阅读说明：本技术 番茄病原性真菌的检测装置及使用了该检测装置的检测方法 (Tomato pathogenic fungus detection device and detection method using same ) 是由 石堂太郎 狩集庆文 于 2019-04-05 设计创作，主要内容包括：本公开提供简易并且可靠的、番茄病原性真菌的选择性检测装置和检测方法。本公开涉及的番茄病原性真菌的检测装置,其特征在于,具有：人工细胞壁；设置在上述人工细胞壁的上部的试验试样液投入部；以及设置在上述人工细胞壁的下部的培养液储存部,在上述培养液储存部中,培养液包含15～30mM的柠檬酸盐缓冲液,并且上述培养液的pH值为5～5.5。(The present disclosure provides a simple and reliable selective detection apparatus and detection method of tomato pathogenic fungi. The present disclosure relates to a device for detecting a tomato pathogenic fungus, comprising: artificial cell walls; a test sample liquid input part arranged on the upper part of the artificial cell wall; and a culture medium storage part arranged at the lower part of the artificial cell wall, wherein the culture medium storage part contains 15-30 mM citrate buffer solution, and the pH value of the culture medium is 5-5.5.)

1. A detection device for a tomato pathogenic fungus, comprising: artificial cell walls; a test sample liquid input part provided above the artificial cell wall; and a culture medium reservoir part provided below the artificial cell wall,

in the culture solution storage part, a culture solution contains 15-30 mM citrate buffer solution, and the pH value of the culture solution is 5-5.5.

2. The apparatus for detecting tomato pathogenic fungi according to claim 1, wherein the artificial cell wall comprises at least:

a substrate having a through hole with a diameter of 2 to 7 μm and a thickness of 5 to 150 μm, and

a cellulose film having a thickness of 0.5 to 2 μm and provided on one surface of the substrate.

3. The apparatus for detecting tomato pathogenic fungi according to claim 1 or 2, wherein the citrate is at least one selected from the group consisting of sodium citrate and potassium citrate.

4. The apparatus for detecting a tomato pathogenic fungus according to any one of claims 1 to 3, wherein the tomato pathogenic fungus to be detected is at least one selected from Botrytis cinerea, tomato Pseudocercospora furigena, and Stachybotrys fulvidraco, Passalora fulva.

5. The apparatus for detecting tomato pathogenic fungi according to any one of claims 1 to 4, which does not detect a fungus of the genus Biscogniauxia, Penicillium, Phoma, and Trichoderma, which are tomato nonpathogenic fungi.

6. The apparatus for detecting tomato pathogenic fungi as claimed in claim 5, wherein the tomato nonpathogenic fungi is Dictyocaulus hainanensis (Biscogenia maritima), Penicillium orellanicum (Penicillium olsonii), Phoma multirhynchophythora (Phoma multirostrata), or Trichoderma asperellum (Trichoderma asperellum).

7. A method for detecting tomato pathogenic fungi, comprising the steps of: the detection device according to any one of claims 1 to 6, which is used for selectively detecting a tomato pathogenic fungus.

Technical Field

The present invention relates to a detection device for tomato pathogenic fungi and a selective detection method using the detection device.

Background

The phytopathogenic fungi have properties related to plant invasion, such as forming an attachment on a plant surface and attaching the attachment, then searching for pores such as stomata and extending hyphae from the pores into a plant body, and secreting a plant cell wall-degrading enzyme (cellulase or pectinase) from the hyphae.

Taking advantage of these characteristics, for example, patent document 1 discloses a fungus metering method using a microporous membrane support. Further, non-patent document 1 discloses: pseudo hyphae of Phytophthora sojae (Phytophthora sojae) of 1 species as a phytopathogenic oomycete seemed to try to sneak downward rather than grow horizontally and penetrated a PET (polyethylene terephthalate) film having pores of 3 μm.

In addition, in view of such properties, the present inventors have already proposed a method for determining phytopathogenic oomycetes (patent document 2).

