Method for determining whether a test sample contains plant pathogenic bacteria

Abstract

The present invention provides a method for selectively determining whether a test sample contains plant pathogenic bacteria from two types of bacteria, plant pathogenic bacteria and plant non-pathogenic bacteria. In the present invention, first, a test sample is arranged on the surface of a substrate having a through hole. The substrate has a cellulose film on its back surface. The through hole of the substrate has a cross-sectional area of ​​7.065 square microns to 19.625 square microns. The cellulose film does not have a through hole and has a thickness of 0.5 microns to 3.7 microns. Next, the test sample is allowed to stand, and the cellulose film is irradiated with ultraviolet rays to contact it with a bacterial color developing reagent. The test sample in which color development is observed is determined to contain plant pathogenic bacteria.

Description

Technical Field

[0001] The present invention relates to a method for determining whether a test sample contains plant pathogenic bacteria. Background Art

[0002] Patent Document 1 discloses a method for measuring filamentous fungi. Fig.10 A cross-sectional view of a microporous membrane support used in the filamentous fungus measuring method disclosed in Patent Document 1 is shown. The purpose of the filamentous fungus measuring method disclosed in Patent Document 1 is to provide a filamentous fungus measuring method that can accurately measure filamentous fungi by measuring the number of filamentous fungi in a short period of culture in order to measure the number of filamentous fungi in a test material. The filamentous fungus measuring method is capable of measuring filamentous fungi 13 by culture in a short period of time by photographing a plurality of extended hyphae of filamentous fungi 13 cultured by liquid culture or cultured on a microporous membrane 1 of a microporous membrane support 4, and then identifying and analyzing the shape, area and luminous brightness by an image analysis unit 10. The microporous membrane 1 is sandwiched between a pressing ring 2 and a base 3.

[0003] Non-Patent Document 1 discloses that pseudohyphae of Phytophthora sojae, a species of plant pathogenic oomycetes, penetrate a PET film having pores of 3 micrometers.

[0004] Patent Document 2 discloses a method for determining whether a test sample contains a plant pathogenic oomycete.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Publication No. 2005-287337

[0008] Patent Document 2: Japanese Patent Application Publication No. 2017-029131

[0009] Non-patent literature

[0010] Non-patent document 1: Paul F.Morris.et.al. "Chemotropic and Contact Responses ofPhytophthora sojae Hyphae to Soybean Isoflavonoids and ArtificialSubstrates", Plant Physiol. (1998) 117:1171-1178 Summary of the invention

[0011] Problems to be solved by the invention

[0012] An object of the present invention is to provide a method for selectively determining whether a test sample contains plant pathogenic bacteria from among two types of bacteria, plant pathogenic bacteria and plant non-pathogenic bacteria.

[0013] Means for solving problems

[0014] The present invention provides a method for determining whether a test sample contains plant pathogenic bacteria. The method comprises the following steps.

[0015] (a) a step of placing the test sample on the front surface of a substrate having a through hole,

[0016] in,

[0017] The substrate has a cellulose film on its back side.

[0018] The through holes of the substrate have a cross-sectional area of ​​7.065 square micrometers to 19.625 square micrometers, the cellulose film has no through holes, and

[0019] The cellulose film has a thickness of 0.5 micrometers to 3.7 micrometers,

[0020] (b) after step (a), a step of allowing the test sample to stand still,

[0021] (c) after step (b), irradiating the cellulose film with ultraviolet rays using an ultraviolet light source disposed so as to face the surface of the cellulose film,

[0022] (d) after step (c), a step of bringing the back surface of the cellulose film into contact with a bacteria coloring reagent, and

[0023] (e) a step of determining that the test sample contains the plant pathogenic bacteria when the bacteria color developing reagent develops color.

[0024] Effects of the Invention

[0025] The present invention provides a method for selectively determining whether a test sample contains plant pathogenic bacteria from two types of bacteria, plant pathogenic bacteria and plant non-pathogenic bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A cross-sectional view of the first container 100 is shown.

[0027] Figure 2 A cross-sectional view of a substrate 170 having a cellulose film 104 on the back surface is shown.

[0028] Figure 3 This is a cross-sectional view showing the first container 100 after the test sample is supplied thereto.

[0029] Figure 4A cross-sectional view of a substrate 170 on the surface of which plant pathogenic bacteria are arranged is shown.

[0030] Figure 5A This is a cross-sectional view showing a state where plant pathogenic bacteria have penetrated through the through-hole 172 and the cellulose film 104 .

[0031] Figure 5B This is a cross-sectional view in step (c).

[0032] Figure 5C This is a cross-sectional view after step (c).

[0033] Figure 6 A cross-sectional view showing an example of a method for accelerating bacterial culture.

[0034] Figure 7 Then Figure 6 A cross-sectional view showing an example of a method for accelerating bacterial culture.

[0035] Figure 8 This is a cross-sectional view showing a state of bacteria observed from the back surface of the cellulose film 104 .

[0036] Fig. 9 This is a cross-sectional view showing a state of bacteria observed from the back surface of the cellulose film 104 .

