Method for evaluating influence of transgenic crops on soil microflora
By collecting and analyzing soil samples from different growth periods and optimizing the experimental process, the problem of time-consuming and costly evaluation of genetically modified crops on soil microbial communities in the existing technology is solved, and a simple, scientific and efficient evaluation method is achieved, ensuring the ecological safety assessment of genetically modified crops.
Patent Information
- Application Number
- CN202510410473.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-02
- Publication Date
- 2025-07-08
AI Technical Summary
The prior art has problems of high workload, time-consuming and expensive in evaluating the impact of genetically modified crops on soil microbial communities, making it difficult to comprehensively, quickly and scientifically evaluate their environmental safety.
By collecting soil samples from different growth periods, analyzing the impact of genetically modified crops and non-genetically modified control crops on cultureable microbial communities, a simple and scientific method is adopted, including soil sample processing, microbial culture and data analysis, and the experimental process is optimized to reduce unnecessary steps.
It has achieved simple, scientific and efficient assessment of the potential impact of genetically modified crops on soil microbial communities, reduced workload and economic costs, and provided an efficient ecological security assessment method.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical fields of soil microbiology and safety evaluation of genetically modified organisms, and particularly relates to a method for evaluating the impact of genetically modified crops on soil microbial communities. Background Art
[0002] Genetically modified crops have been widely used globally. However, after their foreign genes and expression products enter the soil, they may interact with soil microorganisms, thereby affecting the activities of microorganisms. Therefore, the impact of genetically modified crops on the quantity, community function, and structural diversity of soil microorganisms is one of the important indicators for evaluating their environmental safety.
[0003] Soil microbial communities play a crucial role in the ecosystem and directly affect plant growth and soil health. Therefore, evaluating the impact of genetically modified crops on soil microbial communities is an important link in ensuring ecological safety. Currently, common methods for evaluating the impact of genetically modified crops on soil microbial communities include molecular biology techniques, traditional microbial culture techniques, as well as biochemical analysis and functional assessment, etc. However, each of these methods has its own advantages and disadvantages and needs to be used in combination in practical applications in order to comprehensively evaluate the potential impact of genetically modified crops from multiple perspectives. Therefore, establishing a more simple, systematic, and scientific evaluation process and method can more quickly and accurately evaluate the impact of genetically modified crops on soil microbial communities and clarify the environmental safety of genetically modified crops.
[0004] Developing an evaluation method for the impact of genetically modified crops on soil microbial communities provides theoretical support and practical guidance for the ecological safety of genetically modified crops, which not only helps to comprehensively understand the potential impact of genetically modified crops on the soil ecosystem but also promotes the safe and sustainable application of genetically modified technology in agriculture. Summary of the Invention
[0005] The present invention provides a method for evaluating the impact of genetically modified crops on soil microbial communities, especially a simple, scientific, and highly operationally feasible evaluation process and technical method. This method optimizes and improves the problems of large workload, long time consumption, and high cost in existing evaluation methods, and can effectively evaluate the potential impact of genetically modified crops on soil microbial communities.
[0006] To achieve the above object, the present invention adopts the following technical solutions to be realized: The present invention provides a method for evaluating the impact of genetically modified crops on soil microbial communities, by collecting soil samples at different growth stages and analyzing the impact of genetically modified crops and non-genetically modified control crops on culturable microbial communities to comprehensively evaluate their potential impact on the soil ecosystem.
[0007] The present invention is applicable to various types of genetically modified crops, including but not limited to: RNAi crops, gene-edited crops, and crops into which exogenous genes are transferred.
[0008] The key steps of the method include: (1) Soil sample collection: Collect root soil samples before the planting of genetically modified crops, at different growth stages, and after harvest. Preferably, collect rhizosphere soil and rhizoplane soil samples of genetically modified crops; the sampling depth is 10 to 15 cm from the ground surface. Preferably, the growth stage is the critical period of crop growth.
[0009] (2) Sample treatment: Pass the soil sample through a 60-mesh sieve, add sterile water, and prepare a 10% soil suspension, and shake and mix it on a constant temperature shaker.
