Bacillus thuringiensis ZR-560 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus thuringiensis ZR-560 and application thereof, the bacillus thuringiensis ZR-560 is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.30586. The invention further discloses a preparation method of the bacillus thuringiensis ZR-560. The bacillus thuringiensis ZR-560 strain provided by the invention has high insecticidal toxicity, and the parasporal crystal protein preparation of the bacillus thuringiensis ZR-560 strain has a corrected death rate of 100% for spodoptera frugiperda, ostrinia nubilalis, prodenia litura and beet armyworm, and is a Bt strain with a relatively broad-spectrum insecticidal effect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Bacillus thuringiensis ZR-560 and an application thereof. Background Art

[0002] Bacillus thuringiensis (Bt) is a Gram-positive bacterium, a species of the genus Bacillus. Bt produces crystal proteins (ICPs) at the same time as spore formation. Crystal proteins are mainly encoded by crystal protein genes (cry) and cytolytic (cyt) genes. It is reported that there are many types of crystal forms of Bacillus thuringiensis, mainly including: square, rhombus, polygon, spherical, elliptical, rice-shaped, irregular, etc. The types and quantities of insecticidal crystal proteins contained in different subspecies and even different strains are different. Some strains contain more than one crystal form, generally a combination of rhombus and sphere, rhombus and square, etc. The difference in crystal form also indicates the difference in genes in the strain, resulting in differences in the insecticidal activity of the strain.

[0003] Crystalline proteins have specific insecticidal activity, and are highly efficient, specific, non-toxic, and do not pollute the environment. Therefore, they are widely used as microbial insecticides in agriculture, forestry, fruits and vegetables, health, and other fields. In recent years, they have also been widely promoted and applied in genetically modified plants, and have achieved good results.

[0004] Noctuidae is the largest family in the order Lepidoptera, with about 35,000 species in the world. Spodoptera frugiperda, Spodoptera exigua, Spodoptera litura Fabricius and others belong to the same family, and are major agricultural and forestry pests in my country. In recent years, the fall armyworm has caused the most serious damage. According to a comprehensive analysis by the National Agricultural Technology Extension Center, the area of ​​fall armyworm damage in the country reached 45 million mu in 2023, which fully demonstrates that the prevention and control of fall armyworm in my country is very serious and faces major challenges.

[0005] The prior art CN112695001B (publication date: 2021-10-08) discloses a strain of Bacillus thuringiensis NBI-646 with high toxic activity against Noctuidae pests and its application. When the concentration of its fermentation broth is 0.5 μL / mL, the toxic activity against the larvae of the fall armyworm is the highest, reaching 89%.

[0006] However, there are currently few Bt products registered in China for the control of fall armyworm, and there is still a lack of microbial pesticides for sustainable and green control of fall armyworm. Summary of the invention

[0007] The purpose of the present invention is to provide a highly toxic Bacillus thuringiensis ZR-560 in view of the above shortcomings, enrich the efficient strain resources for controlling crop pests mainly including fall armyworm in agricultural production applications, and provide a new option for solving the above problems.

[0008] The objective of the present invention is achieved through the following technical solutions:

[0009] The Bacillus thuringiensis provided by the present invention is classified and named as Bacillus thuringiensis, collected from soil 5-10 cm deep from the surface in Chengdu, Sichuan, and named as Bacillus thuringiensis ZR-560. It has been deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with a deposit number of CGMCC No. 30586, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit date of May 17, 2024.

[0010] The morphological characteristics of the Bacillus thuringiensis ZR-560 provided by the present invention are as follows: the colonies on the LB medium are round or oval, light yellow, with irregular and opaque edges, and slightly raised and waxy. The strain is purple oval rod-shaped under a microscope, the spores are oval, grow close to the middle, and the spore capsules are slightly swollen.

[0011] Based on the results of homologous genome analysis in whole genome sequencing, a phylogenetic tree was constructed using the neighbor-joining method. The results showed that Bacillus thuringiensis ZR-560 belonged to Bacillus thuringiensis serovar chinensis CT-43, and was most closely related to the serotype subspecies (aizawai) of the same branch.