[0037] Fig.10 This is a cross-sectional view showing a microporous membrane support used in the filamentous fungus measurement method disclosed in Patent Document 1. DETAILED DESCRIPTION

[0038] First, the term "fungus" used in this specification is explained. The term "fungus" includes fungi and oomycetes. Fungi are roughly divided into two types of fungi: plant pathogenic fungi and plant non-pathogenic fungi. Plant pathogenic fungi include, for example, Fusarium, Colletotrichum, Botrytis, Passalora or Pseudocercospora. Examples of plant pathogenic fungi are Fusarium avenaceum, Colletotrichum gloeosporioides, Botrytis cinerea, Passalora fulva or Pseudocercospora fuligena. These plant pathogenic fungi cause root rot disease, blast, Anthrax, Gray mold, etc. These plant pathogenic bacteria cause the plants to wilt. Examples of plant non-pathogenic bacteria are Saccharomyces cerevisiae, Penicillium chysogeum, or Aspergillus oryzae.

[0039] The term "phytopathogenic" means pathogenic to plants. The term "non-plant pathogenic" means non-pathogenic to plants. Even if a fungus is pathogenic, if it is not pathogenic to plants, the fungus is "non-plant pathogenic". In other words, as long as the fungus does not cause a bad effect on plants, the fungus is "non-plant pathogenic". The linker "non" contained in the term "non-plant pathogenic" does not modify "plant". The linker "non" modifies "pathogenic".

[0040] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. Japanese Patent No. 6167309, U.S. Patent No. 9695459, European Patent No. 3128001, Japanese Patent No. 6167310, U.S. Patent No. 9689020, European Patent No. 3128002, International Patent Application PCT / JP2016 / 004417, and International Patent Application PCT / JP2017 / 008407 are cited in this specification as references.

[0041] (Step (a))

[0042] In step (a), a test sample is placed on the front surface of the substrate 170 having the through hole 172. The cellulose film 104 is adhered to the back surface 170b of the substrate 170. In other words, the front surface 104a of the cellulose film 104 is in contact with the back surface 170b of the substrate 170. The front surface 104a refers to the upper surface of the cellulose film 104.

[0043] Specifically, Figure 1 As shown, a container 100 is prepared. It is desirable that the container 100 has a flange 102 at the upper end. The bottom surface of the container 100 is formed by a base plate 170.

[0044] like Figure 2 As shown, the substrate 170 has a cellulose film 104 on its back surface 170b. The substrate 170 has a through hole 172 that penetrates from its front surface 170a to the back surface 170b. The through hole 172 has a diameter of 3 microns to 5 microns. Please refer to PCT / JP2016 / 004417 and PCT / JP2017 / 008407. In other words, the through hole 172 has a cross-sectional area of ​​7.065 square microns to 19.625 square microns. Please refer to PCT / JP2016 / 004417 and PCT / JP2017 / 008407. In addition, please note that unlike the substrate 170, the cellulose film 104 does not have a through hole.

[0045] like Figure 3 As shown in FIG. 1 , the test sample 200 is supplied to the interior of the container 100. In this way, the test sample 200 is arranged on the surface 170a of the substrate 170. In the case where the test sample 200 contains plant pathogenic bacteria 202, as shown in FIG. Figure 4 As shown, plant pathogenic bacteria 202 are arranged on the surface 170 a of the substrate 170 .

[0046] The test sample 200 is a solid, a liquid, or a gas. It is desirable that the test sample 200 is a solid or a liquid. Examples of solid test samples 200 are soil or crushed plants. Other examples are agricultural materials such as vermiculite, rock wool, or urethane. Examples of liquid test samples 200 are agricultural water, liquid used for hydroponic cultivation, liquid after use for washing plants, liquid extracted from plants, liquid after use for washing agricultural materials, or liquid after use for washing clothes or shoes of operators.

[0047] (Step (b))

[0048] In step (b), after step (a), the test sample 200 is left to stand for a predetermined culture time. It is desirable to leave the test sample 200 to stand for about 4 to 150 hours (more preferably about 24 to 120 hours). In this way, the bacteria are cultured. In other words, the culture time is expected to be about 4 to 150 hours (expectedly about 24 to 120 hours). The importance of the thickness of the cellulose film 104 and the size of the through hole 172 is described below.

[0049] In step (b), various bacteria contained in the test sample 200 grow. As also confirmed in applications 1 to 4, when the following (I) is satisfied, the plant pathogenic bacteria 202 grows as follows: Figure 5A As shown, the plant pathogenic bacteria 202 grows through the cellulose film 104. As a result, the plant pathogenic bacteria 202 appear on the back surface 104b of the cellulose film 104.

[0050] Condition (I) The cellulose film 104 has a thickness of 0.5 μm to 3.7 μm.

[0051] When the above-mentioned condition (I) is satisfied, the plant non-pathogenic bacteria basically do not penetrate the cellulose film 104. Therefore, the plant non-pathogenic bacteria are basically absent from the back surface 104b of the cellulose film 104. On the other hand, the plant pathogenic bacteria 202 selectively appear on the back surface 104b. In this way, the plant pathogenic bacteria 202 selectively appear on the outside of the container 100. The back surface 104b refers to the lower side of the cellulose film 104.

[0052] In the case where the cellulose film 104 has a thickness exceeding 3.7 microns, not only plant non-pathogenic bacteria but also plant pathogenic bacteria do not penetrate the cellulose film 104. Therefore, in the case where the cellulose film 104 has a thickness exceeding 3.7 microns, selectivity is lost. In the case where the cellulose film 104 has a thickness of less than 0.5 microns (including the case where the cellulose film 104 is not provided), not only plant pathogenic bacteria but also plant non-pathogenic bacteria penetrate the cellulose film 104 (or are found on the back surface 170b of the substrate 170). Therefore, in the case where the cellulose film 104 has a thickness of less than 0.5 microns, selectivity is lost.

