CN115572688A

CN115572688A - Bacillus cereus with high poisoning activity on plant parasitic nematodes and application thereof

Info

Publication number: CN115572688A
Application number: CN202210841985.5A
Authority: CN
Inventors: 孙明; 代大东; 聂鑫; 姚浩哲; 张书荣; 郑金水; 彭东海
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2022-07-18
Filing date: 2022-07-18
Publication date: 2023-01-06

Abstract

The invention belongs to the technical field of microorganisms, and provides bacillus cereus with high poisoning activity on plant parasitic nematodes and application thereof. The strain is preserved in China center for type culture collection of Wuhan university in flood mountain area, wuhan City, hubei province, china at 2022, 06 months and 15 days, and the preservation number is CCTCCNO: m2022893. The separated strain has high poisoning effect on meloidogyne incognita and other meloidogyne incognita and cyst nematode such as soybean cyst nematode, and can be used for biological control of meloidogyne incognita and cyst nematode diseases.

Description

Bacillus cereus with high poisoning activity on plant parasitic nematodes and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a novel strain, in particular to bacillus cereus with high poisoning activity on plant parasitic nematodes and application thereof.

Background

The phylum nematoda is one of the largest phyla in the animal kingdom, has huge abundance, is visible everywhere in environments such as soil and fresh water, even has the existence of the phylum nematoda in extreme environments, can parasitize on animals and plants, or freely live in soil and fresh water. Currently, the first of the ten most harmful plant parasitic nematodes in the world is meloidogyne. The host range of the root-knot nematode is very wide, almost all fruits and vegetables can be infected, and plants infected by the root-knot nematode can form root knots on roots, so that the plants are short and small, leaves are yellow, and even the plants die. The root-knot nematode disease seriously threatens agricultural production and causes the reduction of yield of a large amount of economic crops.

The root-knot nematodes are mainly distributed in surface soil of 3-10 cm, and the existing control method mainly comprises the steps of eradicating the nematodes by means of burning, flooding and high-temperature shed braising during crop rotation planting and stubble changing, but cannot achieve the effect of radical treatment, and still causes certain loss to the economic efficiency income of farmers. At present, the most widely applied method is to spray chemical pesticide on soil before planting to achieve the purpose of preventing and treating in advance. However, the method has high requirement on the spraying amount of chemical pesticides, and long-time application of the chemical pesticides not only affects the soil quality, but also causes the resistance of nematodes to the pesticides, so that the control effect is increasingly poor.

At present, biological pesticides become a research hotspot of novel pesticides due to the advantages of safety, low toxicity, good effect, difficulty in causing pest resistance, environmental friendliness and the like.

Disclosure of Invention

In view of the defects of the prior art, the first purpose of the invention is to provide a biocontrol bacterium with high poisoning activity on plant parasitic nematodes, which has high poisoning effect on meloidogyne incognita, and can be used for biological control of meloidogyne diseases.

In order to achieve the technical purpose, the inventor unexpectedly discovers that the bred nematodes die collectively after hatching in tomato pots planted in repeated continuous cropping in a greenhouse, and based on the discovery, the inventor separates a strain with high toxic activity to plant parasitic nematodes in a benthic species in further research, and determines that the strain is Bacillus, particularly Bacillus cereus, and is named as BMB2600 after molecular biology experiments and genome sequencing. The strain is preserved in China center for type culture collection of Wuhan university in flood mountain area, wuhan City, hubei province, china at 2022, 6 months and 15 days, and the preservation number is CCTCC NO: m2022893.

The Bacillus cereus BMB2600 isolated according to the present invention has the following morphological and cultural characteristics:

morphological characteristics: bacillus cereus BMB2600 is gram-positive bacillus, 1.0-1.2X 3.0-5.0 micron, facultative aerobic, spore-forming, round or cylindrical, and has two smooth ends and most of cells in chain shape. Large colony, rough, flat and irregular surface.

The culture characteristics are as follows: the strain can produce metabolite acrylic acid after fermentation culture in a culture medium containing glucose, and the fermentation liquid has high insecticidal activity on plant parasitic nematodes, especially has high poisoning activity on root-knot nematodes and cyst nematodes.

In addition, the second purpose of the invention is to provide the application of a Bacillus cereus strain, namely the application of the Bacillus cereus BMB2600 in preventing and controlling plant parasitic nematode diseases. And the application of the fermentation liquor of the bacillus cereus in preparing biological pesticide and/or biological organic fertilizer for preventing and treating plant parasitic nematode diseases. Preferably, the plant parasitic nematode is selected from one or more than two of the following: meloidogyne, cyst nematodes. Further preferably, the meloidogyne is meloidogyne incognita, and the cyst nematode is soybean cyst nematode.

