Banana endophyte engineered bacteria based on RNAi technology and targeting pathogenic milRNAs and its application in the prevention and control of fusarium wilt

Abstract

The application discloses banana endophyte engineering bacteria based on RNAi technology and targeting pathogenic milRNAs and application of the banana endophyte engineering bacteria in Fusarium wilt prevention and control. The application takes key pathogenic milRNAs of banana Fusarium wilt as a target, constructs an RNAi silencing vector according to a precursor sequence, and then transforms and introduces the RNAi silencing vector into banana endophytic fungus DJE2023. Through co-culture verification, the engineering bacteria can efficiently deliver small RNAs to pathogenic bacteria, and significantly silence target milRNAs. Potted plant tests show that the engineering bacteria can significantly promote growth indexes such as the height, pseudostem girth and leaf number of Brazilian banana, and efficiently prevent and control banana Fusarium wilt, so that the disease index is significantly reduced; the engineering bacteria also exhibit the same prevention and control effect on celery Fusarium wilt, and have broad spectrum. The engineering bacteria provided by the application have the dual functions of promoting growth and preventing diseases, provide a new microbial inoculant resource for green prevention and control of banana Fusarium wilt, and have a good application prospect of being developed into a green microbial inoculant.

Description

Technical Field

[0001] This invention belongs to the fields of microbiology and plant protection technology, specifically relating to an engineered banana endophytic fungus modified based on RNAi technology and targeting pathogenic milRNAs, and its application in the control of Fusarium wilt. Background Technology

[0002] Banana (Musa spp.) is an important global economic and food crop, widely cultivated in tropical and subtropical regions. However, banana production has long been severely threatened by Fusarium wilt, a soil-borne vascular disease caused by Fusarium oxysporum f. sp. cubense (Foc). Physiological race 4 (Foc TR4) is the most damaging, leading to widespread plant death and significant economic losses. Currently, the control of Fusarium wilt relies mainly on chemical pesticides and the breeding of resistant varieties. However, chemical control presents challenges such as environmental pollution and pathogen resistance, while resistant breeding is time-consuming and slow-progressing. Furthermore, existing major varieties are highly susceptible to physiological race 4, posing a serious challenge to disease control.

[0003] microRNA (miRNA) is a non-coding, single-stranded small RNA molecule, approximately 19–24 nucleotides in length, produced in eukaryotes. A similar small RNA molecule has also been found in fungi, whose precursors possess a typical stem-loop structure, resembling miRNAs in plants and animals; hence, it is called miRNA-like small RNA (milRNA). It has been shown to play an important regulatory role in the pathogenesis of plant pathogenic fungi.

[0004] RNA interference (RNAi)-based gene silencing technology is emerging in the fields of combating pathogenic stress and plant protection. The principle of RNAi is that exogenous double-stranded RNA or endogenous single-stranded RNA capable of forming hairpin structures is recognized and cleaved into small interfering RNAs (21-23 bp in length) by the intracellular enzyme Dicer. These interfering RNAs then form a silencing complex with proteins such as Argonaute, specifically degrading the target gene mRNA according to base pairing principles, thereby inhibiting the expression of the target gene at the post-transcriptional level. The application of RNAi in plant disease control mainly manifests in two forms: host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS). HIGS involves expressing RNAi vectors targeting the pathogenic genes of pests or pathogens in the host plant. By silencing the target genes of pathogens through RNAi, the host is conferred resistance to the pathogen. This method is not only effective against viral infection but also shows good results in the control of pests, pathogenic bacteria, and fungi. For example, HIGS (Highly Activated Sequencing Glycosides) was used to silence the Dicer enzyme synthesis gene in *Botrytis cinerea* in *Arabidopsis thaliana* and tomato, reducing the growth and pathogenicity of *Botrytis cinerea*. Transgenic cotton stably expressing RNAi vectors targeting the pathogenic genes of *Verticillium wilt* was able to use HIGS to silence pathogenic genes in *Verticillium dahliae*, thereby reducing the incidence of *Verticillium wilt*. SIGS is a biotechnology that inhibits key target genes of pathogens by spraying in vitro transcribed double-stranded RNA or small interfering RNA (siRNA). This application method is similar to traditional chemical pesticides, hence siRNA is also figuratively called "RNA pesticide." Due to its easily degradable characteristics, SIGS greatly reduces the risk of pesticide residues and environmental pollution, making it one of the most promising new green control technologies in agricultural production. It has been successfully applied to the control of various pathogenic fungi. For example, by applying small RNA specifically targeting cytochrome b of *Magnaporthe oryzae* in vitro, the pathogenicity of *Magnaporthe oryzae* can be inhibited. These findings demonstrate the development potential and significant value of RNAi-based siRNAs as novel, highly efficient, and environmentally friendly bactericides.