[0053] When the through hole 172 has a cross-sectional area of ​​less than 7.065 square microns (i.e., a diameter of less than 3 microns), it becomes difficult for not only non-plant pathogenic bacteria but also plant pathogenic bacteria to penetrate the cellulose film 104. On the other hand, when the through hole 172 has a cross-sectional area of ​​more than 19.625 square microns (i.e., a diameter of more than 5 microns), the number of invasion points tends to decrease compared to the case where the through hole 172 has a cross-sectional area of ​​19.625 square microns (i.e., a diameter of 5 microns).

[0054] The cellulose film 104 is stretched tightly on the back surface 170 b of the substrate 170 . In this way, the substrate 170 supports the cellulose film 104 .

[0055] like Figure 2 As shown, the substrate 170 has a plurality of through holes 172. The thickness of the substrate 170 is not limited, but is 1 micrometer to 500 micrometers as an example. The cellulose film 104 is very thin. However, if the cellulose film 104 is arranged on such a substrate 170, the handling of the cellulose film 104 becomes easy.

[0056] In order to accelerate the cultivation of bacteria, a culture medium may be supplied to the test sample 200. Specifically, a culture medium may be supplied inside the container 100 containing the test sample 200. The culture medium is preferably liquid. The culture medium may be supplied in step (b). Instead of supplying in step (b), the culture medium may be supplied before step (b). In other words, the culture medium may be supplied in step (a). The culture medium may also be supplied to the inside of the container 100 before step (a).

[0057] Figure 6 Other methods to accelerate bacterial cultivation are shown. Figure 6 As shown, it is desired to make the back surface 104b of the cellulose film 104 contact with the liquid culture medium 302. First, prepare the second container 300 having the liquid culture medium 302 inside. Hereinafter, in order to distinguish it from the second container 300, the container 100 is also referred to as the "first container 100". The first container 100 is overlapped with the second container 300 in such a manner that the bottom surface of the flange 102 contacts the upper end of the second container 300. In other words, the first container 100 is supported by the upper end of the second container 300. In this way, the liquid culture medium 302 is sandwiched between the back surface 104b of the cellulose film 104 and the bottom surface of the second container 300.

[0058] Alternatively, after the first container 100 is overlapped with the second container 300 , the liquid culture medium 302 is supplied between the back surface 104 b of the cellulose film 104 and the bottom surface of the second container 300 .

[0059] A viscous solid culture medium may also be used instead of the liquid culture medium 302. Figure 6 As shown, both a solid culture medium 304 and a liquid culture medium 302 may be used. In this case, the liquid culture medium 302 is sandwiched between the solid culture medium 304 and the cellulose membrane 104. Figure 5A As shown, the cultivation of plant pathogenic bacteria appearing on the back surface 104 b is accelerated by at least one of the liquid culture medium 302 and the solid culture medium 304 .

[0060] (Step (c))

[0061] In step (c), after step (b), if Figure 5B As shown, the cellulose film 104 is irradiated with ultraviolet rays using an ultraviolet light source 400 disposed so as to face the front surface 104 a of the cellulose film 104 .

[0062] Through the sterilization effect of ultraviolet rays, such as Figure 5C As shown, some of the plant pathogenic bacteria 202 located on the surface 104a of the cellulose film 104 are killed. Figure 5C Similarly, the plant non-pathogenic bacteria on the surface 104a on the front side of the cellulose film 104 are also killed. On the other hand, the cellulose film 104 shields ultraviolet rays, so the part of the plant pathogenic bacteria 202 on the surface 104b on the back side of the cellulose film 104 (i.e., the part of the plant pathogenic bacteria 202 that penetrates the cellulose film 104) still survives. The substrate 170 also shields ultraviolet rays.

[0063] The intensity and irradiation time of ultraviolet rays can be appropriately selected by those skilled in the art who read this specification. Ultraviolet rays with too high intensity cannot be fully shielded by the cellulose film 104 and the substrate 170, thereby killing the part of the plant pathogenic bacteria 202 located on the surface 104b of the back side of the cellulose film 104. As a result, the selectivity of the bacteria is lost. Too long ultraviolet irradiation time also causes the same problem.

[0064] (Step (d))

[0065] In step (d), after step (c), the back surface 104b of the cellulose film 104 is brought into contact with a bacterial coloring reagent. Thus, a portion of the plant pathogenic bacteria 202 (see Figure 5C ) to make the bacterial color developing reagent develop color. The bacterial color developing reagent reacts with live bacteria to develop color, but does not react with dead bacteria and does not develop color. The bacterial color developing reagent is generally water-soluble.

[0066] Theoretically, since the second container 300 and the first container 100 are separated by the cellulose film 104, the bacterial color developing reagent does not spread into the interior of the first container 100. Therefore, the plant pathogenic bacteria contained in the first container 100 do not cause the bacterial color developing reagent to develop color.

[0067] However, in reality, the bacteria coloring reagent can spread into the first container 100 through the small gaps formed around the plant pathogenic bacteria 202 that penetrate the cellulose film 104. In addition, the bacteria presenting reagent is generally water-soluble and can spread through the inside of the cellulose film 104 to the surface 104a of the cellulose film 104.

[0068] Therefore, part of the plant pathogenic bacteria 202 located on the surface 104a of the cellulose film 104 also causes the bacteria coloring reagent to develop color. Furthermore, the plant non-pathogenic bacteria (not shown) located on the surface 104a of the cellulose film 104 also causes the bacteria coloring reagent to develop color. Therefore, the selectivity of the bacteria is lost.

