Pest control engineering bacterium as well as construction method and application thereof

Abstract

The invention discloses a pest control engineering bacterium as well as a construction method and application thereof, and belongs to the technical field of engineering bacteria and gene interference. The engineering bacteria disclosed by the invention are obtained by exogenous transfer of dsRNA fragments of pest target genes into plant probiotics. The engineering bacterium disclosed by the invention can stably express dsRNA fragments of pest target genes, can interfere the pest target genes when being used for pest control, reduces pupation rate, and has a good control effect on pests; the engineering bacterium disclosed by the invention can be colonized in various host plants of polyphagous insect fall webworms, such as poplars and mulberries, so that broad-spectrum prevention and control can be realized. Meanwhile, the engineering bacterium retains the original advantages of strong environmental adaptability, convenient genetic manipulation, high product stability and the like of plant probiotics, can exert the plant probiotic characteristics of the plant probiotics, assists the healthy growth of plants while realizing accurate prevention and control of pests, and has a good application prospect. And a promising tool is provided for pest control and synchronous and sustainable protection of various host plants.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of engineering bacteria and gene interference technology, and particularly relates to a pest control engineering bacteria and a construction method and application thereof. BACKGROUND

[0002] RNA interference (RNAi) is a conserved mechanism in organisms to regulate gene expression through double-stranded RNA. This technology has become an important tool for studying gene function and has shown unique advantages in pest control: compared with traditional pesticides, RNAi has sequence specificity and uses natural dsRNA molecules for precise targeted control. Double-stranded RNA delivery technology is the key to application. Among the existing delivery methods, spray-induced gene silencing (SIGS), host-induced gene silencing (HIGS), and microbe-induced gene silencing (MIGS) have great application potential. However, SIGS has poor drug efficacy persistence and safety problems; HIGS is limited by the difficulty of plant transgenesis and single protection range; MIGS can be used for sustained control, but it relies on insect endosymbiotic bacteria as carriers, and the stable colonization and genetic modification of such strains still have challenges.

[0003] Due to the long replacement cycle of forest trees compared to crops, the existing transgenic plant control strategies have limited effect on polyphagous pests (which can harm multiple host plants). For example, the global invasive species Hyphantria cunea can harm more than 600 plant species and has strong reproductive ability, disrupting ecological balance. Existing control relies on chemical pesticides, and new control methods are urgently needed. Therefore, it is particularly important to provide a technology that can stably and effectively control polyphagous pests on multiple host plants. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a pest control engineering bacteria that can stably express dsRNA of a target gene of a pest, has good control effect on the pest, and can protect multiple host plants from pest damage.

[0005] Another purpose of the present application is to provide a construction method of the engineering bacteria.

[0006] Another purpose of the present application is to provide a preparation.

[0007] Another purpose of the present application is to provide an application of the engineering bacteria, the engineering bacteria constructed by the construction method, or the preparation in pest control.

[0008] Another purpose of the present application is to provide a pest control method.

[0009] In order to achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0010] The application provides a pest control engineering bacterium, which is obtained by exogenous transfer of a dsRNA fragment of a pest target gene to a plant probiotic bacterium.

[0011] Preferably, the plant probiotic bacterium comprises Bacillus paralicheniformis, Bacillus licheniformis, Bacillus subtilis or Bacillus amyloliquefaciens.

[0012] Preferably, the plant probiotic bacterium comprises Bacillus paralicheniformis SH-F8, and the preservation number of the Bacillus paralicheniformis SH-F8 is CGMCC No. 30890.

[0013] Preferably, the dsRNA comprises a dsRNA of a vATPaseA gene of Hyphantria cunea.

[0014] Preferably, the sequence of the dsRNA of the vATPaseA gene of Hyphantria cunea comprises a sequence as shown in SEQ ID No. 2, 77th-469th.

[0015] The application further provides a construction method of the engineering bacterium, which comprises the following steps: knocking out a rnc gene of the plant probiotic bacterium, inserting a bidirectional T7 promoter and a dsRNA fragment at the position of knocking out, and starting a T7 RNA polymerase of the T7 promoter to obtain the engineering bacterium.