[0005] However, both HIGS and SIGS face significant challenges in controlling the soil-borne disease Fusarium wilt in bananas. While there are successful examples of using HIGS in Fusarium resistance to Fusarium wilt, such as expressing RNAi vectors targeting Fusarium genes in bananas effectively enhancing resistance, the widespread application of HIGS is difficult due to the complexity of banana genetic manipulation. Furthermore, SIGS has not yet been used in banana Fusarium wilt control due to limitations in the persistence of small RNAs in the soil. Therefore, effectively delivering small RNAs targeting the pathogen to banana roots remains a key challenge for future disease control.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] To overcome the shortcomings and deficiencies of existing banana wilt disease control technologies, such as chemical pesticide pollution, long disease-resistant breeding cycles, and limited application of RNAi technology (HIGS and SIGS) in soil-borne diseases, this invention aims to provide an engineered banana endophytic fungus based on RNAi technology that targets pathogenic milRNAs and its application in wilt disease control. This invention targets the key pathogenic milRNAs of the banana wilt pathogen, constructs an RNAi vector using the natural stem-loop structure of the pathogen's milRNA precursor, and introduces the RNAi vector targeting the key pathogenic milRNAs into endophytic fungi. Utilizing the colonization ability of the banana host, efficient delivery of small RNAs is achieved, thereby developing a targeted control strategy for wilt disease based on RNAi principles and obtaining an engineered strain with both disease prevention and growth-promoting functions.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] An engineered banana endophytic fungus based on RNAi technology and targeting pathogenic milRNAs was obtained by transferring an RNAi vector targeting key pathogenic milRNAs of Fusarium wilt in banana endophytic fungi.

[0010] Furthermore, the milRNAs include, but are not limited to, Foc-milR87 or Foc-milR138.

[0011] The mature sequence of the Foc-milR87 is shown in SEQ ID NO.1, and its precursor sequence is shown in SEQ ID NO.2.

[0012] The mature sequence of Foc-milR138 is shown in SEQ ID NO.3, and its precursor sequence is shown in SEQ ID NO.4.

[0013] Furthermore, the banana endophytic fungus includes Fusarium oxysporum DJE2023.

[0014] Furthermore, the starting vector of the RNAi vector includes vectors of the pSilent series, and more preferably, includes pSilent-1.

[0015] Preferably, the key pathogenic milRNAs of Fusarium wilt of banana can be replaced with key pathogenic target genes of Fusarium wilt of banana.

[0016] A microbial agent that combines disease prevention and growth promotion functions contains the aforementioned engineered endophytic bacteria of bananas.

[0017] The above-mentioned method for constructing banana endophytic bacteria engineered based on RNAi technology and targeting pathogenic milRNAs includes the following steps:

[0018] S1, milRNA target selection

[0019] The mature and precursor sequences of key pathogenic milRNAs of Fusarium wilt of banana (such as Foc-milR87 and Foc-milR138) are shown in SEQ ID NO.1-4.

[0020] S2, Construction of RNAi silencing vector

[0021] Based on the precursor sequences of the above milRNAs, an RNAi silencing vector was constructed. Specifically, the reverse precursor sequences of Foc-milR87 and Foc-milR138 were amplified respectively, and cloned into the RNAi vector by enzyme digestion and ligation technology to construct a recombinant silencing vector.

[0022] S3. Obtaining engineered strains

[0023] The above-mentioned recombinant silencing vector was transformed into banana endophytic fungi using PEG-mediated protoplast transformation technology. After resistance screening and PCR verification, engineered banana endophytic fungi were obtained.

[0024] S4. Functional verification of engineered strains

[0025] Small RNA delivery capability verification: The above-mentioned engineered banana endophytic bacteria were co-cultured with Fusarium wilt of banana. qPCR detection showed that the expression levels of the targets Foc-milR138 and Foc-milR87 in Fusarium wilt of banana were significantly reduced, indicating that the engineered banana endophytic bacteria can efficiently deliver small RNA to Fusarium wilt of banana and silence the expression of the target milRNA.

[0026] Furthermore, the banana wilt pathogen includes physiological race 4 of the banana wilt pathogen.

[0027] The above-mentioned applications of banana endophytic bacteria engineered based on RNAi technology and targeting pathogenic milRNAs or microbial agents in promoting plant growth; further, applications in the preparation of products that promote plant growth.

[0028] Preferably, the plant includes bananas and / or celery, etc.