Finally, a third object of the present invention is to provide a highly active plant nematode-poisoning preparation, the active ingredient of which is prepared from the fermentation broth of Bacillus cereus BMB2600 as described above.

Compared with the prior art, the bacillus cereus BMB2600 provided by the invention has the following advantages and remarkable progress:

(1) The strain has high poisoning effect on Meloidogyne incognita and Soy cyst nematode.

(2) Compared with the characteristic of slow effect of biological pesticides, the strain fermentation liquor has good poisoning effect within 60 minutes when the biological activity of meloidogyne incognita and soybean cyst nematode is measured.

(3) The strain can produce acrylic acid which has high toxicity and killing activity to plant parasitic nematodes when fermented.

(4) The industrial fermentation liquid of the strain BMB2600 is applied to tomato seedlings seriously infected by root-knot nematodes, and the result shows that the industrial fermentation liquid has good control effect on the root-knot nematodes in field tests, and has equivalent effect to chemical pesticides.

Drawings

FIG. 1 is a graph of nematicidal activity of fermentation supernatants at different concentrations, wherein Mi is Meloidogyne incognita; hg: soybean cyst nematode; and Dd: rot stem nematodes; ce: caenorhabditis elegans.

FIG. 2 is a growth curve of Bacillus cereus strain BMB2600.

FIG. 3 shows the effect of shake flask fermentation broth of Bacillus cereus strain BMB2600 on root node number of potted tomato.

FIG. 4 shows the effect of industrial fermentation broth of Bacillus cereus strain BMB2600 on root node number of potted tomato roots (. Beta.p <0.05,. Beta.p < 0.001).

FIG. 5 is a GC-MS plot of the metabolite acrylic acid of Bacillus cereus BMB2600.

FIG. 6 shows the tomato root knot in field experiment; the method comprises the following steps: a control group; the following: a treatment group is implemented.

FIG. 7 is a photograph of tomato roots in the treatment group of the field experiment.

Detailed Description

The present invention is further illustrated by the following detailed description, wherein the technical steps or conditions not specified in the examples are performed according to the technical or conditions described in the literature in the field or according to the product specification. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: discovery process and activity verification experiment of bacillus cereus BMB2600 strain

1. Experimental methods

1.1 culture of root-knot nematodes and hatching eggs

1) Root-knot nematode breeding and egg hatching under greenhouse condition

(1) Planting a susceptible variety of tomato plants, directly sowing seeds into nutrient soil and mixing the nutrient soil and sandy soil according to the volume ratio of 2:1, and germinating under proper conditions;

(2) Pulling out tomato seedlings growing to about 10cm high from the soil, and washing the soil on roots with running water;

(3) Injecting the meloidogyne incognita to the clean root surface by a liquid transfer device, and then transplanting to soil in a large basin, wherein the inoculation amount of each basin is about 2000-3000;

(4) After about 30-45 days, the tomato root system is infected by root-knot nematodes to a proper extent, and diseased plants can be pulled out to pick egg masses;

(5) Firstly, picking the egg mass to a container filled with sterilized ddH ₂ And placing the bottle in O into an incubator at 20 ℃ for 3 days, and harvesting more root-knot nematodes J2s after a proper time.

(6) If cleaner root-knot nematodes J2s are to be obtained, tomato root systems which are seriously infected by the root-knot nematodes and have large and more root knots can be selected and cut up as much as possible.

(1) Immersing the whole into 1% sodium hypochlorite solution, and stirring for about 8 min.

(2) And respectively sieving the suspension liquid through a 60-mesh, 200-mesh, 100-mesh and 500-mesh sieve set, and collecting the dispersed root-knot nematode egg-dispersing granules on a 500-mesh sieve.

(3) And hatching the collected root knot nematode egg suspension on a 500-mesh membrane, and putting the membrane in an incubator at 20 ℃ for 3 days, wherein the time is properly prolonged, and more root knot nematodes J2s can be obtained.

1.2 Strain culture method and Medium

Activating strains: transferring the strain preserved on the inclined plane to an LB flat plate, and culturing for 24h at 37 ℃ for later use; preparing a seed solution: selecting a ring of activated strains, inoculating the strains into 50mL of LB liquid culture medium, and performing shaking culture at the temperature of 37 ℃ and the rpm of 220 for 12 hours for later use; shake flask culture of fermentation liquor: inoculating the strain seed liquid into a fermentation medium according to the inoculation amount of 1%, and carrying out shaking culture at 37 ℃ and 220rpm for a specified time.

Seed medium (LB liquid medium): 0.5% of yeast extract, 1% of peptone, 1% of NaCl, pH7.0, 121 ℃, and sterilizing for 30min.

