CN119242516A

CN119242516A - A strain of Bacillus faecalis GXUN3754 and its application in controlling root-knot nematodes of elephant-ear bean

Info

Publication number: CN119242516A
Application number: CN202411541186.1A
Authority: CN
Inventors: 周燕; 姜明国; 谢晓莹
Original assignee: Guangxi University for Nationalities
Current assignee: Guangxi University for Nationalities
Priority date: 2024-10-31
Filing date: 2024-10-31
Publication date: 2025-01-03

Abstract

The present invention discloses a strain of Empedobacter stercoris GXUN3754 and its application in preventing and controlling root-knot nematodes of elephant ear beans, belonging to the field of microbial application technology. Empedobacter stercoris GXUN3754 provided by the present invention was deposited in the China General Microbiological Collection Management Center on February 27, 2024, with a deposit number of CGMCC No. 29873. Experiments show that Empedobacter stercoris GXUN3754 has the ability to produce siderophore, _NH3 , protease and phytase, and the bacterial liquid and volatile substances of Empedobacter stercoris GXUN3754 have the effect of promoting plant growth, and have the ability to kill root-knot nematodes of elephant ear beans. Therefore, Empedobacter stercoris GXUN3754 is of great significance for the research and development of new and efficient biological preparations and plant growth promoters for killing root-knot nematodes of elephant ear beans.

Description

Eimeria faecalis GXUN and 3754 and application thereof in prevention and control of meloidogyne incognita

Technical Field

The invention relates to the technical field of microorganism application, in particular to a strain of Eimeria faecalis GXUN and 3754 as well as application thereof in preventing and controlling root-knot nematode of like beans.

Background

The early discovery of the root-knot nematode (Meloidogyne enterolobii) of the elephantopus was similar to that of the root-knot nematode found in the root knot of the elephantopus (Enterolobium cyclocarpum) tree in Hainan region of China by Yang et al in 1983, and identified as a new species of the root-knot nematode of the elephantopus according to morphological characteristics. In 1988 Rammah found a root knot nematode on eggplant root knots and named it as maya root knot nematode (Meloidogyme mayaguensis). Through analysis of esterase phenotypes, mtDNA COII, lrRNA and the like of the two nematodes, and analysis and description of model species slides, the maya root-knot nematode is considered to be an synonym species like the Meloidogyne incognita.

The root-knot nematode of the like auricularia auricula is an important pathogenic nematode in tropical and subtropical areas of the world, has strong pathogenicity and reproductive potential, has wide host distribution range, mainly comprises leguminous plants, cucurbitaceae, solanaceae and the like, can be parasitic on crops such as capsicum (N gene) carrying resistance genes, tomatoes (Mi-1 gene) and the like for reproduction, and often causes serious loss of the yield of vegetable crops or fruit trees. At present, the prevention and treatment of the root-knot nematode of the like beans mainly depend on chemical medicines, and the chemical method is efficient and convenient and has good prevention and treatment effects. However, at the same time, excessive use of chemical pesticides is easy to cause environmental pollution, and accumulation of pesticides can destroy the balance of soil ecology and even harm human health, so that the implementation of green and environment-friendly prevention and treatment measures is urgent.

In recent years, biological control is becoming an important point of current research because of the advantages of environmental protection, cost saving, innocuity and innocuity. Biocontrol strains can be obtained from plant rhizosphere, soil and animals and plants, and they generally have the function of dissolving inorganic phosphorus, organic phosphorus, nitrogen fixation and the like, while secreted extracellular enzymes can also resist infection of pathogenic fungi by destroying fungal cell walls. In 2015, thorsten was first separated from a sample from a biogas plant to obtain a strain of Equilibrium faecalis (Empedobacter stercoris) SCVM0123, and its classification status was determined. In 2020, cheng et al screened a strain of stationary bacillus faecalis ES183 with a Tet variant gene from faecal samples of 6 animal farm, strain ES183 had a new tigecycline resistance gene Tet (X14), and predicted homology relationship of Tet (X14) by structural model. At present, reports on the Eimeria faecalis are still few, and no intensive research on the biocontrol function of the Eimeria faecalis is carried out. However, equilibrium brevicum (Empedobacter brevis) belonging to the same genus as Equilibrium faecalis has been a microbial pesticide which has been used more widely in recent years. The Emamectin brevis can enter insect bodies through feeding and mass propagation of insects, and protein toxins are generated to interfere metabolism of the insect bodies, so that insects are killed, and the Emamectin brevis is generally applied to control of various biological pests such as rice leaf rollers, prodenia litura, tea geometrid, cauliflower, maca plutella xylostella and the like.

