CN115369057B - Bacillus thuringiensis B172 and application thereof - Google Patents

Abstract

The invention discloses a bacillus thuringiensis B172 and application thereof, wherein the bacillus thuringiensis B172 is preserved in China Center for Type Culture Collection (CCTCC), the preservation date is 2022, 3 and 7 days, and the preservation number is CCTCC No: m2022205. The strain has high-efficiency, specific and stable insecticidal activity on nocturnal moth pests, effectively kills nocturnal moth larvae through stomach toxic activity, has the advantages of low preparation cost, low environmental pollution and the like, has the potential of being developed into biological control agents for controlling nocturnal moth pests, and meets the important requirements of China on green sustainable comprehensive control of the pests.

Description

Bacillus thuringiensis B172 and application thereof

Technical Field

The invention relates to a bacillus thuringiensis strainBacillus thuringiensis) B172 and application thereof, belonging to the fields of microorganism and biological control.

Background

Noctuidae (notuisae) belongs to the noctuid superfamily (notuoidea) of Lepidoptera, is widely distributed in asia, europe, africa, oceangoin and other areas, is the most abundant one of the Lepidoptera species, and contains a plurality of major pests with great damage to agricultural production, such as spodoptera exigua, spodoptera litura, spodoptera frugiperda, cotton bollworm, armyworm and the like. Most of the insects have the characteristics of edibility impurities, large food consumption, high propagation speed, strong migratory property and the like, and have serious harm to various crops such as rice, wheat, corn, sorghum, tomatoes, peppers, soybeans, potatoes and the like which are closely related to human production and life. Therefore, prevention and control of the pest of this family and disaster reduction have been urgent. For a long time, the prevention and control of noctuidae pests at home and abroad mainly depends on chemical pesticides, so that the population quantity increase of the noctuidae pests can be effectively controlled, and the beneficial effect of protecting the agricultural industry is achieved. However, with the abuse of chemical pesticides, unreasonable application has caused a number of negative effects on the soil, water and other environments, and also has led to the development of pest resistance to some extent. Therefore, it has been urgent to explore a novel environment-friendly disease control method. Compared with the synthetic chemical pesticide, the bacterial pesticide developed by natural pathogenic bacteria is used for biological control of pests, has the characteristics of strong insecticidal specificity, environmental protection, low research and development cost and the like, is an important mode in the field of green pest control, and has great application potential.

Bacillus thuringiensis @Bacillus thuringiensisShort for shortBt) Is a gram-positive bacterium widely distributed in nature, and is mainly characterized in that a large amount of insecticidal crystal proteins (comprisingCryAndCytcrystallin) is produced, and the crystal protein is produced,Btthe companion crystal protein has poisoning effect on lepidoptera, coleopteran, dipteran and other pests. To be used forBtAs a microbial pesticide, the microbial pesticide has the characteristics of high efficiency, specificity, no toxicity, no environmental pollution and the like, and is widely applied to the control of agricultural, forestry and sanitary pests. However, with the followingBtLarge scale use of insecticidal crystal protein based insecticidesBtThe large-scale popularization of transgenic insect-resistant plants, the problem of pest resistance is continuously confirmed in laboratories and field experiments, and at least more than ten insect pairs are foundBtAnd insecticidal crystal proteins thereof. The main reason is that although a plurality of types of products are separated and reported at presentBtSubspecies or serotypes, but the high virulence wild-type strain still has less resources, resulting in the presence ofBtCommercialization of resources developedBtThe strain of the preparation is single in species and is difficult to bearBtThe transgenic insect-resistant plants are planted on a large scale to effectively prevent and control pests. Thus, there is a constant need to isolate more highly virulence from the fieldBtStrains to supplement existingBtScreening and separating out new high virulence from resourceBtThe insecticidal gene enriches the resource library of the insecticidal gene and provides a new gene source for commercialization of transgenic crops and engineering strains, thereby improvingBtThe insect-resistant effect of the transgenic product. Thus, a new search is soughtBtThe resource has very important significance for biological control in China.

Disclosure of Invention

Against the prior artBtThe preparation is easy to generate resistance and the resource shortage of wild strains is caused, and the invention aims to provide a wild bacillus thuringiensis with high-efficiency insecticidal activity on cotton bollworms and armyworms.