Strain preservation medium (LB solid medium): 0.5% of yeast extract, 1% of peptone, 1% of NaCl, 2% of agar, pH7.0, 121 ℃, and sterilizing for 30min.

Fermentation medium: ICPM medium: glucose 0.5%, peptone 0.5%, KH ₂ PO ₄ 0.05％，MgSO ₄ Sterilizing at 115 deg.C for 20min and 0.05%.

1.3 fermentation broth treatment

Fermenting the fermentation liquor at 4 ℃ and 10000rpm; centrifuging for 15min, sucking supernatant, filtering with 0.22 μm pore size filter to obtain supernatant, collecting fermented supernatant, diluting the stock solution by 2 times, 5 times, 10 times, and 100 times, and determining the lethality to nematode.

1.4 determination of the biological Activity of plant parasitic nematodes

The specific steps of carrying out the in vitro bioassay on the plant parasitic nematodes by utilizing a 96-well plate are as follows:

(1) Sucking a certain amount of hatched nematode J2s into a 1.5ml centrifuge tube, centrifuging for 1min at 5000rpm, sucking the upper liquid by a pipette and discarding;

(2) Adding appropriate amount of ddH ₂ O resuspending the nematodes and determining the density of the larvae by microscopic examination；

(3) Preparing fermentation liquid to be detected with different concentrations, adding the fermentation liquid and the nematodes into corresponding holes (20-40J 2s per hole) in a 96-hole bioassay plate by using a liquid transfer device in a superclean workbench, and sterilizing ddH ₂ O is used as a blank control, and the total volume of each hole is 100 mu L; each treatment was repeated three times;

(4) Sealing a 96-hole bioassay plate with a sealing film, placing the bioassay plate in an incubator at 20 ℃, and counting the total number of nematodes in each hole and the death number after 4 hours.

1.5 Bacillus BMB2600 experiment for controlling tomato root-knot nematode

Shake flask culture fermentation in laboratory:

1) Tomato seedlings with consistent growth and size are selected as test seedlings. Inoculating 1000 second-instar larvae at the root of a tomato seedling, adding 20mL of bacterial suspension, and inoculating sterile water and abamectin with the same volume as the reference, and obtaining a Loufuchada diluent. After the treatment, the cells were left to stand for culture. And counting the number of root knots after 30-40 days. The pot experiment had 3 treatments in total, which were: CK. BMB2600 ICPM suspension, positive control (abamectin, lou fuda diluent), 3 replicates per treatment setup.

2) Counting the number of root knots: gently pull up the tomato plant, take out complete root system, avoid hindering the root as far as possible. Then the roots are washed under running water, and after the roots are washed clean, the root knots are visually counted. The number of root knots is expressed by adopting the number of root knots of each tomato plant.

Fermentation in an industrial fermentation tank:

1) The culture medium formula used for industrial fermentation is bacillus industrial fermentation culture medium ingredients, and the formula is calculated according to 1L of culture medium: 18g of soybean cake powder; 5g of corn flour; 36g of corn steep liquor; 5g of glucose; 7.5g of starch; 3g of fish meal; k ₂ HPO ₄ 2.16 g；MgSO ₄ ·7H ₂ O 0.75g；CaCO ₃ 1.5 g；(NH ₄ ) ₂ SO ₄ 2 g; supplementing distilled water to 1L; adjust pH to 7.0).

2) Inoculating according to 0.5% of the culture medium, and fermenting for 32-36h.

3) The plant used for potting is a golden greenhouse third tomato seedling with 3-4 weeks of growth and consistent size, 800-head root-knot second-instar larvae are inoculated at the root of the tomato seedling, and the treatment groups comprise 50 ml/plant, 150 ml/plant, 300 ml/plant industrial fermentation liquor and a sterile water control group. And (4) irrigating the soil with fermentation liquor before transplanting the tomato seedlings for one week, and counting the number of root knots after 30 days. Six replicates of each treatment were processed.

1.6 GC-MS (gas chromatography-Mass spectrometer) detection of strain metabolites in fermentation liquor

The treated and concentrated fermentation supernatant (the treatment method of the fermentation broth supernatant is the same as that in section 1.3 above) was subjected to mass identification by Agilent's GC-MS gas mass spectrometry using a DB-WAX polar column.

1.7 field test

The fermentation liquor used in the field test is the same as the industrial fermentation liquor used in the pot experiment, and is carried out in soil disease areas seriously infected by root knot nematodes in vegetable institute of agricultural science and research institute of Wuhan city, and the used plants are golden greenhouse third tomato seedlings with the same growth vigor and size. The fermentation liquor treatment group comprises 150 ml/plant, 300 ml/plant, CK (water) and 10% fosthiazate of chemical agent, the soil is treated by the fermentation liquor before the tomato seedlings are transplanted for one week, and the second pesticide application is carried out after 30 days. Each treatment was repeated 10 times, and the root-knot status and yield were counted for about 90 days.