[0012] The present invention also provides application of the above-mentioned Bacillus thuringiensis ZR-560 in preventing and controlling crop pests.

[0013] Furthermore, the crop pests include fall armyworm, corn borer, fall armyworm and / or beet armyworm.

[0014] The present invention also provides a microbial preparation for preventing and controlling crop pests, wherein the microbial preparation comprises the parasporal crystal protein of Bacillus thuringiensis ZR-560.

[0015] Furthermore, the concentration of parasporal crystal protein of Bacillus thuringiensis ZR-560 in the microbial preparation is not less than 450 mg / mL.

[0016] The present invention also provides application of the above-mentioned microbial preparation in preventing and controlling crop pests.

[0017] Furthermore, the crop pests include fall armyworm, corn borer, fall armyworm and / or beet armyworm.

[0018] The beneficial effects of the present invention are:

[0019] 1. The Bacillus thuringiensis ZR-560 strain provided by the present invention has high insecticidal toxicity, and the parasporal crystal protein preparation thereof has a corrected mortality rate of 100% against fall armyworm, corn borer, Spodoptera litura, and beet armyworm, and is a Bt strain with a relatively broad-spectrum insecticidal effect.

[0020] 2. The microbial agent prepared with the parasporal crystals of the ZR-560 strain is environmentally friendly and has strong resistance to adversity. The biological activity of detached leaves, potted plant efficacy and field efficacy tests all show that the strain has high efficacy against pests such as the fall armyworm, can significantly reduce their harm to crops, and has high application potential in agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Attached Figure 1 This is a morphological characteristic diagram of Bacillus thuringiensis ZR-560, in which:

[0022] A. Colonies of ZR-560 on LB medium are round or oval, light yellow, with irregular edges, opaque and slightly raised waxy appearance;

[0023] B. The bacteria of ZR-560 are oval rod-shaped under the microscope, the spores are oval, growing close to the middle, and the spore capsule is slightly swollen;

[0024] C, parasporal crystals of strain ZR-560, with a rhombus shape;

[0025] Attached Figure 2 This is the GO (Gene Ontology) annotation distribution bar graph of Bacillus thuringiensis ZR-560;

[0026] Attached Figure 3 It is the classification bar chart of the metabolic pathway (KEGG Pathway) of Bacillus thuringiensis ZR-560;

[0027] Attached Figure 4 is the phylogenetic tree of Bacillus thuringiensis ZR-560;

[0028] Attached Figure 5The SDS-PAGE electrophoresis results of parasporal crystals of Bacillus thuringiensis ZR-560;

[0029] Attached Figure 6 This is a picture of a corn leaf in the in vitro leaf bioactivity assay of Bacillus thuringiensis ZR-560 spores;

[0030] Attached Figure 7 This is a picture of corn leaves after the field control effectiveness test of the present invention is completed;

[0031] Attached Figure 8 This is a picture of corn plants after the field prevention efficacy test of the present invention was completed. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings, but the protection scope of the present invention is not limited to the following.

[0033] Example 1: Screening of Bacillus thuringiensis ZR-560

[0034] Soil samples: collected in Chengdu, Sichuan, from soil 5-10 cm deep from the surface, with a total of 857 soil samples collected.

[0035] Strain screening: Sodium acetate-antibiotic separation method was used for separation. The specific steps were as follows: 15g soil sample was weighed and poured into a 50mL triangular flask containing glass beads and sodium acetate-antibiotic culture medium, and cultured on a shaking table (200r / min, 30°C) for 4h. After the culture was completed, 10mL of soil suspension was taken and added to a sterile centrifuge tube, centrifuged at 300r / min for 15min, 2mL of the upper turbid liquid was taken and placed in a 65°C water bath for 15min, and 0.1mL of the heat-treated turbid liquid was taken and coated on an LB solid plate. After the LB solid plate was cultured at 30°C for 48h, colonies similar to Bt were selected from the plate, sliced, stained with fuchsin, and observed for crystal formation under a microscope (oil lens). Bt strains detected from the same isolate were selected based on their colony morphology, color, vegetative body and spore crystal morphology, and one strain of each morphology was isolated and purified, and then the strain crystals were observed under a scanning electron microscope. The selected single colony was inoculated into 3 mL LB liquid culture medium, cultured in a shaking constant temperature incubator at 220 r / min and 30°C for 16-18 h, mixed thoroughly with 50% glycerol in a 1:1 ratio, and stored in a -70°C ultra-low temperature refrigerator.

