Bacillus megaterium with multiple functions and application thereof

Abstract

The application belongs to the field of crop prevention technology and pesticide residue management, and discloses a bacillus megaterium with multiple functions and application thereof. Bacillus megaterium The bacillus megaterium is bacillus megaterium (ZIGUI 6588) with a preservation number of CCTCC NO: M20242075, and the strain can be used for preventing and controlling citrus penicillium disease, root knot nematode and the like, and has the function of degrading fruit and soil abamectin, and has a wide application prospect in agricultural production.

Description

Technical Field

[0001] This invention belongs to the field of crop pest control technology and pesticide residue management, specifically involving a multifunctional Bacillus megaterium and its applications. Background Technology

[0002] Citrus, also known as mandarin orange, orange, and tangerine, is a small tree belonging to the genus Citrus in the family Rutaceae. Citrus cultivation is widespread throughout the world and is a pillar industry of agriculture in many regions.

[0003] Postharvest blue mold and root-knot nematode disease are major diseases of citrus, seriously threatening the development of the citrus industry. Blue mold is a disease that primarily affects citrus fruit, typically occurring on fruit ripening in the field and during storage. Symptoms include softening of the peel, water-soaked discoloration, and easy cracking under light pressure. Subsequently, numerous aerial hyphae grow from the center of the lesions, forming a thick layer of white mold that rapidly expands into nearly circular white mold spots. Then, bluish or green powdery substances, namely conidiophores and conidia, grow from the center of the mold spots. The disease develops rapidly, spreading to the entire fruit and causing wet rot within days. Root-knot nematode disease is caused by microorganisms that damage the plant's roots and stems, forming root nodules of varying sizes on the root tips. These nodules are spindle-shaped or irregular, ranging in size from a sesame seed to a mung bean, initially milky white, later turning yellowish-brown to dark brown, with sparse and small root hairs. Chemical agents are commonly used in production to control these two diseases. However, the frequent use of chemical agents leads to problems such as pesticide residues, resistance, and environmental pollution. There is an urgent need for safer and more effective control methods in production. Biological control has been widely used in recent years due to its advantages such as safety, no residue, and low likelihood of developing resistance.

[0004] Avermectin is a common insecticide used to control spider mites on crops, and it is widely used in citrus production. This results in pesticide residues in citrus fruits and soil. Currently, the main methods for treating antibiotics include physical adsorption, advanced oxidation, and microbial degradation. Due to increasing environmental awareness, microbial degradation has become a preferred technology for researchers. To date, reported microorganisms capable of degrading avermectin include Bacillus subtilis, Acinetobacter lwoffi, Shigella, and white-rot fungi. These typically utilize macrolide degrading enzymes produced through metabolism to degrade avermectin.

[0005] To meet production needs, this invention provides a multifunctional Bacillus megaterium strain that can control citrus Penicillium rot and root-knot nematode diseases, while also degrading abamectin in fruits and soil. Summary of the Invention

[0006] The purpose of this invention is to provide an isolated Bacillus species, namely Bacillus megaterium ZIGUI6588, with accession number CCTCC NO: M20242075.

[0007] Another objective of this invention is to provide the application of Bacillus megaterium ZIGUI6588 in the preparation of microbial inoculants for citrus cultivation.

[0008] To achieve the above objectives, the present invention adopts the following technical measures:

[0009] The applicant isolated a strain of Bacillus from the rhizosphere soil of citrus plants in a citrus field in Zigui County. The strain was identified as Bacillus megaterium and deposited at the China Center for Type Culture Collection (CCTCC) on September 25, 2024. The classification name is Bacillus megaterium ZIGUI6588; the accession number is CCTCC NO:M20242075; and the location is Wuhan University, Wuhan, China.

[0010] Bacillus megaterium ZIGUI6588 grew well on LA medium, exhibiting a pale yellow colony appearance. Figure 1 ).

[0011] The scope of protection of this invention includes:

[0012] Fermentation broth of Bacillus megaterium ZIGUI6588, wherein the fermentation broth contains live Bacillus megaterium ZIGUI6588.

[0013] A composition containing Bacillus megaterium ZIGUI6588 or its fermentation broth.