[0069] On the other hand, in this embodiment, if Figure 5C As shown, by irradiation with ultraviolet rays, the plant pathogenic bacteria 202 and the plant non-pathogenic bacteria (not shown) are killed on the surface 104a of the surface side of the cellulose film 104. However, a part of the plant pathogenic bacteria 202 still survives on the surface 104b on the back side of the cellulose film 104, and this part causes the bacterial color developing reagent to develop color. Of course, the plant non-pathogenic bacteria do not penetrate the cellulose film 104, and therefore do not cause the bacterial color developing reagent to develop color. In this way, in this embodiment, a part of the plant pathogenic bacteria 202 that penetrates the cellulose film 104 selectively causes the bacterial color developing reagent to develop color.

[0070] An example of a bacterial colorimetric reagent is a chemical substance having the following chemical structure: 1. This chemical substance is called WST-8.

[0071]

[0072] Specifically, after step (c), the liquid culture medium 302 and the solid culture medium 304 are removed from the second container 300. Next, a bacterial color development reagent is added to the interior of the second container 300. Next, as Figure 7 As shown, the first container 100 is overlapped with the second container 300 having the bacteria coloring reagent therein. Alternatively, after the first container 100 is overlapped with the second container 300, a solution 402 containing the bacteria coloring reagent is supplied between the back surface 104b of the cellulose film 104 and the bottom surface of the second container 300. In addition, since the liquid seeps out from the cellulose film 104, the solution 402 containing the bacteria coloring reagent can also be supplied into the first container 100.

[0073] (Step (e))

[0074] In step (e), when the bacteria color developing reagent develops color in step (d), it is determined that the test sample contains plant pathogenic bacteria.

[0075] The present invention also includes an apparatus used in carrying out the above-mentioned method of selectively determining whether or not plant pathogenic bacteria are contained.

[0076] The device of the present invention comprises at least a substrate and a cellulose film. The substrate comprises a through hole, and the through hole has a cross-sectional area of ​​7.065 square micrometers to 19.625 square micrometers, in other words, a diameter of 3 micrometers to 5 micrometers. The substrate can also be a container whose bottom surface is constituted by the substrate and has a flange. A cellulose film is provided on one side of the substrate, and the cellulose film has a thickness of 0.5 micrometers to 3.7 micrometers and does not have a through hole.

[0077] The device of the present invention may also include a culture medium for accelerating the cultivation of bacteria, a bacterial coloring reagent, etc. In addition, the device of the present invention may also include an ultraviolet light source for irradiating ultraviolet light on the cellulose membrane, an optical sensor for optically detecting the color of the bacterial coloring reagent, a display element for displaying the detection result, etc.

[0078] As long as the effect of the present invention is not hindered, other layers may be sandwiched between the cellulose film and the substrate. However, it is desirable that the cellulose film is in contact with the substrate. In other words, as long as the effect of the present invention is not hindered, the cellulose film does not have to be in contact with the substrate, and the position of the cellulose film on the substrate may be arranged in such a manner that other layers are sandwiched between the cellulose film and the substrate. However, although it is a repeated statement, it is desirable that the position of the cellulose film on the substrate is arranged in such a manner that the cellulose film is in contact with the substrate.

[0079] (Example)

[0080] The present invention is described in more detail with reference to the following examples.

[0081] (Preparation of culture medium)

[0082] First, the inventors dissolved 0.56 mM iron sulfate (III), 1.53 mM zinc nitrate, and 1.1 mM manganese sulfate (II) in pure water (1 liter) to prepare an aqueous solution. Next, the inventors mixed and dissolved pure water (1 liter), lactose (37.5 g, obtained from SIGMA-ALDRICH), casein hydrate (3.0 g, obtained from Difco), potassium dihydrogen phosphate (1.0 g, obtained from Wako Pure Chemical Industries, Ltd.), and magnesium sulfate (0.5 g, obtained from Wako Pure Chemical Industries, Ltd.) with the aqueous solution (2 ml) to prepare a mixture. The inventors sterilized the mixture using an autoclave. The pH of the obtained mixture was 6.0. The inventors diluted the mixture 10 times with pure water to obtain a culture medium. Hereinafter, the culture medium obtained in this way will be referred to as "10% LCH culture medium".

[0083] (Preparation of WST reaction solution)

[0084] The present inventors dissolved duroquinone (obtained from Tokyo Chemical Industry Co., Ltd., CAS No.: 527-17-3) in ethanol (obtained from Wako Pure Chemical Industries, Ltd.) to obtain a duroquinone ethanol solution (50 mM). Separately, the present inventors dissolved 2-cyclohexylaminoethanesulfonic acid (obtained from Tokyo Chemical Industry Co., Ltd., CAS: 103-47-9, hereinafter referred to as "Chem") in pure water to obtain a Chem solution (100 mM). The present inventors adjusted the pH of the Chem solution to 9.5 by dropping a NaOH aqueous solution (1 M, obtained from Wako Pure Chemical Industries, Ltd.).

[0085] The present inventors mixed the reagents shown in the following Table 1 to obtain a WST reaction solution.

[0086] Table 1

[0087] Reagent Name Volume ratio 10% LCH medium 170 WST-8 solution 9 Duroquinone ethanol solution (50mM) 1 Chem solution (100mM) 20

[0088] The WST-8 solution is contained in a commercial product obtained from Dojin Chemical Laboratories, Ltd. as a trade name: WST-8 Microbial Viability Assay Kit-WST. WST-8 has the chemical structure described above as "Chemical 1".