At present, few reports on the prevention and treatment of the biological control fungus object Earbean root knot nematode are in China, and no study on application of Eardrop bacillus to biological control is seen. The functional microbial inoculum prepared by the method can help crops resist plant diseases and insect pests and improve crop yield due to the diversity of the biocontrol microbial function and the green pollution-free characteristic of the biocontrol microbial inoculum, and accords with the sustainable development road of green new agriculture. Therefore, the biocontrol bacteria with poisoning activity for screening and researching the object otobean root knot nematode have good research significance and broad agricultural application prospect.

Disclosure of Invention

The invention aims to provide a Eimeria faecalis GXUN3754 and application thereof in preventing and controlling Eimeria mulation root-knot nematode, so as to solve the problems in the prior art, and the Eimeria faecalis GXUN3754 and the microbial inoculum thereof can effectively kill Eimeria mulation root-knot nematode and have growth promoting effect on plants.

In order to achieve the above object, the present invention provides the following solutions:

The invention provides a strain of Eimeria faecalis (Empedobacter stercoris) GXUN3754, wherein the Eimeria faecalis GXUN3754 is preserved in China general microbiological culture Collection center (China center for type collection) for 2 months and 27 days, the preservation address is North Star, the national academy of sciences of China, and the preservation number is CGMCC No.29873.

The invention also provides a microbial inoculum containing the Equilibrium faecalis GXUN and 3754, and the preparation method of the microbial inoculum comprises the following steps:

Inoculating the Equilibrium GXUN3754,3754 to LB culture medium, and shake culturing until OD _600nm is 1.0.

The invention also provides a sterile fermentation filtrate prepared according to the Equilibrium faecalis GXUN and 3754, and the preparation method of the sterile fermentation filtrate comprises the following steps:

Inoculating the Equilibrium GXUN3754,3754 to LB culture medium, shake culturing until the bacterial liquid OD _600nm is 1.0, centrifuging, collecting supernatant, and filtering with 0.22 μm filter membrane.

The invention also provides application of the Eimeria faecalis GXUN and 3754 or the microbial inoculum or the sterile fermentation filtrate in preparation of biological agents for preventing and controlling the root-knot nematode of the like auricularia auricula.

The invention also provides a biological agent for preventing and controlling the root-knot nematode of the like auricularia auricula, and the effective components comprise the Eimeria faecalis GXUN and 3754 or the microbial inoculum or the sterile fermentation filtrate.

The invention also provides an application of the Eimeria faecalis GXUN3754 or the microbial inoculum or the sterile fermentation filtrate or the biological agent in preventing and controlling the root-knot nematode of the like.

The invention also provides a method for preventing and controlling the root-knot nematode of the like auricularia auricula, which comprises the step of applying the Eimeria faecalis GXUN3754,3754 or the microbial agent or the sterile fermentation filtrate or the biological agent to plant rhizosphere soil.

The invention also provides application of the Eimeria faecalis GXUN and 3754 or the microbial inoculum in any one of the following:

(1) Use in promoting plant growth;

(2) The application in preparing microbial preparation for promoting plant growth.

The invention also provides a microbial preparation for promoting plant growth, and the effective components comprise the Equilibrium faecalis GXUN and 3754 or the microbial preparation.

The invention also provides a bacterial fertilizer containing the microbial preparation, and the bacterial fertilizer also comprises organic fertilizer and/or chemical fertilizer commonly used in agriculture.

