阅读说明：本技术 载体中蓟马聚集信息素释放速率的测定方法 (Method for determining release rate of thrips aggregation pheromone in carrier ) 是由 李晓维 吕要斌 章金明 张治军 林文彩 于 2018-08-22 设计创作，主要内容包括：本发明属于农业虫害防治生理指标测定技术领域,具体涉及一种蓟马聚集信息素neryl-(S)-2-methylbutanoate释放速率的测定方法。该方法将固相微萃取技术和气相色谱-质谱联用技术相结合,通过测定载体中neryl-(S)-2-methylbutanoate的释放量进行释放速率的测定,其中载体为橡胶塞载体或PVC管载体,固相微萃取的萃取时间为30min,萃取剂量为10000ng或100000ng。固相微萃取所使用的萃取头为100μm的Polydimethylsiloxane Coating萃取头。本发明所述的方法能够实现蓟马信息素neryl-(S)-2-methylbutanoate在载体中释放速率的快速、准确测定,该方法为明确信息素组分在载体上的释放模式提供了可行方法。(The invention belongs to the technical field of determination of physiological indexes for agricultural pest control, and particularly relates to a method for determining release rate of thrips aggregatory pheromone nyl- (S) -2-methylbutanoate. The method combines a solid phase microextraction technology and a gas chromatography-mass spectrometry combined technology, and measures the release rate by measuring the release amount of the neryl- (S) -2-methylbutanoate in a carrier, wherein the carrier is a rubber plug carrier or a PVC pipe carrier, the extraction time of the solid phase microextraction is 30min, and the extraction dose is 10000ng or 100000 ng. The extraction head used for solid phase microextraction was a 100 μm polydimethysiloxane Coating extraction head. The method can realize the rapid and accurate determination of the release rate of the thrips pheromone nyl- (S) -2-methylbutanoate in the carrier, and provides a feasible method for determining the release mode of the pheromone component on the carrier.)

1. A method for measuring release rate of a carrier-derived thrips pheromone nyl- (S) -2-methylbutanoate combines a solid phase microextraction technology and a gas chromatography-mass spectrometry combined technology, and measures the release rate by measuring the release rate of the carrier-derived thrips pheromone- (S) -2-methylbutanoate, and is characterized in that the carrier is a rubber plug carrier or a PVC pipe carrier, the extraction time of the solid phase microextraction is 30min, and the extraction dose is 10000ng or 100000 ng.

2. The method as claimed in claim 1, wherein the solid phase micro-extraction uses a 100 μm polydimethysiloxane Coating extraction head.

3. The method of claim 1, wherein the solid phase microextraction is performed under the conditions of 25 + 1 deg.C of temperature, 60% + -5% of relative humidity, and 14L:10D of light period.

4. A method according to any of claims 1-3, characterized by the steps of:

(1) placing the prepared lure in a climatic chamber with the temperature of 25 +/-1 ℃, the relative humidity of 60% +/-5% and the illumination period of 14L:10D, and extracting for 30min by adopting a 100-micron polydimethysiloxane Coating extraction head;

(2) analyzing the extracted sample by a gas chromatography-mass spectrometer, and calculating the release amount according to a standard curve;

(3) and calculating the release rate according to the release amount and the time.

5. Use of the method of claim 1 to determine the amount or rate of release of a styryl- (S) -2-methylbutanoate from a rubber stopper carrier or a PVC tube carrier.

Technical Field

The invention belongs to the technical field of measurement of physiological indexes for agricultural pest control, and particularly relates to a method for measuring the release rate of thrips aggregation pheromone in a carrier.

Background

Thrips is a common and important pest on crops, which causes huge economic losses to crops by direct feeding of crops and indirect transmission of viruses. Currently, the types of Thrips which are widely distributed in the world and cause great harm to crops include Thrips (Frankliniella intensa) and Thrips occidentalis (f.occidentalis) of the genus Thrips, Thrips palmi (Thrips palmi) of the genus Thrips, and the like. As thrips has the characteristics of tiny individuals, strong secrecy, strong fecundity, easy generation of drug resistance and the like, the chemical prevention, control and prevention effects are not very ideal. In addition, when a large amount of pesticides are used for controlling insect pests, a series of problems such as pesticide residue exceeding standard, environmental pollution, pest drug resistance and rampant again are brought, and the development of a green prevention and control technology is urgent.

