Field sampling method for leaf-dwelling arthropods

Abstract

The invention provides a field sampling method for leaf arthropods, and belongs to the technical field of agricultural plant protection. According to the field sampling method for the leaf-dwelling arthropods, sampling is carried out with the leaves as sampling units, the unit leaves are directly sleeved with the self-sealing bags, then the leaves and plants are separated, arthropod escape caused by leaf picking is avoided, species except the leaf-dwelling arthropods can be captured, and the capturing efficiency of most species is high. Samples obtained through a field sampling method are subjected to species identification, the samples are taken back to a laboratory, then species identification is conducted in combination with morphology and molecular biology, guarantee is provided for accuracy and scientificity of survey results, the requirement for the professional knowledge level of arthropod taxonomy of experimenters is relatively low, and the application range of people is large. The method is simple, convenient and rapid to operate, the sample is easy to carry, transport and store, and the application range is wide.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural plant protection, and particularly relates to a field sampling method for leaf-dwelling arthropods. Background Art

[0002] Arthropod communities are indispensable for maintaining healthy ecosystems and agricultural production. However, the composition of arthropods in ecosystems is extremely complex, mainly including herbivorous pests such as aphids and thrips, predatory and parasitic natural enemies such as ladybugs, spiders, and parasitic wasps, saprophagous arthropods, economic insects such as bees and butterflies, and neutral insects such as mosquitoes. Survey sampling and species identification are key links in the study and analysis of the ecological habits, species diversity, and community structure of arthropods. In the sampling survey of arthropods in the fields of crops such as cotton, soybeans, and potatoes, direct observation, sweeping nets, vibration drop methods, and whole plant bagging sampling methods are often used. The direct observation and counting method requires investigators to have sufficient taxonomic expertise, is labor-intensive and time-consuming, and is prone to errors and omissions, making it difficult to meet the needs of large-scale experiments. The sweeping net method is greatly affected by weather conditions (temperature, humidity, wind force), plant growth and development period (size, height, canopy density), and operation methods (sweeping net height, speed, and technique), and it is difficult to capture slow-moving species hidden in the leaves. Because some arthropods do not fall or fall into the collecting cloth when the plants are patted, the number of arthropods obtained by the shaking method is often less than that of other methods. The whole plant bagging sampling method is to cover the entire plant with a bag, cut it along the ground plane, and bring the whole plant back to the laboratory to investigate the number of arthropods on the plant. The whole plant bagging sampling method is a relatively comprehensive and complete method for collecting arthropods, but because it requires the whole plant to be brought back, it has defects such as being unsuitable for sampling in multiple places and at a distance, and requires a large space to store samples to be tested. Therefore, it is of great significance to develop a sampling method that is convenient and fast, less affected by environmental conditions and operators, has a wide range of applications, and can representatively describe the composition and population density of arthropod communities, in order to improve the efficiency of field arthropod sampling surveys. Summary of the invention

[0003] The purpose of the present invention is to provide a field sampling method for leaf-dwelling arthropods, aiming to solve the problems of complex and time-consuming sampling operations and difficulty in species identification of field arthropods, and to provide technical support for the study of arthropod communities.

[0004] The invention provides a field sampling method for leaf-dwelling arthropods, comprising the following steps: using a ziplock bag as a field sampling tool, putting the ziplock bag on plant leaves, and separating the plant and the leaves at the petiole.

[0005] Preferably, the length and width of the leaves of the plant are both less than 30 cm.

[0006] Preferably, field sampling is performed on the upper, middle and lower parts of the plant respectively. When dividing the plant, the node from the bottom of the plant with fully expanded leaves to the node of the top of the plant with fully expanded leaves is regarded as a whole, and the whole is divided vertically into three parts, the top third is the upper part, the middle third is the middle part, and the bottom third is the lower part.

[0007] Preferably, during the field sampling, one leaf from the upper part, the middle part and the lower part of the plant are respectively selected for field sampling, and one leaf is placed in each ziplock bag.

[0008] Preferably, when sampling in the field, the specification of the sampling ziplock bag is selected according to the growth of the leaves, the ziplock bag is opened, and the bag is gently put on a single leaf of the plant from the tip of the leaf, and cut at the petiole;

[0009] Seal the bag quickly after cutting at the petiole and expel any excess air from the bag.

[0010] The present invention also provides a method for identifying leaf-dwelling arthropods, comprising bringing samples obtained by the field sampling method back to a laboratory and identifying the species by combining morphology with molecular biology.

