An evaluation method for the impact of cosmos extract on soil ecological environment based on the distribution changes of small and medium-sized soil animals

Abstract

The present invention discloses an evaluation method of the effect of peacock grass extract on soil ecological environment based on the distribution change of small and medium-sized soil animals, comprising the following steps: preparing a solution of thiophene derivatives of peacock grass roots, watering peacock grass soil samples in a dark environment until water drips from the lower end, and dividing each treatment into two groups: one group is left to stand for 24 hours, and the other group is left to stand for 48 hours, and the surface is covered with a 60-mesh nylon net during the standing period; the peacock grass soil samples are respectively placed in dry and wet funnels in an insect drying box to separate small and medium-sized soil animals, and a light-insulating but heat-insulating dyeing film is pulled between the funnel in the insect drying box and the incandescent lamp; the number of each type of small and medium-sized soil animals is counted and data processing is performed. The present invention conducts a wider and higher ecological evaluation of the effect of peacock grass thiophene derivatives based on small and medium-sized soil animals, and avoids the influence of light in the process of separating soil animals, so that the evaluation results are closer to the actual situation.

Description

Technical Field

[0001] The invention belongs to the technical field of soil ecology, and in particular relates to an evaluation method for the impact of a cosmos extract on the soil ecological environment based on the distribution changes of small and medium-sized soil animals. Background Art

[0002] Marigold is a garden plant with large number, long flowering period, special smell and few diseases and pests. It is widely used as potted flowers, flower beds or cut flowers in the southwest region. Marigold has direct or indirect impact on the natural ecosystem in which it is located by releasing chemicals into the soil environment. As the most biodiverse biological environment on earth, soil plays an important role in maintaining the balance of terrestrial ecosystems. Soil animals living in the soil where Marigold is rooted are the bridge and link between the aboveground / underground ecosystems. They participate in almost all ecological processes of the soil ecosystem and play an extremely important role in promoting material circulation, producing soil nutrients, improving soil structure and purifying the underground environment. Evaluating the ecological impact of thiophene derivatives of Marigold's roots on small and medium-sized soil animals is of great significance for evaluating the ecological role of Marigold in its rooted soil.

[0003] Over the past few decades, research on the impact of cosmos on soil animal ecology has gradually deepened. However, current research methods still show two shortcomings.

[0004] On the one hand, people's research methods mainly use the extracted chemical substances secreted by the roots of cosmos to directly treat large soil animals (earthworms, ants, etc.) collected or purchased from other regions that are unrelated to the soil environment of the plant. Only the changes in indicators related to the activity of the soil animals can be observed. The evaluation perspective is limited to individual species and is at a low level. It is impossible to conduct an ecological evaluation of the effects of the plant's allelopathic substances from a higher population or community dimension.

[0005] On the other hand, because the thiophene derivatives that play a role in the ecological impact of cosmos on soil animals have extremely high photoactivation properties, their effectiveness on soil animals will be greatly improved under the action of light. It is a conventional method to separate and count soil animals in the insect drying box by using the characteristics of soil animals that tend to move towards darkness, moisture, and avoid high temperatures, but light will affect the effect of cosmos thiophene derivatives, and in reality, the underground soil is a lightless environment, so it is not conducive to simulating the ecological impact of cosmos thiophene derivatives on soil animals in actual situations. Summary of the invention

[0006] In response to the above-mentioned prior art, the present invention provides an evaluation method for the impact of cosmos extract on the soil ecological environment based on the distribution changes of small and medium-sized soil animals, so as to solve the problem that the existing evaluation method can only observe the changes in indicators related to the activity of the soil animals, the evaluation angle is limited to the individual species, the level is low, and it is unable to simulate a lightless environment.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a method for evaluating the impact of cosmos extract on soil ecological environment based on the distribution changes of small and medium-sized soil animals, comprising the following steps:

[0008] (1) extracting cosmos and preparing the extract into a solution with a concentration of 0.005 g / L to 0.5 g / L, and using distilled water as a control treatment;

[0009] (2) The soil samples of peacock grass were irrigated with peacock grass extract solution in a dark environment until water dripped from the bottom, and each treatment was divided into two groups: one group was left to stand for 24 hours, and the other group was left to stand for 48 hours. During the standing period, the surface was covered with a 60-mesh nylon mesh;

[0010] (3) After standing, the soil samples from the peacock grassland were placed in the dry and wet funnels in the insect drying box to separate the small and medium-sized soil animals. A light-isolating but not heat-insulating dye film was placed between the funnel and the incandescent lamp in the insect drying box, and the surface of the dry funnel was covered with a 60-mesh nylon mesh. Water was added during the separation of the wet funnel to keep the soil sample moist. The temperature in the insect drying box was 35-40℃, and the separation time was 48 hours. The wet funnel was separated and the samples were collected once at 4h, 12h, 24h, and 48h; the dry funnel was separated and the samples were collected once at 24h and 48h.