Insect pheromones are non-chemical control measures, and can be used for monitoring and controlling pest populations and improving pest control effects by being combined with other control measures. Aggregation pheromones are a class of pheromones produced by both female and male insects or insects of either sex, and are capable of causing aggregation of insects of the same species, male and female. The aggregation pheromones of three major thrips pests have been isolated and identified. The aggregation pheromone of Frankliniella occidentalis and Frankliniella occidentalis of the genus Frankliniella has the same major active ingredients, namely, nyl- (S) -2-methylbutanoate ( carnosol (S) -2-methylbutyrate) and (R) -lavandalyl acetate ((R) -lavender acetate). The gathering pheromone active component of thrips palmi of thrips is (R) -lavandulyl3-methyl-3-butenoate ((R) -3-methyl-3-butenoic acid lavender ester).

Because the components of the insect pheromone are volatile and are easily volatilized and degraded under natural conditions, the pheromone is protected from environmental degradation by virtue of a slow release carrier, and the stable and uniform release of the pheromone is kept. The duration of the pheromone in the carrier is closely related to its release rate. Currently, no stable method for determining the release rate of thrips aggregators in different sustained release carriers is disclosed. The Solid Phase Microextraction (SPME) is a novel sample pretreatment method, integrates sampling, extraction and concentration, does not need an organic solvent, has the advantages of high speed, high efficiency, low cost, high safety, capability of being used with other instruments and the like, and is widely applied to insect pheromone research. The successful application of the solid phase microextraction method is closely related to the selection of the conditions during extraction. Factors influencing solid phase microextraction comprise a large amount of samples, a pH value, extraction time, extraction temperature, types of extraction coatings and the like, extraction conditions for artificially synthesizing the aggregation pheromone components in the lure carrier are not clear, and a determination method which is simple to operate, quick and high in sensitivity is crucial to research of the release rule of pheromone in the carrier.

Disclosure of Invention

In order to solve the above technical problems, the present invention aims to provide a method for measuring the release rate of thrips aggregators in carriers, which can quickly and accurately measure the release amount of pheromones in different carriers. The invention adopts the solid phase microextraction technology and the gas chromatography-mass spectrometry (GC-MS) technology to research the release rates of three pheromones of thrips in two carriers, namely a rubber plug and a PVC tube, and screens the optimal extraction conditions suitable for different pheromones by comparing the release rates under different extraction times and different dosages.

The present inventors have surprisingly found that solid phase microextraction, at a temperature of 25. + -. 1 ℃ and a relative humidity of 60. + -. 5%, is not suitable for the determination of the release rate of any pheromone at any dose in any carrier, for different pheromones, which have different adaptations for different carriers.

One of the purposes of the invention is to provide a method for measuring the release rate of the (R) -lavandulyl acetate in the carrier, which combines a solid phase microextraction technology and a gas chromatography-mass spectrometry combined technology, and measures the release rate by measuring the release rate of the (R) -lavandulyl acetate in the carrier, wherein the carrier is a rubber plug carrier, the extraction time of the solid phase microextraction is 30min, and the extraction dosage is 1000ng, 10000ng or 100000 ng.

The group of the invention finds that in a rubber plug carrier, the release rates of (R) -lavandulyl acetate with the doses of 1000ng, 10000ng and 100000ng do not change obviously when the extraction time is respectively 30min, 1h and 2h, namely the experimental result is relatively stable and reliable when the extraction time is 30min, 1h and 2h, and the extraction time is preferably 30min for realizing quick detection; in the PVC pipe carrier, the release rates of the three doses of (R) -lavandulyl acetate have obvious changes at the extraction time of 30min, 1h and 2h respectively, which indicates that the extraction time of solid phase microextraction has obvious great influence on the release rate measurement result, and the result obtained by using the method for measurement is unstable, thereby indicating that the method is not suitable for measuring the release rate of the (R) -lavandulyl acetate taking the PVC pipe as the carrier.

Preferably, the extraction head used in the solid phase micro-extraction is a 100 μm polydimethysiloxane Coating extraction head.

Preferably, the extraction conditions of the solid-phase micro-extraction are that the temperature is 25 +/-1 ℃, the relative humidity is 60% +/-5%, and the illumination period is 14L: 10D.

Preferably, the measuring method comprises the following steps:

(1) placing the prepared lure in a climatic chamber with the temperature of 25 +/-1 ℃, the relative humidity of 60% +/-5% and the illumination period of 14L:10D, and extracting for 30min by adopting a 100-micron polydimethysiloxane Coating extraction head;

(2) analyzing the extracted sample by a gas chromatography-mass spectrometer, and calculating the release amount according to a standard curve;

(3) and calculating the release rate according to the release amount and the time.