The invention discloses the following technical effects:

The invention provides a strain of Eimeria faecalis GXUN and 3754, and the volatile gas substance can be used for efficiently killing the root-knot nematode of the like, and has good growth promoting effect on arabidopsis thaliana. In addition, the Eimeria faecalis GXUN and the metabolite of the Eimeria faecalis object Eimeria otophylla have good lethal effect, and can remarkably prevent and treat the infection of crops by nematodes. Therefore, the Eimeria faecalis GXUN and 3754 have very important significance for researching and developing a novel efficient biological agent for killing the Eimeria praecox.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a colony morphology of Eimeria faecalis GXUN and 3754;

FIG. 2 is a graph showing the results of gram staining of Eimeria faecalis GXUN and 3754 by light microscopy (100X oleoscopy);

FIG. 3 is a phylogenetic tree of Eimeria faecalis GXUN3754 based on the 16S rRNA gene sequence;

FIG. 4 is a graph showing the results of detection of antibacterial extracellular enzymes and detection of indexes related to growth promotion characteristics of Equilibrium faecalis GXUN and 3754, wherein A is protease, B is phytase, C is acidophilic ferrite, D is ammonia, CK is a control group, E is cellulase, and F is amylase;

FIG. 5 is a view for observing the germination promotion condition of Equilibrium faecalis GXUN and 3754 on water spinach seeds, wherein A is the germination condition of soaking the seeds in a blank culture medium for 30min, and B is the germination condition of soaking the seeds in Equilibrium faecalis GXUN and 3754 for 30 min;

FIG. 6 is a view showing the growth promotion of volatile matters of Eimeria faecalis GXUN and 3754 on Arabidopsis thaliana, wherein A is blank medium treatment and B is Eimeria faecalis GXUN and 3754 treatment;

FIG. 7 is a microscopic view of the insecticidal effect of Eimeria tenella GXUN and 3754 on Eimeria tenella, wherein A is the insect body state of Eimeria tenella under blank culture medium treatment, and B is the insect body state of Eimeria tenella after Eimeria tenella GXUN and 3754 treatment;

FIG. 8 is a microscopic view of the insecticidal effect of Eimeria tenella GXUN and 3754 volatile substances on Eimeria otophylla, wherein A is the insect body state of Eimeria otophylla after the blank culture medium treatment, and B is the insect body state of Eimeria otophylla after the volatile gas treatment of Eimeria tenella GXUN and 3754;

FIG. 9 is a microscopic view of the insecticidal effect of Eimeria tenella GXUN3754 on Eimeria tenella in a sterile fermentation filtrate, wherein A is the insect body state of Eimeria tenella under the treatment of a blank culture medium, and B is the insect body state of Eimeria tenella after the treatment of the sterile fermentation filtrate of Eimeria tenella GXUN 3754;

Fig. 10 shows the pot control effect of the Eimeria tenella GXUN and 3754 on the plant root system, wherein A is a negative control plant root system, (a) and (B) are typical areas and enlarged views thereof, B is a positive control plant root system (chemical pesticide fluopyram is applied), (C) and (d) are typical areas and enlarged views thereof, C is a plant root system treated by Eimeria tenella GXUN375, (e) and (f) are typical areas and enlarged views thereof, and red spots are root knots formed by Eimeria tenella.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The experimental materials used in the embodiment of the invention comprise LB solid culture medium, MS solid culture medium, protease detection culture medium, amylase detection culture medium, cellulase detection culture medium, abbe's nitrogen fixation culture medium, peptone water culture medium, nessler reagent, calcium phytate culture medium, PKO inorganic phosphorus culture medium, ferriphilic detection culture medium, L-tryptophan, salkowski colorimetric solution, IAA standard substance and used Izoma Erycibe root-knot nematode which are all derived from Guangxi Zhang autonomous region polysaccharide materials and modified key laboratories.

Example 1 isolation of Strain GXUN and 3754

Plant rhizosphere soil was collected from the mountain heads of the mountain harbour area green mountain (109 ° 25 'east longitude, 21 ° 36' north latitude) of northern sea city, a guangxi Zhuang nationality autonomous region for bacterial isolation. Weighing 5g of soil sample, adding the soil sample into 50mL of sterile water, vibrating for 4 hours at 30 ℃ and 180r/min, then carrying out gradient dilution to obtain 10 ^-3、10^-4、10^-5 soil suspension, respectively sucking 100 mu L of soil suspension, uniformly coating the soil suspension on an LB solid culture medium, carrying out inverted culture at 30 ℃, picking single bacterial colonies with different sizes and forms after 24 hours, carrying out streak purification on the single bacterial colonies in the LB solid plates, inoculating the purified bacterial strains on the LB solid plates, and carrying out streak standby.