[0089] (Experimental Example 1)

[0090] (Cultivation of Colletotrichum gloeosporioide)

[0091] Colletotrichum gloeosporioide, a plant pathogen, was inoculated into a potato dextrose agar medium. The medium was then left to stand at 25 degrees Celsius for 1 week. Colletotrichum gloeosporioide was donated by Associate Professor Shimizu of the Department of Applied Biological Sciences at Gifu University.

[0092] Next, the portion including the tip of the hyphae was cut into pieces with a size of 1 cm x 1 cm together with the culture medium, and the cut portion was immersed in a 10% LCH culture medium arranged on a 12-well plate. The volume of each pure water was 1 ml.

[0093] The water placed on the 12-well plate was observed using an optical microscope. As a result, the release of spores of Colletotrichum gloeosporioide was confirmed in the water placed on the 12-well plate. Thus, an aqueous solution containing Colletotrichum gloeosporioide was obtained. Hereinafter, the aqueous solution is referred to as a "plant pathogenic bacteria aqueous solution".

[0094] (Experimental Example 1)

[0095] Experimental Example 1 includes Example 1A to Example 1D.

[0096] (Example 1A)

[0097] Prepare as follows Figure 1 The first container 100 is shown.

[0098] First, cellulose (available from SIGMA-ALDRICH, trade name: AvicEl pH-101) was dissolved in an ionic liquid to prepare a cellulose solution having a concentration of 1%. The ionic liquid was 1-butyl-3-methylimidazole chloride (available from SIGMA-ALDRICH).

[0099] The cellulose solution is heated to 60 degrees Celsius. Next, the cellulose solution is applied to the back surface of a container having a polyethylene terephthalate film on the bottom surface (obtained from Millipore, trade name: Millicell PISP 12R48) by spin coating for 30 seconds at a rotation speed of 2000 rpm. The polyethylene terephthalate film functions as a substrate 170. The polyethylene terephthalate film randomly has a plurality of through holes 172 with a diameter of 3 microns. In this way, a cellulose film 104 with a thickness of 0.5 microns is formed on the back surface of the polyethylene terephthalate film. According to Millipore, the diameter of the through hole 172 can have an error range of about plus or minus 10%.

[0100] The container was left to stand in ethanol at room temperature for 12 hours. Thus, 1-butyl-3-methylimidazole chloride was replaced with ethanol. In other words, 1-butyl-3-methylimidazole chloride was removed from the cellulose film 104.

[0101] Finally, the container is dried in a vacuum dryer. Figure 1 The first container 100 is shown. Please note Figure 1 The polyethylene terephthalate film functioning as the substrate 170 is not shown in the figure.

[0102] Then, if Figure 6As shown, the first container 100 is overlapped with the second container 300. A 24-well plate (obtained from Corning) is used as the second container 300. The back surface 104b of the cellulose membrane 104 is in contact with the liquid culture medium 302. As the liquid culture medium 302, a 10% LCH culture medium is used. A plant pathogen aqueous solution containing 1000 spores of Colletotrichum gloeosporioide is added to the interior of the first container 100.

[0103] The first container 100 was left to stand at a temperature of 25 degrees Celsius for 24 hours. In other words, in Example 1A, the incubation time was 24 hours.

[0104] (Bacteria Detection)

[0105] Next, the inventors separated the first container 100 from the second container 300. Furthermore, the inventors used a pipette to remove the solution contained in the first container 100. Then, the inventors surrounded the first container 100 with a black 24-well plate (obtained from Zell-Kontakt, trade name: Imaging Plate FC) to shield the first container 100 from light.

[0106] like Figure 5B and Figure 5C As shown, ultraviolet rays were irradiated onto the cellulose film 104 using an ultraviolet light source 400 (manufactured by Panasonic Corporation, trade name: UV-EC910ZA). The interval between the ultraviolet light source 400 and the cellulose film 104 was 1.5 cm. A current of 0.1 ampere was passed through the ultraviolet light source 400 for 10 minutes.

[0107] Next, the present inventors brought the WST reaction solution (100 μl) into contact with the back surface 104 b of the cellulose film 104. Next, the first container 100 was shielded from light at 25 degrees Celsius for 1 hour so that the cellulose film 104 was not irradiated with light.

[0108] The inventors transferred the WST reaction solution (50 μl) in contact with the back surface 104b of the cellulose membrane 104 into a 96-well flat-bottom transparent plate (obtained from Greiner). Next, the inventors measured the absorbance of the WST reaction solution at a wavelength of 460 nm using an ELISA reader (obtained from TECAN, trade name: M1000Pro).

[0109] Separately, as a reference, the WST reaction solution was left to stand at 25 degrees Celsius for 1 hour, and then its absorbance was measured in the same manner. Of course, the WST reaction solution used as a reference was not in contact with the back surface 104b of the cellulose membrane 104. Example 1A was repeated twice.

[0110] The present inventors calculated the chromaticity according to the following formula (I).

[0111] Color development degree = (absorbance of the WST reaction solution in contact with the back surface 104b of the cellulose membrane 104) - (absorbance of the WST reaction solution as a reference) (I)

[0112] The results are shown in Table 2 below.

[0113] Table 2

[0114] Absorbance of WST reaction solution WST reaction solution in contact with the back surface 104b (first time) 0.428 Reference WST reaction solution (first time) 0.046 WST reaction solution in contact with the back surface 104b (second time) 0.308 Reference WST reaction solution (second time) 0.044

[0115] According to Table 2, the average value of the color development of Colletotrichum gloeosporioide in Example 1 was 0.32 (=((0.428-0.046)+(0.308-0.044)) / 2).

