Breeding method of leaf miner parasitic wasps and method for biological control by using leaf miner parasitic wasps

Abstract

The invention belongs to the field of plant protection and biological control, and particularly discloses a method for raising leaf miner parasitic wasps and a method for biological control by using the leaf miner parasitic wasps. Clematis orientalis is used as a host plant to feed clematis orientalis leaf miners, and then the clematis orientalis leaf miners living in the host plant are used as hosts of parasitic wasps of the leaf miners. According to the method, biological control can be particularly carried out in a greenhouse, the fed clematis orientalis leaf miners are used as host plants to feed the clematis orientalis leaf miners, then the whole hosts of the clematis orientalis leaf miners living in the host plants as leaf miner parasitic wasps are placed in the greenhouse, and the fed parasitic wasps do not need to be additionally collected to be independently released; therefore, the parasitic wasps can be fed and released at the same time in the release stage, the leaf miner can be continuously prevented and controlled for a long time, and practical value is achieved.

Description

Technical Field

[0001] The invention belongs to the field of plant protection and biological control, and particularly relates to a method for raising leafminer parasitic wasps and a method for using the method for biological control thereof. Background Art

[0002] Leaf miners (Agromyzidae) are important agricultural and forestry pests that cause serious damage worldwide. Their larvae bore into plant leaves to form white tunnels, which seriously damage photosynthesis, cause leaf shedding and even plant death, and cause significant economic losses. They have a strong reproductive capacity, and their eggs, larvae, pupae, and adults often coexist in the same crop. The temporal and spatial distribution of each insect stage in leaf tissue, soil, leaf surface, and plant canopy is highly heterogeneous, and populations overlap in both developmental stages and ecological niches, greatly increasing the difficulty of integrated prevention and control techniques. Leaf miners are also a serious threat in my country, with more than 130 species recorded, of which 6 are important pests, including the pea leafminer ( Phytomyza horticola )、Lycophora spp. Liriomyza chinensis ), Liriomyza sativae ( L. sativae ), Liriomyza huidobrensis ( L. huidobrensis ), Liriomyza truncatula ( L. trifolii ) and Liriomyza sativae ( L. bryoniae ). With its wide host (can harm more than 300 species of plants such as Leguminosae and Solanaceae), strong reproductive capacity (single female lays 300-600 eggs), short generation cycle (12.0-15.0 days in the immature stage) and rapid development of drug resistance, it poses a continuous threat to my country's vegetable industry. Traditional prevention and control methods (chemical pesticides, physical trapping, etc.) have the disadvantages of high cost, heavy pollution and unstable prevention effect, and it is urgent to develop green prevention and control technology.

[0003] Biological control has become a research hotspot due to its advantages such as environmental friendliness and long-lasting effectiveness. International experience shows that parasitic wasps are effective in controlling leaf miners: Siberian Braconid wasp ( Dacnusa sibirica ) achieves early control of greenhouse tomato leafminers by overwintering in the pupal stage; pea leafminer Diplocera fasciatus ( Diglyphus isaea ) in Malatya Province, Türkiye, had a parasitism rate of up to 48.55% against leaf miners; Neochrysocharis formosa ) was confirmed as the dominant natural enemy of the clover leafminer in Argentina. However, related research in my country is lagging behind, and the natural enemy species used on a large scale are scarce, which restricts the promotion of biological control technology. Summary of the invention

[0004] This study innovatively constructed the “Oriental Clematis ( Clematis orientalis ) - Clematis orientalis leafminer ( Phytomyza vitalbae)-leafminer parasitoid "vector plant system, revealing the interaction mechanism and pest control potential of system components through multidisciplinary methods. First, based on field surveys in the northwest region of China, oligophagous leafminers ( P.vitalbae )(referred to as Clematis orientalis leafminers) and their parasitoid communities were found on Clematis orientalis. The Clematis orientalis leafminers specifically feed on plants of the Ranunculaceae family and do not harm economic crops, providing an ideal alternative host for natural enemy breeding. Through systematic surveys in 9 cities of 5 provinces in China and combined with morphological identification techniques, the species diversity of parasitoids on Clematis leafminers was clarified. A total of 19 species of parasitoids were identified, belonging to 4 families such as Eulophidae and Braconidae. Among them, Neochrysocharis formosa ( N. formosa ) was the dominant species (accounting for 26.79%), and its occurrence dominance showed significant geographical heterogeneity. The parasitoid diversity was the highest in Jiuquan City (15 species), followed by Urumqi City and Kizilsu Kirgiz Autonomous Prefecture (6 - 7 species).

[0005] Aiming at the problem of large-scale propagation of vector plants, the propagation technology of Clematis orientalis was systematically optimized. Cuttage experiments showed that the rooting rate of cuttings was increased to 62.50% (525.00% higher than the control) after treatment with 1000 mg / L auxin (IBA), and the rooting time was shortened to 15.00 d; the substrate of peat:perlite:vermiculite (8:1:1) significantly improved the survival rate (92.50%) and biomass accumulation (vine length 102.73 cm, 117.73 pairs of leaves). In the study of seed germination, mechanical shell-breaking combined with 20 mg / L gibberellin (GA3) treatment increased the germination rate to 78.33% (276.00% higher than the control), and the germination time was shortened to 14.08 d. The above parameters provided key technical support for industrialized seedling cultivation.

[0006] Under a controlled environment (25±1 °C, RH 40 - 50%, photoperiod L:D = 14:10), the life history characteristics of Clematis leafminers on vector plants were analyzed through a two-sex life table. It was found that the immature development duration was 20.31 d, the male and female adult lifespans were 18.32 d and 9.90 d respectively, the fecundity per female was 197.32 eggs, and there was a strong oviposition preference for the back of the leaves (the oviposition amount on the back was 6.03 times that on the front). Population parameters showed that its intrinsic rate of increase ( r = 0.1495) and net reproductive rate ( R o = 85.16) had strong expansion potential, verifying the feasibility of vector plants to maintain pest populations.

[0007] Focusing on the pest control efficacy of the arrhenotokous strain of Neochrysocharis formosa, it was found that it achieved efficient pest control through triple effects of oviposition parasitism (3.94 individuals per day on average), feeding to death (11.20 individuals), and direct killing (1.45 individuals), and the total lethal amount per female reached 283.69 individuals. The immature stage of this strain was 15.31 d, the adult lifespan was 17.10 d, and the finite rate of increase of the population ( λ = 1.1892) was significantly higher than that of the host. Age-stage specific analysis showed that the peak period of pest control efficacy was at 29 days old, providing a theoretical basis for the selection of the timing of field release.

[0008] The present invention provides a method for raising leafminer parasitoids, using Clematis orientalis as the host plant to raise the leafminer of Clematis orientalis, and then using the leafminer of Clematis orientalis living in the host plant as the host of the leafminer parasitoid.

[0009] Specifically, the leafminer parasitoid is selected from Dacnusa sibirica ( Dacnusa sibirica ), Diglyphus isaea ( Diglyphus isaea ), and Neochrysocharis formosa ( Neochrysocharis formosa ).

[0010] In a specific embodiment, the Clematis orientalis is obtained by artificial propagation, and the specific method is as follows: S1. The cuttings are selected from the current-year tender branch cuttings with strong growth potential, plump buds, being robust and free from diseases and pests, and the apical buds and old branches at the roots are discarded; they are cut flat at 0.8 - 1.2 cm above the node and obliquely cut at 40 - 50 degrees at 7 - 10 cm below the node. Each cutting has one node, one side branch of the node is retained, and 1 - 3 leaves are left at the top; S2. The cuttings are soaked in a potassium permanganate solution with a mass percentage of 4 - 6% for 5 - 15 min, and then thoroughly rinsed with water to remove the residual solution; S3. The cuttings are treated with 800 - 1200 mg / L auxin to ensure that the soaking depth of the cuttings is maintained at 4 - 8 cm, the soaking time is 5 - 15 s, and after taking them out, they are immersed in clean water and maintained until rooting; S4. When the cuttings germinate new buds and the root system develops to ≥2 and the average length is ≥1 cm, they are inserted into the substrate seedling raising containing peat: perlite: vermiculite with a volume ratio of 8:1:1; Preferably, an appropriate amount of water is poured into the substrate to ensure that the humidity around the cuttings is maintained above 85%. As the cuttings gradually grow and stabilize, to prevent the cuttings from rotting, the amount of sprayed water should be appropriately reduced; 8 - 12 days after cutting, the seedling pots are transferred to a greenhouse with a temperature controlled at 25 ± 1 °C, the daylight lamp illumination duration is set to 14 h, and the dark time is 10 h; and periodic prevention and control are carried out after cutting, and 600 - fold solution of 50% carbendazim is sprayed every 7.0 d for disinfection.

