WO2005049854A1 - 土壌微生物を格納したバイオセンサーおよびその利用 - Google Patents
土壌微生物を格納したバイオセンサーおよびその利用 Download PDFInfo
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- WO2005049854A1 WO2005049854A1 PCT/JP2004/016422 JP2004016422W WO2005049854A1 WO 2005049854 A1 WO2005049854 A1 WO 2005049854A1 JP 2004016422 W JP2004016422 W JP 2004016422W WO 2005049854 A1 WO2005049854 A1 WO 2005049854A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- the present invention relates to a biosensor using a soil microorganism, and an evaluation of a growth ability of a soil microorganism, an evaluation of a risk of occurrence of a soil disease, and an evaluation of a biological control effect using the biosensor. .
- Soil microorganisms particularly soil-borne phytopathogenic microorganisms and general soil microorganisms (including antagonistic microorganisms), have a huge number and variety of bacteria, and it is difficult to investigate all of them.
- the number of bacteria in soil using a selective medium, direct or indirect microscopy using a microscope, detection using an antibody, detection using a DNA probe, etc. are used. Then, it was confirmed whether or not the target microorganism was present at the time of sampling in the test soil, or it was not possible to obtain information on the number of bacteria.
- detection of soil microorganisms requires several days or weeks of cultivation, requires a considerable amount of time to learn techniques, and requires complicated pretreatment. Have a problem I
- Field fields include (1) soil types such as sand, clay, heavy clay, volcanic soil, alluvial soil, and (2) latitude, topography, etc. (3) Crop cultivation history should be cereals such as rice and wheat, fruit and vegetables such as tomato and eggplant, leafy vegetables such as broccoli, cabbage and lettuce, and root vegetables such as radish and carrot. (4) The usage history of fertilizers, pesticides, composts, and other materials varies depending on the site. It is difficult to find a general solution for disease prediction in such a complex field.
- a general method of predicting disease incidence has been to estimate whether there is an increasing tendency or a decreasing tendency, such as the results of a disease severity survey over the past several years.
- the disease was suddenly confirmed on a soil that had no disease until last year, or suddenly stopped on a field that was troubled by the disease until last year because the weather conditions changed.
- sufficient results have not been obtained with respect to the prediction of disease outbreaks, tests on various soil disease control have been conducted in response to requests from on-site producers.
- tests are conducted on disease-producing fields, such as changing cropping types, crop rotation, breeding, pesticides, fertilizers, soil improvement materials including microbial materials, and soil disinfection.
- disease-producing fields such as changing cropping types, crop rotation, breeding, pesticides, fertilizers, soil improvement materials including microbial materials, and soil disinfection.
- this requires a large test field, and it is impossible to perform many tests at once in a limited test field, and considering that the reproducibility is difficult due to environmental changes, it may take several years. It was extremely inefficient because it took time, for example, the reproducibility had to be checked over time, and it was not possible to earn enough test points.
- Biosensors mainly use biochemical oxygen demand (BOD) measurement (Patent Documents 13) and cyan (Patent Documents 4 and 5) as environmental measurement techniques.
- Mercury Patent Document 6
- alcohol Patent Documents 7 and 8
- surfactant Patent Document 9
- phosphoric acid Patent Documents 10 and 11
- ammonia etc.
- Patent Document 12 It has been developed as a sensor for detecting components or nutrients or measuring the concentration. Furthermore, applications of this technology to simple detection of components in food and measurement of blood glucose level in blood and urea concentration in urine for medical diagnosis have been considered (Non-Patent Documents 4 and 5). ing. In other words, the purpose of the biosensor technology up to now has been to detect components in samples and to measure concentrations.