Disclosure of Invention

Problems to be solved by the invention

Tomato, which is a target plant of the present invention, has a high rate of diseases caused by fungi, and the pathogenic fungi causing the diseases are mostly 3 kinds of Botrytis cinerea (Botrytis cinerea), Pseudocercospora solanacearum (Pseudocercospora fuligena), and alternaria fulva (Passalora fulvva). Among these pathogenic fungi, Botrytis cinerea (Botrytis cinerea) has a lot of offence and infects other plants, but Pseudocercospora coalescens (Pseudocercospora fuligena) and Neurospora fulva (Passalora fulva) are only examples of infection with tomato and are pathogenic fungi with high plant specificity. The present inventors considered that it is necessary to detect and study tomato pathogenic fungi at a stage where it is not clear which species of fungi is actually present in tomato leaves, i.e., at a stage before onset of disease, even if these fungi are specific to tomato.

On the other hand, the technique of selecting pathogenic fungi using an artificial cell wall, which is a basic technique for selective fungi detection used by the present inventors as described in patent document 2, has a possibility of "not being detected as tomato pathogenic fungi as long as it is a plant pathogenic fungus". That is, it is possible to detect other plant pathogenic fungi as tomato pathogenic fungi, provided that they adhere to the leaves of tomato. Tomato cultivation is mostly carried out not from seeds but from seedlings, in nursery fields, because of mixed cultivation with other plants, sharing of a plurality of plants in the same facility, and the like, and thus the possibility of attachment of plant pathogenic fungi other than the tomato pathogenic fungi to tomato seedlings cannot be denied. In addition, in actual cultivation sites and cultivation facilities such as vinyl houses, there is a possibility that pathogenic fungi of plants other than tomatoes may attach to tomato seedlings in the same manner as in the above-described nursery sites. If this is not the case, a false positive may be suggested in the technique of selecting a pathogenic fungus using an artificial cell wall by a plant pathogenic fungus other than the tomato pathogenic fungus, which may cause a great problem in cultivation such as unnecessary administration and shoot renewal.

As a result of research and investigation on the possibility of the occurrence of this false positive, fungi other than the tomato pathogenic fungi actually encountered a false positive in the detection method using an artificial cell wall during the research. These are 4 species of fungi of the genus Dictyocaulus (genus Biscogeniauxia), Penicillium (genus Penicillium), Phoma (genus Phoma), Trichoderma (genus Trichoderma), and studies are required in order not to detect them.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a selective detection apparatus and a detection method for a tomato pathogenic fungus.

Means for solving the problems

As a result of intensive studies, the present inventors have found that the above problems can be solved by a detection device having the following configuration, and further, the present invention has been completed by further conducting extensive studies based on such findings.

That is, the apparatus for detecting a tomato pathogenic fungus according to one aspect of the present invention includes: artificial cell walls; a test sample liquid input part arranged on the upper part of the artificial cell wall; and a culture medium storage part arranged at the lower part of the artificial cell wall, wherein the culture medium storage part contains 15-30 mM citrate buffer solution, and the pH value of the culture medium is 5-5.5.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, it is possible to provide an apparatus and a method capable of selectively detecting tomato pathogenic fungi easily and safely. The present invention is useful in industrial applications because it can detect the presence of bacteria in the pre-pathogenic stage of tomato pathogenic fungi, and can avoid false positive indications caused by phytopathogens other than tomato.

Drawings

Fig. 1 is a schematic cross-sectional view showing an example of the detection device of the present embodiment.

FIG. 2 is a schematic cross-sectional view showing an example of an artificial cell wall provided in the detection device of the present embodiment.

Fig. 3 is a schematic cross-sectional view showing an example of the detection device of the present embodiment.

FIG. 4 is a photomicrograph of the back surface of an artificial cell wall showing the penetration of Botrytis cinerea (Botrytis cinerea) into the artificial cell wall.

Fig. 5 is a graph showing the results of comparative example 1.

Fig. 6 is a graph showing the results of comparative example 2.

Fig. 7 is a graph showing the results of comparative example 3.

Fig. 8 is a graph showing the results of example 1.

Detailed Description

The embodiments of the present invention will be described below in detail, but the present invention is not limited thereto.

As shown in fig. 1, the apparatus 1 for detecting a tomato pathogenic fungus according to the present embodiment is characterized by including: an artificial cell wall 2; a test sample liquid loading part 3 provided above the artificial cell wall 2; and a culture medium reservoir part 4 provided below the artificial cell wall 2, wherein a culture medium 5 contains a citrate buffer solution of 15 to 30mM and has a pH of 5 to 5.5 in the culture medium reservoir part 4.

