JP4395712B2 - Pesticide-degrading bacteria and purification method using the same - Google Patents

Description

  The present invention relates to a method for decomposing an amide pesticide using at least one microorganism belonging to any of the genus Nocardioides, Rhodococcus or Stenotrophomonas, a purification material retained in the microorganism carrier, and the The present invention relates to a decomposing apparatus for amide-based pesticides containing a purification material. Furthermore, the present invention relates to a microorganism having the resolution of mefenacet used as a herbicide.

  In modern agriculture, pesticides have become an indispensable technique for preventing yield loss and stabilizing yields, or for reducing farm work and lowering production costs. However, it has been pointed out that some of the used pesticides flow out of the farmland, affecting the biodiversity in the general environment. From this point of view, research into the dynamics of agricultural chemicals in agricultural land, especially in paddy fields where almost half of the agricultural chemical usage in Japan has been consumed, and some of the agricultural chemicals used were transferred from agricultural drainage channels to rivers, etc. It was revealed that the water had flowed into the general water system. According to the survey results in Chiba Prefecture from 1980 to 1984, dimethamethrin, piperophos, butachlor, clomethoxynil and bencho curve are among the 25 target pesticides whose efflux rate exceeds 1%. Further, those exceeding 3% include molinate, simethrin, oxadiazone and dimulon (Non-patent Documents 1 and 2).

  A survey conducted in Saitama Prefecture in 1986 showed that the spillage rates of molinate, simethrin, pyrazolate and butachlor were as high as 25.6, 18.4, 7.3 and 3.4%, respectively (non- Patent Document 3). Some of these pesticides are no longer sold because their registration has expired, but many are still being used in paddy fields and are therefore considered to continue to flow into rivers. .

  Even after these surveys have been conducted, new herbicidal ingredients have been put on the market one after another. In particular, after the emergence of a one-time treatment agent that lasted the herbicidal effect until harvesting by applying it once immediately after planting rice, it came to be used frequently. One of the ingredients of such a one-time treatment agent is mefenacet (2- (benzothiazol-2-yloxy) -N-methylacetanilide). . Currently, there are 31 types of registered pesticides containing mefenacet, and the amount shipped in Japan in the 2001 pesticide year exceeds 400t. Mefenacet is a pesticide mainly used immediately after transplanting rice, and it has been reported that mefenacet is detected in rivers and lakes from May to July (Non-patent Documents 4, 5, and 6). The outflow rate is 8 to 14.5% (Non-Patent Document 7) with respect to the application rate, and there is an example (Non-Patent Document 8) that reaches 25.8%. Contamination exceeding the environmental standard value (0.009mg / L) established in the water quality evaluation guidelines for agricultural chemicals in public waters (April 15, 1994, Notification of Director General of Water Quality Conservation Bureau, Environment Agency of the Environment, April 15, 1994) Although there are no reports, the measurement itself is carried out on relatively large rivers and lakes, so there is a higher concentration in small rivers where wastewater from agricultural drainage channels and agricultural drainage channels directly flows The environmental impact is concerned.

  As a method for reducing the outflow of agricultural chemicals from paddy fields, the method that is generally considered to be the easiest and most effective is the optimization of water management in paddy fields. As a countermeasure against water leakage, prevent dredged shore leakage, and if pesticides are used in paddy fields, stop water management and drainage after the pesticides have been sufficiently decomposed will reduce pesticide runoff. It is supposed to be possible. However, it is not possible to cope with runoff at the time of water increase due to rain, and the labor force is imposed on the workers.

  In addition, research has been conducted to draw agricultural wastewater into a fallow paddy field, decompose and remove agricultural chemicals using the soil purification ability, and reduce the amount of agricultural chemicals released to the general environment (Non-patent Document 9). According to the report, in the field experiment using molinate and simethrin, the outflow of agricultural chemicals was reduced by 50 to 60%. However, in this method, the ratio of the area of the fallow paddy field that can be secured as a purification site and the ratio of the planted area and the arrangement thereof are problematic, and many problems still need to be solved before this technology can be implemented in an actual paddy field.

  In addition, research has been conducted to install a pesticide removal device in the agricultural drainage channel for purification. Hokkaido Prefectural Central Agricultural Experiment Station has investigated a pesticide removal device that uses charcoal or coffee grains as adsorbents and neutral calcium hypochlorite as a water purification agent for pretilachlor, esprocarb and molinate (non-patent literature). 9). According to the report, although these pesticide removal devices showed a continuous removal effect on molinate, the removal effect on esprocarb depends on the amount of neutral calcium hypochlorite added and almost no on pretilachlor. It was shown that there was no removal effect. When the removal of agricultural chemicals depends on the adsorption to the adsorbent or the decomposition by the chemical reaction with the water purification agent as in this experimental example, the processing capacity of the apparatus is limited by the amount of the adsorbent or the purification agent. In addition, it is known that beniocarb, butachlor, bensulfuron methyl, pyrazolate, triflurane and the like are subject to decomposition by soil microorganisms in paddy field herbicides (Non-patent Document 10).

However, a technology for reducing the concentration of pesticides in the environment using the isolated pesticide-degrading bacteria has not yet been established. Only a few attempts to decompose and remove applied simazine by burying charcoal with simazine-degrading bacteria settled directly under turf grass at golf courses. There are no reports of the degrading bacteria themselves for mefenacet.
Although several attempts have been made so far to control the spilled pesticides as described above, none of them have been in widespread use because they are still in the research stage and there are problems with their performance and effectiveness. Furthermore, the effect on mefenacet has not been studied at all.

Maru 1985 Ecochemistry, 8, 3-10, Maru 1991 Special Report on Chiba Agricultural Experiment Station, 18, 1-62 Nakamura 1986 Environment Agency pesticide residue countermeasure survey, water quality residue survey results report Tanabe et al. 1996J. Chromatogr. A, 754, 159-168 Nishizawa et al. 1997 Fukui Environmental Science Center Annual Report, 27, 104-111 Ebinase et al. 1999 National Institute for Environmental Studies, 144, 37-47 Numabe et al. 1992 Journal of Water Environment, 10, 30-39 1998 Ibaraki Agricultural Research Institute Information, 166, 5-6 Hokkaido Agricultural Experiment Conference Report 2001 Environmental Activities and Runoff Mitigation of Pesticides Applied to Paddy Fields (Study on Dynamics of Pesticides in Environment and Diminishing Environmental Impact) Sasazuka and other 1998 soils and pesticides

Mefenacet, which is one of the general-purpose herbicides in paddy fields, is thought to have caused a load on the natural environment by flowing out from farmland to the general environment such as rivers. As a means for improving such a situation, a means for efficiently decomposing and removing mefenaset and making it harmless is desired.
In addition, many other amide-based pesticides are likely to be spilled from farmland and pollute the natural environment in the same way as mefenacet, and a method for detoxifying them is desired.
The present invention provides a microorganism capable of degrading amide pesticides such as mefenacet, and a method and apparatus for degrading and removing amide pesticides such as mefenacet by the microorganism.

