Soil field in-situ remediation method based on crop rhizosphere indigenous microorganism population

Abstract

The invention discloses a soil field in-situ remediation method based on crop rhizosphere indigenous microorganism populations, which comprises the following steps: carrying out integral in-situ propagation on the crop rhizosphere indigenous microorganism populations by utilizing in-situ soil so as to directionally enrich and proliferate the indigenous microorganism populations in the soil; the microbial population urgently needed by crops can be accurately supplemented by utilizing healthy microbial populations in the soil and directionally enriching and proliferating indigenous microbial populations in the soil, so that the problem of deficiency of microorganisms in the soil is effectively solved, and the number and activity of the microorganisms in the soil, especially the core microbial functional group needed by the roots of the crops, are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of soil remediation, and in particular to a microbial in-situ remediation method for compensating for the damage of root soil microbial communities caused by abuse of pesticides and herbicides, continuous cropping obstacles and land salinization. Background Art

[0002] Microorganisms are an indispensable part of the ecosystem and play a vital role in the material cycle of the earth. In the soil, a common habitat, plants and microorganisms form a close mutualistic and symbiotic relationship. Sugars, organic acids, amino acids and other nitrogen-containing and non-nitrogen-containing compounds secreted by plant roots are the nutrient sources of microorganisms. Through their powerful decomposition and transformation capabilities, microorganisms provide plants with valuable nutrient resources and beneficial substances such as growth stimulants, further promoting the growth and development of plants. At the same time, a healthy rhizosphere microbial community can effectively inhibit the growth and spread of plant pathogens and provide a natural protective barrier for plant growth. However, in actual farming processes, factors such as excessive use of pesticides, abuse of herbicides, and metabolic waste generated by continuous cropping obstacles have caused serious damage to soil microorganisms, which not only disrupted the normal structure of the microbial community, but also greatly weakened the fertility of the soil. More seriously, these unfavorable factors also destroyed the balance of the microbial community in the roots of crops, resulting in limited growth of crops and susceptibility to disease. To address this issue, the current common practice is to apply a single (or composite) exogenous microorganism in liquid or powder form to the soil to increase its number. These single exogenous microbial preparations cannot meet the natural environment's demand for microbial population diversity. It is difficult for exogenous organisms to integrate into native microbial communities and work synergistically with indigenous microorganisms, making it difficult for them to effectively colonize and survive in the soil for a long time. In addition, exogenous microbial products often face challenges of reduced activity and high costs during long-distance transportation and storage. "A Method for Improving Saline-Alkali Land" (Application No.: 202110853586.) proposes an innovative strategy that aims to prepare a composite bacterial agent by isolating and culturing microorganisms that are dominant in number from the original soil, and then compounding them with conventional strains. Although this method is only for saline-alkali land, it can also solve the limitations of single exogenous microbial preparations and the challenges of increased long-distance transportation costs and low activity. However, this method has several defects in actual operation: first, although separation technology is a basic operation in microbiology, it requires the operator to have rigorous training and long-term laboratory experience; second, most microorganisms in nature cannot be isolated and cultured by existing pure culture methods of microorganisms, so many microorganisms are uncultivable microorganisms; third, although there is a certain concept of complex microorganisms, its method cannot restore the overall microbial community in the soil; fourth, it fails to consider the rhizosphere microbial functional groups in the soil, especially the growth-promoting population, and the core functional microbial groups in the rhizosphere are the purpose of agricultural soil remediation, and their complexity and diversity are crucial to plant growth and development. Therefore, this method only focuses on a large number of cultivable microorganisms, ignoring those uncultivable microorganisms in the soil that are difficult to reproduce by separation methods, and these microbial groups may be important functional groups in the construction of microbial communities and play an indispensable role in the functioning of plants.In addition, due to their different growth characteristics and physiological needs, different crops will secrete unique compounds from their roots, thereby attracting and enriching specific microbial communities to serve them. To meet the personalized needs of different crops, this method is difficult to adapt to the relatively fixed composite microbial agents, which to some extent limits its efficiency and universality in practical applications.