- Patent Document 1 Japanese Patent Publication No. 58-30537
- Patent Document 2 JP-A-7-167824
- Patent Document 3 JP-A-5-137597
- Patent Document 4 JP-A-8-211011
- Patent Document 5 JP-A-9-297105
- Patent Document 6 JP-A-5-023198
- Patent Document 7 JP-A-5-041999
- Patent Document 8 Japanese Patent Publication No. 6-041928
- Patent Document 9 JP-A-8-196295
- Patent Document 10 JP-A-5-093692
- Patent Document 11 Japanese Patent Publication 8-020401
- Patent Document 12 JP-A-11-125600
- Patent Document 13 JP-A-2-193059
- Patent Document 14 JP-A-53-137198
- Patent Document 15 Tokuhei 1-22898
- Patent Document 16 JP-A-55-16203
- Patent Document 17 JP-A-59-27255
- Patent Document 18 Japanese Patent Publication No. 57-15696
- Patent document 19 Japanese Patent Publication No. 57-54742
- Patent Document 20 Japanese Patent Publication No. 61-7258
- Patent Document 21 JP-A-57-173745
- Patent Document 22 JP-A-59-133454
- Patent Document 23 JP-A-5-252994
- Patent Document 24 Japanese Patent Publication No. 58-36736
- Non-patent document l Biosensors & Bioelectronics, 16 (2001) 337-353
- Non-Patent Document 4 Methods in Enzymology, 137 (1988) 131-138
- Non-Patent Document 5 Biosensors & Bioelectronics, 16 (2001) 337-353
- Non-Patent Document 6 Microbial. EcoL, 45 (3) (2003) 226-236
- Non-Patent Document 7 Soil.Sci.Soc.Am.J., 66 (2) (2002) 498-506
- Non-Patent Document 8 Analyst, 127 (1) (2002) 5-7
- Non-Patent Document 9 Biosensors & Bioelectronics, 16 (2001) 667-674
- Non-Patent Document 10 Environ Pollut 113 (2001) 19-26
- Non-Patent Document 11 Field Anal.Chem.Tech., 4 (5) (2000) 239-245
- Non-Patent Document 12 Soil Biol. Biochem., 32 (5) (2000) 639-646
- Non-Patent Document 13 Soil Biol. Biochem., 32 (3) (2000) 383-388
- Non-Patent Document 14 Appl.Environ.Microbiol, 69 (6) (2003) 3333-3343
- Non-Patent Document 15 Appl.Environ.Microbiol, 60 (8) (1994) 2869-2875
- Non-Patent Document 16 Can.J.Microbiol, 47 (2001) 302-308
- Non-Patent Document 17 Appl.Environ.Microbiol, 67 (3) (2001) 1308-1317
- the present invention has been made in view of such circumstances, and an object of the present invention is to provide a method capable of easily measuring the growth ability of soil microorganisms in a short period of time. Furthermore, this method can be used in a simple and short term to evaluate the risk of the occurrence or spread of soil diseases caused by soil-borne phytopathogenic microorganisms. It is also an object of the present invention to provide a method for evaluating the control effect of soil microorganisms.
- the present inventors have determined that microbial sensors used for environmental measurement technology and the like can be used for environmental component detection and concentration measurement for various types of on-site soil that can solve the above-mentioned problems.
- a comparative study was carried out using this as a means to evaluate the environmental adaptability of the soil microorganisms intended for the field to various field soil environments.
- the present inventors can examine the growth ability of general soil microorganisms and pathogenic microorganisms in the ecosystem by using the microbial sensor, thereby observing the balance of the soil ecosystem, and furthermore, the risk of disease occurrence and the like. It has been found that the biological control effect of general soil microorganisms can be determined.
- the electrode response ratio of general soil microorganisms Z pathogenic microorganisms measured using a sensor unit containing soil-borne plant pathogenic microorganisms and a sensor unit containing general soil microorganisms is effective for this determination. It was a good indicator.
- the electrode response ratio is low, it is expected that the growth rate of pathogenic microorganisms will exceed that of other general soil microorganisms. It is anticipated that there will be an increase in the direction in which it exists, and the risk of disease occurrence or spread is expected to be high. In addition, it can be expected that the biological control effect of the general soil microorganisms on the soil is low.
- the prediction of the risk of disease occurrence can be made more precise.
- This technique is very useful in that it enables early soil diagnosis, especially for the possibility of disease outbreaks in uninfected fields.
- a plurality of sensors each having a storage unit storing an oxygen electrode and a soil microorganism and a unit serving as a fixing member, and a plurality of sensors each storing a different soil microorganism in the storage unit of each sensor. Contact with the soil suspension of the soil to be measured,
- a storage unit containing oxygen electrodes and general soil microorganisms and a fixing member A sensor having a unit that functions as a unit, and a storage unit containing oxygen electrodes and soil-borne phytopathogenic microorganisms. Contacting a sensor having a unit that is also a member with a soil suspension of the soil to be measured,
- a sensor that has a unit that serves as a fixing member and a storage unit that contains an oxygen electrode and general soil microorganisms, and a sensor that has a unit that also contains a storage unit and a fixing member that contains an oxygen electrode and soil-borne plant pathogenic microorganisms. Is brought into contact with the soil suspension of the soil to be measured,
- the amount or rate of decrease in output current in a general soil microorganism is significantly higher than the amount or rate of decrease in output current in a soil-borne plant pathogenic microorganism, the soil-borne plant-borne pathogen in the soil being measured.