In order to achieve the purpose, a large amount of soil samples are collected from jungles in mountain areas of Liuyang, dafeng mountain, hunan province, bacillus strains are screened in a concentrated mode, the screened bacillus is numbered and named, strains are preserved, cotton bollworms and armyworms are used as control targets, and finally a bacillus strain 172 with efficient insecticidal activity is screened.

The strain 172 is identified as bacillus thuringiensis through colony morphology observation, gram staining, physiological and biochemical characteristics and 16S rRNA gene homology analysisBacillus thuringiensis) Designated Bacillus thuringiensis strain B172. China Center for Type Culture Collection (CCTCC) with a collection date of 2022, 3 months and 7 days, and a collection number of No: m2022205.

The application of the bacillus thuringiensis B172 in the prevention and control of the nocturnal major agricultural pests belongs to the technical field of production technicians, and the bacillus thuringiensis B172 can be prepared into a microbial pesticide for preventing and controlling the nocturnal major agricultural pests.

The invention also provides application of the bacillus thuringiensis strain in prevention and control of nocturnal moth pests. Preferably, the noctuidae pest is selected from at least one of asparagus caterpillar, prodenia litura, spodoptera frugiperda, cotton bollworm and armyworm.

More preferably, the noctuidae pest is selected from at least one of cotton bollworms and armyworms.

The invention also provides a biological pesticide for preventing and controlling nocturnal moth pests, which takes the bacillus thuringiensis B172 as an active ingredient.

Specifically, it is a solid residue obtained by fermenting the bacillus thuringiensis B172 with a culture solution, centrifuging, and removing the supernatant, namely a spore crystal mixture. More specifically, the culture broth was fermented for 3 days, centrifuged on a centrifuge at 8000 rpm/min for 4 min, and the supernatant was removed to obtain a solid residue, namely, a spore mixture, and the Bacillus thuringiensis B172 was used as a main active ingredient.

The biopesticide comprises bacillus thuringiensis B172 as described in one of the present applications. In one embodiment, the biopesticide may be in solid form or in liquid form. The biopesticide may also contain other substances that have a synergistic effect with bacillus thuringiensis B172 of the present application. For example, the substance may be a substance derived from a microorganism and inducing disease resistance in plants, a compound inducing disease resistance in plants, other microorganisms inducing disease resistance in plants, or the like; and the substance can be a microorganism source antibacterial substance, can also be an antibacterial compound, can also be other microorganisms with antibacterial effect, and the like. The biopesticide may further comprise at least one of an adjuvant, a thickener and a dispersant which is coordinated with the bacillus thuringiensis of the present application, for example, the adjuvant may be at least one selected from cotton seed oil, castor oil, tung oil, liquid paraffin, soybean oil, A1, A2, dimethyl phthalate, dibutyl phthalate, etc.; the thickener is at least one of bentonite, aluminum stearate, QH gel, dragon rubber powder, F1, xanthan gum, etc.: the dispersant is at least one of nekal, NNO, LFS, B1, carbon black, etc.

In one embodiment, the formulation of the biopesticide is at least one of a suspending agent, an oil suspension, a powder, a wettable powder and a granule.

Compared with the prior art, the invention has the following beneficial effects:

(1) The bacillus thuringiensis strain B172 is separated from non-cultivated soil of the jungle in the mountain and mountain area of Liuyang city of Hunan province, and finally is determined to be bacillus thuringiensis through morphological observation and molecular identificationBacillus thuringiensisB172; secondly, the applicant also determines the toxicity of the bacillus thuringiensis strain B172 to cotton bollworms and armyworms which are agricultural major pests, and the bacillus thuringiensis strain B172 has higher pathogenicity to the major agricultural pests of the noctuid family, so that the major agricultural pests of the noctuid family can be effectively killed. At presentBtIn the case of a relatively poor availability of the wild strain, the B172 strain according to the invention is a strain of the genusBtYet another supplement of biocontrol strain wild resourcesAnd (5) filling.

(2) The bacillus thuringiensis strain B172 is a biological control strain with potential significance in control of cotton bollworms and armyworms, can be developed into a microbial pesticide, and provides technical support for green and efficient control of serious agricultural pests in nocturnal moth.

The strain of the inventionBacillus thuringiensisstrain B172 with preservation number of CCTCC No: m2022205, the preservation date is 2022, 3 and 7 days, the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation address is university of Wuhan in China, and the culture Collection is detected and survived.

