A method for efficient breeding of tomato leafminer based on a tobacco-tomato dual-host model

Abstract

The application discloses a method for efficiently breeding tomato leafworm based on tobacco tomato dual host mode, and belongs to the field of biological control of agricultural pests. The method comprises the following steps: taking a tobacco plant as a primary host to provide an oviposition site for the tomato leafworm; separating the tobacco plant with insect eggs after the tomato leafworm lays eggs; replacing the tobacco plant with a tomato plant as a secondary host to provide the larvae with a growth environment after the insect eggs hatch into larvae; replacing part of the tomato plants with new tomato plants when the damage rate of the tomato plants reaches 80%; and using the replaced part of the tomato plants for the larvae to continue development, so that the tomato leafworm after eclosion is finally obtained. Compared with the number of the tomato leafworm bred by a single tomato plant, the number of the tomato leafworm bred by the method is larger, the development stage of the larvae is shorter, the eclosion rate of the adult is higher, and the oviposition amount is larger. The application lays a foundation for further management and research of the tomato leafworm under laboratory conditions.

Description

Technical Field

[0001] This invention relates to the field of biological control of agricultural pests, and in particular to a method for the efficient breeding of tomato leafminer moth based on a tobacco-tomato dual-host model. Background Technology

[0002] The tomato leafminer (Tuta absoluta), belonging to the order Lepidoptera and family Gelechiidae, is commonly known as the tomato leafminer. Its larvae are the primary damagers. After hatching, the larvae burrow into the host plant tissue, feeding on the leaf tissue and creating tiny tunnels in the leaves. They can also bore into the fruit, creating holes, or feed at the junction of the calyx and fruit, ultimately causing severe yield reduction or even crop failure and replanting. This pest is most attracted to tomatoes, causing yield reductions of 80%–100% in severe infestations. It can also damage other Solanaceae plants such as potatoes, eggplants, and tobacco. It poses a serious threat to the yield of important Solanaceae crops, primarily tomatoes and potatoes.

[0003] Research on agricultural pest control is of great significance to the study of natural enemy resources. Firstly, it not only provides a deeper understanding of the life cycle of pests but also allows for the identification of critical control periods by understanding their habits. Furthermore, it enables the prediction of pest occurrence in the field based on their responses to different environmental conditions. Secondly, experimental studies of the interaction between host insects and natural enemies can assess the effectiveness of natural enemies in reducing agricultural pest populations. Applying effective research findings to practice leads to efficient control of pests. Thirdly, it provides a scientific basis for the rational utilization and protection of natural enemy resources and the development of more efficient and environmentally friendly biological control methods. Therefore, to solve the problem of tomato leafminer damage to solanaceous crops, the first step is to effectively address the breeding problem of the tomato leafminer, and providing a sufficient number of experimental insects for laboratory testing is the primary foundation.

[0004] Due to the voracious feeding period and large appetite of tomato leafminer larvae, large-scale rearing of adults under laboratory conditions is quite difficult. Current research on the breeding of this pest mainly focuses on feed development and single-larval rearing, as illustrated by patents ZL202310145504.1, ZL202310556990.6, and ZL202411302823.X. While feed rearing reduces a significant amount of preliminary preparation, it also presents several problems. Because this pest is a leafminer, feed feeding can negatively impact its growth and development, such as increasing the hatching time of tomato leafminer eggs, reducing the hatching rate of tomato leafminer larvae, and decreasing the number of eggs laid compared to single-host tomato plant rearing. While single-larval rearing facilitates the study of individual larval growth stages, it is not conducive to conducting large-scale host egg experiments. Therefore, solving the breeding problem of tomato leafminer under laboratory conditions is a pressing issue. Summary of the Invention

[0005] During the larval stage of the tomato leafminer, it exhibits characteristics such as a long transition period and voracious feeding. In a laboratory environment, a large number of tomato plants are needed to sustain the larvae's normal survival. However, due to factors such as temperature and humidity not being at their optimal values ​​at specific times, as well as the sudden onset of pests and diseases, the required number of plant hosts is often difficult to guarantee.

[0006] The purpose of this invention is to provide a method for efficiently breeding tomato leafminer moths based on a tobacco-tomato dual-host model, thereby solving the problems existing in the prior art. This invention uses tobacco as a transitional plant due to its advantages such as large leaf area, higher tolerance, and shorter growth cycle. The tomato-tomato dual-host breeding system provides more living space, maximizes the utilization rate of the quantity, and appropriately reduces competition among tomato leafminer species, improving larval survival rate and thus obtaining tomato leafminer moths of better quantity and quality.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a method for efficiently breeding tomato leafminer moths based on a tobacco-tomato dual-host model, comprising the following steps:

[0009] Using tobacco plants as primary hosts, they provide oviposition sites for the tomato leafminer.