[0011] Preferably, the morphological identification includes carefully taking the leaves out of the ziplock bag under a stereoscope, identifying and counting the arthropods on the front and back of the leaves, observing the empty ziplock bag after the leaves are taken out under a stereoscope, and identifying and counting the species that fall into the bag.

[0012] Preferably, for species that cannot be identified by morphology, the mitochondrial cytochrome oxidase COI gene of the species is amplified and then sequenced, and the obtained sequence is compared with the database to obtain the species information of the sample arthropod.

[0013] Preferably, the primers used for amplification include nucleotide sequences such as COI-F shown in SEQ ID No.1 and COI-R shown in SEQ ID No.2.

[0014] Preferably, the PCR reaction procedure for the amplification includes: denaturation at 98°C for 10s; annealing at 52°C for 5s; and extension at 68°C for 1s, for 45 cycles.

[0015] Beneficial effects: The present invention provides a field sampling method for leaf-dwelling arthropods (referred to as the single-leaf bagging method in the embodiment), which takes leaves as sampling units, directly puts environmentally friendly polyethylene ziplock bags on unit leaves, and then separates the leaves from the plants, thereby avoiding the escape of arthropods caused by picking leaves, and can capture species other than leaf-dwelling arthropods, with high capture efficiency for most species. Compared with the whole-plant bagging method, the sampling method described in the present invention saves the cost of sampling bags, sample storage and transportation costs; and the sampling method of the present invention ensures the comprehensiveness and representativeness of the sampling, and most of the captured samples are complete in morphology, which is convenient for subsequent identification, photography, specimen preparation, breeding and other research.

[0016] The present invention also performs species identification on the samples obtained after the field sampling method, and combines morphology and molecular biology to identify the species after the samples are brought back to the laboratory, which provides a guarantee for the accuracy and scientificity of the survey results, and requires relatively low professional knowledge of arthropod taxonomy for the experimenters, and is applicable to a wide range of people. The method of the present invention is simple and fast to operate, and the samples are easy to carry, transport and store, and have a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of vertical partitioning of soybean plants and sampling by single leaf bag capture method;

[0018] Figure 2 Agarose gel electrophoresis detection diagram of PCR products. In the figure, M represents DNA Marker. Lanes 1-6 are PCR products of different samples. The red frame is the target band. Lane 5 is an invalid sample, and the rest are valid samples.

[0019] Figure 3 Statistical graph of the number of arthropods of various families captured by the two sampling methods in soybean fields;

[0020] Figure 4 Statistical graphs of arthropod taxa and abundance captured by the two sampling methods in peanut fields. DETAILED DESCRIPTION

[0021] The invention provides a field sampling method for leaf-dwelling arthropods, comprising the following steps: using a ziplock bag as a field sampling tool, putting the ziplock bag on plant leaves, and separating the plant and the leaves at the petiole.

[0022] The present invention uses a ziplock bag as a field sampling tool, and the ziplock bag is preferably an environmentally friendly polyethylene ziplock bag, which is directly put on a unit leaf. The field sampling method of the present invention is preferably applicable to various types of leaves, and more preferably applicable to plant leaves with a length and width of less than 30 cm.

[0023] The present invention preferably uses leaves as sampling units for sampling, and when sampling, selects one leaf each from the upper, middle and lower parts of the plant for field sampling, and samples are taken from the upper, middle and lower parts of the plant in the vertical direction, respectively, to ensure the comprehensiveness and representativeness of the sampling. In the present invention, it is preferred that the node with fully expanded leaves at the bottom of the plant to the node with fully expanded leaves at the top of the plant is regarded as a whole, and the node is vertically divided into three equal parts, that is, the top third is the upper part, the middle third is the middle part, and the bottom third is the lower part, that is, when sampling in the field, only 3 leaves are sampled from a single plant, saving the cost of sampling bags, sample storage and transportation costs.

[0024] The present invention preferably selects the specifications of the sampling ziplock bag according to the growth conditions of the leaves, and uses an environmentally friendly polyethylene ziplock bag to directly cover the unit leaf. When the leaf is covered, the mouth of the ziplock bag is opened, and it is gently covered on a single leaf of the plant from the tip of the leaf, and cut off at the petiole; and the bag is quickly sealed after the cutting, and it is noted that the excess air in the bag is discharged for easy carrying, but the arthropods in the ziplock bag cannot be crushed.