[0016] Preferably, the sequence of the rnc gene comprises a sequence as shown in SEQ ID No. 1.

[0017] Preferably, the sequence of the bidirectional T7 promoter and the dsRNA fragment comprises a sequence as shown in SEQ ID No. 2.

[0018] Preferably, the sequence of the T7 RNA polymerase comprises a sequence as shown in SEQ ID No. 3.

[0019] The application further provides a preparation comprising the engineering bacterium or the engineering bacterium obtained by the construction method.

[0020] The application further provides application of the engineering bacterium or the engineering bacterium obtained by the construction method or the preparation in pest control.

[0021] The application further provides a pest control method, which comprises: applying the engineering bacterium or the engineering bacterium obtained by the construction method or the preparation to a plant.

[0022] The application has the following beneficial effects:

[0023] The dsRNA fragment of the pest target gene is introduced into the plant probiotic bacteria, and the obtained engineering bacteria can stably express the dsRNA fragment of the pest target gene, can interfere with the pest target gene when used for pest control, reduces the pupation rate, and has good prevention and control effect on polyphagous pests; the engineering bacteria of the present application can colonize in various host plants such as poplar and mulberry, thereby achieving broad-spectrum prevention and control. At the same time, the engineering bacteria of the present application retain the original advantages of the plant probiotic bacteria, such as strong environmental adaptability, convenient genetic operation, high product stability, etc., can play its plant probiotic characteristics, realize precise pest control, and help plant healthy growth, and provide a promising tool for synchronous and sustainable protection of pests and various host plants.

[0024] Biological preservation information

[0025] The present application is bacillus paralicheniformis SH-F8, which is classified as bacillus paralicheniformis, preserved in the China General Microbiological Culture Collection Center (CGMCC), preserved on June 7, 2024, located at No. 3, Beichen West Road, Beijing Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, and has a preservation number of CGMCC No. 30890. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is the electrophoresis map of the dsRNA expressed by the engineering bacteria strain in Example 1.

[0027] Figure 2 It is the performance of the engineering bacteria after being fed by the American white moth in Example 2, wherein CK represents normal feeding of feed, WT represents feeding of feed wild type strain, and Δ:: represents feeding of engineering bacteria strain.

[0028] Figure 3 It is the colonization of the engineering bacteria strain on the leaves of two trees in Example 3, wherein A is the colonization in the interior of mulberry leaves, B is the colonization on the surface of mulberry leaves, C is the colonization in the interior of poplar leaves, and D is the colonization on the surface of poplar leaves. DETAILED DESCRIPTION

[0029] The present application provides a pest control engineering bacteria, which is obtained by exogenous introduction of a dsRNA fragment of a pest target gene into a plant probiotic bacteria.

[0030] In the present application, the plant probiotics preferably include Bacillus bacteria; Bacillus bacteria are commonly used biocontrol agents, widely distributed and highly adaptable to the environment, and most Bacillus bacteria are safe to humans, animals and plants as biological control agents, and have good biological safety. As an implementable manner, the plant probiotics preferably include Bacillus paralicheniformis, Bacillus licheniformis, Bacillus subtilis or Bacillus amyloliquefaciens. In some embodiments, the plant probiotics preferably include Bacillus paralicheniformis SH-F8, and the preservation number of the Bacillus paralicheniformis SH-F8 is CGMCC No. 30890. The Bacillus paralicheniformis SH-F8 of the present application is isolated from the natural environment and has environmental friendliness.

[0031] In the present application, the type of pests is not particularly limited, and preferably includes polyphagous pests; in some embodiments, the pests preferably include Hyphantria cunea.

[0032] In the present application, the type of target genes is not particularly limited, and in some embodiments, the target genes preferably include the vATPaseA gene of Hyphantria cunea.

[0033] In the present application, the dsRNA fragment preferably includes the dsRNA fragment of the vATPaseA gene of Hyphantria cunea; the sequence of the dsRNA of the vATPaseA gene of Hyphantria cunea is preferably shown in SEQ ID No. 2, positions 77-469.

