Bacillus megaterium with multiple functions and application thereof

Abstract

The invention belongs to the field of crop prevention and control technologies and pesticide residue treatment, and discloses a bacillus megaterium strain with multiple functions and application of the bacillus megaterium strain. The bacillus megaterium is bacillus megaterium ZIGUI6588, the preservation number is CCTCC (China Center For Type Culture Collection) NO: M20242075, and the bacillus megaterium can be used for preventing and controlling diseases such as citrus penicilliosis root-knot nematode and the like, has the function of degrading abamectin in fruits and soil, and has a wide application prospect in agricultural production.

Description

Technical Field

[0001] The present invention belongs to the fields of crop prevention and control technology and agricultural residue treatment, and specifically relates to a Bacillus megaterium with multiple functions and its application. Background Art

[0002] Citrus, also known as tangerine, mandarin orange, etc., is a small tree of the genus Citrus in the Rutaceae family. Citrus cultivation is widely distributed around the world and is the agricultural pillar industry in many regions.

[0003] Postharvest green mold and root-knot nematode diseases of citrus are major diseases of citrus, seriously threatening the development of the citrus industry. Citrus green mold is a disease that mainly harms citrus fruits and usually occurs on stored fruits and field-ripened fruits. Its symptoms include softening of the fruit peel, water-soaked discoloration, and easy rupture when gently pressed by hand. Subsequently, many aerial hyphae grow in the center of the lesion surface, forming a thick white mold, which quickly expands into a white nearly circular mold spot. Then, blue or green powdery substances, namely conidiophores and conidia, grow from the middle of the mold spot. The diseased part develops rapidly and can spread to the whole fruit and become wet and rotten within a few days. Citrus root-knot nematode disease is a microorganism that harms the roots and rhizomes of plants and forms root nodules of different sizes on the root tips of plants. The root nodules are spindle-shaped or irregular, nearly the size of sesame seeds to mung beans, initially milky white, and then turning yellowish brown to blackish brown, with sparse root hairs. In production, chemical agents are usually used to control the above two diseases. However, the frequent use of chemical agents causes problems such as pesticide residues, drug resistance, and environmental pollution. There is an urgent need for safer and more effective prevention and control means in production. Biological control has been widely used in recent years due to its advantages such as safety, no residues, and difficulty in generating drug resistance.

[0004] Avermectin is a common insecticide for controlling spider mites on crops and is widely used in citrus production, causing certain pesticide residues in citrus and the soil. Currently, the treatment methods for antibiotics mainly include physical adsorption method, advanced oxidation method, and microbial degradation method. Due to the continuous strengthening of people's environmental protection awareness, the microbial degradation method has become a technology favored by researchers. Nowadays, the microorganisms reported to be able to degrade avermectin mainly include Bacillus subtilis, Acinetobacter lwoffi, Shigella, white rot fungi, etc. They usually use the macrolide degrading enzymes produced by metabolism to degrade avermectin.

[0005] In response to the needs in production, the present invention provides a Bacillus megaterium with multiple functions, which has the functions of preventing and controlling citrus green mold and root-knot nematode diseases, and can simultaneously degrade avermectin in fruits and the soil. Summary of the Invention

[0006] The object of the present invention is to provide a separated Bacillus, which is Bacillus megaterium ZIGUI6588, and its preservation number is CCTCC NO: M20242075.

[0007] Another object of the present invention is to provide the application of Bacillus megaterium ZIGUI6588 in the preparation of a microbial inoculant for citrus cultivation.

[0008] In order to achieve the above object, the present invention takes the following technical measures:

[0009] The applicant isolated a strain of Bacillus from the rhizosphere soil of citrus plants in the citrus fields of Zigui. After identification, it was Bacillus megaterium, and it was sent to the China Center for Type Culture Collection for preservation on September 25, 2024. The taxonomic name: Bacillus megaterium ZIGUI6588; the preservation number is CCTCC NO: M20242075; the location: Wuhan University, Wuhan, China.

