Bacillus cereus with high toxicity to plant parasitic nematodes and application thereof

Abstract

The application belongs to the technical field of microorganisms, and provides a bacillus cereus with high toxic killing activity to plant parasitic nematodes and application thereof. The strain has been preserved in the China Typical Culture Collection Center of Wuhan University in Hongshan District of Wuhan City in Hubei Province on June 15, 2022, and the preservation number is CCTCC NO: M2022893. The isolated strain of the application has high toxic killing effect on root-knot nematodes such as southern root-knot nematode and cyst nematodes such as soybean cyst nematode, and can be used for biological control of root-knot nematode and cyst nematode diseases.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and more specifically, relates to a new strain, particularly to a Bacillus cereus with high toxicity against plant parasitic nematodes and its application. Background Technology

[0002] Nematodes are one of the largest phyla in the animal kingdom, with enormous abundance. They are ubiquitous in soil and freshwater environments, and even exist in extreme conditions. They can parasitize plants and animals or live freely in soil and freshwater. Currently, the genus *Root-knot nematodes* ranks first among the ten most harmful plant parasitic nematodes in the world. Root-knot nematodes have a very wide host range, infecting almost all fruits and vegetables. Plants infected with root-knot nematodes will develop root knots, leading to stunted growth, yellowing leaves, and even plant death. Root-knot nematode diseases seriously threaten agricultural production, causing significant yield reductions in economic crops.

[0003] Root-knot nematodes are mainly distributed in the top 3-10 cm of soil. Existing control methods mainly include crop rotation, burning, flooding, and high-temperature fumigation during crop change to eradicate nematodes, but these methods cannot achieve complete eradication and still cause some economic losses for farmers. Currently, the most widely used method is to spray the soil with chemical pesticides before planting to achieve early prevention. However, this method requires a high dosage of chemical pesticides, and long-term use of chemical pesticides not only affects soil quality but also causes nematodes to develop resistance to pesticides, leading to increasingly poor control effects.

[0004] Currently, biopesticides have become a research hotspot for new pesticides due to their advantages such as safety, low toxicity, good efficacy, low likelihood of inducing pest resistance, and environmental friendliness. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the first objective of the present invention is to provide a biocontrol bacterium with high toxicity against plant parasitic nematodes. This strain has a high toxicity against root-knot nematodes such as Southern root-knot nematode and can be used for the biological control of root-knot nematode diseases.

[0006] To achieve the aforementioned technical objectives, the inventors unexpectedly discovered that nematodes bred in potted tomatoes grown under repeated cropping conditions in a greenhouse experienced mass mortality after hatching. Based on this discovery, the inventors further isolated a strain with high toxicity against resident plant-parasitic nematodes. Through molecular biology experiments and genome sequencing, this strain was identified as belonging to the genus Bacillus, specifically Bacillus cereus, and named BMB2600. This strain was deposited on June 15, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Hongshan District, Wuhan City, Hubei Province, China, with accession number CCTCC NO: M 2022893.

[0007] It should be noted that the Bacillus cereus BMB2600 isolated in this invention has the following morphological and cultural characteristics:

[0008] Morphological characteristics: Bacillus cereus BMB2600 is a Gram-positive bacillus, 1.0–1.2 × 3.0–5.0 μm in size, facultatively aerobic, producing spores that are round or cylindrical. The bacterial cells have relatively smooth ends and mostly form chains. Colonies are large, with a rough, flat, and irregular surface.

[0009] Culture characteristics: After fermentation in a glucose-containing medium, this strain can produce the metabolite acrylic acid, and the fermentation broth has high insecticidal activity against plant parasitic nematodes, especially root-knot nematodes and cyst nematodes.

[0010] Furthermore, a second objective of this invention is to provide the application of a strain of Bacillus cereus, specifically the application of the aforementioned Bacillus cereus (BMB2600) in the control of plant parasitic nematode diseases. Also, the application of the fermentation broth of the aforementioned Bacillus cereus in the preparation of biopesticides and / or bio-organic fertilizers for the control of plant parasitic nematode diseases. Preferably, the plant parasitic nematode is selected from one or more of the following: root-knot nematodes and cyst nematodes. More preferably, the root-knot nematode is *Strombus heterophylla*, and the cyst nematode is *Strombus sacchariformis*.

[0011] Finally, a third objective of the present invention is to provide a formulation with high toxicity against plant parasitic nematodes, the active ingredient of which is prepared from the fermentation broth of the aforementioned Bacillus cereus BMB2600.

