Bacillus thuringiensis ZR-198 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus thuringiensis, the name of which is bacillus thuringiensis ZR-198, which is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number of which is CGMCC No.30587, the preservation address of which is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation date of which is May 17, 2024. The bacillus thuringiensis ZR-198 has good virulence activity to nematodes, the corrected death rate to caenorhabditis elegans reaches 84%, and the corrected death rate to meloidogyne incognita reaches 86%.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Bacillus thuringiensis ZR-198 and an application thereof. Background Art

[0002] Root-knot nematodes are highly specialized omnivorous plant pathogenic nematodes. As of 2022, there are more than 100 species of root-knot nematodes, of which more than 40 have been reported in my country. Root-knot nematodes are the most destructive plant parasitic nematodes and one of the most important soil-borne pests. They can cause serious yield and quality losses to a variety of agricultural crops, causing economic losses of US$80 billion to US$157 billion to global agricultural production each year. As of October 1, 2023, a total of 334 nematicide pesticides have been registered in my country, including 313 chemical nematicides and 21 microbial nematicides; 263 single agents and 71 compound preparations. Root-knot nematodes are tiny and difficult to detect, and most of them are parasitic in plants. Conventional pesticides find it difficult to reach their parasitic niches, resulting in few microbial strains that are toxic to root-knot nematodes.

[0003] Bacillus thuringiensis (Bt) has been discovered for 100 years and has played a huge role in pest control. It is the most intensively studied, rapidly developed and widely used microbial insecticide in recent years. The insect control principle of Bacillus thuringiensis is that its strains can produce two types of toxins, endotoxins (parasporal crystals) and exotoxins, which can kill pests due to starvation, blood spoilage and nerve poisoning.

[0004] However, since my country started the screening research of nematicidal Bt strains relatively late, the research results in the United States in this regard have basically been patented, and there are very few publicly published documents, so the available resources are very limited. At present, the prevention and control of root-knot nematodes in farmland is still at a stage where it is difficult to prevent and control, so it is of great significance to find Bt strains with high toxicity to root-knot nematodes, which will have a positive role in promoting the economic development of agriculture. Summary of the invention

[0005] The purpose of the present invention is to provide Bacillus thuringiensis ZR-198, enriching the efficient strain resources for controlling crop pests such as root-knot nematodes in agricultural production applications, and providing a new option for solving the above problems. Another purpose of the present invention is to provide the application of the above Bacillus thuringiensis ZR-198 in controlling crop pests.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] A strain of Bacillus thuringiensis, named Bacillus thuringiensis ZR-198, is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No. 30587. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is May 17, 2024.

[0008] The colony morphology of the Bacillus thuringiensis ZR-198 provided by the present invention on the LB medium is usually milky white or milky yellow round or irregular shaped colonies, with a smooth surface, neat edges, and sometimes slight bumps.

[0009] Through whole genome sequencing, it was found that the strain had 5 genes related to Bacillus thuringiensis insecticidal protein, one of which had a similarity of only 51.8%. This gene was named 198-X.

[0010] The present invention also provides the use of the above-mentioned Bacillus thuringiensis ZR-198 in preventing and controlling nematodes.

[0011] In some embodiments, the crop pest includes Caenorhabditis elegans and / or Meloidogyne incognita.

[0012] The present invention also provides a microbial preparation for controlling crop pests, wherein the microbial preparation comprises the parasporal crystal protein of Bacillus thuringiensis ZR-198.

[0013] The present invention also provides application of the above-mentioned microbial preparation in preventing and controlling nematodes.

[0014] In some embodiments, the crop pest includes Caenorhabditis elegans and / or Meloidogyne incognita.

[0015] The beneficial effects of the present invention are:

[0016] The Bacillus thuringiensis ZR-198 of the present invention has good toxic activity against nematodes, and the corrected mortality rate against Caenorhabditis elegans reaches 84%, and the corrected mortality rate against southern root-knot nematodes reaches 87%. The killing of root-knot nematodes helps to down-regulate CLE-related genes in plants, thereby reducing the production of nodules caused by CLE gene expression, and ultimately protecting the normal growth and development of plant roots. In addition, the Bacillus thuringiensis ZR-198 has simple culture conditions, rapid growth and easy preservation, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Attached Figure 1 This is the colony morphology of Bacillus thuringiensis ZR-198;

[0018] Attached Figure 2This is the gene length distribution diagram of Bacillus thuringiensis strain ZR-198;

