KR100743525B1 - A method for removing remained agricultural chemicals in soil using amino acid liquid fertilizer - Google Patents

Abstract

A method for removing remaining agricultural chemicals in soil by using an amino acid liquid fertilizer is provided to promote microorganism degradation of the remaining agricultural chemicals in soil by activating useful microorganisms. The remaining agricultural chemicals in soil such as farmland or lawn are removed by spraying the amino acid liquid fertilizer on the soil in the amount of 0.1-1 ml/m^2 before or after spraying the agricultural chemicals, wherein the spraying of the amino acid liquid fertilizer is performed 2-3 times at an interval of 1 week.

Description

A Method for Removing Remained Agricultural Chemicals in Soil Using Amino Acid Liquid Fertilizer}

1 is a control (KD ₁ ), untreated (KD ₂ ), amino acid liquid ratio 1 times treatment (KD ₃ ), amino acid liquid ratio 2 times treatment (KD ₄ ) samples in order to investigate the microbial community structure of the grass turf soil. This is the result of RFLP.

Figure 2 Alphaproteobacteria in grassy soil Phylogenetic diagram based on 16S rDNAs of (KD ₁ : control, KD ₂ : nontreated, KD ₃ : LFcAA 1x, KD ₄ : LFcAA 2x).

Figure 3 Gammaproteobacteria in the grass turf soil Phylogenetic diagram based on 16S rDNAs of (KD ₁ : control, KD ₂ : nontreated, KD ₃ : LFcAA 1x, KD ₄ : LFcAA 2x).

FIG. 4 is a phylogenetic diagram based on 16S rDNAs of Betaproteobacteria (KD ₁ : control, KD ₂ : nontreated, KD ₃ : LFcAA 1x, KD ₄ : LFcAA 2x) in grass turf soil.

The present invention relates to a novel use of amino acid fertilizers, and more particularly to a method for removing residual pesticides that promote microbiological degradation of soil residual pesticides using amino acid fertilizers.

Pesticides are used to control weeds and pests in farmland as well as lawns such as golf courses, various sports fields, and parks. However, long-term repeated use of chemical pesticides, including herbicides, has a negative effect on soil and plants through prolonged retention of highly toxic agents in the soil, and the continued use of these chemicals is known to adversely affect plant root development. Emmons, 1995; Turgeon, 1991). In particular, weeding on the lawn used as a golf course or various sports venues, unlike farmland is carried out all year, this problem is more serious.

Currently, grass cultivation technology is using a variety of selective herbicides to implement an efficient and reasonable weed control system, and most of the pesticides on the market in Korea are low-toxic pesticides. However, although there may be some differences depending on the chemicals, once the pesticide is injected into the soil, it primarily affects the activity of the soil microorganisms, which affects the residue of the pesticides in the soil and the process of change of the nutrients in the soil. , 1997).

Recently, in fertilization management, not only facility cultivation but also lawn care is used amino acid fertilizer. However, there is little research on the effects of amino acid fertilizers on grass growth. The research on amino acid liquid fertilization was mainly conducted on indoor experiments. Recently, experiments are applied to outdoor forge, but there are difficulties due to various environmental factors. Laboratory experiments are mainly focused on nitrogen absorption and metabolism mechanisms (Muller, 1991; Rodgers, 1993; Causin, 1993), and field forge experiments are mainly focused on amino acid and nitrogen absorption. Reports of impacts are difficult to find. Only a few amino acids are present in the soil and may be a potential plant nutrient in the boreal forest (Perssom, 2002; Perssom, 2001).

Young-sun Kim (2003) investigated the effect of fertilizing amino acid fertilizer on turfgrass on the growth and soil of the turf. As a result of comparing the chemical properties of the soil, the nitrogen utilization rate of soil in the amino acid fertilizer treatment treatment increased compared to the control. As a result of comparing the chemistry of the crops, the nitrogen, koto and chlorophyll contents of the amino acid fertilizer treatments were higher than those of the control, and the fresh weight and dry weight of the grass were reported. In addition, fertilizing amino acid fertilizers increased the utilization of nitrogen in the turf soil, which contributed to the growth of grass.

The Republic of Korea Patent No. 392886 describes that the grass disease control composition which contains amino acids and urea in the main components consisting of charcoal, sulfur, and silicic acid is effective for the control of large fat diseases, and is also effective for the rapid recovery of grass and soil improvement agents. Doing. However, in this document, amino acids are used only as one of the nutritional ingredients, and the control effect is confirmed only for the main composition consisting of charcoal, sulfur, and silicic acid.

Republic of Korea Patent No. 522894 describes a method for producing an environmentally friendly plant growth enhancer containing a natural chitosan as a main component and added to the vinegar solution, complex amino acids and the like. Also in this document, amino acids are used as auxiliary components, and the effect of improving the plant growth of the whole composition containing chitosan as a main component is confirmed.

