CN117535169B - Bacillus licheniformis ZLP-81 and its application - Google Patents

Abstract

The present invention relates to a Bacillus licheniformis ZLP-81, with a deposit number of CGMCC No.25028. The strain has the functions of preventing diseases, killing insects and promoting plant growth. The present invention also discloses the use of the strain in the preparation of biocontrol agents, biological pesticides, soil remediation agents or biological fertilizers.

Description

Bacillus licheniformis ZLP-81 and its application

Technical Field

The invention relates to Bacillus licheniformis ZLP-81 and application thereof.

Background technique

Plant diseases and insect pests bring serious losses to agricultural production. Conventional chemical pesticide control not only kills a large number of natural enemies and beneficial microorganisms and destroys the ecological balance, but also causes a sharp increase in pest resistance, causing great harm to the environment and human health. Therefore, research and development of safe, efficient, and environmentally friendly products and fertilizer and pesticide replacement technologies have become a strategic need to ensure food security, food safety, and environmental safety.

Bacillus licheniformis is a type of aerobic, spore-forming rod-shaped bacteria that is non-toxic and harmless to humans and animals, environmentally friendly, highly resistant to stress, and has a fast reproduction rate. It can also secrete a variety of active substances. Studies have shown that this type of microorganism has a strong inhibitory effect on Rhizoctonia solani, gray mold, fusarium, wilt, early blight, leaf mold, black spot, anthracnose, downy mildew, black spot, ring spot, and small spot, and can promote crop growth.

However, there are no reports on strains that have the functions of preventing diseases, killing insects and promoting plant growth.

Summary of the invention

The purpose of the present invention is to provide a Bacillus licheniformis ZLP-81 having the functions of preventing diseases, killing insects and promoting plant growth and its application.

The present invention adopts the following technical solution:

A licheniformis ( Bacillus licheniformis ) ZLP-81, with a deposit number of CGMCC No.25028, and the depositor is the General Microbiology Center of China Culture Collection Administration, the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, China, and the deposit date is June 9, 2022.

An application of the above-mentioned Bacillus licheniformis ZLP-81 in preventing and controlling pests such as aphids, diamondback moths, Spodoptera litura, white grubs, crickets, pillbugs, cockroaches, etc.

An application of the above-mentioned Bacillus licheniformis ZLP-81 in preventing and controlling plant pathogens, aquatic pathogens and Staphylococcus aureus.

Furthermore, the plant pathogens include: tomato leaf mold, tomato gray mold, pear black spot, fusarium, cotton wilt, cucumber gray mold, grape gray mold, cucumber anthracnose, cucumber downy mildew, cucumber black spot, apple ring spot, Rhizoctonia solani, corn leaf blight and tomato early blight.

Furthermore, the aquatic pathogens include Aeromonas hydrophila, Edwardsiella tarda and Aeromonas veronii.

An application of the above-mentioned Bacillus licheniformis ZLP-81 in the preparation of biocontrol agents, biological pesticides, soil remediation agents or biological fertilizers.

Furthermore, the biocontrol agent, biological pesticide, soil remediation agent or biological fertilizer contains Bacillus licheniformis ZLP-81 bacteria and/or fermentation liquid; or surfactin, iturin, fennel or spermin separated from the fermentation liquid.

A fungicide comprises Bacillus licheniformis ZLP-81 bacteria or fermentation liquid.

A fungicide comprises surfactin, iturin, fennectin or spermin separated from the fermentation liquid of Bacillus licheniformis ZLP-81.

The beneficial effects of the present invention are:

The Bacillus licheniformis ZLP-81 of the present invention is an aerobic, spore-forming rod-shaped microorganism, which has the characteristics of being environmentally friendly, having strong stress resistance, and having a fast reproduction speed, and can secrete a variety of active substances to promote plant growth.

The Bacillus licheniformis ZLP-81 of the present invention has strong insecticidal activity, can effectively kill diamondback moth, Spodoptera litura, aphids, white grubs, crickets, woodlice and other pests, and has broad-spectrum antibacterial properties, and has obvious inhibitory effects on various plant pathogens such as gray mold, sickle disease, wilt, early blight, leaf mold, black spot, anthrax, downy mildew, black spot, ring rot, small spot and the like, and has good application prospects in the development of functional microbial preparations such as insecticides or disease prevention, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a colony image of Bacillus licheniformis ZLP-81.

FIG. 2 is a diagram of the Bacillus licheniformis ZLP-81 strain.

Figure 3 shows the control group (left) and treatment group (right) killing cockroaches by Bacillus licheniformis ZLP-81.

Figure 4 shows the killing of aphids by Bacillus licheniformis ZLP-81 in the treatment group (left) and the control group (right).

Detailed ways

The technical solution of the present invention is described in detail below in conjunction with the preferred embodiments. The following embodiments are only used to illustrate and explain the present invention, and do not constitute a limitation on the technical solution of the present invention.