[0012] Compared with the prior art, the Bacillus cereus BMB2600 provided by the present invention has the following advantages and significant progress:

[0013] (1) This strain has a high toxicity to southern root-knot nematode and soybean cyst nematode.

[0014] (2) Compared with the slow-acting nature of biological pesticides, the fermentation broth of this strain showed a good killing effect on southern root-knot nematodes and soybean cyst nematodes within 60 minutes when the bioactivity was tested.

[0015] (3) This strain can produce acrylic acid, a compound with high toxicity to plant parasitic nematodes, during fermentation.

[0016] (4) The industrial fermentation broth of strain BMB2600 of the present invention was applied to tomato seedlings severely infested with root-knot nematodes. The results showed that it had a good control effect on root-knot nematodes in the field trial, which was comparable to the effect of chemical pesticides. Attached Figure Description

[0017] Figure 1 The graph shows the nematicidal activity curves of fermentation supernatants at different concentrations, where Mi represents Southern Root-knot Nematode; Hg represents Soybean Cyst Nematode; Dd represents Stem Rot Nematode; and Ce represents Caenorhabditis elegans.

[0018] Figure 2 The growth curve of Bacillus cereus strain BMB2600 is shown.

[0019] Figure 3 The effect of shake-flask fermentation broth of Bacillus cereus strain BMB2600 on the number of root knots in potted tomato roots.

[0020] Figure 4 The effect of industrial fermentation broth of Bacillus cereus strain BMB2600 on the number of root knots in potted tomato roots (* for p<0.05, *** for p<0.001).

[0021] Figure 5 This is a GC-MS image of acrylic acid, a metabolite of Bacillus cereus BMB2600.

[0022] Figure 6 Tomato root knots in a field experiment; top: control group; bottom: treatment group.

[0023] Figure 7 Photographs of tomato roots from the treatment groups in a field experiment. Detailed Implementation

[0024] The present invention will be further described in detail below through specific embodiments. Where specific technical operation steps or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0025] Example 1: Discovery process and activity verification experiment of Bacillus cereus strain BMB2600

[0026] 1. Experimental Methods

[0027] 1.1 Culture and hatching of root-knot nematodes

[0028] 1) Cultivation and egg hatching of root-knot nematodes under greenhouse conditions

[0029] (1) To plant susceptible tomato varieties, seeds can be directly sown into a mixture of nutrient soil and sand in a 2:1 volume ratio and allowed to germinate under suitable conditions.

[0030] (2) Pull the tomato seedlings that have grown to about 10cm tall out of the soil and wash the soil off the roots with running water.

[0031] (3) Use a pipette to inject the southern root-knot nematodes onto the clean root surface, and then transplant them into the soil of a large pot. The inoculation amount per pot is about 2,000-3,000 nematodes.

[0032] (4) After about 30-45 days, when the root system of the tomato is infected to a suitable degree by root-knot nematodes, the diseased plants can be pulled up and the egg masses can be picked out.

[0033] (5) First, pick the egg mass into a bottle containing sterilized ddH2O and place it in a 20℃ incubator for 3 days. If the time is extended appropriately, more root-knot nematodes J2s can be harvested.

[0034] (6) If you want to get cleaner root-knot nematodes J2s, you can select tomato roots that are severely infected by root-knot nematodes and have large and numerous root knots, and cut them into pieces as much as possible.

[0035] ① Immerse it completely in a 1% sodium hypochlorite solution and stir for about 8 minutes.

[0036] ② Pass the suspension through a series of sieves with a mesh size of 60, 200, 100 and 500 mesh respectively. The scattered root-knot nematode eggs can be collected on the 500-mesh sieve.

[0037] ③ The collected root-knot nematode egg suspension was incubated on a 500-mesh membrane and placed in a 20℃ incubator for 3 days. The incubation time can be extended appropriately to harvest more root-knot nematodes J2s.

[0038] 1.2 Bacterial Culture Methods and Culture Media

[0039] Strain activation: Transfer the strain preserved on slant agar plates to LB plates and incubate at 37°C for 24 hours for later use; Preparation of seed culture: Pick one loopful of activated strain and inoculate it into 50 mL of LB liquid medium, and incubate at 37°C with shaking at 220 rpm for 12 hours for later use; Fermentation broth shake flask culture: Inoculate the seed culture into the fermentation medium at an inoculation rate of 1%, and incubate at 37°C with shaking at 220 rpm for the specified time.

[0040] Seed culture medium (LB liquid medium): yeast extract 0.5%, peptone 1%, NaCl 1%, pH 7.0, sterilized at 121℃ for 30 min.