- a plurality of sensors each having an oxygen electrode, a storage section for storing soil microorganisms, and a cut as a fixing member, wherein a plurality of different soil microorganisms are stored in the storage section of each sensor.
- [5] Including a sensor having an oxygen electrode, a storage section storing general soil microorganisms, and a unit serving as a fixing member, and a sensor having an oxygen electrode, a storage section storing soil-transmitting plant pathogenic microorganisms, and a sensor having a fixing member unit, A kit for use in the method according to [2] or [3].
- FIG. 1 Microbial sensor containing Fusarium (Eusil) genus using substrate broth (PD broth) It is a figure showing one response.
- the vertical axis indicates the current value (nA) of the microorganism sensor, and the horizontal axis indicates the amount of PD broth added.
- FIG. 2 is a diagram showing a response of a microorganism sensor containing a clubroot fungus to yeast extract.
- the vertical axis indicates the current value (nA) of the microorganism sensor, and the horizontal axis indicates the amount of immobilized cells (ml).
- FIG. 3 is a view showing a structure of a microorganism sensor for predicting soil disease occurrence.
- FIG. 4 is a diagram showing a structure of a microorganism sensor unit.
- the present invention provides a method for measuring the growth ability of soil microorganisms in soil.
- the method comprises the steps of: (a) a plurality of sensors each having a unit that also serves as a fixing member and a storage unit that stores each oxygen electrode and soil microorganisms, wherein different soil microorganisms are stored in the storage units of each sensor. Contacting the plurality of sensors with the soil suspension of the soil to be measured, and (b) measuring the amount of decrease in the output current of each sensor or the difference in the rate of decrease.
- the type of soil microorganism used is not particularly limited, and may be any microorganism that can be separated and cultured by conventional techniques.
- microorganisms that cannot be cultured such as clubroot fungi can be used if they are grown and isolated by using a plant-based growth method.
- the types of soil microorganisms preferably include the microorganisms described below.
- the soil-borne plant pathogenic microorganisms of the present invention are classified into bacteria, actinomycetes, and filamentous fungi.
- soil-borne phytopathogenic bacteria examples include bacterial wilt (Ralstonia solanacearum), soft rot (genus Erwinia, Pseudomonas), and root g.
- Examples of soil-borne phytopathogenic actinomycetes include scabs (StreDtomvces)
- Soil-borne phytopathogenic fungi include seedling blight (Pvthium spp., Rhizoctonia spp., Etc.), te. Clubroot disease Plasmodiophora brassicae, -fe-fe fungus (Phvtophutora spp., Verticillium spp., Fusarium.
- Rhizoctonia genus Rhizoctonia genus, Crest ⁇ ⁇ fungus (HdicobasidiumJ3 ⁇ 4, Rosellinia genus, white silk rot fungus (Corticium genus), brown root rot fungus (Pyrenochaeta genus), streak rot fungus (Cephalosporiumfe,), grass rot fungi (Cvlindrocarpon) and the like.
- the general soil microorganisms of the present invention are classified into antagonistic bacteria, antagonistic filamentous fungi, (above, antagonistic microorganisms), general soil bacteria, general soil actinomycetes, general soil filamentous fungi, and the like. (Soil and Microorganisms, (1981) Soil Microorganisms Research Association, edited by Hakutosha).
- Examples of the antagonistic bacteria include Bacillus spp. And non-pathogenic agrobacterium.
- Genus (Agurobacterium), genus Enterobacter, genus Pseudomonas (Agurobacterium), genus Enterobacter, genus Pseudomonas (Agurobacterium), genus Enterobacter, genus Pseudomonas (Agurobacterium), genus Enterobacter, genus Pseudomonas (Agurobacterium), genus Enterobacter, genus Pseudomonas (Agurobacterium), genus Enterobacter, genus Pseudomonas (Agurobacterium), genus Enterobacter, genus Pseudomonas (Agurobacterium), genus Enterobacter, genus Pseudomonas (Agurobacterium), genus Enterobacter, genus Pseudomonas (Agurobacterium), genus Enterobacter, genus Pseudomonas (
- Examples of the antagonistic filamentous fungi include the genus Aspergillus, the non-pathogenic genus Fusarium, the genus Gliocladium, the genus PenicilliuTn, and the genus Pvthium. , Trichoderma genus, Phoma storm, and Talaromvces genus.