[0011] In another embodiment, the Clematis orientalis is obtained by artificial seed production and cultivation. The specific method is as follows: S1. Put the mature Clematis seeds into sandpaper for polishing to obtain seeds with a thinner seed coat. The polishing degree is that the seed coat becomes thinner but the seeds are not damaged. Soak the seeds after seed coat treatment in a gibberellin GA3 solution with a concentration of 15 - 25 mg / L for 0.5 - 1.5 h and then sow them; S2. The sowing depth is 1.0 - 1.5 cm. When sowing, water the sowing substrate thoroughly. After sowing, put the seedling plug tray into the greenhouse. The temperature in the greenhouse is maintained at 25 ± 1 °C, and the humidity is maintained between 80 - 100%. Determine the watering amount every day according to the dry and wet degree of the substrate surface. The sowing substrate is peat:perlite:vermiculite with a volume ratio of 8:1:1, and the pH value is between 5.5 - 5.6. When the roots of the seedlings to be planted can hold most of the medium, transplant the seedlings into a large seedling pot and cultivate them in the greenhouse. Adopt the watering method of watering when it is dry and not watering when it is wet, and apply water-soluble fertilizer appropriately; The water-soluble fertilizer is a 3000-fold water-soluble compound fertilizer with a nitrogen - phosphorus - potassium ratio of 20 - 20 - 20. The fertilization frequency is once every two weeks. Spray it onto the leaf surface with a sprayer. At the same time, set up a climbing support frame, lead and wind the vines germinated from the Clematis seedlings around the climbing support frame. During the cultivation period, apply the slow-release fertilizer of the special fertilizer for promoting seedling growth and root growth of Aoli A5 appropriately. When fertilizing the Clematis in the medium-sized seedling pot, apply 50 slow-release fertilizers per pot and place them around the roots, avoiding direct placement at the root base.

[0012] After moving to a suitable place, when raising Neochrysocharis formosa, first construct a parthenogenetic population of Neochrysocharis formosa. Put the larvae at the end of the second instar to the early third instar of Liriomyza huidobrensis on Clematis orientalis together with the Clematis leaves or plants into an insect rearing cage, and then introduce the parthenogenetic and female-producing strain females of the parasitoid of Liriomyza huidobrensis for rearing until the parasitoid can be released and then release it.

[0013] Among them, according to the research results, the average daily number of eggs laid and killed by the parthenogenetic strain of Neochrysocharis formosa on the larvae at the end of the second instar to the early third instar of Liriomyza huidobrensis on Clematis orientalis is 3.94, the number of killed by feeding is 11.20, and the direct number of killed is 1.45. The total number of killed reaches 16.59. The peak total number of killed is about 28. Therefore, preferably, the amount of the introduced parasitoid of Liriomyza huidobrensis is in the ratio of parasitoid:Liriomyza huidobrensis on Clematis orientalis of 16 - 28:1.

[0014] The present invention particularly provides a biological control method for Liriomyza. Release the parasitoid of Liriomyza obtained by the method among plants to control Liriomyza; Specifically, it is released when it is predicted that leafminers are about to occur on the crops or in the early stage of leafminer occurrence. And Neochrysocharis formosa is normally released at the 3rd to 5th day of the adult stage, reaching the peak of lethality, and the effect of releasing Neochrysocharis formosa at this time is the best.

[0015] Preferably, the control is carried out in a greenhouse. Clematis orientalis is reared as a host plant to rear the leafminer of Clematis orientalis, and then the whole of the leafminer of Clematis orientalis living in the host plant as the host of the leafminer parasitoid is placed in the greenhouse.

[0016] Preferably, the whole is covered by a gauze net, and the leafminer parasitoid can freely enter and exit through the mesh size of the gauze net. The whole includes a device (such as a cultivation pot) for cultivating the host plant Clematis orientalis, on which the leafminer of Clematis orientalis is inoculated. When the larvae reach the end of the 2nd instar to the early stage of the 3rd instar, the leafminer parasitoid is inoculated again. During the control period, the leafminer parasitoid can freely leave the gauze net covering, and at the same time, the host plant Clematis orientalis is maintained to grow to continuously provide the reared leafminer parasitoid.

[0017] The innovation of this study lies in: (1) constructing a carrier plant system by using the three-level trophic relationship of Clematis orientalis - leafminer of Clematis orientalis - parasitoid to avoid the risk of pest spread; (2) analyzing the community structure of parasitoids and the biological characteristics of dominant species, and clarifying that Neochrysocharis formosa is the core pest control factor; (3) integrating plant propagation, pest-natural enemy interaction and population regulation technologies to form a chain ecological regulation model of "carrier conservation - natural enemy supply - target control". This system provides a new paradigm for the green control of leafminers and is of great significance for promoting the sustainable development of agriculture.

[0018] In terms of practical application, the construction of this carrier plant system provides a new way for the green control of leafminers. The leafminer of Clematis orientalis in the system does not harm economic crops, and even if it flies to the field or greenhouse, it will not harm the crops, so the control risk is reduced. Therefore, the whole of the host plant, the leafminer of Clematis orientalis and the parasitoid can be placed in the control area, such as in a greenhouse (which also avoids the problems of the difficulty of collecting and releasing the reared parasitoid alone and the duration problem in the control area). In this way, due to the long growth cycle of Clematis orientalis, long-term continuous control of leafminers on crops can be achieved. The parasitoid can freely enter and exit the system, making it a "reserve warehouse" for parasitoids and enhancing the biological control effect on leafminers. This not only helps to reduce the use of chemical pesticides, reduce environmental pollution, ensure the quality and safety of agricultural products, but also promotes the wide application of biological control technologies in agricultural production and promotes the sustainable development of agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings of the specification, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0020] Figure 1 The population growth trend and age structure change of Neochrysocharis formosa on Phytomyza clematidis. Specific embodiments

[0021] For the instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, they are all conventional instruments, reagents, materials, etc. existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, they are all conventional experimental methods, detection methods, etc. existing in the prior art.

[0022] Example 1: Study on the species of parasitoid wasps of Phytomyza clematidis orientalis During the period from June 2016 to October 2024, a total of 3,483 parasitoid wasps of leafminers were successfully emerged and collected from the samples of Clematis orientalis collected in 6 cities / states (Jiuquan City, Urumqi City, Kashgar City, Kizilsu Kirgiz Autonomous Prefecture) under the jurisdiction of 5 provinces in China. According to the occurrence quantity of parasitoid wasps in each region from high to low, they are: Jiuquan City (3,305 heads), Urumqi City (101 heads), Kizilsu Kirgiz Autonomous Prefecture (68 heads), and Kashgar Region (9 heads).

[0023] Through morphological identification, a total of 19 species of parasitoid wasps were collected and identified on the randomly collected Clematis samples in 4 prefecture-level urban areas. These parasitoid wasps belong to the families Eulophidae, Braconidae, Encyrtidae, and Scelionidae. Among the sampling regions, the species of parasitoid wasps found on Clematis orientalis in Jiuquan City are the most abundant, up to 15 species, including common species such as Neochrysocharis formosa, Diglyphus isaea, and Neochrysocharoides formosalis; followed by Clematis orientalis in Kizilsu Kirgiz Autonomous Prefecture and Urumqi City, on which 7 species and 6 species of parasitoid wasps were found respectively; only 3 species of parasitoid wasps were found on Clematis orientalis in Kashgar Region.

