Bionidal xy001 of nilaparvata lugens and application thereof

Abstract

The present application relates to the field of agricultural pest control, and particularly relates to a brown planthopper symbiotic bacterium XY001 and its application, and the preservation number is CCTCC NO: M 2023097. The culture process of the brown planthopper XY001 is simple, and the brown planthopper can be artificially infected by a simple and low-cost method, so that the whole body and continuous transmission can be realized, and the exogenous gene can be efficiently expressed in the brown planthopper. The brown planthopper XY001 has a rapid spreading ability in the brown planthopper population, and a specific exogenous gene can be expressed to realize the pest control strategy.

Description

Technical Field

[0001] This invention relates to the field of agricultural pest control, specifically to a symbiotic bacterium for brown planthoppers, XY001, and its application. Background Technology

[0002] Arsenophonus are a class of symbiotic bacteria found in insects. They have a wide host range and diverse transmission methods, playing a vital role in insect nutrition, reproduction, and environmental adaptation. Arsenophonus was first discovered in the pupae of the parasitic blowfly, *Arsenophonus spp.*, and its anandrogenic properties were demonstrated, spreading within the population through maternal transmission. Other studies have reported that symbiotic anandrogenic bacteria can also be transmitted within parasitic wasp populations at the level of shared fruit fly hosts. During the identification process, it was found that Arsenophonus differs from symbionts such as Wolbachia, which are strictly dependent on the insect cellular environment, and can be cultured in pure form in vitro using suitable media.

[0003] Brown planthoppers are among the most destructive pests in rice production, and due to their significant importance in control, they have been included in the "List of Class A Crop Diseases and Pests" by the Ministry of Agriculture and Rural Affairs. Previous studies, using PCR and amplicon sequencing, have confirmed the widespread presence of the anandrogenic bacterium *Arsenophonus* within brown planthopper populations. This bacterium is primarily found in the fat body and other tissues of brown planthoppers, such as the capsule, ovary, intestine, and salivary glands. Furthermore, research has demonstrated that the anandrogenic bacterium within brown planthoppers can be vertically transmitted, enabling cross-generational infection. These characteristics suggest that the anandrogenic bacterium of brown planthoppers has the potential to be engineered into microorganisms for the control of this pest. Using these microorganisms as vectors to express exogenous genes, such as toxins and dsRNA, would be of significant value in the control of brown planthoppers.

[0004] However, to date, the anthropomorphic bacteria of the brown planthopper has not been cultured in vitro, and its research and application have not been formally published, which seriously limits its research and application. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a symbiotic bacterium for brown planthoppers, XY001, and its applications. This invention achieves, for the first time, pure in vitro culture of XY001, a symbiotic bacterium for brown planthoppers; the reintroduced XY001 will stably colonize within the brown planthopper and achieve continuous cross-generational infection through eggs. Furthermore, XY001 can continuously express exogenous genes, and cross-generational expression is achieved through the transgenerational spread of the bacteria.

[0006] To achieve the above objectives, the technical solution designed by this invention is as follows:

[0007] This invention provides a symbiotic bacterium for brown planthoppers, Arsenophonus nilaparvatae XY001, with accession number CCTCC NO: M 2023097.

[0008] The aforementioned symbiotic bacterium *Arsenophonus nilaparvatae*, found in brown planthoppers, exhibits host specificity and can widely infect brown planthopper populations. Identified as belonging to the genus *Arsenophonus* within the family Enterobacteriaceae of the phylum Proteobacteria, it was named *Arsenophonus nilaparvatae* XY001. This bacterium was deposited on February 7, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC M2023097.

[0009] The present invention also provides the application of the above-mentioned brown planthopper symbiotic bacterium Arsenophonus nilaparvatae XY001 in pest control.

[0010] The present invention also provides the application of the above-mentioned brown planthopper symbiotic bacterium Arsenophonus nilaparvatae XY001 in the control of brown planthopper pests.