- Examples of general soil bacteria include Acetobacter genus and Alcaligenes (
- Alcalieenes Bacillus, Burkholderia, Corvnebacterium rat, Flavonobacterium, Gluconobacter, Lactobacillus bacteria, Mycobacterium lactobacillus (Micobacterium) rot, Micrococcus rat, Proteus, Pseudomonas, Rhizobium, Rhodococcus, Sphingomonas, Streptococcus, Streptococcus Eggplant (Zvmomonas) genus and the like.
- Examples of general soil fungi include Aphanomvces, Aspergillus, Candida, CladosQorium, and Muco. Genus Mucor, Penicillium, Phvtophthora, Rhizopus, Trichoderma, and I ⁇ mk.
- the medium used is, for example, a meat extract medium, an LB medium, a potato dextrose medium (PD medium), and the culture method is, for example, standing and shaking in a container such as a petri dish, a test tube, a flask, or a jar fermenter. It is not necessary to carry out under special culture conditions that can be carried out under conditions such as stirring. In addition, even microorganisms that cannot be cultured can be used if they can be grown in a specific part of a plant such as a root-knot and isolated in a large amount.
- any commonly used oxygen electrode may be used, such as a galvanic type or a polar opening type, and a commercially available oxygen electrode may be used.
- microorganisms do not leak from the storage part as much as possible, such as water, volatile substances such as oxygen dissolved in water, and the respiration activity of microorganisms.
- a membrane with a mesh size permeable to organic substances that affect water and substances that inhibit respiratory activity may be used.
- a 0.45 ⁇ m-trocellulose membrane acetyl cellulose membrane or the like is used.
- a suspension of microorganisms is dropped on trocellulose, acetyl cellulose, nylon membrane, or the like, and is sucked from below or pressurized from above to obtain water and water-soluble.
- a substance is sucked or removed under pressure to adsorb and fix microorganisms on a filter
- the microorganism storage section can be fixed as closely as possible to the electrode, and the respiratory activity of volatile substances such as oxygen, which is a soil suspension power, and micro-organisms.
- volatile substances such as oxygen
- the electrode does not hinder diffusion to the electrode.
- immobilizing microorganisms The lower part of the membrane is made of nylon net or wire mesh so that water-soluble substances or fine granular substances can be permeable, and the side of the fixture is made of plastic and screwed in.
- the electrode is fixed to the electrode using a 0-ring, a tube, or the like.
- an appropriate amount for example, 10 g to l, 000 g
- an appropriate amount for example, 1 to 100 times the amount of soil sample 1).
- a supernatant obtained by adding water, a solvent or a buffer, stirring well, mixing, and then performing centrifugation or filtration or the like is preferable.
- the greater the amount or rate of decrease in the output current the higher the respiratory activity of the soil microorganisms and the greater the oxygen consumption, that is, the higher the growth capability of the microorganisms in the soil (suitable for the growth of the microorganisms).
- the soil environment When the amount or rate of decrease of the output current is small, it means that the respiratory activity of the soil microorganism is low and oxygen consumption is small, and the content of organic matter serving as the food of the soil microorganism is small or the soil microorganism is low. It can be seen that the growth ability of the microorganism is low due to the presence of a substance that inhibits the respiratory activity of the microorganism (for example, the soil environment is unfavorable for the growth of the microorganism).
- the present invention relates to (a) a storage member for storing an oxygen electrode and general soil microorganisms and a fixing member.
- a sensor having a unit consisting of a unit consisting of an oxygen electrode and a storage unit containing an oxygen electrode and soil-borne phytopathogenic microorganisms and a unit serving as a fixing member. Also provided is a method for assessing the risk of the occurrence or spread of a soil disease caused by soil-borne phytopathogenic microorganisms, including measuring the amount or rate of decrease in sensor output current.
- a sensor having a unit that also serves as a fixing member and a storage unit that stores oxygen electrodes and general soil microorganisms, and a storage unit that stores oxygen electrodes and soil-borne plant pathogenic microorganisms is brought into contact with a soil suspension of a specific soil
- the comparison between the amount or rate of decrease in output current in general soil microorganisms and the amount or rate of decrease in output current in soil-borne plant pathogenic microorganisms is evaluated as the electrode response ratio (general soil microorganisms Z pathogenic microorganisms). It is possible to do. For example, if the electrode response of a microorganism sensor containing general soil microorganisms (including antagonistic microorganisms) is significantly higher than that of a microorganism sensor containing pathogenic microorganisms, the general soil microorganisms (antagonistic microorganisms) It is expected that the risk of future disease outbreaks will be reduced in the future. In this case, the general soil microorganisms can be determined to have a controlling effect on the soil.