[0010] (3) Microbial culture: Using the culturable microorganism analysis method, dilute the soil suspension to an appropriate concentration according to the target microorganism category; the preferred dilution is: bacteria 10 -6 , fungi 10 -3 , actinomycetes 10 -4 .
[0011] (4) Colony counting: Uniformly coat the diluted soil suspension on an appropriate medium, observe and count the number of colonies after cultivation. Preferably, count after culturing bacteria for 48 hours, count after culturing fungi for 96 hours, and count after culturing actinomycetes for 120 hours.
[0012] (5) Data analysis: Use the analysis of variance method to significantly compare the differences in soil microbial numbers between genetically modified crops and non-genetically modified control crops at different growth stages.
[0013] The method of the present invention is also applicable to genetically modified crops with herbicide tolerance traits. By collecting rhizosphere soil and rhizoplane soil samples of herbicide-tolerant genetically modified crops at different growth stages before and after spraying target herbicides or non-target herbicides, a comparative analysis of soil microbial communities is carried out to further clarify the impact of herbicides on the soil microorganisms of herbicide-tolerant genetically modified crops.
[0014] The present invention is applicable to a variety of crops and their critical growth stages. For example: cotton: seedling stage, budding stage, flowering and boll stage, boll opening stage; rice: seedling stage, tillering stage, heading stage, flowering stage, yellow ripening stage; corn: early whorl stage, late whorl stage, ear stage, filling stage; soybean: trifoliate stage, flowering stage, podding stage, pod filling stage; alfalfa: seedling stage, budding stage, early flowering stage, podding stage; rape: seedling stage, seven-leaf stage, full flowering stage, podding stage, maturity stage.
[0015] Compared with the prior art, the present invention has good simplicity, scientificity and efficiency. Based on culturable microorganism analysis, the experimental process is optimized, the operation steps are clear, unnecessary operation steps are reduced, and the workload and economic cost are effectively reduced.
[0016] The present invention solves the problems of long time consumption and high cost of traditional methods through systematic design and scientific technical solutions. It not only provides a method for evaluating the impact of genetically modified crops on soil microbial communities, but also can evaluate the impact of spraying herbicides on the soil microbial communities of herbicide-tolerant crops. This technology provides an efficient and reliable evaluation method for the ecological safety assessment of genetically modified crops, and is of great significance for the sustainable use of genetically modified crops and environmental protection. Detailed implementation mode
[0017] The following is a detailed description of a method for evaluating the impact of genetically modified crops on soil microbial communities according to the present invention in conjunction with the embodiments. Embodiment
[0018] This embodiment provides a method for evaluating the impact of high-yield and high-quality genetically modified cotton on soil microbial communities, and the steps are as follows: (1) Collection of soil samples Plant high-yield and high-quality genetically modified cotton and its non-genetically modified control cotton. The plot area is 300 m 2 , with 3 replicates. Sow according to the local conventional sowing method and seeding rate, and carry out cultivation management according to the local conventional tillage management mode after sowing. Soil samples are collected respectively before cotton sowing, at the seedling stage, budding stage, flowering and boll stage, boll opening stage and after harvest.
[0019] (2) Sample treatment For each plot, use the diagonal 5-point sampling method to collect soil samples, cotton rhizosphere soil and rhizosphere soil, pass through a 60-mesh sieve and put them into polyethylene bags, and store them at 4°C. Before microbial cultivation, mix the soil samples, weigh 10 grams of soil samples on a super-clean workbench, add 90 ml of sterile water, and shake on a constant temperature shaker at 25°C and 150 rpm for 60 minutes to prepare a soil suspension.
[0020] (3) Microbial cultivation For the cultivation of soil bacteria, fungi and actinomycetes, use the plate dilution isolation and cultivation method. Use nutrient broth peptone medium for cultivating bacteria, Martin-Bengal red medium for cultivating fungi, and Gao's No. 1 medium for cultivating actinomycetes.
[0021] Dilute the soil suspension to an appropriate concentration, 10 -6 dilution for bacteria, 10 -3 dilution for fungi, 10 -4 dilution for actinomycetes.
[0022] (4)Colony counting Using a micropipette, 200 μL of the diluted soil suspension was aspirated and evenly spread onto the culture medium in a laminar flow hood. After allowing the Petri dishes to stand for 30 minutes, they were inverted and incubated at 25 °C. The number of bacterial colonies was counted after 48 hours, the number of fungal colonies was counted after 96 hours, and the number of actinomycete colonies was counted after 120 hours.