[0010] Bacillus megaterium ZIGUI6588 grows well on LA medium, showing a light yellow color ( Figure 1 ).

[0011] The protection scope of the present invention includes:

[0012] The fermentation broth of Bacillus megaterium ZIGUI6588, and the fermentation broth contains viable Bacillus megaterium ZIGUI6588.

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

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

[0015] For the above-mentioned application, preferably, the prevention and control method of the application includes: irrigating the roots of citrus with Bacillus megaterium ZIGUI6588, the fermentation broth of Bacillus megaterium ZIGUI6588, or a composition containing Bacillus megaterium ZIGUI6588 or its fermentation broth, or directly spraying.

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

[0017] Application of Bacillus megaterium ZIGUI6588, fermentation broth of Bacillus megaterium ZIGUI6588, or composition containing Bacillus megaterium ZIGUI6588 or its fermentation broth in degrading abamectin.

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

[0019] Preferably, the above-mentioned application is to degrade abamectin in citrus soil or on the fruit surface.

[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 biocontrol functions of antibacterial and anti-nematode, and has no cross-resistance with existing fungicides and insecticides.

[0022] (2) Compared with the current use of chemically synthesized pesticides for control, since it comes from the natural environment, it has the advantages of high efficiency, low toxicity, non-pollution to the environment, and difficulty in generating drug resistance.

[0023] (3) The Bacillus megaterium screened by the present invention also has the characteristic of degrading abamectin. Description of the Drawings

[0024] Figure 1 Colony morphology of ZIGUI6588 strain.

[0025] Figure 2 Phylogenetic tree of ZIGUI6588

[0026] Figure 3 Schematic diagram of ZIGUI6588 controlling citrus blue mold. Detailed Embodiments

[0027] In order to better explain the present invention, the following further clarifies the main content of the present invention in combination with specific embodiments, but the content of the present invention is not limited to the following embodiments. The technical solutions described in the present invention are conventional technologies in the art unless otherwise specified, and the reagents or materials are from commercial channels unless otherwise specified.

[0028] Example 1:

[0029] Obtaining of Bacillus megaterium ZIGUI6588:

[0030] The applicant isolated a Bacillus strain from the rhizosphere soil of healthy citrus plants. After 16S sequencing and combined physiological and biochemical identification, it belongs to Bacillus megaterium ( Figure 1 , Table 1), and it was sent to the China Center for Type Culture Collection for preservation on September 25, 2024. The taxonomic name is Bacillus mucilaginosus ZIGUI6588; the preservation number is CCTCC NO: M20242075; the address is Wuhan University, Wuhan, China.

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

[0032] Morphological characteristics: light yellow, flat, and circular 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]

[0037] Example 2:

[0038] Fermentation of Bacillus megaterium ZIGUI6588 (all percentages in the following culture medium formulations are mass fractions):

[0039] Preparation of seed liquid: Activate the ZIGUI6588 strain from the slant, inoculate a single colony into NB liquid medium, with a liquid loading of 0.6 L / 3 L, shake culture at 28 °C and 180 r / min for 18 h, and then set aside.

[0040] Fermentation in a 10000 L tank, the culture medium is: sucrose 3%, corn flour 1.5%, MgCl2 0.3%, 0.1%, peptone 0.5%, and the rest is water. The sample loading is 5000 L, the initial pH is 7.5, the culture temperature is 32 °C, inoculate 2.5 L of seed liquid, the ventilation volume is 180 m 3 / h. After 40 h of fermentation, the number of spores in the fermentation broth was measured to be 34×10 8 cfu / ml.

[0041] Example 3:

[0042] Control effect of Bacillus megaterium ZIGUI6588 on citrus blue mold:

[0043] After culturing the citrus blue mold pathogen (Penicillium italicum) at 28 °C for 10 d to produce spores, it was adjusted to 1×10 with sterile water6 Prepare 20 μL of the ZIGUI6588 fermentation broth diluted 50 times per milliliter of spores for standby. Take fresh citrus fruits, use a scalpel to pierce wounds of 3 mm × 3 mm at the waist of the fruits, and drop 20 μL of the diluted ZIGUI6588 fermentation broth at the wound. The control is treated with clear water. After natural air drying, drop 10 μL of the spore suspension of Penicillium digitatum Sacc. into the wound. Each treatment has 3 replicates, and each replicate has 15 fruits. Incubate under moisturized conditions at 25 °C. After 7 days of inoculation, measure the lesion diameters of the control and the treated groups, and calculate the control efficacy.