[0011] This invention also provides a method for controlling brown planthoppers by reapplying the male-killing fungus XY001, comprising the following steps:

[0012] 1) Add the above-mentioned brown planthopper anthraxanthin XY001 to BHI liquid medium for enrichment, culture, and centrifugation to collect bacterial cells;

[0013] 2) Add sterile water to the bacterial cells and disperse them evenly to obtain a bacterial dispersion;

[0014] 3) The bacterial dispersion was reinjected into the brown planthopper via microinjection.

[0015] Furthermore, in step 1), the cultivation conditions are as follows:

[0016] The temperature of the shaker was 28℃, and the rotation speed was 220 rpm; the incubation time was 6 days, and the OD of the cultured antifungal bacterial solution was... 600 >0.2; centrifugation speed was 10,000 rpm, and centrifugation time was 5 min.

[0017] The beneficial effects of this invention are:

[0018] The culture process of the *Brachystomum anthropotoxicum* strain of this invention is simple, and it can be artificially infected into brown planthoppers in a simple and low-cost manner to achieve systemic and continuous spread, and efficiently express exogenous genes within the brown planthoppers. *Brachystomum anthropotoxicum* has a rapid dispersal ability within brown planthopper populations, and its expression of specific exogenous genes can be used to implement pest control strategies. Attached Figure Description

[0019] Figure 1 A colony appearance diagram of the male-killing fungus for brown planthoppers;

[0020] Figure 2 Image of the bacterial cell structure of *Bacillus anthracis* (scanning electron microscope image);

[0021] Figure 3 Temperature growth curve of male-killing fungus for brown planthoppers;

[0022] Figure 4 A fluorescent photograph of brown planthopper eggs infected with GFP-expressing androgynous bacteria;

[0023] Figure 5 A fluorescent photograph of brown planthopper eggs infected with GFP-expressing androgynous bacteria;

[0024] Figure 6 Fluorescent photographs showing widespread expression of GFP in brown planthoppers.

[0025] In the diagram, A: insect body appearance; B: head; C: wings; D: forelegs; E: midlegs; F: hind legs. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0027] Example 1

[0028] Screening and identification of the brown planthopper symbiotic bacterium *Arsenophonus nilaparvatae* XY001

[0029] 1. Bacterial morphological characteristics:

[0030] Brown planthopper field samples were collected, and eggs were collected using micromanipulation. One hundred brown planthopper eggs were placed in sterile water and surface-immersed twice with 75% alcohol (approximately 20 seconds). The surface was then rinsed clean with sterile water. The eggs were then directly crushed using a pestle in 0.1 mL of sterile water. The crushing solution was evenly spread onto DNase agar medium and incubated at 28±2℃ for six days. Colonies were collected afterward. Colonies were covered with mucus, had a smooth surface, raised areas, and were transparent. Figure 1 The bacteria are rod-shaped, measuring 5-10 μm in length and 0.5 μm in width. Figure 2), white, mature fungi are gray.

[0031] The DNase agar medium formula is as follows: each liter of medium contains 15.0g tryptone, 5.0g soybean peptone, 0.02g calcium chloride, 5.0g sodium chloride, 2.0g DNA, and 12.0g agar. The pH value is adjusted to 7.3±0.2 at 25℃.

[0032] 2. Physiological and biochemical characteristics:

[0033] The brown planthopper killer fungus (Arsenophonus nilaparvatae) XY001 grows well at pH 5-7 and 25-30℃. It can be synthesized using dextrin, N-acetyl-D-glucosamine, α-D-glucose, D-mannose, D-fructose, D-glucose-6-phosphate, D-fructose-6-phosphate, L-aspartic acid, L-glutamic acid, L-histamine, D-galacturonic acid, L-galacturonic acid lactone, D-gluconic acid, D-glucuronic acid, glucuronide, mucoacid, α-keto-glutaric acid, L-malic acid, acetoacetic acid, and acetic acid.

