Method for releasing Trichogramma with soybean seedlings as carrier and soybean aphid honeydew as supplementary nutrition

Abstract

The present invention discloses an optimized method for artificially releasing Trichogramma with soybean seedlings as the carrier and soybean aphid honeydew as supplementary nutrition, comprising the following steps: (1) planting and cultivating potted soybeans; (2) inoculating soybean aphids and accumulating honeydew; (3) fixing and releasing Trichogramma. The present invention uses living soybean seedlings as the carrier and the honeydew produced by soybean aphids as the supplementary nutrition source for releasing Trichogramma, which can significantly improve the parasitism rate of Trichogramma. Within a certain period of time after release, the plants and aphids can continue to grow and reproduce, continuously providing nutritional supplements for Trichogramma and providing a nutritional source and habitat for other natural enemy insects. The dosage and release position of the carrier plants can be flexibly adjusted according to the release requirements, and the apoptotic soybean plants can also be turned into green manure and incorporated into the field soil. The host range of soybean aphids is limited and it will not damage other crops. This method is simple to operate, low in cost, environmentally friendly, high in release efficiency, and is also beneficial to conserving natural enemies.

Description

Technical Field

[0001] The present invention belongs to the field of insect rearing, and specifically relates to an application technology for improving the artificial release efficiency of Trichogramma spp. with potted soybean seedlings (Glycine max) as the carrier and the honeydew of Aphis glycines as supplementary nutrition. Background Art

[0002] Trichogramma spp. belongs to the family Trichogrammatidae of the order Hymenoptera and is a typical egg parasitoid wasp. Trichogramma spp. lays eggs inside the host eggs, absorbs the nutrients in the host eggs to complete growth and development, and only gnaws out from the host eggs until the adult wasps emerge. The parasitism of Trichogramma spp. causes the host eggs to be unable to develop and hatch normally, so the pest control effect is achieved before the host causes damage, and it is widely used in the prevention and control of farmland pests.

[0003] Although the industrial production level of Trichogramma spp. is high and it can parasitize a variety of pests in 7 orders and 44 families such as Lepidoptera, Diptera and Coleoptera, due to the short lifespan of the tiny Trichogramma spp. individuals, the parasitism efficiency is limited in the field environment, which to a certain extent restricts the improvement and development of the field application effect of Trichogramma spp. products. Parasitoid wasps can use the plant nectar sources in the field or the honeydew produced by Hemiptera insects to supplement nutrition, extend their lifespan, make up for the energy consumption during the host searching process, promote ovarian development and egg maturation, and improve the field parasitism ability and prevention and control efficiency. Therefore, developing an efficient and simple technology for enhancing the release of Trichogramma spp. in the field and strengthening nutritional supplementation is of great significance for improving the field biological control effect of Trichogramma spp.

[0004] Soybean is an important crop for both food and feed, and is widely planted worldwide. Intercropping soybeans can also improve soil nutrition, provide nectar sources and conserve natural enemies, enhance crop resistance and the pest control effect of natural enemies. Aphis glycines has a narrow host range and mainly damages soybeans, black soybeans and wild soybeans, and will not cause significant damage to other crops; the honeydew produced by Aphis glycines can provide supplementary nutritional sugar sources for parasitic natural enemies, and Aphis glycines itself can also attract the inhabitation and feeding of predatory natural enemy insects. Summary of the Invention

[0005] The object of the present invention is to provide an artificial release technology of Trichogramma spp. for improving the prevention and control efficiency of Trichogramma spp. with potted soybean seedlings as the carrier and the honeydew of Aphis glycines as supplementary nutrition, which is easy to prepare, low in cost, simple to operate and remarkable in effect.

[0006] On the one hand, the present application provides a method for releasing Trichogramma with soybean seedlings as the carrier and soybean aphid honeydew as supplementary nutrition; in this method, soybean seedlings are used as the carrier for releasing Trichogramma, and the honeydew produced by soybean aphids growing on the soybean seedlings is used as the supplementary nutrition source for Trichogramma; the method includes (1) planting and cultivating potted soybean seedlings; (2) inoculating soybean aphids and accumulating honeydew; (3) fixing and releasing Trichogramma.

[0007] Further, in step (1) of the method, the soybean seedlings are cultivated until two true leaves are unfolded before inoculating soybean aphids.

[0008] At this time, the soybean seedlings are the most tender, which is beneficial to the feeding and growth of soybean aphids and the accumulation of honeydew; in addition, at this time, the soybean plants are in the seedling stage and grow vigorously, which can maintain the reproduction of aphids and the continuous supply of honeydew, and can be used as a carrier for releasing Trichogramma multiple times.