[0019] Attached Figure 3 This is the circle map of the genome of Bacillus thuringiensis strain ZR-198. From the inside to the outside, they are: GC content, GC skew value, gene distribution (positive and negative chains are divided into two circles), and scaffold distribution. The color of the gene is filled with the classification of COG;

[0020] Attached Figure 4 It is the full-length gene identification diagram of 198-X, wherein, 1-3: 198-XF / 198-YR PCR product; M: Trans5KPlus DNA marker;

[0021] Attached Figure 5 The results of SDS-PAGE analysis of Bacillus thuringiensis ZR-198 crystal protein;

[0022] Attached Figure 6 This is a picture showing the effect of Bacillus thuringiensis ZR-198 on the embryonic development of southern root-knot nematode;

[0023] Attached Figure 7 These are comparison pictures of tomato roots in four treatment groups after the pot experiment in Example 5, where A is the CK group; B is the biological control group; C is the chemical control group; and D is the experimental group. DETAILED DESCRIPTION

[0024] 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.

[0025] Example 1: Screening of Bacillus thuringiensis ZR-198

[0026] In the early stage of the present invention, 944 strains of Bacillus thuringiensis were isolated from 1769 soil samples collected from the ecological zone of Sichuan according to the general screening method in the field, and 283 strains of crystal-producing Bt bacteria were preliminarily identified from them using an optical microscope. By conducting toxicity tests on the preliminarily identified strains, it was found that 9.89% of the 283 strains of Bt strains had the activity of killing Caenorhabditis elegans, but only more than 20 strains had the toxic effect on southern root-knot nematodes, and one of them was named Bacillus thuringiensis ZR-198.

[0027] As attached Figure 1 As shown, the colony morphology of Bacillus thuringiensis ZR-198 on LB medium is usually milky white or milky yellow round or irregular shaped colonies. The colony surface is smooth, with neat edges, and sometimes slightly uneven.

[0028] Example 2: Identification of Bacillus thuringiensis ZR-198

[0029] Genome Sequencing and Analysis:

[0030] Through whole genome sequencing, the total length of the ZR-198 strain genome sequence is 6,784,341bp, the total length of the annotated genes is 5,664,450bp, and the GC content is 35.63%, including 112 tRNAs, 22 rRNAs, and 7,180 coding genes. The gene length is mainly distributed in the range of 100-500bp, of which 277 genes longer than 2,000bp are predicted. Figure 2 The gene length distribution diagram of ZR-198 strain is shown in Figure 2. Figure 3 This is the circle map of the genome of strain ZR-198.

[0031] It was found that the strain had 5 genes related to insecticidal proteins (see Table 1 below), one of which had a similarity of only 51.8%, and this gene was named ZR198-Kn.

[0032] Table 1

[0033]

[0034] Prediction and analysis of ZR198-Kn gene structure

[0035] (1) Amplification of the full length of the ZR198-Kn gene

[0036] The genome of ZR-198 strain was used as a template for PCR amplification. The results showed that ZR198-Kn amplified a fragment of about 1.5 kb, as shown in the attached Figure 4 shown.

[0037] (2) 198-X protein structure prediction and analysis

[0038] The protein sequence encoded by gene 198-X was analyzed using the software NCBI Conserved Domain Summary, which contained three conserved regions (Domains). The software predicted the structure and showed that amino acids 12-508 encoded three domains, Bac_thur_toxin consisted of 233 amino acid residues from amino acid 12 to amino acid 244; amino acids 274 to 508 encoded two RICINs, the first RICIN from amino acid 247 to amino acid 360, a total of 114 amino acid residues, and the second RICIN from amino acid 424 to amino acid 508, a total of 85 amino acid residues.

[0039] Example 3: Extraction and analysis of parasporal crystal proteins from Bacillus thuringiensis ZR-198

[0040] (1) Bacillus thuringiensis ZR-198 was inoculated into 1 / 2 LB medium, cultured at 28°C and 220 r / min in a shaking incubator for 72 h, the culture solution was placed in a 50 mL centrifuge tube, centrifuged at 4°C and 5000 r / min for 15 min, and the supernatant was discarded;

[0041] (2) Wash the cells with 1 mol / L NaCl and sterile distilled water, centrifuge at 4°C and 5000 rpm for 15 min, discard the supernatant, and repeat three times;

[0042] (3) Dissolve the precipitate with 200 mL of 50 mmol / L Na2CO3, pH = 9.5, and 10 mmol / L dithiothreitol (DTT), centrifuge the mixture at 5000 r / min for 15 min, take the supernatant, add 1:3 3 mol / L sodium acetate solution, pH = 4.5 to the supernatant, shake continuously, let stand at 4°C overnight, centrifuge at 12000 r / min for 10 min, and take the precipitate.