[0014] The application of Bacillus megaterium ZIGUI6588, fermentation broth of Bacillus megaterium ZIGUI6588, or a composition containing Bacillus megaterium ZIGUI6588 or its fermentation broth in the control of citrus diseases; the diseases include citrus blue mold and / or citrus root-knot nematode disease.

[0015] The preferred application of the above-described control method includes: applying Bacillus megaterium ZIGUI6588, the fermentation broth of Bacillus megaterium ZIGUI6588, or a combination of Bacillus megaterium ZIGUI6588 or its fermentation broth to the roots of citrus trees through root irrigation or direct spraying.

[0016] The use of Bacillus megaterium ZIGUI6588, fermentation broth of Bacillus megaterium ZIGUI6588, or a composition containing Bacillus megaterium ZIGUI6588 or its fermentation broth in the preparation of citrus biocontrol agents.

[0017] The use of Bacillus megaterium ZIGUI6588, fermentation broth of Bacillus megaterium ZIGUI6588, or a composition containing Bacillus megaterium ZIGUI6588 or its fermentation broth in the degradation of avermectin.

[0018] Application of Bacillus megaterium ZIGUI6588, fermentation broth of Bacillus megaterium ZIGUI6588, or a composition containing Bacillus megaterium ZIGUI6588 or its fermentation broth in citrus cultivation.

[0019] The preferred application described above is avermectin for degrading the surface of citrus soil or fruit.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) The present invention provides a Bacillus megaterium. Compared with the prior art, the Bacillus megaterium provided by the present invention has both antibacterial and antinematode biocontrol functions and has no cross-resistance with existing fungicides and insecticides.

[0022] (2) Compared with the current use of chemically synthesized pesticides for pest control, it has the advantages of being highly efficient, low in toxicity, not polluting the environment, and not easily developing resistance, because it comes from the natural environment.

[0023] (3) The Bacillus megaterium screened in this invention also has the ability to degrade avermectin. Attached Figure Description

[0024] Figure 1 Colony morphology of strain ZIGUI6588.

[0025] Figure 2 ZIGUI6588 phylogenetic tree

[0026] Figure 3 Schematic diagram of ZIGUI6588 for controlling Penicillium citrus. Detailed Implementation

[0027] To better explain the present invention, the main contents of the present invention are further illustrated below with reference to specific embodiments, but the contents of the present invention are not limited to the following embodiments. Unless otherwise specified, the technical solutions described in the present invention are conventional techniques in the art, and the reagents or materials described, unless otherwise specified, are all from commercial sources.

[0028] Example 1:

[0029] Obtaining Bacillus megaterium ZIGUI6588:

[0030] The applicant isolated a Bacillus strain from the rhizosphere soil of healthy citrus plants. After 16S sequencing and physiological and biochemical identification, it was determined to be Bacillus megaterium. Figure 1 (Table 1) was deposited at the China Center for Type Culture Collection on September 25, 2024, and classified as: Bacillus megaterium ZIGUI6588; accession number: CCTCC NO: M20242075; address: Wuhan University, Wuhan, China.

[0031] In this invention, Bacillus megaterium is referred to as ZIGUI6588.

[0032] Morphological characteristics: pale yellow, flat, with round colonies.

[0033] Physiological and biochemical characteristics of the strain: as shown in Table 1

[0034] Table 1: Physiological and biochemical characteristics of strain ZIGUI6588

[0035] .

[0036] Example 2:

[0037] Fermentation of Bacillus megaterium ZIGUI6588 (all percentages in the following culture medium formulations are by mass):

[0038] Seed culture preparation: ZIGUI6588 strain was activated from slant culture, and a single colony was inoculated into NB liquid medium at a volume of 0.6L / 3L. The culture was then incubated at 28℃ and 180r / min for 18h before use.

[0039] Fermentation was carried out in a 10,000L tank. The culture medium consisted of 3% sucrose, 1.5% corn flour, 0.3% MgCl2, 0.1% MgCl2, 0.5% peptone, and the remainder water. The sample loading volume was 5,000L, the initial pH was 7.5, the culture temperature was 32℃, 2.5L of seed culture was inoculated, and the aeration rate was 180m³ / h. 3 / h, after 40h of fermentation, the number of spores in the fermentation broth was measured to be 34×10 8 cfu / ml.