[0009] Further, in step (1) of the method, 3 - 5 soybean seedlings are planted in each pot, and 100 soybean aphids are inoculated in each pot in step (2).

[0010] Further, in step (2) of the method, after inoculating soybean aphids and allowing them to produce honeydew on the soybean seedlings for 24 hours, the soybean seedlings serve as the carrier for releasing Trichogramma.

[0011] Further, in step (2) of the method, the above-ground part of the soybean seedlings on which soybean aphids were previously cultured is cut off with scissors and transferred to newly cultured soybean seedlings together with the soybean aphids; after inoculating soybean aphids, they are cultured for 3 hours under the environmental conditions of 25 ± 1°C, 16L:8D, 30000 lx, and RH = 65%, and then the old seedlings used for inoculation are removed; the soybean aphids are continuously cultured under the environmental conditions of 25 ± 1°C, 16L:8D, 30000 lx, and RH = 65% until they produce honeydew for 24 hours, and then the soybean seedlings serve as the carrier for releasing Trichogramma.

[0012] If the number of inoculated aphids is too small or the time for accumulating honeydew is too short, the provided honeydew nutrition is insufficient; if the number of inoculated aphids is too large or the time for accumulating honeydew is too long, it will cause Trichogramma to stick and affect its normal activities.

[0013] Further, in step (3) of the method, the Trichogramma card is fixed on the soybean seedlings serving as the carrier for releasing Trichogramma and carried to the release destination for release among the crops.

[0014] During the release period, soybean aphids can also serve as prey for natural enemies such as ladybugs to conserve natural enemies. After the release is completed, the soybean plants can be directly plowed into the field soil as green manure to improve soil nutrition. When releasing, adjust the release position of the carrier plants according to the height of the crops, and it is generally better to release at the middle position of the crops. For the specific release period, release density, and release position, refer to DB14 / T 918—2014 and DB44 / T 2219—2019.

[0015] On the other hand, the present application provides the application of the above method in the biological control method using Trichogramma

[0016] Further, the pest control target of the biological control method is selected from pests parasitizing Lepidoptera

[0017] Further, the pest control target of the biological control method is selected from pests of Noctuidae, Tortricidae, Arctiidae, Pyralidae, Geometridae or Hesperiidae; more preferably, Ostrinia furnacalis, Saccharcane borer and fruit tree heartworms

[0018] The advantages of the present invention are as follows

[0019] (1) Soybeans are convenient to purchase and easy to grow. After the Trichogramma release work is completed, the soybean seedlings can be directly plowed into the field soil as green manure to improve soil nutrition

[0020] (2) Aphis glycines is a common pest on soybeans and is easily obtained. However, the host range of Aphis glycines is narrow and it will not damage other crops. In addition, Aphis glycines is also the prey of common natural enemy insects such as ladybugs, green lacewings and hoverflies in the field, which can attract and increase the number of natural enemies in the field and improve the utilization rate of natural enemies

[0021] (3) Soybean seedlings have a certain continuous growth period. As long as the seedlings are in good condition, the aphids on them will also multiply continuously and maintain the honeydew level. Therefore, the carrier plant has a certain continuous use period and can be used for multiple Trichogramma releases during the control of one generation of pests Specific embodiments

[0022] The following examples facilitate a better understanding of the present invention, but are not limited thereto. These examples are only for illustrative purposes and in no way limit the protection scope of the present invention

[0023] All data statistics of the present invention were analyzed using the SPSS Statistics package 20 software. The difference statistics between different treatments were performed using one-way ANOVA Tukey or Games-Howell tests (significance level P < 0.05)

[0024] The Corcyra cephalonica used for mass-rearing Trichogramma dendrolimi comes from the population artificially reared in the IPM Laboratory, Department of Entomology, College of Plant Protection, China Agricultural University. Newly laid eggs of Corcyra cephalonica can be collected daily to obtain 1-day-old eggs. These eggs are evenly spread on A4 paper with non-toxic double-sided tape attached. The paper is then irradiated for 1 hour approximately 10 cm directly under a 30-watt ultraviolet lamp in a laminar flow hood to arrest the embryonic development of Corcyra cephalonica. The A4 paper is cut into egg cards of 3 cm × 2 cm with scissors for use as hosts for Trichogramma parasitization. Trichogramma dendrolimi comes from the population that has been continuously reared on Corcyra cephalonica eggs in the IPM Laboratory, Department of Entomology, College of Plant Protection, China Agricultural University. The rearing environment is an artificial climate chamber (PXZ-430B, Jiangnan Instrument Factory, Ningbo, China) with a photoperiod of 14 h light: 10 h dark, a temperature of 25 ± 1°C, and a relative humidity of 70 ± 5%.

