Detection method of pine needle disease infected with pine wood nematode using 7-dhc as detection product

Abstract

The invention discloses a method for detecting pine wood nematode infection in Masson pine using 7-dhc as a detection product, comprising the following steps: S1: sample pretreatment: quantitatively weigh a Masson pine branch sample, place it in a flask, add anhydrous ethanol and potassium hydroxide solution to mix, then use magnetic stirring to heat an electric heating jacket for saponification reflux, and pass the extract through anhydrous sodium sulfate in a 150mL flat-bottomed flask, rotary evaporation to dry the fixed volume, and then measure it on a machine after passing through a microporous filter membrane. The method for detecting pine wood nematode infection in Masson pine using 7-dhc as a detection product can obtain a measurement sample by collecting Masson pine branches and carrying out S1 stage treatment, then the sample is tested through multiple items in S2 stage, and the test data is obtained thereby, and finally the test data is brought into the formula W=(C-C0)*V*N / m to obtain the 7-dhc content, and the presence or absence of pine wood nematode disease is judged by whether the 7-dhc content exists, the operation is simple, and the result can be obtained in the early stage of pine wood nematode onset, which is convenient for immediate treatment.

Description

Technical Field

[0001] The invention relates to the technical field of pine wood nematode disease detection methods, in particular to a method for detecting pine wood nematode disease infection in masson pine using 7-dhc as a detection product. Background Art

[0002] Pine wilt disease is a major and devastating forest disease. In order to curb the spread and serious occurrence of pine wilt disease, effective and precise prevention and control measures must be implemented. Early and accurate diagnosis becomes particularly important. Traditional disease detection methods can only be used for sampling and detection when symptoms appear after plants are infected with nematodes, fungi, bacteria and viruses, i.e. in the middle and late stages. For example, the publication number CN117994649A is a method for detecting early pine wilt disease, which obtains an early remote sensing image set and a late remote sensing image set, and identifies plants with pine wilt disease and normal plants in the late remote sensing image set; identifies plants with early pine wilt disease and normal plants in the early remote sensing image set; pre-processes the classified early remote sensing image set to obtain a pre-processed image set; inputs the pre-processed image set into a hyperspectral reconstruction network to obtain a hyperspectral image data set; its main feature is to extract spectral reflectance curves based on the hyperspectral image data set; uses a support vector machine method to classify the spectral reflectance curves, and establishes an early pine wilt disease detection model;

[0003] For example, the publication number CN101334363B discloses a method for detecting pinewood nematode disease based on the flagellin of fluorescent Pseudomonas carried by pinewood nematodes. First, the fluorescent Pseudomonas strain GcM5-1A (preservation number CCTCC No: M204065) carried by pinewood nematodes is cultured in vitro, and flagellin is separated and purified from the fermentation broth of the strain, and used as an antigen to immunize rabbits to prepare rabbit anti-flagellin antiserum; then, sodium dodecyl sulfate solution is used to extract the whole protein of healthy black pine and black pine tissue infected with pinewood nematode disease, and a small amount of extract is spotted on a polyvinylidene fluoride membrane, and the protein of black pine tissue is detected by Western blot prepared with rabbit anti-flagellin antiserum. The protein sample extracted from black pine infected with pinewood nematodes is positive, and the extract of healthy black pine tissue is negative. The method has fast detection speed and low detection cost, is suitable for large-scale investigation of pinewood nematode disease, and is a beneficial supplement to the existing pinewood nematode disease detection method.

[0004] For example, the publication number CN112464705A is a method and system for detecting pine wood nematode diseased trees based on YOLOv3-CIoU, which includes: constructing a target detection model based on the YOLOv3-CIoU algorithm, training the target detection model using a training set, and obtaining a trained target detection model; using a test set to detect the trained target detection model, if any of the multiple preset performance indicators of the trained target detection model does not meet the standard, then continue to train the trained target detection model until the multiple preset performance indicators of the target detection model meet the standard. Its main feature is that when training the target detection model, the compatible overlap loss function (CIoU) is used to improve the loss function of the YOLOv3 detection algorithm, so that the trained target detection model can achieve short-time, high-precision, and low missed detection rate color-changing wood detection, which is suitable for occasions with high accuracy and real-time requirements.

