A method for discriminating between species of the planthopper genus nilaparvata

Abstract

The application discloses a method for identifying brown planthopper species, and relates to the technical field of biology.The application is based on bioinformatics analysis, and a kit is developed by screening three planthopper species-specific genes and designing primers, and the kit has high species specificity.The method adopts loop-mediated isothermal amplification technology to perform isothermal amplification of target genes, adopts hydroxynaphthol blue (HNB) discoloration method, realizes visual detection of the amplification products, reduces requirements on instruments and equipment, reduces operation difficulty, and gets rid of dependence on professional classification skills.In summary, the planthopper rapid detection and identification method has the advantages of accuracy, simple operation, rapidness, low requirement on operators, higher repeatability, easy operability and timeliness compared with a traditional morphological identification method.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for identifying planthopper species of the genus *Brown Planthopper*. Background Technology

[0002] Brown planthoppers are a major pest of rice, causing damage not only by sucking sap but also by transmitting viral diseases. Accurate pest forecasting is crucial for pest control. The initial sighting date, migration volume, and migration peak of brown planthoppers under monitoring lamps are key information for forecasting and for formulating control strategies. False brown planthoppers and pseudo-brown planthoppers are closely related species within the same genus as brown planthoppers. They do not feed on rice but also exhibit the habit of attracting light. Brown planthoppers (Nilaparvata lugens, N1), false brown planthoppers (Nilaparvata muiri, Nm), and pseudo-brown planthoppers (Nilaparvata bakeri, Nb) are very similar in morphology, body color, and external characteristics, making them difficult to distinguish with the naked eye. Professional identification using a microscope based on facial and genital features is required. Manual identification of closely related species within the genus *Nilaparvata* is time-consuming, labor-intensive, and requires highly skilled personnel, and cannot identify incomplete planthopper individuals.

[0003] Nucleic acid detection based on polymerase chain reaction (PCR) technology provides a new means for species identification, making up for many shortcomings of traditional morphological identification. Literature reports (Cui Yali. Biological and molecular marker study of brown planthopper and two closely related species [D]. Beijing: Chinese Academy of Agricultural Sciences, 2012, pp: 26-39.) that primers were designed using the internal transcribed spacer (ITS) gene to preliminarily establish a multiplex PCR detection technique for brown planthopper, pseudo-brown planthopper, and false brown planthopper (requiring a PCR instrument + electrophoresis instrument + gel imaging instrument). However, because it uses pure genomic DNA as a template, it has disadvantages such as long processing time and high cost. Based on this, Liu et al. (Liu SH, Luo J, Liu R, et al. Identification of Nilaparvata lugens and its two sibling species (N. bakerian and N. muiri) by direct multiplex PCR[J]. Journal of Economic Entomology, 2018, 111(6): 2869-2875.) redesigned primer pairs and established a direct multiplex PCR detection method for three planthoppers using crude tissue fluid ground with water as a template (requiring a PCR instrument + electrophoresis instrument + gel imaging instrument). This method does not require DNA extraction, greatly shortening the detection time. Although both of these methods can accurately identify brown planthoppers, they rely on expensive instruments and equipment, making them difficult to promote and apply. Luo Ju et al. (Luo Ju, Tang Jian, Wang Aiying, et al. Rapid identification method of brown planthopper based on recombinase polymerase amplification-lateral flow chromatographic test strip. Chinese Journal of Rice Science, 2022, 36: 96-104.) established a rapid identification technique for brown planthopper based on recombinase polymerase amplification (RPA) and lateral flow chromatographic test strip (LFD), which can complete the detection within 12 minutes without the aid of any instruments. The RPA-LFD method can achieve rapid and accurate identification of brown planthoppers and eliminate the dependence on precision instruments, but it is costly because it requires the use of test strips for visual detection. Moreover, this method can only distinguish between brown planthoppers and non-brown planthoppers, and cannot accurately identify pseudo-brown planthoppers or false brown planthoppers.

[0004] Therefore, it is necessary to further develop economical and rapid detection technologies that can accurately identify brown planthoppers, false brown planthoppers, and pseudo-brown planthoppers. Summary of the Invention

[0005] This invention addresses the challenge of rapidly distinguishing and identifying brown planthoppers and their similar species under insect-attracting lamps by providing a simple, rapid, and visually-based on-site detection method and kit for identifying brown planthoppers and their similar species.