[0023] The soil water content was measured, and the number of culturable microorganisms was calculated according to the following formula:
[0024] The significance of the difference in the number of culturable soil microorganisms between the transgenic cotton fields and the corresponding non-transgenic cotton fields was compared using analysis of variance.
[0025] (5)Data analysis The analysis results showed that there were no significant differences in the numbers of culturable bacteria, fungi, and actinomycetes in the rhizosphere soil and rhizoplane soil between the transgenic cotton fields and the corresponding non-transgenic cotton fields before cotton sowing, at the seedling stage, budding stage, flowering and boll stage, boll opening stage, and after harvest (Table 1).
[0026] Table 1 Numbers of culturable microorganisms (CFU) in the fields of high-yield and high-quality transgenic cotton and non-transgenic cotton
[0027] Note: The data in the table are presented as mean ± standard deviation; different lowercase letters after the data in the same column at the same growth stage indicate significant differences between different varieties ( p < 0.05).
[0028] Example 2 This example provides a method for evaluating the impact of herbicide-tolerant transgenic soybeans on the soil microbial community, and simultaneously evaluates the impacts of target and non-target herbicides on the soil microbial community in herbicide-tolerant transgenic soybean fields. The steps are as follows: (1)Collection of soil samples Herbicide-tolerant transgenic soybeans and their non-transgenic control soybeans were planted with a 1 m isolation belt between plots. The plot area was 150 m 2 2, with 3 replicates. Sowing was carried out according to the conventional sowing method and seeding rate, and conventional tillage management was applied. Soil samples were collected before soybean sowing, at the trifoliate stage, flowering stage, pod-setting stage, grain-filling stage, and after harvest of the soybeans. Target herbicide (glyphosate isopropylammonium salt) and non-target herbicide (quizalofop-p-ethyl) were sprayed during the seedling stage (before the trifoliate stage) of soybean growth at the recommended application rates.
[0029] The treatment of soil samples, microbial culture, and colony counting were the same as in Example 1.
[0030] (2)Data analysis The analysis results show that before sowing the herbicide-tolerant genetically modified soybeans, at the trifoliate stage, flowering stage, pod-setting stage, grain-filling stage of soybeans, and after harvest, there were no significant differences in the numbers of culturable bacteria, fungi, and actinomycetes between the genetically modified soybean fields and the corresponding non-genetically modified soybean fields (Table 2).
[0031] Before and after spraying the target and non-target herbicides, at the sowing stage, trifoliate stage, flowering stage, pod-setting stage, grain-filling stage of soybeans, and after harvest, there were no significant differences in the numbers of culturable bacteria, fungi, and actinomycetes between the genetically modified soybean fields and the corresponding non-genetically modified soybean fields (Tables 3 and 4).
[0032] Table 2 Numbers of culturable microorganisms (CFU) in the fields of herbicide-tolerant genetically modified soybeans and non-genetically modified soybeans
[0033] Note: The data in the table are mean ± standard deviation; at the same growth stage, different lowercase English letters after the same column of data indicate significant differences between different varieties ( p < 0.05).
[0034] Table 3 Numbers of culturable microorganisms (CFU) in the fields of herbicide-tolerant genetically modified soybeans before and after spraying target herbicides
[0035] Note: The data in the table are mean ± standard deviation; at the same growth stage, different lowercase English letters after the same column of data indicate significant differences between different varieties ( p < 0.05).
[0036] Table 4 Numbers of culturable microorganisms (CFU) in the fields of herbicide-tolerant genetically modified soybeans before and after spraying non-target herbicides
[0037] Note: The data in the table are mean ± standard deviation; at the same growth stage, different lowercase English letters after the same column of data indicate significant differences between different varieties ( p < 0.05). Example
[0038] This example provides a method for evaluating the impact of insect-resistant genetically modified maize on soil microbial communities, and the steps are as follows: (1)Collection of soil samples Plant insect-resistant genetically modified maize and its non-genetically modified control maize, with a plot area of 150 m 2, repeated 3 times, arranged randomly, with a 1 m isolation belt set in the plot. Sow according to the conventional sowing method and sowing rate, and conduct conventional tillage management. Collect soil samples before sowing maize, at the early stage of the maize heart leaf, at the late stage of the heart leaf, at the ear stage, at the filling stage, and after harvest respectively.