[0025] The present invention uses a ziplock bag to cover leaves and then separates them from the plants, thereby preventing arthropods from escaping due to leaf picking, and can capture species other than leaf-dwelling arthropods, with high capture efficiency for most species. The sampling method described in the present invention allows most of the captured samples to be in complete morphology, which is convenient for subsequent identification, photography, specimen preparation, breeding and other research.

[0026] The present invention also provides a method for identifying leaf-dwelling arthropods, comprising bringing samples obtained by the field sampling method back to a laboratory and identifying the species by combining morphology with molecular biology.

[0027] The present invention preferably takes the sample back to the laboratory and combines morphology and molecular biology to identify the species, which provides a guarantee for the accuracy and scientificity of the survey results, requires relatively low professional knowledge of arthropod taxonomy for the experimenter, and is applicable to a wide range of people. The morphological identification of the present invention preferably includes carefully taking the leaf out of the ziplock bag under a stereoscope, identifying and counting the arthropods on the front and back of the leaf, observing the empty ziplock bag after the leaf is taken out under a stereoscope, and identifying and counting the species dropped into the bag.

[0028] For species that cannot be identified by morphology, the present invention uses molecular biological methods for identification, preferably including amplifying the mitochondrial cytochrome oxidase COI gene of the species and then sequencing it, comparing the obtained sequence with the database, and obtaining the species information of the sample arthropod. The present invention preferably transfers the species that cannot be directly identified to a 1.5mL centrifuge tube (the same species can be placed in one tube, numbered, and the number of individuals, collection time and location are recorded) for DNA identification; after rinsing the sample in the centrifuge tube with 75% alcohol, extract the total genomic DNA of the sample and store it at -20°C for standby; select the primers of the mitochondrial cytochrome oxidase COI gene for PCR amplification; the obtained PCR product is purified and sequenced.

[0029] The primers used for amplification of the present invention are preferably as follows:

[0030] COI-F(SEQ ID No.1):5'-ATTCAACCAATCATAAAGATATTGG-3';

[0031] COI-R (SEQ ID No. 2): 5'-TAAACTTCTGGATGTCCAAAAAATCA-3'.

[0032] The PCR reaction system used in the amplification of the present invention preferably includes, in 50 μL, KOD One TM PCRMasterMix-Blue 25μL, DNA template about 200ng / 50μL, forward and reverse primers (0.3μM) 1.5mL each, add distilled water to 50μL. PCR reaction conditions are as follows: 98℃ denaturation 10s; 52℃ annealing 5s; 68℃ extension 1s, 45 cycles.

[0033] The present invention detects the PCR product obtained after the PCR amplification by 1% agarose gel electrophoresis, cuts the target band and purifies and recovers it using a FastPure Gel DNA Extraction Mini Kit, and then performs sequencing; the sequence obtained in the sequencing result is compared with the public databases of BOLD (https: / / www.boldsystems.org / ) and NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) websites; if the sequence similarity is greater than or equal to 95%, it is confirmed to be the species.

[0034] In order to further illustrate the present invention, a field sampling method for leaf-dwelling arthropods provided by the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] Sampling and species identification of arthropods in soybean fields using single leaf bag trapping

[0037] 1) Wandou 28 (National Approved Bean 2008004) was used as the test material, and samples were taken at the soybean seedling stage, podding stage, and maturity stage. The test site was the South China Breeding Base of Zhejiang University in Sanya, Hainan Province.

[0038] 2) The experiment adopts a 5-point sampling method. On each sampling date, 5 points were taken diagonally, and 10 consecutive plants were collected at each point. The opening of a 12cm×18cm self-sealing bag was gently inserted from the tip of the leaf. After the entire leaf was in the bag, it was cut off from the petiole and the bag was quickly sealed. Pay attention to exhaust the air in the bag for easy carrying, but do not crush the arthropods in the self-sealing bag; take one leaf from the upper, middle and lower parts of each plant, mark it, and bring it back to the laboratory for identification and counting ( Figure 1 ).