[0029] The above-mentioned applications of engineered banana endophytic bacteria or microbial agents based on RNAi technology and targeting pathogenic milRNAs in the fight against Fusarium wilt; further, their application in the preparation of products resistant to Fusarium wilt.

[0030] Preferably, the wilt pathogens include banana wilt pathogens and / or celery wilt pathogens, etc.

[0031] The above-mentioned applications of engineered banana endophytic bacteria or microbial agents based on RNAi technology and targeting pathogenic milRNAs in the control of Fusarium wilt; further, their application in the preparation of products for the control of Fusarium wilt.

[0032] Preferably, the wilt disease includes banana wilt and / or celery wilt, etc.

[0033] A method for promoting plant growth includes the following steps: treating the plant with the aforementioned engineered banana endophytic bacteria or microbial agents.

[0034] Specifically, the steps include the following:

[0035] The plants were treated by immersing them in a bacterial suspension of the aforementioned engineered banana endophytic bacteria, then transplanted and cultured to promote plant growth.

[0036] Furthermore, the concentration of the bacterial suspension is 1×10⁻⁶. 6-8 per mL.

[0037] Furthermore, the processing time is 0.5-1 h.

[0038] Furthermore, the culture time is 20–60 days.

[0039] Evaluation of growth-promoting effect: Pot experiment showed that after 60 days of treatment with engineered bacteria, the growth indicators of Brazilian bananas, such as plant height, pseudostem circumference, and number of leaves, were significantly higher than those of the control group.

[0040] Evaluation of growth-promoting effect: Pot experiment showed that after 20 days of treatment with engineered bacteria, the plant height and other growth indicators of celery were significantly higher than those of the control group.

[0041] Evaluation of biocontrol efficacy: The engineered strain was inoculated first, followed by Fusarium wilt pathogens (such as Fusarium wilt of banana XJZ2 and Fusarium wilt of celery FOA). After 30 days, the disease index was counted. The results showed that the disease index of the engineered strain treatment group was significantly lower than that of the group inoculated with pathogens alone, and the control efficacy was significant.

[0042] The present invention has the following advantages and effects compared with the prior art:

[0043] (1) This invention uses banana endophytic fungi as chassis strains and utilizes their natural colonization ability to achieve efficient and continuous delivery of small RNAs, overcoming the technical bottlenecks of difficult genetic manipulation of HIGS in bananas and short duration of effect of SIGS in soil.

[0044] (2) The engineered strain constructed in this invention can specifically silence the key pathogenic milRNA of Fusarium wilt in bananas, significantly reduce the pathogenicity of the pathogen, and achieve efficient control of Fusarium wilt.

[0045] (3) The engineered strain of this invention can significantly promote the growth of banana and celery plants while controlling diseases, and has the dual functions of disease prevention and growth promotion. It provides a new microbial agent resource for the green control of banana wilt disease and has good application prospects for development into a green microbial agent. Attached Figure Description

[0046] Figure 1 A schematic diagram of constructing silent Foc-milR138 and Foc-milR87 vectors based on RNAi technology; where a: pSilent::Foc-milR138-; b: pSilent::Foc-milR87-.

[0047] Figure 2 The engineered strain DJE2023 138- PCR verification; where, a: using primers pSilent-F and Foc-milR138-R to validate DJE2023; 138- a: Amplification results of the strain; b: Amplification of DJE2023 using primers HYG-F and HYG-R. 138- Amplification results; lanes 1-3: DJE2023 138- Lane 4: Positive control pSilent::Foc-milR138- plasmid; Lane 5: Strains DJE2023; Lane 6: Negative control ddH2O.

[0048] Figure 3 The engineered strain DJE2023 87- PCR verification; where, a: using primers HindIII-jcr-F and Foc-milR87-R to validate DJE2023; 87-a: Amplification results of the strain; b: Amplification of DJE2023 using primers HYG-F and HYG-R. 87- Amplification results; lanes 1-3: DJE2023 87- Lane 4: Positive control pSilent::Foc-milR87- plasmid; Lane 5: Strains DJE2023; Lane 6: Negative control ddH2O.

[0049] Figure 4 The expression levels of Foc-milR87 and Foc-milR138 of *Fusarium wilt* XJZ2 and engineered strain were determined by co-culturing; where a: XJZ2+DJE2023 87- b: XJZ2+DJE2023 138- .

[0050] Figure 5 Phenotypic (a) and statistical (b) results of plant height, pseudostem circumference and leaf number of Brazilian bananas after treatment with engineered strains.

[0051] Figure 6 This study evaluates the potted plant control efficacy of engineered strains against banana wilt disease; including: a: disease incidence on Brazilian banana leaves and roots; b: disease severity statistics; and c: disease index statistics.