[0010] After the tomato leafminer lays its eggs, the tobacco plants carrying the eggs are separated. After the eggs hatch into larvae, the tomato plants are used as secondary hosts to replace the tobacco plants for the larvae to grow.

[0011] Once the damage rate of the tomato plants reaches 80%, some of the tomato plants are replaced with new tomato plants.

[0012] The replaced tomato plants are used for the larvae to continue developing, eventually resulting in the adult tomato leafminer moth.

[0013] Optionally, when using the tobacco plant as a primary host to provide an oviposition site for the tomato leafminer, the release rate of the tomato leafminer is 20 pairs of tomato leafminers per tobacco plant.

[0014] The leaf area ratio between suitable tomato plants and suitable tobacco plants is approximately 1:2. This invention uses tobacco plants as the primary host, and tobacco leaves provide tomato leafminer moths with more selective oviposition sites compared to tomato leaves.

[0015] Optionally, the tobacco plant containing insect eggs may be separated 72 hours after the tomato leafminer lays its eggs.

[0016] Optionally, the hatching of the insect eggs into larvae refers to hatching into first-instar larvae.

[0017] Optionally, the replacement of some tomato plants means replacing 1 / 3 of the tomato plants.

[0018] Optionally, the larvae continue to develop into mature larvae that have reached the fourth instar.

[0019] Optionally, after the larvae continue to develop into fourth-instar mature larvae, the fourth-instar mature larvae are transferred to a new container, and after they spin cocoons and pupate, the pupae are transferred to another new container, and the tomato leafminer is obtained after the flowers bloom.

[0020] Optionally, the breeding conditions for the tomato leafminer are a temperature of 24–26°C, a humidity of 55–65%, and a photoperiod of 16L:8D.

[0021] Optionally, the tobacco plant is a tobacco plant with 6 to 8 true leaves.

[0022] Optionally, the tomato plant is a tomato plant with 8 to 10 true leaves.

[0023] The present invention discloses the following technical effects:

[0024] This invention uses tobacco as a transitional plant due to its advantages such as large leaf area, higher tolerance, and shorter growth cycle. Therefore, the tomato-tobacco dual-host breeding system can provide a larger living space, maximize the utilization rate, appropriately reduce the competition among tomato leafminer species, improve the survival rate of larvae, shorten the larval development period, and ensure the quality and quantity of adults.

[0025] The tomato leafminer larvae bred in this invention produce a greater number of larvae compared to those bred from a single tomato plant, with a shorter larval development stage, a higher adult emergence rate, and a larger egg production. This lays the foundation for further control and research of the tomato leafminer under laboratory conditions. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] All embodiments of this invention were carried out in the greenhouse facilities of the Institute of Biological Control and the R&D Base for Natural Enemy Insects of Jilin Agricultural University in March 2024.

[0032] Example 1: Mass breeding of adult tomato leafminer moths using a tobacco and tomato dual-host model.

[0033] The tested insect was the tomato leafminer (Tuta absoluta), which was collected in 2024 from a vegetable base in Nong'an County, Changchun City, Jilin Province, and bred for 20 generations at the Institute of Biological Control, Jilin Agricultural University.

[0034] Test host plants: Tobacco (Beijing Tobacco) plants, tested when the tobacco plants have grown to 6-8 true leaves; Tomato (Ricky No. 1) plants, tested when the tomato plants have grown to 8-10 true leaves.

[0035] (1) Cultivation of tobacco host plants

[0036] Tobacco seeds were sown in seedling trays (single seed, 72 cells per tray). Seedling growth was monitored daily. Once the seedlings had four true leaves, they were transplanted into 1.5L pots (one seedling per pot). The potting mix used for both sowing and transplanting was humus-rich black soil, sourced from Jilin Shenzhibei Fertilizer Co., Ltd. The soil was then used for testing when the tobacco seedlings had 6-8 true leaves.

[0037] (2) Cultivation of tomato host plants

[0038] Tomato seeds were sown in seedling trays (105 cells each). Seedling growth was monitored daily. Once the seedlings had four true leaves, they were transplanted into 1.5L pots (one seedling per pot). The potting mix used for both sowing and transplanting was humus-rich black soil, sourced from Jilin Shenzhibei Fertilizer Co., Ltd. The soil was tested when the tomato plants had 8-10 true leaves.