[0034] The present application transfers the dsRNA fragment of the pest target gene into the plant probiotics, and the obtained engineering bacteria can stably express the dsRNA fragment of the pest target gene, which can interfere with the pest target gene and affect its oviposition when used for pest control, and has good prevention and control effect on polyphagous pests; the engineering bacteria of the present application can colonize in various host plants, thereby achieving broad-spectrum prevention and control. At the same time, the engineering bacteria of the present application retain the original advantages of the plant probiotics, such as strong environmental adaptability, convenient genetic manipulation, high product stability, etc., can exert the plant probiotic characteristics, achieve precise pest control, and help the healthy growth of plants, which is in line with the concept of green control.

[0035] The present application also provides a construction method of the engineering bacteria, which comprises: knocking out the rnc gene of the plant probiotics, inserting a bidirectional T7 promoter and a dsRNA fragment at the knockout position, and starting the T7 RNA polymerase of the T7 promoter to obtain the engineering bacteria.

[0036] In the present application, the rnc gene is a gene encoding RNase III, and since RNase III can degrade dsRNA, the rnc gene in the plant probiotic is preferably knocked out and then used to construct the engineered bacteria. In some embodiments, the sequence of the rnc gene preferably comprises the sequence shown in SEQ ID No. 1.

[0037] In the present application, the method for knocking out can be routinely selected according to actual needs.

[0038] In the present application, the sequence of the bidirectional T7 promoter and the dsRNA fragment preferably comprises the sequence shown in SEQ ID No. 2. In the sequence of SEQ ID No. 2, positions 1-23 are a T7 promoter, positions 77-469 are a dsRNA fragment, and positions 483-505 are a T7 promoter; since the dsRNA is double-stranded RNA, the bidirectional T7 promoter can form positive and negative strands and combine into double-stranded RNA, thereby ensuring the exertion of the efficacy of the dsRNA.

[0039] In the present application, the sequence of the T7 RNA polymerase preferably comprises the sequence shown in SEQ ID No. 3; the T7 RNA polymerase can specifically initiate the T7 promoter, thereby ensuring the expression of the double-stranded RNA.

[0040] The construction method of the present application can obtain the engineered bacteria by directly inserting the dsRNA expression system into the genome of the bacterial strain, and compared with the strategy of using a plasmid vector, the method can avoid the instability of the expression of the dsRNA caused by the loss of the plasmid.

[0041] The present application also provides a preparation, which preferably comprises the engineered bacteria or the engineered bacteria constructed by the construction method.

[0042] In some embodiments of the present application, the preparation method preferably comprises: inoculating the single colony of the engineered bacteria into a culture medium for culture; inoculating the culture solution into a new culture medium at a volume ratio of 0.5-5:100 for culture; and collecting the bacterial bodies, resuspending, and obtaining the preparation.

[0043] The type of the culture medium preferably comprises an LB liquid culture medium.

[0044] The single colony is preferably cultured by oscillation; the culture temperature is preferably 30-40℃, for example, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40℃; and the culture time is preferably 8-16h, for example, 8, 9, 10, 11, 12, 13, 14, 15, or 16h.

[0045] The volume ratio of the culture medium to the new culture medium is preferably 0.5:5 to 100, for example 0.5:100, 1:100, 2:100, 3:100, 4:100 or 5:100.

[0046] The preferred types of the new culture medium include LB liquid medium.

[0047] The new culture medium preferably employs shaking culture, and the culture temperature is preferably 30–40°C, for example 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40°C; the culture time is preferably 16–32 h, for example 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 h; the culture rotation speed is preferably 150–200 rpm, for example 150, 160, 170, 180, 190 or 200 rpm.

[0048] The method of collecting the bacterial cells is not particularly limited, but centrifugation is preferred. After collecting the bacterial cells, washing is also preferred, preferably with sterile water, and the number of washes is preferably 2 to 4 times, for example 2, 3 or 4 times.

[0049] The resuspension is preferably performed using sterile water. There is no particular limitation on the bacterial concentration after resuspension. In some embodiments, the OD600 is preferably adjusted to 0.1 to 0.5 after resuspension, for example, 0.1, 0.2, 0.3, 0.4 or 0.5.