Example 2 identification of Strain GXUN and 3754

1. Morphological observation of strains

The strain GXUN and 3754 obtained in example 1 was streaked on LB solid medium and cultured at 30℃for 24 hours, and morphological characteristics such as color, size, transparency and the like of single colony were observed. After picking single colonies and gram staining, the strain morphology was observed by optical microscopy.

The morphological observation of strain GXUN and 3754 is shown in FIG. 1, and the result shows that the strain is yellow on LB solid medium, smooth in surface, creamy and has bulges. The results of gram staining are shown in FIG. 2, and the cells are rod-shaped under an optical microscope and positive in gram staining test.

2. Physiological and biochemical characteristics

The physiological and biochemical indexes of bacterial strain GXUN and 3754 oxidase, starch hydrolysis, gelatin liquefaction, methyl red and the like are identified by referring to the common bacterial System identification handbook and the Berger's bacterial identification handbook.

The physiological and biochemical characteristic analysis results are shown in Table 1, and the results show that the strain is aerobic bacteria, and has negative effects on oxidase, methyl red test, citrate utilization test and V-P test, and positive effects on gelatin hydrolysis and malonate utilization test.

TABLE 1 results of physiological and biochemical characterization

3. Molecular biological identification

After activating the strain GXUN and 3754, extracting total DNA, and using the total DNA as a template, respectively using bacterial universal primers:

27F:5’-AGAGTTTGATCCTGGCTCAG-3’(SEQ ID NO.1);

1492R:5’-TACGGCTACCTTGTTACGAGTT-3’(SEQ ID NO.2);

PCR amplification was performed on the strain GXUN375416S rRNA gene.

PCR amplification system (50. Mu.L) 1. Mu.L template, ddH ₂ O22. Mu.L, 2 XTaq PCR Mix 25. Mu.L forward primer 1. Mu.L reverse primer 1. Mu.L.

PCR amplification procedure 95℃pre-denaturation 5min, 95℃denaturation 1min,55℃annealing 1min,72℃extension 2 mm, 32 cycles, 72℃extension 10min, 4℃storage.

The amplified products were sent to October biosciences (Wuhan) Inc., sequenced, and the sequences were aligned for homology to sequences in the NCBI database, and phylogenetic tree was constructed using MEGA 11.0 software.

As shown in FIG. 3, the molecular biology identification result shows that the 16S rRNA gene sequence of the strain GXUN and 3754 are subjected to homology analysis with the existing data in NCBI database, and a phylogenetic tree is constructed, and the similarity between the 16S rRNA sequence of the strain GXUN3754 and the Etabacillus faecalis (Empedobacter stercoris) SCVM0123 is found to be 99.65%, and the phylogenetic tree is gathered at the same branch.

The strain is determined to be the Eimeria faecalis (Emp edobacter stercoris) by combining morphological characteristics, physiological and biochemical characteristics and molecular biological identification results of the strain. The inventor has preserved the strain GXUN3754 in 2024, 2 months and 27 days in China general microbiological culture Collection center with a preservation address of North Star, qing dynasty district North Star, national academy of sciences of China, and a preservation number of CGMCC No.29873.

EXAMPLE 3 measurement of enzyme production ability of Eimeria faecalis GXUN3754

Respectively dripping 5 mu L of fermentation liquid (OD ₆₀₀ apprxeq 1.0) of Equilibrium faecalis GXUN3754 into the centers of a protease detection culture medium, an amylase detection culture medium and a cellulase detection culture medium, culturing for 3d at 30 ℃, dyeing the cellulase detection culture medium for 30min by using 1mol/L Congo red, decoloring for 30min by using 1mol/LNaCl solution, and developing the amylase detection culture medium by using iodine solution.

Ammonia production, namely 50 mu L of a bacterial solution (OD ₆₀₀ apprxeq.1.0) of Equilibrium faecalis GXUN3754 is inoculated into a test tube filled with 4mL of peptone water culture medium, after shaking culture is carried out for 48 hours at the temperature of 30 ℃, 0.5MLNESSLER reagent is added, the color of the solution changes from yellow to reddish brown, and precipitation is generated, so that the solution has the capability of producing NH ₃.

