CN108056104B - Poplar compound for inhibiting growth of fall webworm and application thereof - Google Patents
Poplar compound for inhibiting growth of fall webworm and application thereof Download PDFInfo
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Abstract
The invention relates to a poplar compound for inhibiting growth of fall webworms and application thereof, belonging to the technical field of auxiliary breeding of insect-resistant poplar and fall webworm control. According to the invention, through relatively comprehensive analysis of compound differences of insect-resistant and insect-susceptible materials and combination of related analysis, stigmasterol is identified as one of main compounds of poplar resistant to fall webworms, and the stigmasterol has an obvious inhibiting effect on development of fall webworm larvae. The compound can be used as a metabolic marker for the auxiliary selection of breeding of insect-resistant poplar; can also be used as a pesticide component for preventing and controlling the fall webworm, and has important significance for breeding poplar with insect resistance and effectively controlling the fall webworm. The stigmasterol auxiliary selection is effectively combined with the conventional breeding technology, so that the breeding time can be shortened, the breeding speed is accelerated, and the insect-resistant breeding efficiency of the poplar is improved.
Description
Technical Field
The invention relates to a poplar compound for inhibiting growth and development of fall webworms and application of the compound in prevention and control of the fall webworms. In particular to application of stigmasterol in breeding of insect-resistant poplars and prevention and control of fall webworms, belonging to the technical field of auxiliary breeding of insect-resistant poplars and prevention and control of fall webworms.
Technical Field
Populus L is a main tree species for the construction of the protection forest, the farmland forest network and the greening of four sides in the three north of China, is also an important raw material of industrial materials such as paper pulp materials, artificial boards and the like, and plays an important role in economic construction. The cultivation area of poplar in China is wide, but due to the influence of a plurality of factors such as environment, climate and the like, the cultivation of artificial poplar forest faces serious insect damage.
The fall webworm is an important international quarantine pest and has the characteristics of eating impurities, large breeding amount, strong adaptability, wide propagation path and the like. Since the first discovery in Dandong of China in 1979, the traditional Chinese medicine has been delivered to provinces and cities such as Hebei, Tianjin, Shandong and the like, and causes great loss and potential threats to forestry production and ecological safety of China. Zhao Tiezhen et al (2004 national white moth epidemic area non-economic loss assessment [ J ]. forestry economic problem, 2006, 26 (4): 321-. Due to the large-area cultivation of poplar, the fall webworm is one of the important leaf-eating pests harming poplar in China at present.
In the common control measures of the fall webworms, the chemical control has quick effect and high efficiency, but has great environmental pollution and is not beneficial to sustainable development. The biological control has little pollution to the environment, can effectively protect natural enemies and play a role of continuous disaster control, but has slower insecticidal effect, and can not completely achieve the purpose of quickly reducing insect population when used under the condition of high insect population density. The physical prevention is simple and practical, but has the defects of time and labor waste, low efficiency, difficulty in thorough treatment and the like.
The compound is a terminal executor of insect-resistant reaction of plants, and plays a more important role in the insect-resistant process. The plant insect-resistant compound can inhibit the growth and development of insects and even poison insects, thereby reducing the harm of insects to hosts. The insecticidal compound of the plant is utilized for preventing and controlling, and the aims of high insecticidal efficiency on target pests and small influence on non-target pests and environment are expected to be fulfilled.
Currently, as poplar insect-resistant compounds against leaf-eating pests, chlorogenic acid (chlorogenic acid) having an inhibitory effect on growth and development of gypsy moth (Lymantriadispar L.), condensed tannins (condensed tannins) having a resistance to phyllotreta, trillin (tremellin) having an inhibitory effect on feeding of trichosanthes tenuissima (Malacosoma distria), and the like have been reported mainly.
However, no poplar insect-resistant compound against fall webworm has been reported at present. Plant anti-insect compounds effective against different insects may vary due to differences in their physiological and feeding characteristics. In addition, the insect-resistant compounds of plants are various in types and different in structure, and related research needs to be carried out to mine more insect-resistant compounds of poplar.