As a result of intensive research on microorganisms collected from various places in order to solve the above problems, the present inventors have found that effective microorganisms exist among microorganisms obtained from mefenacet continuous soil, etc. the novel Nocardioides from the microorganisms (Nocardioides) genus Rhodococcus (Rhodococcus) species, and found that Stenotrophomonas (Stenotrophomonas) microorganism of the genus has a high mefenacet resolution. These microorganisms were also confirmed to degrade amide pesticides other than mefenacet, and other microorganisms belonging to these genera were found to have the resolution of amide pesticides including mefenacet as well. Furthermore, the purification material in which these microorganisms are fixed in a porous substance has been found to maintain the resolution of amide-based pesticides such as mefenacet for a long time, and the present invention has been completed.

  The present invention relates to a method for degrading an amide-based pesticide characterized by using at least one microorganism belonging to any of the genus Nocardioides, Rhodococcus, or Stenotrophomonas. More specifically, the present invention uses at least one microorganism belonging to any of the genus Nocardioides, Rhodococcus, or Stenotrophomonas, which is characterized by the following general formula (I):

(In the formula, A represents a hydrogen atom, a halogen, a C ₁ -C ₈ linear or branched alkoxy group, an optionally substituted phenyl group, an optionally substituted phenoxy group, a substituted group; A benzhydryl group which may have a group, or a benzothiazolyloxy group which may have a substituent (wherein the substituents are independently halogen, C ₁ -C ₈ chain or branched alkyl _group, a linear or branched haloalkyl group having C 1 -C _8, a carboxyl group, a hydroxyl group, a nitro group, or a cyano group.), ^{a 1} is _a straight of C 1 -C ₈ A chain or branched alkylene group, or a single bond, B ¹ is a hydrogen atom, a C ₁ -C ₈ linear or branched alkyl group, a C ₂ -C ₈ linear or branched alkenyl group , the _C 2 -C ₈ linear or branched A alkynyl group, a phenyl group which may have a substituent, or a phenylalkyl group which may have a substituent in which the alkyl group part is a C1-C8 alkyl group (herein, the substituent is each independently, _halogen, C linear or branched alkyl group having 1 -C _8, linear or branched alkoxy group C 1 -C _8, haloalkyl group of _{C 1 -C 8, C 1 -C} 8 A linear or branched haloalkylsulfonylamino group, a hydroxyl group, a nitro group, or a cyano group, and B ² is a hydrogen atom, a C ₁ -C ₈ linear or branched alkyl group, C _2- C ₈ linear or branched alkenyl group, C ₂ -C ₈ linear or branched alkynyl group, C ₁ -C ₈ linear or branched alkoxy-C ₁ -C ₈ linear or Branched alk Group, or a C ₁ of -C ₈ represents a linear or straight-chain or branched alkyl group branched alkoxycarbonyl -C ₁ -C _8, Z is an oxygen atom or a sulfur atom.)
It is related with the decomposition | disassembly method of amide type agricultural chemicals represented by these. In more detail, the present invention uses at least one microorganism belonging to any of the genus Nocardioides, Rhodococcus or Stenotrophomonas, mefenacet, mepronil, flutolanil, metolachlor and pretilachlor. The present invention relates to a method for decomposing one or more amide-based pesticides selected from the group.

The present invention also decomposes an amide pesticide in which at least one microorganism selected from the microorganisms belonging to any of the genus Nocardioides, Rhodococcus, or Stenotrophomonas is held in a microorganism carrier. The present invention relates to a purification material for purifying and a decomposition apparatus containing the purification material. More specifically, the present invention comprises a microorganism carrier that holds at least one microorganism selected from microorganisms belonging to any of the genus Nocardioides, Rhodococcus, or Stenotrophomonas. The present invention relates to a purification material for decomposing and purifying the amide pesticide represented by the general formula (I), and a decomposition apparatus containing the purification material. More specifically, the present invention allows the microorganism carrier to retain at least one microorganism selected from microorganisms belonging to any of the genus Nocardioides, Rhodococcus, or Stenotrophomonas. The present invention relates to a purification material for decomposing and purifying one or more amide pesticides selected from the group of mefenacet, mepronil, flutolanil, metolachlor and pretilachlor, and a decomposition apparatus containing the purification material .
Furthermore, the present invention provides a novel and useful Nocardioides sp. MFC-A (FERMP-19396), Rhodococcus rhodochrous MFC-B (FERM P-19397), or Stenotrophomonas sp. The present invention relates to MFC-C (FERM P-19398), or a microorganism having mefenacet resolution having part or all of the mycological properties of these strains.

  In order to prevent the outflow of amide-based pesticides such as mefenacet, the present inventors have studied methods for biological degradation of these pesticides. Various microorganisms have been collected and biological decomposition methods for these pesticides have been studied. We have collected various microorganisms and examined the availability of these pesticides. We succeeded in isolating new microorganisms with these pesticides from bark compost and mefenacet continuous soil. There were three types of isolated strains, and these were designated as MFC-A strain, MFC-B strain, and MFC-C strain, respectively. The morphological and physiological properties of these three strains were investigated. The results are shown in Table 1 below. In Table 1, “+” indicates presence or positive, and “−” indicates absence or negative.

  In addition, the biochemical properties of these strains were examined. The biochemical properties of MFC-A strain and MFC-B strain among the three isolated strains are shown in Table 2 below, and the biochemical properties of MFC-C strain are shown in Table 3 below.

  In Table 2, the API system (BioMerieux, France) API 20NE measurement method (20057) was followed. Moreover, the method of Table 3 followed the API system API Coryne measurement method (20907). In addition, none of the strains formed aerial hyphae, and did not exhibit anaerobic growth and casein hydrolyzability.

Based on the above mycological properties, the taxonomic positions of MFC-A strain, MFC-B pearl and MFC-C strain were searched with reference to “Bergery's Manual of Systematic Bacteriology”. However, Nocardioides sp. ( Nocardioides sp. ), Rhodococcus rhodochrous and Stenotrophomonas sp. It was determined to be a strain belonging to ( Stenotrophomonas sp. ). These Nocardioides sp. MFC-A strain, Rhodococcus rhodochrous MFC-B strain, and Stenotrophomonas sp. The MFC-C strains are deposited as FERM P-19396, FERM P-19397, and FERM P-19398, respectively, at the National Institute of Advanced Industrial Science and Technology.

These new species of microorganisms are respectively Nocardioides sp. ( Nocardioides sp. ), Rhodococcus rhodochrous , and Stenotrophomonas sp. Since it was found to be a strain belonging to ( Stenotrophomonas sp. ), The strain belonging to these genera was also examined for pesticide degradability. That is, Nocardioides sp. (Nocardioides sp.) In addition to the MFC-A strain for the genus, Nocardioides simplex (Nocardioides simplex) JCM1363 strain, Nocardioides Jenseni (Nocardioides jensenii) JCM1364 strain and Nocardioides Furubusu (Nocardioides fulvus) chose the JCM3335 strain It is.
The Rhodococcus, in addition to the MFC-B strain, Rhodococcus rhodochrous (Rhodococcus rhodochrous) JCM2156 strain, Rhodococcus rhodochrous (Rhodococcus rhodochrous) JCM2157 strain, Rhodococcus rhodochrous (Rhodococcus rhodochrous) JCM2158 strain, Rhodococcus rhodochrous (Rhodococcus rhodochrous) JCM3010 strain, Rhodococcus rhodochrous (Rhodococcus rhodochrous) JCM3202 strain, Rhodococcus coprophilus (Rhodococcus coprophilus) JCM3200 strain, Rhodococcus ruber (Rhodococcus ruber) JCM 3205 strain and Rhodococcus Zopufi chose (Rhodococcus zopfii) JCM9919 strain.
For Stenotrophomonas (Stenotrophomonas) genus, in addition to the MFC-C strain, it chose Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) ATCC13637 strain.