[0116] (Example 1B)

[0117] In Example 1B, the same experiment as in Example 1A was conducted except that the cellulose solution had a concentration of 3%, and the cellulose film 104 had a thickness of 3.7 micrometers. The results are shown in Table 3 below.

[0118] Table 3

[0119] Absorbance of WST reaction solution WST reaction solution in contact with the back surface 104b (first time) 0.361 Reference WST reaction solution (first time) 0.054 WST reaction solution in contact with the back surface 104b (second time) 0.257 Reference WST reaction solution (second time) 0.043

[0120] (Example 1C)

[0121] In Example 1C, the same experiment as in Example 1A was conducted except that a waterproof tape (obtained from Nitto Denko Corporation, trade name: 57130SB) was adhered to the entire surface of the back surface 104b of the cellulose film 104. The waterproof tape suppressed the extension of bacteria to the outside of the back surface 104b of the cellulose film 104. The results are shown in Table 4 below.

[0122] Table 4

[0123] Absorbance of WST reaction solution WST reaction solution in contact with the back surface 104b (first time) 0.078 Reference WST reaction solution (first time) 0.046 WST reaction solution in contact with the back surface 104b (second time) 0.064 Reference WST reaction solution (second time) 0.044

[0124] (Example 1D)

[0125] In Example 1D, the same experiment as in Example 1A was conducted except that the cellulose solution had a concentration of 3%, the cellulose film 104 had a thickness of 3.7 μm, and a waterproof tape was adhered to the entire back surface 104 b of the cellulose film 104. The results are shown in Table 5 below.

[0126] Table 5

[0127] Absorbance of WST reaction solution WST reaction solution in contact with the back surface 104b (first time) 0.071 Reference WST reaction solution (first time) 0.054 WST reaction solution in contact with the back surface 104b (second time) 0.062 Reference WST reaction solution (second time) 0.043

[0128] (Experimental Example 2)

[0129] Experimental Example 2 includes Examples 2A to 2D. In Experimental Example 2, the same experiment as in Experimental Example 1 was performed except that Fusarium avenaceum was used instead of Colletotrichum gloeosporioide.

[0130] The results for Example 2A are shown in Table 6 below.

[0131] Table 6

[0132] Absorbance of WST reaction solution WST reaction solution in contact with the back surface 104b (first time) 0.273 Reference WST reaction solution (first time) 0.046 WST reaction solution in contact with the back surface 104b (second time) 0.273 Reference WST reaction solution (second time) 0.056

[0133] The results for Example 2B are shown in Table 7 below.

[0134] Table 7

[0135] Absorbance of WST reaction solution WST reaction solution in contact with the back surface 104b (first time) 0.213 Reference WST reaction solution (first time) 0.054 WST reaction solution in contact with the back surface 104b (second time) 0.204 Reference WST reaction solution (second time) 0.063

[0136] The results for Example 2C are shown in Table 8 below.

[0137] Table 8

[0138] Absorbance of WST reaction solution WST reaction solution in contact with the back surface 104b (first time) 0.060 Reference WST reaction solution (first time) 0.061 WST reaction solution in contact with the back surface 104b (second time) 0.077 Reference WST reaction solution (second time) 0.056

[0139] The results for Example 2D are shown in Table 9 below.

[0140] Table 9

[0141] Absorbance of WST reaction solution WST reaction solution in contact with the back surface 104b (first time) 0.057 Reference WST reaction solution (first time) 0.062 WST reaction solution in contact with the back surface 104b (second time) 0.071 Reference WST reaction solution (second time) 0.063

[0142] Table 10 below shows the results of Examples 1A to 1D and 2A to 2D.

[0143] Table 10

[0144]

[0145] As shown in Table 10 above, plant pathogenic bacteria that had penetrated the cellulose membrane 104 were detected.

[0146] (Experimental Example 3)

[0147] Regarding Botrytis cinerea (tomato gray mold fungus), which is one type of plant pathogenic bacteria, a plant pathogenic bacteria aqueous solution containing spores of Botrytis cinerea was obtained in the same manner as in Experimental Example 1.

[0148] Experimental Example 3 includes Example 3 and Comparative Examples 3A to 3C.

[0149] (Example 3)

[0150] Prepare in the same manner as in Example 1 Figure 1 The first container 100 is shown.

[0151] Then, if Figure 6 As shown, the first container 100 is overlapped with the second container 300. The second container 300 and the liquid culture medium 302 are the same as those in Example 1. An aqueous plant pathogen solution containing 1000 spores of Botrytis cinerea is added to the first container 100.

[0152] The first container 100 was left to stand at a temperature of 25 degrees Celsius for 40 hours.

[0153] Next, similarly to the above-described first embodiment, the first container 100 is separated from the second container 300 , and after the solution contained in the first container 100 is removed, the first container 100 is shielded from light.

[0154] like Figure 5B and Figure 5C As shown, similarly to the first embodiment, the cellulose film 104 is irradiated with ultraviolet rays using the ultraviolet light source 400 .

[0155] Next, the WST reaction solution (100 μl) was brought into contact with the back surface 104 b of the cellulose film 104. Next, the first container 100 was shielded from light at 25 degrees Celsius for 3 hours so that the cellulose film 104 was not irradiated with light.

[0156] Next, in the same manner as in Example 1, the absorbance of the WST reaction solution (50 μl) in contact with the back surface 104 b of the cellulose membrane 104 was measured.

