Empedobacter faecalis GXUN3754 and application thereof in prevention and treatment of elephant trunk root-knot nematode

Abstract

The invention discloses Empedobacter faecalis GXUN3754 and an application thereof in prevention and treatment of root-knot nematode of Penniospermum erythrorhizon, and belongs to the technical field of microorganism application. The Empedobacter faecalis GXUN3754 provided by the invention is preserved in the China General Microbiological Culture Collection Center on February 27, 2024, and the preservation number of the Empedobacter faecalis GXUN3754 is CGMCC (China General Microbiological Culture Collection Center) No. 29873. Experiments show that the Empedobacter faecalis GXUN3754 has the capabilities of producing siderophore, NH3, protease and phytase, and the Empedobacter faecalis GXUN3754 bacterial liquid and volatile substances have the effect of promoting plant growth, and also have the capability of killing the root-knot nematode of Penicillium spp. Therefore, the Empedobacter faecalis GXUN3754 has very important significance in research and development of novel biological preparations and plant growth promoters capable of efficiently killing the Pennisetum alopecuroides root-knot nematode.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, and in particular to a strain of Bacillus faecalis GXUN3754 and application thereof in preventing and controlling root-knot nematodes of elephant ear beans. Background Art

[0002] Meloidogyne enterolobii was first discovered in 1983, when Yang et al. found a root-knot nematode on the root knot of Enterolobium cyclocarpum trees in Hainan, China. Based on morphological characteristics, it was identified as a new species of Meloidogyne enterolobii. In 1988, Rammah found a root-knot nematode on the root knot of eggplant and named it Meloidogyme mayaguensis. Through the analysis of the esterase phenotype, mtDNA COⅡ and lrRNA of the two nematodes, as well as the analysis and description of the slides of the model species, it is believed that the Mayaguensis should be a synonym of the elephant ear bean root-knot nematode.

[0003] The root-knot nematode of elephant ear bean is an important pathogenic nematode in tropical and subtropical regions around the world. It has strong pathogenicity and reproductive potential. Its host distribution range is wide, mainly in Leguminosae, Cucurbitaceae, Solanaceae, etc. It can also parasitize on crops such as pepper (N gene) and tomato (Mi-1 gene) carrying resistance genes for reproduction, often causing serious losses in vegetable crops or fruit tree yields. At present, the prevention and control of the root-knot nematode of elephant ear bean mainly relies on chemical drugs. Chemical methods are efficient and convenient, and have good prevention effects. However, the excessive use of chemical pesticides is likely to cause environmental pollution. The accumulation of pesticides will destroy the balance of soil ecology and even endanger human health. It is urgent to promote green and environmentally friendly prevention and control measures.

[0004] In recent years, biological control has gradually become the focus of current research because of its advantages such as green environmental protection, cost saving, non-toxicity and harmlessness. Biocontrol strains can be obtained from plant rhizosphere, soil and animals and plants. They usually have the functions of dissolving inorganic phosphorus, organic phosphorus and fixing nitrogen. At the same time, the secreted extracellular enzymes can also resist the infection of pathogenic fungi by destroying the fungal cell wall. In 2015, Thorsten isolated a strain of Empedobacterstercoris SCVM0123 for the first time from samples of a biogas plant and determined its taxonomic status. In 2020, Cheng et al. screened a strain of Empedobacterstercoris ES183 with a Tet variant gene from feces samples from 6 livestock farms. Strain ES183 has a new tigecycline resistance gene Tet (X14), and predicted the homology relationship of Tet (X14) through a structural model. At present, there are still few reports on Empedobacterstercoris, and there has been no in-depth research on its biocontrol function. However, Empedobacter brevis, which belongs to the same genus as Bacillus faecalis, is a widely used microbial pesticide in recent years. Empedobacter brevis can enter the insect body through insect feeding and multiply in large numbers, producing protein toxins to interfere with the metabolism of the insect body, thereby killing the insect. It is usually used to control various biological pests such as rice leaf roller, Spodoptera litura, tea looper, cauliflower worm, and maca diamondback moth.