Soils play an essential role in ecosystem metabolism. Therefore, in order to maintain the ecosystem, it is necessary to take a biological approach to evaluate, manage and use the soil environment in consideration of biological capacity such as soil self-cleaning capacity and material circulation capacity. The first areas to be considered in soil management are the physicochemical properties of the soil, but a large emphasis should also be placed on the soil microorganisms. The reason for this is that ecosystems must have living organisms and material circulation must proceed efficiently in order for the ecosystems to remain intact, as soil microbes are deeply involved in this process. However, the trends of domestic research on soil microorganisms have been performed in a fragmentary and mainly applied manner. In the future, it is necessary to find ways to biologically evaluate soil quality in terms of environmental conservation.

Recent rapid development of molecular biological methods, including polymerase chain reaction (PCR), has led to the continued development of bacterial identification methods based on these techniques. Among the various molecular biology techniques, the most widely used for bacterial identification is the method of targeting 16S rRNA gene (16S rDNA) (El Amin, 2000). The use of 16S rDNA sequences in soil microbial ecology is more meaningful when combined with direct strain isolation (Amann, 1995).

Many studies have been conducted on the effects of amino acid fertilizers, but all are related to their effects as fertilizers. The direct effects of amino acid fertilizers on the removal and removal of residual pesticides are unknown.

It is an object of the present invention to provide a novel use of amino acid liquor related to the decomposition and removal of soil residue pesticides. In the present invention, the effect of the amino acid fertilizer on the decomposition of soil residual pesticides is found. In particular, in the present invention, the effect of the amino acid liquid ratio on soil microorganisms associated with decomposition of residual pesticides is found.

Other objects and advantages of the present invention will be described below and will be better understood by practice of the present invention.

In the present invention, among the herbicides currently used, the herbicides with the residual risks of continuous repetitive use are selected and sprayed on the turfgrass ( Zoysia japonica ), and then treated with amino acid liquid ratio, and the residual pesticide detection test and microbial community structure are identified. Understand the effects of amino acid fertilizers on the degradation of residual pesticides in the soil.

Based on these findings, the present invention provides a method for removing soil residue pesticides that promotes microbiological decomposition of residue pesticides in soil for new applications of amino acid fertilizers.

Moreover, in this invention, the removal elimination agent of the residual herbicide in soil which has an amino acid liquid ratio as an active ingredient is provided.

In the present invention, the herbicide and the grass turf soil having residual risk were tested, but the new use of the amino acid fertilizer according to the present invention is not limited thereto. The present invention can be applied to all uses that are apparent to those skilled in the art by the novel efficacy of the amino acid liquids disclosed in the present invention. In particular, the new use of amino acid fertilizers for the decomposition of residual pesticides in the soil according to the present invention can be used on the lawn, such as a golf course, various athletic fields, and can be used for all cropland regardless of facility cultivation and open field cultivation. In the present invention, the amino acid liquor can be treated to the soil before or after the spraying of the pesticide. The amino acid liquid ratio is used by diluting 100 to 1,500 times in water, preferably diluting to 300 to 1,000 times, more preferably 400 to 600 times. The amino acid liquid ratio (stock solution) per 1,000 m 2 of soil may be treated to about 100 to 1,000 ml (0.1 to 1 ml / m 2), preferably 300 to 800 ml (0.3 to 0.8 ml / m 2), more preferably 500 to 700 ml (0.5-0.7 ml / m 2). The number of treatments may be from one to four or more times if necessary, but generally two to three treatments per week are appropriate. As a treatment method, a spray using a manual sprayer or a power sprayer may be generally used, but is not limited thereto, and may be used in the present invention as long as it is a pesticide treatment method known to those skilled in the art.

The term "pesticide" in the present invention means a chemical pesticide used to control diseases, pests, and weeds occurring before or during cultivation and storage of crops, and specifically includes herbicides.

In the present invention, "herbicide" means a chemical agent used to remove weeds unless specifically limited.

In the present invention, the following experiment was conducted to determine the effect of amino acid liquid ratio on the microbiological degradation of the soil pesticides. The experiments were conducted on the field grasses of Gap-dong, Yuseong-gu, Daejeon, Korea, and treated with untreated, liquid fertilizer 1x, and liquid fertilizer 2x. Was detected, and the results were as follows.

(1) Saturn of the soil in the experiment area consisted mainly of high quality sand, and the soil pH was 5.8-6.6, and the nitrogen, effective phosphorus, substitutional Ca, and Mg contents of the soil except for organic matter were different by treatment. There was no big difference. Nitrogen, phosphate, kali, and calcium content in the grass roots did not differ significantly between treatments. As a result, it was confirmed that the treatment of the liquid fertilizer containing the amino acid did not significantly affect the chemical properties of the soil and the plant.