[0005] In summary, although the existing detection methods can detect pathogens at this time, they are usually unable to provide effective treatment, and the nematode body must be isolated and the nematode body must carry the bacteria for verification. With the development of new detection technologies, many researchers are currently using liquid chromatography-mass spectrometry-related technologies to detect changes in metabolites after plant infection, and screen out specific marker metabolites as indicators for disease detection. For example, liquid chromatography-mass spectrometry, i.e., LC-MS, is used to detect asymptomatic plants infected with tomato late blight at an early stage, and it is found that tomatidine is an important biomarker of infection, and saponins are metabolites in the early stages of infection. For example, mass spectrometry imaging (MSI) can identify changes in metabolites in tissue samples infected with citrus Huanglongbing at the cellular or subcellular level. 7-dhc (7-dehydrocholesterol), as a sterol metabolite, plays a vital role in the metabolic process of plants and animals. It can absorb ultraviolet b (UVB; λ = 280–320nm) and photochemically convert it into vitamin D3. Vitamin D3 not only regulates calcium homeostasis in animals, but also has pleiotropic activities, including stimulating differentiation and inhibiting proliferation of cells of different lineages, acting as an anticancer agent. It also suppresses adaptive immunity and inflammation, and stimulates epidermal barrier and hair growth. In addition, it can act as a chemopreventive agent in proliferative, malignant and inflammatory skin diseases. Plants can also use 7-dehydrocholesterol to produce vitamin D3 and participate in photosynthesis.

[0006] 7-dhc is an important cholesterol and a quality standard. It is currently mainly used in the quality inspection of milk and honey. That is, HPLC can be used to accurately detect the content of this substance in food, but the extraction and detection methods in plants are still immature. According to research, 7-dhc is a specific metabolite of pine wood nematodes infecting Masson pine. Exploring a suitable condition and using HPLC technology to detect the content of 7- in Masson pine can be used for early detection of pine wood nematode disease. Therefore, a method of determining whether Masson pine is infected with pine wood nematode disease by detecting the content of 7-dhc is proposed.

[0007] Traditional molecular detection methods can only be used in the middle and late stages of Masson pine infection, at which time it is often impossible to treat pine wilt disease. Bm7 and FCBM are both pseudopine wood nematodes, belonging to different species from pine wood nematodes. Among them, FCBM does not have pathogenicity, but Bm7 can also cause pine tree death and lead to the same external symptoms as pine wood nematode disease. This feature makes it impossible to determine whether the death of Masson pine is caused by infection with pine wood nematode disease by phenotype or appearance (such as existing technology hyperspectral detection, etc.). In response to this problem, a method for early detection of Masson pine infected with pine wood nematode disease using 7-dhc as a detection product is proposed. Summary of the invention

[0008] The object of the present invention is to provide a method for detecting pine wood nematode infection in Masson pine using 7-dhc as a detection product, so as to solve the problem that the pine wood nematode detection method proposed in the above-mentioned background technology can only be used for detection when pine wood nematodes proliferate in large numbers in the plant body, causing the host plant to wilt. Although pine wood nematodes can be detected at this time, effective treatment is usually no longer possible.

[0009] To achieve the above object, the present invention provides the following technical solution: a method for detecting pine needle disease infected with pine wood nematode using 7-dhc as a detection product, comprising the following steps:

[0010] S1: Sample pretreatment:

[0011] Quantitatively weigh 1g of Masson pine branch sample and place it in a 250mL flat-bottom flask, then add anhydrous ethanol and potassium hydroxide solution to mix, then use magnetic stirring to heat the electric heating jacket for saponification reflux, and indirectly shake. After the saponification is completed, rinse the inside of the condenser tube with 5mL of anhydrous ethanol from the top, remove the round-bottom flask, cool it to room temperature with cold water, transfer it to a 250mL separatory funnel, rinse the round-bottom flask with 30mL of water, and then rinse the round-bottom flask with 40mL of petroleum ether-anhydrous ether, shake it for 2min, let it stand for stratification, transfer the aqueous phase, combine the organic phases three times, wash with 100mL of water each time until neutral, pass the extract through anhydrous sodium sulfate in a 150mL flat-bottom flask, rotary evaporate to dryness, then make up to volume and pass through a microporous filter membrane before measuring on the machine;

[0012] S2: Sample injection test:

[0013] Test the samples on chromatographic columns, detectors, injection volume, wavelength, flow rate, mobile phase, etc. and record the results;

[0014] S3: Content calculation:

[0015] The 7-dhc content is monitored using the formula, and the calculation formula is:

[0016] W=(C-C0)*V*N / m

[0017] The content is calculated and used to determine whether pine wood nematode disease exists;

[0018] S4: Insect disease determination:

[0019] Whether the plant is infected with pine wood nematode disease is determined based on the content data calculated in the S3 stage.

