阅读说明：本技术 番茄病原性真菌的检测装置和使用该装置的检测方法 (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：本公开提供简易且确切的番茄病原性真菌的选择性检测装置和检测方法。本公开的番茄病原性真菌的检测装置的特征在于,具有人工细胞壁、在所述人工细胞壁的上部设置的试验试样液投放部、和在所述人工细胞壁的下部设置的培养液存积部,在所述试验试样液投放部,试验试样液包含50～70mM的柠檬酸盐缓冲液,并且所述试验试样液的pH为5～5.5。(The present disclosure provides a simple and precise selective detection apparatus and detection method of tomato pathogenic fungi. The disclosed device for detecting a tomato pathogenic fungus is characterized by comprising an artificial cell wall, a test sample solution application part provided on the upper part of the artificial cell wall, and a culture solution storage part provided on the lower part of the artificial cell wall, wherein the test sample solution contains a citrate buffer solution of 50-70 mM in the test sample solution application part, and the pH of the test sample solution is 5-5.5.)

1. A detection device for tomato pathogenic fungi is characterized in that,

comprising an artificial cell wall, a test sample solution application part provided on the upper part of the artificial cell wall, and a culture solution storage part provided on the lower part of the artificial cell wall,

the test sample solution feeding unit contains a 50 to 70mM citrate buffer solution, and the pH of the test sample solution is 5 to 5.5.

2. The apparatus for detecting a tomato pathogenic fungus according to claim 1, wherein the artificial cell wall has a through hole having a pore diameter of 2 to 7 μm, and at least comprises a substrate having 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.

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

4. The apparatus for detecting tomato pathogenic fungi according to any one of claims 1 to 3, wherein the tomato pathogenic fungi to be detected is at least one selected from Botrytis cinerea (Botrytis cinerea), Blastomyces solani (Pseudocercospora fuligena) and Blastomyces lycopersici (Passalora fulva).

5. The apparatus for detecting tomato pathogenic fungi according to any one of claims 1 to 4, wherein as tomato nonpathogenic fungi, any of the fungi of the genera Dictyocaulus (Biscogniauxia), Penicillium (Penicillium), Phoma (Phoma), and Trichoderma (Trichoderma) cannot be detected.

6. The apparatus for detecting tomato pathogenic fungi according to claim 5, wherein the tomato nonpathogenic fungi is Dictyocaulus hainanensis (Biscogniauxia maritima), Penicillium olgensis (Penicillium olsonii), Phoma multirhynchophylla (Phoma multirosetrata) or Trichoderma asperellum (Trichoderma asperellum).

7. A method for detecting tomato pathogenic fungi comprising: 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 same.

Background

The plant pathogenic fungi have properties related to plant invasion, such as forming adherent cells on the surface of a plant and then adhering the cells, finding pores such as stomatal tissues from which hyphae are inserted into the plant body, or secreting plant cell wall-degrading enzymes (cellulase and pectinase) from the hyphae.

Taking advantage of these features, for example, patent document 1 discloses a fungus metering method using a microporous membrane support. Further, non-patent document 1 discloses a PET (polyethylene terephthalate) film in which pseudohyphae of 1 species of Phytophthora sojae (Phytophthora sojae), which is a plant pathogenic oomycete, grow horizontally as if they are going to invade downward and penetrate through a hole having a diameter of 3 μm.

In addition, the present inventors have already proposed a method for determining a plant pathogenic oomycete in view of the above properties (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 is said to cause 3 or more species of pathogenic fungi, including Botrytis cinerea (Botrytis cinerea), Botrytis cinerea (Pseudocercospora fuligena), and Botrytis cinerea (Passalora fulva). Among these pathogenic fungi, Botrytis cinerea (Botrytis cinerea) is a dominant parasite and infects other plants, but the germs of the rhizopus oryzae (Pseudocercospora fuligena) and the germs of the phyllorum (Passalora fulva) are only examples of infection of tomato, and are pathogenic fungi having high plant specificity. The present inventors considered that it is necessary to examine tomato pathogenic fungi at a stage where it is not known what kind of fungi are actually present on tomato leaves, that is, at a stage before onset of disease, and studied them.