[0011] (4) After the separation is completed, the small and medium-sized soil animals are classified and identified, the number of each type of small and medium-sized soil animals is counted and data processing is performed to evaluate the impact of cosmos extract on the soil ecological environment.

[0012] Based on the above technical solution, the present invention can also be improved as follows.

[0013] Furthermore, the cosmos extract is a thiophene derivative.

[0014] Furthermore, thiophene derivatives are prepared by the following steps: taking the root powder of marigold and mixing it with petroleum ether in a solid-liquid ratio of 1:10, stirring and reflux extraction in a 65°C water bath for 1 hour, reflux once, then filtering, and distilling the filtrate to a viscous state to obtain a thiophene extract paste crude product, which is then separated and purified.

[0015] Further, the separation and purification includes the following steps: separation and purification adopts high-speed countercurrent chromatography, rotation speed: 800rpm, mobile phase flow rate: 2mL·min -1The solvent system was a mixture of n-hexane, dichloromethane and acetonitrile in a volume ratio of 10:3:7, and the loading amount and the solid-liquid ratio of the mixed solution of the stationary phase and the mobile phase were 200 mg:10 mL.

[0016] Furthermore, soil samples from the peacock grassland were prepared by the following steps: after taking soil from the sample site with a ring knife, the ring knife was sealed with a 60-mesh nylon net, and then the ring knife was placed in a black cloth bag and stored in a fresh-keeping box.

[0017] Furthermore, small and medium-sized soil animals include rotifers, nematodes, mites, collembolans, and protoceras.

[0018] Furthermore, the data processing includes collating the richness index, diversity index, dominance index, evenness index and dominance index of small and medium-sized soil animals.

[0019] The beneficial effects of the present invention are:

[0020] (1) Compared with large soil animals, small and medium-sized soil animals are more sensitive to environmental changes and respond more quickly. The use of small and medium-sized soil animals can provide more timely feedback and more comprehensive reflection of the impact of plant allelopathic substances on soil animals and soil ecology.

[0021] (2) The small and medium-sized soil animals used were isolated from the original soil rather than from the soil animals in unrelated soils. The cumulative effect of the soil itself on the small and medium-sized soil animals of the experimental objects was taken into account, making the experimental materials closer to the natural state of soil animals in the soil environment where cosmos grows.

[0022] (3) The results obtained by the evaluation method of the present invention include parameters that include both the changes in the number of individuals of small and medium-sized soil animal species and the changes in the comprehensive number of small and medium-sized soil animal populations. Compared with the current observation angle that is limited to the activity changes of a certain large soil animal individual, it can more intuitively reflect the ecological impact of thiophene derivatives from the roots of cosmos on soil animals.

[0023] (4) The present invention is carried out in darkness throughout the indoor operation process, especially adding a layer of dye film with light-isolating but not heat-isolating properties in the insect drying box to simulate the underground soil environment to create a lightless condition, thereby avoiding the situation where the photoactivation characteristics of the thiophene derivatives of cosmos cause a significant deviation from the actual data and inaccurate evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figures 1 to 4 The diversity of small and medium-sized dry soil animals after the treatment of thiophene extracts from the roots of cosmos;

[0025] Figures 5 to 8 The diversity of small and medium-sized wetland soil animals after treatment with thiophene extracts from the roots of Marigold. DETAILED DESCRIPTION

[0026] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0027] Example 1

[0028] A method for evaluating the impact of cosmos extract on soil ecological environment based on the distribution changes of small and medium-sized soil animals, comprising the following steps:

[0029] (1) Three urban green spaces with basically the same site conditions and planted with cosmos were selected to set up sample plots, with three replicates in each location, for a total of nine sample plots;

[0030] (2) Take 48 soil samples from each plot using a 100 ml ring cutter and number them. Seal the ring cutter with a 60-mesh nylon net and then put the ring cutter into a black cloth bag and store it in a fresh-keeping box.