[0005] At present, there are few reports on the control of the ear bean root knot nematode by biocontrol bacteria in China, and there is no research on the application of Bacillus faecalis in biological control. Due to the diversity of biocontrol bacteria and its green and pollution-free characteristics, it is in line with the sustainable development path of green new agriculture. The functional bacterial agents made from it can help crops resist pests and diseases and increase crop yields. Therefore, screening and studying biocontrol bacteria with toxic activity against ear bean root knot nematodes has good research significance and broad agricultural application prospects. Summary of the invention

[0006] The purpose of the present invention is to provide a strain of Bacillus faecalis GXUN3754 and its application in preventing and controlling root-knot nematodes of elephant ear beans, so as to solve the problems existing in the above-mentioned prior art. The Bacillus faecalis GXUN3754 and its bacterial agent provided by the present invention can effectively kill the root-knot nematodes of elephant ear beans and have a growth-promoting effect on plants.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a strain of Empedobacter stercoris GXUN3754, which was deposited in the China General Microorganism Collection Center on February 27, 2024, with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 29873.

[0009] The present invention also provides a bacterial agent containing the Bacillus faecalis GXUN3754, and the preparation method of the bacterial agent comprises the following steps:

[0010] The faecalis GXUN3754 was inoculated into LB medium and cultured with shaking until OD 600nm is 1.0, that is.

[0011] The present invention also provides a sterile fermentation filtrate prepared according to the Stibacillus faecalis GXUN3754, and the preparation method of the sterile fermentation filtrate comprises the following steps:

[0012] The faecalis GXUN3754 was inoculated into LB medium and cultured with shaking until the bacterial solution OD 600nm The concentration of 1.0 was adjusted, and the supernatant was taken and filtered using a 0.22 μm filter membrane.

[0013] The present invention also provides the use of the Bacillus faecalis GXUN3754 or the bacterial agent or the sterile fermentation filtrate in preparing a biological preparation for preventing and controlling the root-knot nematode of elephant ear bean.

[0014] The present invention also provides a biological preparation for preventing and treating root-knot nematodes of elephant ear beans, wherein the effective ingredients include the faecalis GXUN3754 or the bacterial agent or the sterile fermentation filtrate.

[0015] The present invention also provides the use of the Bacillus faecalis GXUN3754 or the bacterial agent or the sterile fermentation filtrate or the biological agent in preventing and controlling the root-knot nematode of elephant ear bean.

[0016] The present invention also provides a method for preventing and controlling root-knot nematodes of elephant ear beans, comprising the step of applying the faecalis GXUN3754 or the bacterial agent or the sterile fermentation filtrate or the biological agent to the rhizosphere soil of plants.

[0017] The present invention also provides the use of the Bacillus faecalis GXUN3754 or the bacterial agent in any of the following:

[0018] (1) Application in promoting plant growth;

[0019] (2) Application in the preparation of microbial preparations for promoting plant growth.

[0020] The present invention also provides a microbial preparation for promoting plant growth, wherein the effective ingredients include the Stibacillus faecalis GXUN3754 or the bacterial agent.

[0021] The present invention also provides a bacterial fertilizer comprising the microbial preparation, and also comprising organic fertilizer and / or chemical fertilizer commonly used in agriculture.

[0022] The present invention discloses the following technical effects:

[0023] The present invention provides a strain of Bacillus faecium GXUN3754, whose volatile gas substances can be used to efficiently kill the root-knot nematode of elephant ear bean, and has a good growth-promoting effect on Arabidopsis thaliana. In addition, the Bacillus faecium GXUN3754 and its metabolites also have a good lethal effect on the root-knot nematode of ear bean, and can significantly prevent and control nematode infection of crops. Therefore, Bacillus faecium GXUN3754 is of great significance for the research and development of new and efficient biological agents for killing the root-knot nematode of elephant ear bean. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is the colony morphology of Bacillus faecalis GXUN3754;

[0026] Figure 2 This is the result of Gram staining of Staphylococcus faecalis GXUN3754 under optical microscope (100× oil objective);

[0027] Figure 3 This is the phylogenetic tree of Bacillus faecalis GXUN3754 based on the 16S rRNA gene sequence;

[0028] Figure 4 This is a graph showing the results of the detection of antibacterial extracellular enzymes and growth-promoting characteristics of Bacillus faecalis GXUN3754; A is protease, B is phytase, C is siderophore production, D is ammonia production, CK is the control group, E is cellulase, and F is amylase;