[0052] Figure 7 The figures show the phenotypic (a) and statistical (b) results of celery plant height and tiller number after treatment with engineered strains.

[0053] Figure 8 This is an evaluation of the potted control efficacy of engineered strains against celery wilt; where a: celery disease incidence; b: disease severity statistics; c: disease index statistics. Detailed Implementation

[0054] The present invention will now be described in further detail with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0055] The *Fusarium wilt* fungus XJZ2 involved in the examples is strain XJZ2 of physiological race 4 of *Fusarium wilt* (*Fusarium oxysporum* f. sp. *cubense tropical race 4), and the endophytic fungus DJE2023 is *Fusarium oxysporum* DJE2023. Both XJZ2 and DJE2023 are disclosed in the literature "Jin LQ, Huang R, Zhang J, et al. Identification and Characterization of Endophytic Fungus DJE2023Isolated from Banana (Musa sp. cv. Dajiao) with Potential for Biocontrol of Banana Fusarium Wilt[J]. J. Fungi 2024, 10, 877."

[0056] Example 1: Obtaining the milRNA sequence

[0057] In our previous study, we compared the sRNA expression profiles of the banana wilt pathogen XJZ2 strain under pure culture conditions and in the early infection stage using high-throughput sequencing and phenotypic analysis. Based on this, we identified a class of milRNAs that play a key pathogenic role in the early stage of infection, including Foc-milR80, milR87, milR106, milR133, milR138, and milR148 (separately mentioned in our previous research, Xie et al., 2025). Further knockout of the precursors of these milRNAs revealed a significant decrease in the pathogenicity of the banana wilt pathogen. In this study, we focused on Foc-milR87 and Foc-milR138, and their sequence information is shown in Table 1.

[0058] Table 1 Sequence information of milRNA

[0059]

[0060] Example 2: Construction of silencing targets Foc-milR138 and Foc-milR87 vectors based on RNAi technology

[0061] 2.1 Purification of the target fragment

[0062] The amplification reaction was carried out using primers Foc-milR138-reverse-F / R and Foc-milR87-reverse-F / R (SEQ ID NO.5-8) (Table 2) containing specific restriction endonuclease adapters. The target banana wilt pathogen DNA was used as a template, and PCR amplification was performed using a high-fidelity DNA polymerase (such as Phanta Max Super-Fidelity) to ensure the accuracy of the product. The amplification system is shown in Table 3.

[0063] Table 2. Primer sequences for amplifying reverse precursors of Foc-milR138 and Foc-milR87 and their colony verification.

[0064]

[0065] Table 3 High-fidelity enzyme amplification system

[0066]

[0067] PCR program: Pre-denaturation 95℃, 3 min; denaturation 95℃, 15 s; annealing 58℃, 15 s; extension 72℃, 60 s / kb; 35 cycles; final extension 5 min. Electrophoresis analysis of the amplified products specifically amplified the reversed precursor sequences of Foc-milR138 and Foc-milR87 (248 bp and 233 bp in length) from *Fusarium wilt of banana* XJZ2. DNA fragment recovery was performed using an OMEGA gel purification kit; detailed instructions are provided in the product manual. After concentration testing, the recovered products were stored at -20℃.

[0068] 2.2 Plasmid linearization

[0069] The pSilent-1 vector was linearized by double digestion with XhoI and KpnI enzymes at 37℃ for 2-3 hours. After digestion, electrophoresis was used to confirm successful digestion. If successful, the digestion product was recovered using an OMEGA agarose gel extraction kit to ensure high-purity fragments. The digestion system is shown in Table 4.

[0070] Table 4 Enzyme digestion system

[0071]

[0072] 2.3 Linearized vector and target fragment connection

[0073] Using the Novizan Rapid Cloning Kit (ClonExpresII One Step Cloning Kit), the enzyme-digested vector was ligated with the reverse precursor fragments of Foc-milR138 and Foc-milR87 obtained by amplification as described above. The specific ligation system is detailed in Table 5.

[0074] Table 5 Connection System

[0075]

[0076] Connection conditions: 37℃, 1 h, then placed on ice to cool.

[0077] 2.4 Escherichia coli transformation

[0078] (1) Take 100 μL of Escherichia coli DH5α competent cell suspension from the -80℃ ultra-low temperature freezer and thaw it on ice. Add 10 μL of ligation product to the tube in a sterile operating table, mix it repeatedly by aspiration and then let it stand on ice for 30 minutes.

[0079] (2) Transfer the mixture to a 42°C constant temperature water bath and perform heat shock stimulation for 90 seconds. Then quickly transfer it back to ice and let it stand for 5 minutes to stabilize the cell membrane structure.