[0003] Therefore, there is an urgent need for a method that can effectively solve the lack of microorganisms in the soil and significantly increase the number and activity of soil microorganisms, especially the core microbial functional groups required by crop roots. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an on-site in-situ soil remediation method based on indigenous microbial populations in the crop rhizosphere, which utilizes the healthy microbial population in the soil and, through the targeted enrichment and proliferation of indigenous microbial populations in the soil, can accurately supplement the microbial populations urgently needed by crops, effectively solving the problem of microbial deficiency in the soil, and significantly improving the number and activity of soil microorganisms, especially the core microbial functional groups required by crop roots.

[0005] The soil in-situ remediation method based on the indigenous microbial population in the crop rhizosphere of the present invention utilizes the in-situ soil to carry out the overall in-situ expansion of the indigenous microbial population in the crop rhizosphere, so as to enrich and proliferate the indigenous microbial population in the soil in a targeted manner;

[0006] Furthermore, the in-situ soil is used to quickly cultivate a soil remediation solution on-site that can compensate for the lack of soil rhizosphere microorganisms;

[0007] Further, the following steps are included:

[0008] S1, soil in situ activation;

[0009] S2, preparing bacterial suspension: preparing bacterial suspension using activated soil; or preparing bacterial suspension using root washing water of plants in activated soil;

[0010] S3, fermentation of bacterial suspension to obtain soil remediation solution;

[0011] S4, applying the repair solution to the soil;

[0012] Furthermore, in step S1, a soil activator is sprayed on the soil for in-situ activation.

[0013] Further, in step S2, soil sampling or plant sampling of the target bacterial suspension is performed one week after soil activation;

[0014] Further, in step S2, sterile water is added to the sampled soil to prepare a bacterial suspension, or the roots of the plants are washed with sterile water to prepare a bacterial suspension;

[0015] Further, in step S3, the bacterial suspension and the soil activator are mixed for fermentation;

[0016] Further, in step S3, a small-scale fermentation device is first used for fermentation, and then a large-scale fermentation device is used for fermentation to achieve proliferation and expansion of a large number of microorganisms;

[0017] Furthermore, in step S4, the soil remediation solution is diluted and drip-irrigated to the agricultural operation area where the in-situ soil belongs.

[0018] Beneficial effects of the present invention: The soil in-situ remediation method based on the indigenous microbial population in the crop rhizosphere of the present invention utilizes healthy microbial communities in the soil, especially specific microbial communities that are attracted and enriched by the roots of different crops due to their different growth characteristics and physiological needs. By directional enrichment and proliferation of indigenous microbial communities in the soil, the microbial populations urgently needed by crops can be accurately supplemented, effectively solving the problem of microbial deficiency in the soil, and significantly improving the number and activity of soil microorganisms, especially the core microbial functional groups required by the roots of crops. Through the directional promotion and optimal utilization of local microbial resources, the native synergy and co-amplification of indigenous microbial populations can meet the needs of different crops for different microbial communities, and also avoid the problem that foreign microbial communities are difficult to adapt and colonize in complex agricultural environments. Through the method of the present invention combined with an in-situ automatic expansion device and commercial organic preparations, replenishable beneficial indigenous microbial preparations can be immediately cultivated on site, achieving rapid and accurate supplementation of urgently needed microorganisms, and efficiently improving biomass and biological functions for agricultural soils. It is a "liquid composting technology" idea. The operation is easy to operate, economical, efficient and precise. It can supplement the problems of soil salinization, compaction and so on caused by the lack of microorganisms due to the application of pesticides and herbicides. It meets the actual needs of most agricultural planting scenarios and has wide versatility.

[0019] The advantages of the present invention are:

[0020] (1) No artificial assembly and manipulation is required, and the biomass that is largely compensated is the complete indigenous microbial community required by crops, avoiding interference from foreign organisms and restoring the overall microbial ecological community, ecological niche and ecological relationship, including core functional groups and redundant functional groups.