(2) Residual pesticides in field grasses treated with Ventazone and MCPP were found to be the lowest in the two-fold treatments than in the untreated and one-fold treatments.

(3) The microorganisms in the grass turf soil were found to be more diverse and effective in plant microorganisms in 2-fold treatment than in untreated and 1-drain treatments. In particular, the microorganisms irradiated in the two-fold treatment group were identified as bacteria that decompose various organic molecules and play a very important role in mineralization processes in nature and in sewage treatment. Phenylobacterium immobile , a herbicide-degrading herbicide found when fertilizing fertilizer containing amino acids, was found to be a Gram-negative bacterium and known as a microorganism that degrades herbicide chloridazon.

Hereinafter, the present invention will be described in more detail with reference to specific experiments and examples. However, the scope of the present invention is not limited by the following experiments and examples, and the technical scope of the present invention and equivalent scope of the claims to be described below by those skilled in the art to which the present invention pertains. Of course, various modifications and variations are possible.

I. Experimental Materials and Methods

1. Survey Area

This experiment was carried out on experimental forge, Gap-dong, Yuseong-gu, Daejeon, and grass was used as Z. japonica . The herbicides used were Bentazone (light agriculture) (8 mL / 2 L (H ₂ 0) / 5 m ₂ ), MCPP (Mecoprop, Youngil Chemical) (7 mL / 2 L (H ₂ 0) / 5 m ₂ ). As a fertilizer, liquid fertilizer (LFcAA) containing amino acids was used. The liquid fertilizer was purchased from amino Wang Gold ™ (Bionel), which contains many essential amino acids and total amino acids in the commercial liquid fertilizer, and was used in the experiment.The chemical characteristics of the test fertilizer were 3.7% nitrogen and 2.78 water-soluble phosphoric acid. %, Water soluble galley 4.94%, water soluble boron 0.15%, and water soluble molybdenum 0.0011%. The amino acid content was 41.7 g / l (Kim Young-sun, 2003). The test packaging was 100 m 2 in size, arranged according to the liquid fertilization treatment and carried out in 5 repetitions. The experiment was conducted on March 10, 2005, and the mixed preparation treatment was performed on April 25. After the treatment of amino acid fertilization on May 5, and then after 15 days, 30 days and 45 days, soil residue pesticide components and microbial community structure and diversity were investigated. A 1-fold amino acid solution ratio (LFcAA 1X) was diluted to 1,000-fold solution in one treatment and sprayed 1.5ℓ of dilutions per 5㎡ of soil using a manual sprayer and a power sprayer (1.5mL / 1.5ℓ (H ₂ O) / 5 m 2), which was treated once more on May 12 and was treated twice. LFcAA 2X treatment solution (LFcAA 2X) was diluted 500 times in one treatment and sprayed 1.5ℓ of dilutions per 5m2 of soil using manual sprayer and power sprayer (3mL / 1.5ℓ (H ₂ O) / 5 m 2), which was treated once more on May 12 and was treated twice. Non-treated, treated with pesticides only, no amino acid solution. The control was left untreated without both pesticide and amino acid fertilization.

2. Sample Collection

For the analysis, Z. japonica soil and roots were collected from May 2005 and June of the same year. Soil samples were collected from the field of grass turf for five treatment areas as representative sampling sites and collected from 0 to 10 cm deep from the surface. Soil samples for soil microorganism identification were collected from 0 to 10 cm deep from the surface at the same point as sampling for soil analysis. Samples of grass turf roots were collected from the experimental area by selecting one place (20cm × 20cm) for each treatment area.

3. Analysis method

1) Grassgrass Soil Sample and Root Analysis

During Daejeon, the grass turf soil of 0-10cm depth was collected once from Gabdong material test for Yuseong-gu, and dried for 5-10 days. For the grass roots, the foreign substances on the surface of the collected roots were washed with running water, washed again with distilled water, and dried in an oven until they reached a constant temperature of 80 ° C or lower.

Soil organic matter content was determined by Wakely-Black wet oxidation method, total nitrogen was determined by Kjeldahl method, effective phosphoric acid by Lancaster method, and substitutional K was analyzed by ICP. Soil pH was analyzed by the method of ultrasonography diluted to 1: 5 and cation substitution capacity was analyzed by Brown method.

The average values of grass turf samples and root nutrients were compared at the 0.05 level, and were analyzed using SPSS (ver. 12.0) for all statistical analysis.

2) field grass Bentazone  Residual Pesticide Analysis

Field grasses were collected three times from May 2005 to June of the same year, and soil samples from 0 to 10 cm deep from the surface of the field grass were used.