[0024] Among the Eulophidae, Braconidae, Encyrtidae and Scelionidae found in the investigation, Eulophidae was the absolutely dominant family, with the occurrence quantity accounting for 97.82% of the total occurrence quantity. Scelionidae ranked second, accounting for 1.55%. The proportions of Encyrtidae and Braconidae were relatively small, being 0.34% and 0.29% respectively. Further subdividing each family, among the dominant family Eulophidae, Sympiesis soriceicornis had the most significant occurrence dominance, with the occurrence quantity accounting for 27.44% of the total occurrence quantity of Eulophidae. Among Braconidae, Dacnusa sibirica was the dominant species, with the occurrence quantity accounting for 70.00% of the total occurrence quantity of Braconidae. Among Encyrtidae, Aprostocetus atratus was the dominant species, accounting for 83.33%. Only one parasitoid, Aprostocetus atratus, was found in Scelionidae.

[0025] Example 2: Large-scale propagation of the carrier plant Clematis orientalis

[0026] 1. Materials and methods Unless otherwise specified, the test materials used are conventional experimental materials in this field and can be obtained through commercial channels.

[0027] The test base is located at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. The test was carried out in the greenhouse of the Key Laboratory for the Prevention and Control of Alien Invasive Organisms, Institute of Plant Protection, Chinese Academy of Agricultural Sciences. During the test, the air temperature was 25 ± 1 °C, and the relative humidity was maintained between 80 - 100%.

[0028] 1) Cutting seedling propagation technology

[0029] The test material was Clematis orientalis growing naturally in Jingqiao Village, Dunhuang City, Jiuquan City, Gansu Province ( C. orientalis), the cuttings selected are the tender branch cuttings of the current year with strong growth potential, plump axillary buds, robustness, and no pests and diseases. Discard the apical buds and old branches at the roots and send them back to the laboratory for standby. Considering that the material collection process takes a long time, to avoid excessive water loss of the cuttings, after receiving the samples, the cut cuttings will be immediately placed in a container filled with clean water for moisture preservation treatment for subsequent experiments. Use a sharp pair of scissors to make a horizontal cut at 1 cm above the node at a 90-degree angle and a 45-degree oblique cut at 7 - 10 cm below the node to increase the contact area with the soil. Each cutting has one node, one side branch is retained at the node, and two leaves are left at the top. Before the cuttings are used, soak them in a potassium permanganate solution with a mass percentage of 5% for 10 minutes, and then thoroughly rinse them with tap water to remove the residual solution. According to the requirements of the experimental design, prepare auxin (IBA) solutions with different concentration gradients. Put the treated samples into auxin (IBA) solutions with different concentrations for treatment, ensuring that the immersion depth of the samples is kept at about 6 cm, and the immersion time is 10 s. After taking them out, immerse them in clean water and maintain this treatment until rooting. When the cuttings germinate new buds and the root system develops to ≥2 (average length ≥1 cm), transplant them into seedling pots filled with different substrates.

[0030] 2) Seed production and seedling propagation techniques

[0031] The experimental materials were collected from the mature Clematis seeds in Jingqiao Village, Dunhuang City, Jiuquan City, Gansu Province. Remove impurities at room temperature and screen out shriveled seeds by water selection method. The remaining seeds are put into a self-sealing bag and stored in a 4 °C refrigerator for standby.

[0032] 3) Auxin (IBA) screening experiment

[0033] In this experiment, treatment groups of auxin (IBA) solutions with different concentration gradients were set, and the treatment with clean water was used as the blank control. During the treatment process, the experimental materials were soaked in auxin (IBA) solutions of each concentration for 10 s. Three biological replicates were set for each treatment group, and each replicate contained 40 cuttings. Observe and record the germination dynamics of the radicles at the base of the cuttings at a fixed time (10:00) every day, record the specific time when the radicles first break through the epidermis, and use the radicle protrusion length ≥2 mm as the rooting judgment criterion.

[0034] Table 1. Treatment of auxin (IBA) concentration and soaking time

[0035] 4) Substrate screening experiment The experiment adopted a single-factor design, fixed the perlite-vermiculite ratio at 1:1 to control variable interaction, focused on different volume ratios of peat treatment, and the dominant effects on substrate water retention, aeration, and structural stability, so as to optimize the configuration of functional components, and set river sand as the control group. Three biological replicates were set for each treatment group, and each replicate contained 40 cuttings for rooting and germination. The cuttings were selected and treated by soaking in an auxin (IBA) solution for 10 s, then soaked in a water cup filled with clean water. When the cuttings germinated new buds and the root system developed to ≥2 (average length ≥1 cm), they were inserted into the pre-prepared small transparent seedling pots. The small transparent seedling pots had a square top opening with a side length of 7 cm, a square bottom with a side length of 5.5 cm, a height of 10 cm, and hydrophobic and breathable holes at the bottom. The required cutting depth was 6 cm. The whole process was kept moist. The survival rate, vine length, and number of leaves were measured in the same year.

[0036] Table 2. Treatments of different substrates and their ratios

[0037] 5) Gibberellin (GA3) concentration screening experiment The experiment was carried out with the following different concentrations of gibberellin (GA3), and clean water was used as the control group. The treatment time of the gibberellin (GA3) solution was 1 h. Three replicate groups were set under each treatment condition, and 40 seeds were used for independent testing in each group. The germination status of the seeds was monitored at 10:00 every day, and the criterion for determining germination was when the radicle grew to a clearly visible state.

[0038] Table 3. Treatments of gibberellin (GA3) concentration and soaking time

[0039] 6) Synergistic experiment of mechanical treatment of seed coat and gibberellin (GA3) The experiment selected mature, plump, healthy and dry Clematis seeds with uniform size, and treated the seed coats of the experimental Clematis seeds. Three biological replicates were set for each treatment, and each replicate sample contained 40 seeds. Specifically, the seed coats were put into sandpaper for grinding to obtain seeds with thinner seed coats, and the grinding degree was that the seed coats became thinner without damaging the seeds. The seeds after seed coat treatment were soaked in a 20 mg / L gibberellin (GA3) solution for 1 h; the germination status of the seeds was monitored at 10:00 every day, and the time point when the radicle first broke through the seed coat was recorded, and the criterion for determining germination was when the radicle grew to a clearly visible state.

[0040] Table 4. Combination of mechanical treatment of seed coat and gibberellin (GA3)

[0041] 7) Cutting and post-cutting management Before cutting, the medium was watered thoroughly with a potassium permanganate solution with a mass percentage of 5% for sterilization. After the sterilization operation, to prevent bacteria from growing due to excessive humidity in the substrate and affecting the subsequent test results, it was placed in a well-ventilated environment and dried for 1.0 d. One day before cutting, the substrate was mixed according to the ratio. Before cutting, to prevent damage to the newly grown roots of the cuttings, the cuttings were first placed horizontally into a small transparent seedling-raising pot with a square top opening side length of 7.0 cm, a square bottom side length of 5.5 cm, a height of 10.0 cm, and hydrophobic and breathable holes at the bottom using the direct insertion method. Then, the thoroughly stirred substrate was filled into the small transparent seedling-raising pot and gently pressed to compact the substrate. Finally, they were neatly placed in a tray with a length of 56.0 cm, a width of 38.0 cm, and a height of 8.0 cm and watered thoroughly. After cutting, the tray was placed in a wire mesh cage with a length of 60.0 cm, a width of 40.0 cm, and a height of 180.0 cm in the greenhouse, and watering operations were carried out daily with a watering can. The amount of water sprayed was adjusted flexibly according to the wilting condition of the leaves of the Clematis orientalis cuttings. At the same time, an appropriate amount of water was also poured into the substrate to ensure that the humidity around the cuttings was maintained at about 90%. In the first few days of the initial cutting stage, since the cuttings had a large demand for water, the amount of water sprayed was relatively large. As the cuttings gradually grew and stabilized, to prevent the branches and leaves of the cuttings from rotting, the amount of water sprayed should be appropriately reduced. After 10.0 d of cutting, the seedling-raising pots were transferred to a greenhouse with a temperature controlled at 25 ± 1 °C, and the lighting duration of the fluorescent lamp was set to 14 h and the dark time to 10 h. Periodic prevention and control were carried out after cutting. Every 7.0 d, a 600-fold solution of 50% carbendazim was sprayed for disinfection to prevent the occurrence of brown spot disease and anthracnose. Diseased plants and rotten plants were promptly removed every day to prevent the spread of diseases. When the roots in the small transparent seedling-raising pot could hold most of the medium, they could be transplanted into a large seedling-raising pot with a bottom side length of 13.0 cm, a top side length of 17.0 cm, and a height of 15.5 cm, and the special fertilizer for promoting seedling growth and root growth of Osmocote A5 was added for conventional seedling management.