[0050] In this invention, the formulation preferably also includes formulation-acceptable adjuvants. There are no special limitations on the types of adjuvants, including but not limited to one or more of carriers, diluents, excipients, preservatives, surfactants, and antioxidants.

[0051] In this invention, the dosage form of the preparation is not particularly limited, and can be a liquid preparation, a solid preparation, a semi-solid preparation, or a gaseous preparation, etc., preferably a liquid preparation.

[0052] The present invention also provides the application of the engineered bacteria or the engineered bacteria constructed by the construction method or the preparation thereof in pest control.

[0053] The present invention also provides a method for controlling pests, wherein the method preferably includes: applying the engineered bacteria or the engineered bacteria constructed by the construction method or the preparation to plants.

[0054] In this invention, the method of application is not particularly limited, but spraying is preferred; in some embodiments, it is preferred to spray on the above-ground parts of the plant, and more preferably on the leaves of the plant.

[0055] In this invention, the application period is not particularly limited, but in some embodiments, application during the seedling stage is preferred.

[0056] In this invention, the types of plants are not specifically limited, but preferably include plants that are susceptible to pests; the plants preferably include trees, and in some embodiments, the plants preferably include mulberry or poplar.

[0057] When the engineered bacteria of this invention are applied to plants, they can stably colonize on the plant surface and inside the plant, and can accompany the plant's growth for a long time, exerting their beneficial effects on the plant; at the same time, they can also stably express the dsRNA of pest target genes, exerting a toxic effect through the pest's feeding process, effectively controlling pests.

[0058] The control method of this invention has good gene silencing persistence and a wide range of pest and host protection. Compared with host-induced gene silencing (HIGS), it expands the types of plants protected, and compared with spray-induced gene silencing (SIGS), it provides a more lasting control effect.

[0059] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0060] Unless otherwise specified, the following embodiments are all conventional methods.

[0061] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0062] Example 1

[0063] This embodiment illustrates the construction of the engineered bacteria described in this invention, using the expression of the vATPaseA gene dsRNA from the fall webworm as an example and Bacillus paralichrysogenum SH-F8 as the starting strain. The specific steps are as follows:

[0064] 1. Cloning and knockout of RNase III in Bacillus paralichrysiformis SH-F8

[0065] A 0.2g sample of mulberry leaves, a preferred host of the fall webworm, was immersed in 100ml of sterile water and shaken at 30℃ and 160 rpm for 60 minutes. Subsequently, a 100µL serially diluted suspension was plated onto tryptone-soybean agar (TSA) plates (containing 1.5% tryptone, 0.5% soybean peptone, 0.5% sodium chloride, and 1.5% agar, pH 7.0) and incubated at 30℃ for 48 hours. Single colonies were picked for isolation and purification, and a preliminary screening revealed an epiphytic Bacillus strain from mulberry leaves, named SH-F8. This strain was identified as *Bacillus paralicheniformis* and deposited on June 7, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 30890.

[0066] RNase III degrades dsRNA, so the gene encoding RNase III in SH-F8 needs to be knocked out in order for it to express dsRNA as a chassis strain. To obtain the gene encoding RNase III from SH-F8, the COI gene of SH-F8 was first amplified and sequenced. Next, BLAST analysis of the COI sequence of SH-F8 was used to identify bacterial strains with similar genomes. Based on the RNase III encoding genes of these similar strains, degenerate primers were designed, and the gene encoding RNase III in SH-F8, namely the rnc gene, was successfully identified, with its sequence shown in SEQ ID No. 1.

[0067] To knock out the rnc gene in the SH-F8 strain, a deletion mutant was constructed using the previously developed temperature-sensitive suicide plasmid pMAD-tet (Li, Y., et al. (2019) Surfactin and fengycin contribute to the protection of aBacillus subtilis strain against grape downy mildew by both direct effect and defense stimulation. Molecular plant pathology 20, 1037-1050). The specific steps are as follows:

[0068] First, the upstream fragment (sequence shown in SEQ ID No. 4) and downstream fragment (sequence shown in SEQ ID No. 5) of the rnc gene were amplified from the genomic DNA of SH-F8. Then, the upstream and downstream fragments of the rnc gene were ligated into a single fragment by PCR and inserted into the pMAD-tet plasmid to generate the pMAD-tet-updn plasmid. This plasmid was introduced into *E. coli* DH5α and confirmed by sequencing. Next, the pMAD-tet-updn plasmid was transformed into the SH-F8 strain by electroporation. After culturing on LB plates containing tetracycline for 2 days, tetracycline-resistant (TetR) transformants were obtained. Subsequently, a two-step allele exchange was performed: the first step was incubation at 42°C for 10 h, and the second step was incubation at 25°C for 24 h. Finally, the gene deletion clone was confirmed by PCR and sequencing, and this mutant strain was named SH-F8Δrnc.

