filter (0.22 μm membrane) to obtain the fermentation supernatant. The fermentation supernatant was diluted with sterile water to obtain 50%, 20%, and 10% dilutions for later use.

[0053] 2. The root-knot nematodes used in this example were second-instar larvae, isolated from diseased rice roots infected with the root-knot nematode. Specifically, the diseased rice roots were cleaned and thoroughly rinsed, and then the root knots were picked under a microscope. The roots were disinfected with 1% sodium hypochlorite for 5 minutes and rinsed three times with sterile water. Fresh, plump, and mature egg masses were picked from the diseased roots with dissecting forceps and collected by sieving to obtain an egg suspension. The egg masses were placed in a sterilized plate filled with sterile water and incubated at 28°C. After three days, the second-instar larvae were separated using the Baermann funnel method for later use.

[0054] 3. Direct toxicity of the fermentation supernatant of strain CG-7 to plant nematodes

[0055] (1) The lethality of the fermentation supernatant of strain CG-7 against the second-instar larvae (J2) of the root-knot nematode Pseudomonas graminicola

[0056] The contact killing method was used to determine the biocontrol effect of the fermentation supernatant of the strain on the root-knot nematode Pseudomonas graminicola. The details are as follows:

[0057] To each well of a 12-well plate, 2 mL of fermentation supernatant from the test strain CG-7 at varying concentrations and approximately 100 nematodes were added. Sterile H2O and LB broth were used as controls. Each treatment was replicated six times, with three independent replicates. The 12-well plates were incubated at 28°C and observed under a stereomicroscope after 24 and 48 hours. Dead and surviving nematodes were counted (observed 2 minutes after instillation of 2% NaCl solution; dead nematodes exhibited rigidity, while live nematodes exhibited curled and wriggling behavior). Adjusted mortality was calculated using the following formula.

[0058] Mortality (%) = number of dead nematodes / total number of observed nematodes × 100

[0059] Corrected mortality (%) = (treatment nematode mortality - control nematode mortality) / (1 - control nematode mortality) × 100

[0060] The experimental results are shown in Table 1

[0061] Table 1 Determination of lethal activity of fermentation supernatant against root-knot nematode J2 of Poaceae

[0062]

[0063] Note: Different letters in the table indicate significant differences (SPSS17.0t test, P≤0.05)

[0064] As shown in Table 1, the results of the indoor contact toxicity test showed that the lethality of 50%, 20%, and 10% dilutions of the fermentation supernatant of strain CG-7 for 24 hours against root-knot nematode J2 of the Poaceae family could reach 100%, which was significantly different from the blank control CK and LB culture medium treatments.

[0065] The results showed that the fermentation supernatant of strain CG-7 had significant nematicidal activity against the root-knot nematode J2 of the Poaceae family.

[0066] (2) Inhibition rate of CG-7 fermentation supernatant on the hatching of root-knot nematode eggs

[0067] The egg hatching inhibition assay was performed in sterile 12-well cell culture plates. 2 mL of 50% CG-7 fermentation supernatant and 100 eggs were added to each well. Controls were treated with HO and LB medium. Each treatment was replicated six times, with three independent replicates. The 12-well plates were incubated at 28°C. Nematode hatching rates were observed under a stereomicroscope at 24, 48, 72, and 96 hours.

[0068] Note: Hatching rate (%) = (number of hatched J2 / total number of eggs) × 100

[0069] Hatching inhibition rate (%) = (1-hatching rate of treatment group / hatching rate of control group) × 100

[0070] The experimental results are shown in Table 2.

[0071] Table 2 Effects of strain CG-7 on the hatching rate of root-knot nematode eggs in the Poaceae family

[0072]

[0073] Note: Different letters in the table indicate significant differences (SPSS17.0t test, P≤0.05)

[0074] As shown in Table 2, the hatching test results show that the hatching inhibition rate of 50% dilution of the fermentation supernatant of strain CG-7 on root-knot nematode eggs reached its highest level at 50.9% at 72 hours, significantly higher than that of the LB medium control (P < 0.05). Strain CG-7 inhibited egg hatching and had a significant inhibitory effect on nematode development during the egg stage.

[0075] (3) Fumigation effect of the fermentation supernatant of strain CG-7 on plant nematodes

[0076] The fumigation effect of the fermentation supernatant of strain CG-7 on root-knot nematodes of the Poaceae family was tested by the sterile double-septum plate method. Specifically:

[0077] 2 ml of CG-7 fermentation supernatant at varying concentrations (50%, 20%, and 10%) was added to the first compartment of a two-divided culture dish. 200 root-knot nematodes (Meloidogyne graminicola) were added to the second compartment of the dish. In the control group, the first compartment contained sterile water and LB medium, respectively. Each treatment was replicated four times, with three independent replicates. The dishes were sealed with double-layer parafilm and incubated in a 28°C incubator. The number of dead nematodes and their condition were observed and recorded every 24 hours. Nematode mortality was recorded at 24 and 48 hours.