Therefore, the research on the poplar compound with poisoning or development inhibiting effect on the fall webworm has important significance on the effective control of the fall webworm and the cultivation of the poplar insect-resistant variety.
Disclosure of Invention
One of the objects of the present invention is to provide a poplar compound having a poisoning or growth inhibiting effect on fall webworms, so as to effectively control the occurrence of fall webworms.
The above object of the present invention is achieved by the following technical solutions:
the application of a compound for inhibiting growth and development of fall webworms in preparing products for preventing and treating the fall webworms is disclosed, wherein the compound is stigmasterol.
Preferably, the stigmasterol is used as a main component of a chemical pesticide for chemical control of fall webworm.
The invention also aims to provide a poplar compound with poisoning or development inhibiting effect on fall webworms, which can shorten the breeding time, accelerate the breeding speed and improve the breeding efficiency of poplar insect-resistant breeding when being used for poplar insect-resistant auxiliary breeding.
The above object of the present invention is achieved by the following technical solutions:
the application of a compound for inhibiting the growth and development of fall webworms in the auxiliary breeding of poplar insects-resistant, wherein the compound is stigmasterol.
The compound for inhibiting the growth and development of the fall webworm is identified by the following steps:
(1) insect resistance assay
Firstly, carrying out fall webworm insect resistance determination on a poplar variety, and screening an insect-resistant poplar variety and an insect-susceptible poplar variety;
(2) metabolic analysis
Grinding materials of insect-resistant poplar varieties and insect-susceptible poplar varieties under a freezing condition, respectively extracting total metabolites, drying, performing derivatization, and performing GC-TOF/MS (gas chromatography-time of flight/mass spectrometry) determination on the compounds;
(3) identification of differential Compounds
The method comprises the steps of deconvolution, peak area extraction, mass spectrum comparison and the like, so as to obtain the composition and relative content of compounds in the insect-resistant poplar variety and the insect-susceptible poplar variety, screening differential compounds between the insect-resistant poplar and the insect-susceptible poplar through single-dimensional and multi-dimensional statistical analysis, and carrying out correlation analysis on the relative content of the compounds and the development period of larvae, so as to identify the compound with high correlation with the resistance of fall webworms as stigmasterol in poplar leaves.
After the step (3), the following steps can be added:
(4) validation of differential Compounds
The content of stigmasterol in leaves of 36 # poplar and 50 # poplar of insect-resistant poplar variety, the Zhonglin2025 of insect-sensitive poplar variety and the Nankang poplar is determined by GC/MS and an external standard method, and the metabonomics analysis result is verified, namely the content of the stigmasterol in the insect-resistant material is obviously higher than that of the insect-sensitive material, and the content of the stigmasterol is positively correlated with the development period of larvae.
The number of the poplar varieties in the step (1) is 14, the screened insect-resistant varieties are 36 poplar (Populus delipides cv. '2 KEN 8') and 50 poplar (P.delipides cv. '55/56'), and the screened insect-susceptible varieties are Zhonglin2025 (P.delipides cv. 'Zhonglin 2025') and Nankang poplar (P.delipides cv. 'Nankang').
The method for determining the compound in the step (2) is GC-TOF/MS (Agilent 6890 gas chromatography for GC, and Pegasus III mass spectrometry for LECO in series).
Advantageous effects
The invention identifies that the stigmasterol is one of the main compounds of the resistance of the poplar to the fall webworm by comprehensively analyzing the compound difference of the insect-resistant material and the insect-susceptible material and combining related analysis. The artificial feed addition experiment shows that stigmasterol has obvious inhibition effect on the development of hyphantria cunea larvae. The compound can be used as a metabolic marker for auxiliary selection of insect-resistant poplars, can also be used as a pesticide component for control of fall webworms, and has important significance for insect-resistant breeding of poplars and effective control of fall webworms.