And (3) dissolving phosphorus, namely taking 5 mu L of a bacterial solution (OD ₆₀₀ is approximately equal to 1.0) of the Equilibrium faecalis GXUN, respectively inoculating the bacterial solution to the centers of a Meng Jinna culture medium and a PKO inorganic phosphorus culture medium, and culturing for 5 days at a temperature of 30 ℃, wherein if transparent rings are produced around the strain, the bacterial strain has the capability of dissolving organic phosphorus or inorganic phosphorus.

And (3) producing the siderobacterium faecalis GXUN and 3754 strains are inoculated in the center of a ferrite detection medium and cultivated for 7 days at a constant temperature of 30 ℃, if orange-yellow halos are produced around the strains, the strains are proved to have the sideroblasts, and the sideroblasts of antagonistic strains are judged according to the size of the halos.

As shown in FIG. 4, qualitative detection results of the growth promoting function and enzyme producing characteristics of Equilibrium faecalis GXUN and 3754 show that Equilibrium faecalis GXUN3754 produces orange-yellow halo on CAS detection medium, after Nessler reagent is added, peptone water medium turns reddish brown and has precipitate production, transparent rings are produced on protease detection medium and phytase detection medium, but no transparent rings are produced on cellulase detection medium and amylase detection medium, which means that Equilibrium faecalis GXUN3754 has the capability of producing ferrite, NH ₃, protease and phytase, and no capability of producing amylase and cellulase.

EXAMPLE 4 Protoffee of Equilibrium faecalis GXUN3754

1. Promoting growth of Equilibrium faecalis GXUN and 3754 on water spinach

The seeds are subjected to seed surface disinfection. Soaking in sterile water for 1h to pick out bad seeds, washing with 75% ethanol for 1min for 1 time, soaking with 1% sodium hypochlorite for 3min for 2 times, and washing with sterile water for 3-5 times. The Eimeria faecalis GXUN and 3754 were inoculated into 50mL of LB medium at 2% (V/V), and cultured with shaking at 30℃and 180rpm for 24 hours until OD _600nm was about 1.0 for use. And (3) taking an LB blank medium as a control, putting 25 water spinach seeds with sterilized surfaces into the LB blank medium, and soaking the water spinach seeds in a bacterial liquid for 1h. The water spinach seeds are placed in a sterile culture dish paved with wet filter paper, and under the conditions of 30 ℃ and 16:8 hours (light culture: dark culture), the germination rate and bud length are observed after 5 days.

The calculation formula of the germination rate is as follows:

germination percentage (%) =number of germinated seeds/number of test seeds×100%;

As shown in FIG. 5, the germination rate of the strain GXUN and 3754 on the water spinach seeds is 68% in the control group, the germination rate of the water spinach seeds treated by GXUN and 3754 is 88%, and the germination bud length of the water spinach seeds after 5d treatment is obviously improved compared with that of the control group.

2. Growth promoting effect of volatile substances of Eimeria faecalis GXUN and 3754 on Arabidopsis thaliana

The Eimeria faecalis GXUN and 3754 are inoculated into 50mL LB culture medium at 2% (V/V), and are subjected to shaking culture at 30 ℃ and 180r/min for 24 hours until the OD ₆₀₀ nm is about 1.0 for later use. Arabidopsis thaliana was vernalized at 4℃for 72h, then dibble seeded onto MS solid medium and cultured for 4-5d with two leaves in one heart. And respectively pouring an MS solid culture medium and an LB solid culture medium into the left and right of the two partition plates, and solidifying and sterilizing the culture medium by ultraviolet rays on an ultra-clean bench for later use. Transplanting Arabidopsis thaliana growing to two leaves and one heart on an MS solid culture medium of a bipartite partition board, repeating the treatment for three times each group, transplanting 5 Arabidopsis thaliana seedlings each time, dripping 5 mu L of fungus on the center of an LB solid culture medium on the other side of the bipartite partition board, and observing the growth condition of Arabidopsis thaliana after 14 days under the conditions of 30 ℃ and 16:8 hours (light culture: dark culture) by taking the LB liquid culture medium as a control.