[0039] The treatment of soil samples, the cultivation of microorganisms, and the colony counting are the same as in Example 1.
[0040] (2)Data analysis The analysis results show that there are no significant differences in the numbers of culturable bacteria, fungi, and actinomycetes between the transgenic maize fields and the corresponding non-transgenic maize fields before sowing maize, at the early stage of the maize heart leaf, at the late stage of the heart leaf, at the ear stage, at the filling stage, and after harvest (Table 5).
[0041] Table 5 Culturable microorganism numbers (CFU) in the fields of insect-resistant transgenic maize and non-transgenic maize
[0042] Note: The data in the table are mean ± standard deviation; at the same growth stage, different lowercase English letters after the data in the same column indicate significant differences between different varieties ( p < 0.05).
[0043] The above examples are only for the description of the preferred embodiments of the present invention and do not constitute a limitation on the scope of the present invention. Without departing from the design concept of the present invention, various modifications, deformations, or improvements made by those skilled in the art to the technical solutions of the present invention should be included within the protection scope defined by the claims of the present invention.
Claims
1. A method for evaluating the impact of genetically modified crops on soil microbial communities, characterized in that, It includes the following steps: (1) Soil sample collection: Collect rhizosphere soil and rhizoplane soil samples before the planting of genetically modified crops, at different growth stages and after harvest, with the soil sample collection depth being 10 to 15 cm from the ground surface; (2) Soil sample treatment: Pass the soil sample through a 60-mesh sieve, add sterile water to prepare a 10% (mass fraction) soil suspension, and shake and mix it evenly on a constant temperature shaker; (3) Microbial culture: According to the target microbial category, the soil suspension is diluted to an appropriate concentration respectively. For bacteria, a dilution of 10 -6 is used; for fungi, a dilution of 10 -3 is used; for actinomycetes, a dilution of 10 -4 is used; (4) Colony counting: Uniformly coat the diluted soil suspension onto the culture medium for cultivation. Count the number of colonies after 48 hours of bacterial cultivation, 96 hours of fungal cultivation, and 120 hours of actinomycete cultivation; (5) Data analysis: Use the analysis of variance method to significantly compare the differences in the number of soil microbial communities between genetically modified crops and non-genetically modified control crops at different growth stages.
2. The method according to claim 1, wherein The genetically modified crops mentioned include, but are not limited to, RNAi crops, gene-edited crops, and genetically modified crops into which exogenous genes are introduced.
3. The method according to claim 1, wherein The growth stages for soil sample collection are the key growth stages of the crops, including, but not limited to: the seedling stage, budding stage, flower-boll stage, boll-opening stage of cotton; the seedling stage, tillering stage, heading stage, flowering stage, yellow-ripening stage of rice; the early whorl stage, late whorl stage, heading stage, filling stage of maize; the trifoliate stage, flowering stage, pod-setting stage, pod-filling stage of soybean; the seedling stage, budding stage, early flowering stage, pod-setting stage of alfalfa; the seedling stage, seven-leaf stage, full-bloom stage, pod-setting stage, maturity stage of rapeseed.
4. The method according to claim 1, characterized in that For herbicide-tolerant genetically modified crops, collect rhizosphere soil and rhizoplane soil samples at different growth stages of herbicide-tolerant genetically modified crops before and after spraying target herbicides and non-target herbicides, and evaluate the impact of herbicides on the soil microbial communities of herbicide-tolerant genetically modified crops through comparative analysis of the number of microbial communities.
5. The method according to claim 1, characterized in that, The diluted soil suspension is inoculated onto the surface of the culture medium by the uniform coating method, the number of colonies is observed and recorded, and finally, the impact of genetically modified crops and non-genetically modified control crops on soil microbial communities is evaluated through significance analysis.
6. The method according to claim 1, characterized in that This method is applicable to the ecological safety assessment of various crops, especially applicable to the key growth stages of crops.