[0039] In steps (1) and (2), the host plants were cultivated in the greenhouse of the Institute of Biological Control, Jilin Agricultural University, from March to November each year. The greenhouse was artificially lit with more than 12 hours of light per day, and the temperature was artificially controlled between 20 and 35°C, with a relative humidity of 50% to 60%.

[0040] (3) Preparation of adult tomato leafminer

[0041] One tobacco plant cultivated in step (1) was placed in an insect rearing cage, and 20 pairs (one female and one male per pair) of adult tomato leafminers were released for testing at a ratio of 20 pairs per tobacco plant. 72 hours after the adult tomato leafminers laid their eggs, the tobacco plants cultivated in step (1) were used to replace the tobacco plants in the insect rearing cage for later use. The tobacco plants with tomato leafminer eggs that were replaced were used to continue hatching the eggs.

[0042] When the insect eggs hatch into first-instar larvae, replace the tobacco plants with tomato plants cultivated in step (2) and transfer them to a new insect rearing cage. When the damage rate of the tomato plants in the insect rearing cage reaches 80%, replace 1 / 3 of the tomato plants in the insect rearing cage with new tomato plants cultivated in step (2). Place the replaced tomato plants in a new insect rearing cage. When the tomato leafminer larvae develop into fourth-instar mature larvae, transfer the larvae to a plastic box lined with plastic film. After they spin cocoons and pupate, pick all the pupae into test tubes. After the adults emerge, they become experimental insects.

[0043] The breeding climate conditions are 24-26℃, 55-65%RH, and 16:8L:D.

[0044] Example 2: Comparison of the development time of tomato leafminer larvae

[0045] The tested insect was the tomato leafminer (Tuta absoluta), which was collected in 2024 from a vegetable base in Nong'an County, Changchun City, Jilin Province, and bred for 20 generations at the Institute of Biological Control, Jilin Agricultural University.

[0046] Test host plants: Tobacco (Beijing Tobacco) plants, tested when the tobacco plants have grown to 6-8 true leaves; Tomato (Ricky No. 1) plants, tested when the tomato plants have grown to 8-10 true leaves.

[0047] (1) Comparison of hatching rates of tomato leafminer eggs: Tomato leafminer adults (first emergence <6h) bred from single-host tomato plants and tomato and tobacco dual-host breeding methods as described in Example 1 were obtained by the Biological Control Research Group of Invasive Pests at Jilin Agricultural University. The tomato leafminer adults (first emergence <6h) were fully mated by both breeding methods to provide the required number of eggs for the experiment. After 72h of oviposition, tobacco and control plants were removed. Eggs were randomly selected from both plants, 30 eggs per group, with 3 replicates. The number of tomato leafminer eggs laid on the plants was counted, and the number of hatched larvae was observed daily, along with the number of unhatched eggs, until all eggs successfully hatched. The hatching rates of larvae eggs under the two rearing methods were compared. Hatching rate (%) = Number of hatched larvae / Total number of eggs laid × 100%.

[0048] (2) Comparison of hatching time of tomato leafminer eggs: Tomato leafminer adults (initial emergence <6h) bred from single-host tomato plants and tomato and tobacco dual-host breeding methods as described in Example 1 were obtained by the Biological Control Research Group of Invasive Pests at Jilin Agricultural University. The number of eggs required for the experiment was provided after sufficient mating of the tomato leafminer eggs obtained from both breeding methods. Eggs were randomly selected, with 30 eggs per group, and repeated for 3 groups. The eggs were placed in test tubes and observed and recorded daily until all eggs hatched into first-instar larvae. The hatching time of the larvae in the two rearing systems was compared.

[0049] (3) Comparison of the larval stages of the tomato leafminer: Adult tomato leafminers (first emergence <6h) bred from single-host tomato plants and leafminers obtained using the tomato-tobacco dual-host breeding method described in Example 1 were obtained by the Biological Control Research Group of Invasive Pests at Jilin Agricultural University. Eggs from both breeding methods were provided after sufficient mating. The larvae developed into the larval stage required for the experiment. Eggs were transferred from the tomato plants to new tobacco plants using a brush, with only 30 eggs retained per group, repeated three times. Eggs were checked daily, and records were made when the eggs hatched into first-instar larvae until the larvae developed to the pupal stage. The larval stages under the two rearing systems were compared.