[0029] Figure 5 This is an observation diagram of the promotion of water spinach seed germination by Bacillus faecalis GXUN3754; A is the germination of seeds soaked in blank medium for 30 minutes; B is the germination of seeds soaked in Bacillus faecalis GXUN3754 for 30 minutes;

[0030] Figure 6 This is an observation diagram of the growth promotion of Arabidopsis thaliana by volatile substances of Bacillus faecalis GXUN3754; A is the blank culture medium treatment, and B is the Bacillus faecalis GXUN3754 treatment;

[0031] Figure 7 The microscopic observation diagram of the insecticidal effect of Bacillus faecalis GXUN3754 on the root-knot nematode of Elephant's ear bean; wherein A is the body state of the root-knot nematode of Elephant's ear bean under the treatment of blank culture medium; B is the body state of the root-knot nematode of Elephant's ear bean after being treated with Bacillus faecalis GXUN3754;

[0032] Figure 8 The microscopic observation diagram of the insecticidal effect of the volatile substances of Bacillus faecalis GXUN3754 on the root-knot nematode of Elephanthus chinensis, wherein A is the state of the worm body of the root-knot nematode of Elephanthus chinensis after being treated with the blank culture medium; B is the state of the worm body of the root-knot nematode of Elephanthus chinensis after being treated with the volatile gas of Bacillus faecalis GXUN3754;

[0033] Fig. 9 The microscopic observation diagram of the insecticidal effect of the sterile fermentation filtrate of Bacillus faecalis GXUN3754 on the root-knot nematode of Elephant's ear, wherein: A is the body state of the root-knot nematode of Elephant's ear under the treatment of blank culture medium; B is the body state of the root-knot nematode of Elephant's ear after being treated with the sterile fermentation filtrate of Bacillus faecalis GXUN3754;

[0034] Fig.10 Figure 3 shows the control effect of Bacillus faecalis GXUN3754 on the root-knot nematode of Elephanthus chinensis potted plants. A is the root system of the negative control plant, (a) and (b) are typical areas and their magnified images; B is the root system of the positive control plant (treated with the chemical pesticide fluopyram), (c) and (d) are typical areas and their magnified images; C is the root system of the plant treated with Bacillus faecalis GXUN375, (e) and (f) are typical areas and their magnified images; the red dots are the root knots formed by the root-knot nematode of Elephanthus chinensis. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] The experimental materials used in the examples of the present invention: LB solid medium, MS solid medium, protease detection medium, amylase detection medium, cellulase detection medium, Axubei nitrogen fixation medium, peptone water medium, Nessler reagent, calcium phytate medium, PKO inorganic phosphate medium, siderophore detection medium, L-tryptophan, Salkowski colorimetric solution, IAA standard and the elephant ear bean root knot nematode used are all from the Guangxi Zhuang Autonomous Region Key Laboratory of Polysaccharide Materials and Modification.

[0041] Example 1 Isolation of strain GXUN3754

[0042] Plant rhizosphere soil was collected from Qingshantou, Tieshan Port District, Beihai City, Guangxi Zhuang Autonomous Region (109°25' east longitude, 21°36' north latitude) for bacterial isolation. The specific method is as follows: 5 g of soil sample was weighed and added to 50 mL of sterile water. After shaking at 30°C and 180 r / min for 4 h, the soil sample was diluted to 10 -3 , 10 -4 , 10 -5 100 μL of soil suspension was taken and evenly spread on LB solid culture medium, inverted and cultured at 30°C. After 24 hours, single colonies of different sizes and morphologies were picked out and streaked on LB solid plates for purification. The purified strains were inoculated on LB solid plates and streaked for later use.

[0043] Example 2 Identification of strain GXUN3754

[0044] 1. Observation of strain morphology

[0045] The strain GXUN3754 obtained in Example 1 was streaked and inoculated into LB solid medium and cultured at 30° C. for 24 h, and the morphological characteristics of the single colony such as color, size, transparency, etc. A single colony was selected and Gram-stained, and the strain morphology was observed under an optical microscope.

[0046] The results of morphological observation of strain GXUN3754 are shown in Figure 1 The results showed that the strain was yellow on LB solid medium, with a smooth, creamy surface and ridges. The results of Gram staining are shown in Figure 2 , the bacteria are rod-shaped under an optical microscope and the Gram staining test is positive.