[0021] (2) Some rare populations or “unculturable microbial” populations using traditional pure culture techniques, which are likely to be too important to be isolated and studied, can be cultured synchronously and in the same proportion using the new method, thus avoiding the omission of unrecognized populations and forming a stable and balanced ecosystem.

[0022] (3) The microbial separation step is omitted, so that it does not require the operation of people with specialized technical training, which lowers the technical threshold and avoids human operation errors; it reduces the tedious separation process, saves a lot of separation time, and can quickly support agricultural applications.

[0023] (4) The use of rhizosphere bacterial suspension can meet the different needs of different crops and obtain targeted microbial populations, especially a variety of growth-promoting populations that regulate nitrogen cycles and plant hormones (such as auxins).

[0024] (5) In-situ expansion has high activity, low cost and easy operation. Liquid fermentation can immediately turn it into water-soluble fertilizer and can enter the fruit and vegetable liquid fertilizer system, which is crucial to the support and application of the agricultural industry. DETAILED DESCRIPTION

[0025] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples.

[0026] In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0027] Embodiment 1

[0028] (1) Select healthy tomatoes and add liquid beef extract medium to the soil around them as an activator to awaken the dormant microorganisms in the soil. Beef extract medium: 0.3g beef extract, 1.0g peptone, 0.5g sodium chloride, 100ml water.

[0029] (2) One week after applying the soil activator, pull out the tomatoes, remove the loose soil on the roots, immerse the roots in the prepared sterile water and wash for 5 minutes. After standing, take the supernatant for later use;

[0030] (3) Use the soil activation station that has been set up on site, which includes a level I expansion unit (a 5L aerated and stirred small bucket) and a level II expansion unit (a 100L aerated large bucket). Add 950mL of liquid culture medium and 50mL of bacterial suspension to the level I expansion system, add 49L of liquid culture medium to the level II expansion system, inoculate the level I fermentation liquid into the level II expansion unit, and cultivate it under aeration at room temperature for 3 days to obtain a strongly reduced culture solution;

[0031] (4) The plate dilution counting method showed that the process could increase the biomass to 10 8 CFU / g or more. After the strong reduction culture solution was diluted 50 times, spraying it on tomatoes also compensated for the lack of soil flora. See Table 1 for details.

[0032] (5) Compared with the control group, tomato yield increased by about 28%, and the fruit tasted richer; soil microbial detection showed that the biomass in the soil increased significantly. Compared with the microbial dormancy caused by long-distance transportation, the amount of active microorganisms in the soil after in-situ cultivation increased significantly. See Table 1 for details.

[0033] Through the above steps, the chemo-organic aerobic bacteria, which are the majority of decomposers in the soil and are important groups that maintain the soil microbial community, including bacteria, actinomycetes and yeast groups of fungi, can rapidly proliferate by using the oxygen-rich environment and organic liquid culture medium. It can be used in the base fertilizer and topdressing period, and is also suitable for the application of pesticides, herbicides and crop rotation barriers.

[0034] Table 1. Comparison of biomass and yield

[0035]

[0036] Note: The tomato varieties, light and rainfall and other natural growth environments of the experimental group and the control group were the same

[0037] Embodiment 2

[0038] (1) Select healthy kiwifruit roots, add liquid beef extract culture medium (same as in Example 1) as an activator to the surrounding soil to awaken the dormant microorganisms in the kiwifruit roots;

[0039] (2) One week after applying the soil activator, cut 10 cm from the root of the kiwifruit with a sterile knife. 2 , remove the floating soil on the roots, immerse the roots in the prepared sterile water for 5 minutes, let it stand and take the supernatant for later use;

[0040] (3) Establish an on-site soil flora activation station, which includes a level I expansion unit (5L aerated and stirred small barrel), a level II expansion unit (100L aerated large barrel) and a level III expansion tank (1 ton tank, aerated). 50 mL of bacterial suspension was added to 950 mL of liquid culture medium for level I expansion (3 days), 1L of mother stock solution was added to 49L of liquid culture medium for level II expansion (3 days), and 50L of level II expansion solution was transferred to 450L for level III expansion. After 3 days of aeration cultivation at room temperature, it became a strongly reduced culture medium;

[0041] (4) The plate dilution counting method showed that the process could increase the biomass by 10 6 -10 8 CFU / g or more. After the strong reduction culture solution was diluted 50 times, it was used to irrigate the roots of kiwifruit to make up for the lack of kiwifruit soil flora. See Table 2 for details.