- the electrode response of a microorganism sensor containing general soil microorganisms is significantly higher than that of a microorganism sensor containing pathogenic microorganisms
- the electrode response ratio is 0.4 or more, preferably 0.6 or more. , More preferably 2.0 or more.
- this numerical value can be further improved by correction based on the results of measurement of the current disease severity, the bacterial density of the pathogenic bacteria and the general soil microorganisms or the abundance ratio.
- the electrode response of a microorganism sensor containing general soil microorganisms is remarkably small. Since the growth rate of common soil microorganisms (including antagonistic microorganisms) is low, it is difficult to control pathogenic microorganisms. In the future, the risk of disease occurrence can be expected to increase further. In this case, it can be determined that the general soil microorganism has a low control effect on the soil.
- the degree of disease on the soil at the site can be evaluated by planting a crop and investigating the disease occurrence status.
- the number of pathogenic microorganisms and general soil microorganisms (including antagonistic microorganisms) in soil can be determined by a general dilution plate method using a selective medium, a direct microscopy method, an antibody method, a method using a DNA probe, etc.
- colony-causing pathogenic microorganisms and priority species obtained by the dilution plate method can be selected and isolated, and immobilized on the microorganism sensor of the present invention.
- the number of antagonistic microorganisms to be introduced into the field can be easily measured by a general dilution plate method or the like, although the bacterial density is described for some materials.
- the site soil is packed in pots, and fertilizers, pesticides, soil improvement materials, etc. are input, the soil pH, the content of organic substances, etc. are adjusted, and the soil improvement such as water permeability, water retention, air permeability, and biological properties is performed.
- the chemical, physical and biological properties of the soil can be improved, but at the same time, the growth rates of pathogenic microorganisms and general soil microorganisms (including antagonistic microorganisms) also change.
- a sensor containing pathogenic microorganisms it is possible to easily determine which material is added and to what extent the growth rate of the pathogenic microorganisms decreases.
- kits used for a method for measuring the growth ability of soil microorganisms has a plurality of sensors each having an oxygen electrode, a storage unit for storing soil microorganisms, and a unit serving as a fixing member. Includes multiple sensors containing different soil microorganisms.
- the kit used includes a sensor having a unit that functions as a fixing member and a storage unit that stores an oxygen electrode and general soil microorganisms, and a unit that includes a storage unit and a fixing member that stores the oxygen electrode and soil-borne plant pathogenic microorganisms.
- a sensor having a unit that functions as a fixing member and a storage unit that stores an oxygen electrode and general soil microorganisms and a unit that includes a storage unit and a fixing member that stores the oxygen electrode and soil-borne plant pathogenic microorganisms.
- kits may further include instructions for use.
- lettuce root rot fungus (Fusarium oxvsporum f. Sp.
- the current value (nA) generated in the membrane of the microbial electrode was measured.
- Bacillus cereus (MU ⁇ mK12N) strain was used as a representative antagonistic microorganism.
- a microbial sensor containing Bacillus sp. was inserted and gently stirred with a stirrer.
- the current value (nA) generated in the membrane of the microbial electrode was measured.
- Bacterial cells of the genus Bacillus (MI) and spore suspension of the genus Fusarium in the logarithmic growth phase cultured in the same manner as in Examples 1 and 2 were collected, washed, and the absorbance at 660 nm was measured.
- the concentration of each bacterium was 0.43 for Bacillus and 0.1 for Fusarium.
- 1.0 ml of each of the bacteria was aspirated on a trocellulose filter to be immobilized, and then contained in a microorganism sensor cut.
- the viable cell count was measured by the dilution plate method, and the Bacillus spp. Stored in the sensor unit was calculated as 1.4 ⁇ 10 7 cfo I filter, and the Fusarium spp. 1.0 ⁇ 10 5 clu I filter.
- Bacillus spp. Is equivalent to about 14-140g in soil. For Fusarium spp., It is equivalent to about 100 to 1000 g of bacteria in soil.
- Table 5 shows the results of examining the response to various substrates. As is clear from the results in Table 5, it was found that Bacillus spp. Had better electrode responsiveness to yeast extract, L broth, and ripe compost "Biotech Bioace” than Fusarium spp.!
- Example 4 Influence of Extraction Time of Soil or Compost on Responsiveness of Microbial Sensor
- Example 5 Microbial Sensor Containing Various Biological Pesticides, Microbial Materials, and Soil Microorganisms Creation and responsiveness
- the absorbance at 660 ° was measured with an absorbance meter to calculate the bacterial density. Then, the cells were collected, washed, aspirated and immobilized on a filter, and stored in a sensor unit.
