Method for improving resistance of tea trees to ectropis grisescens

Abstract

The invention provides a method for improving the resistance of tea trees to ectropis grisescens, and belongs to the technical field of green prevention and control of insect pests of tea trees, the method comprises the following steps: carrying out linalool exposure treatment on the tea trees; the concentration of the linalool exposure treatment is 50-150ng / h / strain, the daily treatment time is 2-6 hours, and the linalool exposure treatment is continuously performed for 3-6 days. According to the method, linalool exposure treatment is carried out on tea trees, and after the tea trees are damaged by ectropis grisescens, linalool can remarkably increase the content of jasmonic acid and jasmonic acid-isoleucine which are defense related hormones in tea tree leaves; meanwhile, the expression of jasmonic acid signal channel related genes is directly induced or activated after the tea trees are damaged by ectropis grisescens, so that the insect resistance of the tea trees is enhanced.

Description

Technical Field

[0001] This invention belongs to the field of green pest control technology for tea trees, and in particular relates to a method for improving the resistance of tea trees to the gray tea geometrid moth. Background Technology

[0002] The tea plant (Camellia sinensis) is a perennial woody crop of significant economic value. The grey tea looper (Ectropis grisescens), belonging to the family Geometridae (Lepidoptera), is one of the most damaging chewing pests in tea gardens, occurring in almost all tea-growing regions. The grey tea looper has a high reproductive rate, with 6 to 7 generations per year. It overwinters as pupae in the shallow soil around the tea plant roots, emerging as adults to lay eggs the following spring. First to third instar larvae feed on the leaf tissue, leaving only the midrib; fourth and fifth instar larvae are voracious, consuming all the tender leaves, old leaves, and even tender stems, leaving only bare branches. Severe infestations can lead to reduced yields, crop failure, or even plant death, seriously threatening the tea garden ecosystem and tea quality.

[0003] Currently, the control of the tea geometrid moth still relies mainly on chemical pesticides. However, this method has significant drawbacks: first, it easily leads to excessive pesticide residues in tea, endangering food safety and affecting tea export trade; second, the overuse of chemical pesticides can induce pesticide resistance in pests, reducing control effectiveness and increasing control costs; and third, the use of broad-spectrum pesticides can also damage the tea garden ecosystem, kill natural enemy populations, and exacerbate pest outbreaks. Therefore, there is an urgent need to develop efficient, safe, and environmentally friendly green control technologies.

[0004] Plant-derived attractants, as a novel green biological control method, enhance the plant's resistance to pests, diseases, and abiotic stresses by activating its own immune system. They offer advantages such as high efficiency, low toxicity, broad spectrum, long duration, no pollution, and low likelihood of developing resistance. Developing safe and efficient plant-derived attractants for tea tree pest control has become a current research and application hotspot.

[0005] Linalool is a natural acyclic monoterpene compound that is widely distributed in the flowers, fruits, leaves, stems and roots of various monocotyledonous and dicotyledonous plants. It has an aroma similar to lily of the valley and is widely used in food, fragrance, daily chemical and pharmaceutical fields. Currently, there are no reports on the effects of linalool on tea plant resistance. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a method for improving the resistance of tea trees to the tea geometrid moth. The method involves exposing tea trees to linalool. After tea trees are damaged by the tea geometrid moth, linalool can significantly increase the content of defense-related hormones jasmonic acid and jasmonic acid-isoleucine in the leaves of tea trees. At the same time, it can directly induce or activate the expression of genes related to the jasmonic acid signaling pathway after tea trees are damaged by the tea geometrid moth, thereby enhancing the insect resistance of tea trees.

[0007] This invention provides a method for improving the resistance of tea trees to the gray tea geometrid moth, comprising the following steps: exposing tea trees to linalool;

[0008] The concentration of linalool for the exposure treatment was 50–150 ng / h / plant, the daily treatment time was 2–6 hours, and the treatment was carried out continuously for 3–6 days.

[0009] Preferably, the concentration of linalool exposure treatment is 70-90 ng / h / plant, the daily treatment time is 3-5 hours, and the treatment is carried out continuously for 4-5 days.

[0010] Preferably, the linalool exposure treatment is performed using a sustained-release device.

[0011] Preferably, the slow-release device is suspended on the tea tree.

[0012] Preferably, the linalool exposure treatment can induce the synthesis of defense-related hormones in tea leaves.

[0013] Preferably, the defense-related hormones include jasmonic acid and jasmonic acid-isoleucine.

[0014] Preferably, the linalool exposure treatment can induce upregulation of the expression of jasmonic acid pathway-related genes in the leaves.

[0015] Preferably, the jasmonic acid pathway-related genes include CsLOX1, CsOPR3, and CsJAR.