[0050] (4) Comparison of survival rates of tomato leafminer larvae to pupae: Tomato leafminer adults (initial emergence <6h) bred from single-host tomato plants and those obtained using the tomato-tobacco dual-host breeding method described in Example 1 were obtained by the Biological Control Research Group of Invasive Pests at Jilin Agricultural University. Both types of tomato leafminer adults (initial emergence <6h) were bred from single-host tomato plants. After sufficient mating, the eggs were provided for the experiment. The eggs hatched and developed into larvae, which were then used for the experiment. Mature larvae were randomly selected using a brush, with 30 larvae per group, and three replicates were performed. The number of mature larvae pupating daily was recorded, and the larval stages under the two rearing systems were compared.

[0051] Example 3: Comparison of the reproductive capacity of adult tomato leafminer moths.

[0052] The tested insect was the tomato leafminer (Tuta absoluta), which was collected in 2024 from a vegetable base in Nong'an County, Changchun City, Jilin Province, and bred for 20 generations at the Institute of Biological Control, Jilin Agricultural University.

[0053] Test host plants: Tobacco (Beijing Tobacco) plants, tested when the tobacco plants have grown to 6-8 true leaves; Tomato (Ricky No. 1) plants, tested when the tomato plants have grown to 8-10 true leaves.

[0054] (1) Determination of the size of the tomato leafminer: Adult tomato leafminers (newly emerged <6h) bred from single-host tomato plants and adults (newly emerged <6h) bred from tomatoes and tobacco plants according to the dual-host breeding method in Example 1 were obtained by the Biological Control Research Group of Invasive Pests at Jilin Agricultural University. The body length of female and male adults under the two rearing methods was measured using a micrometer.

[0055] (2) Determination of the emergence rate of the tomato leafminer: Tomato leafminer pupae were obtained from Case 1, with 30 pupae per group, repeated three times. The number of emerging moths was investigated after the pupae had fully emerged. Similarly, tomato leafminer pupae bred from single-host tomato plants were obtained from the Biological Control Research Group of Invasive Pests at Jilin Agricultural University, with 30 pupae per group, repeated three times. The number of emerging moths was investigated after the pupae had fully emerged. The above studies were conducted in an artificial climate chamber with the following climatic conditions: (25±1)℃, RH (60±5)%, and photoperiod 14L:10D. Emergence rate (%) = number of emerging moths / 30 × 100%.

[0056] (3) Determination of the number of eggs laid by female tomato leafminers: Tomato leafminer adults (dual host) were obtained from Case 1, and tomato leafminer adults bred from single-host tomato plants were obtained from the Biological Control Research Group of Invasive Pests at Jilin Agricultural University (single host). The number of adult tomato leafminers raised on single hosts and dual hosts was collected using an insect suction device. One pot of cultivated tobacco plants was placed in the insect rearing cage, and 20 pairs of adult tomato leafminers were released into the cage. After 72 hours of egg laying, the tobacco plants were removed and placed in the insect rearing cage containing tomato plants. The insects were observed regularly every day, and the total number of eggs laid was recorded after 72 hours of egg laying. The above experiment was repeated for three groups.

[0057] (4) Comparison of the lifespan of adult tomato leafminers: Adult tomato leafminers (dual-host) (initial emergence <6h) were obtained from Case 1, and adult tomato leafminers (single-host) (initial emergence <6h) bred from single-host tomato plants were obtained from the Biological Control of Invasive Pests Research Group of Jilin Agricultural University. The lifespans of male and female adults under the two breeding systems were compared.

[0058] Results and Analysis

[0059] (1) Comparison of hatching rates of tomato leafminer larvae

[0060] By comparing the hatching rates of larvae, it was found that the hatching rate of tomato leafminer larvae bred on two hosts was as high as 85.7%, which was significantly higher than the 81.4% hatching rate of tomato leafminer larvae bred on a single host tomato plant (Table 1).

[0061] Table 1 Comparison of hatching rates of tomato leafminer larvae bred using two methods

[0062]

[0063] (2) Comparison of hatching time of tomato leafminer larvae

[0064] By comparing the hatching time of larval eggs, it was found that the hatching time of tomato leafminer larvae bred on two hosts was 4.83 days, while the hatching time of tomato leafminer larvae bred on a single host tomato plant was 4.87 days, and the difference between the two was not significant (Table 2).

[0065] Table 2 Comparison of hatching time of tomato leafminer larvae bred by two methods

[0066]

[0067] (3) Comparison of the development time of tomato leafminer larvae

[0068] By comparing the larval development time, it was found that the development time of tomato leafminer larvae bred on two hosts was 13.8 days, which was shorter than the development time of tomato leafminer larvae bred on a single host tomato plant (15.7 days) (Table 3).