① Create calibration curve

Dissolve 101.52mg of Bentazone standard in acetonitrile to make 1,000ppm stock solution, dilute it with acetonitrile to prepare 0.1, 0.5, 1, 2, 3ppm, and inject 20μ into HPLC-UV / vis A calibration curve was prepared based on the criteria.

② Sample analysis process

end. Sample Preparation

2 kg of each soil was collected and ground, followed by 25g of two repetitions. 100 ml of acetone and 1 tablespoon of celite 545 were added and extracted with a sonicator for 30 minutes, using Kiriyama funnel. 2 filtered under reduced pressure and concentrated under reduced pressure until it became 100 ml at 40 degreeC.

I. extraction

The concentrated solution was transferred to a 1 liter separatory filter, and then 450 ml of distilled water, 50 ml of saturated saline and 100 ml of dichloromethane were added thereto, followed by vigorous shaking for 30 minutes using a shaker. 20 ml of 1M-HCl was added thereto to adjust the pH to 1-2, and then 100 ml of dichloromethane was added thereto, followed by vigorous shaking. The dichloromethane layer was separated, and 50 ml of dichloromethane was further shaken vigorously for 10 minutes. It was left to stand, the dichloromethane layer was separated, and it concentrated under reduced pressure at 40 degreeC or less and dried.

All. Clean up

5 g of activated florisil (60-100 mesh) was placed in a column (I. D 10 × H 400 mm), filled with 1 cm of anhydrous sodium sulfate, prewashed with 50 ml of n-hexane, and the above dried substance was mixed with acetone. : n-hexane (v / v) = 3: 7) It melt | dissolved in 50 ml and flowed. Again eluting with 30 ml of a mixed solvent (acetone: n-hexane (v / v) = 8: 2), and drying under reduced pressure at 40 ° C., 20 ml of acetonitrile was injected into 20 ml and analyzed by injection.

3) field grass MCPP  Residual Pesticide Analysis

Field grasses were collected three times from May 2005 to June of the same year, and soil samples from 0 to 10 cm deep from the surface of the field grass were used.

① Create calibration curve

103.2 mg of MCPP standard was correctly dissolved in acetonitrile to prepare 1,000 ppm of stock standard solution. 1 ml of the stock solution was aliquoted, the hexane layer was dehydrated with anhydrous sodium sulfate, concentrated under reduced pressure, washed in a 100 ml volumetric flask, and filled to the mark to prepare a 10 ppm working standard solution. 0.005ppm, 0.01ppm, 0.025ppm, 0.05ppm, 0.1ppm, 0.25ppm, 0.5ppm, 0.75ppm standard among working standards and then 0.01ng, 0.02ng, 0.05ng, 0.1ng, 0.2ng, 0.5ng, 1.0 20ng of ng and 1.5ng were injected into HPLC to prepare a calibration curve based on the peak height on the chromatogram.

② Extraction and Purification

25 g of soil samples were collected, sonicated for 30 minutes by adding 50 ml of 0.1N-HCl, and extracted for 5 minutes after addition of 100 ml of acetone. The extract was filtered using Buchner filter (thin celite 545 on the filter head and filtered), and the residue was washed with acetone, suctioned and filtered. The filtrate was transferred to a 1,000 ml separatory filter, and 300 ml of distilled water and 30 ml of saturated NaCl were added, followed by alkaline treatment by adding 1.5 ml of 3N-NaOH. After extracting once with 50 mL of dichloromethane, acid treatment was performed by adding 25 mL of 1M-HCl to the dichloromethane layer, followed by further fractionation of 100 mL of hexane. The fractionated layer was concentrated under reduced pressure again, dissolved in 5 ml of hexane, and then purified by putting the sample in 5 g of florisil. 100 ml of the purified liquid hexane: ethyl ether (90:10) was added, and the first 20 ml were discarded sequentially, and the remaining 80 ml was received, concentrated under reduced pressure, and dried. The solution was dissolved in 10 ml of acetonitrile, and 20 µl of this solution was added and analyzed by injection into HPLC.

4) Identifying grass turf microorganisms

Field grass collection was carried out in May 2005, and soil samples from 0 to 10 cm deep from the surface of the grass turf were used.

① Extract DNA from soil, 16S rDNA  Purification and Amplification

и72 thermal block(Bioneer, Daejeon, Korea)을 이용하여 증폭하였다. 토양 세균의 16S rDNA를 증폭하기 위하여 고안된 universal primer인 27F(E. coli numbering 8∼27 : AGAGTTTGATCMTGGCTCAG)와 1492R(E. coli numbering 1492∼1510 : GGYTACCTTGTTACGACTT)가 사용 되었다. PCR 반응조건은 94℃에서 1분, 50℃에서 1분, 72℃에서 2분씩 25회 반복하고 마지막에는 72℃에서 8분간 처리하였다.Total DNA was extracted from 0.2 g of soil. Total DNA was used with a QIAamp DNA stool mini kit (Qiagen, German), and extraction was performed according to the protocol included in the manual. 16S rDNA gene from the collected soil was MyGenie from 20μl reaction solution through polymerase chain reaction (PCR).