[0069] 2. Construct an engineered strain of dsRNA expression mutant based on SH-F8Δrnc strain.

[0070] To ensure stable expression of dsRNA and avoid plasmid loss, a self-expressing dsRNA system was constructed in the SH-F8Δrnc strain, comprising a T7-dsRNA-T7 fragment (sequence shown in SEQ ID No. 2) and a T7 RNA polymerase fragment (sequence shown in SEQ ID No. 3).

[0071] (1) Fragment amplification:

[0072] The T7-dsRNA-T7 fragment was amplified from the L4440 plasmid constructed in a previous study (Zhang, X., et al. (2023) Bacteria-mediated RNAi for managing fall webworm,: screening target genes and analyzing lethal effect. Pest Management Science 79, 1566-1577), which contains a dual T7 promoter and a dsRNA sequence targeting the fall webworm (H. cunea) vATPase A. The T7 RNA polymerase fragment was amplified from E. coli HT115(DE3).

[0073] (2) Fragment assembly:

[0074] By using overlap extension PCR, the upstream fragment of the rnc gene, T7-dsRNA-T7, T7 RNA polymerase, and the downstream fragment of the rnc gene were sequentially ligated to form a long fragment udTd. The udTd fragment was inserted into the pMAD-tet vector to construct the pMAD-tet-udTd plasmid, which was then confirmed by sequencing.

[0075] (3) Plasmid transformation and screening:

[0076] The pMAD-tet-udTd plasmid, confirmed by sequencing, was transformed into the SH-F8Δrnc strain, and tetracycline-resistant (TetR) transformants were screened. Through two-step allele exchange, the mutant engineered strain SH-F8Δrnc::T7dsT7-T7p was finally obtained. This strain contains T7 RNA polymerase, a double T7 promoter, and the dsRNA sequence of vATPase A from the fall webworm.

[0077] 3. Detection of dsRNA expression in SH-F8Δrnc::T7dsT7-T7p strain

[0078] To detect the expression level of dsRNA in the SH-F8Δrnc::T7dsT7-T7p strain, the following experiment was conducted.

[0079] (1) Cultured strains:

[0080] Single colonies of the SH-F8Δrnc::T7dsT7-T7p strain were inoculated into LB liquid medium and cultured at 37°C with shaking at 200 rpm for 16 h. 0.5 mL of the culture was then inoculated into 50 mL of fresh LB liquid medium and cultured at 37°C for 8 h or 24 h to determine the optimal harvest time. Two conditions were set for each culture time: with and without IPTG (inducer). IPTG was added when the OD600 of the bacterial culture reached approximately 0.4, with a final IPTG concentration of 1 mM.

[0081] (2) RNA extraction:

[0082] After culture, cells were collected by centrifugation (7000×g, 10 min). The cell pellet was washed four times with sterile water and then treated with lysozyme (50 mg / mL) at 37°C for 1 h. Total RNA was extracted using TRIzol Plus reagent.

[0083] (3) dsRNA isolation and detection:

[0084] The extracted RNA was treated with DNase and RNase to degrade single-stranded RNA, retaining dsRNA. dsRNA expression was confirmed by 1% agarose gel electrophoresis, and the results are as follows: Figure 1 As shown.

[0085] Depend on Figure 1 The results showed that the target gene dsRNA band was visible in all four groups. The brightest band was in the group that was shaken for 24 hours and did not receive IPTG induction treatment. This indicates that the obtained SH-F8Δrnc::T7dsT7-T7p strain can successfully express dsRNA without IPTG induction.