[0006] To achieve the above objectives, the solution of the present invention is:

[0007] A target for identifying species of brown planthoppers, the target being a combination of three sequences, the specific nucleotide sequences of which are as follows:

[0008] (1) Nl: The sequence is as shown in SEQ ID No. 1;

[0009] (2) Nm: The sequence is shown in SEQ ID No. 2;

[0010] (3) Nb: The sequence is shown in SEQ ID No.3.

[0011] The present invention also provides the application of the target in identifying species of the genus *Brown planthopper*.

[0012] Preferably, the planthopper species of the genus *Nilaparvata* are *Nilaparvata lugens*, *Nilaparvata muiri*, and *Nilaparvata bakeri*.

[0013] This invention also provides a primer combination for identifying species of the genus *Brown Planthopper*, comprising the following three sets, the nucleotide sequences of which are as follows:

[0014] (1) Nl: The outer primer sequence is shown in SEQ ID NO.4-5, and the inner primer sequence is shown in SEQ ID NO.6-7;

[0015] (2) Nm: The outer primer sequence is shown in SEQ ID NO.8-9, and the inner primer sequence is shown in SEQ ID NO.10-11;

[0016] (3) Nb: The outer primer sequence is shown in SEQ ID NO.12-13, and the inner primer sequence is shown in SEQ ID NO.14-15.

[0017] The present invention also provides a kit for identifying planthopper species of the genus *Brown Planthopper*, comprising the primer combination described above.

[0018] The present invention also provides a method for identifying planthopper species of the genus *Brown planthopper*, comprising the following steps:

[0019] (1) Prepare crude tissue solution of the sample to be tested;

[0020] Take a single planthopper to be tested, place it in a centrifuge tube, add distilled water, and gently grind it with a grinding rod or pipette tip; the grinding solution does not need to be centrifuged and is already crude tissue fluid;

[0021] (2) Preparation of primer reaction units for identifying planthopper species of the genus *Brassica oleracea*; wherein, the nucleotide sequences of the outer primers used for detection in the *Brassica oleracea* primer reaction unit are shown in SEQ ID No. 4-5, and the nucleotide sequences of the inner primers are shown in SEQ ID No. 6-7; the nucleotide sequences of the outer primers used for detection in the *Pseudo-Brassica oleracea* primer reaction unit are shown in SEQ ID No. 8-9, and the nucleotide sequences of the inner primers are shown in SEQ ID No. 10-11; the nucleotide sequences of the outer primers used for detection in the *Pseudo-Brassica oleracea* primer reaction unit are shown in SEQ ID No. 12-13, and the nucleotide sequences of the inner primers are shown in SEQ ID No. 14-15;

[0022] (3) The HNB color change method is used for detection. If the brown planthopper primer reaction unit is sky blue, it indicates that the sample to be tested is brown planthopper; if the pseudo brown planthopper primer reaction unit is sky blue, it indicates that the sample to be tested is pseudo brown planthopper; if the pseudo brown planthopper primer reaction unit is sky blue, it indicates that the sample to be tested is pseudo brown planthopper; if all reaction units are purple, it indicates that the sample to be tested is not one of brown planthopper, pseudo brown planthopper or pseudo brown planthopper.

[0023] Specifically, the HNB colorimetric reaction system is as follows: 1 μL of 100 mM magnesium sulfate, 2.5 μL of magnesium ion-free 10× Buffer, 3.5 μL of 10 mM dNTP, 3 μL of 5 M Betaine, 4 μL of 10 μM inner primer, 1 μL of 5 μM outer primer, 1 μL of 1.25 mM hydroxynaphthol blue, 1 μL of 8 U / μL Bst 2.0, 1 μL of crude tissue solution of the sample to be tested, and ddH2O to be added to 25 μL.

[0024] The inner and outer primers in the HNB colorimetric reaction system are mixtures, wherein the inner primers are a mixture of two inner primers in a volume ratio of 1:1, and the outer primers are a mixture of two outer primers in a volume ratio of 1:1.

[0025] The reaction conditions for the HNB colorimetric method are: constant temperature amplification at 65℃ for 30-50 minutes.

[0026] The method for identifying planthopper species of the genus *Brown Planthopper* also includes the determination of negative controls and positive standards; the results are valid when the negative control is purple and the positive standard is sky blue.