[0080] (3) Inject 800 μL of sterile LB liquid culture medium into a centrifuge tube, gently vortex to mix, and place in a constant temperature shaker. Shake at 37°C and 180 rpm for 1 hour to restore bacterial activity.

[0081] (4) Centrifuge the culture medium at 6000 rpm for 2 minutes, discard the supernatant and keep only 100 μL of bacterial culture, fully resuspend the precipitate and spread it evenly on the surface of LB solid medium containing ampicillin, invert the plate and incubate it in a 37℃ incubator for 12-16 hours.

[0082] 2.5 Validation of Colony PCR

[0083] If a single colony grows on the surface of the culture medium, the single E. coli colony is used as a template. The upstream primer on the vector and the downstream primer of the target gene (SEQ ID NO.9-12) (Table 2) are used to verify the single colony on the plate. The amplification system is prepared using Novizan Green Taq Mix. The specific amounts are shown in Table 6.

[0084] Table 6 Colony PCR System

[0085]

[0086] PCR program: Pre-denaturation 94℃, 5 min; denaturation 94℃, 30 s; annealing 60℃, 30 s; extension 72℃, 60 s / kb, for 30 cycles, followed by a final extension of 5 min. The bands were checked for accuracy by agarose gel electrophoresis. Positive colonies matching the expected molecular weight were selected from the electrophoresis results and transferred using a sterile inoculating loop to LB broth containing 100 μL / mL ampicillin for enrichment culture. DNA sequencing services were ultimately provided by Sangon Biotech (Shanghai) Co., Ltd.

[0087] 2.6 Large plasmid extraction

[0088] The correctly identified recombinant plasmid-containing *E. coli* monoclonal strain was placed in 200 mL of LB broth containing 100 μL / mL ampicillin and amplified at 37°C and 180 rpm for 12 hours. Subsequently, plasmid purification was performed using the TIANGEN toxin-free large-scale plasmid extraction kit according to standard operating procedures. Finally, the RNAi-based silencing vectors pSilent::Foc-milR138- and pSilent::Foc-milR87- were successfully constructed. Figure 1 ).

[0089] Example 3 Engineered strain DJE2023 138- and DJE2023 87- Screening and verification

[0090] Previously, our research group isolated and purified an endophytic fungus, designated DJE2023, from the pseudostem tissue of healthy bananas. Based on morphological characteristics and multigene phylogenetic analysis, this strain was identified as *Fusarium oxysporum*. We then used PEG-mediated protoplast transformation technology to introduce the aforementioned silencing vector into the endophytic fungus DJE2023 to obtain an engineered strain.

[0091] 3.1 Preparation of protoplasts

[0092] (1) Scrape the mycelium of strain DJE2023 from the PDA plate after 5 days of culture, rinse it 3 times with sterile ddH2O and filter it into a 50 mL centrifuge tube.

[0093] (2) Centrifuge at 4℃ and 3500 rpm for 10 min and discard the supernatant.

[0094] (3) Add 1 mL of YPD liquid medium to resuspend the mycelium, transfer it to 150 mL of YPD medium, and culture at 28℃ and 180 rpm for 8 h.

[0095] (4) Centrifuge at 4℃ and 3500rpm for 5min to collect the mycelial precipitate, and wash it once.

[0096] (5) Resuspend the precipitate in 50 mL of 0.8 M NaCl solution and centrifuge for 5 min under the same conditions.

[0097] (6) Add 5 mL of enzyme hydrolysate and hydrolyze at 32°C and 120 rpm for 4 h.

[0098] (7) Add 20 mL of 0.8M NaCl solution to terminate the reaction, centrifuge at 4℃ and 3500 rpm for 5 min and discard the supernatant.

[0099] (8) Resuspend the precipitate with 20 mL of STC buffer, centrifuge at 4°C and 3500 rpm for 5 min and discard the supernatant.

[0100] (9) The precipitate was resuspended in 800 μL STC buffer, 200 μL PTC solution and 20 μL DMSO, and the protoplast concentration was adjusted to 1×10⁻⁶. 7 Aliquots per mL into 1.5 mL centrifuge tubes and store at -80°C for later use.

[0101] 3.2. PEG-mediated protoplast transformation

[0102] (1) Take 100 μL of protoplast suspension and add it to a pre-cooled 50 mL centrifuge tube, and incubate on ice for 10 min.

[0103] (2) Add 20 μL of silencing vector, mix gently, and then incubate on ice for 30 min.

[0104] (3) Add 100 μL, 300 μL and 600 μL of 50% PEG 4000 solution in sequence, mix gently at 5 min intervals between each step, and let stand at room temperature for 20 min.