[0086] Example 2: Experiment of feeding American white moth (H. cunea) larvae with SH-F8Δrnc::T7dsT7-T7p strain

[0087] This example illustrates that the engineered bacteria SH-F8Δrnc::T7dsT7-T7p constructed in Example 1, when fed to the American white moth, can interfere with target genes and affect its oviposition.

[0088] To evaluate the RNA interference effect of the SH-F8Δrnc::T7dsT7-T7p strain on the fall webworm (H. cunea), fall webworm larvae were fed an artificial diet containing a mixture of SH-F8Δrnc::T7dsT7-T7p strain. The experiment was divided into three groups:

[0089] (1) Control group (CK): Normal feed, no bacteria added.

[0090] (2) Wild type group (WT): SH-F8 wild type strain culture solution was added to the feed.

[0091] (3) Engineered microbial group (Δ::): SH-F8Δrnc::T7dsT7-T7p strain culture medium was added to the feed.

[0092] Culture methods for SH-F8 wild-type strain and SH-F8Δrnc::T7dsT7-T7p strain: The preserved strains were inoculated onto LB agar plates and cultured overnight. Single colonies that grew were picked and cultured in 5 mL of fresh liquid LB medium at 180 rpm for about 10 hours to prepare a stock solution. Then, 0.5 mL of the stock solution was inoculated into 50 mL of fresh liquid LB medium and cultured at 180 rpm for 24 hours. All the above processes were carried out at 37℃.

[0093] The cultures of the wild-type SH-F8 strain and the SH-F8Δrnc::T7dsT7-T7p strain were centrifuged (7000×g, 10 min) and resuspended in sterile water to a concentration of 1×10⁻⁶. 7 CFU / mL. Cut freshly prepared artificial feed (about 30g) into small pieces (0.5×0.5×0.5cm) and soak them in 5mL of the corresponding bacterial solution.

[0094] Each group had 5 replicates, with 50 larvae per replicate. Larvae were reared in petri dishes (20 cm in diameter) at 26 ± 1℃, relative humidity 75% ± 10%, and a 14h:10h light / dark cycle. Feed was changed daily for each group from the second instar larval stage to the pupal stage. The number of larval deaths and pupae was recorded until pupation was complete. Results are shown in Table 1 and [Table data missing]. Figure 2 As shown.

[0095] Table 1. Larval pupation status in each group

[0096] Treatment Number of dead larvae (only) Number of failed pupation (only) Number of successful pupation (only) Control group (CK) 2.82±1.30 4.10±0.66 38.41±3.42 Wild type group (WT) 4.45±1.31 10.87±2.15 26.57±2.20 Engineered bacteria group (Δ::) 11.81±2.31 21.21±3.25 13.32±1.58

[0097] From Table 1 and Figure 2 The results showed that, compared with the control group and the wild-type group, feeding the feed containing the culture medium of the SH-F8Δrnc::T7dsT7-T7p strain significantly increased the pupation failure rate of fall webworm larvae. The SH-F8Δrnc::T7dsT7-T7p strain can interfere with the target genes of fall webworm larvae and affect their pupation.

[0098] Example 3: Labeling and colonization ability experiment of SH-F8Δrnc::T7dsT7-T7p strain

[0099] This embodiment illustrates that the engineered bacteria described in this invention can colonize on a variety of plants.

[0100] To facilitate identification of the SH-F8Δrnc::T7dsT7-T7p strain, the tetracycline resistance plasmid pGFP78 was transformed into the SH-F8Δrnc::T7dsT7-T7p strain and the SH-F8 wild-type strain, respectively, and labeled as "marked mutant" and "marked wild-type" for subsequent colonization experiments.

[0101] Single colonies of the labeled mutant and labeled wild-type strains were inoculated into 5 mL of LB broth containing 10 μg / mL tetracycline and cultured overnight at 37°C with shaking at 180 rpm. The culture was then transferred 1:100 to 100 mL of LB broth containing tetracycline and cultured at 37°C with shaking at 180 rpm for 24 h. The bacterial cells were collected by centrifugation, washed three times with sterile water, resuspended in sterile water, and OD adjusted. 600 The concentration was reduced to approximately 0.3 for subsequent experiments.