Further, as a control bacteria, as usually a bacteria such as E. coli, Escherichia coli (Escherichia coli) NIHJJC-2 strain, Bacillus subtilis (Bacillus subtilis) ATCC 6633 strain, Staphylococcus aureus (Staphylococcus aureus) 209PJC-1 strain, Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) P-2 strain and Proteus vulgaris IID874 strain were selected.
These fungal species were tested for pesticide degradability using mefenacet. A liquid medium containing mefenacet at a rate of 1 mg / L was prepared and each bacterial species was inoculated. After completion of the culture, each culture solution was analyzed by HPLC, and the mefenacet concentration was determined by an absolute calibration curve method. The results are shown in Table 4 below.

  As a result, it was found that all of the bacteria other than those used as control bacteria had mefenacet degradation activity. This is not only about the three types of MFC-A strain, MFC-B strain and MFC-C strain that were found this time, but also about various degradability of pesticides such as mefenacet. This means that the fungus has a pesticide-degrading activity, and the method for decomposing the pesticide of the present invention is limited to the three types of MFC-A strain, MFC-B strain and MFC-C strain. Rather, many strains of the genus to which these bacteria belong have similar degrading activity. That is, the present invention provides an amide-based pesticide, preferably mefenacet, mepronil, flutolanil, metola, characterized by using at least one microorganism belonging to any of the genus Nocardioides, Rhodococcus or Stenotrophomonas. It provides a method for degrading chlor and pretilachlor, more preferably mefenacet.

Next, the dependency of pH on the degradation of mefenacet was examined for three strains, MFC-A strain, MFC-B strain and MFC-C strain.
Add mefenacet to a liquid medium adjusted to pH 5, 6, 7, 8 and 9 so that the final concentration is 1 mg / L, and then add MFC-A strain, MFC-B strain, and MFC-C strain. Each was added, and cultured with shaking at 30 ° C. and 120 rpm for 5 days. A part of the culture solution was sampled over time, and the mefenacet concentration was measured. The concentration of mefenacet in the medium was determined by an absolute calibration curve method using HPLC.
The result is shown in FIG. 1A shows the case of MFC-A strain, FIG. 1B shows the case of MFC-B strain, and FIG. 1C shows the case of MFC-C strain. The vertical axis of each graph in FIG. 1 shows the concentration of mefenacet (mg / L), and the horizontal axis shows the culture time (days). In each graph of FIG. 1, the black circle (●) indicates the case where the initial pH of the medium is 4, the white triangle mark (Δ) indicates the case where the initial pH is 5, and the white circle mark (◯) indicates the initial pH of the medium. 6, the black square mark (■) indicates the case where the initial pH of the medium is 7, and the black triangle mark (▲) indicates the case where the initial pH of the medium is 8.
As a result, in MFC-A strain and MFC-B strain, mefenacet degradation activity was not observed when the initial pH of the medium was 4 or 5 (see FIGS. 1 (A) and (B)). Then, it was found that when the initial pH of the medium was strong acidity of 4, the mefenacet degradation activity was not observed. (See FIG. 1C). That is, it can be seen that the mefenacet decomposition requires mildly acidic to alkaline conditions of about pH 5 to pH 6.

Next, about three types of MFC-A strain, MFC-B strain, and MFC-C strain, the resolution | decomposability with respect to four types of mepronil, a flutolanil, a metolachlor, and a pretilachlor was investigated as amide type agricultural chemicals other than a mefenacet.
Add the pre-cultured MFC-A strain, MFC-B strain or MFC-C strain to the liquid medium containing the pesticides, and perform shaking culture at 26 ° C. The concentration of pesticide was measured by sampling. The measurement of the pesticide concentration in the medium was determined by the absolute calibration curve method using HPLC.
The result is shown in FIG. 2 (A) shows the case of mepronil, FIG. 2 (B) shows the case of flutolanil, FIG. 2 (C) shows the case of metolachlor, and FIG. 2 (D) shows the case of pretilachlor. Indicates. The square mark (■) in each graph of FIG. 2 indicates the case of MFC-A strain, the circle mark (●) indicates the case of MFC-B strain, and the triangle mark (▲) indicates the case of MFC-C strain. . Furthermore, a broken line shows the case of the control which has not been inoculated.
As a result, when the MFC-C strain was inoculated in the medium supplemented with mepronil (see FIG. 2A), the medium supplemented with flutolanil or metolachlor was inoculated with the MFC-A strain (FIG. 2). (See (B) and (C)), when any strain was inoculated in the medium added with pretilachlor (see FIG. 2D), a decrease in the concentration of each pesticide was observed compared to the control. . That is, it was confirmed that the microorganisms of the present invention have the ability to decompose not only mefenacet but also amide pesticides such as flutolanil, metolachlor and pretilachlor, preferably amide pesticides represented by the general formula (I). It was.

Next, the resolution of mefenaset was examined when these strains were retained on a microorganism carrier to produce a purification material. MFC-B strain was used as the microorganism, and charcoal, brick, and vermiculite were used as the microorganism holder.
First, a purification material prepared by holding a mefenacet on a microorganism holder was prepared. This was added to a liquid medium containing mefenacet at a rate of 10 mg / L, cultured with shaking at 30 ° C. and 120 rpm, and the concentration of mefenacet in the culture was measured over time by HPLC. The same operation was performed on the autoclaved sterilization material, and the results were compared. In addition, the thing which did not add material was set as the control.
The results are shown in FIG. The vertical axis of the graph in FIG. 3 indicates the concentration of mefenacet (mg / L), and the horizontal axis indicates the culture time (days).
In Fig. 3, rhombus marks (◇, ◆) indicate the case of using brick, square marks (□, ■) indicate the case of using charcoal, and circle marks (○, ●) indicate the case of using vermiculite. The triangle marks (Δ, ▲) indicate the case where the microorganism carrier is not used as a control. Each white mark indicates a case where each material is autoclaved.
As a result, in any case, when the purification material is sterilized by autoclaving, no decomposition activity is observed, but when it is not sterilized, it can show strong decomposition activity even if any purification material is used. Indicated. That is, it was confirmed that the strain having the resolution of the amide pesticide of the present invention can be used as a purification material using a microorganism carrier.