[0078] The results are shown in Table 3.

[0079] Table 3. Nematicidal activity of VOCs in the fermentation supernatant of strain CG-7

[0080]

[0081] Note: Different letters in the table indicate significant differences (SPSS17.0t test, P≤0.05)

[0082] As shown in Table 3, the results showed that the volatile nematicidal active substances were present in the CG-7 supernatant. The lethality rate of 10% CG-7 supernatant dilution in 48 hours could reach 73.17%, indicating significant fumigation nematicidal activity.

[0083] The above results indicate that Enterobacter sp. strain CG-7 controls nematodes mainly by producing active fermentation products, and has a significant toxic effect on root-knot nematodes of the Poaceae family.

[0084] Example 4 Effects of spraying strain CG-7 at the seedling stage on root binding capacity and growth of rice seedlings

[0085] The strain CG-7 was fermented according to the method of Example 2, and the fermentation culture was collected after 48 hours of fermentation. The number of viable bacteria was 1×10 8 cfu / ml. Rice seedlings were raised in trays using a matrix. Whitened seeds were grown in the matrix. Three days before transplanting, the rice seedbed was sprayed with 50 L / mu of fermented liquid of the biocontrol bacteria CG-7. The control group CK was sprayed with 50 L / mu of clean water. Each treatment was repeated 4 times and three independent experiments were conducted. Conventional seedling raising methods were used for seedling raising. The perforated seedling trays were 60 cm × 25 cm × 3 cm in size. About 100 g of wet rice seeds were evenly sown in each tray and cultured in a greenhouse at a temperature of 25-30°C, a photoperiod of 14 h light / 10 h dark, and a relative humidity of 70%-90%.

[0086] On the day of transplanting, 5 rice seedlings were randomly selected from each seedling tray. The number of root tips, root length, plant height, maximum leaf width, stem base width, root weight, fresh weight, fresh weight of 100 seedlings and chlorophyll SPAD content of each plant were recorded. After the seedlings were taken out of the whole tray, a 10cm×10cm sample area was cut out, and the number of seedlings in the sample area was washed and counted. At the same time, the root plate binding force was measured: a 10cm×10cm seedling block was fixed at one end, and the other end was hooked with a spring dynamometer, and pulled horizontally. When the seedling block broke, the maximum value displayed on the spring scale was the root plate binding force value. Each treatment was repeated 3 times. Calculation formula:

[0087] Seedling rate = (number of seedlings / total number of seeds sown) × 100%

[0088] Seedling density = total number of seedlings / cultivation area (plants / cm 2 )

[0089] The results are shown in Tables 4 and 5.

[0090] Table 4 Effects of spraying the fermentation liquid of strain CG-7 on the root characteristics and seedling rate of seedlings

[0091]

[0092] Note: Different letters in the table indicate significant differences (SPSS17.0t test, P≤0.05)

[0093] As shown in Table 4, the root-binding capacity of the group treated with the fermentation liquid of strain CG-7 increased by 44.5% compared to the CK, the maximum leaf width increased by 14.0%, and the stem base width increased by 20.5%. The chlorophyll SPAD value reached 29.69, and the stem base width reached 3.33 mm. This indicates that spraying the fermentation liquid of strain CG-7 on the roots of rice seedbeds significantly promoted rice growth, increased seedling establishment rate, and increased root-binding capacity.

[0094] As shown in Table 5, the fresh weight of the treated group increased by 53.9% and the dry weight increased by 56.3% compared with the CK. In summary, it can be seen that the fermentation liquid of the biocontrol fungus CG-7 significantly improved the growth of rice seedlings.

[0095] Table 5 Effects of the fermentation liquid of strain CG-7 on the quality of rice seedlings

[0096]

[0097]

[0098] Note: Different letters in the table indicate significant differences (SPSS17.0t test, P≤0.05)

[0099] Example 5 Effect of spraying strain CG-7 at the seedling stage on rice planting quality

[0100] On the day after planting, the survey was conducted in the middle 6 rows of each plot, with 10 consecutive holes in each row, totaling 60 holes. The number of missed holes, floating seedlings, injured seedlings, and turned seedlings was recorded, and the missed planting rate, injured seedling rate, floating seedling rate, and turned seedling rate were calculated.