[0102] Select mulberry (Morus alba) and poplar (Populus alba) seedlings of uniform growth. Spray the prepared labeled mutant or labeled wild-type bacterial suspensions onto the leaves until a dense, non-drip mist forms on the leaf surface. The suspensions of labeled mutants are denoted as Mut, and those of labeled wild-types as wt. Each experiment has three replicates. The epiphytic and endophytic colonization abilities of the strains are assessed on days 1, 7, 14, and 21 post-inoculation.

[0103] Endophytic colonization detection: Fresh leaves were taken, washed, and surface-sterilized (immersed in 75% ethanol for 3 min, then in 2% NaClO for 5 min), and rinsed three times with sterile water. The leaves were blotted dry with sterile filter paper, ground into a suspension, diluted, and spread onto LB agar plates containing tetracycline. The final sterile water rinse solution was also spread as a control to exclude residual bacteria on the surface. Incubation was carried out at 37°C until single colonies appeared.

[0104] Epiphytic colonization detection: Fresh leaves were placed in Erlenmeyer flasks containing 100 mL of sterile water and shaken for 1 hour to wash away surface bacteria. 1 mL of the suspension was diluted and spread onto LB agar plates containing tetracycline. Each experiment was performed in triplicate, and the plates were incubated upside down at 37°C until single colonies appeared. Colony levels were quantified by colony counting.

[0105] The results are as follows Figure 3 As shown, the SH-F8Δrnc::T7dsT7-T7p strain can colonize both the surface and interior of mulberry and poplar leaves, and its colonization is not significantly different from that of the wild-type strain. Therefore, the SH-F8Δrnc::T7dsT7-T7p strain of this invention can be directly applied to the surface of plant leaves. It not only has the ability to stably colonize both on and inside the plant, allowing it to accompany the plant's growth for a long time and exert its beneficial effects on the plant, but also exerts a toxic effect through the feeding process of pests, effectively controlling pests.

[0106] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An engineered microorganism for pest control, characterized in that, The engineered bacteria were obtained by exogenously transferring dsRNA fragments of pest target genes into plant probiotics.

2. The engineered bacteria according to claim 1, characterized in that, The plant probiotics include Bacillus paralicheniformis, Bacillus licheniformis, Bacillus subtilis, or Bacillus amyloliquefaciens.

3. The engineered bacteria according to claim 2, characterized in that, The plant probiotics include Bacillus paralicheniformis SH-F8, whose preservation number is CGMCC No. 30890.

4. The engineered bacteria according to claim 1, characterized in that, The dsRNA includes the dsRNA of the vATPaseA gene from the American white moth.

5. The engineered bacteria according to claim 4, characterized in that, The dsRNA sequence of the vATPaseA gene of the American white moth includes the sequence shown in positions 77-469 of SEQ ID No.

2.

6. The method for constructing engineered bacteria according to any one of claims 1 to 5, characterized in that, The construction method includes: knocking out the rnc gene of the plant probiotic, inserting a bidirectional T7 promoter and dsRNA fragment at the knockout site, and a T7 RNA polymerase to start the T7 promoter, to obtain engineered bacteria.

7. The construction method according to claim 6, characterized in that, The sequence of the rnc gene includes the sequence shown in SEQ ID No. 1; the sequence of the bidirectional T7 promoter and dsRNA fragment includes the sequence shown in SEQ ID No. 2; and the sequence of the T7 RNA polymerase includes the sequence shown in SEQ ID No.

3.

8. A formulation, characterized in that, The formulation comprises the engineered bacteria according to any one of claims 1 to 5 or the engineered bacteria constructed by the construction method according to any one of claims 6 to 7.

9. The application of the engineered bacteria according to any one of claims 1 to 5, or the engineered bacteria constructed by the construction method according to any one of claims 6 to 7, or the preparation according to claim 8, in pest control.

10. A method for controlling pests, characterized in that, The prevention and control method includes: applying the engineered bacteria according to any one of claims 1 to 5, or the engineered bacteria constructed by the construction method according to any one of claims 6 to 7, or the preparation according to claim 8 to the plant.

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