[0036] After screening in the above steps, a Bacillus strain was isolated and named Bacillus thuringiensis ZR-560.

[0037] The morphological characteristics of Bacillus thuringiensis ZR-560 are shown in the attached Figure 1As shown: The colony is round or oval, light yellow, with irregular, opaque edges, and slightly raised waxy. The strain appears purple oval rod-shaped under a microscope, the spores are oval, growing close to the middle, and the spore capsules are slightly swollen.

[0038] Example 2: Identification of Bacillus thuringiensis ZR-560:

[0039] The Bacillus thuringiensis ZR-560 was sequenced and assembled to obtain a total base number of 1348734478 bp, a GC (guanine and cytosine) content of 36.23%, and 7291 coding genes, of which nearly 56% of the genes were longer than 500 bp, and nearly 20% of the genes were longer than 1000 bp.

[0040] GO (Gene Ontology) functional classification annotations show that genes are enriched in 52 pathways. In biological processes, the gene functions corresponding to metabolic processes and cellular processes in organisms are more enriched. Genes involved in cell composition are mainly enriched in cells, cell components, and membrane processes. In molecular functions, gene annotations are more enriched in the catalytic activity, binding, and molecular transducer activity pathways. Figure 2 This is a bar graph showing the GO annotation distribution of Bacillus thuringiensis ZR-560.

[0041] The results of KEGG (Kyoto Encyclopedia of Genes and Genomes) functional annotation showed that the genes were enriched in 34 pathways in total, most of which were attributed to the metabolic process (74.3%), followed by the environmental information process (14.88%). In the metabolic process, the genes were mainly enriched in carbohydrate metabolism (Carbohydrate metabolism) and amino acid metabolism (Amino acidmetabolism). Figure 3 This is a classification bar chart of the metabolic pathway (KEGG Pathway) of Bacillus thuringiensis ZR-560.

[0042] Based on the results of homologous genome analysis in whole genome sequencing, the phylogenetic tree was constructed using the neighbor-joining method. The results showed that strain ZR-560 belongs to Bacillus thuringiensis serovar chinensis CT-43 and is most closely related to the serotype subspecies (aizawai) of the same branch. Figure 4 This is the phylogenetic tree of Bacillus thuringiensis ZR-560.

[0043] Example 3: Insecticidal activity of fermentation broth

[0044] The insecticidal activity of Bacillus thuringiensis ZR-560 fermentation liquid against Spodoptera frugiperda was determined according to the method of GB / T19567.2-2004, and the LC 50 The value is 10.21μg / g.

[0045] Example 4: Extraction of parasporal crystals of Bacillus thuringiensis ZR-560

[0046] The following steps are used:

[0047] (1) Streak culture of Bacillus truncatus ZR-560 on LB solid medium for 12 h, pick a single colony and inoculate it into 1 / 2 LB medium and culture it at 37°C in a shaking incubator at 200 r / min for 3 days;

[0048] (2) Collect the bacteria in a 50 mL tube and centrifuge at 8000 rpm and 4°C for 5 min;

[0049] (3) Wash the bacteria twice with 1 mol / L NaCl and centrifuge at 8000 rpm at 4°C for 5 min;

[0050] (4) Wash the bacteria twice with sterile water and centrifuge at 8000 rpm and 4°C for 5 min;

[0051] (5) Dissolve the cells in 50 mmol / L sodium carbonate solution (pH = 9.5) with an appropriate amount of 20 mmol DTT (the ratio of DTT to sodium carbonate is approximately 1:3-1:4) and shake at 37°C, 200 r / min, for 45 min;

[0052] (6) Centrifuge at 8000 r / min, 4°C for 5 min, collect the supernatant, add 1 / 10 volume of sodium acetate solution (4 mol / L, pH = 4.5) to the supernatant, and place in a 4°C refrigerator for more than 2 h;

[0053] (7) Centrifuge at 12000 r / min, 4°C for 5 min and collect the precipitate;

[0054] (8) Wash twice with sterile water and centrifuge at 12000 rpm and 4°C for 3 min.