[0047] 2. Physiological and biochemical characteristics

[0048] With reference to the Manual of Identification of Common Bacterial Systems and the Bergey's Manual of Identification of Bacteria, the physiological and biochemical indicators of strain GXUN3754, such as oxidase, starch hydrolysis, gelatin liquefaction, and methyl red, were identified.

[0049] The results of physiological and biochemical characteristics analysis are shown in Table 1. The results showed that the strain was an aerobic bacterium, with negative results in oxidase test, methyl red test, citrate utilization test and VP test, and positive results in gelatin hydrolysis and malonate utilization test.

[0050] Table 1 Results of physiological and biochemical characteristics analysis

[0051]

[0052] 3. Molecular Biological Identification

[0053] After activating strain GXUN3754, total DNA was extracted and used as a template to generate the following PCR products using bacterial universal primers:

[0054] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1);

[0055] 1492R: 5'-TACGGCTACCTTGTTACGAGTT-3' (SEQ ID NO. 2);

[0056] The S rRNA gene of strain GXUN375416 was amplified by PCR.

[0057] PCR amplification system (50 μL): template 1 μL, ddH 2 O 22μL, 2×Taq PCR Mix 25μL, forward primer 1μL, reverse primer 1μL.

[0058] PCR amplification program: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 2 min, 32 cycles; extension at 72°C for 10 min; storage at 4°C.

[0059] The amplified products were sent to Aoke Biotechnology (Wuhan) Co., Ltd. for sequencing, and the sequences were compared with those in the NCBI database for homology, and the phylogenetic tree was constructed using MEGA 11.0 software.

[0060] The results of molecular biological identification are as follows Figure 3 As shown, the 16S rRNA gene sequence of strain GXUN3754 was analyzed for homology with the existing data in the NCBI database and a phylogenetic tree was constructed. It was found that the 16S rRNA sequence of strain GXUN3754 was 99.65% similar to that of Empedobacter stercoris SCVM0123, and the phylogenetic tree was clustered in the same branch.

[0061] Based on the morphological characteristics, physiological and biochemical properties and molecular biological identification results of the strain, it was determined that the strain was Emp edobacter stercoris. The inventor has deposited the strain GXUN3754 in the China General Microbiological Collection Center on February 27, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 29873.

[0062] Example 3 Determination of enzyme production capacity of Bacillus faecalis GXUN3754

[0063] 5 μL of the fermentation broth of Bacillus faecalis GXUN3754 (OD 600 ≈1.0) in the center of protease detection medium, amylase detection medium and cellulase detection medium, and culture at 30℃ for 3d. The cellulase detection medium was stained with 1mol / L Congo red for 30min and then decolorized with 1mol / L NaCl solution for 30min. The amylase detection medium was developed with iodine solution.

[0064] Ammonia production: Take 50 μL of Bacillus faecalis GXUN3754 bacterial solution (OD 600 ≈1.0) was inoculated into a test tube containing 4 mL of peptone water culture medium and cultured at 30°C for 48 h. Then, 0.5 mL of Nessler reagent was added. The color of the solution changed from yellow to reddish brown and a precipitate was produced, indicating that it had the ability to produce NH 3 ability.

[0065] Phosphate dissolution: Take 5 μL of Bacillus faecalis GXUN3754 bacterial solution (OD 600≈1.0), and were inoculated in the center of Montgena medium and PKO inorganic phosphorus medium, respectively. After culturing at 30℃ for 5 days, if a transparent zone was produced around the strain, it indicated that it had the ability to degrade organic or inorganic phosphorus.

[0066] Production of siderophore: Inoculate the GXUN3754 strain of Bacillus faecalis in the center of the siderophore detection culture medium and culture it at 30°C for 7 days. If an orange-yellow halo is produced around the strain, it indicates that it has the ability to produce siderophores. The siderophore production ability of the antagonistic strain can be judged based on the size of the halo.

[0067] The qualitative test results of the growth-promoting function and enzyme-producing characteristics of Bacillus faecalis GXUN3754 are as follows Figure 4 As shown in the figure, GXUN3754 produced an orange-yellow halo on the CAS test medium. After adding Nessler reagent, the peptone water medium turned reddish brown and precipitated. Transparent circles were produced on the protease test medium and the phytase test medium, but no transparent circles were produced on the cellulase test medium and the amylase test medium, indicating that GXUN3754 has the ability to produce siderophore and NH 3 It has the ability to produce protease and phytase, but does not have the ability to produce amylase and cellulase.