The growth promoting effect of the strain GXUN and 3754 on the arabidopsis is shown in figure 6, and various index data are shown in table 2, so that the effect of the volatile substance of GXUN and 3754 on the growth promoting effect of the volatile substance on the arabidopsis can be found to be obvious under the condition that no obvious difference exists between the tiller number and the root length, and the leaf length, the leaf width and the fresh weight of the arabidopsis are obviously improved after the volatile substance of GXUN and 3754 is treated.

TABLE 2GXUN3754 volatile substances Protoffee Arabidopsis thaliana

Note that "+" indicates significant difference between the same groups (p < 0.05), and "+" indicates significant difference (p < 0.01);

EXAMPLE 5 insecticidal Activity of Eimeria faecalis GXUN and 3754 against Eimeria otophylla

The method comprises the steps of artificially raising the root-knot nematode of the green Chinese cabbage to a height of 15cm-20cm, transplanting the green Chinese cabbage to the infected soil containing the root-knot nematode of the green Chinese cabbage, and culturing for 15 days to enable the root-knot nematode of the green Chinese cabbage to adhere to the root system of the green Chinese cabbage to form a knot. And (3) selecting egg masses, incubating at the normal temperature of 30 ℃, and selecting larvae of the meloidogyne incognita J ₂ for biological activity measurement.

Equilibrium faecalis GXUN and 3754 are inoculated into 50mL of LB culture medium at 2% (V/V), the temperature is 30 ℃, shaking culture is carried out at 180rpm for about 24 hours until the bacterial liquid OD _600nm is approximately equal to 1.0, 200 mu L of bacterial liquid is taken in a six-hole plate, 600 mu L of sterile water is added, 30 heads of larvae of Izoma otophyllum J ₂ (200 mu L of insect suspension) are placed, and the total volume of experimental liquid is 1mL. And (3) taking LB blank medium as a negative control, taking the chemical pesticide fluopyram as a positive control, diluting thirteen gradients, repeating 3 times for each group, culturing at a constant temperature of 28 ℃ for 72 hours, observing the death condition of the root-knot nematode of the like auricularia auricula, and calculating the death rate, correcting the death rate and LC ₅₀ under the correction of the death rate.

The formulas for mortality and corrected mortality are as follows:

Mortality (%) = number of dead nematodes per total number of nematodes×100%;

Corrected mortality (%) = [ (treatment group mortality-control group mortality)/(1-control group mortality) ]x100%.

Results of the insecticidal activity of Eardrop's root knot nematode on Eardrop's bacillus GXUN3754 subject are shown in FIG. 7, data are shown in Table 3, and LC ₅₀ data are shown in Table 4.

Table 3 insecticidal Activity of Eimeria faecalis GXUN and 3754 against Eimeria otophylla

As shown in Table 3, the insecticidal activity of Earbean root-knot nematode in Eardrop-vine bacillus GXUN and 3754 can reach 88.39%.

Table 4LC ₅₀ comparison of results

As can be seen from table 4, LC ₅₀ of the steady bacillus faecalis GXUN and 3754 was smaller compared to that of the steady bacillus faecalis GXUN and 3754, and the insecticidal activity of the root knot nematode of the otobean was higher than that of the chemical pesticide fluopyram.

EXAMPLE 6 determination of insecticidal Activity of Eimeria faecalis GXUN3754 volatile Material and of the sterile fermentation filtrate object Eimeria

1. Insecticidal activity of Eimeria faecalis GXUN and 3754 volatile substance object Eimeria otophylla

Inoculating Equilibrium faecalis GXUN3754,3754 into 50mL LB culture medium at 2% (V/V), and shake culturing at 30deg.C and 180r/min for about 24 hr until bacterial liquid OD _600nm is about 1.0 for use. And pouring LB solid culture medium into the center of the sterile six-hole plate, and performing ultraviolet sterilization in an ultra-clean workbench for standby. And (3) 100 mu L of bacterial liquid is coated on a culture medium in a six-hole plate, 30 larvae of the like-eared bean root-knot nematode J ₂ are put in adjacent holes, each group is repeatedly cultured for 72 hours at the constant temperature of 28 ℃ for 3 times by taking LB blank culture medium as a control, the death condition of the like-eared bean root-knot nematode is observed, and the death rate and the correction death rate are calculated.