[0041] Culture medium for preserving microorganisms (LB solid medium): 0.5% yeast extract, 1% peptone, 1% NaCl, 2% agar, pH 7.0, sterilized at 121℃ for 30 min.

[0042] Fermentation medium: ICPM medium: glucose 0.5%, peptone 0.5%, KH2PO4 0.05%, MgSO4 0.05%, sterilized at 115℃ for 20 min.

[0043] 1.3 Fermentation Broth Treatment

[0044] The fermentation broth was centrifuged at 4℃ and 10,000 rpm for 15 min. The supernatant was then collected and filtered through a 0.22 μm pore size filter to obtain the supernatant. The fermentation supernatant was then diluted 2, 5, 10, and 100 times to determine the lethality against nematodes.

[0045] 1.4 Determination of the bioactivity of plant-parasitic nematodes

[0046] The specific steps for in vitro bioassays of plant parasitic nematodes using a 96-well plate are as follows:

[0047] (1) Take a certain number of hatched nematodes J2s into a 1.5ml centrifuge tube, centrifuge at 5000rpm for 1min, and use a pipette to remove and discard the upper liquid.

[0048] (2) Add an appropriate amount of ddH2O to resuspend the nematodes and examine them under a microscope to determine the nematode density.

[0049] (3) Prepare different concentrations of the fermentation broth to be tested. In a clean bench, use a pipette to add the fermentation broth and nematodes to the corresponding wells of the 96-well bioassay plate (20-40 J2s per well). Use sterile ddH2O as a blank control. The total volume of each well is 100 μL. Each treatment is repeated three times.

[0050] (4) After sealing the 96-well bioassay plate with sealing film, place it in a 20℃ incubator. After 4 hours, count the total number of nematodes and the number of dead nematodes in each well.

[0051] 1.5 Experiment on the control of tomato root-knot nematodes by Bacillus spp. BMB2600

[0052] Laboratory shake-flask fermentation:

[0053] 1) Select tomato seedlings of uniform growth and size as test seedlings. Inoculate the roots of the tomato seedlings with 1000 second-instar larvae, adding 20 mL of bacterial suspension. The control is inoculated with an equal volume of sterile water and diluted avermectin and levofloxacin. After treatment, place them in the culture. The number of root knots is counted after 30-40 days. The pot experiment has 3 treatments: CK, BMB2600 ICPM bacterial suspension, and positive control (avermectin and diluted levofloxacin), with 3 replicates for each treatment.

[0054] 2) Counting the number of root knots: Gently pull up the tomato plant and remove the entire root system, avoiding root damage as much as possible. Then rinse the roots under running water, and after rinsing them clean, visually count the number of root knots. The number of root knots is expressed as the number of root knots per tomato plant.

[0055] Industrial fermentation tank fermentation:

[0056] 1) The culture medium formula used for industrial fermentation is the Bacillus industrial fermentation culture medium formula. Calculated per 1L of culture medium, the formula is: 18g soybean meal powder; 5g corn flour; 36g corn steep liquor; 5g glucose; 7.5g starch; 3g fish meal; 2.16g K2HPO4; 0.75g MgSO4·7H2O; 1.5g CaCO3; 2g (NH4)2SO4; add distilled water to 1L; adjust pH to 7.0).

[0057] 2) Inoculate at 0.5% of the culture medium mass and ferment for 32-36 hours.

[0058] 3) The potted plants were Jinpeng No. 3 tomato seedlings of uniform size and growth, 3-4 weeks old. 800 second-instar root-knot larvae were inoculated at the base of the tomato seedlings. Treatment groups included 50ml / plant, 150ml / plant, and 300ml / plant industrial fermentation liquid, and a control group of sterile water. The soil was treated with the fermentation liquid one week before transplanting, and the number of root knots was counted 30 days later. Each treatment was replicated six times.

[0059] 1.6 GC-MS Detection of Strains' Metabolites in Fermentation Broth

[0060] The processed and concentrated fermentation supernatant (the fermentation broth supernatant was processed in the same way as described in Section 1.3 above) was identified by Agilent GC-MS gas chromatography-mass spectrometry using a DB-WAX polar column.

[0061] 1.7 Field Trials

[0062] The fermentation broth used in the field experiment was the same as the industrial fermentation broth used in the pot experiment. It was conducted in a soil disease area severely infested with root-knot nematodes at the Vegetable Research Institute of Wuhan Academy of Agricultural Sciences. The plants used were Jinpeng No. 3 tomato seedlings of uniform size and growth. The fermentation broth treatment groups included 150 ml / plant, 300 ml / plant, control (CK) (water), and the chemical agent 10% thiazophos. The soil was treated with the fermentation broth one week before transplanting the tomato seedlings, and a second application was made 30 days later. Each treatment had 10 replicates, and root knot characteristics and yield were recorded approximately 90 days later.