[0020] Preferably, the anhydrous ethanol in the S1 stage is 30 ml, and the potassium hydroxide solution in the S1 stage is 10 mL of 60% potassium hydroxide solution.

[0021] Preferably, the temperature of the magnetic stirring heating in the S1 stage is 100° C., and the saponification reflux time in the S1 stage is 1 hour.

[0022] Preferably, in the S1 stage, the round-bottom flask is rinsed with 30 mL of water 2-3 times, and the washing liquid of the round-bottom flask in the S1 stage is added to the separating funnel.

[0023] Preferably, in the S1 stage, 40 mL of petroleum ether-anhydrous ether is used to rinse the round-bottom flask 2-3 times and then added to the separatory funnel, and the ratio of petroleum ether to anhydrous ether is 1:1.

[0024] Preferably, the S1 stage is dissolved with anhydrous ethanol and fixed to 1 mL, and the microporous filter membrane of the S1 stage is 0.45 μm.

[0025] Preferably, the chromatographic column of the S2 stage is SHISEIDOC18 (4.6 mm*250 mm*5 μm), the detector of the S2 stage is DAD, and the detection column temperature is 38°C.

[0026] Preferably, the injection volume of the S2 stage is 10 μL, and the wavelength of the S2 stage is 270 nm.

[0027] Preferably, the flow rate of the S2 stage is 1.0 mL / min, and the mobile phase of the S2 stage is methanol, with gradient elution.

[0028] Preferably, in the S3 stage content calculation formula, W is the content of the target in the sample, in mg / L; C is the concentration of the target in the sample assay solution, in mg / L; C0 is the concentration of the target in the blank control, in mg / L; V is the fixed volume, in mL; N is the dilution factor; and m is the sample volume, in g.

[0029] Compared with the prior art, the outstanding feature of the present invention is that the use of 7-dhc as a detection product for masson pine infected with pine wood nematode disease can achieve the effect of early diagnosis.

[0030] The present invention detects whether the Masson pine is infected with pine wood nematode disease by detecting whether the Masson pine contains 7-dhc content. The Masson pine branches are collected and subjected to S1 stage processing to obtain a test sample. The sample is then subjected to S2 stage chromatographic column, detector, injection volume, wavelength, flow rate, mobile phase and other items for detection to obtain test data. Finally, the test data is brought into a formula W=(C-C0)*V*N / m to obtain the 7-dhc content. The content is used to judge whether the pine wood nematode disease exists, and the result can be obtained in the early stage of infection, so as to facilitate targeted treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a detection flow chart of the present invention;

[0032] Figure 2 This is a schematic diagram of the processing items in the S1 stage of the present invention;

[0033] Figure 3 PCA score diagrams of positive ion and negative ion modes of the present invention;

[0034] Figure 4 This is the total clustering heat map of the present invention;

[0035] Figure 5 is a Venn diagram of the differentially expressed metabolites of the present invention;

[0036] Figure 6 It is a bubble diagram of the influencing factors of the metabolic pathway of the present invention;

[0037] Figure 7 It is the standard curve diagram of 7-dhc of the present invention;

[0038] Figure 8 This is a comparison of the appearance changes of Masson pine within one month after different treatments. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] See also Figure 1-8 The present invention provides a technical solution: a method for detecting pine wilt infection in Masson pine using 7-dhc as a detection product, comprising the following steps:

[0041] S1: Sample pretreatment:

[0042] Quantitatively weigh 1g of Masson pine branch sample and place it in a 250mL flat-bottom flask, then add anhydrous ethanol and potassium hydroxide solution to mix, then use magnetic stirring to heat the electric heating jacket for saponification reflux, and indirectly shake. After the saponification is completed, rinse the inside of the condenser tube with 5mL of anhydrous ethanol from the top, remove the round-bottom flask, cool it to room temperature with cold water, transfer it to a 250mL separatory funnel, rinse the round-bottom flask with 30mL of water, and then rinse the round-bottom flask with 40mL of petroleum ether-anhydrous ether, shake it for 2min, let it stand for stratification, transfer the aqueous phase, combine the organic phases three times, wash with 100mL of water each time until neutral, pass the extract through anhydrous sodium sulfate in a 150mL flat-bottom flask, rotary evaporate to dryness, then make up to volume and pass through a microporous filter membrane before measuring on the machine;

[0043] The anhydrous ethanol in the S1 stage is 30 ml, and the potassium hydroxide solution in the S1 stage is 10 mL of a 60% potassium hydroxide solution.

[0044] The temperature of the magnetic stirring heating in the S1 stage was 100° C., and the saponification reflux time in the S1 stage was 1 h.