[0157] (Comparative Example 3A)

[0158] In Comparative Example 3A, the same experiment as in Example 3 was conducted, except that a cellulose solution having a concentration of 5% was used instead of a cellulose solution having a concentration of 1% when preparing the first container 100 in Example 3, and that as the container having a polyethylene terephthalate film having a plurality of through holes on the bottom surface, a through hole having a diameter of 1 micron (Millipore product name: Millicell PIRP 12R 48) was used instead of a through hole having a diameter of 3 microns (Millipore product name: Millicell PISP 12R 48).

[0159] (Comparative Example 3B)

[0160] In Comparative Example 3B, the same experiment as in Example 3 was conducted except that the cellulose film 104 was not irradiated with ultraviolet rays.

[0161] (Comparative Example 3C)

[0162] In Comparative Example 3C, the same experiment as in Comparative Example 3A was conducted except that the cellulose film 104 was not irradiated with ultraviolet rays.

[0163] (Experimental Example 4)

[0164] Regarding Passalora fulva (tomato leaf mold fungus), which is one of the plant pathogens, a plant pathogen aqueous solution containing spores of Passalora fulva was obtained in the same manner as in Experimental Example 1.

[0165] Experimental Example 4 includes Example 4 and Comparative Examples 4A-4B.

[0166] (Example 4)

[0167] Prepare in the same manner as in Example 1 Figure 1 The first container 100 is shown.

[0168] Then, if Figure 6 As shown, the first container 100 is overlapped with the second container 300. The second container 300 and the liquid culture medium 302 are the same as those in Example 1. An aqueous plant pathogen solution containing 1000 spores of Passalora fulva is added to the first container 100.

[0169] The first container 100 was left to stand at a temperature of 25 degrees Celsius for 120 hours.

[0170] Next, similarly to the above-mentioned Example 1, the first container 100 is separated from the second container 300, and after the solution contained in the interior of the first container 100 is removed, the first container 100 is shielded from light.

[0171] like Figure 5B and Figure 5C As shown, similarly to the first embodiment, the cellulose film 104 is irradiated with ultraviolet rays using the ultraviolet light source 400 .

[0172] Next, the WST reaction solution (100 μl) was brought into contact with the back surface 104 b of the cellulose film 104. Next, the first container 100 was shielded from light at 25 degrees Celsius for 3 hours so that the cellulose film 104 was not irradiated with light.

[0173] Next, in the same manner as in Example 1, the absorbance of the WST reaction solution (50 μl) in contact with the back surface 104 b of the cellulose membrane 104 was measured.

[0174] (Comparative Example 4A)

[0175] In Comparative Example 4A, the same experiment as in Example 4 was conducted, except that a cellulose solution having a concentration of 5% was used instead of a cellulose solution having a concentration of 1% when preparing the first container 100 in Example 4, and that as the container having a polyethylene terephthalate film having a plurality of through holes on the bottom surface, a through hole having a diameter of 1 micron (Millipore product name: Millicell PIRP 12R 48) was used instead of a through hole having a diameter of 3 microns (Millipore product name: Millicell PISP 12R 48).

[0176] (Comparative Example 4B)

[0177] In Comparative Example 4B, the same experiment as in Example 4 was conducted except that the cellulose film 104 was not irradiated with ultraviolet rays.

[0178] (Experimental Example 5)

[0179] Regarding Pseudocercospora fuligena (tomato black mold fungus) which is one of the plant pathogens, a plant pathogen aqueous solution containing spores of Pseudocercospora fuligena was obtained in the same manner as in Experimental Example 1.

[0180] Experimental Example 5 includes Example 5 and Comparative Examples 5A-5B.

[0181] (Example 5)

[0182] Prepare in the same manner as in Example 1 Figure 1 The first container 100 is shown.

[0183] Then, if Figure 6 As shown, the first container 100 is overlapped with the second container 300. The second container 300 and the liquid culture medium 302 are the same as those in Example 1. An aqueous plant pathogen solution containing 1000 spores of Pseudocercospora fuligena is added to the first container 100.

[0184] The first container 100 was left to stand at a temperature of 25 degrees Celsius for 75 hours.

[0185] Next, similarly to the above-mentioned Example 1, the first container 100 is separated from the second container 300, and after the solution contained in the interior of the first container 100 is removed, the first container 100 is shielded from light.

[0186] like Figure 5B and Figure 5C As shown, similarly to the first embodiment, the cellulose film 104 is irradiated with ultraviolet rays using the ultraviolet light source 400 .

[0187] Next, the WST reaction solution (100 μl) was brought into contact with the back surface 104 b of the cellulose film 104. Next, the first container 100 was shielded from light at 25 degrees Celsius for 1 hour so that the cellulose film 104 was not irradiated with light.

[0188] Next, in the same manner as in Example 1, the absorbance of the WST reaction solution (50 μl) in contact with the back surface 104 b of the cellulose membrane 104 was measured.

[0189] (Comparative Example 5A)

[0190] In Comparative Example 5A, the same experiment as in Example 5 was conducted, except that a cellulose solution having a concentration of 5% was used instead of a cellulose solution having a concentration of 1% when preparing the first container 100 in Example 5, and that as the container having a polyethylene terephthalate film having a plurality of through holes on the bottom surface, a through hole having a diameter of 1 micron (Millipore product name: Millicell PIRP 12R 48) was used instead of a through hole having a diameter of 3 microns (Millipore product name: Millicell PISP 12R 48).