Furthermore, in order to confirm whether the above-described purification material of the present invention can be used continuously for a long period of time, mefenacet is included as a sole carbon source at a rate of 1 mg / L and the pH is set to 7. The conditioned basic inorganic salt medium was continuously tested. Cultivation culture was carried out at 30 ° C. and 120 rpm for 2.6 days, and thereafter, culture was continued under the same conditions while changing the medium with the same composition every 2 days, and the mefenacet concentration during that time was measured over time by HPLC. .
The results are shown in FIG. 4A shows the results for the first 2.6 days, FIG. 4B shows the results for the next 2 days, and FIG. 4C shows the results for the next 2 days. Show. The vertical axis of each graph in FIG. 4 indicates the concentration of mefenacet (mg / L), and the horizontal axis indicates the culture period (days). In each graph in Fig. 4, diamond marks (◇, ◆) indicate the case of using brick, square marks (□, ■) indicate the case of using charcoal, and circle marks (○, ●) use vermiculite. The triangle marks (Δ, ▲) indicate the case where the microorganism carrier is not used as a control. Each white mark indicates a case where each material is autoclaved.
As a result, a weak activity was observed in the first 2.6 days in the control group (black triangle (▲)) that did not use the microorganism carrier, but the activity decreased when continuously used. I understood. In addition, when a microbial support is used, continuous use is possible, but the activity tends to decrease little by little. However, when charcoal was used as the microorganism carrier (black square mark (■)), it was found that the activity could be maintained for a long time even if it was continuously used. Charcoal is a porous wood carbonized material, and it can be seen that a porous wood carbonized material such as charcoal is extremely excellent as the microorganism-supporting body of the present invention.

As described above, the present invention provides a method for decomposing an amide-based pesticide characterized by using at least one kind of microorganism belonging to any of the genus Nocardioides, Rhodococcus or Stenotrophomonas. It is to provide. The microorganism of the present invention is a microorganism belonging to any of the genus Nocardioides, Rhodococcus, or Stenotrophomonas, as long as it has at least one resolution of amide pesticides including mefenacet. Preferably, the microorganism preferably has a mefenacet resolution.
The “genus” used in the present invention basically follows the taxonomy defined in biology (see, for example, the Bergery's Manual of Systematic Bacteriology), but is not necessarily limited thereto. Any microorganism having the bacteriological properties similar to those of these genera and having at least one resolution of amide pesticides including mefenacet is included as the microorganism of the present invention.
As an example of the microorganism of the present invention, as a microorganism classified into the genus Nocardioides, Nocardioides sp. MFC-A strain, Nocardioides simplex JCM1363 strain, Nocardioides jensenii JCM1364 strain and Nocardioides fulvus ( Nocardioides fulvus 35 strain) is Rhodococcus rhodochrous MFC-B strain, Rhodococcus rhodochrous (Rhodococcus rhodochrous) JCM2156 strain, Rhodococcus rhodochrous (Rhodococcus rhodochrous) JCM2157 strain, Rhodococcus rhodochrous (Rhodococcus rhodochrous) JCM2158 strain, b Dokokkasu rhodochrous (Rhodococcus rhodochrous) JCM3010 strain, Rhodococcus rhodochrous (Rhodococcus rhodochrous) JCM3202 strain, Rhodococcus coprophilus (Rhodococcus coprophilus) JCM3200 strain, Rhodococcus ruber (Rhodococcus ruber) JCM3205 strain and Rhodococcus Zopufi (Rhodococcus zopfii) such as JCM9919 strain and the like, Steno Torofomonasu (Stenotrophomonas) the microorganisms that are classified in the genus, Stenotrophomonas sp. Examples include MFC-C strain and Stenotrophomonas maltophilia ATCC13637 strain.

Among these microbial groups, Nocardioides sp. MFC-A, Rhodococcus rhodochrous MFC-B, and Stenotrophomonas sp. MFC-C is a novel microorganism isolated from nature. All of these three species have high amide pesticide resolution including high mefenacet.
Therefore, the present invention provides these new types of microorganisms. The new species of microorganism of the present invention is Nocardioides sp. MFC-A (FERM P-19396), Rhodococcus rhodochrous MFC-B (FERM P-19397), and Stenotrophomonas sp. There are three types of MFC-C (FERM P-19398). These microorganisms of the present invention include not only these microorganisms themselves but also microorganisms having some or all of the mycological properties of these microorganisms and having amide-type pesticide-degrading ability including mefenacet. is there. The part of the mycological property is preferably 50% or more and 70% of the above-mentioned mycological property. Or it is 80% or more, and it is not common in all mycological properties, but it is common in many parts. Therefore, “having part or all of mycological properties” as used in the present invention does not refer to a completely different bacterial species, but is common in many respects to the new bacterial species of the present invention. It is a bacterial species having bacteriological properties and similarities.

  The amide pesticide in the present invention is one that is decomposed by at least one microorganism of the genus Nocardioides, Rhodococcus, or Stenotrophomonas, and has at least one in the molecule. Any one having an amide group or a thioamide group may be used. Specific examples of the amide pesticide of the present invention include the following general formula (I)

(In the formula, A represents a hydrogen atom, a halogen, a C ₁ -C ₆ linear or branched alkoxy group, a phenyl group which may have a substituent, a phenoxy group which may have a substituent, a substituted group. A benzhydryl group which may have a group, or a benzothiazolyloxy group which may have a substituent (wherein the substituents are independently halogen, C ₁ -C ₆ A chain or branched alkyl group, a C ₁ -C ₃ linear or branched haloalkyl group, a carboxyl group, a hydroxyl group, a nitro group, or a cyano group.), A ¹ is a straight chain of C ₁ -C ₆ . A chain or branched alkylene group, or a single bond, B ¹ is a hydrogen atom, a C ₁ -C ₆ linear or branched alkyl group, a C ₂ -C ₆ linear or branched alkenyl group , the _C 2 -C ₆ linear or branched A alkynyl group, a phenyl group which may have a substituent, or a phenylalkyl group in which the alkyl group part may have a substituent which is a C1-C6 alkyl group (herein, the substituent is each independently, _halogen, C 1 -C linear or branched alkyl group of _6, linear or branched alkoxy group C 1 -C _6, haloalkyl group of _{C 1 -C 3, C 1 -C} 3 A linear or branched haloalkylsulfonylamino group, a hydroxyl group, a nitro group, or a cyano group.), B ² is a hydrogen atom, a C ₁ -C ₆ linear or branched alkyl group, C _2- A C ₆ linear or branched alkenyl group, a C ₂ -C ₆ alkynyl group, a C ₁ -C ₃ linear or branched alkoxy-C ₁ -C ₆ linear or branched alkyl group, or _C 1 -C Of a linear or straight-chain or branched alkyl group branched alkoxycarbonyl -C ₁ -C _5, Z is an oxygen atom or a sulfur atom.)
An amide-based pesticide represented by