[0101] The results are shown in Table 6.

[0102] Table 6 Effects of spraying the fermentation liquid of strain CG-7 on the quality of rice seedlings during transplanting

[0103] deal with Missed insertion rate (%) Damaged seedling rate (%) Floating rice seedling rate (%) Rice seedling turning rate (%) CK 5.5±1.91a 7.73±1.96a 7.93±1.66a 7.76±3.86a CG-7 2.2±1.51b 4.40±1.91b 4.30±1.61b 4.80±1.51b

[0104] As shown in Table 6, compared with CK, the missing transplanting rate, seedling injury rate, seedling floating rate and seedling turning rate of the CG-7 treatment group were significantly reduced. Spraying the CG-7 strain at the seedling stage significantly improved the quality of rice transplanting.

[0105] Example 6: Potted control effect of strain CG-7 on rice root-knot nematodes

[0106] Greenhouse potting experiments were conducted in the greenhouse of the Plant Nematode Research Laboratory at Hunan Agricultural University. Soil from the cultivated layer of the experimental base was dried in an oven at 160°C for 2 hours to obtain sterile soil. Rice seedlings were raised in the same substrate as in Example 4. After reaching 25 days of age, the seedlings were transplanted into perforated flower pots (11 cm in diameter and 10 cm in height). 500 g of sterile soil was placed in each pot, and five uniformly sized seedlings were transplanted (per pot). The seedlings were then cultured in a greenhouse at 25-30°C, with a photoperiod of 14 h / 10 h and a humidity of 70-90%.

[0107] The number of viable bacteria in the fermentation broth of strain CG-7 was 1.0×10 8 CFU / mL, according to Example 4, during the matrix seedling raising period, bacterial liquid spraying (50L / mu) and CK clear water spraying (50L / mu). This test sets 3 treatments, namely, bacterial strain CG-7 fermentation liquid seedling raising, CK (clear water seedling raising) and 33.36mg / L of fluopyram (spraying treatment two days after transplanting, 30mL is used for every pot), four repetitions are set, and three independent experiments are performed. After transplanting 48h, 200 root-knot nematodes (J2) of the gramineous family are inoculated around the root system of each rice plant, and 1000 J2 are inoculated for every pot. After inoculation of the gramineous family root-knot nematodes for 30d, the number of rice root knots and the root knot index are investigated, and the control effect is calculated. At the same time, five points of pot soil are sampled, and the number of nematodes and eggs in every 100g soil is counted by sucrose centrifugation, and the decline rate of second-instar larvae and eggs is calculated.

[0108] Root knot index = ∑ (values ​​at each level × number of plants at each level) × 100 / highest level value × number of surveyed plants.

[0109] Control effect (%) = (root knot index of control - root knot index of treatment) × 100 / root knot index of control.

[0110] The results are shown in Table 7.

[0111] Table 7 Control effect of strain CG-7 fermentation liquid on root knot nematode disease

[0112]

[0113] Note: Different letters in the table indicate significant differences (SPSS17.0t test, P≤0.05)

[0114] As shown in Table 7, the results from rice potted plants show that treatment with the fermentation broth of strain CG-7 has a significant control effect on root-knot nematodes of the Poaceae family. Compared to the blank control, treatment with the fermentation broth of strain CG-7 significantly reduced the number of root knots in rice. The root knot index was not significantly different from the control, fluopyram treatment. The control efficacy reached 56.96% after seedling treatment with the fermentation broth. The J2 reduction rate in the rhizosphere soil of rice seedlings treated with the fermentation broth of strain CG-7 reached 62.39%, which was not significantly different from the control, fluopyram treatment at 33.36 mg / L. The corresponding egg reduction rate was 72.24%, demonstrating significant reduction in the number of second-instar larvae and eggs of root-knot nematodes of the Poaceae family in the rhizosphere soil.

[0115] Example 7 Field Control of Rice Root-Knot Nematodes by Fermentation Broth of Strain CG-7