[0055] The extracted parasporal crystals were analyzed by SDS-PAGE electrophoresis to preliminarily predict the molecular weight of the protein expressed by the strain. Figure 5 As shown in the figure, the strain expresses proteins with a size of 70-130 kDa, which have typical characteristics of Bt insecticidal crystal proteins.

[0056] Example 5: Determination of the biological activity of Bacillus thuringiensis ZR-560 spores in vitro

[0057] Preparation of cytosolic mixture and protein quantification:

[0058] Pick a single colony of activated Bt bacteria ZR-560 and inoculate it into LB liquid. After culturing for 1 day, aspirate 200 μL and transfer it to 50 mL 1 / 2 LB liquid and culture it for 3 days. Centrifuge at 8000 r / min, discard the supernatant, add 5 mL of sterile water to the precipitate, weigh the wet weight of the precipitate and prepare a 472 mg / mL cytosolic mixture solution, and ultrasonically break it (total working time: 20 min, power: 150 W, ultrasonic on time: 10 s, ultrasonic off time: 15 s) to prepare a cytosolic mixture preparation sample, which is stored in a -80°C refrigerator for later use.

[0059] Cut the corn leaves into pieces about 1 cm long. 2 Then soak the leaves in the above-mentioned cell crystal mixture for 10s and then take them out to dry. The leaves soaked in clean water were used as blank controls (CK group). They were placed in 12-well culture plates, two sections were placed in each well, and a newly hatched larva of the fall armyworm was placed in each well. A blow-molded plate was placed in the cover of the 12-well cell culture plate and tied with a rubber band. They were placed in an insect breeding room with a temperature of 28±1℃ and a relative humidity of 60%±5%. The damage to the corn leaves was observed every day. Depending on the freshness of the corn tissue, freshly treated leaves were replaced every two days, and the number of deaths was counted after 3 days.

[0060] The experimental results are as attached Figure 6 As shown, it can be seen that after 3 days, the corn leaves in the CK group were seriously damaged, while the corn leaves in the experimental group were basically not damaged, and all the fall armyworm larvae that fed on the leaves in the experimental group died, with a corrected mortality rate of 100%.

[0061] The corrected mortality of Bt strain ZR-560 spores against corn borer, beet armyworm and Spodoptera litura was determined using the same method. The results are shown in Table 1 below.

[0062] Table 1

[0063] pests Corn borer Beet Armyworm Spodoptera litura Adjusted mortality rate (%) 100 100 100

[0064] According to Table 1, the Bt strain ZR-560 cytoplasms have high insecticidal activity against corn borer, beet armyworm and Spodoptera litura.

[0065] Example 6: Potted plant protection effect determination

[0066] Seedlings were raised in a tissue culture room (temperature 28°C), 2 wild-type corn seeds were sown in each pot, 3 replicates were used for each treatment, and when the corn seedlings grew to the 3-4 leaf stage, 2 newly hatched fall armyworm larvae were inoculated into each corn plant. The whole corn plant was sprayed with the Bt strain ZR-560 spore crystal mixture prepared in Example 5 using a spray bottle, until both sides of the leaves were wetted. A commercial Bt biopesticide (32000 IU / mg Bacillus thuringiensis, G033A Guard) was used as a positive control, and clean water was used as a negative control. Each treatment was placed in an insect cage to prevent the fall armyworm larvae from escaping.

[0067] The damage to corn in each group was observed after 3 days, 5 days and 7 days respectively.