[0027] The beneficial effects of this invention are:

[0028] This invention, based on bioinformatics analysis, screens species-specific genes of three planthopper species and designs primers to develop a kit with high species specificity. The method employs loop-mediated isothermal amplification (LAMP) technology for isothermal amplification of target genes and uses the hydroxynaphthol blue (HNB) colorimetric method to achieve visual detection of the amplified products, reducing the requirements for instrumentation and operational difficulty, and eliminating reliance on specialized taxonomic skills. In summary, the rapid detection and identification method for planthoppers of this invention is accurate, simple to operate, rapid, and requires less skilled operators. Compared with traditional morphological identification methods, it has higher reproducibility, ease of operation, and timeliness. Attached Figure Description

[0029] Figure 1 The image shows the results of species-specific primer screening in Example 1, where the sample templates include ultrapure water, brown planthopper, pseudo-brown planthopper, false brown planthopper, white-backed planthopper, gray planthopper, barnyard planthopper, and smoke-winged white-backed planthopper.

[0030] Figure 2 The image shows the HNB color change detection results of the sample in Example 2, where: Nl represents the brown planthopper detection reaction tube; Nm represents the pseudo-brown planthopper detection reaction tube; Nb represents the pseudo-brown planthopper detection reaction tube; 1 to 8 represent: blank control, positive control, brown planthopper body 1, brown planthopper body 2, pseudo-brown planthopper body 1, pseudo-brown planthopper body 2, pseudo-brown planthopper body 1, and pseudo-brown planthopper body 2, respectively. Detailed Implementation

[0031] Example 1

[0032] (1) Preparation of sequencing test insects

[0033] Live *Pseudobrown planthopper* were provided by Zhejiang Normal University, while live *Pseudobrown planthopper* were collected from weeds along field edges in Zhaoping County, Guangxi. Both planthopper species were further reared for 10–15 generations in the insect rearing laboratory of the China National Rice Research Institute. In each generation, single-female-single-male pairings were conducted to purify the genetic background and establish near-isogenic populations.

[0034] (2) Sequencing sample preparation

[0035] Several male and female adults of the near-isogenic line population of *Pseudobrown planthopper* or *Pseudobrown planthopper* were collected from the indoor environment. The midgut was removed under a microscope, and RNA was extracted from the remaining insects using the Trizol method. After verification, the RNA was sent to the company for sequencing.

[0036] (3) Transcriptome sequencing and assembly

[0037] Transcriptome sequencing was commissioned to Beijing Novogene Technology Co., Ltd. Standard procedures were followed for library construction and sequencing reactions. The sequencing platform was Illumina NovaSeq 6000, and the sequencing strategy was PE150. The raw reads obtained from sequencing were filtered to remove reads containing adapters, N (indicating undetermined base information), and low-quality reads, resulting in high-quality clean reads. Trinity (v2.1.4) was used to assemble the clean reads de novo to obtain the transcriptome sequences of *Pseudobrown planthopper* and *Pseudobrown planthopper*.

[0038] (4) Species-specific gene screening

[0039] A local planthopper CDS database was established, containing five species: brown planthopper, pseudo-brown planthopper, false brown planthopper, white-backed planthopper, and gray planthopper. Species-specific nucleotide sequences for brown planthopper, pseudo-brown planthopper, and false brown planthopper were screened using a local BLAST program (alignment parameter: -qcov_hsp_perc 90-evalue 1e-5). The resulting target sequences are shown in SEQ ID Nos. 1–3.

[0040] (5) LAMP primer design and screening

[0041] LAMP primers were designed using the online software PrimerExplorer V5 with default parameters (https: / / primerexplorer.jp / e / ), with 5 sets for each species. Pure genomic DNA from *Ixodes spp.*, *Ixodes pseudo-brown*, *Ixodes simianus*, *Ixodes spp.*, and *Ixodes auricula* were used as templates (10 ng / μL), and one set of specific primers was selected for each species using the LAMP-Sybr system (as shown in Table 1). Figure 1 The selected Nl primer combinations are shown in SEQ ID No. 4-7, the Nm primer combinations are shown in SEQ ID No. 8-11, and the Nb primer combinations are shown in SEQ ID No. 12-15.