[0042] 8) Seed production and seedling breeding management The seeds after the above-mentioned experimental treatments were sown into small transparent seedling pots containing sowing substrate, with the depth ranging from 1.0 to 1.5 cm, and numbered. The small transparent seedling pot has a square top opening with a side length of 7.0 cm, a square bottom with a side length of 5.5 cm, and a height of 10.0 cm. There are hydrophobic and breathable holes at the bottom. The sowing depth is 1.0 - 1.5 cm. When sowing, the sowing substrate needs to be watered thoroughly. After sowing, the seedling tray is placed in a greenhouse. The temperature in the greenhouse is maintained at 25 ± 1 °C, and the humidity is maintained between 80 - 100%. The amount of watering is determined daily according to the dry and wet degree of the substrate surface. The sowing substrate is peat:perlite:vermiculite with a volume ratio of 8:1:1, and the pH value is between 5.5 - 5.6. When the roots of the seedlings to be planted can hold most of the medium, the seedlings are transplanted into large seedling pots and cultivated in the greenhouse. The watering method of seeing dry and watering is adopted, and water-soluble fertilizer is applied appropriately. The medium-sized seedling pot has a square top opening with a side length of 17.0 cm, a square bottom with a side length of 13.0 cm, and a height of 15.5 cm. There are hydrophobic and breathable holes at the bottom. When the seedlings are transplanted into the medium-sized seedling pot, the planting depth of the seedlings is such that the cultivation substrate just covers the new roots. The cultivation substrate in the medium-sized seedling pot is peat:perlite:vermiculite with a volume ratio of 8:1:1. The water-soluble fertilizer is a 3000-fold water-soluble compound fertilizer with a nitrogen-phosphorus-potassium ratio of 20-20-20, and the fertilization frequency is once every 2 weeks. It can be sprayed onto the leaf surface with a sprayer. At the same time, a climbing support frame is set up, and the emerging vines of the clematis seedlings are led and wound around the climbing support frame. During the cultivation period, a special slow-release fertilizer for promoting seedling growth and increasing roots, OSMOCOTE A5, is applied appropriately. When applying fertilizer to the clematis in the medium-sized seedling pot, 50 slow-release fertilizers are applied to each pot and placed around the roots, avoiding direct placement at the root base.

[0043] 2. Experimental Results

[0044] 1) The effects of indole-3-butyric acid (IBA) concentration on the rooting of Clematis orientalis are shown in Table 5. Exogenous IBA treatment significantly affected the rooting efficiency of C. orientalis cuttings. In the control group without IBA application, the rooting rate of cuttings was only 10%, and the average rooting time was as long as 36.0 d, indicating significant regeneration obstacles in this species under natural conditions. 1000 mg / L IBA was the optimal treatment concentration. When the IBA concentration was 1000 mg / L, the rooting rate of cuttings reached 62.50%, and the average rooting time was shortened to 15.0 d, which were 6.25 times and 2.40 times that of the control group, respectively (Table 5). Compared with other concentrations, the 1000 mg / L treatment group showed the best performance in both rooting time and rooting rate: compared with the 500 mg / L group, the rooting time was shortened by 1.21 times (from 20.0 d to 15.0 d), and the rooting rate increased by 109.00% (from 30.00% to 62.50%); compared with the 1500 mg / L group, the rooting time was shortened by 1.40 times (from 21.0 d to 15.0 d), and the rooting rate increased by 208.00% (from 20.00% to 62.50%); compared with the 2000 mg / L group, the rooting time was shortened by 1.75 times (from 26.0 d to 15.0 d), and the rooting rate increased by 291.00% (from 15.00% to 62.50%).

[0045] In terms of rooting time, the 1000 mg / L group was significantly better than other concentration groups (P < 0.05). In terms of rooting rate, the 1000 mg / L group was significantly higher than the 1500 mg / L, 2000 mg / L and control groups (P < 0.05), but there was no significant difference compared with the 500 mg / L group (P > 0.05). It should be noted that there was no significant difference in the rooting rate between the 2000 mg / L group (15%) and the control group (10%) (P > 0.05), indicating that high-concentration IBA may have an inhibitory effect. The promoting effect of 1000 mg / L IBA may be related to its effective regulation of the endogenous hormone balance of cuttings. Appropriate concentration of IBA can induce the expression of rooting-related genes and promote the differentiation of vascular tissues. While high concentrations (≥1500 mg / L) may lead to rooting inhibition by inhibiting cell division.

[0046] Table 5. Comparison of rooting time and rooting rate of Clematis orientalis under different IBA concentrations (mean ± standard deviation)

[0047] Note: If there are no same lowercase letters after the data in the same column, it indicates that there is a significant difference between the two at the P < 0.05 level. 2) Effects of substrates on the survival of Clematis orientalis cuttings during rooting The volume ratios of peat: perlite: vermiculite are 10:1:1, 8:1:1, 5:1:1, 3:1:1, 1:1:1 respectively, and six substrates including river sand are represented by substrates of type A, B, C, D, E, and F. As can be seen from (Table 6), under the same greenhouse and water management conditions, substrate B (peat: perlite: vermiculite = 8:1:1) shows the best performance in terms of cutting survival rate (92.50 %), vine length (102.73 cm), and number of leaf pairs (117.73 pairs), which are 1.06 times, 1.10 times, and 1.26 times that of substrate A respectively, 1.26 times, 1.24 times, and 1.33 times that of substrate C, 1.42 times, 1.30 times, and 1.39 times that of substrate D, 1.54 times, 1.35 times, and 1.41 times that of substrate E, and 4.63 times, 6.47 times, and 4.32 times that of substrate F (river sand), significantly higher than those of substrates C, D, E, and F. This result indicates that the water and fertilizer retention capacity and air permeability of substrate B reach the best balance. The relatively high peat content (8 parts) provides sufficient organic matter and mineral nutrients for the cuttings, while the reasonable ratio of perlite to vermiculite (1:1) ensures the loose structure of the substrate, promoting aerobic respiration of roots and water absorption, indicating that the substrate ratio significantly affects the cutting survival rate, vine length, and number of leaf pairs.

[0048] The number of leaf pairs of substrates D (3:1:1) and E (1:1:1) are 84.54 pairs and 83.50 pairs respectively (Table 6), and there is no significant statistical difference between them (P > 0.05). This phenomenon may be related to the insufficient nutrient supply caused by the decrease in peat proportion. When the peat proportion is lower than 3 parts, the water holding capacity and nutrient release rate of the substrate decrease significantly, restricting the differentiation and growth of leaves. In addition, the excessive proportion of perlite and vermiculite in substrate E (1:1:1) may lead to too large pores in the substrate, weakening the root fixing ability and indirectly affecting the development of the above-ground part.

[0049] The cutting survival rate (20.00 %), vine length (15.88 cm), and number of leaf pairs (27.25 pairs) of substrate F are significantly lower than those of other treatment groups (Table 6). Although river sand has good air permeability, it lacks organic matter and has poor water retention capacity, resulting in the inability of the cuttings' roots to obtain sufficient water and nutrients, ultimately inhibiting vegetative growth.

[0050] This study clarified that the substrate ratio of peat: perlite: vermiculite = 8:1:1 is the optimal scheme for the cutting of Clematis orientalis. By optimizing the physical structure and nutrient supply of the substrate, it significantly promoted the vegetative growth of the cuttings. The equivalence of substrates D and E indicates that when the peat proportion is lower than 3 parts, other measures (such as adding slow-release fertilizers) are needed to supplement nutrients.