[0040] Example 3:

[0041] The control effect of Bacillus megaterium ZIGUI6588 on Penicillium mold in citrus:

[0042] After the citrus pathogen (Penicillium italicum) was cultured at 28℃ for 10 days to produce spores, it was adjusted to a concentration of 1×10⁻⁶ with sterile water. 6Prepare spores per ml. Take fresh citrus fruits and make a 3mm × 3mm incision in the middle of the fruit using a scalpel. Add 20μL of ZIGUI6588 fermentation broth diluted 50 times to the incision. The control is treated with water. After air drying, add 10μL of spore suspension of Penicillium citrinum pathogen. Each treatment is replicated 3 times, with 15 fruits per replicate. Incubate at 25℃ with humidity. After 7 days of inoculation, count the diameter of lesions in the control and treatment, and calculate the control efficacy.

[0043] Control efficacy = (diameter of control lesion - diameter of treated lesion / diameter of control lesion) × 100%;

[0044] ZIGUI6588 fermentation broth has a good control effect on citrus Penicillium wilt, with a control effect of 89.57%. Figure 3 ).

[0045] Example 4:

[0046] The control effect of Bacillus megaterium ZIGUI6588 on citrus root-knot nematodes

[0047] The field trial was conducted in Zigui County, Hubei Province. The tested citrus variety was Yichang Honey Tangerine, with trees aged 7 years. The roots were irrigated with 15 L of ZIGUI6588 fermentation liquid prepared in Example 2 at a dilution of 200 times. A second irrigation was performed 15 days later. The control was irrigated with plain water. Each treatment was replicated three times, with five trees per replicate. The root knot index for each treatment was recorded after three months, and the control efficacy was calculated.

[0048] According to the disease grading standards, the occurrence of diseases is as follows: Grade 0: no root knots; Grade 1: root knots account for 1% to 10% of the total root system; Grade 2: root knots account for 11% to 25% of the total root system; Grade 3: root knots account for 26% to 50% of the total root system; Grade 4: root knots account for 51% to 75% of the total root system; Grade 5: root knots account for 69% to 90% of the total root system; Grade 6: root knots account for 91% to 100% of the total root system.

[0049] Root knot index = ∑ (number of plants at each level × level) / (total number of plants surveyed × highest representative level) × 100.

[0050] Control efficacy = (Control root knot index - Treatment root knot index) / Control root knot index × 100%

[0051] The experimental results showed that ZIGUI6588 had a certain control effect on citrus root-knot nematodes, with a control efficacy of 69.17% (Table 2).

[0052] Table 2. Field control efficacy of ZIGUI6588 inoculant against citrus root-knot nematodes.

[0053] .

[0054] Example 5:

[0055] The effect of Bacillus megaterium ZIGUI6588 on the degradation of avermectin

[0056] Weigh 20g of citrus field soil that has not been treated with abamectin and place it in a 250mL Erlenmeyer flask. Add abamectin at a rate of 50mg / kg, stir well with a glass rod, and seal the flask. Add 100μL of ZIGUI6588 fermentation broth to the soil. The control strain is 10 billion / g Bacillus megaterium inoculum (Yiqiang Bio). Isolate single colonies of the product and ferment according to Example 2. Add 100μL of the control Bacillus megaterium fermentation broth to the soil. Add water to the blank control.

[0057] The samples were incubated at 25°C under light and culture, and samples were taken on day 20. 1 g of soil sample was added to 1 mL of ultrapure water, followed by 2 mL of acetonitrile. The mixture was vortexed for 3 min, centrifuged at 5000 rpm for 10 min, and the supernatant was collected and filtered through a 0.45 μm microporous membrane. The filtrate was transferred to a 5 mL centrifuge tube, and 0.25 g of NaCl was added. The mixture was vortexed for 90 s, allowed to stand for 30 min, and the upper layer was transferred to a 1.5 mL guide tube for HPLC determination of avermectin concentration. Each treatment was repeated three times.