2 results

2.1 The BMB2600 fermentation supernatant has high poisoning activity on root-knot nematode

As can be seen from figure 1, the fermentation supernatant of Bacillus BMB2600 has strong lethal effect on the second-instar larvae of Meloidogyne incognita (Mi) and Soy cyst nematode. The highest mortality rate of the fermentation stock solution to nematodes can reach 100%, and the mortality rate to nematodes is gradually reduced along with the increase of dilution times. But the 5-fold diluent still has stronger nematicidal activity, and the lethality rate to the nematodes is 55.1 percent

2.2 BMB2600 Strain growth Curve

As can be seen from FIG. 2, the strain is in the growth retardation stage within 0-6h, and the strain grows more slowly; the growth exponential phase is 6-12h, the strain grows exponentially, the strain enters the growth stable phase within 18-26h, and the strain amount is basically kept stable; after 26h, the thallus grows into a decline stage.

2.3 fermentation supernatant potting experiment with Strain

Treating the supernatant of the BMB2600 strain fermentation liquor to be a treatment group, taking Loufida and abamectin as a control group, taking an ICPM culture medium as CK, carrying out a potting experiment on each group, and counting the root knot number after 30-45 days. The experimental results are shown in fig. 3: compared with CK, the tomato root system root knot number is obviously reduced after the supernatant is treated by the shake flask fermentation liquor of the BMB2600 bacterial strain, which indicates that the insecticidal activity of the fermentation supernatant has certain influence on the infection and reproduction of nematodes in the plant growth process.

2.4 Industrial fermentation broth potting experiment

The fermentation broth (fermentation broth is not required to be treated and is directly applied) after the industrial fermentation of the BMB2600 strain comprises 50 ml/strain, 150 ml/strain and 300 ml/strain, and the potting experiment is carried out by taking sterile water as CK. After 30d, counting the number of the root knots. The results of the experiment are shown in FIG. 4: compared with the control, after the BMB2600 bacterial strain fermentation liquor is treated, the root knot number is obviously reduced after 150ml and 300ml treatment, which shows that the insecticidal activity of the industrial fermentation liquor inhibits the infection and the permanent planting of the root knot nematode to the plant, and is consistent with the pot experiment result of using the culture medium in a laboratory.

2.5 GC-MS results

After gas chromatography analysis, it was found that the strain BMB2600 co-produced acrylic acid, a small acid metabolite, in the fermentation (see FIG. 5).

2.6 field test

As shown in the field results of Table 1, in the field test, 300ml of BMB2600 industrial fermentation liquor per plant has the effect equivalent to that of 10% fosthiazate, can well inhibit the infection of root-knot nematodes, does not affect the yield of the plants, and has the effect of indirectly promoting the growth of the plants. The comparison between the treated group and the control group is shown in FIGS. 6 and 7.

TABLE 1 Effect of the treatment groups on root-knot nematodes and tomato yields

3 conclusion

The invention separates a strain with high poisoning activity on plant parasitic nematodes from greenhouse soil, which is named as Bacillus cereus BMB2600, and the supernatant of the fermentation liquor has strong lethal ability on Meloidogyne incognita and Soy cyst nematodes. And then, carrying out shake flask fermentation and industrial fermentation by using a culture medium in a laboratory, carrying out a potting experiment on the obtained fermentation liquor of the BMB2600 strain, and verifying the inhibition effect of the BMB2600 strain on nematode infection in practical application, wherein two potting results show that the fermentation liquor of the BMB2600 strain well controls the field planting of the root-knot nematodes and are verified in field experiments.

Claims

1. A Bacillus cereus (Bacillus cereus) BMB2600 with high activity of killing plant parasitic nematodes, which has a preservation number of CCTCC NO: m2022893.

2. Use of a Bacillus cereus BMB2600 as defined in claim 1 for controlling plant parasitic nematode diseases.

3. Use of a fermentation broth of Bacillus cereus (Bacillus cereus) BMB2600 as defined in claim 1 for the preparation of biopesticides and/or bioorganic fertilizers for controlling plant parasitic nematode diseases.

4. The use of claim 2 or 3, wherein the plant parasitic nematode is selected from one or more of the following: meloidogyne, cyst nematodes.

5. The use according to claim 4, wherein the Meloidogyne is Meloidogyne incognita and the Heterodera is Sojae atricolor.

6. A preparation having high poisoning activity against plant parasitic nematodes, wherein the active ingredient of the preparation is prepared from a fermentation broth of Bacillus cereus (Bacillus cereus) BMB2600 as defined in claim 1.