[0105] (4) Slowly add 1 mL, 3 mL and 6 mL of STC buffer, and centrifuge at 4℃ and 3000 rpm for 10 min.

[0106] (5) Discard 90% of the supernatant, and resuspend the remaining 400 μL of precipitate in 1.6 mL of RM regeneration medium and incubate at 26 °C and 60 rpm for 3 h with shaking.

[0107] (6) Add hygromycin to the culture medium in advance, mix the bacterial solution with the pre-melted RMA solid culture medium at 55℃, pour into plates, and incubate at 28℃ for 48 h.

[0108] 3.3 Engineered strain DJE2023 138- and DJE2023 87- The acquisition

[0109] After culturing in RMA regeneration medium containing hygromycin (20 μg / mL) at 28°C for 2 days, single colonies of the transformed strains were selected and transferred to new hygromycin-resistant plates for rescreening. The plates were then incubated at 28°C for further growth. After 3 days of growth, DNA was extracted from transformants that still grew normally on the resistant plates using a fungal DNA extraction kit (FungalDNAKit, OMEGA) for further validation.

[0110] Based on the target fragment, colony verification primers pSilent-F / Foc-milR138-R, HindIII-jcr-F / Foc-milR87-R, and hygromycin verification primer HYG-F / R (SEQ ID NO. 9-14) were designed (Table 2). PCR verification was performed using transformant DNA as a template, with wild-type strain DJE2023 DNA and recombinant silencing plasmids used as controls. Figure 2 As can be seen from 'a', primers pSilent-F and Foc-milR138-R were used to target DJE2023. 138- The strains (lanes 1-3) were amplified, and all showed a specific band at approximately 705 bp, consistent in size with the amplified band of the positive control pSilent::Foc-milR138- plasmid (lane 4); Figure 3 As indicated by 'a' in the text, primers HindIII-jcr-F and Foc-milR87-R were used to target DJE2023. 87- Amplification of strains (lanes 1-3) all showed a specific band at approximately 284 bp, consistent with the size of the amplified band from the positive control pSilent::Foc-milR87- plasmid (lane 4); however, no target band was observed in the wild-type strain DJE2023 (lane 5) or the negative control ddH2O (lane 6). These results indicate that the target fragment in the recombinant plasmid was successfully integrated into strain DJE2023. Figure 2 b and Figure 3 The results in section b showed that using the hygromycin resistance gene primers HYG-F and HYG-R on DJE2023... 138- and DJE2023 87- Amplification was performed, and a specific band appeared at approximately 554 bp in both strains, consistent with the size of the amplified band in the positive control recombinant silencing plasmid (lane 4). However, no target band was observed in the wild-type strain DJE2023 (lane 5) or the negative control ddH2O (lane 6). These results further confirm that the hygromycin resistance gene carried by the recombinant plasmid has been successfully introduced into DJE2023.

[0111] Example 4: Expression levels of Foc-milR87 and Foc-milR138 after co-culturing banana wilt pathogen XJZ2 with engineered strain

[0112] The banana wilt pathogen XJZ2 was separately combined with the engineered strain DJE2023. 138- DJE2023 87- Co-culture was performed in YPD medium. After 2 days, XJZ2 mycelia were collected from each co-culture system, and total RNA was extracted using an RNA extraction kit. The RNA concentration and purity were detected using a micro spectrophotometer. An equal volume of total RNA was used for reverse transcription of small RNA using an AG reverse transcription kit (Evo M-MLV reverse transcription kit) with stem-loop primers (SEQ ID NO.15-16) (Table 7). The reaction system is shown in Table 8. The reaction conditions were: 37℃ for 15 min, 85℃ for 5 s, and storage at 4℃ to obtain cDNA template. The cDNA template was then diluted to 200 ng / μL and stored at -20℃.

[0113] Table 7. Stem-loop primers and quantitative primer sequences for Foc-milR138 and Foc-milR87.

[0114]

[0115] Table 8 Stem-Loop Reverse Transcription System

[0116]

[0117] Quantitative real-time PCR was performed on the cDNA of the test strain using ChamQ Universal SYBR qPCR Master Mix (Vazyme) (primer sequences are shown in SEQ ID NO. 17-19). The specific system preparation is shown in Table 9, and the amplification program was run according to the standard procedure. Relative quantification was then performed using method 2. -ΔΔCt The difference in expression levels between the target gene and the internal reference gene was calculated using the C(t) values.