[0031] (3) Weigh the coarse powder of the root of cosmos and place it in a round-bottom flask, add petroleum ether in a ratio of 1:10 between solid and liquid, stir and reflux in a water bath at 65°C for 1 hour, reflux once, filter with a Buchner funnel while hot, collect the filtrate, and distill the filtrate under reduced pressure in a rotary evaporator until it becomes viscous to obtain a paste of crude thiophene extract of the root of cosmos. Use high-speed countercurrent chromatography to separate and purify the thiophene substances in the paste to obtain thiophene substances with a purity of more than 99% (the instrument is GS10AB- A semi-preparative HSCCC, column volume: 230mL, rotation speed: 800rpm, mobile phase flow rate: 2mL·min-1. Solvent system: n-hexane. dichloromethane, acetonitrile (10:3:7, v / v), sample load is 200mg, dissolved in 10mL mixed solution of stationary phase and mobile phase) and prepared into three concentration gradient solutions of 0.005g / L, 0.05g / L, and 0.5g / L, with distilled water as control treatment, and the above steps were carried out in the dark;

[0032] (4) 48 soil samples from peacock grass were irrigated with 0.005 g / L, 0.05 g / L, and 0.5 g / L peacock grass root thiophene derivative solutions or distilled water in a dark environment until water dripped from the bottom. The soil samples after each concentration treatment were divided into two groups: one group was left to stand for 24 h, and the other group was left to stand for 48 h. During the standing period, the surface was covered with a 60-mesh nylon net to prevent soil animals from escaping; (3 concentrations × 3 repetitions × 2 durations × 2 funnel experimental groups + 3 repetitions × 2 durations × 2 funnel blank controls)

[0033] (5) After standing, the circular cutter containing the soil sample of peacock grassland was placed in the dry and wet funnels in the insect drying box to separate small and medium-sized soil animals, and a light-proof but heat-proof dye film (closely fitting with the drying box) was pulled between the funnel in the insect drying box and the incandescent lamp, and the surface of the dry funnel was covered with a 60-mesh nylon mesh; water was added to keep the soil sample moist during the separation of the wet funnel to facilitate the smooth separation of hygroscopic soil animals; the temperature in the insect drying box was 37°C, and the separation time was 48 hours, during which the wet funnel was separated and collected once at 4 hours, 12 hours, 24 hours, and 48 hours; the dry funnel was separated and collected once at 24 hours and 48 hours.

[0034] (6) After separation, small and medium-sized soil animals were classified and identified, and the number of each type of small and medium-sized soil animals was counted and the data were sorted using Microsoft Excel and SPSS13.0. The diversity of soil animal community structure was calculated using the Margalef richness index (M), Shannon-Wiener diversity index (H'), and Simpson dominance index (C).

[0035] Table 1 is a classification table of treatment methods for 48 soil samples in Example 1.

[0036] Table 1 Soil sample treatment and separation methods of small and medium-sized soil animals

[0037]

[0038] The treatment data obtained in Example 1 show the diversity of small and medium-sized dry soil animals after treatment with thiophene extracts from the roots of cosmos. Figures 1 to 4 As shown, the influence trends of thiophene extracts from the roots of cosmos on the individual density, density group index DG and diversity index H' of small and medium-sized dry soil animals treated for 24h and 48h were generally consistent, and gradually decreased with the increase of the concentration of thiophene extracts from the roots of cosmos; the dominance index C showed an opposite trend, and gradually increased with the increase of the concentration of thiophene extracts from the roots of cosmos.

[0039] Variance analysis showed that when the treatment time was 24h, the individual density of small and medium-sized dry soil animals was significantly different at different concentrations. The density group index DG, diversity index H' and dominance index C under the control group treatment were significantly different from those under 0.005g / L, 0.05g / L and 0.5g / L treatments (P<0.05). When the treatment time was 48h, the individual density, density group index DG and diversity index H' under the control group treatment were significantly different from those under 0.005g / L, 0.05g / L and 0.5g / L treatments (P<0.05). The dominance index C under 0.05g / L and the control group and 0.5g / L treatments was significantly different (P<0.05).

[0040] The treatment data obtained in Example 1 show the diversity of small and medium-sized wetland soil animals after treatment with thiophene extracts from the roots of cosmos. Figures 5 to 8 As shown, the influence trends of thiophene extracts from the roots of cosmos on the individual density, density group index DG and diversity index H' of small and medium-sized dry soil animals treated for 24h and 48h were generally consistent, and gradually decreased with the increase of the concentration of thiophene extracts from the roots of cosmos; the dominance index C showed an opposite trend, and gradually increased with the increase of the concentration of thiophene extracts from the roots of cosmos.