The stigmasterol auxiliary selection is effectively combined with the conventional breeding technology, so that the breeding time can be shortened, the breeding speed is accelerated, and the insect-resistant breeding efficiency of the poplar is improved.
The invention is further illustrated by the following figures and detailed description of the invention, which are not meant to limit the scope of the invention.
Drawings
FIG. 1 is a graph showing the effect of artificial feed supplemented with stigmasterol on the development history of fall webworm larvae.
Detailed Description
Example 1
The identification method of the compound associated with the anti-fall webworm character of the poplar comprises the following steps:
(1) insect resistance assay
The method is characterized in that 14 poplar varieties (shown in the following table 1) widely cultivated in production are planted in the same nursery in a cuttage mode, and the insect resistance of the fall webworms is determined when the seedlings grow to 2 years. The poplar leaves were placed in an ice box, brought back to an insect culture room (25. + -. 1 ℃ C.; 14 hours light), and placed in an insect culture flask (bottom diameter: 12 cm; height: 15 cm). Putting the 2 nd-old primary larva of the fall webworm into a culture bottle with poplar leaves, and putting 30 heads in each bottle. Changing fresh leaves every 3 days, and feeding fall webworm until pupation. During the period, the weight, the development stage, the pupation rate and the death rate of each age of larva of the fall webworm are counted. Each variety was set for 3 replicates. Statistical results show that the death rate of the hyphantria cunea larvae is not obviously different among the varieties to be tested, but the larval development index, the pupation rate and the pupal weight are obviously different (see the following table 2). Judging the resistance degree of the poplar variety to the hyphantria cunea according to the development index of the hyphantria cunea. From these, Populus deltoids cv. '2 KEN 8' and Populus 50 (P.deltoids cv. '55/56') were selected as being more resistant to fall webworm, Zhonglin2025 (P.deltoids cv. 'Zhonglin 2025') and Nankang (P.deltoids cv. 'Nankang').
TABLE 1 Populus varieties for insect resistance testing
| Name of breed | Latin name | Origin of origin |
| 36 # poplar | Populus deltoides cv.‘2KEN8’ | China forestry scientific research institute forestry institute preservation |
| No. 50 poplar | P.deltoides cv.‘55/56’ | China forestry scientific research institute forestry institute preservation |
| All-grass of south Populus | P.deltoides cv.‘Nanyang’ | Cultivation of China forestry scientific research institute |
| Zhonglin2025 | P.deltoides cv.‘Zhonglin 2025’ | Chinese forestCultivation of institute of Industrial science and forestry |
| Red box | P.deltoides cv.‘Danhong’ | Cultivation of China forestry scientific research institute |
| All-red poplar | P.deltoides cv.‘Quanhong’ | Cultivation of scientific research institute of forestry in Henan province |
| South China poplar | Deltoides cv. 'Nankang No. 3' | Cultivation of China forestry scientific research institute |
| European black poplar | Populus nigra L. | China forestry scientific research institute forestry institute preservation |
| 107 Populus tremula | P.×euramericana cv.‘Neva’ | China forestry scientific research institute forestry institute preservation |
| 108 Populus | P.×euramericana cv.‘Guariento’ | China forestry scientific research institute forestry institute preservation |
| Populus olgensis 2012 | P.×euramericana cv.‘Por’ | China forestry scientific research institute forestry institute preservation |