Examples of the halogen in the general formula (I) include fluorine, chlorine, bromine and iodine. In the general formula (I), the alkyl group is a linear or branched alkyl group having 1 to 8, preferably 1 to 6, and optionally 1 to 3 carbon atoms. Preferred alkyl group is C _1. include straight-chain or branched alkyl group -C _6, more specifically, for example, a methyl group, an ethyl group, n- propyl group, i- propyl, n- butyl, t- butyl group, 2 , 2-dimethylpropyl group and the like. In the general formula (I), the alkoxy group is an alkyloxy group derived from a linear or branched alkyl group having 1 to 8, preferably 1 to 6, and optionally 1 to 3 carbon atoms, Preferred alkoxy groups include C ₁ -C ₆ linear or branched alkoxy groups, and in some cases, C ₁ -C ₃ linear or branched alkoxy groups. More specifically, for example, a methoxy group Ethoxy group, n-propoxy group, i-propoxy group, n-butoxy group, t-butoxy group and the like. In the general formula (I), the alkenyl group is a linear or branched alkenyl group having 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, and the preferred alkenyl group is a C ₂ -C ₆ straight chain or Examples thereof include a branched alkenyl group, and more specifically, for example, vinyl group, n-1-propenyl group, n-2-propenyl group, propen-2-yl group, n-1-butenyl group, n-2. -Butenyl group, n-1-pentenyl group, etc. are mentioned. In the general formula (I), the alkynyl group is a straight chain or branched alkynyl group having 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, and the preferred alkynyl group is a C ₂ -C ₆ straight chain or Examples thereof include branched alkynyl groups, and more specifically, for example, ethynyl group, n-1-propynyl group, n-2-propynyl group, propyn-2-yl group, n-1-butynyl group, n-2 -Butynyl group, n-1-pentynyl group, etc. are mentioned.

In the general formula (I), the alkylene group is a divalent group derived from a linear or branched alkyl group having 1 to 8, preferably 1 to 6, and optionally 1 to 3 carbon atoms. Preferred alkylene groups include C ₁ -C ₆ linear or branched alkylene groups, and more specifically, for example, methylene group, 1,2-ethylene group, 1,1-ethylene group, 1, 1-n-propylene group, 1,2-n-propylene group, 1,3-n-propylene group, 2,2-n-propylene group, 1,1-n-butylene group, 1,2-n-butylene Group, 1,3-n-butylene group, 1,4-n-butylene group, derivatives thereof and the like. In the general formula (I), the phenylalkyl group is a group formed by substituting a phenyl group at an arbitrary position of the above-described alkyl group of the present invention. As a preferable phenylalkyl group, the alkyl group moiety is C1. -C6 alkyl group is a phenylalkyl group, and more specifically, for example, benzyl group, 2-phenylethyl group, 1 phenylethyl group, 3-phenylpropyl group, 2-phenylpropyl group, 1-phenylpropoxy group. Group, α, α-dimethylbenzyl group and the like.

In the general formula (I), the alkoxyalkyl group is a group formed by substituting the above-described alkoxy group of the present invention at any position of the above-described alkyl group of the present invention. Preferred alkoxyalkyl groups include “C ₁ -C ₃ linear or branched alkoxy-C ₁ -C ₆ linear or branched alkyl group” can be mentioned, and more specifically, for example, methoxymethyl group, 2-methoxyethyl group 1-methoxyethyl group, 3-methoxypropyl group, 2-methoxypropyl group, 1-methoxypropyl group, 2-methoxy-1-methyl-ethyl group, ethoxymethyl group, 2-ethoxyethyl group, 1-ethoxyethyl Group, 3-ethoxypropyl group, 2-ethoxypropyl group, 1-ethoxypropyl group, 2-ethoxy-1-methyl-ethyl group, n-propyl group Xymethyl group, 2- (n-propoxy) ethyl group, 1- (n-propoxy) ethyl group, 3- (n-propoxy) propyl group, 2- (n-propoxy) propyl group, 1- (n-propoxy) Propyl group, 2- (n-propoxy) -1-methyl-ethyl group, i-propoxymethyl group, 2- (i-propoxy) ethyl group, 1- (i-propoxy) ethyl group, 3- (i-propoxy) ) Propyl group, 2- (i-propoxy) propyl group, 1- (i-propoxy) propyl group, 2- (i-propoxy) -1-methyl-ethyl group and the like. In the general formula (I), the alkoxycarbonylalkyl group is a group formed by substituting an alkoxycarbonyl group derived from the alkoxy group of the present invention at any position of the alkyl group of the present invention. And preferred alkoxycarbonylalkyl groups include “C ₁ -C ₃ linear or branched alkoxycarbonyl-C ₁ -C ₅ linear or branched alkyl groups”, and more specifically, for example, , Methoxycarbonylmethyl group, 2-methoxycarbonylethyl group, 1-methoxycarbonylethyl group, 3-methoxycarbonylpropyl group, 2-methoxycarbonylpropyl group, 1-methoxycarbonylpropyl group, ethoxycarbonylmethyl group, 2-ethoxycarbonyl Ethyl group, 1-ethoxycarbonylethyl 3-ethoxycarbonylpropyl group, 2-ethoxycarbonylpropyl group, 1-ethoxycarbonylpropyl group, n-propoxycarbonylmethyl group, 2- (n-propoxycarbonyl) ethyl group, 1- (n-propoxycarbonyl) ethyl group 3- (n-propoxycarbonyl) propyl group, 2- (n-propoxycarbonyl) propyl group, 1- (n-propoxycarbonyl) propyl group, i-propoxycarbonylmethyl group, 2- (i-propoxycarbonyl) ethyl Group, 1- (i-propoxycarbonyl) ethyl group, 3- (i-propoxycarbonyl) propyl group, 2- (i-propoxycarbonyl) propyl group, 1- (i-propoxycarbonyl) propyl group and the like. The haloalkyl group in the general formula (I) is a group formed by substituting the halogen of the present invention at any position of the alkyl group of the present invention, and preferred haloalkyl group is C. _1- C ₆ linear or branched haloalkyl group, C ₁ -C ₃ linear or branched haloalkyl group, and more specifically, for example, fluoromethyl group, chloromethyl group, bromomethyl group, Dichloromethyl group, trifluoromethyl group, 2-fluoroethyl group, 1-fluoroethyl group, 2-chloroethyl group, 1-chloroethyl group, 2-bromoethyl group, 1-bromoethyl group, 2,2,2-trifluoroethyl Groups and the like.