[0045] In the S1 stage, the round-bottom flask was rinsed 2-3 times with 30 mL of water, and the washing liquid from the round-bottom flask in the S1 stage was added to the separatory funnel.

[0046] In the S1 stage, the round-bottom flask was rinsed with 40 mL of petroleum ether-anhydrous ether 2-3 times and the mixture was added to the separatory funnel, and the ratio of petroleum ether to anhydrous ether was 1:1.

[0047] The S1 stage is dissolved with anhydrous ethanol and fixed to 1 mL, and the microporous filter membrane of the S1 stage is 0.45 μm.

[0048] S2: Sample injection test:

[0049] Test the samples on chromatographic columns, detectors, injection volume, wavelength, flow rate, mobile phase, etc. and record the results;

[0050] The chromatographic column of the S2 stage is SHISEIDOC18 (4.6 mm*250 mm*5 μm), the detector of the S2 stage is DAD, and the detection column temperature is 38°C.

[0051] The injection volume at the S2 stage was 10 μL, and the wavelength at the S2 stage was 270 nm.

[0052] The flow rate of the S2 stage was 1.0 mL / min, and the mobile phase of the S2 stage was methanol, with gradient elution.

[0053] S3: Content calculation:

[0054] The 7-dhc content is monitored using a formula, and the calculation formula is W = (C-C0) * V * N / m to calculate the content, and the content is used to determine whether pine wood nematode disease exists.

[0055] In the S3 stage content calculation formula, W is the content of the target in the sample, in mg / L; C is the concentration of the target in the sample test solution, in mg / L; C0 is the concentration of the target in the blank control, in mg / L; V is the fixed volume, in mL; N is the dilution factor; m is the sample sampling amount, in g.

[0056] S4: Insect disease determination:

[0057] Whether the plant is infected with pine wood nematode disease is determined based on the content data calculated in the S3 stage.

[0058] Specifically, pine wilt disease is a rapidly occurring and devastating disease. After being infected with pine wilt nematodes, Masson pine will quickly die within 1-2 months. Traditional appearance observation methods can only be used when Masson pine shows symptoms, and traditional molecular detection methods can only be used in the middle and late stages of Masson pine infection. At this time, pine wilt disease is often no longer treatable. The present invention uses 7-dhc for HPLC detection to perform rapid and accurate detection in the early stage of infection of Masson pine (within 21 days). At this time, pine wilt disease is still in the early stage and Masson pine does not show symptoms. Targeted treatment can be carried out, thereby achieving early prevention and control of Masson pine wilt disease.

[0059] 1.2 Test methods

[0060] 1.2.1 Nematode inoculation and sampling

[0061] An inoculation experiment was carried out on July 10, 2022. The three nematode suspensions of BM7, FCBM, and FCBX were adjusted to 2000 nematodes / ml. The bark grafting method was used to inoculate Pinus massoniana. The inoculation point was located in the lower middle part of the stem of Pinus massoniana. The specific method was as follows: Use a sterilized scalpel to make an oblique cut into the xylem on the stem of Pinus massoniana, insert a sterilized cotton ball into the wound, and use sealing film to wrap the lower part of the wound close to the wound into a funnel shape. Inject 1 ml of nematode suspension into the cotton ball in the funnel, and keep the cotton ball moist within 24 hours after inoculation to prevent nematode death. For the LC-MS metabolomics detection of Masson pine, both the treatment group and the control group were inoculated with 5 trees. After 3 days, the stems of the 5 Masson pine trees were mixed and sampled. A total of 5 biological replicates were collected and tested 5 times. For the HPLC verification of Masson pine specific metabolites, both the treatment group and the control group were inoculated with 12 trees. After 3, 7 and 21 days, the stems of 3 Masson pine trees were mixed and tested, and the other 3 trees were photographed to observe the changes in the disease course after inoculation.

[0062] 1.2.2 Detection of the Masson pine metabolome

[0063] Sample pretreatment: Use a balance to accurately weigh the sample, place it in a centrifuge tube, add methanol, and shake; add glass beads and grind with a tissue grinder; ultrasonically extract at 25°C for 15 minutes; centrifuge for 10 minutes, then filter the supernatant and add the filtrate to the test bottle for later detection.

[0064] Liquid phase conditions: ultra-high performance liquid phase system, flow rate adjusted to 0.25 mL / min, column temperature set to 40°C, injection volume of 2 μL; mobile phase A was acetonitrile containing 0.1% formic acid, mobile phase B was 0.1% formic acid water, and gradient elution was performed.