[0118] Table 9 Real-time quantitative PCR system

[0119]

[0120] Figure 4 The results showed that the banana wilt pathogen XJZ2 was related to the engineered strain DJE2023. 87- DJE2023 138- After co-culture, the relative expression levels of Foc-milR87 and Foc-milR138 in the pathogen were significantly lower than those in the XJZ2 control group cultured alone. Analysis using the Duncans Multiple Range test showed that the differences between the engineered bacteria co-culture treatment group and the control group were highly significant (p < 0.0001 was marked as ****).

[0121] The above results indicate that the engineered strain DJE2023 87- DJE2023 138- Both methods can efficiently deliver artificially designed small RNA molecules into the banana wilt pathogen XJZ2 during co-culture, achieving silencing of the target small RNA.

[0122] Example 5: Evaluation of the growth-promoting effect of engineered strains on banana (Brazilian banana) and their biocontrol effect on banana wilt disease.

[0123] A pot experiment was conducted, setting up a wild-type DJE2023 strain treatment group and an engineered strain DJE2023. 87- DJE2023 138- Treatment group and blank control group (CK). Healthy Brazilian banana (AAA) tissue culture seedlings were rinsed at the roots with sterile water and then immersed in a solution of 1×10⁻⁶ water. 6 The plants were treated with a bacterial suspension of 1000 cells / mL for 1 hour. Subsequently, the inoculated plants were transplanted into sterilized sand and placed in a greenhouse at 28°C. At 60 days after inoculation, the plant height, pseudostem circumference, and number of leaves of the Brazilian banana plants were measured (n=15) to evaluate the promoting effect of the engineered strain on the growth phenotype of Brazilian banana.

[0124] Figure 5 The results showed that, compared with the control group (CK), the plant height, pseudostem circumference, and leaf number of the Brazilian banana plants treated with each engineered strain all showed varying degrees of increase, indicating a significant growth-promoting effect. However, inoculation with the wild-type strain DJE2023 showed no significant growth-promoting effect. One-way ANOVA was used for difference analysis, and Duncan's method was employed for post-hoc multiple comparisons. All differences between the engineered strain treatment groups and the control group (CK) were statistically significant (p < 0.0001 was marked as ****, p < 0.001 as ***, and p < 0.01 as **).

[0125] To evaluate the biocontrol efficacy of the engineered strain against banana wilt disease, this study employed a strategy of inoculating with the engineered strain first, followed by inoculation with the pathogen. Specifically, the engineered strain was first inoculated with a concentration of 1×10⁻⁶, and the pathogen was then inoculated with the pathogen. 6 Wild-type DJE2023 and engineered DJE2023 per mL 138- DJE2023 87- Banana plant roots were soaked in spore suspension for 1 hour, and then the inoculated plants were transplanted into sterilized sand. After they were fully established (approximately 7 days later), they were inoculated with the same concentration of 1×10⁻⁶ spores via root drenching. 6 The experiment included a control group (CK) inoculated with water, a group inoculated solely with the pathogen XJZ2, and a group inoculated with wild-type strain DJE2023 and engineered strain DJE2023.138- DJE2023 87- After inoculation with the pathogen, the XJZ2 treatment group was then inoculated. Thirty days after inoculation, the disease incidence in each treatment group was statistically analyzed (n=15).

[0126] Figure 6 The results showed that plants inoculated with XJZ2 alone exhibited obvious browning symptoms at the base of the pseudostem, with more than half of the plant showing browning. Banana seedlings in the DJE2023+XJZ2 treatment group had greener leaves, and the browning area of ​​the root vascular bundles was generally less than half. (DJE2023...) 138- DJE2023 87- The banana seedlings in the co-inoculation group with XJZ2 showed good growth, with greener leaves, no obvious browning of vascular bundles, and plant phenotypes close to the control group (CK).

[0127] In terms of disease severity, plants in the XJZ2 treatment group were mainly at Grade 3 (severe disease) and Grade 2 (moderate disease), exhibiting the strongest pathogenicity; most plants in the DJE2023 treatment group were at Grade 1 (mild disease) and Grade 2 (moderate disease); and most plants in each engineered strain treatment group were at Grade 0 (no disease) and Grade 1 (mild disease).

[0128] Disease index statistics showed that the disease index of the XJZ2-only treatment group was significantly higher than that of the other groups. Compared with the XJZ2-only treatment, the disease index of all treatment groups co-inoculated with wild-type strains and engineered strains was significantly reduced, with the engineered strain treatment group showing the most significant decrease, indicating that the engineered strain has a highly effective biocontrol effect against banana wilt.

[0129] Example 6: Evaluation of the growth-promoting effect of engineered strains on celery and their biocontrol effect on celery wilt disease.