[0051] Table 6. Effects of cutting substrates on the survival rate, vine length, and number of leaves of Clematis orientalis cuttings (mean ± standard deviation)

[0052] Note: The volume ratios of peat: perlite: vermiculite are 10:1:1 (A); 8:1:1 (B); 5:1:1 (C); 3:1:1 (D); 1:1:1 (E); CK river sand (F) in sequence. If there are no same lowercase letters after the data in the same column, it indicates that there are significant statistical differences between the two at the P < 0.05 level. 3) Effect of gibberellin (GA3) concentration on seed germination of Clematis orientalis As shown in Table 7, exogenous GA3 treatment significantly shortened the germination time of Clematis orientalis seeds and increased the germination rate. In the control group without GA3 application, the average germination time of seeds was 27.09 d, and the germination rate was only 27.50%, indicating that this species has strong physiological dormancy characteristics. When the GA3 concentration was 20 mg / L, the seed germination time was shortened to 17.90 d, and the germination rate reached 72.50%, which were 66.00% and 264.00% of the control group respectively. Compared with other concentrations, the 20 mg / L group showed significant advantages: compared with the 10 mg / L group, the germination time was shortened by 1.06 times (from 18.96 d to 17.90 d), and the germination rate was increased by 116.00% (from 65.00% to 72.50%); compared with the 40 mg / L group, the germination time was shortened by 1.44 times (from 20.33 d to 17.90 d), and the germination rate was increased by 138.00% (from 52.50% to 72.50%); compared with the 80 mg / L group, the germination time was shortened by 1.32 times (from 23.67 d to 17.90 d), and the germination rate was increased by 164.00% (from 44.17% to 72.50%).

[0053] Analyzed from the perspective of germination time, the 20 mg / L group was significantly superior to other concentration groups (P < 0.05). The promoting effect of 20 mg / L GA3 may be related to its effective regulation of the expression of seed dormancy-related genes. GA3 promotes endosperm degradation and embryo elongation by activating the gibberellin signaling pathway. In terms of germination rate, the 20 mg / L group was significantly higher than the 10 mg / L, 40 mg / L, 80 mg / L and control groups (P < 0.05), but there was no statistical difference between the 40 mg / L and 80 mg / L groups (P > 0.05). The equivalence between the 40 mg / L and 80 mg / L groups may be due to the saturation effect of GA3 receptors.

[0054] This study shows that 20 mg / L GA3 is the optimal treatment concentration for breaking the dormancy of Clematis orientalis seeds, and it significantly shortens the germination time and increases the germination rate by regulating the hormone balance. The equivalence of the 40 mg / L and 80 mg / L groups suggests that over-reliance on high-concentration hormones should be avoided in production practice.

[0055] Table 7. Comparison of the germination time and germination rate of Clematis orientalis seeds with different concentrations of gibberellin (GA3) (mean ± standard deviation)

[0056] Note: If there are no same lowercase letters after the data in the same column, it indicates that there is a statistically significant difference between the two at the P < 0.05 level. 4) Effects of the synergistic action of mechanical treatment of the seed coat and gibberellin (GA3) on the germination of Clematis orientalis seeds As shown in Table 8, the synergistic action of mechanical treatment of the seed coat and gibberellin (GA3) significantly improved the germination efficiency of Clematis orientalis seeds. Treatment group d (seed polishing + GA3) showed the best performance, with an average germination time of 14.08 d and a germination rate of 78.33%, which were 52.00% and 376.00% of the control group (group a, non-polished + water), respectively.

[0057] The germination time of treatment group d was shortened by 1.92 times compared with group a (from 27.03 d to 14.08 d), 1.44 times compared with group b (polished + water) (from 20.31 d to 14.08 d), and 1.27 times compared with group c (non-polished + GA3) (from 17.90 d to 14.08 d). There were significant differences between treatment group d and the other three groups (P < 0.05), indicating that the synergistic action of seed coat polishing and GA3 effectively broke the physical and physiological double barriers of seed dormancy. Mechanical treatment of the seed coat promoted water absorption by destroying the seed coat structure, while GA3 might accelerate endosperm degradation by activating the activity of hydrolytic enzymes.

[0058] The germination rate of treatment group d was increased by 2.76 times compared with group a (from 28.33% to 78.33%), 1.84 times compared with group b (from 42.50% to 78.33%), and 1.08 times compared with group c (from 72.50% to 78.33%). Group d was significantly better than group a and group b (P < 0.05), but there was no statistical difference with group c (P > 0.05). This indicates that GA3 treatment alone (group c) could partially relieve physiological dormancy but did not completely overcome the mechanical limitation of the seed coat; while polishing treatment (group b) improved physical permeability, but the germination rate was limited due to the lack of hormone regulation. There was no significant difference between group a and group b (P > 0.05), indicating that simple mechanical polishing could not replace the role of hormone regulation.

[0059] The synergistic effect of treatment group d verified the synergistic mechanism of physical shell breaking and hormone regulation. Seed shell polishing increases seed coat permeability, making it easier for GA3 to penetrate into the embryo, thereby activating downstream signaling pathways. The equivalence of germination rates between groups c and d (78.33% and 37.78%) suggests that in the absence of physical shell breaking, GA3 may partially compensate for the seed shell barrier through the concentration gradient effect, but the effect is limited.

[0060] This study confirmed that the synergistic application of seed husk mechanical treatment and GA3 is an effective method to break the dormancy of Clematis orientalis seeds. Treatment group d (polishing + GA3) significantly shortened the germination time and increased the germination rate through a dual mechanism (physical husk breaking and hormone regulation), providing a theoretical basis for the large-scale seedling cultivation of this species.

[0061] Table 8. Effects of seed husk mechanical treatment and gibberellin (GA3) on the germination process and germination rate of Clematis orientalis seeds (mean ± SD)

[0062] Note: Unpolished seeds + clean water (a); polished seeds + clean water (b); unpolished seeds + gibberellin (GA3) (c); polished seeds + gibberellin (GA3) (d); If there is no same lowercase letter after the data in the same column, it means that there is a statistically significant difference between the two at the P<0.05 level Example 3: Clematis plantminer has a high reproductive potential on Clematis orientalis

[0063] 1. Materials and methods

[0064] 1) Overview of the trial site The experiment was conducted at Yunzhiduo Inn in Dunhuang, Jiuquan City, Gansu Province, in a constant temperature room built with air conditioning. During the entire test period, the indoor environmental parameters were managed: the air temperature was kept stable at 25±1 ℃; the relative humidity was maintained in the range of 40-50%; the photoperiod was set to 14 hours of light and 10 hours of darkness.

[0065] 2) Test plants

[0066] Throughout the study period, the experimental materials were taken from Clematis orientalis growing naturally in Jingqiao Village, Dunhuang City, Jiuquan City, Gansu Province. C. orientalis. To ensure that the number of leaves can meet the test requirements, samples were collected every 2.0 days. The selection criteria for cuttings are young branches that are grown in the current year, have vigorous growth and are strong, and the terminal buds and old branches at the roots are discarded. In view of the long time taken to collect materials, in order to prevent the cuttings from losing too much water and affecting the subsequent test results, the cut cuttings will be immediately placed in a bucket filled with clean water for moisturizing. In the cutting processing stage, use sharp scissors to make a 90-degree flat cut 1 cm above the node and a 45-degree oblique cut 10-15 cm below the node. This treatment method can increase the contact area between the cuttings and clean water. Only one node is retained for each cutting, and fresh and tender leaves without pests and diseases are selected for retention, and the retained leaves are thoroughly rinsed. The processed cuttings will be placed in a plastic transparent water cup with a lid of a specific specification for cultivation in clean water for standby use. The height of the water cup is 10.7 cm, the upper diameter is 9.8 cm, and the lower diameter is 5.6 cm. The cup cover has two specifications, one with a 1 cm hole and one with 5 1 cm holes. All seedlings cultured in clean water must undergo an observation period of at least one week. During this period, the seedlings are closely observed for diseases and insect pests to reduce experimental errors. Only seedlings cultured in clean water that are confirmed to be free of diseases and insect pests and have good growth conditions can be used for subsequent experimental research.

[0067] 3) Insect source for testing

[0068] A special fluke tube was used to capture the Clematis plant leafminer. The specific structure of the fluke tube is: two 1 mL gun tips are connected at both ends, one end is used as the entrance for collecting insects, and the other end is for the experimenter to hold in the mouth during the operation. The gun tips at both ends are connected by a yellow soft rubber tube to ensure the flexibility and operability of the fluke tube. It is worth noting that a gauze net is specially set at the connection between the two gun tips at the front end to prevent the insect body from escaping and foreign objects from entering. The host plant of the Clematis plant leafminer captured this time is Oriental Clematis. The captured Clematis plant leafminer was transferred to an insect-raising gauze cage (specifications are 20 cm×20 cm×20 cm, mesh size is 100 mesh), and the Oriental Clematis seedlings were cultured in clean water to carry out life activities such as egg laying and development. At the same time, in order to prolong the survival time of leaf miners and improve their egg-laying efficiency, white absorbent cotton pieces were hung inside the insect cages, and 20% concentration of Sophora japonica honey water was sprayed on the cotton pieces at a fixed period. Through continuous multi-generation cultivation, an indoor population was established to provide a stable insect source for subsequent indoor experiments.

