JP2006329639A - Method of diagnosing soil environment - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of generally diagnosing a soil environment for specifying the selection of a soil additives or the like fitted to the growth of a plant in soil to be used or the optimum mixing condition of the soil additives or the like and soil from various combinations of a soil improving agent and soil in an extremely simple manner in a short time without planting a plant. <P>SOLUTION: The activity of microorganisms present in soil is measured, specifically a microorganism culture medium is added to a soil suspension to perform culture and a mediator is added to the obtained culture supernatant to perform reaction before the current value obtained by the application of voltage is measured. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a soil environment diagnostic method. More specifically, the present invention relates to a soil environment diagnosis method that enables selection of a soil conditioner suitable for various types of soil.

  In plant cultivation, an appropriate soil environment is important. For example, excessive fertilization not only adversely affects the growth of crops, but excessive nutrients that flow out of the agricultural system also cause environmental pollution, which is undesirable. Therefore, the soil environment is diagnosed before planting in order to keep the soil environment healthy and ensure stable crop production.

There are a variety of methods for analyzing soil nutrient status by diagnosing the soil environment, including simple diagnosis centered on soil acidity and electrical conductivity, and diagnosis of nutrient balance by multi-item analysis using expensive analytical machines. .
“Method and application of soil diagnosis” “Soil, Water Quality and Crop Body Analysis”, Ministry of Agriculture, Forestry and Fisheries, Agriculture and Horticulture Bureau, Agriculture Division (1979) Takashige Higuchi et al., Doi 54, 377-82, 1983 JP-A-9-178735 JP 2004-337027 A

  However, the simple diagnosis only extracts one element of soil chemistry, and in order to apply to soil diagnosis, a plurality of methods must be used in combination for the same specimen. In addition, it is necessary to weigh the soil, dispense the extract, and filter for each extracted component of each analysis target substance, and depending on the component, complicated operations and labor are required. It has been. On the other hand, in other item analysis using expensive equipment, time and cost are problems.

  The object of the present invention is to select a soil additive suitable for the growth of plants under the soil to be used from among various soil improver and soil combinations, or to optimally mix the soil with the soil additive. An object of the present invention is to provide a versatile soil environment diagnostic method for specifying conditions and the like in a short time and in a very simple manner without planting a plant.

  The purpose of the present invention is to measure the activity of microorganisms present in the soil, specifically, to perform culture by adding a microorganism culture medium to the soil suspension, and to add a mediator to the obtained culture supernatant. This is accomplished by measuring the current value obtained by applying an applied voltage after the reaction.

  In the conventional method, chemical analysis of soil components and physical analysis of soil conditions can only evaluate the properties of the soil only in one aspect, and the environment in which microorganisms in the soil are easy to grow is suitable for the growth of fertile and plants. Although evaluation of the aspect was difficult, according to the method of the present invention, the activity of soil microorganisms was evaluated in the presence of soil and one or more types of soil improvers. Can be judged comprehensively.

  In addition, farmers and specialists have determined the quality of each soil while relying on empirical intuitions to find the optimum soil conditioner and its addition amount. By quantifying empirical and subjective judgments and quantitatively comparing each condition, it is possible to objectively and quickly determine the optimum amount and optimum mixing ratio of the soil conditioner and the like.

  This method can be applied not only to the effect of the soil conditioner on the soil environment but also to the evaluation of the soil environment for all substances that can affect microorganisms in the soil, such as fungicides and agricultural chemicals. A database can be created by quantifying the examination results under each condition.

  The soil used for diagnosis is not particularly limited, but preferably the soil around the position where the root of the target plant grows and absorbs nutrients is used. In addition to the collected soil itself, the soil affects the soil environment. For example, a product to which at least one of a soil conditioner, a pesticide, a disinfectant and the like is added is used.

  The activity of microorganisms present in soil is measured by redox ability, turbidity, respiration (carbon dioxide generation), coloring of cells by pigment, intensity by fluorescent staining, etc. depending on the number of living microorganisms. The numerical value obtained by measuring the enzyme activity related to respiration is preferably used. In general, microorganisms in soil are said to be essential for plant growth, and soil in which many microorganisms are actively active is considered to be fertile land, so the activity of microorganisms is quantified. As a result, the degree of fertilization of the soil to be diagnosed can be estimated, and changes in the soil state due to the addition of additives that affect the soil environment can be quantified.