[0068] Example 4 Growth-promoting effect of Bacillus faecalis GXUN3754

[0069] 1. Growth-promoting effect of Bacillus faecalis GXUN3754 on water spinach seeds

[0070] The seeds were disinfected on the surface. The bad seeds were picked out by soaking in sterile water for 1 hour, washed once with 75% ethanol for 1 minute, washed twice with 1% sodium hypochlorite for 3 minutes, and washed 3-5 times with sterile water. The faecal steatosis GXUN3754 was inoculated into 50 mL LB medium at 2% (V / V) and cultured at 30°C and 180 rpm for 24 hours until the OD 600nm About 1.0 for standby use. Take LB blank medium as control, put in 25 surface-sterilized water spinach seeds, soak in bacterial solution for 1 hour. Put water spinach seeds in a sterile culture dish covered with moist filter paper, at 30℃, 16:8h (light culture: dark culture), observe germination rate and sprout length after 5 days.

[0071] The calculation formula for germination rate is as follows:

[0072] Germination rate (%) = number of germinated seeds / number of tested seeds × 100%;

[0073] The growth-promoting effect of strain GXUN3754 on water spinach seeds Figure 5As shown, it can be found that the germination rate of the control group is 68%, and the germination rate of the water spinach seeds treated with GXUN3754 is 88%, and the length of the water spinach seeds germinated 5 days later in the treatment group is significantly improved compared with the control group.

[0074] 2. Growth-promoting effect of volatile substances from Bacillus faecalis GXUN3754 on Arabidopsis thaliana

[0075] The faecalis GXUN3754 was inoculated into 50 mL LB medium at 2% (V / V) and cultured at 30°C with shaking at 180 rpm for 24 h until the OD 600 nm is about 1.0 for later use. Arabidopsis thaliana was vernalized at 4°C for 72 hours, then planted on MS solid culture medium and cultured for 4-5 days until it had two leaves and one heart. MS solid culture medium and LB solid culture medium were poured on the left and right sides of the two partitions, respectively, and sterilized by ultraviolet solidification in a clean bench for later use. Arabidopsis thaliana that had grown to two leaves and one heart was transplanted to the MS solid culture medium on the two partitions. Each group was treated three times, and 5 Arabidopsis seedlings were transplanted each time; 5 μL of bacterial liquid was added to the center of the LB solid culture medium on the other side of the two partitions, and LB liquid culture medium was used as the control. Under the conditions of 30°C and 16:8h (light culture: dark culture), the growth of Arabidopsis thaliana was observed after 14 days.

[0076] Growth-promoting effect of strain GXUN3754 on Arabidopsis thaliana Figure 6 As shown in Table 2, the data of various indicators show that when there is no significant difference in tiller number and root length, the leaf length, leaf width and fresh weight of Arabidopsis thaliana were significantly increased after treatment with GXUN3754 volatile substances, indicating that GXUN3754 volatile substances have a significant growth-promoting effect on Arabidopsis thaliana.

[0077] Table 2 Growth-promoting effect of GXUN3754 volatile substances on Arabidopsis

[0078]

[0079] Note: “*” indicates significant difference between the same groups (p<0.05), “**” indicates extremely significant difference (p<0.01);

[0080] Example 5 Insecticidal activity of Bacillus faecalis GXUN3754 against root-knot nematodes of ear beans

[0081] The root-knot nematode of elephant ear bean was artificially raised: the water spinach seedlings were planted to 15cm-20cm high, and then transplanted into the soil infected with the root-knot nematode of elephant ear bean. The seedlings were cultivated for 15 days, so that the root-knot nematode of elephant ear bean attached to the root system of the water spinach and formed knots. The egg masses were picked and incubated at room temperature of 30℃. The root-knot nematode of elephant ear bean was picked and then the egg masses were removed. 2 Larvae were subjected to bioactivity assays.