  In the general formula (I), an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, a phenyl group, a phenoxy group, a phenoxyalkyl group, a benzhydryl group, and a benzothiazolyloxy group are substitutable positions of these groups. Optionally have one or more substituents. Examples of such a substituent include the halogen described above, the alkyl group described above, the alkoxy group described above, the haloalkyl group described above, the carboxyl group, the hydroxyl group, the nitro group, the cyano group, and the amino group substituted with the haloalkylsulfonyl described above (haloalkyl). A sulfonylamino group), a carbonyl group substituted with alkoxy as described above (alkoxycarbonyl group), and the like. More specifically, the substituent of the alkyl group preferably includes halogen, an alkoxy group or an alkoxycarbonyl group, and the substituent of the alkenyl group preferably includes halogen, an alkoxy group or an alkoxycarbonyl group, and alkynyl. The substituent of the group is preferably a halogen, an alkoxy group or an alkoxycarbonyl group, and the substituent of the alkoxy group is preferably a halogen, an alkoxy group or an alkoxycarbonyl group. Further, as the substituent of the phenyl group, preferably, a halogen, an alkyl group, an alkoxy group, a carboxyl group, a haloalkyl group, a haloalkylsulfonylamino group, an alkoxycarbonyl group, and the like are mentioned, and as the substituent of the phenylalkyl group, Halogen, alkyl group, alkoxy group, carboxyl group, haloalkyl group, haloalkylsulfonylamino group, alkoxycarbonyl group and the like are mentioned, and the substituent of phenoxy group is preferably halogen, alkyl group, alkoxy group, carboxyl group, haloalkyl group , A haloalkylsulfonylamino group, an alkoxycarbonyl group, and the like. As the substituent of the benzhydryl group, a halogen, an alkyl group, an alkoxy group, a carboxyl group, a haloalkyl group, a haloalkyl group is preferable. Examples of the substituent for the benzothiazolyloxy group include a halogen, an alkyl group, an alkoxy group, a carboxyl group, a haloalkyl group, a haloalkylsulfonylamino group, and an alkoxycarbonyl group. Can be mentioned.

  It is obvious to those skilled in the art that the groups having the substituents described above are not limited to the combinations described above, and each combination is specified and directly and uniquely disclosed. That is. Specific examples of these substituent groups include alkyl substitution such as 2-methylphenyl group, 2,6-dimethylphenyl group, 2-methyl-6-ethylphenyl group, and 2,6-diethylphenyl group. Alkoxy-substituted phenyl groups such as phenyl group, 3-i-propoxyphenyl group, haloalkyl-substituted phenyl groups such as 2-trifluoromethylphenyl group, 3,5-dichlorophenyl group, 3,4-dichlorophenyl group, 2,6-dichlorophenyl Group, halogen-substituted phenyl group such as 2,3-dichlorophenyl group, carboxyl group-substituted phenyl group such as 2,3,4,5-tetrachloro-6-carboxyphenyl group or halogen and carboxy group-substituted phenyl group, 4,6 -Halo such as dimethyl-3- (trifluoromethylsulfonylamino) -phenyl group Alkyl-substituted phenylalkyl groups such as a ruylsulfonylamino-substituted phenyl group, 1-methyl-1-phenylethyl group, halogen-substituted phenoxy groups such as 2,4-dichloro-3-methylphenoxy group, alkyl-substituted phenoxy groups, or halogens And alkyl-substituted phenoxy groups, alkyl-substituted alkynyl groups such as 1,1-dimethyl-3-propynyl group, alkoxy-substituted alkyl groups such as 2- (n-propoxy) -ethyl group, 1-methyl 2-methoxyethyl group, etc. Can be mentioned.

The AA ¹ -moiety in the general formula (I) forms an acid part of an amide group together with an adjacent carbonyl group or thiocarbonyl group, and the preferred AA ¹ -moiety has a substituent. linear or branched alkyl group optionally C ₁ -C ₈ also, may have a substituent group, an optionally substituted phenoxyalkyl group, which may have a substituent A benzhydryl group or a benzothiazolyloxyalkyl group which may have a substituent, and these substituents are each independently halogen, C ₁ -C ₆ linear or branched alkyl. group, a linear or branched alkoxy group C ₁ -C _6, linear or branched haloalkyl group having C ₁ -C _3, a carboxyl group, a hydroxyl group, a nitro group, or a cyano group.

The alkyl group herein is the same as the above-described alkyl group, and may have one or two or more substituents at any substitutable position. Examples of such substituents include independent groups. to, halogen, straight-chain or branched alkoxy group of C ₁ -C _8, a carboxyl group, a hydroxyl group, a nitro group, or a cyano group. More specifically, examples include an alkoxyalkyl group such as a methoxymethyl group, a halogen-substituted alkyl group such as a chloromethyl group, and a 1-bromo-t-pentyl group.
The above-described phenoxyalkyl group is a group formed by substituting a phenoxy group at any position of the above-described alkyl group of the present invention, and a preferable phenoxyalkyl group is a straight chain having an alkyl moiety of C ₁ -C ₆ . Or the phenoxyalkyl group which is a branched alkyl group is mentioned, More specifically, a phenoxymethyl group, 2-phenoxyethyl group, 1-phenoxyethyl group etc. are mentioned, for example. These phenoxyalkyl groups may have one or more substituents at any substitutable position. Examples of such substituents include, independently, halogen, C _1- A C ₆ linear or branched alkyl group, a C ₁ -C ₆ linear or branched alkoxy group, a C ₁ -C ₃ linear or branched haloalkyl group, a carboxyl group, a hydroxyl group, a nitro group, Or a cyano group etc. are mentioned. More specifically, examples include 1- (2,4-dichloro-3-methyl-phenoxy) -ethyl group.
The above-mentioned benzothiazolyloxyalkyl group is a group formed by substituting a benzothiazolyloxy group, preferably a benzothiazol-2-yl-oxy group, at any position of the above-described alkyl group of the present invention. Preferred benzothiazolyloxyalkyl groups include benzothiazolyloxyalkyl groups in which the alkyl moiety is a C ₁ -C ₆ linear or branched alkyl group, and more specifically, for example, Examples include benzothiazol-2-yl-oxy-methyl group, 2- (benzothiazol-2-yl-oxy) -ethyl group, 1- (benzothiazol-2-yl-oxy) -ethyl group, and the like. These phenoxyalkyl groups may have one or more substituents at any substitutable position. Examples of such substituents include, independently, halogen, C _1- A C ₆ linear or branched alkyl group, a C ₁ -C ₆ linear or branched alkoxy group, a C ₁ -C ₃ linear or branched haloalkyl group, a carboxyl group, a hydroxyl group, a nitro group, Or a cyano group etc. are mentioned.
In addition, the above-described phenyl group and benzhydryl group may also have one or more substituents at any substitutable position. Examples of such substituents include, independently, a halogen atom. C ₁ -C ₆ linear or branched alkyl group, C ₁ -C ₆ linear or branched alkoxy group, C ₁ -C ₃ linear or branched haloalkyl group, carboxyl group, hydroxyl group , A nitro group, or a cyano group. Examples of such substituents include those described above.

Each option of A, A ¹ , B ¹ , B ² , and Z in the general formula (I) is arbitrarily selected so that each can be combined to form an amide group or a thioamide group. It will be apparent to those skilled in the art that each combination identified above is disclosed directly and unambiguously.
As a preferable example of such a combination, for example, A of the amide pesticide represented by the general formula (I) has a halogen, a phenyl group which may have a substituent, or a substituent. An optionally substituted benzothiazolyloxy group (wherein the substituents are each independently a C ₁ -C ₈ linear or branched alkyl group, a C ₁ -C ₈ linear or branched group, A ¹ represents a C ₁ -C ₈ linear or branched alkylene group or a single bond, and B ¹ represents a phenyl group which may have a substituent. (Here, the substituents are each independently halogen, C ₁ -C ₈ linear or branched alkyl group, or C ₁ -C ₈ linear or branched alkoxy group). B ² is a hydrogen _atom, a linear or branched C 1 -C ₈ Alkyl group, a linear or straight-chain or branched alkyl group branched alkoxy -C ₁ -C ₈ of C ₁ -C _8, there may be mentioned those in which Z represents an oxygen atom, to which It is not limited.
Specific examples of the amide-based pesticide represented by the general formula (I) include the following pesticides having an amide bond in the molecule, but are not particularly limited thereto. Absent.