On the other hand, the technique of sorting pathogenic fungi using artificial cell walls, which is the basic technique for selective fungi detection used by the present inventors as described in patent document 2, is possible to detect not only pathogenic fungi of tomato as long as it is a plant pathogenic fungus. That is, it is assumed that if other plant pathogenic fungi are attached to the leaves of tomato, it is possible to detect the fungi as tomato pathogenic fungi. Tomato cultivation is mostly carried out not by seeds but by seedlings, and in a nursery field, the possibility of adhesion of phytopathogenic fungi other than tomato pathogenic fungi to tomato seedlings cannot be denied due to mixed cultivation with other plants, use of various plants to and fro in the same facility, and the like. In addition, in an actual cultivation site, as in the case of the above-described nursery site, there is a possibility that pathogenic fungi of plants other than tomatoes may adhere to tomato seedlings in cultivation facilities such as vinyl houses. If this is not the case, it is possible that plant pathogenic fungi other than the tomato pathogenic fungi would suggest false positives in sorting techniques for pathogenic fungi using artificial cell walls, and sometimes useless application, seedling renewal, and the like would have a significant adverse effect on cultivation.

The possibility of occurrence of this false positive was investigated, and as a result, the fungus was actually encountered as a fungus other than the tomato pathogenic fungus, and a false positive fungus was suggested in the detection method using an artificial cell wall under investigation. These are 4 species of the genus Dictyocaulus (Biscogniauxia) fungi, Penicillium (Penicillium) fungi, Phoma (Phoma) fungi, Trichoderma (Trichoderma) fungi, and they need to be investigated in order to be undetectable.

The present invention has been made in view of such facts, and an object thereof is to provide a selective detection apparatus and a detection method for tomato pathogenic fungi.

Means for solving the problems

The present inventors have conducted extensive studies and, as a result, have found that the above problems can be solved by a detection device having the following configuration, and further studies have been made based on the findings, thereby completing the present invention.

That is, the present invention relates to an apparatus for detecting a tomato pathogenic fungus, comprising an artificial cell wall, a test sample solution application part provided on an upper part of the artificial cell wall, and a culture solution storage part provided on a lower part of the artificial cell wall, wherein the test sample solution contains 50 to 70mM of a citrate buffer solution in the test sample solution application part, and the test sample solution has a pH of 5 to 5.5.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, it is possible to simply and safely provide a device and a method for selectively detecting tomato pathogenic fungi. The present invention is useful for industrial use because it enables the presence of bacteria to be examined at the pre-disease stage by tomato pathogenic fungi, and in this case, false positive cues caused by plant pathogenic fungi other than tomato can be avoided.

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 the artificial cell wall showing the state where Botrytis cinerea (Botrytis cinerea) penetrates the artificial cell wall in example 1.

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

Fig. 6 is a graph showing the results of example 1.

Fig. 7 is a graph showing the results of comparative example 2.

Fig. 8 is a graph showing the results of comparative example 3.

Fig. 9 is a graph showing the results of comparative example 4.

Fig. 10 is a graph showing the results of comparative example 5.

Fig. 11 is a graph showing the results of comparative example 6.

Detailed Description

The embodiments according to the present invention will be specifically described below, but the present invention is not limited to these.

As shown in fig. 1, the apparatus 1 for detecting a tomato pathogenic fungus according to the present embodiment is characterized by comprising an artificial cell wall 2, a test sample solution application part 3 provided on the upper part of the artificial cell wall 2, and a culture solution storage part 4 provided on the lower part of the artificial cell wall 2, wherein the test sample solution 5 contains a citrate buffer solution of 50 to 70mM in the test sample solution application part 3, and the test sample solution 5 has a pH of 5 to 5.5.

