Application of Bacillus endophyticus fermentation broth in the control of psyllids

Abstract

This invention belongs to the field of microbial fermentation and its pest control technology, specifically involving the application of Bacillus velezensis fermentation broth in the control of psyllids. This invention discloses a Bacillus velezensis NM153, which was deposited at the China Center for Type Culture Collection on March 27, 2025, with accession number CCTCC M 2025605. Treatment of psyllids with the fermentation broth, supernatant, and precipitate of Bacillus velezensis NM153 showed that the fermentation broth, supernatant, and precipitate of Bacillus velezensis NM153 had a significant control effect on psyllids.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and pest control technology, specifically involving the application of Bacillus endophyticus fermentation broth in the control of psyllids. Background Technology

[0002] Psyllids belong to the class Insecta, order Hemiptera, suborder Psyllid. These insects lay their eggs extracellularly; the eggs are elongated and have an egg stalk. They are metamorphic insects, with five nymphal instars, and their individual development progresses through three stages: egg, nymph, and adult. Their bodies are flattened, with the terminal abdominal segments fused together. They often secrete wax and large amounts of honeydew, which covers the insect's body, making pesticide application difficult.

[0003] The rapid outbreak and spread of Huanglongbing (HLB) in citrus worldwide has severely hampered the development of the citrus industry. In 2020, the Ministry of Agriculture and Rural Affairs classified it as a Class I disease. HLB is a major citrus disease caused by the candidate genus *Candidatus Liberibacter* spp. In nature, this disease is primarily transmitted by the citrus psyllid *Diaphorinacitri*. Due to its extremely high reproductive rate and environmental adaptability, the citrus psyllid can rapidly and efficiently spread the HLB pathogen. Summary of the Invention

[0004] To address the technical problem of reducing the spread of citrus Huanglongbing (HLB), this invention provides the following technical solution:

[0005] The Bacillus velezensis NM153 involved in this invention was deposited at the China Center for Type Culture Collection on March 27, 2025, with accession number CCTCC M 2025605, and has been disclosed in CN121320142A.

[0006] This invention discloses a Bacillus velezensis NM153 fermentation broth, the preparation method of which is as follows:

[0007] (1) A single colony of Bacillus velezensis NM153 was inoculated into the liquid culture medium of Bacillus velezensis NM153 fermentation broth and activated by shaking culture to obtain activated Bacillus velezensis NM153; the Bacillus velezensis NM153 was deposited at the China Center for Type Culture Collection on March 27, 2025, with accession number CCTCC M 2025605;

[0008] (2) The activated Bacillus velezensis NM153 was inoculated into the Bacillus velezensis NM153 fermentation broth liquid culture medium for fermentation to obtain the Bacillus velezensis NM153 fermentation broth.

[0009] Preferably, the preparation method of the Bacillus velezensis NM153 fermentation broth liquid culture medium in steps (1) and (2) is as follows: weigh 8-12 g / L of tryptone, 4-7 g / L of yeast extract, 8-12 g / L of sodium chloride, 20 nmol / L of PQQ and 10 μg / L of ferrous sulfate monohydrate, and autoclave at 121℃ for 15-25 min; in step (1), the colony volume ratio of Bacillus velezensis NM153 to Bacillus velezensis NM153 fermentation broth liquid culture medium is 1:200-300.

[0010] Preferably, the conditions for the shake culture activation in step (1) are 28-35℃, 170-250r / min, and culture for 24-48h.

[0011] Preferably, in step (2), the inoculum amount of Bacillus velezensis NM153 is 2-4% of the volume of the Bacillus velezensis NM153 fermentation broth liquid culture medium.

[0012] Preferably, the fermentation conditions in step (2) are: the shaker is set to 30-35℃, 170-250r / min, and cultured for 48-72h.

[0013] Preferably, in step (2), the Bacillus velezensis NM153 fermentation broth is centrifuged to obtain a supernatant and a precipitate, wherein the concentration of Bacillus velezensis NM153 in the Bacillus velezensis NM153 fermentation broth is 2.2-3.0 × 10⁻⁶. 10cfu / ml; the supernatant contains undecyl phthalate and 4-hydroxybenzonitrile as active ingredients, wherein the mass ratio of undecyl phthalate to 4-hydroxybenzonitrile is 1:1-6, and the total mass of undecyl phthalate and 4-hydroxybenzonitrile is 1-4% of the mass of Bacillus velezensis NM153 fermentation broth.