Preferably, the measurement method can be applied to the measurement of the release amount or release rate of (R) -lavandulyl acetate in a rubber stopper carrier.

Another object of the present invention is to provide a method for measuring release rate of a cyclic thrips pheromone nyl- (S) -2-methylbutanoate in a carrier, which combines a solid phase microextraction technique and a gas chromatography-mass spectrometry technique, and measures the release rate by measuring the release rate of the cyclic thrips pheromone- (S) -2-methylbutanoate in the carrier, wherein the carrier is preferably a rubber plug carrier or a PVC pipe carrier, the extraction time of the solid phase microextraction is 30min, and the extraction dose is 10000ng or 100000 ng.

The group of the invention finds that in a rubber plug or a PVC pipe carrier, the release rates of 10000ng and 100000ng of neryl (S) -2-methylbutanate do not change significantly when the extraction time is 30min, 1h and 2h respectively, namely the experimental result is relatively stable and reliable when the extraction time is 30min, 1h and 2h, and the extraction time is preferably 30min for realizing rapid detection; the release rate of 1000ng of the neryl (S) -2-methylbutanoate in the rubber plug carrier cannot be detected, the release rate in the PVC tube carrier has obvious changes at 30min, 1h and 2h of extraction time respectively, which indicates that the extraction time of solid phase microextraction has obvious great influence on the release rate measurement result, and the result obtained by measuring by using the method is unstable, thereby indicating that the method is not suitable for measuring the release rate of the neryl (S) -2-methylbutanoate with the dosage of 1000ng by using the rubber plug or the PVC tube as the carrier.

Preferably, the extraction head used in the solid phase micro-extraction is a 100 μm polydimethysiloxane Coating extraction head.

Preferably, the extraction conditions of the solid-phase micro-extraction are that the temperature is 25 +/-1 ℃, the relative humidity is 60% +/-5%, and the illumination period is 14L: 10D.

Preferably, the measuring method comprises the following steps:

(1) placing the prepared lure in a climatic chamber with the temperature of 25 +/-1 ℃, the relative humidity of 60% +/-5% and the illumination period of 14L:10D, and extracting for 30min by adopting a 100-micron polydimethysiloxane Coating extraction head;

(2) analyzing the extracted sample by a gas chromatography-mass spectrometer, and calculating the release amount according to a standard curve;

(3) and calculating the release rate according to the release amount and the time.

Preferably, the measurement method can be applied to the measurement of the release amount or rate of the neryl- (S) -2-methylbutanoate in the rubber stopper carrier or PVC tube carrier at a dose of 10000ng or 100000 ng.

The third purpose of the invention is to provide a method for measuring the release rate of the thrips pheromone (R) -lavandulyl3-methyl-3-butenoate in the carrier, which combines a solid phase microextraction technology and a gas chromatography-mass spectrometry combined technology, and measures the release rate by measuring the release rate of the (R) -lavandulyl3-methyl-3-butenoate in the carrier, wherein the carrier is a PVC pipe carrier, the extraction time of the solid phase microextraction is 30min, and the extraction dosage is 10000 ng.

The group of the invention finds that in a rubber plug carrier, the release rate of 1000ng of (R) -lavandulyl3-methyl-3-butenoate cannot be detected, while the release rates of 10000ng and 100000ng of (R) -lavandulyl3-methyl-3-butenoate have obvious changes when the extraction time is 30min, 1h and 2h respectively, which indicates that the extraction time of solid phase microextraction can have obvious great influence on the release rate measurement result, and the result obtained by measuring by applying the method is not stable, thereby indicating that the method is not suitable for measuring the release rate of (R) -lavandulyl3-methyl-3-butenoate taking the rubber plug as the carrier; in the PVC tube carrier, the release rates of 1000ng and 100000ng of (R) -lavandulyl3-methyl-3-butenoate change significantly at 30min, 1h and 2h of extraction time, respectively, indicating that the method is also not suitable for the measurement of the release rate of (R) -lavandulyl3-methyl-3-butenoate at 1000ng and 100000ng of dose using PVC tube as carrier, while the release rate of (R) -lavandulyl3-methyl-3-butenoate at 10000ng of dose does not change significantly at 30min, 1h and 2h of extraction time, respectively, i.e. the experimental results are relatively stable and reliable at 30min, 1h and 2h, and the extraction time is preferably 30min for rapid detection.

Preferably, the extraction head used in the solid phase micro-extraction is a 100 μm polydimethysiloxane Coating extraction head.