Example 1 Isolation and screening of Bacillus licheniformis ZLP-81

(1) Soil sampling: Soil samples were collected from Qamdo, Tibet, China, with each sample weighing 50-100 g. The samples were placed in sterilized kraft paper bags, sealed, and the sampling location, environment, and date were recorded.

(2) Isolation and purification of Bacillus: Soil samples were separated using the plate dilution method on PB culture medium. The specific operation is as follows: 10 g of soil sample was accurately weighed and added to a 90 mL 0.9% saline conical flask containing glass beads. The soil sample was shaken at 180 r/min for 30 min to make the soil sample evenly distributed and prepare a soil suspension with a concentration of 10 ^-1 ; the soil suspension was diluted to concentrations of 10 ^-5 , 10 ^-6 , and 10 ^-7 in sequence. The samples were placed in a constant temperature water bath at 80°C for 30 min, and 100 µL of the dilution solution was applied to the PB plate. The plate was incubated inverted in a constant temperature incubator at 30°C for 48 h. Single colonies of different morphologies were picked and identified by spore staining. The Bacillus was purified by streaking and transferred to the slant of a PB culture tube. After culturing at 30°C for 48 h, it was stored in a refrigerator at 4°C for later use.

(3) Screening of strains with insecticide and disease prevention effects

Screening of insecticidal strains: Taking aphids as target organisms, the purified microorganisms were cultured in liquid shake flasks, and the fermentation liquid was centrifuged at a speed of 12000r/min for 10min to remove the bacteria. The supernatant was filtered with a 0.22μm sterile filter membrane to prepare a sterile fermentation filtrate. Take appropriate lengths of broad bean stems and leaves and put them into a conical flask. Spray an appropriate amount of fermentation liquid on the living aphids and the stems and leaves where they are located, then put them into a conical flask, cover the bottle mouth with gauze and fix it with a rubber band. After 2 days, the number of aphids was counted and strains with better insecticidal effects were screened.

Screening of strains with disease prevention effects: Cucumber gray mold was used as the indicator bacteria, and the plate confrontation method was used for initial screening. In the sterile operating room, the gray mold pathogens grown on the PDA medium were punched into d=5mm cakes, and then the cakes were transferred to the center of the PDA medium plate. Then the sterile filtrates of each antagonistic bacteria obtained in the initial screening were respectively connected to 2.5cm away from the pathogens, with 3 replicates. At the same time, the plate with only pathogen cakes was used as the control, and cultured at a constant temperature of 26℃. When CK was cultured to fill the culture medium, the colony diameter was measured and the average inhibition rate was calculated. Screen out strains with strong antagonism to pathogens. The strains with strong insecticide and disease prevention effects were frozen and stored at -80℃.

(4) Identification of strains with insecticide and disease prevention effects

According to the experimental methods in the Manual of Identification of Common Bacteria Systems, the strains with strong insecticide and disease prevention effects were identified by morphology and physiology and biochemistry. The strains were cultured on PB medium plates at 32±1℃ for 14~16h, and the colonies numbered ZLP-81 were observed to be irregularly round, white, dull, and rough on the surface. The bacteria were rod-shaped (as shown in Figures 1~2), and the spores were oval. Its physiological and biochemical identification indicators are shown in Table 1.

Table 1 Physiological and biochemical characteristics of strain ZLP-81

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Through morphological observation and physiological and biochemical experiments, it was determined that the strain numbered ZLP-81 was Bacillus. Then molecular biological classification identification was carried out, and 16S rDNA sequence analysis was used to extract the total genomic DNA of the strain as a template to amplify its 16S rDNA sequence. The target fragment was amplified using universal primers 27F/1492R, and the amplified product was detected by 1% agarose gel electrophoresis. The gene sequencing was performed using a bidirectional sequencing method. The sequencing results were compared by BLAST sequence homology comparison in the GenBank database, and the strain numbered ZLP-81 was identified as Bacillus licheniformis and named Bacillus licheniformis ZLP-81.

Bacillus licheniformis ZLP-81 was sent to the General Microbiology Center of China Microorganism Culture Collection Administration for preservation, the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, the preservation number is CGMCC No. 25028, and the preservation date is June 9, 2022.

Example 2 The killing effect of Bacillus licheniformis ZLP-81 on aphids, diamondback moth, Spodoptera litura, grubs, pillbugs, cockroaches and crickets

Bacillus licheniformis ZLP-81 was fermented and cultured. NB liquid medium was used, with the formula of beef extract 5g/L, peptone 10g/L, NaCl 5g/L, glucose 10g/L, pH 7.0. After inoculation with 5% inoculum, the medium was placed in a shaking incubator at 180r/min and 32°C for 48h to obtain the fermentation broth. The fermentation broth level (viable count) was 1.18×10 ⁷ cfu/mL.

The fermentation liquid of Bacillus licheniformis ZLP-81 (treatment group) and NB liquid culture medium (control group) were used for contact killing. The larvae of aphids (50), diamondback moth (30), grubs (30), Spodoptera litura (30), pillbugs (30), cockroaches (50) and crickets (60) with the same insect size were selected. After 24 hours of feeding in an environment with a temperature of 25℃~28℃, the fermentation liquid was evenly sprayed on the insect surface in the treatment group, and the NB liquid culture medium was evenly sprayed on the insect surface in the control group to ensure that the body surface of each insect could contact the liquid. After spraying, they were placed in a room at room temperature for culture. After 24 hours, the number of deaths was counted and the corrected mortality rate was calculated. The results are shown in Table 2 and Figures 3~4.