[0042] Table 1 LAMP-Sybr system

[0043] Haigene (A3801-01) LAMP-Sybr (10 μL system) <![CDATA[ddH2O]]> 3.3 <![CDATA[MgSO4(100mM)]]> 0.4 <![CDATA[10×Buffer(Mg 2+ 6mM)]]> 1 dNTPs (10mM) 1.4 FIP / BIP Mix (10μM) 1.6 F3 / B3 Mix (5μM) 0.4 10×Sybr Green (10×) 1 Bst 2.0 (8U / μL) 0.4 DNA / crude tissue fluid 1

[0044] Example 2

[0045] (1) Test Sample Acquisition

[0046] Planthopper samples of brown planthopper, pseudo-brown planthopper, and false brown planthopper under monitoring lights in Zhaoping, Guangxi; Shaodong, Hunan; and Fuyang, Zhejiang.

[0047] (2) Test template preparation

[0048] Take a single planthopper to be tested and place it in a 1.5 mL or 2 mL centrifuge tube; add 50–300 μL of distilled water (or mineral water) and gently grind it with a grinding rod or pipette tip; the grinding solution does not need to be centrifuged and is the crude planthopper tissue fluid, which can be used as a DNA template for subsequent processing.

[0049] (3) HNB color change detection method reaction system:

[0050] Haigene (A3801-01) LAMP-HNB (25μL) <![CDATA[ddH2O]]> 7 <![CDATA[MgSO4(100mM)]]> 1 <![CDATA[10×Buffer (Mg-free 2+ )]]> 2.5 dNTPs (10mM) 3.5 Betaine (5M) 3 FIP / BIP Mix (10μM) 4 F3 / B3 Mix (5μM) 1 HNB (1.25mM) 1 Bst 2.0 (8U / μL) 1 coarse tissue fluid 1

[0051] (4) Reaction conditions for HNB color change detection method:

[0052] Amplification at a constant temperature of 65℃ for 35 minutes using a water bath or metal bath.

[0053] (5) Determination of HNB color change test results:

[0054] The template for the negative control is sterile water, and the positive control is the genomic DNA of Nl, Nm, or Nb (i.e., for the Nl test tube, the positive control is the genomic DNA of Nl; for the Nm test tube, the positive control is the genomic DNA of Nm; and for the Nb test tube, the positive control is the genomic DNA of Nb).

[0055] If the negative and positive control results are correct (i.e., the negative control is purple and the positive standard is sky blue), if the brown planthopper primer reaction unit changes from purple to sky blue, the sample to be tested is a brown planthopper; if the pseudo-brown planthopper primer reaction unit changes from purple to sky blue, the sample to be tested is a pseudo-brown planthopper; if the false brown planthopper primer reaction unit changes from purple to sky blue, the sample to be tested is a false brown planthopper; if no change from purple to sky blue is observed in any of the three reaction units, the sample to be tested is not any of the three planthopper species of the genus *Brachystomata*.

[0056] Figure 2 In the diagram, Nl represents the brown planthopper detection reaction tube; Nm represents the pseudo-brown planthopper detection reaction tube; Nb represents the pseudo-brown planthopper detection reaction tube; and 1 to 8 represent: blank control, positive control, brown planthopper body 1, brown planthopper body 2, pseudo-brown planthopper body 1, pseudo-brown planthopper body 2, pseudo-brown planthopper body 1, and pseudo-brown planthopper body 2, respectively.

[0057] from Figure 2 As can be seen from the results, in the N1 tube representing the brown planthopper detection reaction tube, tube 2 (positive control), tube 3 (detecting brown planthopper body 1), and tube 4 (detecting brown planthopper body 2) all changed from purple to sky blue, while the other reaction tubes did not change color and remained purple. On the other hand, in the pseudo-brown planthopper detection reaction tube and the false brown planthopper detection reaction tube, the corresponding reaction tubes 3 and 4 also did not change color and remained purple.

[0058] In the Nm-representing pseudo-brown planthopper detection reaction tubes, tube 2 (positive control), tube 5 (detecting pseudo-brown planthopper body 1), and tube 6 (detecting pseudo-brown planthopper body 2) all changed from purple to sky blue, while the other reaction tubes did not change color and remained purple. On the other hand, in the brown planthopper detection reaction tubes and the pseudo-brown planthopper detection reaction tubes, the corresponding reaction tubes 5 and 6 also did not change color and remained purple.

[0059] In the Nb-representing *Pseudobrown planthopper* detection reaction tubes, tube 2 (positive control), tube 7 (detecting *Pseudobrown planthopper* body 1), and tube 8 (detecting *Pseudobrown planthopper* body 2) all changed from purple to sky blue, while the other reaction tubes did not change color and remained purple. On the other hand, in the *Pseudobrown planthopper* and *Pseudobrown planthopper* detection reaction tubes, the corresponding reaction tubes 7 and 8 also did not change color and remained purple.