[0116] The experimental site was located in Huanghualing Village, Wushan Subdistrict, Wangcheng District, Changsha City, Hunan Province (E 112°43′, N 28°22′), a paddy field severely infested with root-knot nematodes. The soil nematode population was 987.5 per 100 mL of soil. The rice variety was Hanyou 73, and the rice was transplanted using a factory-based centralized seedling raising and machine transplanting system. The plot area was 50 m2. 2 The number of viable cells in the fermentation broth of strain CG-7 was 1.0×10 8 CFU, according to Example 4, during the matrix seedling period, the bacterial liquid was sprayed (50L / mu). Set the CG-7 bacterial liquid seedling treatment, set the concentration of 33.36mg / L of fluopyram as the agent control, and spray the blank control group with clean water. Each treatment was repeated in 4 plots, arranged in random blocks, and the fertilization and pest control were consistent with other local farmlands. Sampling survey of the growth status of rice seedlings at 60d (growing period), according to the five-point sampling method, 5 rice plants were taken at each point, and 25 plants were inspected in each plot. The number of rice root knots, root knot index and control effect were counted. At the same time, 1L of soil was taken from the rice rhizosphere soil (0-20cm) after mixing evenly. After indoor sieving, the second-instar larvae and eggs were separated and counted by sucrose centrifugation.

[0117] The results are shown in Table 8.

[0118] As shown in Table 8, field trial results show that the number of root knots in rice seedlings treated with the fermentation solution of strain CG-7 was significantly lower than that in the blank control. Treatment with the fermentation solution of strain CG-7 at the seedling stage achieved a 65.52% control efficacy against root-knot nematodes, demonstrating significant control effectiveness. These results demonstrate that the strain enhances the stress resistance of seedling rice and maintains a significant control effect against root-knot nematodes.

[0119] Table 8. Field control effect of strain CG-7 fermentation liquid on root-knot nematodes of Poaceae

[0120]

[0121]

[0122] Note: Different letters in the table indicate significant differences (SPSS17.0t test, P≤0.05)

[0123] Example 8 Effect of the fermentation liquid of strain CG-7 on the growth promotion and yield increase of rice in the field

[0124] The experiment was conducted in the rice fields of Huanghualing Village, Wushan Street, Wangcheng District, Changsha City, Hunan Province. The rice variety was Hanyou 73. The factory-based centralized seedling raising machine transplanting model was adopted. The plot area was 50m 2 , each treatment was repeated 4 times, and the fertilization and pest and disease control were consistent with other local farmlands. The same CG-7 bacterial solution seedling treatment (50L / mu) was set as in Example 6, and a concentration of 33.36mg / L of fluopyram was set as the drug control, and the blank control group (clear water) was used. Sampling was taken 60 days after sowing to check the growth status of rice seedlings. Five rice plants were taken at each point in each plot according to the five-point sampling method, and a total of 25 rice plants were checked for plant height, root length, fresh weight, root weight, chlorophyll SPAD and stem base width. 1m 2 The number of rice holes in the sample plot was investigated; the seeds were sown indoors to measure the number of effective panicles, number of grains, number of fruits and thousand-grain weight, and the theoretical yield and yield increase rate were calculated.

[0125] Theoretical yield (kg / mu) = (number of effective ears per square meter × number of grains per ear × thousand-grain weight × fruit setting rate × 667m 2 / mu) / (1000 grains);

[0126] Yield increase rate = (theoretical yield of treatment group - theoretical yield of control group) / theoretical yield of control group × 100%.

[0127] The results are shown in Tables 9 and 10.

[0128] As shown in Table 9, the results of the examination of the growth status of rice seedlings treated with the fermentation liquid of strain CG-7 showed that the fresh weight of rice treated with the fermentation liquid of strain CG-7 at the seedling stage was significantly higher than that of the blank control during the subsequent growth period. The rice plant height, root weight, and stem base width were also higher than those of the control treatment, indicating that it has a significant growth-promoting effect on rice plants.

[0129] Table 9 Effects of strain CG-7 treatment on rice seedling growth

[0130]

[0131] Note: Different letters in the table indicate significant differences (SPSS17.0t test, P≤0.05)

[0132] As shown in Table 10, the statistical results of mature rice yield indicate that treatment with the fermentation solution of strain CG-7 during the seedling stage significantly increased the rice seed set rate at harvest, boosting rice yield. The seed set rate of rice treated with strain CG-7 reached 83.83%, and the rice yield reached 303.85 kg / mu, a yield growth rate of 45.64%. Furthermore, treatment with the fermentation solution of strain CG-7 also significantly increased the dry weight, 1000-grain weight, and number of effective panicles. These results demonstrate that treatment with the fermentation solution of strain CG-7 during seedling cultivation can consistently promote rice growth and significantly increase rice yield.