Drawings

FIG. 1 is a front view of colony morphology of strain B172;

FIG. 2 is a back view of colony morphology of strain B172;

FIG. 3 shows the absorbance of strain B172 at 30℃in different pH environments with stable growth;

FIG. 4 shows the absorbance of strain B172 under neutral conditions at different temperatures for growth stability;

FIG. 5 is a 16S rRNA sequence evolutionary tree of strain B172.

Detailed Description

The conception and technical effects of the present invention will be further clearly and completely described in the following examples to achieve a full understanding of the objects, features and effects of the present invention, which are to be construed as supplementary but not limiting. The described embodiments are only some, but not all, embodiments of the present invention, and other embodiments, which a person skilled in the art would obtain without inventive faculty, are within the scope of protection of the present invention.

The materials and reagents used in the following examples, unless otherwise specified, were all conventional; the materials used, the reagents and the like, unless otherwise specified, are all those which are commercially available.

Example 1: isolation, culture and identification of Strain B172

1. Isolation of strains

The bacillus thuringiensis of the inventionBacillus thuringiensis) The separation and purification method of B172 is as follows: selecting soil in a mountain area of Liuyang, dafengshan and Zhenmountain in Hunan province, selecting soil in a non-cultivated area with a depth of about 5-10cm during sampling, shoveling out surface soil by using a spade to remove impurities on the surface of the soil, collecting 3-5 soil samples at different positions by about 100 g, fully and uniformly mixing, putting into a pollution-free self-sealing bag, and making relevant acquisition records (soil number, sampling place, longitude and latitude, sampling date and the like). Weighing 5 g soil samples, putting the soil samples into a 50 mL sodium acetate liquid culture medium, vibrating the soil samples on a vortex oscillator for 1-3 min, and fully breaking the soil samples; after shaking culture at 30℃at 200 rpm/min for 4h, the soil sample-medium mixture was heat-treated in an oven at 80℃for 30 min, and after the high temperature had killed the bacteria, allowed to stand for cooling. In an ultra clean bench, 1 mL supernatant is diluted to 10 and 10 respectively ² 、10 ³ Three concentration gradient soil suspensions, 100 mu L of diluted supernatant is sucked onto LB solid culture medium, the coating is carried out in a circling way in the same direction, and the culture is carried out for more than 72 hours at 30 ℃ after the coating is uniform, and the observation is carried out for later use.

And (3) separating and purifying the bacterial strains in the cultured flat plate in an ultra-clean workbench. Carrying out four-zone line separation and purification on different types of strains on an LB solid culture medium according to the shape, diameter, color, viscosity and the like of the strains; repeating repeatedly until single colony spots appear, transferring the strain into LB liquid medium, and culturing on a shaker at 30deg.C and 200 rpm/min for 72h to obtain single strain bacterial liquid.

The isolated single strain was streaked again on LB solid plates, cultured in an incubator at 30℃for 48 or more h, and stored in a refrigerator at 4℃when a large amount of plaque formation was observed (see FIG. 1). During recovery, single bacterial plaque on the flat plate is selected and transferred into 5ml of LB liquid medium, and shake cultivation is performed at 200 rpm/min and 30 ℃ to complete activation.

2. Screening of active strains

Preparing feed: cutting artificial feed with consistent size, placing into a test tube subjected to high-temperature sterilization treatment, adding the cultured bacterial liquid, fully and uniformly mixing the bacterial liquid and the feed, spreading the uniformly mixed feed on a clean newspaper, and airing; after being dried, the dried seeds are respectively subpackaged in 24-hole insect breeding plates (the 24-hole insect breeding plates are subjected to advanced sterilization treatment) for later use; and feed without bacterial liquid treatment was used as a control to correct mortality of the insects.

And (3) primary screening: the 1-2-year-old armyworm larvae fed in a laboratory are gently dipped by a writing brush into a worm raising plate filled with feed treated by bacterial liquid, the feed which is not treated by bacterial liquid is used as a blank control, 20 heads are injected into each hole, and the mixture is placed at 27 ℃ and has relative humidity of 60% -70% and illumination of 16L: in an 8D constant temperature illumination incubator, the state of the insects is observed every day, the death number of the larvae is recorded, the death rate is calculated after 3 days, and the death rate is corrected.