[0060] As can be seen from the above, the present invention can accurately detect and identify planthopper species of the genus *Brown Planthopper*. The primers designed in this invention have high species specificity and have higher repeatability, ease of operation and timeliness than traditional morphological identification methods.

Claims

1. A target combination for identifying planthopper species of the genus *Brown Planthopper*, characterized in that, The target combination consists of the following three targets: (1) Nl: The nucleotide sequence is shown in SEQ ID No. 1; (2) Nm: The nucleotide sequence is shown in SEQ ID No. 2; (3) Nb: The nucleotide sequence is shown in SEQ ID No.

3.

2. The application of the target combination according to claim 1 in identifying species of the genus *Brown Planthopper*, wherein the species of *Brown Planthopper* is *Brown Planthopper* (… Nilaparvata lugens ), false brown planthopper ( Nilaparvata muiri ) and brown planthopper ( Nilaparvata bakeri ).

3. A primer combination for identifying planthopper species of the genus *Brown Planthopper*, characterized in that, Includes the following 3 groups: (1) Primers for Nl: The nucleotide sequences of the outer primers are shown in SEQ ID NO.4~5, and the nucleotide sequences of the inner primers are shown in SEQ ID NO.6~7; (2) Primers for Nm: The nucleotide sequences of the outer primers are shown in SEQ ID NO. 8~9, and the nucleotide sequences of the inner primers are shown in SEQ ID NO. 10~11; (3) Primers for Nb: The nucleotide sequences of the outer primers are shown in SEQ ID NO.12~13, and the nucleotide sequences of the inner primers are shown in SEQ ID NO.14~15.

4. A kit for identifying planthopper species of the genus *Brown Planthopper*, characterized in that, Includes the primer combination described in claim 3.

5. A method for identifying planthopper species of the genus *Brown Planthopper*, characterized in that, Includes the following steps: (1) Prepare crude tissue solution of the sample to be tested; (2) Preparation of primer reaction units for identifying planthopper species of the genus *Brassica rapa*; wherein, the nucleotide sequence of the outer primer used for detection in the *Brassica rapa* primer reaction unit is shown in SEQ ID No. 4-5, and the nucleotide sequence of the inner primer is shown in SEQ ID No. 6-7; the nucleotide sequence of the outer primer used for detection in the *Pseudo-Brassica rapa* primer reaction unit is shown in SEQ ID No. 8-9, and the nucleotide sequence of the inner primer is shown in SEQ ID No. 10-11; the nucleotide sequence of the outer primer used for detection in the *Pseudo-Brassica rapa* primer reaction unit is shown in SEQ ID No. 12-13, and the nucleotide sequence of the inner primer is shown in SEQ ID No. 14-15; (3) The HNB color change method is used for detection. If the brown planthopper primer reaction unit is sky blue, it indicates that the sample to be tested is brown planthopper; if the pseudo brown planthopper primer reaction unit is sky blue, it indicates that the sample to be tested is pseudo brown planthopper; if the pseudo brown planthopper primer reaction unit is sky blue, it indicates that the sample to be tested is pseudo brown planthopper; if all reaction units are purple, it indicates that the sample to be tested is not one of brown planthopper, pseudo brown planthopper or pseudo brown planthopper.

6. The method for identifying planthopper species of the genus *Brown Planthopper* as described in claim 5, characterized in that, The HNB colorimetric reaction system consisted of: 1 μL of 100 mM magnesium sulfate, 2.5 μL of magnesium-free 10× Buffer, 3.5 μL of 10 mM dNTPs, 3 μL of 5 M Betaine, 4 μL of 10 μM inner primer, 1 μL of 5 μM outer primer, 1 μL of 1.25 mM hydroxynaphthol blue, 1 μL of 8 U / μL Bst 2.0, 1 μL of crude tissue solution of the sample to be tested, and ddH2O to a final volume of 25 μL.

7. The method for identifying planthopper species of the genus *Brown Planthopper* as described in claim 6, characterized in that, The reaction conditions for the HNB colorimetric method are: constant temperature amplification at 65℃ for 30-50 minutes.

8. The method for identifying planthopper species of the genus *Brown Planthopper* as described in claim 6, characterized in that, Step (3) also includes the determination of negative control and positive standard; the result is valid when the negative control is purple and the positive standard is sky blue.

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