[0044] Control efficacy = ((lesion diameter of the control - lesion diameter of the treatment) / lesion diameter of the control) × 100%;

[0045] The ZIGUI6588 fermentation broth has a good control efficacy against Penicillium digitatum Sacc., and the control effect is 89.57% ( Figure 3 )

[0046] Example 4:

[0047] Control efficacy of Bacillus megaterium ZIGUI6588 against citrus root-knot nematodes

[0048] The field experiment was conducted in Zigui County, Hubei Province. The tested citrus variety was Yichang tangerine with a tree age of 7 years. Irrigate the roots with the ZIGUI6588 fermentation broth prepared in Example 2 at a dilution of 200 times, 15 L per plant, and irrigate again after an interval of 15 days. The control was irrigated with clear water. Each treatment had 3 replicates, and each replicate had 5 trees. After 3 months, count the root-knot index of each treatment and calculate the control efficacy.

[0049] Count the disease occurrence according to the disease grading standard: Grade 0, no root knots; Grade 1, root knots account for 1% - 10% of the total root system; Grade 2, root knots account for 11% - 25% of the total root system; Grade 3, root knots account for 26% - 50% of the total root system; Grade 4, root knots account for 51% - 75% of the total root system; Grade 5, root knots account for 69% - 90% of the total root system; Grade 6: root knots account for 91% - 100% of the total root system.

[0050] Root-knot index = ∑(number of plants at each level × level) / (total number of plants surveyed × highest representative level) × 100.

[0051] Control effect = (root-knot index of the control - root-knot index of the treatment) / root-knot index of the control × 100%

[0052] The test results showed that ZIGUI6588 had a certain control efficacy against citrus root-knot nematodes, and the control effect reached 69.17% (Table 3).

[0053] Table 2 Field control efficacy of ZIGUI6588 bactericide against citrus root-knot nematodes

[0054] Treatment Root-knot index Control efficacy (%) ZIGUI6588 13.66 69.17 Control 44.31 -

[0055] Example 5:

[0056] Degradation effect of Bacillus megaterium ZIGUI6588 on abamectin

[0057] Weigh 20 g of unused abamectin citrus orchard soil and put it into a 250 mL Erlenmeyer flask. Add abamectin at a dose of 50 mg / kg, stir well with a glass rod, and seal the flask. Add 100 μL of ZIGUI6588 fermentation broth to the soil. The control strain is a 10 billion / g Bacillus megaterium bactericide (Yi Qiang Biology). Isolate single colonies of the product and ferment according to Example 2. Add 100 μL of the control Bacillus megaterium fermentation broth to the soil, and add clear water to the blank control.

[0058] Incubate in a light incubator at 25 °C and sample on the 20th day. Take 1 g of soil sample, add 1 mL of ultrapure water, then add 2 mL of acetonitrile, vortex mix for 3 min, centrifuge at 5000 r / min for 10 min. Aspirate all the supernatant and filter it through a 0.45 μm microporous membrane. Transfer the filtrate to a 5 mL centrifuge tube, add 0.25 g of NaCl, vortex mix for 90 s, let it stand for 30 min, and transfer the upper layer liquid to a 1.5 ml guiding tube for HPLC determination of abamectin concentration. Each treatment is repeated three times.

[0059] Dilute 0.5% abamectin emulsifiable concentrate 50 times and spray it on citrus tree fruits using a sprayer. After air drying, dilute the ZIGUI6588 fermentation broth 200 times and spray it on the above fruits. Spray clear water as a control. After air drying, retrieve the fruits and place them indoors at 28 °C. Sample on the 20th day. Take 1 g of citrus peel sample, treat it as above, and determine the abamectin concentration by HPLC. Each treatment is repeated three times.