[0133] Table 10 Effects of strain CG-7 treatment on rice yield and its components

[0134]

[0135] Note: Different letters in the table indicate significant differences (SPSS17.0t test, P≤0.05)

[0136] Example 9: Induction of Rice Resistance by Fermentation Broth of CG-7 Strain

[0137] The greenhouse pot experiment was conducted in the same manner as in Example 5, with three treatments: (1) sterile water (CK), (2) sterile water treatment and inoculation with J2 root-knot nematodes (J2), and (3) CG-7 seedling treatment and inoculation with J2 root-knot nematodes (CG-7+J2). The activities of defense-related enzymes (including phenylalanine ammonia lyase (PAL) and peroxidase (POD)) in rice plants were measured 2, 4, 6, and 8 days after J2 nematode infestation. The concentrations of PAL and POD enzymes in rice roots treated with CG-7 bacterial solution under root-knot nematode stress are shown in Tables 11 and 12.

[0138] Table 11 Effects of strain CG-7 treatment at seedling stage on rice PAL enzyme

[0139] deal with 2dpi 4dpi 6dpi 8dpi CK 20.01±4.41c 18.08±4.27c 20.87±3.34c 17.59±2.49c J2 35.28±2.63b 29.57±2.57b 25.91±2.35b 22.06±1.97b CG-7+J2 99.95±7.87a 76.41±3.51a 61.95±5.92a 46.69±4.99a

[0140] Note: Different letters in the table indicate significant differences (SPSS17.0t test, P≤0.05)

[0141] Compared to the nematode control (J2), the application of the fermented liquid of the CG-7 strain under nematode stress significantly increased PAL enzyme concentrations in rice roots (Table 11). Following application of the CG-7 liquid, POD activity in rice roots gradually increased, reaching a peak on day 4, then slowly decreased and stabilized. Compared to both the negative control (CK) and the nematode group (J2), POD values ​​in the roots of plants treated with the CG-7 liquid were significantly increased (Table 12).

[0142] Table 12 Effects of strain CG-7 treatment at seedling stage on rice POD enzyme

[0143] deal with 2dpi 4dpi 6dpi 8dpi CK 47.31±8.01c 51.36±8.99c 47.64±5.31c 48.35±4.97c J2 81.72±3.85b 93.51±9.38b 73.03±4.95b 55.33±6.78b CG-7+J2 170.71±9.83a 272.73±11.73a 180.58±9.25a 145.62±10.58a

[0144] Note: Different letters in the table indicate significant differences (SPSS17.0t test, P≤0.05)

[0145] In summary, the results show that after the fermentation liquid of strain CG-7 is treated as rice seedlings, it will activate the activity of defense enzymes in the host plant, causing the rice to develop sustained defense resistance to plant nematodes.

[0146] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0147] The above-described embodiments merely illustrate several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that a person skilled in the art would be able to make numerous modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. An Enterobacter sp strain, deposited in the China Center for Type Culture Collection on November 1, 2019, with the accession number CCTCC NO: M2019830.

2. A microbial agent containing the Enterobacter strain according to claim 1.

3. The microbial agent according to claim 2, characterized in that The microbial agent includes the Enterobacter strain or a fermentation culture of the Enterobacter strain.

4. The microbial agent according to claim 3, characterized in that The fermentation culture includes fermentation supernatant and bacterial cells.

5. The method for preparing the microbial agent according to any one of claims 2 to 4, characterized in that: The following steps are involved: Slant seed culture: Streak the Enterobacter strain onto a slant medium to obtain slant seeds; Liquid seed culture: Pick a single activated colony from the slant and inoculate it into the seed culture medium to obtain the seed culture solution; Fermentation culture: inoculate the seed culture solution into the fermentation medium at an inoculation rate of 1%-5%, and shake culture to obtain the fermentation culture; Preparation of fermentation broth: Collect the fermentation broth from the fermentation culture and prepare it into microbial liquid or solid inoculum.

6. The method for preparing the microbial agent according to claim 5, characterized in that: The conditions for the slant seed culture are 30°C and the culture time is 24 hours; the conditions for the liquid seed culture are 180 r / min, 30°C, and the culture time is 24 hours; the conditions for the fermentation culture are 30°C, a rotation speed of 180 r / min, and a fermentation time of 48 hours; the slant culture medium, seed culture medium, and fermentation culture medium are all LB culture medium.

7. Use of the Enterobacter strain according to claim 1 or the microbial agent according to any one of claims 2 to 4 in rice seedling cultivation.

8. Use of the Enterobacter strain according to claim 1 or the microbial agent according to any one of claims 2 to 4 in controlling soil-borne plant nematodes.

9. Use of the Enterobacter strain according to claim 1 or the microbial agent according to any one of claims 2 to 4 in the preparation of a product for promoting plant growth and stress resistance.

10. Use of the Enterobacter strain according to claim 1 or the microbial agent according to any one of claims 2 to 4 in the preparation of microbial pesticides or biofertilizers.

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