[0025] Example 1: Effects of Supplementing Nutrition with Aphis glycines Honeydew Solution on the Longevity and Parasitism Rate of Trichogramma dendrolimi

[0026] First, prepare a 5% agar solution and pour it into the bottom of a petri dish with a diameter of 5.5 cm to form an agar layer with a height of 1 cm. Then, use a hole punch with a diameter of 1.5 cm to make leaf discs from trifoliate leaves of soybean plants. Place the freshly made leaf discs on the agar surface with the back side up to keep them moist. Use a soft brush to transfer 20 wingless Aphis glycines to the leaf discs in the petri dish, and cover the lid of the petri dish with aluminum foil (pre-weighed and recorded as M1). After covering the lid and observing until all aphids are stably feeding on the leaf discs, invert the petri dish and place it in an artificial climate chamber at 25 ± 1°C, 16L:8D, 30,000 lx, RH = 65% to collect honeydew. After 24 hours, take out the aluminum foil, place it in an oven at 40°C for 5 minutes, and then weigh it again (recorded as M2). The weight of the collected honeydew is M2 - M1. Use a micropipette to repeatedly dissolve the honeydew with an appropriate amount of distilled water to prepare a 10% honeydew solution.

[0027] Weigh 10 g of sucrose (analytical grade) with an electronic balance and transfer it to a 100-ml beaker. Add 80 ml of distilled water and stir with a glass rod until the sucrose is completely dissolved. Transfer this sucrose solution to a 100-ml graduated cylinder, rinse it 3 times with a small amount of distilled water, transfer the rinsing solution to the graduated cylinder, and then make up the volume to 100 ml with distilled water to complete the preparation of the control 10% sucrose aqueous solution.

[0028] Use forceps to hold cotton balls made of absorbent cotton with a diameter of 2 mm and dip them respectively into 10% honeydew aqueous solution and 10% sucrose solution, and apply them by dotting inside the bottom of a petri dish with a diameter of 35 mm to prepare two supplementary nutrition devices. Introduce single female Trichogramma dendrolimi that have emerged within 24 hours from the Corcyra cephalonica egg cards and have mated sufficiently into the supplementary nutrition devices with a soft brush. After covering the dish lid, wrap the petri dish around twice with sealing film to seal the periphery of the petri dish to prevent the escape of Trichogramma dendrolimi. Then place them under normal breeding conditions (photoperiod 14L:10D, temperature 25 ± 1°C, relative humidity 70 ± 5%) in an artificial climate chamber for continued single-head cultivation. Supplement fresh honeydew solution droplets every 24 hours until the female wasps in the dish die, record the survival status of each female wasp daily, and calculate the lifespan of each Trichogramma dendrolimi. Each female wasp is one replicate, and each treatment is replicated 20 times.

[0029] The detection treatment for survival rate is basically the same, only place a Corcyra cephalonica egg card (50 eggs / card) in the petri dish at the same time. After parasitism for 24 hours, take out the parasitized egg card and transfer it to a new petri dish for continued cultivation in an artificial climate chamber (25 ± 1°C, 14L:10D, RH = 65%). And on the 5th to 7th day after the eggs are parasitized, observe and record the number of parasitized eggs on each parasitized egg card under a stereomicroscope (the eggs turn black indicating successful parasitism by Trichogramma dendrolimi), and calculate the parasitism rate of Trichogramma dendrolimi using the formula (parasitism rate = number of successfully parasitized eggs / total number of eggs provided). Each female wasp is one replicate, and each treatment is replicated 6 times.

[0030] After the supplementary nutrition treatment, the average lifespan and parasitism rate of Trichogramma dendrolimi are shown in Table 1. Although the lifespan of Trichogramma dendrolimi after treatment with aphid honeydew solution is significantly lower than that of Trichogramma dendrolimi after treatment with sucrose solution, it is significantly longer than that of the water treatment. This shows that using aphid honeydew as supplementary nutrition can significantly extend the lifespan of Trichogramma dendrolimi. In addition, providing honeydew solution as supplementary nutrition can also significantly increase the parasitism rate of Trichogramma dendrolimi, and its promoting effect has no significant difference from that of sucrose solution.