[0065] Mass spectrometry conditions: A mass spectrometer detector and an electrospray ion source (ESI) were used to collect data in positive and negative ion modes.

[0066] 1.2.4 Metabolomics data processing and differential analysis

[0067] The R XCMS software package was used for peak detection, peak filtering, and peak alignment to obtain a quantitative list of metabolites. Public databases such as HMDB, massbank, LipidMaps, mzcloud, KEGG, and self-built substance libraries were selected to identify the substances. The orthogonal partial least squares discriminant analysis (OPLS-DA) method was used to determine the projection importance (VIP) of the variables. If the P value is less than 0.05 and the VIP value is greater than 1, the metabolite can be identified as a differential metabolite.

[0068] 1.2.5 Detection of 7-dhc content in Masson pine stems by HPLC

[0069] Weigh an appropriate amount of mixed sample, add 30mL of anhydrous ethanol and 10mL of 60% potassium hydroxide solution for extraction, mix well and saponify and reflux for 1h. After saponification, transfer to a 250mL separatory funnel, rinse the round-bottom flask with 30mL of water 2-3 times, add the washing liquid to the separatory funnel, let stand and separate, remove the aqueous phase, pass the organic phase through anhydrous sodium sulfate in a 150mL flat-bottom flask, evaporate to dryness by rotary evaporation, dissolve with anhydrous ethanol and make up to 1mL, pass through a 0.45μm microporous filter membrane and measure on the machine. The 7-dhc content in the extracted sample is calculated as follows: 1.2.4 Metabolome data processing and differential analysis

[0070] The RXCMS software package was used to perform peak detection, peak filtering, and peak alignment to obtain a quantitative list of metabolites. Public databases such as HMDB, massbank, LipidMaps, mzcloud, KEGG, and self-built substance libraries were selected to identify the substances. The orthogonal partial least squares discriminant analysis (OPLS-DA) method was used to determine the projection importance (VIP) of the variables. If the P value is less than 0.05 and the VIP value is greater than 1, the metabolite can be identified as a differential metabolite.

[0071] 1.2.5 Detection of 7-dhc content in Masson pine stems by HPLC

[0072] Weigh an appropriate amount of mixed sample, add 30mL of anhydrous ethanol and 10mL of 60% potassium hydroxide solution for extraction, mix well, saponify and reflux for 1h, transfer to a 250mL separatory funnel after saponification, rinse the round-bottom flask with 30mL of water 2-3 times, add the washing liquid to the separatory funnel, let stand and separate, remove the aqueous phase, pass the organic phase through anhydrous sodium sulfate in a 150mL flat-bottom flask, evaporate to dryness by rotary evaporation, dissolve with anhydrous ethanol and make up to 1mL, pass through a 0.45μm microporous filter membrane and measure on the machine. The 7-dhc content in the extracted sample is calculated as follows:

[0073] W=(C-C0)*V*N / m

[0074] Where: W is the content of the target in the sample, in mg / L; C is the concentration of the target in the sample solution, in mg / L; C0 is the concentration of the target in the blank control, in mg / L; V is the fixed volume, in mL; N is the dilution factor; m is the sample size, in g.

[0075] 2. Results and Analysis

[0076] 2.1 Principal component analysis of metabolome

[0077] When conducting metabolomics research based on mass spectrometry technology, quality control (QC) is required to ensure the reliability and high quality of the metabolomics data obtained. Figure 3 As shown in the figure (the red dots are QC samples, the green dots are formal experimental samples, (A) positive ion mode; (B) negative ion mode), this stability is manifested in the dense distribution of QC samples, which indicates that the instrument measurement and experimental process are stable and the data quality obtained is high. In the positive and negative ion modes, the contribution of the first principal component is 19.6% and 8% respectively, and the contribution of the second principal component is 15.1% and 11.3% respectively, indicating that the two principal components can reflect the main characteristic information of the metabolome.

[0078] 2.2 Metabolite clustering heat map analysis

[0079] The metabolite expression of different nematode species after inoculation with Masson pine can be analyzed by the overall cluster heat map (e.g. Figure 4 As shown in (A) positive ion mode; (B) negative ion mode), it was found that the metabolome had little difference in the five replicates between the treatment groups, indicating good homogeneity and data reliability between biological replicates. The metabolome had obvious differences between different treatment groups, and was clustered into three clusters in both positive and negative ion modes, indicating that the inoculation of different types of nematodes would trigger a defensive response in Masson pine, leading to changes in metabolites in Masson pine.