[0043] (4) Wash the precipitate with sterile distilled water, centrifuge at 7000 r / min for 10 min, repeat 3 times, and dissolve the precipitate with 50 mmol / L Na2CO3.

[0044] SDS-PAGE analysis of crystal proteins:

[0045] The analysis results are as attached Figure 5 As shown, it was found that the electrophoretic band sizes of the crystal protein of this strain were mainly 70kDa, 60kDa, 55kDa and 35kDa.

[0046] Example 4: Indoor biological activity experiment of Bacillus thuringiensis ZR-198 crystal protein

[0047] Determination of insecticidal activity in Caenorhabditis elegans:

[0048] (1) Take a sterile 96-well microplate and use a worm picker or a 10 μL pipette to pick C. elegans of uniform size under a microscope. Place 30 second-instar nematodes into each well.

[0049] (2) Add 20 μL of diluted crystal protein sample.

[0050] (3) Five concentration gradients were set up, each concentration was repeated 3 times, and a 0.05 mol / L Na2CO3 solution (pH = 9.5) was used as a negative control. The cells were cultured at 28°C.

[0051] (5) Record the number of live worms once every 24 hours for a total of three times.

[0052] Determination of insecticidal activity against southern root-knot nematodes:

[0053] (1) Take a sterile 96-well microplate and use a worm picker or a 10 μL pipette to pick up second-instar larvae of the southern root-knot nematode of uniform size under a microscope, and suck 30 second-instar larvae into each well.

[0054] (3) Add 20 μL of diluted crystal protein sample and, according to experimental requirements, add 20 μg / mL chloramphenicol to inhibit the growth of bacteria.

[0055] (4) Five concentration gradients were set, each concentration was repeated 3 times, and the 0.05 mol / L Na2CO3 solution (pH = 9.5) was used as a negative control. The cells were cultured at 28°C.

[0056] (5) Record the number of live worms once every 24 hours for a total of three times.

[0057] The corrected mortality rates of Caenorhabditis elegans and R. incognita were calculated respectively, and the regression equation of toxicity and the median lethal concentration (LC) of crystal protein were obtained by probability value and EXCEL software analysis. 50 The results are shown in Table 2 below.

[0058] Table 2

[0059]

[0060]

[0061] Determination of the effects on nematode embryonic development:

[0062] Pick mature oocysts of similar size and color, place them in 0.5% sodium hypochlorite solution for disinfection for 3 minutes, and rinse them repeatedly with sterile water for 3 times. Add 3 oocysts to each well of a sterilized 96-well PCR plate, then add a crystal protein sample (concentration is about 295.4μg / mL), and repeat each treatment 3 times. Use sterile LB liquid or 0.05mol / L Na2CO3 solution (pH=9.5) as a control and place at 28°C. Take samples every 24h for observation. When the observation reaches 72h, dissect the oocysts with a dissecting needle, rinse the oocysts repeatedly with a small amount of sterile water, and draw 50μl of egg suspension for observation under a microscope.

[0063] The results showed that compared with the control group, the crystal protein treatment group of ZR-198 strain had a significant inhibitory effect on the number of root-knot nematode eggs, reducing it by 62.5%. Figure 6 As shown in the figure, after 48 hours of treatment, the material in the eggs of the southern root-knot nematode treated with the crystal protein of the ZR-198 strain showed severe vacuolization, the cell contents showed uneven and chaotic distribution, and some of the contents leaked out, resulting in the inability of the formed larvae to break through the wall and develop normally. This shows that the ZR-198 crystal protein has a serious impact on the eggs of the southern root-knot nematode during the critical development period.

[0064] Example 5: Potted experiment on the control effect of Bacillus thuringiensis ZR-198 suspension on root-knot nematodes

[0065] The tomato varieties to be tested were sown in seedling trays, and when the tomato seedlings grew to 4 true leaves, they were transplanted into plastic pots filled with sterilized soil. Two days after transplanting, the bacterial suspension was watered to a concentration of 10 6 CFU / g soil, with the same amount of LB medium as the blank control (CK), the same concentration of Pseudomonas lilacinus suspension as the biological control group, and 1.8% avermectin emulsifiable concentrate as the chemical control group (purchased from the market, the dosage refers to the drug instructions). At the same time, the nematodes were inoculated into the roots of tomatoes at an inoculation rate of 1000 second-instar larvae of southern root-knot nematodes per plant, and each treatment was repeated 3 times. After inoculation, the pots were randomly placed on the light incubator platform, and the culture conditions were 16h of light, 26℃, 8h of darkness, 20℃, and the plants were taken out after 35d of culture.