And (3) re-screening: laboratory reared 3-year-old armyworm larvae were carefully picked with forceps into a feed-board with bacteria liquid-treated feed, and feed without bacteria liquid treatment was used as a blank control. And injecting 20 heads into each hole, respectively setting 3 repetitions, observing the states of the insects every day under the conditions of the rest culture conditions and the primary screening, recording the death number of the larvae, calculating the death rate after 3 days, and correcting the death rate. Strains with high insecticidal virulence were obtained for subsequent experiments.

Corrected mortality = (treatment mortality-control mortality)/(1-control mortality)

The invention discloses a method for preparing 455 strains from soil samples by co-separation, which uses relatively sensitive bollworm larvae raised indoors as target insects to perform biological activity preliminary screening, and preliminarily screens 4 strains of bacillus strains with high insecticidal activity (see table 1). The biological activity measurement experiment result of the 3-year-old myxoplasma larva shows that: strain 172 had the strongest poisoning effect on the 3-year-old myxoworm larvae, with a corrected mortality rate of 40% after 3 days, while strain 58 had no apparent poisoning effect on the 3-year-old myxoworm larvae (see table 1).

Table 1 insecticidal Activity of four strains against 1-2-year-old Helicoverpa armigera larvae and 3-year-old myxoma larvae

3. Identification of Strain B172

1) Determination of optimal temperature and pH for Strain 172

The isolated strain 172 was inoculated on LB solid medium, pH was adjusted with 1mol/L NaOH and 1mol/L HCl, and five pH gradients of 3, 5, 7, 9, 11 were set, 3 replicates were set for each gradient, and the strain was cultured in a 30℃incubator for 72 hours, and the growth of the strain was observed and recorded every 8 hours and its absorbance was measured with an enzyme-labeled instrument. As a result, strain 172 was found to have a slow growth rate and a substantially stable growth at the time of culturing for 40 hours, and the absorbance measured for 40 hours was shown in FIG. 3. As can be seen in FIG. 3, strain 172 grew better in a neutral environment, with an optimum pH of 7.

The isolated strain 172 was inoculated on a neutral LB solid medium, and cultured in five temperature gradient thermostats at 20, 25, 30, 35 and 40℃for 72 hours, 3 replicates were set for each gradient, and the growth of the strain was observed and recorded every 8 hours and its absorbance was measured using a microplate reader. As a result, strain 172 was found to grow rapidly at 24 hours of cultivation, and to grow exponentially, and after 24 hours, the growth rate was slowed down, and after 40 hours, it was found to reach a substantial equilibrium. The absorbance of strain 172 measured at 24h of culture is shown in FIG. 4. As can be seen from FIG. 4, strain 172 grows best at a temperature of 35 ℃.

2) Cell morphology and culture characteristic observation and physiological and biochemical index measurement

Culturing in a 35 ℃ incubator for three days, observing and recording the characteristics of bacterial colony; simultaneously picking a proper amount of bacterial colonies for gram staining; physiological and biochemical index determination the physiological and biochemical characteristics of strain 172 were identified by reference to related experimental methods of "Berger's Manual of bacteria identification (eighth edition) and" Manual of microbiology experiments ".

As shown in FIGS. 1 and 2, the strain 172 is in the shape of a rod, white colony and circular shape, and has micro-ridges in the middle and edge notches on a common LB solid medium. The physiological and biochemical identification results of the strain are shown in Table 3.

As can be seen from Table 2, strain 172 is a gram-positive bacterium, and its physiological and biochemical characteristics and morphological characteristics conform to the Bojie's Manual of bacteria identification (eighth edition) and the common bacteria identification handBacillus thuringiensis in bookBacillus thuringiensis) Is a classification criterion of (2).

TABLE 2 identification of physiological and biochemical characteristics of strain 172

Note that: "+" indicates a positive reaction, and "-" indicates a negative reaction

3) Molecular biological identification

The specific implementation process comprises the following steps: single colonies were picked from LB plates, transferred to 1.5 mL Ep tubes containing 1.2 mL LB liquid medium, cultured at 20℃at 200 rpm/min with shaking for 24 hours, and then extracted from the total DNA of strain 172 using a bacterial genomic DNA extraction kit according to the procedure. And (3) performing quality inspection on the extracted total DNA, and sending the total DNA to Beijing begrime and Kangbio-technology limited company for high-throughput sequencing after the quality inspection is qualified.

In order to improve the quality of sequencing data, fastQC software is used for carrying out quality control analysis on the original data, and fastp software is used for carrying out quality control on the original data so as to obtain high-quality clean reads. Genome assembly is carried out on the data after quality control by adopting Unicycler software to obtain a scaffolds genome sketch, and the genome sketch is annotated by using Bakta software; 16S rRNA was extracted from the annotated genome.