[0080] 2.3 Screening of differential metabolites

[0081] Differential metabolites were screened using P value < 0.05 and VIP value > 1. A total of 79 differential metabolites were screened in the comparison group between the pine wood nematode strain (FCBX) and the blank control (CK), of which 52 differential metabolites were up-regulated and 27 differential metabolites were down-regulated. A total of 52 differential metabolites were screened in the comparison group between the pine wood nematode strain (FCBX) and the non-pathogenic pine wood nematode strain (FCBM), of which 24 differential metabolites were up-regulated and 28 differential metabolites were down-regulated. A total of 19 differential metabolites were screened in the comparison group between the pine wood nematode strain (FCBX) and the pathogenic pine wood nematode strain (BM7), of which 7 differential metabolites were up-regulated and 12 differential metabolites were down-regulated. Figure 5(Among them, (A) FCBX intersects with BM7, FCBM, and CK differential metabolites, (B) FCBM intersects with BM7, FCBX, and CK differential metabolites, and (B) BM7 intersects with FCBM, FCBX, and CK differential metabolites, respectively) shows the results of Venn diagram analysis of all differential metabolites in each group screened in positive and negative ion modes. The Venn diagrams of FCBX vs FCBM, FCBX vs BM7, and FCBM vs CK show that there are two common differential metabolites: 9(S)-HPOT and 7-dhc; the Venn diagrams of FCBM vs FCBX, FCBM vs BM7, and FCBM vs CK show that there are two common differential metabolites: 21-deoxycortisol and naringenin chalcone; the Venn diagrams of BM7 vs FCBX, BM7 vs FCBM, and BM7 vs CK show that there are no common differential metabolites.

[0082] 2.4 KEGG enrichment analysis of differential metabolites

[0083] The names of the three groups of identified differential metabolites were imported into MetaboAnalyst4.0, and the Arabidopsis metabolic database in KEGG was selected for metabolic pathway analysis. The top 20 pathway enrichment results are shown in Figure 2. Figure 6 ((A) FCBX vs CK, (B) FCBX vs BM7, (C) FCBX vs FCBM) shows that the bubble size represents the number of metabolites enriched in the pathway. The more metabolites enriched in the pathway, the larger the bubble. The bubble color is related to the P value. The darker the color, the smaller the P value, indicating that the detected differential metabolites have a more significant impact on the pathway. Among the top 20 pathways, the common pathways of FCBX vs CK and FCBX vs BM7 are ABC transporters, cysteine ​​and methionine metabolism, zeatin biosynthesis, and sulfur relay system; the common pathways of FCBX vs CK and FCBX vs FCBM are cysteine ​​and methionine metabolism, alpha-linolenic acid metabolism, and arachidonic acid metabolism.

[0084] 2.5 Preparation of 7-dhc standard curve

[0085] In order to ensure the accuracy of 7-dhc content detection by high performance liquid chromatography, the 7-dhc standard solution was diluted to different concentrations of 1, 5, 10, 20, and 50 L mg / L, and the mass concentration of 7-dhc in the standard solution was used as the abscissa and the corresponding peak area as the ordinate to draw a standard curve, as shown in the figure. Figure 7 As shown, the standard curve was linearly regressed using SPSS25.0 to calculate its linear regression equation and determination coefficient, as shown in Table 1. According to the chart, 7-dhc showed a good linear relationship in the range of 1-50Lmg / L, and the linear determination coefficient was 0.9995, indicating that the fit was very good, which showed that the high performance liquid chromatography was extremely accurate in detecting the content of 7-dhc. Table 1 shows the linear regression equation and determination coefficient

[0086]

[0087] 2.6 Verification of virulence of nematodes with different pathogenicity

[0088] After inoculation with sterile water (CK), virulent pine wood nematode (FCBX), virulent pseudo-pine wood nematode (BM7), and non-toxic pseudo-pine wood nematode (FCBM), it was found that the Masson pine inoculated with FCBX and BM7 showed symptoms of water loss at the top of the shoots, wilting, and discoloration of the needles at 25 days / 30 days, respectively, while the Masson pine inoculated with FCBM and sterile water grew well without any disease symptoms ( Figure 8 , among which (A) CK treatment, (B) FCBX treatment, (C) BM7 treatment, (D) FCBM treatment), so the time period for verifying the metabolomics results is more appropriate to select the 3rd, 7th and 21st days after inoculation with different insect strains, which can be used as the detection time when pine wood nematode disease does not show symptoms in the early stage;

[0089] In summary, Bm7 and FBCM are both pseudo-pine wood nematodes, which belong to different species from the pine wood nematode (FCBX is taken as an example in this article); FCBM does not have pathogenicity, while Bm7 can also cause the death of pine trees and lead to the same external symptoms, that is, it is impossible to determine by appearance or external manifestations (such as existing hyperspectral detection) whether the death of Masson pine is caused by infection with pine wood nematode disease.