Among them, adjusted mortality rate = (mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group) × 100%; mortality rate = number of dead insects / total number of insects × 100%.

Table 2 The killing effect of Bacillus licheniformis ZLP-81 on pests

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Example 3 Determination of the antibacterial spectrum of Bacillus licheniformis ZLP-81

Common pathogens such as tomato leaf mold, tomato gray mold, pear black spot, fusarium, cotton wilt, cucumber gray mold, grape gray mold, cucumber anthracnose, cucumber downy mildew, cucumber black spot, apple ring spot, Rhizoctonia solani, corn leaf blight, tomato early blight and other plant pathogens; aquatic pathogens such as Aeromonas hydrophila, Edwardsiella tarda, Aeromonas verticillata and Staphylococcus aureus and were selected as targets, and the plate confrontation method was used to determine the antibacterial spectrum of strain ZLP-81 to determine the inhibitory ability of ZLP-81 strain on the growth of pathogens.

According to the results in Table 3, strain ZLP-81 has strong antagonism against all 18 pathogens tested, and has the strongest inhibitory effect on tomato leaf mold, with the diameter of the inhibition zone reaching 25.20 mm. This indicates that the fungicide with the fermentation liquid of Bacillus licheniformis ZLP-81 as the active ingredient has good application value in agricultural production.

Table 3 Inhibitory effect of Bacillus licheniformis ZLP-81 on different plant pathogens

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Example 4 Growth-promoting effect of Bacillus licheniformis ZLP-81 fungicide

Cucumber was selected as the test crop. 20 cucumber seedlings were planted in pots indoors, with 3 parallels. The treatment group used Bacillus licheniformis ZLP-81 fermentation liquid (the method was the same as in Example 2, with a viable count of ≥10 ⁷ cfu/mL) and carbendazim solution (1g carbendazim dissolved in 800mL water) in a volume ratio of 500:1, which was evenly mixed and sprayed dry, and applied to the soil at a viable count of 10 ⁷ cfu/kg; the control group did not apply any growth-promoting products. Then 10 28-day-old cucumber seedlings were randomly selected, and the plant height of the seedlings was measured with a ruler, and the stem diameter was measured with a vernier caliper. The seeds were sterilized at 105℃ for 15min, dried at 75℃ to constant weight, and the dry mass of the aboveground and underground parts was determined. The chlorophyll content was determined by ethanol-acetone extraction, and the photosynthetic parameters were determined by a portable photosynthetic measurement system. As shown in Table 4, the plant height, stem diameter, fresh weight of the aboveground part of a single plant, dry weight of the aboveground part of a single plant, leaf area, fresh weight of a single root, dry weight of a single root, total root volume of a single plant, total chlorophyll content and net photosynthetic rate of cucumber seedlings were improved by 3.68%, 10.99%, 18.687%, 15.41%, 0.49%, 77.33%, 8.51%, 119.57%, 63.54% and 89.03% compared with CK, and except for the plant height, leaf area and dry weight of a single root, all other differences were significantly different. This shows that the bacillus licheniformis ZLP-81 fungicide of the present invention has a significant growth-promoting effect on cucumber seedlings.

Table 4 Growth-promoting effect of Bacillus licheniformis ZLP-81 fermentation liquid on cucumber seedlings

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The embodiments and methods described above are the best implementation schemes of the present invention. Without departing from the technical principles of the present invention, partial changes, modifications, substitutions and combinations may be made, all of which are included in the protection scope of the present invention.

Claims (5)

1. A bacillus licheniformis (Bacillus licheniformis) ZLP-81 is characterized in that the preservation number is CGMCC No. 25028.

2. Use of bacillus licheniformis ZLP-81 according to claim 1 for controlling plutella xylostella, prodenia litura, aphid, grub, cricket and pillworm.

3. Use of bacillus licheniformis ZLP-81 according to claim 1 for controlling phytopathogens;

The plant pathogenic bacteria are tomato leaf mold bacteria, tomato gray mold bacteria, pear black spot bacteria, fusarium, cotton fusarium, cucumber gray mold bacteria, grape gray mold bacteria, cucumber anthracnose bacteria, cucumber downy mildew bacteria, cucumber cladosporium cucumerinum, apple alternaria alternate bacteria, rhizoctonia solani, corn small spot bacteria and tomato early blight bacteria.

4. Use of bacillus licheniformis ZLP-81 according to claim 1 for preparing products for controlling aquatic pathogens and staphylococcus aureus;

The aquatic pathogenic bacteria are aeromonas hydrophila, edwardsiella tarda and aeromonas veronii.

5. A bactericide comprising the bacillus licheniformis ZLP-81 fermentation broth according to claim 1.