[0031] Table 1 Average lifespan of Trichogramma dendrolimi with supplementary nutrition of different sugar solutions

[0032]

[0033] Example 2 Effect of honeydew produced by different numbers of Aphis glycines on the parasitism rate of Trichogramma

[0034] Plant the soybean seeds purchased from the supermarket in plastic flower pots (diameter 8 cm × height 8.0 cm) filled with a uniform mixture of vermiculite and peat (v / v = 1:1), plant 3 - 5 plants in each pot, place them in an artificial climate chamber (25 ± 1°C, 16L:8D, 30,000 lx, RH = 65%) for cultivation, water once every other day until two true leaves of the soybean plants unfold.

[0035] Cut off the above-ground part of the soybean seedlings on which soybean aphids were cultured previously with scissors, and transfer them together with the soybean aphids to newly cultured soybean seedlings. Treatments with 50, 100, and 150 soybean aphids inoculated per pot were set respectively. After the aphids were inoculated, they were continuously cultured under the environmental conditions of 25±1°C, 16L:8D, 30,000 lx, and RH = 65%. 3 h after inoculation, the aphids had transferred to the new seedlings, and the old seedlings used for inoculation were removed. Let the aphids continuously produce honeydew on the new seedlings for 24 h to obtain soybean plants with different amounts of honeydew.

[0036] Transfer the soybean plants to disposable insect rearing boxes (upper bottom radius 5 cm, lower bottom radius 7 cm, height 8 cm) respectively, and then introduce 10 female Trichogramma dendrolimi that emerged within 24 h from the Corcyra cephalonica egg cards and had mated sufficiently into the insect rearing boxes. Cover the top of the boxes with degreased cotton gauze (110 mesh) to prevent the escape of Trichogramma dendrolimi. After the Trichogramma dendrolimi fed on honeydew for 24 h, introduce 10 female wasps into disposable culture dishes containing Corcyra cephalonica egg cards respectively (1 wasp / dish). Place the culture dishes in an artificial climate chamber for parasitization for 24 h, take out the parasitized egg cards and transfer them to new culture dishes and continue to culture them in the artificial climate chamber (25±1°C, 14L:10D, RH = 65%). From the 5th day to the 7th day after the eggs were parasitized, observe and record the number of parasitized eggs on each egg card under a stereomicroscope (the eggs turn black indicating successful parasitization by Trichogramma dendrolimi), and calculate the parasitization rate of Trichogramma dendrolimi using the formula (parasitization rate = number of successfully parasitized eggs / total number of eggs provided). Each female wasp was a replicate, and each treatment was replicated 10 times.

[0037] Compared with the treatment of soybean plants without inoculating aphids, the parasitization rate of Trichogramma dendrolimi was significantly increased after the treatment of plants inoculated with 100 soybean aphids, and there was no significant difference in the parasitization rate of Trichogramma dendrolimi after the treatment of plants inoculated with 50 and 150 soybean aphids compared with the control (see Table 2). If the number of aphids is too small, the amount of honeydew produced is limited; while inoculating too many aphids will result in too much honeydew, affecting the activities of Trichogramma dendrolimi.

[0038] Table 2 Effects of honeydew produced by different numbers of soybean aphids on the parasitization rate of Trichogramma dendrolimi

[0039]

[0040] Example 3 Determination of the effect of supplementing nutrition with soybean aphid honeydew on the parasitization rate of Trichogramma dendrolimi with soybean plants as the carrier in the greenhouse

[0041] Plant the soybean seeds purchased from the supermarket in plastic flower pots (diameter 8 cm × height 8.0 cm) filled with a uniform mixture of vermiculite and peat (v / v = 1:1), with 3 - 5 plants planted per pot, and place them in an artificial climate chamber (25±1°C, 16L:8D, 30,000 lx, RH = 65%) for cultivation, watering once every other day until two true leaves of the soybean plants unfold.

[0042] Cut off the above-ground part of the soybean seedlings on which Aphis glycines were previously cultured in the laboratory with scissors, and transfer them together with the aphids to newly cultured soybean seedlings. About 100 Aphis glycines were inoculated into each pot. After the aphids were inoculated, they were continuously cultured under the environmental conditions of 25±1°C, 16L:8D, 30,000 lx, and RH = 65%. After 3 hours of inoculation, the aphids had transferred to the new seedlings, and the old seedlings used for inoculation were removed. Let the aphids continuously produce honeydew on the new seedlings for 24 hours.