  As a method for measuring the activity of such microorganisms, for example, the soil to be diagnosed is suspended in a buffer solution such as a PBS buffer solution or a phosphate buffer solution, and a microorganism culture medium is further added at 28 to 45 ° C., Preferably, potassium hexacyanoferrate (III) is added to the culture supernatant obtained by culturing at 30 to 35 ° C. for about 4 to 24 hours, preferably about 16 to 18 hours, and filtering or centrifuging the culture solution. The reaction is carried out by adding a mediator such as resazurin and reacting for 10 minutes to 8 hours, preferably 2 to 5 hours, and then measuring the current value obtained by applying an applied voltage. Incubation other than the above temperature is not preferable because microorganisms do not grow sufficiently. As a result, the reduction catalytic activity by the activity of the electron transfer system in the cytoplasm accompanying the respiration of soil microorganisms in the soil is measured electrochemically through the mediator, and the soil environment is quantified.

  The principle of electrochemical measurement through the mediator of the activity of microorganisms is as follows. When microorganisms are present in sample solutions containing organic and inorganic compounds that they can assimilate, they metabolize them for energy acquisition. In the process, electrons move in the electron transport system of the respiratory chain. At this time, there is a correlation between the concentration of the metabolized substance and the amount of transferred electrons. Therefore, the material environment around the microorganism can be determined by measuring the amount of the moving electrons. Since it is difficult to directly measure the amount of transferred electrons, the present invention evaluates the microbial activity in the sample solution by immersing the electrode and counter electrode in the sample solution and detecting the redox of the mediator.

  The reason why microorganisms in the soil are cultured and propagated before the measurement is to amplify the current response derived from the microorganism activity, and during the cultivation, the microorganisms are propagated, that is, additives for activating the microorganisms. For example, in addition to commonly used microbial growth media such as LB media, potato dextrose media, meat extract media, etc., selectively promote the growth of specific microbial groups such as King'B media, SM media, etc. -Additives that suppress it are used.

  Next, the present invention will be described with reference to examples.

Example 1
5g of basic soil for seeding as a soil sample (modular seed of Sakata seed product), 20ml of PBS solution (Wako Pure Chemicals), LB medium (10g Bacto-Tryptone, 5g Yeast Extract, 10g NaCl in 1L of distilled water Sterilized) 2 ml and (1) Musa compound fertilizer No. 2 (Musashi organic product) 0.5 g or (2) Menedale fertilizer (Mendale product) 0.2 ml are mixed, and 30 ° C using IWAKI Universal Shaker SHK-U4 After shaking overnight at 150 rpm, the suspension was filtered with a cotton plug. Next, potassium hexacyanoferrate (III) as a mediator (Kanto Chemical Product) was added to the obtained filtrate to a final concentration of 80 mM, and the reaction was carried out with stirring using a stirrer. Immerse the working electrode and counter electrode of the carbon electrode into the electrode, apply an applied voltage of 900 mV, and obtain the current value (due to the microbial redox reaction) 3 seconds later using an electrochemical analyzer (ALS, MODEL 1202). It was measured by chronoamperometry. During the electrochemical measurement, the reaction system was kept at 25 ° C. using a thermostatic bath (Cool Thermo Unit, CTU-N, manufactured by Taitec Co., Ltd.).

Comparative Example 1
In Example 1, neither a soil sample nor any fertilizer was used.

  The results obtained in Example 1 and Comparative Example 1 are shown in FIG. Here, the elapsed time indicates the elapsed time after the addition of the mediator. As a result, it was confirmed that the reproducibility of the current response value 4 hours after the addition of the mediator was the best.

Example 2
In Example 1, Musashino organic fertilizer No. 2 was set to 0 g, 0.05 g, 0.15 g, 0.5 g, or 1.5 g as a fertilizer, and the current value was measured 4 hours after adding the mediator. Assuming that the response value obtained without adding fertilizer was 1, the activity ratio of the response value when coexisting with organic fertilizer in the case of only the soil for seed sowing and the organic fertilizer was calculated.

  The calculation result is shown in FIG. The larger the activity ratio, the more active the microbial activity in the soil suspension is, and when 0.5 g of Musashino organic fertilizer 2 is added, the microorganism is most activated (activity ratio 3.0). It has been shown. On the other hand, the activity of microorganisms decreased in a volume-dependent manner when the amount of organic fertilizer was lower or higher. Musa organic fertilizer No. 2 0.5 g activated microorganisms is 10% (w / w) added to 5 g soil sample, which is recommended by the vendor when growing vegetables, flowers and garden trees It was confirmed that it was consistent with the blending amount.

Example 3
In Example 1, 0 ml, 0.02 ml, 0.06 ml, 0.2 ml, 0.6 ml or 1.2 ml of Menedale was used as a fertilizer, and the current value was measured 4 hours after the addition of the mediator. The response value obtained without adding fertilizer was taken as 1, and the activity ratio of the response value when coexisting with Menedale was calculated.