- a ripe chicken manure of a microbial sensor containing various microorganisms (Response to brand name biotech bye)
- Example 6 Preparation of microbial sensor containing cruciferous clubroot and its responsiveness Brassicaceae clubroot (Plasmodiophora brassicae) cannot be cultured! /
- the spore suspension was prepared by collecting the nodules generated on the roots of the wild and wild rhinoceros from the field where the spores were generated, crushing them with a mixer, and collecting and washing. Spore density was measured with a hemocytometer and was 2.8 ⁇ 10 9 spores I ml.
- the spore suspension was diluted 100-fold, 200, 1.0 ml, and 3.0 ml were dropped on a nitrocellulose filter, suctioned, fixed, and stored in a sensor unit.
- the responsiveness of a microbial sensor containing a clubroot fungus prepared using yeast extract as a substrate was confirmed.
- the results are shown in Table 12 and FIG. A high positive correlation was found between the number of stored spores and the response of the microorganism sensor to the yeast extract.
- Plant pathogenic microorganisms to be stored include Brassicaceae clubroot fungus (Plasmodiophora brassicae), lettuce fefej) and fungus (Fusarium oxvsporum f. Sp. Lactucum) SN3B strain.
- Bacillus cereus K12N strain was used as an antagonistic microorganism to be stored.
- the amount of cells to be stored should be about 10-100 times the bacterial density in the general diseased soil for phytopathogenic microorganisms, and about 10-100 times the bacterial density in general soil for antagonistic microorganisms. Doubled.
- Microbial sensors containing clubroot or antagonistic microorganisms (Bacillus sp.)
- Microbial electrode response ratios are as low as 0.32 and 0.66, respectively, from the respiratory activity of antagonistic microorganisms. It was also found that the respiratory activity of pathogenic microorganisms was increasing. In other words, when both microorganisms entered the soil after sterilization, it was expected that the environment would be easier for the pathogenic microorganisms to proliferate than the antagonistic microorganisms, and the risk of disease recurrence would be higher. This result confirms that even if soil disinfection is performed on site, the effect is difficult to achieve, and the recurrence is likely to occur.
- the soil sample of Mr. I House in Nishine-cho, Iwate Prefecture was used.
- the disease-causing soil samples were the root-knot disease-causing soil samples from the Chushin Agricultural Experiment Station in Nagano Prefecture, and the root-causing disease soil samples from Kawakami-mura, Nagano Prefecture.
- Soil microorganisms were separated from each soil sample by the dilution plate method.
- the medium used was an albumin agar medium for bacteria and a rose bengal agar medium for filamentous fungi. For colonies that appeared on the medium, classification was attempted based on the color, outline, surface condition, and size of the colonies.
- Bacteria were classified into 4 types of colors * 2 types of outlines * 4 types of surface conditions * 3 types of sizes, and could be classified into a total of 96 patterns.
- the filamentous fungi were classified into 7 types of colors * 2 types of outlines * 4 types of surface conditions * 3 types of sizes, and could be classified into 168 patterns. The results are shown in Table 16.
- the number in parentheses is the value of the pattern classification (two strain No.)
- Example 10 A microbial sensor containing pathogenic microorganisms for diseased soil and healthy soil Comparison of responsiveness of microbial sensors containing general soil microbes
- the pathogenic microorganisms clubroot and root rot fungi were used.
- the general soil microorganism the microorganism isolated in Example 9 (priority species of each soil sample: 6 strains) was used. After culturing each microorganism, it was stored in each microorganism sensor unit. In addition, since the amount of bacteria cultured for some of the microorganisms was not sufficient, the response to yeast extract was expressed as a relative value when the response to yeast extract was set to 100. The results are shown in Tables 18 and 19.
- Microbial sensors containing clubroot fungi and microbial sensors containing common microorganisms Response ratio, degree of clubroot disease on each soil and pathogen density.
- the electrode response ratio with pathogenic bacteria was determined using antagonistic microorganisms as soil microorganisms or preferential species (general soil bacteria and general soil fungi) isolated from on-site soil. By examining), it was possible to predict whether the soil environment is at a high risk of disease and at a low risk of disease. These predictions were in good agreement with the findings on the field of disease outbreaks.
- the Pvthium oligandrum MMR2 strain is an antagonistic microorganism with bacterial parasitism and resistance induction. By administering this bacterium, the disease severity was reduced to 46.3 and the diseased strain rate to 50.0. However, the control value was 22.9, indicating a control effect against the bacterial wilt of tomato.