[0039] 3) Under a stereoscope, carefully remove the leaf from the ziplock bag, identify and count the arthropods on the front and back of the leaf according to their morphology, and observe the empty ziplock bag after the leaf is removed under a stereoscope to identify and count the species that fall into the bag;

[0040] 4) Using the sweeping net sampling method as a control, on each sampling date, the diagonal five-point sampling method was used, using a portable sweeping net to sweep the plants on both sides of the sampling belt, sweeping each point 5 times (one time from left to right is 1 time, sweeping while walking, and the sampling area covers 10 plants). When sampling, one person sweeps the net, and the other is responsible for using an electric insect sucker to suck the arthropods in the mesh into a collection bottle filled with 75% alcohol (a brush can be used to assist the operation), and bring them back to the laboratory for identification and counting;

[0041] 5) Use a brush to transfer the arthropod samples in the collection bottle to a new culture dish. Pour a small amount of alcohol into the culture dish to keep the insects moist. Place the culture dish under a stereoscope, separate the arthropods by species, and then identify and count them based on their morphological characteristics;

[0042] 6) Transfer the species that cannot be identified and identified based on morphology in step 3) and step 5) into 1.5 mL centrifuge tubes (put the same species into one tube), number them, and wait for subsequent identification;

[0043] 7) After washing the sample in the centrifuge tube in step 6) with 75% alcohol, the total genomic DNA was extracted using Universal Genomic DNA Kit and stored at -20°C;

[0044] 8) Amplify the COI sequence of the sample using universal primers. The primer sequences are as follows:

[0045] COI-F:5'-ATTCAACCAATCATAAAGATATTGG-3'

[0046] COI-R:5'-TAAACTTCTGGATGTCCAAAAAATCA-3'

[0047] 9) PCR reaction system: KOD One TM PCRMasterMix-Blue 25μL, DNA template about 200ng / 50μL, forward and reverse primers (0.3μM) 1.5mL each, add distilled water to 50μL. PCR reaction conditions are as follows: 98℃ denaturation 10s; 52℃ annealing 5s; 68℃ extension 1s, 45 cycles.

[0048] 10) The PCR product obtained in step 10) was detected by 1% agarose gel electrophoresis ( Figure 2 ), the target band was cut out and purified using the FastPure Gel DNA Extraction Mini Kit, and then sequenced;

[0049] 11) Compare the sequence obtained in the sequencing result of step 11) with the public databases of BOLD (https: / / www.boldsystems.org / ) and NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) websites; if the sequence similarity is greater than or equal to 95%, it is confirmed to be the species.

[0050] Two methods were used to sample arthropods in the soybean experimental field in Sanya, Hainan Province. A total of 2,762 arthropods were captured by the single leaf bag method, and a total of 2,322 arthropods were captured by the sweep net method. The total number of arthropods, herbivorous insects, predatory natural enemies, and parasitic natural enemies captured by the single leaf bag method were all greater than those captured by the sweep net method, while the sweep net method captured more neutral insects (Table 1). The results of the analysis of arthropods by family classification showed that compared with the sweep net method, the single leaf bag method had a higher capture efficiency for Thripidae, Aphidoidea, and Aleyrodidae, and could capture a large number of arthropods with weaker activity that were difficult to obtain by the sweep net method, such as Tetranychidae, Pseudococcidae, and Phytoseiidae. The advantage of the sweep net method is that it can capture species with strong flying ability, such as Chironomidae, Muscidae, and Cicadelloidea. Figure 3 ).

[0051] The single leaf bag capture method described in the present invention has low cost, and the species captured are basically the same as those captured by the sweep net method. In addition, it can capture groups that cannot be captured by the sweep net method, and has a high capture efficiency for herbivorous pests, predatory natural enemies, and parasitic natural enemies. This shows that the present invention is suitable for collecting arthropods in soybean fields, and has good effects, and can replace some methods with complex operations and high economic costs.

[0052] Table 1 Number of arthropods of various groups captured by the two sampling methods

[0053]

[0054] Example 2

[0055] Sampling and species identification of arthropods in peanut fields using single leaf bag trapping

[0056] 1) Using Zhejianghua No. 2 (GPD Peanut (2022) 330016) as the experimental material, field arthropods were sampled during the flowering period. The experimental site was Changhe Town, Cixi, Zhejiang.

[0057] 2) Divide the peanut plant vertically into three equal parts, i.e. the top third is the upper part, the middle third is the middle part, and the bottom third is the lower part; gently put a ziplock bag (12×17cm) on a single leaf of the plant from the tip of the leaf, and cut it from the leaf stem; quickly seal the bag, paying attention to exhaust the air in the bag for easy carrying, but do not crush the arthropods in the ziplock bag. Randomly select 3 plots for sampling, and select 10 plants in each plot.