[0034] Carbon source utilization of *Bacillus anthracis* was identified using a Biolog GENIII plate (Cat. No. 1030). *Bacillus anthracis* was added to BHI medium for enrichment and cultured at 28°C and 220 rpm for 6 days (200 mL total volume) until OD600 > 0.2. The bacterial pellet was then obtained by centrifugation at 10,000 g for one minute and resuspended in IF-A inoculum (Biolog, Cat. No. 72401). Following the Biolog GENIII instructions, 100 μL of the bacterial suspension was added to 96 wells of a Biolog GENIII plate. After 48 h of culture, absorbance at 590 nm and 750 nm was measured using a fluorescence microplate reader, and growth efficiency was calculated using software.

[0035] Among them, the brain and heart broth (BHI) has the following culture medium formula: each liter of culture medium contains 10.0g of peptone, 12.5g of dehydrated calf brain extract powder, 5.0g of dehydrated calf heart extract powder, 5.0g of sodium chloride, 2.0g of glucose, 2.5g of disodium hydrogen phosphate, and the pH value is adjusted to 7.4±0.2 at 25℃.

[0036] 3. Characterization by scanning electron microscopy (HITACHI Regulus 8100)

[0037] Arsenophonus nilaparvatae XY001 was enriched in BHI liquid medium, centrifuged (10,000 rpm), washed twice with PBS, and stored in electron microscopy fixative. After dehydration, drying, and gold sputtering, it was characterized by scanning electron microscopy (HITACHI Regulus 8100). Figure 2 As shown: The bacterial cells are long and rod-shaped, break apart and multiply, with a size of 5-10 μm in length and 0.5 μm in width.

[0038] 4.16S rRNA sequence analysis

[0039] The bacterial suspension of *Arsenophonus nilaparvatae* was amplified using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′), and the product was sequenced to obtain the 16S rRNA sequence of *Arsenophonus nilaparvatae*. DNA sequencing analysis showed that the 16S rDNA sequence of this bacterium consisted of 1405 bases, as shown in SEQ ID NO:1. Nucleotide homology comparison of the 16S rDNA sequence of *Arsenophonus nilaparvatae* XY001 with 16S rDNA sequences already registered in GenBank using the BLAST program showed that the homology with most reported sequences of the genus *Arsenophonus* was over 99%, indicating that this isolate is a new species of *Arsenophonus nilaparvatae*.

[0040] Based on the morphological characteristics, physiological and biochemical properties, and 16S rRNA sequence analysis, the bacterium *Arsenophonus nilaparvatae* was identified as belonging to the genus *Arsenophonus* of the family Enterobacteriaceae in the phylum Proteobacteriaceae. It was named *Arsenophonus nilaparvatae* XY001 and deposited on February 7, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, with accession number CCTCC M 2023097.

[0041] Example 2

[0042] The replenishment method for Arsenophonus nilaparvatae XY001, a fungicide for brown planthoppers, includes the following steps:

[0043] The above-mentioned male-killing bacterium (Arsenophonus nilaparvatae) XY001 was enriched using BHI liquid medium and cultured at 28℃ and 220 RPM for 6 days until OD600>0.2. The bacterial pellet was collected by centrifugation at 10,000 RPM for 5 min, and 1 mL of sterile water was added to disperse it evenly. The male-killing bacterium was then reinjected into the brown planthopper through microinjection.

[0044] By enriching the bacteria and microinjecting them back into the brown planthopper, systemic and cross-generational stable colonization was achieved. At the same time, by genetically modifying the bacteria, exogenous genes can be expressed in the insect. Taking green fluorescent protein as an example, the exogenous gene expression mediated by this bacteria will be dispersed throughout the insect, indicating its potential application in insect population control and insect research.

[0045] Example 3: Expression of the exogenous gene of XY001, a male-killing bacterium of the brown planthopper

[0046] 1. Preparation of XY001, a fungicide for killing brown planthoppers:

[0047] The male-killing bacteria of brown planthoppers were enriched using BHI liquid medium and cultured at 28°C and 220 rpm for 6 days until OD600 > 0.2. The bacterial pellet was obtained by centrifugation at 16,000g for one minute. The pellet was resuspended in 15% sterile glycerol and the above process was repeated four times. Finally, the pellet was resuspended in 100 μL of 15% sterile glycerol.