- Tomato bacterial wilt is cultured on a TTC agar medium at 30 ° C for 1-2 days, and Pichidium is shaken at 25 ° C for 1 month in an Erlenmeyer flask containing a V8 juice medium. Culture was continued. After collecting both strains, they were stored in the respective microorganism sensor units.
- the response to the yeast extract was confirmed using an electrode on which both microorganisms were immobilized. (Tomato bacterial wilt: 196 nA, Picidium: 23 nA).
- Example 12 Effect of application of various microbial materials on soil disease and prediction of disease reduction using microbial sensor
- the inoculum density of microbial materials and phytopathogenic microorganisms was calculated from the bacterial concentration before inoculation into soil.
- Bio 21 (Sakata Seed Co., Ltd.) is a bacterium belonging to the genus Bacillus, and MMR3 has bacterial parasite and resistance induction obtained from Hokkaido Agricultural Experiment Station. It is a bacterium belonging to the genus Pisium.
- Bacterial density ⁇ Untreated means the bacterial density of plant pathogenic microorganisms, and others indicate the bacterial density of various microbial materials (antagonistic microorganisms).
- Cell density column Untreated cells indicated the cell density of plant pathogenic microorganisms, and others indicated cell densities of various microbial materials (antagonistic microorganisms).
- Cell density column Untreated cells indicated the cell density of plant pathogenic microorganisms, and others indicated the cell densities of various microbial materials (antagonistic microorganisms).
- the bacteria were cultured with shaking at 30 ° C for 12 days using the medium Lbroth, and the filamentous fungi using the PD broth medium. And cultured with shaking at 25 ° C for 7 days.
- the cells were immobilized on a nitrocellulose filter and stored in a sensor unit.
- Table 28 shows the results of determining the response of each microbial sensor to the potted soil and the electrode response ratio of the antagonistic microorganisms Z pathogenic microorganisms.
- the amount of microbial material input was determined in accordance with the method recommended by each material manufacturer or development stakeholder, but as a result, the amount of microbial microorganisms input as antagonistic microorganisms and the amount of inoculum of pathogenic microorganisms were extremely low. Was sometimes different.
- the disease mitigation effect was determined according to the following table in which the results of the electrode response ratio and the correction of the bacterial density were taken into account.
- control value 10.0 or more was regarded as having a control effect.
- the results are shown in Table 30.
- a control value of 10 or more is evaluated as effective.
- the electrode response ratio is the electrode response ratio of both microorganism sensors for antagonistic microorganisms Z and pathogenic microorganisms. Bacterial density correction was performed when there was a 100-fold or more bacterial count difference.
- the microbial sensor for predicting soil microbial dynamics of the present invention it is possible to analyze the dynamics of soil microbes, and in particular, to predict the dynamics of soil-borne phytopathogenic microorganisms and general soil microorganisms (including antagonistic microorganisms) in the field soil. Become. In addition, it is possible to make an early prediction of the risk of the occurrence of disease on the soil at the site, which can lead to early control measures. In the field where the disease has already occurred, it is possible to predict the risk of future disease occurrence even in the field where the disease is not easily generated. In addition, it is possible to lead to a soil improvement proposal based on the prediction result.
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Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP04799520A EP1700919A4 (en) | 2003-11-19 | 2004-11-05 | BIOSENSOR HAVING A SOIL MICROORGANISM CONTAINED WITHIN THE BIOSPER AND USE THEREOF |
JP2005515577A JP4528939B2 (ja) | 2003-11-19 | 2004-11-05 | 土壌微生物を格納したバイオセンサーおよびその利用 |
US10/579,670 US20070148725A1 (en) | 2004-11-05 | 2004-11-05 | Soil microorganism-housing biosensors and their uses |
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JP2003-390013 | 2003-11-19 | ||