[0016] Preferably, the linalool exposure treatment significantly inhibits the growth rate of the tea geometrid moth larvae and reduces their feeding area on tea leaves.

[0017] Compared with existing technologies, the present invention has the following beneficial effects: The present invention provides a method for improving the resistance of tea trees to the tea geometrid moth by exposing the tea trees to linalool; the concentration of linalool exposure is 50-150 ng / h / plant, the daily treatment time is 2-6 hours, and the treatment is continuous for 3-6 days. Using the linalool treatment concentration and time specified in the present invention, after the tea trees are damaged by the tea geometrid moth, it can induce the synthesis of defense-related hormones in the tea leaves and induce the upregulation of the expression of genes related to the jasmonic acid signaling pathway in the tea leaves. This significantly inhibits the growth rate of the tea geometrid moth larvae and reduces their feeding area on the tea leaves, thereby improving the resistance of the tea trees to the tea geometrid moth. Attached Figure Description

[0018] Figure 1 The release rate of linalool in tea trees under insect stress and in the slow-release device;

[0019] Figure 2 The effect of linalool exposure for 1 day on the content of defense-related hormones in tea leaves;

[0020] Figure 3 The effect of linalool exposure for 4 days on the content of defense-related hormones in tea leaves;

[0021] Figure 4 The effect of linalool exposure on the expression levels of jasmonic acid pathway-related genes in tea leaves;

[0022] Figure 5 The effect of linalool exposure on the body weight of larvae of the tea geometrid moth;

[0023] Figure 6 Comparison of the growth of larvae of the tea geometrid moth in the linalool exposure group and the control group;

[0024] Figure 7 The effect of linalool exposure on the leaf feeding area of ​​the larvae of the tea geometrid moth;

[0025] Figure 8 The feeding behavior of tea tree leaf larvae on tea leaves in the linalool exposure group and the control group. Detailed Implementation

[0026] This invention provides a method for improving the resistance of tea trees to the gray tea geometrid moth, comprising the following steps: exposing tea trees to linalool.

[0027] In this invention, the concentration of linalool exposure treatment is 50-150 ng / h / plant, preferably 70-90 ng / h / plant, more preferably 75-85 ng / h / plant; the daily treatment time of linalool exposure is preferably 2-6 hours, more preferably 3-5 hours; the linalool exposure treatment is preferably continuous for 3-6 days, more preferably continuous for 4-5 days.

[0028] In this invention, the linalool exposure treatment is preferably performed using a sustained-release device; the sustained-release device is suspended on the tea tree. In this invention, the sustained-release device is preferably a threaded injection bottle, and the volume of the sustained-release device is preferably 1-3 mL, more preferably 2 mL; glass wool is placed in the sustained-release device, and then a 98% pure linalool solution is added to the sustained-release device. After sealing with a perforated threaded cap, a microcapillary tube with a length of 15 mm and a pore size of 2 μL is inserted; the linalool is slowly released into the surrounding air through the capillary tube; the microcapillary tube of the above specifications can ensure that the release concentration of linalool is within the range defined by this invention.

[0029] In this invention, linalool exposure can directly induce or induce the synthesis of defense-related hormones in tea leaves after tea trees are damaged by the tea geometrid moth; the defense-related hormones preferably include jasmonic acid and jasmonic acid-isoleucine; after tea trees are damaged by the tea geometrid moth, linalool exposure can induce the upregulation of the expression of jasmonic acid pathway-related genes in the leaves, the jasmonic acid pathway-related genes preferably include CsLOX1, CsOPR3 and CsJAR.

[0030] In this invention, the linalool exposure treatment significantly inhibits the growth rate of the larvae of the tea geometrid moth and reduces their feeding area on tea leaves.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] The test crop was a 3-year-old potted seedling of the tea tree 'Longjing 43', planted at the Tea Research Institute of the Chinese Academy of Agricultural Sciences.

[0033] Test insects: The larvae of the gray tea geometrid moth were collected from a tea garden in Longwu Town, Hangzhou City, Zhejiang Province. They were fed with tender shoots of 'Longjing 43' tea trees and were reared in a culture box (temperature 26±2℃, relative humidity 70±5%, light cycle 12:12h).

[0034] Test reagent: Linalool (CAS: 78-70-6) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0035] Preparation of the linalool sustained-release device: 100 mg of glass wool was wrapped into a small ball and placed into a 2 mL brown screw-on sample bottle. Then, 300 μL of 98% pure linalool solution was added. After capping with a perforated screw cap, a capillary tube was inserted, allowing the sustained-release bottle to release linalool at a rate of 80 ng / h. This concentration represents the physiological concentration of linalool naturally released by tea plants after being fed on by the tea geometrid moth. Figure 1 Simultaneously, a sustained-release bottle containing 100 mg of glass wool but without added linalool was prepared as a control.