[0066] The experimental results are as attached Figure 7 As shown, the symptoms of root-knot nematode disease in the CK group were very obvious, with a large number of root knots on the root system, and heavy root knots appeared. The egg sacs on the root knots were relatively mature, while the root knots of tomatoes treated with the experimental group were smaller.

[0067] The root length of the plants was measured and the number of root nodes was investigated. The specific data are shown in Table 3 below.

[0068] Table 3

[0069] Group CK Experimental Group Biological control group Chemical control group Root length (cm) 30±2d 47±2.88a 37.67±2.52c 42.33±1.53b Number of root knots 25.33±0.58a 2.67±2.52c 10.33±3.51b 3.67±1.53c

[0070] As shown in Table 3, at 35 days, the number of root knots of the tested tomato plants decreased by 89.46%, 74.15% and 27.25% compared with the CK group, biological control group and chemical control group, respectively.

[0071] In summary, it was shown that the ZR-198 strain could effectively reduce the infection of early nematodes and the formation of root knots in tomatoes.

[0072] Eggplant, pepper, cucumber, zucchini and melon potted plants were used as experimental objects. The control effect of Bacillus thuringiensis ZR-198 bacterial suspension on root-knot nematodes was tested in the same way. The results are shown in Table 4.

[0073] Table 4

[0074]

[0075] At the same time, the growth of the plants was measured, and the results showed that compared with the CK group, the aboveground fresh weight and underground fresh weight of the tomato seedlings in the experimental group increased by 104.58% and 106.48% respectively after 35 days, and the plant height increased by 14.55%. Compared with the biological control group, except for plant height and stem diameter, the other indicators were significantly better, among which the underground fresh weight was the most obvious, increasing by 34.18%, followed by the aboveground fresh weight, which increased by 12.70%. Compared with the chemical control group, the underground fresh weight of tomato plants in the experimental group increased by 14.84%.

[0076] Compared with the CK group, the stem diameter of eggplant seedlings in the experimental group increased by 80.00%, the plant height increased by 63.00%, and the underground fresh weight increased by 18.92% after 35 days. Compared with the biological control group, there were no significant differences in indicators such as plant height and stem diameter. Compared with the chemical control group, the growth promotion effect of eggplant plants in the experimental group was slightly lower.

[0077] Compared with the CK group, the aboveground fresh weight and underground fresh weight of pepper seedlings in the experimental group increased by more than 2 times after 35 days, followed by plant height and stem diameter, which increased by 71.34% and 83.33%, respectively.

[0078] Compared with the CK group, the plant height of the cucumber seedlings treated in the experimental group increased by 81.06% after 35 days, and the aboveground fresh weight and underground fresh weight increased by 76.90% and 60.00%, respectively. Compared with the biological control group, except for plant height and stem diameter, the other indicators were significantly better, with aboveground fresh weight increased by 35.27% and underground fresh weight increased by 17.31%; compared with the chemical control group, the aboveground fresh weight and underground fresh weight of the cucumber plants treated in the experimental group increased by 26.01% and 6.09%, respectively.

[0079] Compared with the CK group, the aboveground fresh weight and underground fresh weight of zucchini seedlings treated in the experimental group increased by 34.20% and 69.74% respectively after 35 days. Compared with the biological control group, there was no difference in plant height and stem diameter, and the aboveground fresh weight and underground fresh weight increased by 6.28% and 33.68% respectively; compared with the chemical control group, the underground fresh weight of zucchini plants treated in the experimental group increased by 33.68%.

[0080] Compared with the CK group, the plant height and underground fresh weight of melons in the experimental group increased by 32.23% and 35.86% respectively. There was no significant difference compared with the biological control group and the chemical control group.

[0081] 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-198, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.30587, 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-198 according to claim 1 in controlling nematodes.

3. The use according to claim 2, characterized in that: The nematodes include Caenorhabditis elegans and / or Meloidogyne incognita.

4. A microbial preparation for controlling crop pests, characterized in that: The microbial preparation comprises the parasporal crystal protein of Bacillus thuringiensis ZR-198 according to claim 1.

5. Use of the microbial preparation according to claim 4 in controlling nematodes.

6. The use according to claim 5, characterized in that: The nematodes include Caenorhabditis elegans and / or Meloidogyne incognita.

Images