The homology alignment was performed on the measured 16S rRNA sequences using Basic BLAST on NCBI website, and sequences with higher homology were selected for alignment in MEGA X and a systematic NJ tree was constructed, and the results are shown in FIG. 5.

As can be seen from the phylogenetic tree, strain 172 andBacillus thuringiensisbelongs to one genetic branch and has higher support. The strain 172 is determined to be bacillus thuringiensis by combining morphological characteristics and physiological and biochemical testsBacillus thuringiensis) Classified and named asBacillus thuringiensis strain B172, the preservation unit is China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC No:m2022205, the preservation date is 2022, 3 and 7, and the preservation address is No. 299 of Wuchang district of Wuhan, hubei province.

Example 2: insecticidal Activity of Strain B172

The specific implementation process is as follows: picking single colony from LB plate, inoculating into LB liquid medium of 5mL, activating and culturing at 35deg.C and 200 rpm for 12 hr, transferring strain into 200 mL liquid medium according to 1% inoculum size, taking out culture solution after 3 days, centrifuging on 8000 rpm/min centrifuge for 4 min, removing supernatant and weighing solid residue (spore crystal mixture); respectively adding the spore mixture into culture mediums according to different qualities, diluting to obtain spore solutions with concentration gradients of 10 mg/mL, 50 mg/mL, 100 mg/mL, 200 mg/mL, 500 mg/mL and 1000 mg/mL, treating the feed with the spore solutions, ensuring that the feed is uniformly adhered to the spore solutions, and airing; putting the dried feed into a 24-hole insect-culturing plate, carefully picking 3-year-old myxoma and cotton bollworm larvae raised in a laboratory into the insect-culturing plate with the feed treated by the bacterial liquid, respectively repeating the steps for three times by taking the feed which is not treated by the bacterial liquid as a blank control, observing the survival condition of the larvae every day, counting the death rate of the larvae under different liquid medicine concentration gradients after 3 days, and calculating the death concentration amount every day by using SPSS software.

TABLE 3 determination of the Activity of Strain B172 against armyworm

Table 3 shows the results of the biological activity assay of strain B172 against armyworm and cotton bollworm larvae of three ages. From the results, strain B172 had the highest toxicity to the target pest on the third day, and had LC50 values of 18.36 mg/mL and 66.08 mg/mL for myxoma, respectively; LC50 for cotton bollworms was 51.88 mg/mL and LC95 value was 570.27 mg/mL, respectively.

In conclusion, the bacillus thuringiensis of the inventionBacillus thuringiensis) The B172 strain has strong inhibition effect on both cotton bollworms and armyworms which are agricultural major pests, and is environment-friendly, and cannot cause secondary pollution to ecological environment.The bacillus thuringiensis B172 strain is suitable for biological control of agricultural major pests, namely cotton bollworm and armyworm, and is available at the present stageBtIn the case of lack of wild strainBtYet another supplement to the resource.

Although the invention has been described with reference to specific embodiments, those skilled in the art will appreciate that various modifications might be made without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of materials, and method to the essential scope, spirit, and scope of the present invention. All such modifications are intended to be included within the scope of this invention as defined in the following claims.

Claims (7)

1. Bacillus thuringiensis strainBacillus thuringiensis) B172, the culture medium is preserved in China Center for Type Culture Collection (CCTCC) No: m2022205.

2. The use of a strain of bacillus thuringiensis according to claim 1 for the control of noctuidae pests selected from at least one of cotton bollworm and armyworm.

3. A biopesticide for controlling noctuidae pests selected from at least one of cotton bollworms and armyworms, comprising the bacillus thuringiensis B172 of claim 1 as an active ingredient.

4. A biopesticide according to claim 3, which is obtained by fermenting said bacillus thuringiensis B172, centrifuging and removing the supernatant to obtain a solid residue.

5. The biopesticide according to claim 4, wherein the solid residue is obtained by fermenting for 3 days, centrifuging for 4 minutes in a centrifuge at 8000 rpm/min, and removing the supernatant.

6. The biopesticide according to claim 4 or 5, wherein the dosage form of the biopesticide is at least one of a suspension, a powder and a granule.

7. The biopesticide according to claim 4 or 5, wherein the formulation of the biopesticide is an oil suspension or wettable powder.

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