[0069] 4) Test methods

[0070] The hydroponic seedlings of Clematis orientalis to be tested were randomly placed in a gauze cage with 100 meshes and a specification of 20 cm×20 cm×20 cm. The adults that emerged for 24 h were released in the middle of the plants to allow them to feed and lay eggs naturally for 5 h, and this time point was taken as the starting point of egg development. After that, the tested plants were taken out and observed under a Japanese Olympus SZX-16 stereomicroscope, and the egg-laying positions on the leaves were marked with a marker pen. The hatching of eggs was observed regularly at 8:00 and 20:00 every day. After the eggs hatched, the larvae with the same hatching time were selected, and the label numbers were pasted and recorded. To ensure the accuracy of the test results and minimize experimental errors, during the test process, only 1 larva was retained on each leaf, and the remaining larvae were removed in a timely manner. Thereafter, continuous observation was carried out on the remaining larvae, and their development process and survival status were recorded in detail. When the larvae grew and developed to the third instar stage, a gauze with one end open was tied to the base of the plant to collect the pupae after the larvae pupated. The pupae were placed one by one into a 3.5 cm diameter plastic Petri dish lined with filter paper, and an appropriate amount of water was dropped onto the filter paper every day (just enough so that it did not flow) to maintain the humidity. The emergence dynamics of the pupae were closely monitored, and the sex of the emerged adults was recorded in detail. After the adults emerged, the female and male adults were selected and paired separately. The successfully paired adults were placed into plastic transparent boxes with dimensions of 15.0 cm in length, 10.0 cm in width, and 8.0 cm in height. There was an opening in the middle of the box lid (8.0 cm in length and 5.0 cm in width), and a 100-mesh gauze was covered with hot melt glue. A cotton ball containing a 20% honey solution was placed in the box as a nutritional supplement for the adults. More than 4 leaves of the hydroponic seedlings of Clematis orientalis in 1.5 ml plastic tubes with holes were replaced every day (the leaves retained a 3.0 - 5.0 cm stem and were taken from the tested plants described in 4.1.2). During the test period, the number of eggs laid by the adults and their survival status were observed and recorded in detail every day, and this operation continued until the female insects died. To ensure the reliability of the test data, during the entire test process, if male insects died or escaped, new male insects were immediately added to ensure that the female insects had sufficient mating opportunities. In addition, the number of female insects in each test treatment group was not less than 20 heads.

[0071] 5) Data processing

[0072] This study was based on the theory of the two-sex life table and adopted the data processing method proposed by Qi Xin et al. (Chi et al ., 2020; Chi et al ., 2022). The TWOSEX-MSChart program was used to statistically analyze the growth and development data of Phytomyza clematidis on Clematis orientalis. Through this program, key biological parameters such as the developmental duration, survival rate, adult lifespan, and daily egg production of Phytomyza clematidis were analyzed, and a specific age-stage survival rate (s xj ), age-specific survival rate ( l x ), age-stage fecundity ( f xj ), population age-specific fecundity ( m x ), age-stage life expectancy ( e xj ), and age-stage fertility ( v xj ). In addition, this study further analyzed the population dynamic parameters of *Phytomyza clematidina*, including the intrinsic rate of increase ( r ), finite rate of increase ( λ ), net reproductive rate ( R 0 ), and mean generation time ( T ). The calculation formulas for each parameter are as follows: Age-specific survival rate ( l x ):

[0073] Population age-specific fecundity ( m x ):

[0074] Intrinsic rate of increase ( r ):

[0075] Finite rate of increase ( λ ):

[0076] Net reproductive rate ( R 0):

[0077] Mean generation time ( T ):

[0078] In this study, SPSS 26.0 software was used to analyze the life history parameters of *Phytomyza clematidina* on *Clematis orientalis*, such as development time, oviposition period, and daily oviposition amount. Meanwhile, the Bootstrap method (resampling 100,000 times) was used to calculate the mean and standard error of population parameters, and the paired Bootstrap test was used to analyze the intrinsic rate of increase ( r ), finite rate of increase ( λ ), net reproductive rate ( R 0), mean generation time ( T ).

[0079] 2. Results

[0080] 1) Immature developmental duration of Phytomyza clematidis on Clematis orientalis

[0081] The developmental durations of each instar of Phytomyza clematidis feeding on Clematis orientalis are shown in Table 9. The research results show that Phytomyza clematidis can successfully complete the entire development process from egg to adult on Clematis orientalis plants. Specifically, the average egg stage is 3.02 d; the total larval stage is 6.02 d, and the developmental times of the first, second, and third instar larvae are 2.04 d, 1.97 d, and 2.01 d, respectively; the average pupal stage is 11.30 d; the pre-adult stage is 20.31 d. These data indicate that the developmental duration of Phytomyza clematidis on Clematis orientalis shows obvious stage characteristics, and the pupal stage accounts for the main part of the entire development cycle, which may be related to the physiological transformation and reproductive preparation required during the pupal stage. In addition, the larval stage is relatively short, reflecting the rapid growth and development characteristics of Phytomyza clematidis during the feeding stage.

[0082] Table 9 Developmental durations of Phytomyza clematidis on Clematis orientalis (mean ± standard error)

[0083] 2) Adult lifespan and fecundity of Phytomyza clematidis on Clematis orientalis On Clematis orientalis plants, the lifespan of female Phytomyza clematidis is 18.32 d, the lifespan of male is 9.90 d, the pre-oviposition period is 2.53 d, the total pre-oviposition period is 22.83 d, the oviposition days are 5.00 d, and the average number of eggs laid is 197.32.

[0084] The average lifespan of female Phytomyza clematidis on Clematis orientalis plants is 18.32 d, significantly longer than that of male (9.90 d). The pre-oviposition period of female is 2.53 d, and the total pre-oviposition period is 22.83 d. The oviposition days are 5.00 d, and the average number of eggs laid is 197.32. This result reveals the reproductive characteristics of Phytomyza clematidis on Clematis orientalis.

[0085] 3) Oviposition selectivity of Phytomyza clematidis on different parts of Clematis orientalis leaves

[0086] The leafminer Phytomyza clematidina lays eggs on both the upper and lower surfaces of the leaves of Clematis orientalis, and shows selective differences between the upper and lower surfaces during the egg-laying process. Phytomyza clematidina prefers to lay eggs on the lower surface of the leaves, and the number of eggs laid on the lower surface is 6.03 times that on the upper surface (the number of eggs laid on the upper surface is 28.05±12.96, and that on the lower surface is 169.26±57.82). The results of this study reveal that the environment on the lower surface may be more conducive to the hatching and early growth and development of its larvae. This finding provides a basis for further research on the egg-laying behavior and ecological adaptability of Phytomyza clematidina.

[0087] 4) Population growth trend and instar structure changes of Phytomyza clematidina on Clematis orientalis

[0088] In this study, by setting an initial population of 10 eggs, the population dynamics of Phytomyza clematidina were simulated and predicted within 60.0 days. The results showed that the population of Phytomyza clematidina showed a significant growth trend. By the 60.0th day, the total number of insects reached 27324.06. Among them, the numbers of eggs, larvae, pupae and adults were 11157.51, 12989.36, 2318.02 and 429.74 respectively, specifically including 6100.40 first-instar larvae, 4206.61 second-instar larvae, 2682.35 third-instar larvae, 296.17 female adults and 133.26 male adults. It is worth noting that on the 42.0th day, the second and third generations of eggs of Phytomyza clematidina appeared simultaneously, indicating that there is a generation overlap in its population reproduction, which may be the result of the combined action of differences in individual development rates and environmental suitability.