The test sample liquid loading unit 3 is a container for loading a test sample liquid, and the container is preferably provided with a flange at an upper end thereof. The bottom surface of the test sample liquid loading portion 3 is formed by the artificial cell wall 2.

As shown in fig. 2, the artificial cell wall 2 preferably includes at least a substrate 21 having a through-hole 22 and a cellulose film 23 provided on one surface of the substrate 21. By using such artificial cell walls, it becomes easier to selectively detect the tomato pathogenic fungi as a target.

The through hole 22 penetrates from the surface of the front side to the surface of the back side of the substrate 21, and the diameter of the through hole is preferably 2 to 7 μm (cross-sectional area 4.5 to 38.5 μm)²). By setting the pore diameter to the above range, the pathogenic fungi of interest can be selectively detected more reliably.

Further, in order to selectively detect the pathogenic fungi of interest more reliably, it is preferable to also adjust the thickness of the cellulose film 23. Specifically, the thickness of the cellulose film 23 is preferably 0.5 to 2 μm.

In the artificial cell wall 2 of the present embodiment, the pore diameter of the through-hole 22 of the substrate 21 and the film thickness of the cellulose film 23 are adjusted in the above ranges, so that the tomato nonpathogenic fungi do not penetrate the cellulose film 23 in many cases, and it is considered that some of the tomato nonpathogenic fungi can be excluded at this stage. On the other hand, the tomato pathogenic fungi targeted in this embodiment selectively appear on the back surface of the substrate.

The thickness of the substrate 21 is not particularly limited, but is preferably about 5 to 150 μm, for example.

As shown in fig. 1, a test sample liquid is supplied into the test sample liquid loading portion 3. When the test sample liquid contains a tomato pathogenic fungus, the tomato pathogenic fungus is present on the surface of the substrate 21 on the front surface side.

In the present embodiment, the test sample solution is a solution (bacteria-recovered solution) containing fungi mainly adhering to tomato leaves, and is not particularly limited as long as it has a possibility of containing the target pathogenic fungi. Examples of the liquid used for washing tomato leaves and the liquid impregnated with tomato leaves include water, physiological saline, and surfactant-containing water (Tween 800.01 to 0.1%).

The tomato pathogenic fungi targeted by the detection device of the present embodiment is preferably at least one selected from the group consisting of Botrytis cinerea (Botrytis cinerea), Pseudocercospora solanacearum (Pseudocercospora fuligena), and chrysosporium fulvum (Passalora fulva).

The detection device of the present embodiment preferably does not detect fungi that may be present in tomato leaves but are non-pathogenic fungi in tomato, such as fungi of the genus hypocrea (bismonogouia), Penicillium (Penicillium), Phoma (Phoma), and Trichoderma (Trichoderma). More specifically, the tomato nonpathogenic fungus is xylaria marinus (bisgonococcus maritima), penicillium olgensis (penicillium personatum), Phoma polycephalum (Phoma multirosata) or Trichoderma asperellum (Trichoderma asperellum).

In addition, in the present specification, the term "tomato pathogenic" means having pathogenicity to tomato. The term "tomato nonpathogenic" means that there is no pathogenicity to tomato. Even if a fungus is pathogenic, it is "non-pathogenic" as long as it is not pathogenic to tomato. In other words, a fungus is "non-pathogenic" to tomatoes as long as the fungus does not adversely affect tomatoes. The term "non-pathogenic" of tomato is intended to encompass the prefix "non-modified" tomato "and the prefix" non-modified "pathogenic".

In the detection apparatus of the present embodiment, a culture medium 5 is added to a culture medium reservoir 4 provided below the artificial cell wall 2. The culture solution 5 is not particularly limited as long as it can culture fungi, and a general culture medium or culture solution can be used. For example, a potato glucose medium, a saxashi glucose medium, or the like can be used as a general fungal culture medium. In order to accelerate the culture of fungi, a culture solution may be added not only to the culture solution storage unit 4 but also to the test sample solution.

In this embodiment, it is important that the pH of the culture solution 5 is 5 to 5.5 and that the culture solution 5 contains 15 to 30mM of a citrate buffer. With such a configuration, in the detection of pathogenic fungi, interfering bacteria (tomato nonpathogenic fungi) that show false positives can be excluded, and the target tomato pathogenic fungi can be selectively detected.