  The calculation result is shown in FIG. It was shown that the microorganism was most activated (activity ratio 1.5) when 0.2 ml of Menedale was added. On the other hand, the activity of microorganisms decreased in a dose-dependent manner even when Menedale was below this level and above. 0.2 ml of Menedale with activated microorganisms was added in an amount of 1% (v / v) to 20 ml of PBS solution, and it was confirmed that this was consistent with the recommended amount by the vendor.

Example 4
In Example 1, the same amount of red soil (Shinei product) is used in place of the basic soil for seeding, and compost (Numata Shoten product) is composted as fertilizer / (compost + red soil) x 100 (%) = 0-100% The current value was measured 4 hours after the addition of the mediator.

  The results obtained are shown in FIG. Compost / (compost + red soil) = 20%, that is, compost: red soil = 1: 4, the current response is maximized, indicating that the microorganisms are most activated, which is consistent with the recommended amount by the vendor It was confirmed.

Example 5
In Example 1, instead of the basic soil for sowing seeds, the same amount of mountain soil (collected at Gotenbuchi, Tama area, Tokyo) is used, and humus (manufactured by Numata Shoten) is used as fertilizer. %) = 0 to 100%, and the current value was measured 4 hours after adding the mediator.

The results obtained are shown in FIG. It was shown that the current response was maximized when humus / (humus + mountain) = 40%, that is, humus: mountain = 2: 3, and the microorganisms were most activated. Here, when mixing humus and base soil in the cultivation of vegetables and flowers, the mixing ratio of humus is recommended to be 30 to 40% of the total, and the same mixing ratio is also used in experiential exploration by farmers. The results of this example were shown to be consistent with the soil mixing ratio based on such experience.
“How to select and use soil and fertilizer”

  From the results obtained in Examples 2 to 5 above, by measuring the degree of activation of microorganisms present in the soil, the ratio of fertilizer addition to various soils, which has been determined based on conventional experience, can be easily achieved. The possibility that it could be quantified was suggested.

Example 6
In Example 1, as soil samples, (1) rose roots (collected at the Tokyo Institute of Technology campus rose garden), (2) pine roots (collected at the Tokyo University of Technology campus), (3) mountain soil- 1 (collected at Tama district Gotenbuchi, Tokyo), (4) Yamado-2 (collected at Tama district Gotenbuchi, Tokyo), (5) sowing foundation soil (modular seed) or (6) red soil (Shinei) Product) 5g was used and no fertilizer was used.

  The results obtained are shown in FIG. Generally, microorganisms in soil are said to be essential for plant growth, and soils rich in microorganisms are considered to be fertile land. Therefore, it is suggested that mountain soil-2 and rose roots with high microbial activity, that is, current response of 400 μA or more 4 hours after addition of mediator is fertile land (fertile soil group), while current response The sowing base soil and the root of the pine that have a current of 200 μA or less can be classified into oligotrophic soil groups. Mountain soil-1 and red soil, which are around 300 μA, are judged to correspond to these intermediate soil groups.

Example 7
In Example 1, (1) Rose root soil, (2) Matsune root soil, (3) Mountain soil-1, (4) Mountain soil-2, ( 5) Basic soil for sowing or (6) 5 g of red soil and 0 g, 0.05 g, 0.15 g, 0.5 g or 1.5 g of Musashino organic fertilizer No. 2 as fertilizer are used, and the current value after 4 hours from the addition of the mediator Was measured. Assuming that the response value obtained without adding fertilizer was 1, the activity ratio of the response value when coexisting with the organic fertilizer in the case of the sample soil alone and the mud was calculated. The calculation result is shown in FIG.

  First, in soil-2 (A-1) and rose root (A-2), which were determined to be fertile soil groups in Example 6, there was a change in the microorganism activation effect from the addition of no fertilizer to the addition of 0.15 g of fertilizer. Not seen (activity ratio 1.0-1.1), when 0.5g which is the recommended amount of the vendor is added, the microbial activity is conversely inhibited. This result suggests that the soil judged to be a fertile soil group does not require fertilizer and excessive fertilization may lead to a decrease in soil quality.

  Next, in the soil-1 (B-1) and red soil (B-2), which were determined to be intermediate soil groups in Example 6, the effect of fertilization depends on the properties of the sample itself, and the soil-1 Then, when 0.05 g of fertilizer is added, the maximum activation effect (activity ratio 1.4) is obtained, and when the fertilizer amount exceeds 0.5 g, the microbial activity is inhibited. On the other hand, red soil shows the maximum microbial activity (activity ratio 1.9) at 0.5 g, which is the recommended amount by the vendor, and almost no activation is observed at other fertilizer levels. Therefore, it is considered that the soil judged to be an intermediate soil group can improve the quality of the soil by appropriately selecting the amount of fertilization.