[0069] Table 3 Comparison of development time of tomato leafminer larvae bred by two methods

[0070]

[0071] (4) Comparison of survival rates of tomato leafminer larvae to pupae

[0072] By comparing the larval development time, it was found that the survival rate of tomato leafminer larvae bred on two hosts to the pupal stage was 88.6%, which was higher than the 85.2% survival rate of tomato leafminer larvae bred on a single host tomato plant (Table 4).

[0073] Table 4 Comparison of survival rates from tomato leafminer larvae to pupae bred using two methods.

[0074]

[0075] (5) Comparison of the size of adult tomato leafminer moths

[0076] Comparing the individual sizes of tomato leafminer bred using two different plant host systems revealed that the tomato leafminer bred in the dual-host system produced the largest individuals, with females averaging 7.1 mm in body length and males averaging 6.4 mm. In contrast, the tomato leafminer bred on a single host system had females averaging 6.7 mm in body length and males averaging 5.5 mm in body length (Table 5). Overall, the tomato leafminer bred using the tobacco-tomato dual-host system produced significantly larger individuals.

[0077] Table 5 Comparison of body length of tomato leafminer bred by two methods

[0078]

[0079] (6) Comparison of feathering rate

[0080] By comparing the emergence rate, it was found that the emergence rate of tomato leafminer bred by two hosts was as high as 91%, which was higher than the emergence rate of 86% of tomato leafminer bred by a single host tomato plant (Table 6).

[0081] Table 6 Comparison of emergence rates of tomato leafminer bred using two methods

[0082]

[0083] (7) Comparison of average egg-laying number per female tomato leafminer

[0084] By comparing the number of eggs laid by adult tomato leafminers reared in two different ways, it was found that the number of eggs laid by female tomato leafminers reared on two hosts was as high as 78.4, which was significantly higher than the number of eggs laid by female tomato leafminers reared on a single host tomato plant (52.7 eggs) (Table 7).

[0085] Table 7 Comparison of egg production of female tomato leafminers bred using two methods

[0086]

[0087] (8) Comparison of the lifespan of adult tomato leafminer moths

[0088] By comparing the lifespan of adult tomato leafminers raised in two different ways, it was found that the lifespan of female tomato leafminers bred on two hosts was as high as 25.52 days, which was higher than the lifespan of female tomato leafminers bred on a single host tomato plant (21.23 days) (Table 8).

[0089] Table 8 Comparison of adult lifespan of tomato leafminer moths under two different breeding methods

[0090]

[0091] Based on the combined results of (1), (2), (3), (4), (5), (6), (7), and (8), the dual-host system for rearing tomato leafminer larvae showed a higher hatching rate, shorter larval period, larger adult size, higher emergence rate, and greater egg production, thus enabling more efficient breeding of tomato leafminer.

[0092] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for efficient rearing of tomato leafworms in tobacco-tomato dual host mode, characterized by, The method comprises the following steps: providing a primary host of tobacco plants in a rearing cage to provide oviposition sites for tomato leafminer; after the tomato leafminer lays eggs, separating the tobacco plants with eggs, and after the eggs hatch into first instar larvae, replacing the tobacco plants with tomato plants as secondary hosts and placing the replaced tomato plants in new rearing cages for the larvae to grow; when the damage rate of the tomato plants in the rearing cage reaches 80%, replacing part of the tomato plants with new tomato plants; placing the replaced tomato plants in new rearing cages for the larvae to continue to develop, and finally obtaining the tomato leafminer after eclosion.

2. The method of claim 1, wherein, When the tobacco plants are used as primary hosts to provide oviposition sites for the tomato leafminer, the release amount of the tomato leafminer is 20 pairs of tomato leafminer per tobacco plant.

3. The method of claim 1, wherein, The time for separating the tobacco plants with eggs is 72 hours after the tomato leafminer lays eggs.

4. The method of claim 1, wherein, The replaced part of the tomato plants is 1 / 3 of the tomato plants.

5. The method of claim 1, wherein, The larvae continue to develop into fourth instar mature larvae.

6. The method of claim 5, wherein, After the larvae continue to develop into fourth instar mature larvae, the fourth instar mature larvae are moved to new containers, and after cocooning and pupating, the pupae are moved to another new container, and after eclosion, the tomato leafminer is obtained.

7. The method of claim 1, wherein, The breeding conditions of the tomato leafminer are temperature 24-26℃, humidity 55-65%, and photoperiod 16L:8D.

8. The method of claim 1, wherein, The tobacco plants are tobacco plants with 6-8 true leaves.

9. The method of claim 1, wherein, The tomato plants are tomato plants with 8-10 true leaves.

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