[0090] 2.7 Detection of 7-dhc content using high performance liquid spectrometer

[0091] In order to ensure the credibility of the experimental data, this study inoculated the same batch of indoor cultivated Masson pine again, inoculated with three nematodes FCBX, FCBM, BM7 and blank control, and then collected the stems near the inoculation point on the 3rd, 7th and 21st days to extract metabolites, and checked the content of 7-dhc in the samples. The content of 7-dhc in different treatment groups and control groups is shown in Table 2. It was found that the response peak of 7-dhc could only be detected in the samples treated with FCBX nematodes on the 3rd, 7th and 21st days, and the peak was 10.953 minutes, which was basically consistent with the peak time of the standard solution injection. According to the peak area and calculation formula, the content of 7-dhc in the stems was 4.09μg / g, 66.77μg / g and 28.8μg / g after 3 days, 7 days and 21 days of inoculation of Masson pine with pine wood nematode FCBX, respectively. However, the content of 7-dhc could not be detected in the samples treated with BM7 and FCBM nematodes and the blank control samples, indicating that the content of 7-dhc in the stem samples was lower than 0.02 μg / g (the detection limit of the high-performance liquid chromatography in this experiment) or may be zero. It was proved that 7-dhc is a metabolite specifically produced in the early stage after the Masson pine was inoculated with pine wood nematodes, and its specific response is persistent. Table 2 shows the content of 7-dhc in the samples

[0092]

[0093] 3. Discussion

[0094] Based on the extensive application of LC-MS in plant disease detection in the early stage, this experiment used non-targeted metabolomics to analyze the differential metabolites of Masson pine after early infection by different types of nematodes, in order to explore the metabolites that specifically respond to Masson pine only after pine wood nematode infection, and to verify whether these metabolites can be used as early indicators of pine wood nematode infection in Masson pine, and then explore the possibility of early diagnosis of pine wood nematode disease by detecting these metabolites.

[0095] Quality control analysis of the metabolome of Masson pine after inoculation with different types of nematodes and sterile water control revealed that in the PCA graph, QC samples were densely distributed in both positive and negative ion modes, indicating that the metabolome data had good repeatability. The results of quality control analysis showed that metabolome data can be used to accurately analyze differential metabolites, and these metabolites can be used as potential early specific indicators for subsequent verification. In previous studies, spectral technology and physiological indicator determination were used for early detection of pine wood nematode disease, but these detection methods are often affected by temperature changes, light intensity and growth environment, and accurate detection results cannot be obtained. If pine trees are infected with other pests and diseases, it may also affect the appearance and physiological indicators of needles, which will further affect the test results. Studies have found that when many diseases infect plants, they will stimulate the plant's defense response and induce the production of some specific metabolites, and the production of these substances is hardly affected by environmental factors. High performance liquid chromatography is fast and highly sensitive in detecting metabolites. After optimizing the conditions, the detection time can be shortened to 10 minutes at the fastest. Therefore, the use of this technology to detect specific metabolites and use them as a basis for diagnosing plant diseases has rapid and reliable results, which can save a lot of time and money for the prevention, control and treatment of diseases.

[0096] In this experiment, it was found that there were two common differential metabolites in the three comparison groups of FCBX vs FCBM, FCBX vs BM7, and FCBX vs CK, namely 9(S)-HPOT and 7-dhc, indicating that these two substances may be specifically expressed after the Masson pine is inoculated with pine wood nematode FCBX, so they can be used as potential early metabolite indicators after the pine wood nematode infects the Masson pine. Because there is no mature detection method for 9(S)-HPOT and the price is too expensive, it is not suitable for large-scale promotion and verification in the later market, while the 7-dhc standard product is cheap and has a mature detection method, which is suitable for large-scale promotion and detection. Therefore, 7-dhc was selected to verify whether it can be used as an early indicator after the pine wood nematode infects the Masson pine. The HPLC method was used to accurately detect and verify the content of 7-dhc in the metabolites of Masson pine 3, 7, and 21 days after inoculation with pine wood nematodes. It was found that 7-dhc was only detected in the stems of the FCBX nematode treatment group, while it was not detected in the other two nematode treatment groups and the sterile water control. The highly virulent pine wood nematode BM7 will obtain similar pathogenic reactions to those inoculated with pine wood nematodes after inoculation with Masson pine, such as wilting, dehydration, and wilt, but LC-MS detection showed that Masson pine did not produce 7-dhc after inoculation with BM7, which indicates that these two substances may only respond to the invasion of the pine wood nematode species and have nothing to do with whether Masson pine shows a disease response.