[0043] Prepare a 5% agar solution and pour it into the bottom of a petri dish with a diameter of 5.5 cm to form an agar layer with a height of 1 cm. Then, use a punch with a diameter of 1.5 cm to make leaf discs from the trifoliate leaves of soybean plants, and lay the freshly made leaf discs face up on the agar surface to keep them moist. Use a soft brush to transfer 100 wingless Aphis glycines to the leaf discs in the petri dish, and cover the petri dish with a lid covered with tinfoil. After observing that all aphids were stably feeding on the leaf discs, invert the petri dish and place it in an artificial climate chamber at 25±1°C, 16L:8D, 30,000 lx, and RH = 65% to collect honeydew. After 24 hours, take out the tinfoil, and use a micropipette to repeatedly dissolve the honeydew with 50 μl of distilled water to prepare a honeydew solution.

[0044] In addition to the treatment of the carrier soybean plants with 100 Aphis glycines producing honeydew for 24 hours, simultaneously conduct the treatment of spraying the soybean plants with 50 μl of distilled water and spraying the soybean plants with 50 μl of honeydew solution. Place three pots of soybean carrier plants (one pot for each treatment) equidistantly in a cage (75 cm×75 cm×75 cm), and fix 2 Corcyra cephalonica egg cards (50 eggs / card) on each pot of soybean plants with paper clips. Release 15 female Trichogramma dendrolimi that emerged within 24 hours and had mated sufficiently at the middle position of the cage. After 24 hours of parasitization treatment, take out the parasitized egg cards and place them in a petri dish and culture them in an artificial climate chamber (25±1°C, 14L:10D, RH = 65%). From the 5th day to the 7th day after the eggs were parasitized, observe and record the number of parasitized eggs on each egg card under a stereomicroscope (the eggs turn black indicating successful parasitization by Trichogramma dendrolimi), and calculate the parasitization rate of Trichogramma dendrolimi using the formula (parasitization rate = number of successfully parasitized eggs / total number of eggs provided). Each cage is one replicate, and each treatment is replicated 10 times.

[0045] The measurement results show that the soybean carrier plants directly inoculated with Aphis glycines to produce honeydew can significantly increase the parasitization rate of Trichogramma dendrolimi, while directly spraying the honeydew solution does not promote the parasitization rate of Trichogramma dendrolimi (see Table 3).

[0046] Table 3 Parasitization rate of Trichogramma dendrolimi released by soybean carrier plants measured in greenhouse cages

[0047]

Claims

1. A method for releasing Trichogramma wasps that uses soybean seedlings as a carrier and soybean aphid honeydew as supplementary nutrition to improve the parasitism rate of Trichogramma wasps, characterized in that, In the method, soybean seedlings are used as the release carrier of Trichogramma, and the honeydew produced by Aphis glycines growing on the soybean seedlings is used as the supplementary nutrient source for Trichogramma; the method includes: (1) planting and culturing potted soybean seedlings; (2) inoculating Aphis glycines and accumulating honeydew; (3) fixing and releasing Trichogramma. In step (1), 3 - 5 soybean seedlings are planted in each pot. In step (2), 100 Aphis glycines are inoculated in each pot. The above-ground part of the soybean seedlings on which Aphis glycines were cultured previously is cut off with scissors and transferred to the newly cultured soybean seedlings together with Aphis glycines; after inoculating Aphis glycines, they are cultured under the environmental conditions of 25 ± 1°C, 16L:8D, 30000 lx, and RH = 65% for 3 hours. After the aphids have transferred to the new seedlings, the old seedlings used for inoculation are removed; the Aphis glycines are continuously cultured under the environmental conditions of 25 ± 1°C, 16L:8D, 30000 lx, and RH = 65% for 24 hours to produce honeydew, and then the soybean seedlings are used as the release carrier of Trichogramma. In step (3), the Trichogramma card is fixed on the soybean seedlings serving as the release carrier of Trichogramma and taken to the release destination for release among the crops.

2. According to the method described in claim 1, in step (1) of the method, the soybean seedlings are cultured until two true leaves are unfolded for inoculating Aphis glycines.

3. Application of the method according to claim 1 or 2 in the biological control method using Trichogramma.

4. According to the application described in claim 3, the control target of the biological control method is selected from parasitic Lepidoptera pests.

5. According to the application described in claim 4, the control target of the biological control method is selected from pests of Noctuidae, Tortricidae, Arctiidae, Pyralidae, Geometridae or Hesperiidae.

6. According to the application described in claim 5, the control target of the biological control method is Ostrinia furnacalis or Diatraea saccharalis.

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