[0130] Given that previous studies have confirmed that Foc-milR87 and milR138 are highly conserved in Fusarium oxysporum, in order to systematically evaluate the broad-spectrum biocontrol potential and growth-promoting effects of engineered strains, this study, referring to the banana wilt evaluation system, used the main cultivated celery variety 'Ventura' as material, selected Fusarium oxysporum f. sp. apii (FOA) as the target pathogen, and conducted the verification of the celery wilt control effect using the same experimental method as in Example 5.

[0131] In the growth-promoting effect experiment, unlike Example 5, this example measured the plant height and tiller number of celery plants 20 days after inoculation (n=20). In the biocontrol effect experiment, unlike Example 5, n=20.

[0132] Figure 7-8 The results showed that the engineered strain DJE2023 138- and DJE2023 87- The effects of the engineered strain on celery wilt disease (FUD) in promoting growth and biocontrol were consistent with those in the banana system. Regarding growth promotion, treatment with the engineered strain significantly increased celery plant height, but had little effect on tiller number; the wild-type strain showed no growth-promoting effect. In terms of biocontrol, pretreatment with the engineered strain effectively inhibited disease development, resulting in normal plant growth and significantly lower vascular bundle browning compared to the wild-type treatment group, while the FOA-only treatment group exhibited typical yellow wilt symptoms. Disease index statistics further confirmed that the engineered strain treatment group showed the most significant reduction, indicating its stable and highly effective biocontrol capability against celery FUD. Therefore, the engineered strain also demonstrated equivalent control efficacy against celery FUD, exhibiting broad-spectrum activity.

[0133] The above results indicate that the engineered strain not only possesses the potential to inhibit Fusarium wilt pathogens in crops but also promotes the vegetative growth of host plants, demonstrating its application value as a microbial agent with both disease prevention and growth-promoting functions. Further research, combining physiological and biochemical indicators with rhizosphere microecological analysis, can elucidate its growth-promoting mechanism in greater depth.

[0134] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A banana endophytic bacteria engineered based on RNAi technology and targeting pathogenic milRNAs, characterized in that: The engineered banana endophytic bacteria were obtained by transferring an RNAi vector targeting key pathogenic milRNAs of banana wilt pathogen into banana endophytic fungi. The banana endophytic fungi include Fusarium oxysporum DJE2023.

2. The banana endophytic bacteria engineered based on RNAi technology and targeting pathogenic milRNAs according to claim 1, characterized in that: The milRNAs include, but are not limited to, Foc-milR87 or Foc-milR138; And / or, the starting vector of the RNAi vector includes vectors from the pSilent series.

3. The engineered banana endophytic bacteria based on RNAi technology and targeting pathogenic milRNAs as described in claim 2, characterized in that: The mature sequence of the Foc-milR87 is shown in SEQ ID NO.1; The mature sequence of the Foc-milR138 is shown in SEQ ID NO.

3.

4. The engineered banana endophytic bacteria based on RNAi technology and targeting pathogenic milRNAs as described in claim 2, characterized in that: The precursor sequence of the Foc-milR87 is shown in SEQ ID NO.2; The precursor sequence of the Foc-milR138 is shown in SEQ ID NO.

4.

5. The engineered banana endophytic bacteria based on RNAi technology and targeting pathogenic milRNAs according to claim 1, characterized in that: The key pathogenic milRNAs of Fusarium wilt in bananas are replaced with key pathogenic target genes of Fusarium wilt in bananas.

6. A microbial agent with both disease prevention and growth promotion functions, characterized in that, It includes the banana endophytic bacteria engineered based on RNAi technology as described in any one of claims 1 to 5.

7. The method for constructing banana endophytic bacteria engineered based on RNAi technology and targeting pathogenic milRNAs as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1. An RNAi silencing vector was constructed based on the precursor sequences of key pathogenic milRNAs of Fusarium wilt of banana. S2. The RNAi silencing vector from step S1 was transformed into banana endophytic fungi. After resistance screening and PCR verification, the engineered bacteria were obtained.

8. The application of the banana endophytic bacteria engineered based on RNAi technology and targeting pathogenic milRNAs as described in any one of claims 1 to 5, or the microbial agent as described in claim 6, characterized in that, For any one or more of the following applications: (1) Application in promoting plant growth; (2) Application in combating Fusarium wilt pathogens; (3) Application in the prevention and control of Fusarium wilt disease.

9. The application according to claim 8, characterized in that: (a) Application in the preparation of products that promote plant growth; (b) Application in the preparation of products resistant to Fusarium wilt; (c) Application in the preparation of products for the prevention and control of Fusarium wilt.

10. A method for promoting plant growth, characterized in that, The method includes the following steps: treating the plant with the engineered banana endophytic bacteria as described in any one of claims 1 to 5 or the microbial agent as described in claim 6.

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