[0089] From the perspective of population structure changes, the numbers of eggs and larvae are dominant, accounting for 40.83% and 47.54% of the total number of insects respectively, while the proportions of pupae and adults are relatively low, 8.48% and 1.57% respectively. This distribution characteristic reflects the biological characteristics of the Phytomyza clematidina population with eggs and larvae as the main developmental stages. The significant increase in the numbers of eggs and larvae may be closely related to the rich nutritional resources provided by the leaves of Clematis orientalis and the suitable environmental conditions (such as temperature and humidity). In addition, the number of adults is relatively small, but they play a key reproductive role in population growth. Especially, female adults directly promote the growth of the population by laying eggs. Generally speaking, the population of Phytomyza clematidina shows an exponential growth trend within 60.0 days, indicating that it has high reproductive potential and ecological adaptability on Clematis orientalis.

[0090] The research results revealed the dynamic characteristics of the population development of Phytomyza clematidina, as well as its high reproductive potential and ecological adaptability on Clematis orientalis, providing an important basis for further understanding its reproductive ecological characteristics, population dynamics, and life history strategies. Meanwhile, the research results provided important theoretical guidance and practical basis for subsequent establishment of a banker plant system with Phytomyza clematidina as an alternative food to control leafminer pests.

[0091] The research results showed that Phytomyza clematidina could successfully complete its entire development process on Clematis orientalis. The developmental durations of the egg stage, larval stage, pupal stage, and pre-adult stage were 3.02 d, 6.02 d, 11.30 d, and 20.31 d, respectively. The lifespan of female insects was significantly longer than that of male insects, with an average of 18.32 d. Moreover, female insects showed a high oviposition ability during the reproductive period, with an average oviposition amount of 197.32 eggs. In addition, the oviposition behavior of Phytomyza clematidina on the leaves of Clematis orientalis showed significant preference, and it was more inclined to lay eggs on the back of the leaves. The analysis of population dynamic parameters showed that Phytomyza clematidina had a high reproductive potential on Clematis orientalis, with an intrinsic rate of increase of 0.1495, a finite rate of increase of 1.161, and a net reproductive rate of 85.16, indicating that the leafminer population showed an exponential growth trend under suitable conditions.

[0092] Example 4: Life history and pest control potential of thelytokous strain of Neochrysocharis formosa on Phytomyza clematidina.

[0093] 1. Materials and Methods

[0094] 1) General situation of the test site

[0095] Same as before.

[0096] 2) Test insect sources

[0097] The thelytokous Neochrysocharis formosa used in this study was collected from the vegetable planting base of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences in Beijing. The test insect sources used in the research were all from population individuals reared for 22 generations or more. The whole rearing process was continuously carried out in succession with the "Phaseolus lunatus - Liriomyza sativae larvae at the end of the 2nd instar to the beginning of the 3rd instar system" to ensure population reproduction and the supply of test insects.

[0098] 3) Host insects

[0099] Same as before.

[0100] 4) Test methods

[0101] Developmental duration of the immature stage of thelytokous Neochrysocharis formosa

[0102] In this experiment, larvae at the end of the second instar to the early third instar (about 80 larvae) of the host insects in 5.1.3 were selected and placed in an insect rearing cage together with Clematis leaves. Subsequently, 5 female wasps of the parthenogenetic female-producing strain were introduced and numbered, and this time point was taken as the starting point of egg development. The parasitoid wasps were removed 3 h after introduction, and the parasitized larvae were numbered and marked 72 h later. To control experimental errors, only 1 larva was retained on each leaf, and the rest of the larvae were removed. Since the egg stage of Neochrysocharis formosa (endoparasitoid) is difficult to observe, the egg stage was included in the larval stage for statistics and analysis. There were no less than 20 female wasps in each treatment.

[0103] Lifespan and three host treatment behaviors of Neochrysocharis formosa under parthenogenetic host feeding conditions

[0104] Thirty newly emerged active and healthy parthenogenetic Neochrysocharis formosa were selected and introduced into transparent plastic boxes with specifications of 15 cm (length) × 10 cm (width) × 8 cm (height) respectively, and numbered. An opening of 8 cm × 5 cm was made in the center of the box lid, and a 100-mesh gauze was fixed with hot melt glue to ensure ventilation. A cotton ball soaked with 20% honey solution was placed in the box as a nutritional supplement for the adults. The leaves of Clematis hydroponic seedlings with host larvae at the end of the second instar to the beginning of the third instar (35 - 40 larvae) were replaced daily. The leaves were inserted into a perforated 1.5 mL plastic tube (retaining a 3.0 - 5.0 cm stem segment) until the parasitoid wasps died, and the death time was recorded. After the parasitoid wasps died, they were transferred to an alcohol tube for storage according to the number.

[0105] 72 h after inoculation, the research used a microscope to observe three behaviors of the wasps causing host death, and recorded the number of host deaths corresponding to each behavior. The behaviors that caused host death were mainly divided into the following three categories: (1) Parasitic lethality: There were parasitoid larvae or eggs in the host larvae, and the host showed the characteristic of "rigid death"; (2) Feeding lethality: The parasitoid wasps directly fed on the blood and tissue fluid of the host larvae and caused death; (3) Direct lethality: The host larvae died with a rigid body, accompanied by black spots left by the ovipositor puncture of the parasitoid wasps on the body surface, and the body was full, without parasitoid eggs or larvae inside.

[0106] 2. Experimental results

[0107] 1) Developmental duration of the immature stage of the parthenogenetic strain of Neochrysocharis formosa

[0108] The research results on the developmental duration data of each stage of the immature period of thelytokous line of Neochrysocharis formosa feeding on Phytomyza clematidina showed that the larval period of the thelytokous line of Neochrysocharis formosa was 5.57 d, the prepupal period was 0.63 d, the pupal period was 9.14 d, and the total length of the immature period was 15.31 d. Among them, the pupal period accounted for the main part of the immature period, which might be related to the morphological and physiological transformation of Neochrysocharis formosa from larva to adult during the pupal period. The prepupal period was short, reflecting the rapidity of its transition from larva to pupa. The larval period was relatively long, indicating that Neochrysocharis formosa needed a longer time to accumulate sufficient nutritional reserves during the feeding and growth stages in the host body. This result revealed the developmental characteristics of the thelytokous line of Neochrysocharis formosa in the immature period, providing an important basis for the study of its population dynamics and biological control applications.

[0109] 2) Life history parameters of adults of the thelytokous line of Neochrysocharis formosa

[0110] The research results on the life history parameters of adults of the thelytokous line of Neochrysocharis formosa feeding on Phytomyza clematidina showed that the average survival days of the thelytokous line of Neochrysocharis formosa on the larvae of Phytomyza clematidina from the end of the 2nd instar to the beginning of the 3rd instar were 17.10 d. During its survival period, an average of 67.45 host larvae were killed by oviposition, 191.48 by feeding, and 24.76 directly by a single female wasp, and the total number of killed hosts reached 283.69. Among them, the number of hosts killed by feeding was significantly higher than those killed by oviposition and directly, indicating that Neochrysocharis formosa mainly controlled the host population through feeding behavior. This result revealed the high lethality of the thelytokous line of Neochrysocharis formosa to Phytomyza clematidina in the adult stage, especially the key role played by its feeding behavior in the process of pest control.

[0111] 3) Average number of hosts killed by adults per day

[0112] The research results on the average number of hosts killed by adults per day of the thelytokous line of Neochrysocharis formosa feeding on Phytomyza clematidina showed that the average number of hosts killed by oviposition per day of the thelytokous line of Neochrysocharis formosa on the larvae of Phytomyza clematidina from the end of the 2nd instar to the beginning of the 3rd instar was 3.94, the number killed by feeding was 11.20, and the number killed directly was 1.45, and the total number of killed hosts reached 16.59. Among them, the number of hosts killed by feeding was significantly higher than those killed by oviposition and directly.

[0113] 4) Daily age dynamics of host control behavior of adults

[0114] The lethality of the parthenogenetic strain of the clematis plantminer showed significant age dependence, with the egg-laying lethality, feeding lethality, direct lethality and total lethality all showing a trend of first increasing and then decreasing. Specifically, the egg-laying lethality reached a peak at 25 days of age (6.58 heads) and then gradually decreased. This change may be related to the increase in egg-laying of the clematis wasp during the peak reproductive period, followed by a decrease in egg-laying ability due to the consumption of reproductive resources and physiological aging. The feeding lethality reached a maximum value at 29 days of age (18.35 heads) and then gradually decreased. This may be related to the fact that the feeding activity of the clematis wasp was most active in the middle adult period, and the feeding ability was weakened in the later period due to physical decline. The direct lethality reached a peak at 20 days of age (2.84 heads), and then fluctuated between 21-45 days of age (0-1.86 heads). This fluctuation may reflect the difference in aggressiveness of the clematis wasp under different physiological states. The total lethality reached a maximum value (6.58 heads) at 29 days of age, and then gradually decreased. This trend comprehensively reflects the changes in the lethality of the Fuxinji wasp at different developmental stages. Overall, the lethality of the Fuxinji wasp reached its peak in the middle adult stage, and then gradually weakened due to the decline of physiological functions. This change pattern is closely related to its physiological state and energy allocation strategy during its life cycle.