The insecticidal activity of the volatile substance object Eimeria otophylla is GXUN3754, the result is shown in figure 8, the data is shown in table 5, and the insecticidal activity of the volatile substance object Eimeria otophylla is GXUN3754, namely the Eimeria otophylla can reach 98.85%.

TABLE 5GXUN3754 insecticidal Activity of volatile Material against Meloidogyne incognita

2. Insecticidal activity of Eimeria tenella GXUN3754 in sterile fermentation filtrate object Eimeria tenella

Inoculating Equilibrium faecalis GXUN3754,3754 into 50mL LB culture medium at 2% (V/V), shake culturing at 30deg.C for about 24 hr until bacterial liquid OD _600nm is about 1.0, centrifuging at 8000r/min for 4min, collecting supernatant, filtering with 0.22 μm filter membrane, and preserving at 4deg.C for use. 200 mu L of sterile fermentation filtrate is taken in a six-hole plate, 600 mu L of sterile water is added, 30 head of the larvae of the root-knot nematode J ₂ (200 mu L of worm suspension) of the elephantopus are put in, the experiment is carried out by 5-time dilution, so that the total volume of experimental liquid is 1mL, LB blank culture medium is used as a control, the culture is repeated for 3 times at the constant temperature of 28 ℃, the death condition of the root-knot nematode of the elephantopus is observed, and the death rate and the correction death rate are calculated.

The results of the insecticidal activity of the Eimeria otophylla in the object of the aseptic fermentation filtrate of Eimeria faecalis GXUN and 3754 are shown in figure 9, the data are shown in table 6, and the insecticidal activity of the Eimeria faecalis GXUN and 3754 in the object of the aseptic fermentation filtrate is as high as 86.07%.

Table 6 Eimeria faecalis GXUN3754 insecticidal Activity of the sterile fermentation filtrate against Eimeria otophylla

EXAMPLE 7 Equilibrium faecalis GXUN3754 potted plant control effect

Equilibrium faecalis GXUN and 3754 are cultured in LB culture medium at 30 ℃ and 180r/min until OD _600nm is approximately equal to 1.0 for standby. Planting and raising the water spinach until the water spinach leaves are four leaves and one heart for later use. Mixing the infected soil and the common nutrient matrix soil in a mass ratio of 1:1, taking 200g of the mixed soil into a transparent small flowerpot, and transplanting the water spinach seedlings with consistent growth vigor into the flowerpot filled with the mixed soil. And (3) taking an LB blank medium as a negative control, taking chemical pesticide (fluopyram) as a positive control, pouring 5-fold diluted bacillus faecalis GXUN3754 bacterial liquid into each group of five water spinach seedlings in the 1d, 11d and 21d, harvesting in the 30d, observing the root knot condition of the plants, and calculating the root knot control rate. The calculation formula of the root knot prevention rate is as follows:

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

The control effect of the potted plant of the strain GXUN and 3754 is shown in figure 10, the root knot condition is shown in table 7, the control rate of the root knot of the plant treated by using the Etabacillus faecalis GXUN and 3754 can reach 91.67%, and the control rate is similar to that of the chemical pesticide fluopyram, so that the potted plant has good control effect.

Table 7 potted plant control effect of Meloidogyne incognita (Fr.) Sing of strain GXUN and 3754

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. A strain of Empedobacter stercoris GXUN3754, characterized in that the deposit number of Empedobacter stercoris GXUN3754 is CGMCC No.29873, which was deposited in the China General Microorganism Collection Center on February 27, 2024, and 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 Staphylococcus 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 reached 1.0.

3. A sterile fermentation filtrate of Stibacillus faecalis GXUN3754 according to claim 1, characterized in that the preparation method of the sterile fermentation filtrate comprises the following steps:

The faecalis GXUN3754 was inoculated into LB medium, shaken and cultured until the bacterial solution OD _600nm was 1.0, centrifuged, and the supernatant was taken and filtered using a 0.22 μm filter membrane to obtain the product.

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 preparation for preventing and controlling root-knot nematodes of elephant ear beans, characterized in that the active ingredient comprises the faecal stibacillus GXUN3754 according to claim 1 or the bacterial agent according to claim 2 or the sterile fermentation filtrate according to 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 it comprises the step of applying the faecalis 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 comprises the Bacillus faecalis GXUN3754 according to claim 1 or the bacterial agent according to claim 2.

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