[0097] In summary, 7-dhc can be used as an early specific metabolite indicator after pine wood nematodes infect Masson pine. In the future, the experimental process of detecting 7-dhc will be further simplified, and early diagnosis and treatment will be carried out to achieve the goal of saving pine forest resources.

[0098] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. 7-dhc is used as the detection product for the detection method of pine wilt disease in Masson pine, where the pine wilt disease is caused by the highly virulent pine wilt nematode FCBX, comprising the following steps: S1: Sample pretreatment: Quantitatively weigh 1g of Masson pine branch sample and place it in a 250mL flat-bottom flask, then add anhydrous ethanol and potassium hydroxide solution to mix, then use magnetic stirring to heat the electric heating jacket for saponification reflux, and indirectly shake. After the saponification is completed, rinse the inside of the condenser tube with 5mL of anhydrous ethanol from the top, remove the round-bottom flask, cool it to room temperature with cold water, transfer it to a 250mL separatory funnel, rinse the round-bottom flask with 30mL of water, and then rinse the round-bottom flask with 40mL of petroleum ether-anhydrous ether, shake it for 2min, let it stand for stratification, transfer the aqueous phase, combine the organic phases three times, wash with 100mL of water each time until neutral, pass the extract through anhydrous sodium sulfate in a 150mL flat-bottom flask, rotary evaporate to dryness, then make up to volume and pass through a microporous filter membrane before measuring on the machine; S2: Sample injection test: The content of 7-dhc was detected by high performance liquid spectrometry; S3: Content calculation: The 7-dhc content is monitored using the formula, and the calculation formula is: W=(C-C0)*V*N / m The content is calculated and used to determine whether pine wood nematode disease exists; In the content calculation formula of the S3 stage, W is the content of the target in the sample, in mg / L; C is the concentration of the target in the sample test solution, in mg / L; C0 is the concentration of the target in the blank control, in mg / L; V is the fixed volume, in mL; N is the dilution multiple; m is the sample volume, in g; S4: Insect disease determination: Whether the plant is infected with pine wood nematode disease is determined based on the content data calculated in the S3 stage.

2. The method for detecting pine infection with pine wood nematode disease using 7-dhc as a detection product according to claim 1, characterized in that: The anhydrous ethanol in the S1 stage is 30 ml, and the potassium hydroxide solution in the S1 stage is 10 mL of 60% potassium hydroxide solution.

3. The method for detecting pine needle infection with pine wood nematode disease using 7-dhc as a detection product according to claim 1, characterized in that: The temperature of the magnetic stirring heating in the S1 stage is 100° C., and the saponification reflux time in the S1 stage is 1 hour.

4. The method for detecting pine infection with pine wood nematode disease using 7-dhc as a detection product according to claim 1, characterized in that: In the S1 stage, the round-bottom flask is rinsed with 30 mL of water 2-3 times, and the washing liquid of the round-bottom flask in the S1 stage is added to the separating funnel.

5. The method for detecting pine needle infection with pine wood nematode disease using 7-dhc as a detection product according to claim 1, characterized in that: In the S1 stage, the round-bottom flask was rinsed with 40 mL of petroleum ether-anhydrous ether 2-3 times and the mixture was added to the separatory funnel, and the ratio of petroleum ether to anhydrous ether was 1:

1.

6. The method for detecting pine infection with pine wood nematode disease using 7-dhc as a detection product according to claim 1, characterized in that: The S1 stage is dissolved with anhydrous ethanol and fixed to 1 mL, and the microporous filter membrane of the S1 stage is 0.45 μm.

7. The method for detecting pine infection with pine wood nematode disease using 7-dhc as a detection product according to claim 1, characterized in that: The chromatographic column of the S2 stage is SHISEIDOC18, the detector of the S2 stage is DAD, and the detection column temperature is 38°C.

8. The method for detecting pine infection with pine wood nematode disease using 7-dhc as a detection product according to claim 1, characterized in that: The injection volume of the S2 stage was 10 μL, and the wavelength of the S2 stage was 270 nm.

9. The method for detecting pine infection with pine wood nematode disease using 7-dhc as a detection product according to claim 1, characterized in that: The flow rate of the S2 stage was 1.0 mL / min, and the mobile phase of the S2 stage was methanol, with gradient elution.

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