Amplified using an и72 thermal block (Bioneer, Daejeon, Korea). Universal primers 27F ( E. coli numbering 8-27: AGAGTTTGATCMTGGCTCAG) and 1492R ( E. coli numbering 1492-1510: GGYTACCTTGTTACGACTT), designed to amplify soil bacterial 16S rDNA, were used. PCR reaction conditions were repeated 25 times 1 minute at 94 ℃, 1 minute at 50 ℃, 2 minutes at 72 ℃ and finally treated for 8 minutes at 72 ℃.

② Terminal restriction intercept  Analysis (T- RFLP )

Although the T-RFLP method is insufficient to provide accurate information on qualitative and quantitative analysis of microbial species, it is known to be a relatively fast and effective method to explain the similarity and distribution between microbial communities (CHO Hye-yeon, 2004). Blackwood, 2003; Dunbar, 2000). 50 ng / μl of the purified PCR product was treated with the manufacturer's method with a final volume of 20 μl with restriction enzyme HaeIII of BIONIA (Daejeon, Korea). The terminal restriction fragment was completed using an automatic base sequence determination device (model ABI 3100, automated DNA sequencer, Applied Biosystems Instrument, Foster, USA).

③ 16S rDNA Cloning  And sequencing

PCR products were purified by gel and cloned according to the manufacturer's protocol using a TOPO TA cloning kit PCR2.1-TOPO vector (Invitrogen, Co., California, USA). About 110 colonies were selected for sequencing of 16S rDNA on individual plates. RDNA inserts from PCR2.1 vector clones were PCR amplified using vector primers M13F (GTAAAACGACGGCCAG) and M13R (CAGGAAACAGCTATGAC).

Amplified clones were submitted to genotech (Daejeon, Korea) for sequence analysis using a sequence analyzer of ABI 3700XL DNA sequencer (Applied Biosystems, Foster, USA).

The determined 16S rDNA base sequences were obtained from the BLAST search of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nih.gov/) to obtain the reference sequence by examining the group closest to the clone. Sequencing and alignment were performed using Clustal X (ver. 1.83) (Thompson, 1997), and the tree-view tree was used with the Neighbor-Joining method using TreeView 1.6.6. In order to investigate the stability of branches in the system, 1,000 bootstrap resampling analyzes were performed to indicate the values.

II. Experiment result

1. Chemical Properties of Grass Turf Soil and Roots

Saturn of the experimental forge was mostly composed of high quality sand, and the soil pH was distributed in 5.8-6.6, and there were slight differences among treatments. The results of analyzing the chemical properties of the grass turf soil are shown in Table 1 below. The organic matter content in soil was distributed between 1.24 and 1.95 and showed relatively low content. Nitrogen and effective phosphate in soil did not show a significant difference between treatments, but in the case of substitutional Ca and Mg, the two-fold treatment of amino acid solution ratio showed relatively high content. In addition, the cation substitution capacity (CEC) in the soil was also 4.20 ~ 6.24 did not show a significant difference by treatment.

The nitrogen content in the roots of the grass was found to be almost the same in the four treatments. The results of analyzing the chemical properties of the grass roots are shown in Table 2 below. The contents of phosphate and calcium in the roots were not significantly different between treatments, but potassium was relatively higher in the two-fold treatment of amino acid solution. As a result, the treatment of liquid fertilizer containing amino acids did not appear to have a significant effect on the soil and plant chemical properties.

2. Analysis of Pesticide Residues in Grassy Soil

After the herbicide treatment, soil samples treated with no liquid fertilization, 1 times amino acid fertilization, and 2 times amino acid fertilization were analyzed for soils with a depth of 0 to 10 cm. The average concentrations of Ventazone after 15 days, 30 days, and 45 days of field grass treated with Ventazone were in the order of 2 times amino acid treatment ratio> 1 times amino acid treatment ratio> no liquid treatment. The results are shown in Table 3 below.

Treated with the fastest residual pesticide decomposition in the field grass of Ventazone-treated soil, which was treated with twice the amount of amino acid fertilization, after 30 days, the average value was 0.05 ppm, which was less than 1/7 compared with no liquid fertilization. Compared to this, the value was very low, about 1/2. These results are believed to be due to the addition of amino acid fertilizers, and thus, it can be concluded that the treatment of amino acid fertilizers can improve the soil environment in the field grasses treated with ventazone.