[0191] (Comparative Example 5B)

[0192] In Comparative Example 5B, the same experiment as in Example 5 was conducted except that the cellulose film 104 was not irradiated with ultraviolet rays.

[0193] Table 11 below shows the results of Experimental Examples 3 to 5. The number of penetrating hyphae in Table 11 indicates the result of counting the number of hyphae penetrating the cellulose film 104 by observation under an optical microscope.

[0194] Table 11

[0195]

[0196] The color development in Examples 3 to 5 is caused by killing the bacteria on the substrate 170 by ultraviolet irradiation and only the hyphae penetrating the cellulose film 104 .

[0197] In addition, Comparative Examples 3A, 4A, and 5A were implemented under conditions where it was difficult for bacteria to penetrate, so there was no penetration of bacteria, or the penetration of bacteria was very small. Therefore, the bacteria on the substrate 170 were killed by ultraviolet irradiation, and the color development was very low compared with Examples 3 to 5. From the comparison of Comparative Examples 3A, 4A, and 5A with Examples 3 to 5, it can be understood that the method of the present invention can determine whether there is penetration of hyphae by color development.

[0198] In Comparative Examples 3B, 3C, 4B, and 5B, ultraviolet rays were not irradiated, so the bacteria on the substrate 170 were not killed. Therefore, regardless of the presence of penetrating hyphae, the color development was higher than that of each of Examples 3 to 5, and the presence of penetrating hyphae could not be determined.

[0199] As described above, from the results of Experimental Examples 3 to 5, according to the method of the present invention, the phytopathogenic bacteria that have penetrated the cellulose membrane cause the bacteria coloring reagent to develop color, thereby making it possible to determine whether the test sample contains the phytopathogenic bacteria.

[0200] Industrial Availability

[0201] The present invention can be used to easily determine whether a test sample such as agricultural water, plant fragments, or soil contains plant pathogenic bacteria.

[0202] Explanation of symbols

[0203] 100 Container 1

[0204] 102 Flange

[0205] 104 Cellulose membrane

[0206] 104a Surface side

[0207] 104b Dorsal surface

[0208] 170 substrate

[0209] 170a Surface side

[0210] 170b Dorsal surface

[0211] 200 test specimens

[0212] 202 Plant pathogenic bacteria

[0213] 202a Part of plant pathogenic bacteria

[0214] 300 2nd container

[0215] 302 Liquid culture medium

[0216] 304 Solid culture medium

[0217] 402 Solution containing bacterial colorimetric reagent

[0218] 500 Light Source

[0219] 600 Microscope

Claims

1. A method for determining whether a test sample contains plant pathogenic bacteria, the method comprising the following steps: (a) a step of placing the test sample on the front surface of a substrate having a through hole, in, The substrate has a cellulose film on its back side. The through holes of the substrate have a cross-sectional area of ​​7.065 square micrometers to 19.625 square micrometers, the cellulose film has no through holes, and The cellulose film has a thickness of 0.5 micrometers to 3.7 micrometers, (b) after step (a), a step of allowing the test sample to stand still, (c) after step (b), irradiating the cellulose film with ultraviolet rays using an ultraviolet light source disposed so as to face the surface of the cellulose film, (d) after the step (c), a step of bringing the back surface of the cellulose film into contact with a solution of a bacteria coloring reagent contained in a container, and (e) a step of determining that the test sample contains the plant pathogenic bacteria when the solution-like bacteria color developing reagent contained in the container develops color, The bacterial color development reagent is WST-8.

2. The method according to claim 1, wherein: The plant pathogenic bacteria are plant pathogenic fungi.

3. The method according to claim 2, wherein: The plant pathogenic fungus is at least one fungus selected from the group consisting of Colletotrichum, Fusarium, Botrytis, Passalora and Pseudocercospora.

4. The method according to claim 2, wherein: The plant pathogenic fungus is at least one fungus selected from the group consisting of Colletotrichum gloeosporioides, Fusarium avenaceum, Botrytis cinerea, Passalora fulva and Pseudocercospora fuligena.

5. The method according to claim 1, wherein: The plant pathogenic bacteria are plant pathogenic oomycetes.

6. The method according to claim 1, wherein: Prior to the step (b), the method further comprises supplying a culture medium to the test sample.

7. The method according to claim 6, wherein: The culture medium is a liquid culture medium.

8. The method according to claim 6, wherein: The culture medium is a solid culture medium.

9. The method according to claim 1, wherein: In the step (b), the test sample is allowed to stand while the back surface of the cellulose membrane is in contact with the culture medium.

10. The method according to claim 9, wherein: The culture medium is a liquid culture medium.

11. The method according to claim 9, wherein: The culture medium is a solid culture medium.

12. The method according to claim 1, wherein: The test specimen is a solid.

13. The method according to claim 12, wherein: The solid is at least one solid selected from the group consisting of soil and crushed plants.

14. The method according to claim 12, wherein: The solid is at least one solid selected from the group consisting of a part of a plant body and soil.

15. The method according to claim 1, wherein: The test sample is a liquid.

16. The method according to claim 15, wherein: The liquid is at least one liquid selected from the group consisting of agricultural water, liquid extracted from plants, and liquid used for washing clothes or shoes.

17. The method according to claim 15, wherein: The liquid is at least one liquid selected from the group consisting of a liquid used for hydroponic cultivation, a liquid used for washing plants, a liquid extracted from plants, a liquid used for washing agricultural materials, and a liquid used for washing clothes or shoes.

Images