[0014] The application of a Bacillus velezensis NM153 fermentation broth in the control of psyllids.

[0015] The application of a Bacillus velezensis NM153 fermentation broth in the preparation of microbial agents or biopesticides for controlling psyllids.

[0016] The beneficial effects of this invention are:

[0017] This application describes the treatment of psyllids with fermentation broth, supernatant, and precipitate of Bacillus vesicularis NM153, diluted 10 times. The results show that fermentation broth, supernatant, and precipitate of Bacillus vesicularis NM153, diluted 10 times, have a significant technical control effect on psyllids. Attached Figure Description

[0018] Figure 1 The effects of Bacillus vesiculosus NM153 fermentation broth, supernatant, and precipitate on psyllid control were investigated. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0020] Example 1: Preparation of fermentation broth of Bacillus velezensis NM153

[0021] A fermentation broth of Bacillus velezensis NM153, wherein the preparation method of the Bacillus velezensis NM153 fermentation broth is as follows:

[0022] (1) A single colony of Bacillus velezensis NM153 was inoculated into the liquid culture medium of Bacillus velezensis NM153 fermentation broth and cultured at 28-35℃ and 170-250 r / min for 24-48 h for activation culture. The volume ratio of Bacillus velezensis NM153 to Bacillus velezensis NM153 fermentation broth liquid culture medium was 1:200-300 to obtain activated Bacillus velezensis NM153. The Bacillus velezensis NM153 was deposited at the China Center for Type Culture Collection on March 27, 2025, with the accession number CCTCC. M2025605; The preparation method of liquid culture medium is as follows: Weigh 8-12 g / L tryptone, 4-7 g / L yeast extract, 8-12 g / L sodium chloride, 20 nmol / L PQQ and 10 μg / L ferrous sulfate monohydrate, and autoclave at 121℃ for 15-25 min.

[0023] (2) The activated Bacillus velezensis NM153 was inoculated into the Bacillus velezensis NM153 fermentation broth liquid culture medium for fermentation. The inoculation amount was 2-4% of the volume of the Bacillus velezensis NM153 fermentation broth liquid culture medium. The culture was carried out at 30-35℃ and 170-250r / min for 48-72h to obtain the Bacillus velezensis NM153 fermentation broth.

[0024] The concentration of Bacillus velezensis NM153 in the fermentation broth was 2.2-3.0 × 10⁻⁶. 10The fermentation broth was centrifuged to obtain supernatant and precipitate. Identification revealed that the supernatant of the Bacillus velezensis NM153 fermentation broth contained undecyl phthalate and 4-hydroxybenzonitrile, with a mass ratio of undecyl phthalate to 4-hydroxybenzonitrile of 1:1-6. The total mass fraction of undecyl phthalate and 4-hydroxybenzonitrile in the Bacillus velezensis NM153 fermentation broth was 1-4%. The CAS registry numbers for undecyl phthalate and 4-hydroxybenzonitrile are 96507-86-7 and 767-00-0, respectively.

[0025] Example 3: Application of Bacillus belyss NM153 fermentation broth (stock solution), supernatant, and precipitate

[0026] Experimental Methods: The experiment included the following treatments: *Bacillus vesiculosus* NM153 fermentation broth (30 billion CFU / ml, excluding metabolites), *Bacillus vesiculosus* NM153 fermentation broth (30 billion CFU / ml, excluding metabolites) diluted 10-fold, supernatant (excluding bacterial metabolites), supernatant diluted 10-fold, precipitate (excluding bacterial cells), and precipitate (excluding bacterial cells) diluted 10-fold. Each treatment contained 25 citrus psyllids, with 3 replicates. The specific method was as follows: Absorbent paper towels were completely immersed in each treatment solution for 15 seconds. After removal, excess liquid was drained, and the paper towels were laid flat on the bottom of transparent bioassay cups, ensuring complete coverage. It was ensured that the test insects could not burrow into the gaps between the paper towels and the cup walls. After laying the paper towels, any contact point between the test insects and the surface was considered the drug-loaded surface. 25 healthy, uniformly sized adult psyllids were introduced into each cup. The cup opening was covered with a transparent, breathable sealing film (with microporous membrane) to prevent escape while maintaining air permeability. The experimental conditions and observations were conducted under constant temperature and humidity. The number of dead psyllids was observed and recorded after 12 hours. The experimental results for each treatment group were observed and recorded 12 hours after treatment.