| 109 Populus | P.deltoides×P.alba cv.‘Mincio’ | China forestry scientific research institute forestry institute preservation |
| Sea number 1 | P.deltoides×P.cathayana cv.‘Senhai 1’ | Cultivation of China forestry scientific research institute |
| Sea number 2 | P.deltoides×P.cathayana cv.‘Senhai 2’ | Cultivation of China forestry scientific research institute |
TABLE 2 statistics of development index of fall webworm larvae feeding on different poplar varieties
Note: the capital letters of the shoulder marks in the same column of data are completely different and represent significant differences (P < 0.05)
(2) Metabolic analysis
After 100mg of leaf blades of E.cunningus-sensitized middle forest 2025 (P.deltoids cv. 'Zhonglin 2025') and E.nankang (P.deltoids cv. 'Nankang') were ground in liquid nitrogen, 2mL of extraction mixed solution 1 (methanol: chloroform: water ═ 2.5: 1:1 (v: v: v)) stored at-20 ℃ in advance was added, 10. mu.L of nonadecanoic acid (2mg/mL) and 50. mu.L of ribitol (0.2mg/mL) were added as internal standards of a lipid phase and a methanol/aqueous phase, respectively, and the mixture was shaken on a bed at 220rpm for sufficient mixing for 30min, then 11000g/min for 10min, and the supernatant was transferred to a clean 10mL to obtain a first supernatant; adding 1mL of solution 2 (methanol: chloroform: 1 (v: v)) stored at-20 deg.C into the precipitate, placing on a shaker at 220rpm, mixing thoroughly for 30min, and centrifuging at 11000g/min for 10 min; collecting the second supernatant, mixing with the first supernatant, adding 500 μ L sterilized deionized water, and centrifuging at 5000g/min for 3 min; then, transferring the upper layer methanol/water phase solution and the lower layer chloroform phase solution to a clean centrifugal tube respectively; drying the methanol/water phase solution by using a vacuum freeze dryer, and drying the chloroform phase solution by using nitrogen; placing the dried sample at-80 ℃ for later use, or directly performing derivatization; add 50. mu.L of methoxylamino hydrochloride (20mg/ml) dissolved in pyridine, seal, vortex, centrifuge briefly; bathing at 30 deg.C for 90 min; adding 80 μ L silylation reagent N-methyl-N- (trimethylsilane) -trifluoroacetamide (N-methyl-N- (trimethylsilyl) -trifluoroacetamide, MSTFA), and bathing at 37 deg.C for 30 min; placing the derivatized sample at room temperature for 2h, and performing GC-TOF/MS detection; GC is Agilent6890 gas chromatography, tandem LECO company Pegasus III mass spectrum; the gas chromatographic column was DB-5ms (J & W Scientific, Folsom, CA, USA) with helium as carrier gas at 1.5 ml/min; the chloroform phase adopts a non-split flow mode, the initial column temperature is 80 ℃, the temperature is maintained for 4min, then the temperature is increased to 330 ℃ at the speed of 5 ℃/min, and the temperature is maintained for 5 min; the methanol/water phase heating procedure is that the initial column temperature is 80 ℃, the temperature is maintained for 4min, the temperature is increased to 260 ℃ at the speed of 4 ℃/min, the temperature is maintained for 1min, and then the temperature is increased to 320 ℃ at the speed of 20 ℃/min, and the temperature is maintained for 3 min. A split ratio of 1:10 is adopted; the sample inlet temperature is 250 ℃, the ion source temperature is 200 ℃, the spectrogram is acquired in a delayed mode for 5min, the mass charge m/z detection range is 70-600, 20 spectrograms are scanned every second, the detection voltage is 1700V, and the EI electron bombardment energy is 70 eV.