[0048] 2. pOM1 plasmid transformation:

[0049] Add 1 μL of pOM1-GFP plasmid, incubate on ice for 5 min, perform a single electroporation at 2 KV, add 1 mL of BHI liquid medium, incubate overnight at 28 °C, and plate onto a DNase agar plate containing 50 μg / μL spectinomycin. Green fluorescent colonies were obtained after approximately 5 days. Single colonies were picked, and the successfully transformed GFP-transfected anandrogenic strain GFP-Ars was enriched using liquid BHI.

[0050] 3. GFP-Ars complementation:

[0051] Thirty fourth-instar nymphs of brown planthopper were selected. A glass needle was inserted into the pronotum of the brown planthopper and 50 nL of bacterial solution was injected. After they grew to the adult stage, the brown planthoppers were paired and their eggs were collected in isolated glass tubes. The specific method was as follows: the roots of two 10cm rice seedlings were wrapped with absorbent cotton and filled to the bottom of the glass tube. A pair of brown planthopper adults replenished with GFP-Ars bacterial solution were placed in the glass tube and the tube was sealed with gauze.

[0052] 4. Observation:

[0053] Twenty-four hours after oviposition, the rice seedlings were removed, and the phloem was carefully peeled back with tweezers to collect the eggs. The eggs were then placed on fresh agar plates for daily observation. Under a stereofluorescence microscope (Olympus SZX16), the eggs were observed as follows: Figure 4 As shown, strong green fluorescence is visible in the eggs, proving that GFP-Ars has been colonized and expresses GFP in the brown planthopper and can be transmitted to the next generation of test insects through the eggs.

[0054] like Figures 5-6 As shown, eggs containing green fluorescence continuously express fluorescent proteins during the development of brown planthoppers and widely infect them. Previous biocontrol bacteria, or bacteria with biocontrol potential, were only transmitted through feeding and rarely penetrated the entire body of the test insects. However, the anthropotoxic bacteria of the brown planthopper can infect throughout the entire body via hemolymphatic movement, suggesting that using it as a vector to express insecticidal proteins or to express specific genes (such as dsRNA) would yield better results. Furthermore, its vertical dispersal characteristics will facilitate the rapid preparation of brown planthopper populations carrying this bacterium, demonstrating its potential in entomological research.

[0055] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A symbiotic bacterium of brown planthoppers that kills male bacteria ( Arsenophonus nilaparvatae XY001, its accession number is: CCTCC NO: M 2023097.

2. A symbiotic bacterium for killing male bacteria of the brown planthopper as described in claim 1 ( Arsenophonus nilaparvatae The application of XY001 in the control of brown planthoppers is characterized by: Insecticidal proteins were expressed using XY001, a symbiotic bacterium of brown planthoppers, as a vector.

3. A method for controlling brown planthoppers by reapplying the male-killing fungus XY001, characterized in that: Includes the following steps: 1) The brown planthopper anthelmintic bacterium XY001 described in claim 1 is added to BHI liquid medium for enrichment, culture, and centrifugation to collect the bacterial cells; wherein, the brown planthopper symbiotic bacterium anthelmintic bacterium XY001 is used as a vector to express a protein with insecticidal activity; 2) Add sterile water to the bacterial cells and disperse them evenly to obtain a bacterial dispersion; 3) The bacterial dispersion was reinjected into the brown planthopper via microinjection.

4. The method for controlling brown planthoppers by reapplying the male-killing fungus XY001 according to claim 3, characterized in that: In step 1), the culture conditions are as follows: The temperature of the shaker was 28℃, and the rotation speed was 220 rpm; the incubation time was 6 days, and the OD of the cultured antifungal bacterial solution was... 600 >0.2; centrifugation speed was 10,000 rpm, and centrifugation time was 5 min.

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