The mean concentrations of MCPP after 15 days, 30 days, and 45 days of field turf soil treated with MCPP were in the order of 2 times amino acid fermentation treatment> 1 times amino acid fermentation treatment> no liquid fermentation. The results are shown in Table 4 below. The treatment group showing the fastest residual pesticide decomposition in the field turfgrass treated with MCPP was twice the amino acid fertilization, and the average value was 0.006ppm after 30 days. Showed a very low value of 1/9. These results are believed to be due to the addition of amino acid fertilizer as described above. Thus, the provisional conclusion that the amino acid fertilization can be improved by treating the amino acid fertilizer to promote the decomposition of residual pesticides in field grasses treated with MCPP can be improved. there was.

In the present invention, it was thought that the amino acid fertilization promotes the decomposition of residual pesticides in the grass turf treated with Ventazone and MCPP, because the amino acid fertilization affects the microbial environmental ecosystem of the soil, which can be confirmed by the following experimental results. there was.

3. Identification of microorganisms in grassy soil

① T- RFLP analysis

In order to investigate the microbial community structure of the grass turf soil, T-RFLP was performed on the control (KD ₁ ), untreated (KD ₂ ), amino acid fertilization 1-fold treatment (KD ₃ ) and amino acid fertilization double-treatment (KD ₄ ) samples. It was. The results are shown in FIG.

As a result of the analysis, the peak ratios of control (KD ₁ ), untreated (KD ₂ ), amino acid fertilization 1-fold treatment (KD ₃ ) and amino acid fertilization 2-fold treatment (KD ₄ ), as well as the relative proportion of each T-RFs Showed a difference. In KD ₁ , 69 peaks with an effective bp (base pair) size were found. KD ₂ , which was not treated with amino acid solution ratio, showed 23 peaks with effective bp size, and 32 and 38 peaks gradually increased with KD ₃ and KD ₄ treatment groups. It is believed that the liquid fertilizer containing amino acids affects the microbial environmental ecosystem of the soil and thereby causes various microorganisms to inhabit.

② 16S rDNA  Clone analysis

After the pesticide treatment, clones of control (KD ₁ ), untreated (KD ₂ ), amino acid 1-fold treatment (KD ₃ ) and amino acid 2-fold treatment (KD ₄ ) soils One hundred and ten randomly selected 16S rDNA clone partial sequences were analyzed. The results are shown in Tables 5 to 8 below.

The 110 analyzed partial sequences were sorted by chromas213 program and compared with 16S rRNA sequence using Genebank database. The sequencing of 16S rDNA clones revealed mainly clones belonging to Alpha-, Beta-, Gamma-, Deltaproteobacteria group, Acidobacteria group, Actinobacteria group, Sphingobacteria and Planctomycetacia (Table 7-10). A high proportion of the Proteobacteria group was observed, particularly clones belonging to Planctomycetales , which did not appear in KD _{2 in} the case of KD ₄ . Most of the clones were clones with more than 90% similarity, and most of them showed high similarity over 95%.

Alphaproteobacteria : In this study, the largest number of clones were investigated in Alphaproteobacteria . FIG. 2 is a phylogenetic tree based on 16S rDNAs of Alphaproteobacteria (KD ₁ : control, KD ₂ : nontreated, KD ₃ : LFcAA 1x, KD ₄ : LFcAA 2x) in field grass. Nine clones were examined in the control group KD ₁ , mainly Sphingomonadaceae. Uncultured environmental sample strains belonging to the group were examined and showed a similarity between 95 and 97%.

In the non-treated group KD ₂ , eight clones were found in Sphingomonadaceae uncultured environmental samples and Sphingomonas sp. Showed a flexible relationship of 96-97% (Table 5). Eight clones were examined in KD ₃ , a 1-fold amino acid fermentation treatment group, with 94% similarity to the non-cultivated environmental sample of Sphingomonadaceae and Kaistobacter genus, and 98% similar to the Brevundimonas genus of Caulobacteraceae . And it showed the non-culture of environmental samples and the similarity of 98% of Methylobacteriaceae, the Caulobacteraceae Caulobacter 96% similar to genus. Bacteria belonging to the genus Caulobacter are usually isolated from low-nutrient freshwater or seawater habitats but are also present in the soil. In addition, 14 clones appeared in KD ₄ , which was twice the amino acid solution ratio, and most of the clones had a high similarity of 96-98%. This particular KD ₄ clones of the microorganism belonging to the Rhizobiales, Sphigomonadales, and Caulobacterales was investigated. Four clones of KD ₄ -1 (97%), 5 (96%), 15 (96%) and 78 (98%) belonged to Rhizobiales . These members belonging to the group of the family Rhizobiaceae are Gram-negative and aerobic. Often a motile bacterium with poly-beta-hydroxybutyl particles, the shape varies in adverse environments. Rhizobium is a bacteroid that fixes nitrogen by forming a symbiotic relationship with the root gall cells of legumes.