[0042] (5) Compared with the control group, the leaves of the fruit trees are obviously tender green, and the yield of kiwifruit per mu under the same conditions is increased by more than 25%, with more large fruits and richer taste; soil microbial detection shows that the biomass in the soil has increased significantly, up to tens of thousands times higher than that of the control group. See Table 2 for details.

[0043] Table 2. Comparison of biomass and yield

[0044]

[0045] Note: The kiwifruit varieties, light and rainfall and other natural growth environments of the experimental group and the control group were the same

[0046] Embodiment 3

[0047] (1) Take 0.5 kg of unfertilized soil and add it to 1 L of water. Stir and allow to settle. Then separate the mixture and keep the supernatant as soil extract. Add inorganic substances to form an inorganic liquid culture medium, sterilize it, and cool it for later use.

[0048] The formula of the inorganic salt culture medium can be adjusted as needed, but the basic formula includes the following ingredients: nitrogen source: (NH4)2SO4 1.0 g, or NaNO3 1.0 g; phosphate source: KH2PO4 0.5 g; potassium salt source: KCl 0.5 g; sulfate source: MgSO4·7H2O 0.5 g; trace elements: FeSO4·7H2O 0.01 g, MnSO4·H2O 0.01 g, ZnSO4·7H2O 0.005 g, CuSO4·5H2O 0.005 g, CoSO4·7H2O 0.005 g, NiSO4·6H2O 0.005 g; soil extract 1L.

[0049] (2) Spray the soil activation solution of (1) in the healthy soil area. One week after applying the soil activator, take a soil sample at the activation point, remove the top 10 cm, select 10g of soil as the soil microorganism sample, add 100mL of sterile water and shake well, and use the supernatant as the fermentation liquid mother seed.

[0050] (3) A fermentation system was set up in the target soil area. The system was as described in (3) in Example 2. The culture medium was changed to an inorganic liquid culture medium. Each level of culture lasted for 1 week. After the expansion, the plate dilution counting method showed that the process could increase the biomass to 10 4 -10 5 CFU / g, see Table 3 for details.

[0051] (4) The culture solution was diluted 30 times and used to irrigate or spray the crops to supplement the lack of substance transformation bacteria in the soil, such as nitrite bacteria, nitrifying bacteria, sulfur bacteria and iron bacteria. Compared with the control group, the yield of the tested vegetables and tea increased by 10%-30%. Soil microbial detection showed that the biomass in the soil increased significantly. The number of nitrite bacteria, nitrate bacteria, sulfur oxidizing bacteria, iron bacteria and hydrogen bacteria, which are not large in number in the soil samples but play an important role in substance transformation, has increased significantly.

[0052] Through the above steps, the chemo-inorganic autotrophic bacteria can be rapidly proliferated using oxygen-rich and inorganic liquid culture medium. This group does not contain any crop pathogens and is suitable for the application of pesticides and herbicides and crop rotation obstacles, and can also be used during base fertilizer and topdressing.

[0053] Table 3. Comparison of biomass and yield

[0054]

[0055] Note: The tea varieties, light and rainfall and other natural growth environments of the experimental group and the control group were the same

[0056] Embodiment 4

[0057] (1) Select healthy soybeans and add liquid rhizobium culture medium to the soil around their roots as an activator to awaken the dormant microorganisms in the soil. Rhizobium culture medium: 10g glucose, 0.5g dipotassium hydrogen phosphate, 3g calcium carbonate, 0.2g magnesium sulfate, 0.4g yeast powder, 1000ml water, 1ml 1% crystal violet solution.