[0058] 0.5% abamectin emulsifiable concentrate was diluted 50 times and sprayed onto citrus fruits using a sprayer. After air drying, ZIGUI6588 fermentation broth was diluted 200 times and sprayed onto the same fruits. A control was sprayed with water. After air drying, the fruits were collected and placed indoors at 28℃. Samples were taken on day 20. 1g of citrus peel samples were taken, treated as above, and the abamectin concentration was determined by HPLC. Each treatment was repeated three times.

[0059] Degradation rate % = (Avermectin concentration in blank control group - Avermectin concentration in experimental group) / Avermectin concentration in blank control group * 100%.

[0060] The experimental results are shown in Table 3, indicating that the ZIGUI6588 fermentation broth can effectively degrade avermectin in citrus fruits and soil, while the control Bacillus megaterium has no activity in degrading avermectin.

[0061] Table 3. Effect of Bacillus megaterium ZIGUI6588 on the degradation of avermectin

[0062] .

[0063] Example 6:

[0064] Colonization ability of Bacillus megaterium strain ZIGUI6588 in citrus soil

[0065] ZIGUI6588 was screened for resistant mutants on LA plates containing different gradient concentrations of rifampicin to obtain resistant mutant strains. The control strain was 10 billion / g of Bacillus megaterium inoculum (Yiqiang Bio). Single colonies of the product were isolated, and resistant mutant strains were obtained according to the above method. The two resistant mutant strains mentioned above, except for rifampicin resistance, had the same physiological and biochemical properties as the starting strain.

[0066] Following the fermentation method in Example 2, the concentration of the fermentation broth from both Bacillus megaterium strains was adjusted to 100 million CFU / mL and applied as a root drench to the roots of citrus seedlings at a rate of 10 L / plant. After 14 days, the citrus roots were dug up, rinsed three times repeatedly with sterile water, and 1 g of the root was cut off, ground, diluted, and then spread onto a substrate containing 300 μg / mL of Bacillus megaterium. -1 The colonization ability of ZIGUI6588 and control Bacillus megaterium in citrus roots was determined by counting rifampicin on LB plates, incubated at 37°C for 24 h.

[0067] The experimental results showed that ZIGUI6588 had a root colonization rate of 7.7 × 10⁻⁶ in citrus. 6 The colonization rate of ZIGUI6588 in citrus roots was 0, while the colonization rate of the control Bacillus megaterium was 0. This colonization ability of ZIGUI6588 in citrus helps to control citrus nematode disease.

Claims

1. An isolated strain of Bacillus megaterium ( Bacillus megaterium ZIGUI6588, the preservation number of the Bacillus megaterium is CCTCC NO: M20242075.

2. The fermentation broth of Bacillus megaterium ZIGUI6588 as described in claim 1, wherein the fermentation broth contains live Bacillus megaterium ZIGUI6588.

3. A composition containing Bacillus megaterium ZIGUI6588 as described in claim 1 or the fermentation broth as described in claim 2.

4. The application of Bacillus megaterium ZIGUI6588 as described in claim 1, the fermentation broth of Bacillus megaterium ZIGUI6588 as described in claim 2, or the composition as described in claim 3 in the control of citrus diseases; wherein the disease is caused by… Penicillium italicum Diseases caused by this disease and / or citrus root-knot nematode disease.

5. The application according to claim 4, wherein the method for preventing and controlling the application includes: The Bacillus megaterium ZIGUI6588 of claim 1, the fermentation broth of Bacillus megaterium ZIGUI6588 of claim 2, or the composition of claim 3, can be used for root irrigation or direct spraying of citrus.

6. The application of the Bacillus megaterium ZIGUI6588 of claim 1, the fermentation broth of Bacillus megaterium ZIGUI6588 of claim 2, or the composition of claim 3 in the preparation of a citrus biocontrol agent, wherein the target pest of the biocontrol agent is... Penicillium italicum And / or root-knot nematodes.

7. The use of Bacillus megaterium ZIGUI6588 of claim 1, the fermentation broth of Bacillus megaterium ZIGUI6588 of claim 2, or the composition of claim 3 in the degradation of avermectin.

8. The application according to claim 7, characterized in that, The purpose of the application is to degrade avermectin on the surface of citrus soil or fruit.

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