(3) Screening for differential Compounds
Performing pretreatment such as peak smoothing denoising, baseline correction, peak alignment and the like on mass spectrum data by using ChromaTOF software, and extracting mass spectrum data after deconvolution; submitting the mass spectrum information to a National Institute of Standards and Technology (NIST) metabolite database, comparing the mass spectrum information, identifying metabolites, and obtaining metabolite compositions and relative contents of resistant/sensitive varieties; performing model discrimination analysis on mass spectrum data by using SIMCA-P + software, and screening out different compounds between insect-resistant and insect-susceptible varieties based on a PLS-DA model; statistical analysis is carried out by using SPSS 17.0 software, and the differential compounds are further verified at a single-dimensional statistical level; performing Pearson correlation analysis on the differential metabolite, the development cycle of the larva with the insect-resistant character and the weight of the larva to identify that stigmasterol is one of the compounds with high correlation with the insect-resistant character;
(4) validation of differential Compounds
Absolute quantification is carried out on the stigmasterol content in the leaves of the insect-resistant and insect-susceptible varieties by adopting an external standard method; weighing 1.2mg of the purchased stigmasterol standard substance, adding the weighed stigmasterol standard substance into a 10mL volumetric flask, shaking up and fixing the volume to a scale mark to prepare mother liquor; taking a proper amount of mother liquor, adding ethanol to gradually dilute the mother liquor to 1/10, 1/2, 1, 5 and 10 times of the concentration of the mother liquor, and preparing a stigmasterol gradient solution; drawing a standard curve and measuring the content of the sample by using an Agilent gas mass spectrometer (GC-MS, 6890N-5973N); the gas chromatographic column was DB-5ms (J & WSscientific, Folsom, CA, USA), the carrier gas was helium, and the flow rate was 1.5 mL/min; the split-flow mode of 1:10 is adopted, the column temperature is started to be 80 ℃, the temperature is maintained for 4min, the temperature is increased to 260 ℃ at the speed of 4 ℃/min, the temperature is maintained for 1min, and the temperature is increased to 320 ℃ at the speed of 20 ℃/min, and the temperature is maintained for 3 min. (ii) a The sample inlet temperature is 250 ℃, the ion source temperature is 200 ℃, the spectrogram is acquired in a delayed mode for 5min, the mass charge m/z detection range is 70-600, 20 spectrograms are scanned every second, the detection voltage is 1500V, and the EI electron bombardment energy is 70 eV; carrying out linear regression analysis by using the concentration (C) corresponding to the peak area (A) of the stigmasterol; the result shows that the standard curve is in linear relation in the range of the mass concentration to be inspected (0.0012 mg/mL-1.2 mg/mL), and the correlation coefficient is 0.9992; the method is used for measuring the stigmasterol content in the leaves of the insect-resistant and insect-susceptible varieties; the recovery was calculated as 0.5, 1 and 1.5 times the measured content, respectively, and was between 80.4% and 94.9%. The same samples were subjected to 6 parallel tests under the same conditions, and the results are shown in the following Table 3 for No. 36 Populus delipides cv. '2 KEN 8', No. 50 Populus delipides cv. '55/56', Zhonglin2025 (P. delipides cv. 'Zhonglin 2025'), and Nankan Populus deltoids cv. 'Nankang' in which the mean content of stigmasterol was 0.35mg/g, 0.25mg/g, 0.07mg/g and 0.03mg/g, respectively. The content of the substance in the insect-resistant poplar variety is obviously higher than that of the insect-susceptible poplar variety, the content change trend is consistent with metabonomics, and the substance is positively correlated with the development period of larvae.
TABLE 3 Styrax leaf stigmasterol content and fall webworm resistance index correlation analysis
Note: significant correlation at 0.01 level
Example 2
Application of stigmasterol compound in preparation of product for preventing and treating fall webworm
Ethanol is used as a solvent to prepare stigmasterol solution, and the concentration of the stigmasterol solution is 0.1 time and 1 time of the content of insect-resistant variety No. 36 poplar leaves, namely 35 mu g/ml and 350 mu g/ml. Preparing the artificial feed for the fall webworms, and pouring the artificial feed into the insect breeding cups. Sucking 200 μ L of stigmasterol solution, uniformly coating on the surface of artificial feed, and placing in a ventilated kitchen until ethanol is completely volatilized. Approximately 100 of the first hatched larvae were placed in each insect rearing cup and were reared in an insect rearing room (25. + -. 1 ℃ C.; 14 hours of light). When the insects grow to the early stage of 3 years, randomly reducing the number of the insects in the feeding cup to 30, and subpackaging into 5 bottles after the insects grow to the 4 years. Changing the artificial feed with stigmasterol every 2 days, and feeding the hyphantria cunea until pupation. Each concentration setting was repeated 4 times. During the period, the development period of the hyphantria cunea larva is counted. The results are shown in fig. 1, and the effect of adding artificial feed for feeding stigmasterol of the invention on the development period of fall webworm larvae is shown. Wherein the solvent control is ethanol without stigmasterol, and the treatment 1 is that the stigmasterol addition concentration is 35 μ g/ml; treatment 2 was a stigmasterol addition concentration of 350. mu.g/ml. From the development history of the larvae in the control and different concentration treatments in fig. 1, it can be seen that the difference between the development history of larvae in feeding treatment 2 (supplemented with stigmasterol at a concentration of 350 μ g/ml) and feeding solvent control reached a very significant level (. about. sup. sup.0.01).