[0060] Degradation rate % = (Abamectin concentration in the blank control group - Abamectin concentration in the experimental group) / Abamectin concentration in the blank control group * 100%.

[0061] The test results are shown in Table 3, indicating that the ZIGUI6588 fermentation broth can effectively degrade abamectin in citrus fruits and soil, and the control Bacillus megaterium has no abamectin degradation activity.

[0062] Table 3 Degradation effect of Bacillus megaterium ZIGUI6588 on abamectin

[0063]

[0064]

[0065] Example 6:

[0066] Colonization ability of Bacillus megaterium ZIGUI6588 strain in citrus soil

[0067] ZIGUI6588 was screened for rifampicin-resistant mutants on LA plates containing different gradient concentrations of rifampicin to obtain rifampicin-resistant mutant strains. The control strain was a Bacillus megaterium bacterium agent (Yi Qiang Biology) at 10 billion / g. Single colonies of the isolated products were obtained, and rifampicin-resistant mutant strains were obtained according to the above method. Except for rifampicin resistance, the other physiological and biochemical properties of the two rifampicin-resistant mutant strains described above were the same as those of the starting strain.

[0068] According to the fermentation method of Example 2, the fermentation broth concentrations of the two Bacillus megaterium strains were adjusted to 100 million cfu / mL, and the roots of citrus seedlings were irrigated at 10 L / plant. After 14 days, the citrus roots were dug out, and the roots were repeatedly rinsed 3 times with sterile water. Then, 1 g of the roots was cut off, ground, diluted, and spread on an LB plate containing 300 μg·mL -1 of rifampicin, and the number of colonies was counted after culturing at 37 °C for 24 h to determine the colonization ability of ZIGUI6588 and the control Bacillus megaterium in citrus roots.

[0069] The test results showed that the colonization amount of ZIGUI6588 in citrus roots was 7.7×10 6 cfu / g, while the colonization amount of the control Bacillus megaterium in citrus roots was 0. The colonization ability of ZIGUI6588 in citrus was helpful for the prevention and control of citrus nematode disease.

Claims

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

2. The fermentation broth of Bacillus megaterium ZIGUI6588 according to claim 1, wherein the fermentation broth contains live bacteria of Bacillus megaterium ZIGUI6588.

3. A composition comprising the Bacillus megaterium ZIGUI6588 according to claim 1 or the fermentation broth according to claim 2.

4. Use of the Bacillus megaterium ZIGUI6588 according to claim 1, the fermentation broth of Bacillus megaterium ZIGUI6588 according to claim 2, or the composition according to claim 3 in the prevention and treatment of citrus diseases; the diseases include citrus penicillium and / or citrus root-knot nematode disease.

5. The use according to claim 4, wherein the control method of the use comprises: The Bacillus megaterium ZIGUI6588 described in claim 1, the fermentation liquid of Bacillus megaterium ZIGUI6588 described in claim 2, or the composition described in claim 3 is used to irrigate the roots of citrus or directly spray them.

6. Use of the Bacillus megaterium ZIGUI6588 according to claim 1, the fermentation liquid of Bacillus megaterium ZIGUI6588 according to claim 2, or the composition according to claim 3 in the preparation of a citrus biocontrol agent, wherein the control target is Penicillium and / or root-knot nematodes.

7. Use of the Bacillus megaterium ZIGUI6588 according to claim 1, the fermentation broth of Bacillus megaterium ZIGUI6588 according to claim 2, or the composition according to claim 3 in degrading avermectin.

8. Use of the Bacillus megaterium ZIGUI6588 according to claim 1, the fermentation liquid of Bacillus megaterium ZIGUI6588 according to claim 2, or the composition according to claim 3 in citrus cultivation.

9. The use according to claim 8, characterized in that: The purpose of application is to degrade avermectins in citrus soil or on the surface of fruit.

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