  Preferable amide pesticides of the present invention include mefenacet, mepronil, flutolanil, metolachlor and pretilachlor, more preferably mefenacet, flutolanil and pretilachlor, and more preferably mefenacet.

The present invention also provides a purification material in which at least one microorganism selected from microorganisms belonging to any of the genus Nocardioides, Rhodococcus, or Stenotrophomonas is held in a microorganism holder. To do. At least one microorganism selected from microorganisms belonging to any of the genus Nocardioides, Rhodococcus, or Stenotrophomonas has at least one resolution of amide pesticides such as mefenacet. Hereinafter also referred to as amide-based pesticide-degrading bacteria. The purification material of the present invention is a purification in which amide pesticide-degrading bacteria such as mefenacet are cultured together with a microorganism carrier composed of a porous material, and amide pesticide-degrading bacteria such as mefenacet are fixed on the porous material. The material can be used for decomposing amide-based pesticides, preferably mefenacet, mepronil, flutolanil, metolachlor and pretilachlor, more preferably mefenacet. As described above, the purification material of the present invention holds an amide-based agricultural chemical, preferably mefenacet, mepronil, flutolanil, metolachlor and pretilachlor, more preferably one or more microorganisms that degrade mefenacet in a microorganism carrier. It is possible to purify soil and water contaminated with amide pesticides, preferably mefenacet, mepronil, flutolanil, metolachlor and pretilachlor, more preferably mefenacet.
The microorganism holder in the purification material of the present invention is not particularly limited as long as it is a porous substance and can stably inhabit microorganisms. For example, charcoal and brick stones that are wood carbonaceous substances Vermiculite and the like are exemplified as a preferred microorganism carrier. The microorganism holder of the present invention can be of any size, but has a size of about 1 to 150 mm, 3 to 100 mm, preferably about 3 to 50 mm for convenience of the manufacturing process and transportation process. can do.
In the present invention, the purification material is shown in Table 5 below.

Table 5 Composition of 1/10 LB liquid medium ───────────────────────
Medium content

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Tryptone 1g
Sodium chloride 1g
Yeast extract 0.5g
Milli-Q water 1000m1

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For example, charcoal, brickstone, and vermiculite are sterilized in a medium such as 1/10 LB liquid medium as shown in Fig. 1 and inoculated with the mefenacet-degrading bacterium of the present invention, followed by shaking culture. The purification material of the present invention can be produced by removing the culture solution after the cultivation and fixing the mefenacet-degrading bacteria, but is not limited to this production method. The culture can be performed at a pH of 5 to 10, preferably at a pH of 6 to 9, and at a temperature of 20 ° C. to 50 ° C., preferably about 24 ° C. to 38 ° C. for 10 hours to 100 hours.
By installing the material for purification of the present invention in drains, waterways and rivers contaminated with amide-based pesticides such as mefenacet, or by immersing them in wastewater, amide-based pesticides such as mefenacet can be efficiently used for a long time. It can be decomposed and removed.

The present invention provides an apparatus for decomposing amide-based agricultural chemicals, preferably mefenacet, mepronil, flutolanil, metolachlor and pretilachlor, more preferably mefenacet, comprising the above-described purification material of the present invention. The purification material of the present invention can be used as it is buried in the soil or thrown into the water, but in order to prevent the purification material from scattering or flowing out. The purification material of the present invention can also be used as a decomposition apparatus in a container. Such a container may have various shapes such as a bag shape, a flat plate shape, a rectangular parallelepiped shape, and a spherical shape. By making the surface of the container net-like, it can be used as it is embedded in soil or dropped in water.
Further, as another example of the decomposition apparatus of the present invention, the purification material of the present invention is fixed in a container, and the soil or water in the container is introduced to purify the contaminated soil and water in the container. Then, it can also be set as the batch type or continuous type apparatus which discharges | purifies the purified soil and water.

  The present invention relates to a microorganism belonging to the genus Nocardioides, Rhodococcus and Stenotrophomonas which effectively degrades mefenacet which is one of the herbicides, and a method for degrading amide pesticides including mefenacet using the microorganism Can be provided. Furthermore, the purification material retaining these microorganisms can maintain the resolution of amide pesticides such as mefenacet for a long time.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.

Isolation of mefenacet-degrading bacteria and their bacteriological properties Mefenacet-degrading bacteria were accumulated in an inorganic salt medium using mefenacet-continuous soil as a microbial source and mefenacet as the sole carbon source and nitrogen source, and then 1/10 LB liquid medium Screening is performed in a medium (mefenacet-containing 1/10 LB medium) added with 10 ml of a 0.1 g / L mefenacet acetone solution to a final concentration of 1 mg / L (see Table 5) and has one kind of mefenacet resolution. A colony was obtained. This was named MFC-A strain. Further, from the bark compost, after culturing in an inorganic salt medium, screening was performed in a 1/10 LB medium containing mefenacet to obtain two types of colonies. These were named MFC-B strain and MFC-C strain, respectively. The morphological and physiological properties of these three strains were investigated. The results are shown in Table 1.
In addition, the biochemical properties of these strains were examined. Among the three isolated strains, biochemical properties of MFC-A strain and MFC-B strain are shown in Table 2, and biochemical properties of MFC-C strain are shown in Table 3, respectively.
From the above mycological properties, the taxonomic positions of MFC-A strain, MFC-B pearl and MFC-C strain were searched with reference to the 9th edition of Bergery's Manual of Systematic Bacteriology. ( Nocardioides sp. ), Rhodococcus rhodochrous and Stenotrophomonas sp. ( Stenotrophomonas sp. ). These Nocardioides sp. MFC-A strain, Rhodococcus rhodochrous MFC-B strain and Stenotrophomonas sp. The MFC-C strains were deposited as FERM P-19396, FERM P-19397, and FERM P-19398, respectively, at the National Institute of Advanced Industrial Science and Technology.