[0058] (2) One week after applying the soil activator, pull out the soybeans, remove the loose soil on the roots, immerse the roots in the prepared sterile water for 5 minutes, let them stand and take the supernatant for later use;

[0059] (3) Use the bacterial suspension activation station set up on site, which includes a Class I expansion unit (a 5L aerated and stirred small barrel) and a Class II expansion unit (a 100L aerated large barrel). Add 950mL of liquid culture medium and 50mL of bacterial suspension to the Class I expansion unit, and aerate and culture at room temperature for 3 days; transfer 1L of Class I fermentation liquid as the inoculated mother culture to the 49L liquid culture medium of the Class II expansion unit, and aerate and culture at room temperature for 3 days to obtain a strongly reduced culture medium;

[0060] (4) After the strong reducing culture solution was diluted 30 times, it was injected into the roots of soybeans to strongly reduce the rhizobium group. Compared with the control group, the soybean yield increased by 20-30%, and the number of nodules on the root system increased significantly. See Table 4 for details.

[0061] Table 4. Comparison of biomass and yield

[0062] Serial number Project indicators Control group Experimental Group Remark 1 Soybean production ～123 ～169 (kg / mu)

[0063] Note: The soybean varieties, light and rainfall and other natural growth environments of the experimental group and the control group were the same

[0064] In the above embodiments, based on the method of the present invention, those skilled in the art can make appropriate adjustments according to actual needs. For example, the strong reducing liquid can be sprayed or applied after being carried by a carrier (such as straw, rice husk and biochar powder, etc.), but it does not depart from the protection scope of the present invention; targeted proliferation of single or complex bacterial species can also be carried out to promote these bacterial species to interact with the original microbial communities in the soil, effectively synergize, and enhance the diversity, activity and stability of the soil microbial community; the soil strong reducing liquid can be used for soaking seeds, soaking roots, etc.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A soil in-situ remediation method based on indigenous microbial populations in the crop rhizosphere, characterized by: In situ soil is used to carry out the overall in situ expansion of the indigenous microbial population in the crop rhizosphere, so as to enrich and proliferate the indigenous microbial community in the soil in a targeted manner.

2. The method for soil in-situ remediation based on indigenous microbial populations in the crop rhizosphere according to claim 1, characterized in that: Soil remediation fluid that can compensate for the lack of soil rhizosphere microorganisms is quickly cultivated on-site using in-situ soil.

3. The method for soil in-situ remediation based on indigenous microbial populations in the crop rhizosphere according to claim 2, characterized in that: The following steps are involved: S1, soil in situ activation; S2, preparing bacterial suspension: preparing bacterial suspension using activated soil; or preparing bacterial suspension using root washing water of plants in activated soil; S3, fermentation of bacterial suspension to obtain soil remediation solution; S4, applying the repair solution to the soil.

4. The method for soil in-situ remediation based on indigenous microbial populations in the crop rhizosphere according to claim 3, characterized in that: In step S1, a soil activator is sprayed on the soil for in-situ activation.

5. The method for soil in-situ remediation based on indigenous microbial populations in the crop rhizosphere according to claim 3, characterized in that: In step S2, soil sampling or plant sampling of the target bacterial suspension is performed one week after the soil is activated.

6. The method for in-situ soil remediation based on indigenous microbial populations in the crop rhizosphere according to claim 5, characterized in that: In step S2, sterile water is added to the sampled soil to prepare a bacterial suspension, or the roots of the plants are washed with sterile water to prepare a bacterial suspension.

7. The method for soil in-situ remediation based on indigenous microbial populations in the crop rhizosphere according to claim 3, characterized in that: In step S3, the bacterial suspension and the soil activator are mixed for fermentation.

8. The method for soil in-situ remediation based on indigenous microbial populations in the crop rhizosphere according to claim 7, characterized in that: In step S3, a small-scale fermentation device is first used for fermentation, and then a large-scale fermentation device is used for fermentation to achieve proliferation and expansion of a large number of microorganisms.

9. The method for soil in-situ remediation based on indigenous microbial populations in the crop rhizosphere according to claim 3, characterized in that: In step S4, the soil remediation solution is diluted and drip-irrigated to the agricultural operation area where the in-situ soil belongs.

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