[0036] Linalool exposure treatment of tea plants: Sixty uniformly grown 'Longjing 43' potted seedlings were selected and randomly divided into a linalool exposure treatment group and a control group, with 30 seedlings in each group. Each seedling was placed in a transparent square plastic container after being fitted with a slow-release device (with or without linalool). The 'Longjing 43' potted seedlings were exposed to the linalool slow-release device for 3 hours daily for one day, or for four consecutive days, in a greenhouse. After the exposure period, all slow-release bottles were removed and tightly sealed. The tea seedlings treated with this method were used for subsequent tea plant insect resistance testing or simulated insect pest treatment.

[0037] Simulated pest treatment: The second leaf of the potted tea seedlings that had been exposed to linalool was used as the treatment target. First, two wounds were made on each side of the leaf parallel to the main vein using a sterilized barbed roller to mechanically damage the plant. During the operation, the roller was kept rolling from the base of the leaf to the tip. Then, 10 μL of oral secretions from the tea geometrid moth were taken and evenly applied to the surface of the two wounds.

[0038] Example 1

[0039] Effects of linalool exposure on defense-related hormones in tea leaves

[0040] Tea plants exposed to linalool for 1 day or continuously for 4 days were subjected to simulated insect infestation treatment for 0 hours, 3 hours, and 6 hours, respectively. The treated tea leaves were collected, ground under liquid nitrogen, and 100 mg of tea powder was accurately weighed. Jasmonic acid and jasmonic acid-isoleucine were extracted from the tea powder using ethyl acetate with added isotopic internal standards (d6-JA, d6-JA-Ile). Detection and analysis were performed using an ultra-high performance liquid chromatography (UPLC) and triple quadrupole mass spectrometry (TQS-Micro, Waters, Manchester, UK) system.

[0041] The results are as follows Figure 2 and Figure 3As shown, linalool exposure for 1 day or 4 consecutive days can enhance the resistance of tea trees to the tea geometrid moth by inducing the synthesis of jasmonic acid and jasmonic acid-isoleucine in tea leaves after the tea trees have been damaged by the geometrid moth. Among them, the defense-related hormone response of tea leaves was stronger after 4 days of linalool exposure compared with only 1 day of exposure.

[0042] like Figure 2 As shown, compared with the control, the jasmonic acid content of tea trees exposed to linalool for 1 day increased significantly by 176.57% after 6 hours of simulated pest treatment, while the jasmonic acid-isoleucine content increased significantly by 51.93%.

[0043] like Figure 3 As shown, compared with the control, the jasmonic acid content of tea trees that were continuously exposed to linalool for 4 days increased significantly by 48.88% after 3 hours of simulated pest treatment; and by 6 hours of simulated pest treatment, the jasmonic acid content increased significantly by 332.38%, and the jasmonic acid-isoleucine content increased significantly by 164.33%.

[0044] Example 2

[0045] Effects of linalool exposure on jasmonic acid signaling pathway genes in tea leaves

[0046] Because the levels of defense-related hormones in tea leaves responded strongly after 4 days of linalool exposure, simulated pest treatments were subsequently applied to tea plants exposed to linalool for 0, 3, and 6 hours. The treated tea leaves were then collected and ground into tea powder under liquid nitrogen. Total RNA was extracted from samples in the linalool-exposed group and the control group using a plant polysaccharide and polyphenol total RNA extraction kit (TIANGEN, China). 500 ng of RNA was used as a template for reverse transcription using PrimeScript™ RTMasterMix (TaKaRa, Japan) to obtain cDNA; detailed operating procedures are provided in the manufacturer's product instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to quantify the expression of jasmonic acid pathway genes (CsLOX1, CsOPR3, CsJAR) in tea leaves. The primer sequences used are shown in Table 1 below. qRT-PCR was performed using a LightCycler 480SYBR Green IMaster (Roche, Swizerland) on a LightCycler 384 system (Roche Diagnostics GmbH, Germany). CsGAPDH was used as the internal reference gene, and a 2... -ΔΔCt The method calculates the relative expression levels of each gene, and the detection results are as follows: Figure 4 As shown.

[0047] The qRT-PCR reaction system consisted of: 2 μL cDNA; 5 μL LightCycler480 SYBR Green I Master; 0.8 μL each of forward and reverse primers at a concentration of 10 pmol / μL; and 1.4 μL H2O.

[0048] The qRT-PCR amplification conditions were: 95℃ for 30s; (95℃ for 15s, 57℃ for 30s, 72℃ for 20s) for 40 cycles; 95℃ for 15s; 40℃ for 30s.