[0027] Results Tables 1-3 and Figure 1 The results showed that the fermentation broth, supernatant, and precipitate of *Bacillus belye* NM153 exhibited significant insecticidal activity against psyllids after a 10-fold dilution. Figure 1 As shown in the figure, a large number of psyllids died after about 12 hours of treatment, indicating that the supernatant has good rapid-acting properties and is suitable for practical application scenarios that require rapid control of pests.

[0028] To further confirm that the active ingredients (undecyl phthalate and 4-hydroxybenzonitrile) in the metabolites are key factors affecting insecticidal efficacy, undecyl phthalate and 4-hydroxybenzonitrile were mixed at a mass ratio of 1:2, and verification experiments were conducted according to the method described above. The results are shown in Table 4. The mixture of Bacillus belye containing undecyl phthalate and 4-hydroxybenzonitrile effectively inhibited psyllids in a short time, demonstrating its potential for development into a highly effective pesticide.

[0029] Table 1. Mortality rate of psyllids controlled by fermentation broth of Bacillus belyss NM153

[0030]

[0031] Table 2 Mortality rate of psyllids controlled by supernatant of Bacillus belyss NM153

[0032]

[0033] Table 3. Mortality of psyllids controlled by Bacillus belysin NM153 precipitate.

[0034]

[0035] Table 4. Mortality rates of psyllids controlled by undecyl phthalate and 4-hydroxybenzonitrile

[0036]

[0037] Note: In Tables 1-4, the number before the slash represents the number of psyllids that died, and the number after the slash represents the total number of psyllids.

Claims

1. A fermentation broth of Bacillus velezensis NM153, characterized in that, The preparation method of the Bacillus velezensis NM153 fermentation broth is as follows: (1) A single colony of Bacillus velezensis NM153 was inoculated into the liquid culture medium of Bacillus velezensis NM153 fermentation broth and activated by shaking culture to obtain activated Bacillus velezensis NM153; the Bacillus velezensis NM153 was deposited at the China Center for Type Culture Collection on March 27, 2025, with accession number CCTCC M 2025605; (2) The activated Bacillus velezensis NM153 was inoculated into the Bacillus velezensis NM153 fermentation broth liquid culture medium for fermentation to obtain the Bacillus velezensis NM153 fermentation broth.

2. The Bacillus velezensis NM153 fermentation broth according to claim 1, characterized in that, The preparation method of the Bacillus velezensis NM153 fermentation broth liquid culture medium in steps (1) and (2) is as follows: weigh 8-12 g / L of tryptone, 4-7 g / L of yeast extract, 8-12 g / L of sodium chloride, 20 nmol / L of PQQ and 10 μg / L of ferrous sulfate monohydrate, and autoclave at 121℃ for 15-25 min; in step (1), the colony volume ratio of Bacillus velezensis NM153 to Bacillus velezensis NM153 fermentation broth liquid culture medium is 1:200-300.

3. The Bacillus velezensis NM153 fermentation broth according to claim 1, characterized in that, The conditions for the shake culture activation in step (1) are 28-35℃, 170-250r / min, and culture for 24-48h.

4. The Bacillus velezensis NM153 fermentation broth according to claim 1, characterized in that, In step (2), the inoculum amount of Bacillus velezensis NM153 is 2-4% of the volume of the Bacillus velezensis NM153 fermentation broth liquid culture medium.

5. The Bacillus velezensis NM153 fermentation broth according to claim 1, characterized in that, The fermentation conditions described in step (2) are: the shaker is set to 30-35℃, 170-250r / min, and cultured for 48-72h.

6. The Bacillus velezensis NM153 fermentation broth according to claim 1, characterized in that, In step (2), the Bacillus velezensis NM153 fermentation broth is centrifuged to obtain supernatant and precipitate. The concentration of Bacillus velezensis NM153 in the Bacillus velezensis NM153 fermentation broth is 2.2-3.0 × 10⁻⁶. 10 cfu / ml; the supernatant contains undecyl phthalate and 4-hydroxybenzonitrile as active ingredients, the mass ratio of undecyl phthalate to 4-hydroxybenzonitrile is 1:1-6, and the total mass of undecyl phthalate and 4-hydroxybenzonitrile is 1-4% of the mass of Bacillus velezensis NM153 fermentation broth.

7. The use of Bacillus velezensis NM153 fermentation broth as described in any one of claims 1-6 in the control of psyllids.

8. The use of Bacillus velezensis NM153 fermentation broth as described in any one of claims 1-6 in the preparation of microbial agents or biological pesticides for controlling psyllids.

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