[0082] Inoculate 2% (V / V) of Bacillus faecalis GXUN3754 into 50 mL LB medium and culture at 30°C with shaking at 180 rpm for about 24 h until the bacterial solution OD 600nm ≈1.0, take 200 μL of bacterial solution into a six-well plate, add 600 μL of sterile water, and put 30 R. 2 Larvae (200 μL insect suspension), the total volume of the experimental liquid was 1 mL. LB blank medium was used as negative control, chemical pesticide fluopyram was used as positive control, thirteen dilution gradients were used, each group was repeated 3 times, and cultured at 28°C for 72 h. The death of the root-knot nematode of the elephant ear bean was observed, and the mortality rate, corrected mortality rate and LC under the corrected mortality rate were calculated. 50 .

[0083] The formulas for calculating mortality rate and adjusted mortality rate are as follows:

[0084] Mortality rate (%) = number of dead elephant ear bean root knot nematodes / total number of elephant ear bean root knot nematodes × 100%;

[0085] Corrected mortality rate (%) = [(mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group)] × 100%.

[0086] The results of the insecticidal activity of Bacillus faecalis GXUN3754 against root-knot nematodes of ear beans are as follows Figure 7 As shown in Table 3, LC 50 The data are shown in Table 4.

[0087] Table 3 Insecticidal activity of Bacillus faecalis GXUN3754 against root-knot nematodes of R.

[0088]

[0089] As shown in Table 3, the insecticidal activity of Bacillus faecalis GXUN3754 against root-knot nematodes of R. fulvidraco can reach 88.39%.

[0090] Table 4LC 50 Comparison of operation results

[0091]

[0092] As shown in Table 4, in comparison, the LC 50 The insecticidal activity of Bacillus faecalis GXUN3754 against root-knot nematodes of ear beans is higher than that of the chemical pesticide fluopyram.

[0093] Example 6 Determination of the insecticidal activity of volatile substances of Bacillus faecalis GXUN3754 and sterile fermentation filtrate against root-knot nematodes of ear bean

[0094] 1. Insecticidal activity of volatile substances of Bacillus faecalis GXUN3754 against root-knot nematodes of radix oleraceus

[0095] Inoculate 2% (V / V) of Bacillus faecalis GXUN3754 into 50 mL LB medium and culture at 30°C, 180 rpm for about 24 h until the OD of the bacterial solution reaches 600nm ≈1.0, set aside. Pour LB solid culture medium into the center of a sterile six-well plate, sterilize with ultraviolet light in a clean bench and set aside. Take 100 μL of bacterial solution and spread it on the culture medium in the six-well plate, and place 30 elephant ear bean root knot nematodes J in adjacent wells. 2 The larvae were cultured in LB blank medium as control, and each group was repeated 3 times. The culture was carried out at 28℃ for 72h. The death of the root-knot nematodes of the elephant ear bean was observed, and the mortality rate and corrected mortality rate were calculated.

[0096] The results of the volatile substances of Bacillus faecalis GXUN3754 on the insecticidal activity of root-knot nematodes of ear beans are as follows Figure 8 As shown in Table 5, the insecticidal activity of the volatile substances of Bacillus faecalis GXUN3754 against the root-knot nematode of R. aurantii can reach 98.85%.

[0097] Table 5 Insecticidal activity of GXUN3754 volatile substances against root-knot nematodes of ear beans

[0098]

[0099] 2. Insecticidal activity of the filtrate of sterile fermentation of Bacillus faecalis GXUN3754 against root-knot nematodes of radix oleraceus

[0100] Inoculate 2% (V / V) of GXUN3754 into 50 mL LB medium and culture at 30°C with shaking at 180 rpm for about 24 h until the bacterial solution OD 600nm ≈1.0, centrifuge at 8000r / min for 4min, take the supernatant, filter it with a 0.22μm filter membrane, and store it at 4℃ for later use. Take 200μL of sterile fermentation filtrate into a six-well plate, add 600μL of sterile water, and put in 30 Elephant ear bean root knot nematodes J 2 The larvae (200 μL of insect suspension) were diluted 5 times for the experiment, so that the total volume of the experimental liquid was 1 mL. LB blank medium was used as the control. The experiment was repeated 3 times and cultured at 28°C for 72 hours. The death of the root-knot nematode of the elephant ear bean was observed, and the mortality rate and corrected mortality rate were calculated.

[0101] The results of the sterile fermentation filtrate of Bacillus faecalis GXUN3754 on the target of root-knot nematodes of ear bean are as follows Fig. 9 As shown in Table 6, the insecticidal activity of the aseptic fermentation filtrate of Bacillus faecalis GXUN3754 against root-knot nematodes of ear beans was as high as 86.07%.