If the above-mentioned pH of the culture solution 5 is less than 5, or exceeds 5.5, it may not be possible to completely exclude tomato nonpathogenic fungi that interfere with the detection of tomato pathogenic fungi. Furthermore, if the above culture solution 5 contains a citrate buffer solution at a concentration of less than 15mM, it may not be possible to exclude all tomato nonpathogenic fungi that interfere with the detection of tomato pathogenic fungi. On the other hand, if the concentration of the above citrate buffer exceeds 50mM, part or all of the tomato pathogenic fungi that may be targeted may be excluded.

The citrate is not particularly limited, but is preferably a monovalent citrate salt, and more specifically, sodium citrate, potassium citrate, or the like.

Further, in the test sample liquid, EC (electric conductivity) is preferably about 2 to 4mS/cm in general.

The detection apparatus of the present embodiment detects the presence or absence of the tomato pathogenic fungi in the sample by observing the presence or absence of the tomato pathogenic fungi on the back surface of the cellulose membrane 23 of the artificial cell wall 2 after a certain period of culture has elapsed. The observation method is not particularly limited, and for example, as shown in fig. 3, a microscope 6 may be placed below the artificial cell wall 2, and optical observation may be performed by the microscope 6.

The period of culturing the fungus is not particularly limited, but is preferably 72 hours or more. The culture temperature is preferably about 20 to 28 ℃.

Further, the present invention comprises a method for detecting a tomato pathogenic fungus, comprising the steps of: the tomato pathogenic fungi were selectively detected using the detection device as described above.

The method for detecting a tomato pathogenic fungus according to the present embodiment is not particularly limited as long as the detection apparatus is used, and includes, for example, the following steps: a step of introducing a test sample liquid into a test sample liquid introduction portion 3 of the detection device; a step (step of culturing) in which the test sample solution is allowed to stand in the detection device; a step of observing the back surface of the artificial cell wall 2 (cellulose membrane 23) of the detection device after standing; and a step of judging that the test sample liquid contains a tomato pathogenic fungus when a fungus is observed on the back surface of the cellulose film 23.

The present specification discloses various techniques as described above, but the main techniques thereof are summarized below.

The device for detecting a tomato pathogenic fungus according to one aspect of the present invention is characterized by comprising: artificial cell walls; a test sample liquid input part arranged on the upper part of the artificial cell wall; and a culture medium storage part arranged at the lower part of the artificial cell wall, wherein the culture medium storage part contains 15-30 mM citrate buffer solution, and the pH value of the culture medium is 5-5.5.

With such a configuration, it is possible to provide an apparatus and a method capable of selectively detecting tomato pathogenic fungi easily and safely.

Further, in the detection device, it is preferable that the artificial cell wall includes at least a substrate having a through hole with a pore diameter of 2 to 7 μm and a thickness of 5 to 150 μm, and a cellulose film having a thickness of 0.5 to 2 μm provided on one surface of the substrate. This can be considered to more reliably achieve the above-described effects.

In the detection device, the citrate is preferably at least one selected from sodium citrate and potassium citrate. This can be considered to more reliably achieve the above-described effects.

Further, in the above-mentioned detection apparatus, the tomato pathogenic fungus to be detected is preferably at least one selected from the group consisting of Botrytis cinerea (Botrytis cinerea), Pseudocercospora solanacearum (Pseudocercospora fuligena), and Botrytis fulvidraco (Passalora fulva). In such a case, it is considered that the above-described effects can be more exhibited.

Preferably, the detection device does not detect any fungi of the genera bistrophia (bisgonococcus), Penicillium (Penicillium), Phoma (Phoma), and Trichoderma (Trichoderma) which may be present in tomato leaves but are non-pathogenic fungi of tomato. In such a case, it is considered that the above-described effects can be more exhibited.

The tomato nonpathogenic fungi are more preferably xylaria marinus (bisgonococcus maritima), Penicillium olgensis (Penicillium olsonii), Phoma polycephalum (Phoma polyrotata) or Trichoderma asperellum (Trichoderma asperellum).

Another aspect of the present invention relates to a method for detecting a tomato pathogenic fungus, comprising the steps of: using the above detection apparatus, tomato pathogenic fungi were selectively detected.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited thereto.