JP2003390013 | 2003-11-19 |
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WO2005049854A1 true WO2005049854A1 (ja) | 2005-06-02 |
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PCT/JP2004/016422 WO2005049854A1 (ja) | 2003-11-19 | 2004-11-05 | 土壌微生物を格納したバイオセンサーおよびその利用 |
Country Status (3)
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EP (1) | EP1700919A4 (ja) |
JP (1) | JP4528939B2 (ja) |
WO (1) | WO2005049854A1 (ja) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008187911A (ja) * | 2007-02-01 | 2008-08-21 | National Institute Of Advanced Industrial & Technology | 微生物の活性測定法 |
JP2010143876A (ja) * | 2008-12-19 | 2010-07-01 | National Agriculture & Food Research Organization | 植物病害防除剤 |
CN110093261A (zh) * | 2019-04-28 | 2019-08-06 | 陕西科技大学 | 一种用于沉积物中厌氧菌原位分离的装置及使用方法 |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014076663A1 (en) * | 2012-11-15 | 2014-05-22 | Basf Corporation | Mulch and potting soil compositions containing microorganisms and related methods |
CA2890635C (en) | 2012-11-22 | 2022-12-06 | Basf Corporation | Synergistic pesticidal mixtures comprising bacillus subtilis mbi-600 |
AU2015263304B2 (en) | 2014-05-23 | 2018-11-15 | Basf Se | Mixtures comprising a bacillus strain and a pesticide |
BR112017009282A2 (pt) | 2014-11-07 | 2018-01-30 | Basf Se | misturas fungicidas, composição pesticida, métodos para controlar pragas fitopatogênicas, para melhorar a fitossanidade e para proteção de material de propagação de plantas contra pragas, e, material de propagação de plantas. |
US10905122B2 (en) | 2016-03-16 | 2021-02-02 | Basf Se | Use of tetrazolinones for combating resistant phytopathogenic fungi on cereals |
EP3429358A1 (en) | 2016-03-16 | 2019-01-23 | Basf Se | Use of tetrazolinones for combating resistant phytopathogenic fungi on fruits |
BR112018068705B1 (pt) | 2016-03-16 | 2022-09-06 | Basf Se | Método para controlar fungos fitopatogênicos |
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JPH05123186A (ja) * | 1991-10-31 | 1993-05-21 | Shimadzu Corp | 好気性微生物の測定法および測定装置 |
JPH0947299A (ja) * | 1995-05-31 | 1997-02-18 | Horiba Ltd | 微生物活動状態計測方法 |
JP2000157258A (ja) * | 1998-11-25 | 2000-06-13 | Natl Inst Of Agro-Environmental Sciences Maff | 土壌の病害抑止性の評価方法 |
JP2003116591A (ja) * | 2001-10-15 | 2003-04-22 | Japan Science & Technology Corp | 微生物の代謝機能評価方法及びその装置 |
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DE19714219A1 (de) * | 1997-04-07 | 1998-10-08 | Cornelius Prof Dr Friedrich | Verfahren und Vorrichtung zur Aktivitätsbestimmung immobilisierter Mikroorganismen |
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2004
- 2004-11-05 JP JP2005515577A patent/JP4528939B2/ja not_active Expired - Fee Related
- 2004-11-05 EP EP04799520A patent/EP1700919A4/en not_active Withdrawn
- 2004-11-05 WO PCT/JP2004/016422 patent/WO2005049854A1/ja active Application Filing
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JPH05123186A (ja) * | 1991-10-31 | 1993-05-21 | Shimadzu Corp | 好気性微生物の測定法および測定装置 |
JPH0947299A (ja) * | 1995-05-31 | 1997-02-18 | Horiba Ltd | 微生物活動状態計測方法 |
JP2000157258A (ja) * | 1998-11-25 | 2000-06-13 | Natl Inst Of Agro-Environmental Sciences Maff | 土壌の病害抑止性の評価方法 |
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HASHIMOTO Y. ET AL: "Tsuchi no Kenko Shindan -Dojo Shindan-yo Biosensor no Kaihatsu", BRAIN TECHNO NEWS, vol. 105, 5 September 2004 (2004-09-05), pages 12 - 17, XP002996972 * |
HASHIMOTO Y: "Dojo Shindan-yo Biosensor no Kaihatsu", INSPECTION ENGINEERING, vol. 9, no. 10, 1 October 2004 (2004-10-01), pages 35 - 41, XP002996973 * |
See also references of EP1700919A4 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008187911A (ja) * | 2007-02-01 | 2008-08-21 | National Institute Of Advanced Industrial & Technology | 微生物の活性測定法 |
JP2010143876A (ja) * | 2008-12-19 | 2010-07-01 | National Agriculture & Food Research Organization | 植物病害防除剤 |
CN110093261A (zh) * | 2019-04-28 | 2019-08-06 | 陕西科技大学 | 一种用于沉积物中厌氧菌原位分离的装置及使用方法 |
CN110093261B (zh) * | 2019-04-28 | 2022-11-25 | 陕西科技大学 | 一种用于沉积物中厌氧菌原位分离的装置及使用方法 |
Also Published As
Publication number | Publication date |
---|---|
JPWO2005049854A1 (ja) | 2007-06-07 |
EP1700919A4 (en) | 2007-05-30 |
JP4528939B2 (ja) | 2010-08-25 |
EP1700919A1 (en) | 2006-09-13 |
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