The 1-fold concentration of stigmasterol (350 mug/ml stigmasterol) can be added to obviously prolong the development period of the larva. The larval development history was extended by 5.2 days at 1-fold addition compared to the solvent control.
Example 3
Application of stigmasterol in auxiliary breeding of insect-resistant poplar
In the breeding process of the poplar insect-resistant variety, in order to ensure the effectiveness and accuracy of the test, the insect-resistant test is usually carried out on the basis of trees for more than 1 year, and at least 1 insect generation needs to be observed in the test process, so that the period is long, and time and labor are wasted. The content of the compound in the plant is determined by genotype, and the research finds that the stigmasterol content in the poplar leaves is positively correlated with the resistance of the fall webworms through metabonomics analysis, correlation analysis and insect resistance verification, so that the stigmasterol can be used as a metabolic marker to predict the resistance level of the poplar to the fall webworms.
The implementation process of using stigmasterol to carry out poplar insect-resistant auxiliary selection comprises the following steps: detecting the content of stigmasterol in leaves of the bred individuals by a chromatograph or a mass spectrometer, and comparing the content of the stigmasterol in different individuals to further infer the relative resistance level of the stigmasterol to the fall webworms; the stigmasterol is used as a metabolic marker for auxiliary selection of poplar insect-resistant breeding, so that early selection can be allowed, and the manpower, material resources and financial resources wasted in the cultivation process are saved; the implementation of early selection can also allow more populations to be included in the objects of research selection, thus realizing wider and more intense selection; in addition, the evaluation of insect resistance of individuals is usually destructive and affects later growth and development of individuals; the stigmasterol is used for auxiliary selection, only a small amount of leaves are needed, and the individual can continue to grow, so that a breeding worker can select other properties of the breeding population at the same time; therefore, the stigmasterol auxiliary selection is effectively combined with the conventional breeding technology, so that the breeding time can be shortened, the breeding speed is accelerated, and the insect-resistant breeding efficiency of the poplar is improved.
The invention identifies that the stigmasterol is one of the main compounds of the resistance of the poplar to the fall webworm by comprehensively analyzing the compound difference of the insect-resistant material and the insect-susceptible material and combining related analysis. The artificial feed addition experiment shows that stigmasterol has obvious inhibition effect on the development of hyphantria cunea larvae. The compound can be used as a metabolic marker for auxiliary selection of insect-resistant poplars, can also be used as a pesticide component for control of fall webworms, and has important significance for insect-resistant breeding of poplars and effective control of fall webworms.
Claims (2)
1. The application of stigmasterol in preparing products for preventing and treating fall webworm; the stigmasterol is used as a main component of a chemical pesticide and is used for chemical control of fall webworms; ethanol is used as a solvent to prepare the stigmasterol solution with the concentration of 350 mu g/ml.
2. The application of stigmasterol in assistant breeding of anti-fall webworm poplar is characterized in that: the stigmasterol is used as a metabolic marker in auxiliary breeding of anti-fall webworm poplar.
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