[0063] 2 Results

[0064] 2.1 The fermentation supernatant of BMB2600 has high toxicity against root-knot nematodes.

[0065] Depend on Figure 1 It can be seen that the fermentation supernatant of Bacillus BMB2600 has a strong lethal effect on the second instar larvae of the southern root-knot nematode (Mi) and the soybean cyst nematode. The original fermentation broth showed the highest lethality against nematodes, reaching 100%. The lethality gradually decreased with increasing dilution. However, the 5-fold dilution still exhibited strong nematicidal activity, with a lethality of 55.1% against nematodes.

[0066] 2.2 Growth curve of BMB2600 strain

[0067] Depend on Figure 2 It can be seen that the strain is in the lag phase from 0 to 6 hours, during which the strain grows relatively slowly; from 6 to 12 hours, it is in the exponential growth phase, during which the strain exhibits exponential growth; from 18 to 26 hours, it enters the stationary growth phase, during which the bacterial count remains relatively stable; after 26 hours, the bacterial growth enters the decline phase.

[0068] 2.3 Pot Experiment of Fermentation Supernatant of Strains

[0069] The supernatant of the BMB2600 strain fermentation broth was used as the treatment group, while Lufida and Avermectin were used as control groups. ICPM medium was used as the control (CK). Pot experiments were conducted in each group, and the number of root knots was counted after 30-45 days. The experimental results are as follows: Figure 3 As shown, compared with the control (CK), the number of root knots in tomato roots was significantly reduced after treatment with the supernatant of the shake-flask fermentation broth of strain BMB2600, indicating that the insecticidal activity of the fermentation supernatant had a certain impact on the infection and reproduction of nematodes during plant growth.

[0070] 2.4 Industrial Fermentation Broth Pot Experiment

[0071] The fermentation broth from industrial fermentation of strain BMB2600 (no treatment required, applied directly) was used in pot experiments with treatments including 50ml / strain, 150ml / strain, and 300ml / strain, with sterile water as the control (CK). Root knot counts were recorded after 30 days. Experimental results are as follows: Figure 4As shown: Compared with the control, the number of root knots was significantly reduced after treatment with 150ml and 300ml of BMB2600 strain fermentation broth, indicating that the insecticidal activity of the industrial fermentation broth inhibited the infection and colonization of root-knot nematodes on the plants, which is consistent with the results of pot experiments using laboratory culture medium.

[0072] 2.5 GC-MS Results

[0073] Gas chromatography analysis revealed that strain BMB2600 produced a small molecule acid metabolite—acrylic acid—during fermentation (see [link to gas chromatography]). Figure 5 ).

[0074] 2.6 Field Trials

[0075] Table 1 shows the field results. In the field trial, 300 ml of BMB2600 industrial fermentation broth per plant was comparable in effect to 10% thiazophos, effectively inhibiting root-knot nematode infestation without affecting plant yield, and indirectly promoting plant growth. The comparison of root knots between the treatment group and the control group is shown below. Figure 6 , Figure 7 .

[0076] Table 1. Imitation of root-knot nematodes and tomato yield in each treatment group.

[0077]

[0078] 3. Conclusion

[0079] This invention isolates a strain of Bacillus cereus with high toxicity against plant-parasitic nematodes from greenhouse soil, named Bacillus cereus BMB2600. The supernatant of its fermentation broth exhibits strong lethality against both southern root-knot nematodes and soybean cyst nematodes. Subsequently, using laboratory shake-flask fermentation and industrial fermentation, the fermentation broth of strain BMB2600 was used in pot experiments to verify its inhibitory effect on nematode infection in practical applications. Both pot experiment results showed that the fermentation broth of strain BMB2600 effectively controlled the colonization of root-knot nematodes, a finding also validated in field trials.

Claims

1. A Bacillus cereus species with high toxicity against plant parasitic nematodes (Bacillus cereus) Bacillus cereus BMB2600, with accession number CCTCC NO: M 2022893 at the China Center for Type Culture Collection.

2. The Bacillus cereus of claim 1 ( Bacillus cereus Application of BMB2600 in the control of plant parasitic nematode diseases; the plant parasitic nematodes are selected from one or more of the following: root-knot nematodes and cyst nematodes; the root-knot nematode is Southern Root-knot Nematode, and the cyst nematode is Soybean Cyst Nematode.

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