[0041] Variance analysis showed that when the treatment time was 24h, the individual density of 0.5g / L and the control treatment, 0.05g / L and 0.5g / L treatments were significantly different (P<0.05). The density group index DG and diversity index H' of the control treatment, 0.005g / L and 0.5g / L treatments were significantly different (P<0.05). The dominance index C of the control treatment and 0.5g / L treatment was significantly different (P<0.05). When the treatment time was 48h, the density group index DG of small and medium-sized wet soil animals in the control treatment, 0.05g / L treatment and 0.5g / L treatment was extremely significantly different (P<0.01). The density-group index DG of 0.005g / L and 0.05g / L treatments was significantly different (P<0.05), and was extremely significantly different from the 0.5g / L treatment (P<0.01). The individual density of blank control group, 0.005g / L, 0.05g / L and 0.5g / L treatments were significantly different (P<0.01). There was no significant difference in the diversity index and dominance index of small and medium-sized wetland soil animals under different concentrations of thiophene extracts from the roots of cosmos (P>0.05).

[0042] The results showed that when the treatment time was 24h and 48h, the influence trend of different concentrations of thiophene derivatives from the roots of cosmos on the individual density, density group index DG and diversity index H' of small and medium-sized dry and wet soil animals was consistent, and the overall performance was a decrease with the increase of concentration; the dominance index C showed an increase with the increase of treatment concentration. This is similar to the results of field observation experiments, which shows that thiophene derivatives from the roots of cosmos can directly produce an inhibitory ecological effect on small and medium-sized dry and wet soil animals, and have a negative impact.

[0043] Although the specific implementation of the present invention is described in detail in conjunction with the drawings, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A method for evaluating the impact of cosmos extract on soil ecological environment based on the distribution changes of small and medium-sized soil animals, characterized in that: The following steps are involved: (1) Extract the cosmos and prepare the extract into a solution with a concentration of 0.005 g / L to 0.5 g / L, and use distilled water as a control treatment; (2) The soil samples of peacock grass were irrigated with peacock grass extract solution in a dark environment until water dripped from the bottom. Each treatment was divided into two groups: one group was left to stand for 24 hours, and the other group was left to stand for 48 hours. The surface was covered with a 60-mesh nylon mesh during the standing period. (3) After standing, the soil samples from the peacock grassland were placed in the dry and wet funnels in the insect drying box to separate small and medium-sized soil animals. A light-proof but heat-proof dye film was placed between the funnel and the incandescent lamp in the insect drying box, and the surface of the dry funnel was covered with a 60-mesh nylon mesh. Water was added during the separation of the wet funnel to keep the soil sample moist. The temperature in the insect drying box was 35-40°C, and the separation time was 48 hours. The wet funnel was separated and collected once at 4 hours, 12 hours, 24 hours, and 48 hours; the dry funnel was separated and collected once at 24 hours and 48 hours. (4) After separation, the small and medium-sized soil animals were classified and identified, the number of each type of small and medium-sized soil animals was counted and data was processed to evaluate the impact of cosmos extract on the soil ecological environment.

2. The evaluation method according to claim 1, characterized in that: The cosmos extract is a thiophene derivative.

3. The evaluation method according to claim 2, characterized in that: The thiophene derivatives are prepared by the following steps: mixing the root powder of marigold with petroleum ether at a solid-liquid ratio of 1:10, stirring and refluxing in a 65°C water bath for extraction for 1 hour, refluxing once, then filtering, distilling the filtrate to a viscous state, and obtaining a thiophene extract paste crude product, which is then separated and purified.

4. The evaluation method according to claim 3, characterized in that: The separation and purification comprises the following steps: The separation and purification adopts high-speed countercurrent chromatography, with a rotation speed of 800 rpm and a mobile phase flow rate of 2 mL·min -1 The solvent system was a mixture of n-hexane, dichloromethane and acetonitrile in a volume ratio of 10:3:7, and the loading amount and the solid-liquid ratio of the mixed solution of the stationary phase and the mobile phase were 200 mg:10 mL.

5. The evaluation method according to claim 1, characterized in that: The peacock grassland soil sample is prepared by the following steps: after taking soil from the sample site with a ring knife, the ring knife is sealed with a 60-mesh nylon net, and then the ring knife is placed in a black cloth bag and stored in a fresh-keeping box.

6. The evaluation method according to claim 1, characterized in that: The small and medium-sized soil animals include rotifers, nematodes, mites, collembolans, and protoceras.

7. The evaluation method according to claim 1, characterized in that: The data processing includes collating the richness index, diversity index, evenness index and dominance index of small and medium-sized soil animals.

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