[0068] The experimental results showed that the fall armyworm's appetite was relatively low 3 days after inoculation. After the potted corn in the clean water group was damaged by the fall armyworm larvae, a small number of insect holes appeared on the corn leaves, and the mesophyll tissue of the leaves was eaten, leaving only the epidermis; while the corn leaves in the experimental group and the Bt biological pesticide group were basically not damaged.

[0069] Five days after inoculation, the damage to the corn leaves in the clean water group was further aggravated, while the leaves in the Bt biological pesticide group only had sporadic holes, and the leaves in the experimental group were still not damaged.

[0070] Seven days after the insect inoculation, the fall armyworms began to feed heavily. After the leaves of the water group were damaged by the fall armyworm larvae, the corn leaves appeared striped, and most of the leaves had been completely eaten, with only a few leaves left. The corn leaves of the experimental group and the leaves of the Bt biological pesticide group were slightly damaged.

[0071] The damage degree of corn plants after the above three groups of treatments was graded and statistically analyzed, and the statistical results are shown in Table 2 below.

[0072] Table 2

[0073]

[0074] Example 7: Preliminary determination of field control efficacy

[0075] Corn was inoculated with insects at the trumpet stage of corn that was growing normally in the field, with each plant inoculated with 5 first-instar larvae of fall armyworm. The Bt strain ZR-560 spore crystal mixture prepared in Example 5 was sprayed on the corn plants, and an equal amount of clean water was sprayed as a negative control, and an equal amount of Bt biological pesticide (32000 IU / mg Bacillus thuringiensis, G033A Guard) was sprayed as a positive control. The spray was performed until the corn leaves were moist and the liquid was about to drip. After 14 days of treatment, the growth of corn in the field and the damage of fall armyworm were observed.

[0076] The results showed that dead insects appeared on the corn leaves of the treatment group of the ZR-560 cytoplasm mixture preparation and the positive control group. The insects were black and stiff. No dead insects were found on the corn leaves of the negative control group. The surviving insects grew and developed to the fourth instar (such as attached Figure 7 The corn leaves in the treatment group and the positive control group were not damaged, while the negative control group was seriously damaged, especially the cob and heart leaves. There were a lot of insect holes on the leaves, a lot of leaves were damaged in strips, many leaves were bitten off, and there were a lot of insect feces (such as attached Figure 8 (as shown in the figure), by pulling apart the curled leaves, you can see the larvae of the fall armyworm damaging the corn.

[0077] The field control effect of Bt strain ZR-560 was evaluated according to the degree of fall armyworm damage, and the results are shown in Table 3. The damage degree of corn leaves treated with this strain reached level 1, the damage degree of corn leaves in the positive control Bt G033A Guard treatment group reached level 1, and the damage degree of leaves in the negative control group was level 5. It can be seen that there is a significant difference between the corn leaves treated with Bt strain ZR-560 and the negative control, while there is no significant difference between strain ZR-560 and G033A Guard on the market, indicating that the control effect of this strain is equivalent to that of Bt G033A Guard registered on the market for fall armyworm control.

[0078] Table 3

[0079]

[0080] It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not depart from the spirit and scope of the present invention and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. A strain of Bacillus thuringiensis, named Bacillus thuringiensis ZR-560, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.30586, a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit date of May 17, 2024.

2. Use of Bacillus thuringiensis ZR-560 according to claim 1 in controlling crop pests.

3. The use according to claim 2, characterized in that: The crop pests include fall armyworm, corn borer, fall armyworm and / or beet armyworm.

4. A microbial preparation for controlling crop pests, characterized in that: The microbial preparation comprises the parasporal crystal protein of Bacillus thuringiensis ZR-560 according to claim 1.

5. The microbial preparation for controlling crop pests according to claim 4, characterized in that: The concentration of parasporal crystal protein of Bacillus thuringiensis ZR-560 in the microbial preparation is not less than 450 mg / mL.

6. Use of the microbial preparation according to claim 4 in controlling crop pests.

7. The use according to claim 6, characterized in that: The crop pests include fall armyworm, corn borer, fall armyworm and / or beet armyworm.

Images