[0058] 3) Under a stereoscope, carefully remove the leaf from the ziplock bag, identify and count the arthropods on the front and back of the leaf according to their morphology, and observe the empty ziplock bag after the leaf is removed under a stereoscope to identify and count the species that fall into the bag; transfer the species that cannot be identified and identified based on morphology into a 1.5 mL centrifuge tube (put the same species into one tube), number them, and wait for molecular identification;

[0059] 4) Using the sweeping net method as a control, use a portable sweeping net to sweep the peanut plants on both sides of the sample strip back and forth, sweeping each plot 5 times (one sweep back and forth is counted as one sweep), for a total of 3 plots. When sampling, one person sweeps the net, and the other is responsible for using an electric insect sucker to suck the arthropods in the mesh into a collection bottle filled with 75% alcohol (a brush can be used to assist the operation).

[0060] 5) Use a brush to transfer the arthropod samples in the collection bottle to a new culture dish. Pour a small amount of alcohol into the culture dish to keep the insect body moist. Place the culture dish under a stereoscope, first separate the arthropods by species, and then identify and count them based on morphological characteristics; transfer species that cannot be identified and identified based on morphology to 1.5mL centrifuge tubes (put the same species in one tube), number them, and wait for molecular identification;

[0061] 6) The molecular identification step is the same as step 7) to step 11) in Example 1

[0062] The survey results showed that the total number of arthropods in peanut fields was relatively small, and the total number of arthropods obtained by the single leaf bagging method was more than that obtained by the sweep net method, especially arthropods of Hemiptera (such as Thripidae and Cicadidae pests), Acarina, Arachnida (predatory natural enemies) ( Figure 4 ).

[0063] In summary, the sampling method of the present invention has obvious advantages in investigating dicotyledonous arthropods, and the capture effect of leaf-dwelling arthropods such as Acarina, Aphididae, Cicadidae, etc. is significantly better than the sweeping net method. Compared with the sweeping net method, which requires transferring the insect body to the collection bottle in the field, the operation is more cumbersome and there is a possibility of escape or loss during the transfer process, and the sampling of the lower layer of the plant is incomplete, the single leaf bag capture method can significantly improve the efficiency of field sampling and the accuracy of the survey results.

[0064] The sampling method of the present invention is not only applicable to crops, but also to vegetables, flowers, low shrubs and the like.

[0065] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A field sampling method for leaf-dwelling arthropods, characterized in that: The method comprises the following steps: using a ziplock bag as a field sampling tool, putting the ziplock bag on plant leaves, and separating the plant and the leaves at the petiole.

2. The field sampling method according to claim 1, characterized in that: The length and width of the leaves of the plant are both less than 30 cm.

3. The field sampling method according to claim 1, characterized in that: Field sampling was performed on the upper, middle and lower parts of the plant respectively. When dividing the plant, the node from the bottom of the plant with fully expanded leaves to the node of the top of the plant with fully expanded leaves was regarded as a whole, and the whole was divided vertically into three parts, the top third was the upper part, the middle third was the middle part, and the bottom third was the lower part.

4. The field sampling method according to claim 3, characterized in that: During the field sampling, one leaf from the upper part, the middle part and the lower part of the plant is selected for field sampling respectively, and one leaf is put into each ziplock bag.

5. The field sampling method according to claim 4, characterized in that: When taking samples in the field, the specifications of the sampling ziplock bag are selected according to the growth of the leaves, the ziplock bag is opened, and it is gently put on a single leaf of the plant from the tip of the leaf and cut off at the petiole; Seal the bag quickly after cutting at the petiole and expel any excess air from the bag.

6. A method for identifying leaf-dwelling arthropods, characterized in that: The method comprises bringing the samples obtained by the field sampling method according to any one of claims 1 to 5 back to the laboratory and identifying the species by combining morphology and molecular biology.

7. The identification method according to claim 6, characterized in that: The morphological identification includes carefully taking the leaves out of the ziplock bag under a stereoscope, identifying and counting the arthropods on the front and back of the leaves, observing the empty ziplock bag after taking out the leaves under a stereoscope, and identifying and counting the species dropped into the bag.

8. The identification method according to claim 6, characterized in that: For species that cannot be identified by morphology, the mitochondrial cytochrome oxidase COI gene of the species is amplified and sequenced, and the obtained sequence is compared with the database to obtain the species information of the sample arthropod.

9. The identification method according to claim 8, characterized in that: The primers used for the amplification include COI-F with nucleotide sequences as shown in SEQ ID No.1 and COI-R with nucleotide sequences as shown in SEQ ID No.

2.

10. The identification method according to claim 8, characterized in that: The PCR reaction program for the amplification includes: denaturation at 98° C. for 10 seconds; annealing at 52° C. for 5 seconds; and extension at 68° C. for 1 second, for 45 cycles.

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