Five clones of KD ₄ -71 (96%), 92 (92%), 97 (96%), 103 (94%) and 112 (97%) were found to belong to Sphingomonadales (Table 5). 3 is a phylogenetic tree based on 16S rDNAs of Gammaproteobacteria (KD ₁ : control, KD ₂ : nontreated, KD ₃ : LFcAA 1x, KD ₄ : LFcAA 2x) in grassy soil. One clone of KD ₄ -102 (94%) belonged to Caulobacterales and was investigated with Phenylobacterium immobile . This clone is a gram-negative bacterium and is known as a microorganism that degrades herbicide chloridazon. These results were judged to be beneficial to the survival of various microorganisms in turf environment where the double treatment of amino acid liquid ratio is poor.

Gammaproteobacteria: Gammaproteobacteria has formed a number of clones in the second survey of the Proteobacteria. KD ₁ was examined in 5 clones. This group is Pseudomonas of Pseudomonadaceae Clones in the genus showed 97% similarity, a group of bacteria that rely on oxygen as a receptor, some of which use hydrogen or carbon monoxide as energy sources. Non-cultivated environmental samples and clones with 95% similarity to the genus Rhodanobacter from Xanthomonadaceae were examined. KD ₂ showed two clones and showed 97% significance with Enterobacteriaceae and Pantoea genus, but they were not systematically consistent (FIG. 3).

KD ₃ formed two clones and KD ₄ formed four clones. The clone of untreated KD ₂ was found in the Enterobacteriales tree. Pantoea agglomerans (97%) were investigated (Table 6). Clones of KD ₄ were examined mainly in the genus Pseudomonas . These are the most important genera in Psuedomonadales and Pseudomonadaceae . Bacteria in the genus Pseudomonas are gram-negative bacteria that are straight or slightly bent, are aerobic, and use respiratory metabolism with oxygen as their receptor. Many types can degrade a wide variety of organic molecules. Therefore, it is very important for the mineralization process in nature and in sewage treatment.

Betaproteobacteria: the two in clones KD ₁ belonging to the group, 7 in the KD _2, 5 gae the KD _{3, 4} in the emerged KD five clones. KD ₁ was the clones belonging to the environmental sample, KD ₂ is Burkholderiales, clone 1 having Janthinobacteria in the environmental sample, and 94% similarity Oxalobacteraceae, 1 clones having Acidovorax sp. And 96% similarity dog, Janthinobacterium sp. Four clones with and 97% similarity were examined. Another clone was derived from phylogenetic analysis of Comamonadaceae . 97% similarity to the genus Acidovorax was investigated in the isolates involved in denitrification. The clone of KD ₃ showed 96% similarity to Burkholderia of Burkholderiaceae , and another clone 94% similar to Nitrosospira of Nitrosomonadaceae . In KD ₄ , mainly Nitrosomonadales Nitrosospira genus was mainly investigated. The clones in KD ₄ showed a high 98% similarity to the genus Nitrosospira . This group is ecologically important and lives in soils, sewage treatment systems, freshwater and seawater habitats. These genera oxidize ammonia into nitrites, the nitrogen of which is best used by plants. FIG. 4 is a phlogenetic tree based on 16S rDNAs of Betaproteobacteria (KD ₁ : control, KD ₂ : nontreated, KD ₃ : LFcAA 1x, KD ₄ : LFcAA 2x) in grassy soil. Of Comamonadaceae Acidovorax Clones belonging to the genus showed a high similarity of 99%. KD ₄ -47, 76 formed a group with a bootstrap value of 1,000. Janthinobacteria of Oxalobacteraceae in Nitrosospira sp. Clones with 97% similarity to L115 were shown. In KD ₄ , the clones were mainly involved in nitrogen assimilation than the untreated group.

Deltaproteobacteria : In this study, clones belonging to Deltaproteobacteria were found to have 92% similarity to the uncultured delta proteobacterium clone AKYH836 in KD ₁ alone. It was not investigated in other experiments.

Acidobacteria : This group was tested for 7 clones in KD ₁ , ₁ in KD ₂ , and ₂ in KD ₄ . In general, this test a group appears mainly in the anaerobic environment was much from KD _1. This group is a Gram-negative bacterium that grows in anaerobic environments using various organic acids such as acetate and propinate, and is chemically heterotrophic in acidophilic environments.

Actinobacteria : Three clones in this group were examined in KD ₁ and KD ₂ and one clone in KD ₃ and 4, respectively. Most of the genus belonging to this group have an irregular shape and are gram-positive bacilli that do not form spores and have aerobic or conditional aerobic metabolism.