[0102] Table 6 Insecticidal activity of sterile fermentation filtrate of Bacillus faecalis GXUN3754 against root-knot nematodes of Auricularia aurantii

[0103]

[0104] Example 7 Control effect of Bacillus faecalis GXUN3754 in potted plants

[0105] The strain GXUN3754 was cultured in LB medium at 30°C and 180 rpm until the OD 600nm ≈1.0 for later use. Plant and raise water spinach seedlings until they have four leaves and one heart for later use. Mix the infected soil and ordinary nutrient matrix soil in a mass ratio of 1:1, take 200g of the mixed soil in a transparent small flowerpot, and transplant the water spinach seedlings with consistent growth into the flowerpot filled with the mixed soil. Using LB blank culture medium as the negative control and chemical pesticide (fluopyram) as the positive control, each group of five water spinach seedlings was watered with a 5-fold diluted bacterial solution of Bacillus faecalis GXUN3754 on the 1st, 11th, and 21st days, and harvested on the 30th day, the root knot condition of the plants was observed, and the root knot control rate was calculated. The calculation formula for the root knot control rate is as follows:

[0106] Root knot control rate = [(number of root knots in the negative control group - number of root knots in the treatment group) / number of root knots in the negative control group] × 100%.

[0107] The control effect of strain GXUN3754 in potted plants is as follows Fig.10 The root knot situation is shown in Table 7. The root knot control rate of plants treated with Bacillus faecalis GXUN3754 can reach 91.67%, which is similar to the control rate of the chemical pesticide fluopyram, and has a good potted plant control effect.

[0108] Table 7 Effect of strain GXUN3754 on potted control of root-knot nematodes in ear beans

[0109]

[0110] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Empedobacter stercoris GXUN3754, characterized in that: The deposit number of the faecal stibacillus GXUN3754 is CGMCC No. 29873. It was deposited in the China General Microorganism Collection Center on February 27, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A bacterial agent containing the Bacillus faecalis GXUN3754 according to claim 1, characterized in that: The preparation method of the bacterial agent comprises the following steps: The faecalis GXUN3754 was inoculated into LB medium and cultured with shaking until OD 600nm is 1.0, that is.

3. A sterile fermentation filtrate of Bacillus faecalis GXUN3754 according to claim 1, characterized in that: The method for preparing the sterile fermentation filtrate comprises the following steps: The faecalis GXUN3754 was inoculated into LB medium and cultured with shaking until the bacterial solution OD 600nm The concentration of 1.0 was adjusted, and the supernatant was taken and filtered using a 0.22 μm filter membrane.

4. Use of the Bacillus faecalis GXUN3754 according to claim 1, the bacterial agent according to claim 2, or the sterile fermentation filtrate according to claim 3 in the preparation of a biological agent for controlling root-knot nematodes of elephant ear beans.

5. A biological agent for controlling root-knot nematodes of elephant ear beans, characterized in that: The active ingredient includes the faecal stibacillus GXUN3754 described in claim 1 or the bacterial agent described in claim 2 or the sterile fermentation filtrate described in claim 3.

6. Use of the faecal stibacillus GXUN3754 according to claim 1, the bacterial agent according to claim 2, the sterile fermentation filtrate according to claim 3, or the biological preparation according to claim 5 in controlling root-knot nematodes of elephant ear beans.

7. A method for controlling root-knot nematodes of elephant ear beans, characterized in that: The method comprises the step of applying the faecal stibacillus GXUN3754 of claim 1 or the bacterial agent of claim 2 or the sterile fermentation filtrate of claim 3 or the biological agent of claim 5 to the rhizosphere soil of the plant.

8. Use of the Bacillus faecalis GXUN3754 according to claim 1 or the bacterial agent according to claim 2 in any of the following: (1) Application in promoting plant growth; (2) Application in the preparation of microbial preparations for promoting plant growth.

9. A microbial preparation for promoting plant growth, characterized in that: The active ingredient includes the faecal stibacterium GXUN3754 described in claim 1 or the bacterial agent described in claim 2.

10. A bacterial fertilizer comprising the microbial preparation according to claim 9, characterized in that: It also includes organic fertilizers and / or chemical fertilizers commonly used in agriculture.

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