[0049] Table 1 Primer sequences

[0050]

[0051] Figure 4 This indicates that linalool exposure treatment can enhance the resistance of tea trees to the tea looper by directly inducing or inducing the expression of jasmonic acid pathway-related genes in tea leaves after the tea tree has been damaged by the tea looper.

[0052] like Figure 4 As shown, compared with the control, linalool exposure directly induced the expression of the 12-oxo-phytodienoic acid reductase gene CsOPR3. After 3 hours of simulated insect pest treatment, linalool exposure significantly induced the transcription of the lipoxygenase gene CsLOX1; and after 6 hours of simulated insect pest treatment, linalool exposure significantly induced the expression of both the lipoxygenase gene CsLOX1 and the jasmonic acid amino acid conjugation enzyme gene CsJAR.

[0053] Example 3

[0054] Tea tree insect resistance test

[0055] The first leaf of a 'Longjing 43' potted seedling, exposed to linalool for 4 days, was selected and inserted into pre-soaked potting soil to maintain its condition. The leaf and potting soil were then placed in square petri dishes, and five 4-day-old larvae of the tea geometrid moth (L. a.m.) that had been starved for 3 hours were introduced into each dish. The larvae were fed leaves from either the linalool-exposed control group or the treatment group for 6 consecutive days, with the leaves replaced every 1-2 days depending on the larvae's feeding behavior. The larvae were weighed and recorded on days 4 and 6 after feeding. The leaf area of ​​the tea leaves before and after feeding was scanned and analyzed using a leaf area analyzer. The results are shown below. Figures 5-8 As shown.

[0056] Figures 5-8 This indicates that feeding tea looper larvae with leaves treated with linalool can effectively inhibit the growth rate of the larvae and reduce their feeding area on tea leaves, thereby enhancing the tea tree's resistance to the tea looper.

[0057] Compared to the control, the body weight of *Tea geometrid moth* larvae that fed on tea leaves exposed to linalool for 4 and 6 days was significantly reduced. Figure 5 As shown, the weight of *Tea geometrid moth* larvae in the linalool-exposed group decreased by 25.17% after 4 days of feeding and by 28.40% after 6 days. A comparison of the growth of *Tea geometrid moth* larvae in the linalool-exposed group and the control group after 6 days of feeding is shown in the figure. Figure 6 Meanwhile, compared with the control, the leaf area consumed by the larvae of the tea geometrid moth after exposure to linalool was significantly or extremely significantly reduced. Figure 7 As shown, after 2, 4, and 6 days of feeding, the linalool-exposed leaf area of ​​tea leaves decreased by 26.17%, 26.43%, and 37.47%, respectively. For details on the feeding of tea leaves, see [link to relevant data]. Figure 8 .

[0058] As can be seen from the above embodiments, the method involves exposing tea trees to linalool. Linalool can significantly increase the content of defense-related hormones jasmonic acid and jasmonic acid-isoleucine in tea leaves after tea trees are damaged by the tea geometrid moth. At the same time, it can directly induce or activate the expression of genes related to the jasmonic acid signaling pathway after tea trees are damaged by the tea geometrid moth, thereby enhancing the insect resistance of tea trees.

[0059] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for improving the resistance of tea trees to the gray tea geometrid moth, characterized in that, Includes the following steps: Tea plants were exposed to linalool. The concentration of linalool for the exposure treatment was 50–150 ng / h / plant, the daily treatment time was 2–6 hours, and the treatment was carried out continuously for 3–6 days.

2. The method according to claim 1, characterized in that, The concentration of linalool for the exposure treatment was 70–90 ng / h / plant, the daily treatment time was 3–5 hours, and the treatment was carried out continuously for 4–5 days.

3. The method according to claim 1, characterized in that, The linalool exposure treatment was carried out using a sustained-release device.

4. The method according to claim 3, characterized in that, The slow-release device is suspended on the tea tree.

5. The method according to claim 1, characterized in that, The linalool exposure treatment can induce the synthesis of defense-related hormones in tea leaves.

6. The method according to claim 5, characterized in that, The defense-related hormones include jasmonic acid and jasmonic acid-isoleucine.

7. The method according to claim 1, characterized in that, The linalool exposure treatment can induce upregulation of the expression of jasmonic acid pathway-related genes in leaves.

8. The method according to claim 8, characterized in that, The jasmonic acid pathway-related genes include CsLOX1, CsOPR3, and CsJAR.

9. The method according to claim 4, characterized in that, The linalool exposure treatment significantly inhibited the growth rate of the larvae of the tea geometrid moth and reduced their feeding area on tea leaves.

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