Planctomycetes: KD ₃ in this clone with the environment of the sample Planctomycetales bacterium and 89% similarity of Planctomycetales gae 1, KD ₄ was investigated in dogs clone 2 clone and environmental samples Planctomycetales planctomycete having a similarity of 91%.

Sphingobacteria:.. Sphingobacteria of the group belonging to the Bacteroidetes is a clone having Sphingobacteriaceae of Spingobacterium sp and 97% of clones 1 having a similarity, Flexibacteaceae of Taxeobacter sp and 92% similarity at the KD ₁ 1, Adhaeribacter in the Adhaeribacter aquaticus strain 90 1 clone with% similarity, Hymenobacter of the genus Hymenobacter sp., and the two clones with a similarity of 90%, Sphingobacteriaceae of Pedobacter sp. Six clones were examined, including one clone showing 97% similarity to. The KD ₄ was examined by a single clone having Hymenobacter sp. And 93% similarity Flexibacteraceae.

③ Cleanup

This experiment, published as herbicides Ventana zone _{(8㎖ / 2ℓ (H 2 0} ) / 5㎡), MCPP after using _{(7㎖ / 2ℓ (H 2 0} ) / 5㎡) and process the manure that contains the amino acid The analysis of 16S rDNA gene in grassy soil was carried out to identify changes in microbial community structure and diversity. TD-RFLP analysis of soil microorganisms of KD ₁ , KD ₂ , KD ₃ , and KD ₄ confirmed that the microbial community structure was different in the experimental group treated with the liquid fertilization doubling solution containing amino acids compared to the non-treated group (FIG. 1). As a result of 16S rDNA sequencing analysis, the microorganisms in the grass turf treated with and without the amino acid fertilization showed different distributions, and most of the clones showed inconsistent with the cultivable microorganism group (FIGS. 2 to 4). ).

After handling the pesticides deuljandi was when a KD ₃ to KD ₄ processing amino acids manure compared with KD ₂ not treated with acids manure KD _3, KD ₄ is more diverse and know that effective microorganisms inhabit the plant. In particular, KD ₄ inhabited more microorganisms than KD ₃ . In Alphaproteobacteria Rhizobiales, Sphigomonadales, and was a clone of a microorganism belonging to the Caulobacterales neck irradiation, Gamma proteobacteria were born they appear mostly in the genus Pseudomonas bacteria, Betaproteobacteria in Nitrosomonadales of Nitrosospira The genus was mainly investigated. In addition, the Acidobacteria group, Actinobacteria group, planctomycetes and Sphingobacteria groups were examined. The microorganisms investigated in these KD ₄ are bacteria that decompose various types of organic molecules and play an important role in mineralization processes in nature and in sewage treatment. In addition, these microorganisms oxidize ammonia into nitrite, and nitrogen of nitrate is inhabited mainly by microorganisms that play an important role in helping plants grow. In particular, when fertilizers containing amino acids were fertilized, Phenylobacterium immobile was found to degrade herbicides. This clone is a gram-negative bacterium and is known as a microorganism that breaks down herbicide chloridazon.

According to the novel efficacy revealed in the present invention, the amino acid liquid ratio can be usefully used for the removal of chemical pesticides remaining in the soil. In particular, the new use of the amino acid liquid fertilizer according to the present invention can be used on the lawn, such as various sports stadiums, such as golf courses, regardless of facility cultivation, outland cultivation can be used for all cropland arable land. In addition, researches on biological control have been actively conducted to reduce the damage of chemical pesticides. In order to enable biological control by microorganisms, active microorganisms must be active to suppress pathogens. Therefore, even in this case, the treatment of the amino acid liquid fertilizer according to the present invention is expected to grow a variety of microorganisms by the activation of the effective microorganisms is expected to further increase the effect of biological control.

Claims (8)

A method for removing soil residual pesticides characterized by promoting the microbiological decomposition of residual pesticides in the soil by the amino acid liquid ratio. The method of claim 1, wherein the amino acid fertilizer is treated to the soil before or after the spraying of the pesticide. The method of claim 1 wherein the soil is farmland or lawn. The method of claim 3 wherein the soil is a lawn in a golf course. The method according to any one of claims 1 to 4, wherein the amino acid liquid ratio is sprayed at 0.1 to 1 ml / m 2 on the soil on the basis of the stock solution. 6. The method according to claim 5, wherein the amino acid liquid ratio is sprayed at 0.5 to 0.7 ml / m 2 on the soil based on the stock solution. 7. The method according to claim 6, wherein the amino acid liquid ratio is treated 2-3 times at weekly intervals. Decomposition and removal of residual herbicides in soil containing amino acid fertilizer as an active ingredient.

Images