[0115] 5) Population life table analysis

[0116] Population growth trend and age structure changes of Fuxin parthenogenetic fly on Clematis oleracea Figure 1 As shown. In this study, by setting the initial population to 10 eggs, the population dynamics of Fuxin parthenogenetics within 60.0 days were simulated and predicted. The results showed that the population of Fuxin parthenogenetics showed a significant growth trend. By the 60.0 day, the total number of insects reached 88394.34. Among them, the number of egg-larvae, prepupae, pupae and female adults was 63135.73, 3326.83, 16831.74 and 5100.04, respectively. It is worth noting that on the 26.0 day, the second and third generation eggs of Fuxin parthenogenetics appeared at the same time, indicating that its population reproduction showed a generation overlap phenomenon, which may be the result of the combined effect of differences in individual development rates and the suitability of environmental conditions.

[0117] From the perspective of population structure changes, the number of individuals in the egg-larval stage dominated, accounting for 71.42% of the total number of insects, indicating that the population of the Fuxinji wasp mainly develops in the egg and larva stages. The number of pre-pupae and pupae accounted for 3.76% and 19.04% of the total number of insects, respectively, reflecting the stage characteristics of the Fuxinji wasp in the development process. The number of female adults accounted for 5.77% of the total number of insects. Although the proportion is relatively low, it plays a key reproductive role in population growth, especially directly promoting the growth of population through egg laying.

[0118] The results of the population dynamic parameters of Fuxin parthenogenesis on Phytomyza clematidina are shown in Table 10. Its maximum instantaneous growth rate is 0.1732, indicating that this strain has a high population growth potential when parasitizing and killing host larvae. After each female wasp goes through one generation (with an average of 22.7230 d), the net increase in the number of offspring is 51.7637, further reflecting the strong reproductive ability of Fuxin parthenogenesis under parasitic conditions. In addition, the population number increases by 1.1892 times every 1.0 d, indicating that its population shows a significant exponential growth trend. These parameters comprehensively show that Fuxin parthenogenesis exhibits efficient population expansion ability when parasitizing the larvae of Phytomyza clematidina, which may be closely related to its full utilization of host resources and strong environmental adaptability.

[0119] Table 10. Population dynamic parameters of Fuxin parthenogenesis on Phytomyza clematidina (mean ± standard error)

[0120] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for raising a leafminer parasitic wasp, characterized in that: Oriental clematis leafminer is raised with oriental clematis as a host plant, and the oriental clematis leafminer living in the host plant is used as the host of the leafminer parasitic wasp.

2. The feeding method according to claim 1, characterized in that The leafminer parasitoid wasp is selected from **Dacnusa sibirica** ( Dacnusa sibirica ), **Diglyphus isaea** ( Diglyphus isaea ), and **Neochrysocharis formosa** ( Neochrysocharis formosa ).

3. The feeding method according to claim 1, characterized in that, The oriental clematis is obtained by artificial propagation, and the specific method is as follows: S1, select the tender branches of the current year with vigorous growth, full buds, robustness, and no diseases and insect pests, and discard the terminal buds and old branches at the roots; cut flatly at 0.8-1.2 cm above the node, and cut obliquely at 40-50 degrees at 7-10 cm below the node, leaving one node on each cutting, retaining a branch on one side of the node, and leaving 1-3 leaves on the top; The S2 cuttings were immersed in a 4-6% potassium permanganate solution for 5-15 min, and then rinsed thoroughly with water to remove the residual solution; S3: Use 800-1200 mg / L auxin to treat the cuttings, ensure that the cuttings are immersed at a depth of 4-8 cm for 5-15 seconds, and then soak them in clean water until they take root; When S4 cuttings sprouted new shoots and developed ≥2 roots with an average length of ≥1 cm, they were planted in a substrate seedling culture containing peat: perlite: vermiculite in a volume ratio of 8:1:1; Preferably, water the substrate in an appropriate amount to ensure that the humidity around the cuttings is maintained at more than 85%. As the cuttings gradually grow and stabilize, the amount of water sprayed should be appropriately reduced to prevent the branches and leaves of the cuttings from rotting. After 8-12 days of cutting, the seedling pots are transferred to a greenhouse with a temperature controlled at 25±1°C, and the fluorescent light duration is set to 14 h and the dark time is 10 h. Periodic prevention and control is carried out after cutting, and 600 times diluted 50% carbendazim is sprayed for disinfection every 7.0 d.

4. The feeding method according to claim 1, characterized in that, The oriental clematis is obtained by artificial seed production and cultivation, and the specific method is as follows: S1. Mature Clematis seeds are put into sandpaper for grinding to obtain seeds with thinner seed shells. The grinding degree is such that the seed shells become thinner but the seeds are not damaged. The seeds after seed shell treatment are soaked in a 15-25 mg / L gibberellin GA3 solution for 0.5-1.5 seconds and then sown. The sowing depth of S2 is 1.0-1.5 cm. The sowing substrate is irrigated with water during sowing. After sowing, the seedling tray is placed in a greenhouse. The temperature of the greenhouse is maintained at 25±1℃, and the humidity is maintained between 80-100%. The amount of watering is determined every day according to the dryness and wetness of the substrate surface. The sowing substrate is peat: perlite: vermiculite with a volume ratio of 8:1:1, and the pH value is 5.5-5.

6. When the roots of the planted seedlings can cover most of the medium, the seedlings are moved into large seedling pots and cultured in the greenhouse. The watering method is used when the soil is dry, and water-soluble fertilizers are applied in appropriate amounts. The water-soluble fertilizer is a 3,000-fold water-soluble compound fertilizer with a nitrogen-phosphorus-potassium ratio of 20-20-20. The fertilization frequency is once every two weeks, and it can be sprayed onto the leaf surface with a sprayer. At the same time, a climbing support frame is set up, and the vines germinated from the Clematis seedlings are led and wound around the climbing support frame. During the cultivation period, a slow-release fertilizer, namely, the special fertilizer for promoting seedling growth and root growth of Osmocote A5, is applied appropriately. When fertilizing the Clematis in the medium-sized seedling pots, 50 slow-release fertilizer granules are applied to each pot and placed around the roots, avoiding direct placement at the root base.

5. The feeding method according to claim 1, characterized in that, When rearing Neochrysocharis formosa, first construct a parthenogenetic population of Neochrysocharis formosa. The larvae at the late second instar to the early third instar of Liriomyza clematidis, together with the Clematis leaves or plants, are placed in an insect rearing cage, and then a parthenogenetic and female-producing strain female wasp of the leaf miner parasitoid is introduced for rearing (preferably, the ratio of parasitoid to leaf miner is 16-28:1), and released until the parasitoid can be released.

6. A biological control method for leafminers, characterized in that, Release the leaf miner parasitoid obtained by the method according to any one of claims 1 to 5 in the plants that need biological control to control the leaf miner.

7. The biological control method for leafminers according to claim 6, characterized in that, Release when Neochrysocharis formosa parthenogenetic females reach the adult stage at 3 to 5 days old.

8. The biological control method according to claim 7, wherein Release at the time when leaf miners are about to occur or in the early stage of leaf miner occurrence on the predicted crops.

9. The biological control method according to claim 6, wherein The control is carried out in a greenhouse. The Clematis orientalis is reared as a host plant for Liriomyza clematidis, and then the whole, with Liriomyza clematidis living in the host plant as the host of the leaf miner parasitoid, is placed in the greenhouse.

10. The biological control method according to claim 9, characterized in that, The whole is covered by a gauze net, and the free entry and exit of the leaf miner parasitoid are controlled by the mesh size of the gauze net.

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