Measurement of mefenacet resolution Nocardioides sp. Classified as Nocardioides sp. MFC-A strain, Nocardioides simplex JCM1363 strain, Nocardioides jensenii JCM1364 strain and Nocardioides Fulboc strain J , Rhodococcus rhodochrous (Rhodococcus rhodochrous) JCM2156 strain, Rhodococcus rhodochrous (Rhodococcus rhodochrous) JCM2157 strain, Rhodococcus rhodochrous (Rhodococcus rhodochrous) JCM2158 strain, Rhodococcus rhodochrous (Rh dococcus rhodochrous) JCM3010 strain, Rhodococcus rhodochrous (Rhodococcus rhodochrous) JCM3202 strain, Rhodococcus coprophilus (Rhodococcus coprophilus) JCM3200 strain, Rhodococcus ruber (Rhodococcus ruber) JCM3205 strain and Rhodococcus Zopufi (Rhodococcus zopfii) JCM9919 strain, Stenotrophomonas (Stenotrophomonas) genus Stenotrophomonas sp. MFC-C strain and Stenotrophomonas maltophilia ATCC13637 strain were each inoculated into L-shaped tubes containing 5 ml of mefenacet-containing 1/10 LB medium (see Table 5 and Example 1) and allowed to stand at room temperature for 5 days. Cultured with shaking. Also, Escherichia coli (Escherichia coli) as a control strain NIHJJC-2 strain, Bacillus subtilis (Bacillus subtilis) ATCC 6633 strain, Staphylococcus aureus (Staphylococcus aureus) 209PJC-1 strain, Pseudomonas aeruginosa (Pseudomonas aeruginosa) P-2 strain and Proteus The same operation was performed for the Proteus vulgaris IID874 strain.
After completion of the culture, each culture solution was analyzed by HPLC, and the mefenacet concentration was determined by an absolute calibration curve method. The results are shown in Table 4. The HPLC measurement conditions are shown in Table 6.

Table 6 HPLC measurement conditions ────────────────────────────────────
Column Shiseido Capcell Pak C18 UG120 S3, 3mm id x 250mm

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Mobile phase Water / acetonitrile (1: 1) mixture
Flow rate 1mg / L
Column temperature 40 ° C
Sample injection volume 10μl
Detection UV (220nm)

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Examination of pH A mefenacet-containing 1/10 LB liquid medium (see Example 1) having a pH adjusted to 5, 6, 7, 8, or 9 was prepared. In this liquid medium, Nocardioides sp. MFC-A strain, Rhodococcus rhodochrous MFC-B strain and Stenotrophomonas sp. Each MFC-C strain was diluted in physiological saline and added. This was cultured with shaking in a culture tank at 30 ° C. and 120 rpm for 5 days, and a portion of the culture solution was sampled over time to measure the mefenacet concentration. For measuring the mefenacet concentration in the medium, HPLC was used in the same manner as in Example 2, and the concentration was determined by the absolute calibration curve method. The result is shown in FIG.

Degradation of mepronil, flutolanil, metolachlor and pretilachlor Nocardioides sp. MFC-A strain, Rhodococcus rhodochrous MFC-B strain and Stenotrophomonas MFC-C strain were each pre-cultured for 1 week in 1/10 LB liquid medium (see Table 5). This culture solution was added to a liquid medium prepared by adding mepronil, flutolanil, metolachlor or pretilachlor acetone solution to a 1/10 LB liquid medium to a final concentration of 4 to 10 mg / L. This was cultured in a culture tank at 26 ° C. for about one week, and the concentration of the pesticide in the medium was measured over time. For the measurement, HPLC was used, and the concentration was determined by the absolute calibration curve method. The result is shown in FIG.

Resolution of mefenaset when using purification material Rhodococcus Rhodoclaus MFC-B strain fixed on charcoal, brick or vermiculite was thoroughly washed with sterilized physiological saline. Then, 10 ml of a 1 g / L mefenacet acetone solution is added to a 1/10 LB liquid medium (see Table 5) to a final concentration of 10 mg / L and added to 20 ml of the medium, and cultured with shaking at 30 ° C. and 120 rpm. The mefenacet concentration in the culture was measured over time by HPLC. The same operation was performed on the autoclaved sterilization material, and the results were compared. In addition, the thing which did not add material was set as the control. The results are shown in FIG.

Mefenacet resolution when using the purification material Among the mefenacet-degrading microorganisms, the purification material in which Rhodococcus rhodochrous MFC-B strain was fixed on charcoal, brick or vermiculite was thoroughly washed with sterilized physiological saline. Then, the mixture was shaken at 30 ° C./120 rpm for 2.6 days in 20 ml of a basic inorganic salt medium in which pH was adjusted to 7 and 10 ml of a 0.1 g / L mefenacet acetone solution was added to a final concentration of 1 mg / L. Culture was performed. Thereafter, the culture was continued under the same conditions while changing the medium every other day with a medium having the same composition, and the mefenacet concentration during that time was measured over time by HPLC. The results are shown in FIG.

FIG. 1 is a graph showing changes in mefenacet concentration with time in the culture solution at each pH. FIG. 2 is a diagram showing the resolution of amide pesticides other than mefenacet. FIG. 3 is a diagram showing changes in mefenacet concentration with time in the culture solution using various purification materials in which the MFC-B strain has been established. FIG. 4 is a diagram showing the sustainability of mefenaset resolution when using a purification material.

Claims (8)

Nocardioides sp. MFC-A, Nocardioides simplex JCM1363, Nocardioides Jenseni JCM1364, or Bruno Nocardioides genus selected from Cardi Oy des Furubusu JCM3335; Rhodococcus rhodochrous MFC-B, Rhodococcus rhodochrous JCM2156, Rhodococcus rhodochrous JCM2157, Rhodococcus rhodochrous JCM2158, Rhodococcus rhodochrous Rhodococcus sp. JCM3010, Rhodococcus rhodochrous JCM3202, Rhodococcus coprofylus JCM3200, Rhodococcus luber JCM3205, or Rhodococcus zopphy JCM9919 ; or Stenotrophomonas sp. Decomposition of mefenacet using at least one microorganism belonging to the genus Stenotrophomonas selected from MFC-C or Stenotrophomonas maltophilia ATCC 13637 ; Nocardioides sp. Degradation of flutolanil, metolachlor or pretilachlor using MFC-A; degradation of pretilachlor using Rhodococcus rhodochrous MFC-B; or Stenotrophomonas sp. A method for decomposing amide-based pesticides, comprising decomposing mepronil or pretilachlor using MFC-C . The decomposition method according to claim 1 , wherein the decomposition method is based on a purification material in which microorganisms are held in a microorganism holder. Nocardioides sp. MFC-A (FERM P-19396) or an amide-based pesticide selected from mefenacet, flutolanil, metolachlor, or pretilachlor in which a microorganism carrier having a microbiological property of the strain is retained; Rhodococcus rhodochrous MFC-B ( FERM P-19397) or an amide-based pesticide selected from mefenacet or pretilachlor in which a microorganism having the mycological properties of the strain is retained in a microorganism carrier; or Stenotrophomonas sp. A purification material for decomposing an amide-based pesticide selected from mefenacet, mepronil, or pretilachlor in which MFC-C (FERM P-19398) or a microorganism having the mycological properties of the strain is held in a microorganism carrier . The purification material according to claim 3 , wherein the microorganism holding body is a porous substance. The purification material according to claim 3 or 4 , wherein the microorganism holding body is charcoal, brick, or vermiculite. An apparatus for decomposing an amide-based agricultural chemical selected from mefenacet, mepronil, flutolanil, metolachlor, or pretilachlor, comprising the purification material according to any one of claims 3 to 5 .   Nocardioides sp. MFC-A (FERM P-19396) or a microorganism having the mycological properties of the strain and having mefenacet resolution.   Stenotrophomonas sp. MFC-C (FERM P-19398) or a microorganism having the mycological properties of the strain and having mefenacet resolution.

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