  Furthermore, in the eutrophic soil group, the microbial activation effect by fertilization is larger than in the intermediate soil group, and in the basic soil for seeding (C-1), the amount of fertilizer added is from 0 g to 0.5 g in a dose-dependent manner. The activity increased and the maximum activation effect was obtained when 0.5 g of fertilizer was added (activity ratio 3.0). In addition, although the activation is seen at the fertilizer amount of 1.5 g, the activation effect is greatly reduced compared to the case where 0.5 g is added (activity ratio 1.5). It was suggested to bring about a decrease. In addition, since the activity ratio of fertilizer application amount 0.5g is three times that of the case without fertilizer, the oligotrophic soil group clearly demonstrates the soil improvement effect with the same amount of fertilizer than the intermediate soil group. It has been shown. In addition, in the soil of Matsune (C-2), the microbial activity increased in a dose-dependent manner up to 1.5 g of fertilizer addition, and the maximum activation effect was when 1.5 g of fertilizer was added (activity ratio 1.4) .

  From the results of Examples 6 and 7, by measuring the microbial activity of the soil itself, it is possible to estimate the fertility of the soil and to estimate the amount of fertilizer required. It was shown that by examining the microbial activity when the fertilizer concentration was varied, it was possible to estimate the type and amount of fertilizer appropriate for various soils in more detail.

Comparative Example 2
In Example 1, no mediator was used. The results obtained are shown in the table.
table
Current response (μA)
0.5 min 30 min 1 hr 2 hr 3 hr 4 hr Musashino fertilizer No. 2 0.07 0.03 0.08 0.07 0.01 0.08
Menedale 0.02 0.06 0.08 0.02 0.03 0.06

  By using the method of the present invention, by examining the microbial activity when various fertilizers are used with varying concentrations in advance with respect to the soil used for plant cultivation, the types and amounts of fertilizers appropriate for various soils are determined. It becomes possible to guess. Therefore, the method of the present invention can be effectively used as a soil environment diagnostic method.

It is a graph which shows the time-dependent change of the electric current response value after mediator addition. It is a graph which shows the microbial activity ratio at the time of using the predetermined amount of mix fertilizer No. 2 of the seedling for the basic soil for sowing. It is a graph which shows the microbial activity ratio at the time of using predetermined amount of Menedale for the basic soil for sowing. It is a graph which shows microbial activity at the time of using a predetermined amount of compost for red soil. It is a graph which shows microbial activity at the time of using a predetermined amount of humus for mountain soil. It is a graph which shows the microbial activity of various soil samples. It is a graph which shows the microbial activity ratio at the time of using the predetermined amount Musashino compound fertilizer No. 2 for various soil samples.

Claims (12)

  A method for diagnosing a soil environment, comprising measuring the activity of microorganisms present in soil.   The method for diagnosing a soil environment according to claim 1, wherein the activity of the microorganism is determined by measuring a respiration rate.   The method for diagnosing a soil environment according to claim 1 or 2, wherein soil containing an additive that affects the soil environment is used.   The method for diagnosing a soil environment according to claim 3, wherein the additive affecting the soil environment is at least one of a soil conditioner, a pesticide and a fungicide.   The activity of microorganisms is measured by adding a mediator to the culture supernatant obtained by culturing microorganisms in a soil suspension, and then applying the applied voltage to determine the current value obtained. The method for diagnosing a soil environment according to claim 1, which is carried out by measurement.   The soil environment diagnostic method according to claim 5, wherein the soil suspension is obtained by mixing soil with a buffer solution.   The method for diagnosing a soil environment according to claim 5, wherein the microorganism is cultured at 28 to 45 ° C.   The method for diagnosing a soil environment according to claim 5, wherein an additive for activating the microorganism is used in culturing the microorganism.   The method for diagnosing a soil environment according to claim 8, wherein a microorganism growth medium is used as an additive for activating microorganisms.   The method for diagnosing a soil environment according to claim 9, wherein the microorganism growth medium is an LB medium, a potato dextrose medium, or a meat extract medium.   The method for diagnosing a soil environment according to claim 8, wherein an additive for selectively promoting or suppressing the growth of a specific group of microorganisms is used as an additive for activating microorganisms. The method for diagnosing a soil environment according to claim 1, wherein the fertility of the soil is measured by measuring the activity of microorganisms present in the soil.

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