Preferably, the extraction conditions of the solid-phase micro-extraction are that the temperature is 25 +/-1 ℃, the relative humidity is 60% +/-5%, and the illumination period is 14L: 10D.

Preferably, the measuring method comprises the following steps:

(1) placing the prepared lure in a climatic chamber with the temperature of 25 +/-1 ℃, the relative humidity of 60% +/-5% and the illumination period of 14L:10D, and extracting for 30min by adopting a 100-micron polydimethysiloxane Coating extraction head;

(2) analyzing the extracted sample by a gas chromatography-mass spectrometer, and calculating the release amount according to a standard curve;

(3) and calculating the release rate according to the release amount and the time.

Preferably, the assay is applied to determine the amount or rate of release of 10000ng of (R) -lavandulyl3-methyl-3-butenoate from PVC tube carrier.

Advantageous effects: the release rate of the artificially synthesized aggregated pheromone component in the carrier was found to be closely related to the dose of the pheromone component, the extraction time and the carrier by measuring the release rate of the artificially synthesized aggregated pheromone component using SPME and GC-MS. Through comparison experiments, the solid phase microextraction is selectively used for measuring the release rate in the carrier aiming at different pheromones, different carriers and different dosages under the conditions that the temperature is 25 +/-1 ℃ and the relative humidity is 60% +/-5% to obtain accurate and reliable measurement results. The method provided by the invention can realize rapid and accurate determination of the release rate of the thrips pheromone in the carrier, provides a feasible method for determining the release mode of the pheromone component on the carrier, and lays a foundation for further determining the long-term release rule of the thrips aggregation pheromone component and developing an attractant based on the aggregation pheromone.

Drawings

FIG. 1 is a graph of the release rate of the seed aggregated pheromone component from rubber stopper carriers at different dosages and for different extraction times. Wherein, A: component (R) -lavandulyl acetate; b: the component neryl (S) -2-methylbutanoate; c: the component (R) -lavandulyl 3-methyl-3-butenoate.

FIG. 2 is a graph of the release rates of three aggregated pheromone components at different doses and at different extraction times in PVC tube carriers. Wherein, A: component (R) -lavandulyl acetate; b: the component neryl (S) -2-methylbutanoate; c: the component (R) -lavandulyl 3-methyl-3-butenoate.

Detailed Description

The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.

In the invention, all parts and percentages are weight units, and all equipment, raw materials and the like can be purchased from the market or are commonly used in the industry, if not specified. Unless otherwise indicated, the examples employ methods that are within the ordinary skill in the art.

The aggregation pheromone (R) -lavondulyl acetate (purity is more than or equal to 98 percent and optical isomer is more than or equal to 99 percent) and the neryl (S) -2-methylbutanoate (purity is more than or equal to 98 percent and optical isomer is more than or equal to 99 percent) applied by the invention are artificially synthesized by Chinese agriculture university according to the method reported by Hamilton et al (2005); the aggregation pheromone (R) -lavondulyl 3-methyl-3-butenoate (the purity is more than or equal to 98 percent, and the optical isomer is more than or equal to 99 percent) of thrips palmi was artificially synthesized by the method reported by Akella et al (2014) at the university of agriculture in China.

For the sake of distinction, the three components (R) -lavandulyl acetate, neryl (S) -2-methylbutanoate and (R) -lavandulyl3-methyl-3-butenoate are each represented by R, S, Z.

The carrier used in the invention is a green rubber plug carrier and a PVC pipe carrier. Green rubber plug carriers (diameter. times. length. 1 cm. times.1.5 cm) were purchased from Beijing Zhongjie Site Biotech, Inc. Soaking in chromatographic pure n-hexane for 24 hr, oven drying at 80 deg.C for 2 hr to volatilize n-hexane, and packaging in sealed glass bottle. PVC pipe carriers (1.5 mm. times.8 cm. in. diameter. times.length) were purchased from Yanglingliang forest, science and technology, chemical Co., Ltd. Soaking in anhydrous ethanol for 24 hr before use, air drying, and storing in sealed bag.

A manual sample feeding handle and an extraction head (100 μm) special for solid phase micro-extraction are purchased from Supelco, USA. When in use, the extraction head is activated at a gas chromatography sample inlet at 200 ℃ until no impurity peak exists, the core-attracting carrier to be detected is placed into a 20ml sample bottle, and the extraction head is directly inserted into a release space (20ml sample bottle) through a bottle cap to extract for a certain time. Then, the components on the SPME column were separated and analyzed by a gas chromatograph-mass spectrometer (GC-MS).