The test sample liquid pouring section 3 is a container for pouring the test sample liquid 5, and it is desirable that the container has a flange at the upper end. The bottom surface of the test sample solution application part 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 targeted tomato pathogenic fungi.

The through-hole 22 penetrates from the front side surface to the back side surface 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 making the pore size in the range, the pathogenic fungi as a target can be detected more accurately and selectively.

In addition, in order to more accurately selectively detect the pathogenic fungi as a target, 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, it is considered that many of the tomato nonpathogenic fungi do not penetrate the cellulose film 23 by adjusting the pore diameter of the through-hole 22 of the substrate 21 and the film thickness of the cellulose film 23 to the above ranges, and therefore, part of the tomato nonpathogenic fungi can be excluded at this stage. On the other hand, the tomato pathogenic fungi targeted in the present 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 as an example.

As shown in fig. 1, a test sample liquid 5 is supplied into the test sample liquid loading portion 3. In this way, when the test sample solution 5 contains the tomato pathogenic fungi, the tomato pathogenic fungi are present on the surface of the substrate 21 on the front side.

In the present embodiment, the test sample liquid 5 is a liquid (bacteria-recovered liquid) containing fungi mainly adhering to tomato leaves, and is not particularly limited as long as it is a liquid that may contain a target pathogenic fungus. For example, the liquid after use for washing the leaves of tomatoes, the liquid for soaking the leaves of tomatoes.

In this embodiment, it is important that the test sample solution 5 has a pH of 5 to 5.5 and the test sample solution 5 contains 50 to 70mM of a citrate buffer. With such a configuration, it is possible to exclude an interfering bacterium (tomato nonpathogenic fungus) showing false positive in the detection of pathogenic fungi, and to selectively detect a target tomato pathogenic fungus.

If the pH of the test sample solution 5 is less than 5, or exceeds 5.5, tomato nonpathogenic fungi that interfere with the detection of tomato pathogenic fungi may not be completely excluded. In addition, if the test sample solution contains a citrate buffer solution at a concentration of less than 50mM, tomato nonpathogenic fungi that interfere with the detection of tomato pathogenic fungi may not be completely excluded. On the other hand, if the concentration of the citrate buffer exceeds 70mM, part of the tomato pathogenic fungi that may be linked as targets is also excluded.

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

Further, in the test sample liquid 5, EC (electric conductivity) is preferably about 7 to 15mS/cm in general.

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

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

In addition, in the present specification, the term "tomato pathogenic" means having a pathogenic effect on tomatoes. The term "tomato nonpathogenic" means that there is no pathogenicity to tomato. Even if the fungus is pathogenic, it is "non-pathogenic" if 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 prefix "non-modified" tomato "is included in the term" non-pathogenic tomato "and the prefix" non-modified "pathogenic".

In the detection apparatus of the present embodiment, a culture medium is added to the culture medium reservoir 4 provided below the artificial cell wall 2. The culture medium is not particularly limited as long as it is a culture medium capable of culturing fungi, and a general medium or culture medium can be used. For example, a potato glucose medium, a Sabouraud's glucose medium, or the like, which is a general culture medium for fungi, can be used. In order to accelerate the culture of fungi, a culture medium may be added to the test sample solution 5 in addition to the culture medium reservoir 4.

In the detection apparatus of the present embodiment, after a certain period of culture, the presence or absence of the tomato pathogenic fungus in the sample is detected by observing whether or not the tomato pathogenic fungus appears on the back surface of the cellulose membrane 23 of the artificial cell wall 2. The observation means 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, the method comprising: 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 above-described detection apparatus is used, and includes, for example: a step of charging a test sample liquid in a test sample liquid charging section 3 of the detection device; a step (culture step) of allowing the test sample solution to stand in the detection device; a step of observing the back surface of the artificial cell wall 2 (cellulose film 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 the techniques of the various modes described above, and the main techniques thereof are summarized below.

The apparatus for detecting a tomato pathogenic fungus according to one aspect of the present invention includes an artificial cell wall, a test sample solution application unit provided above the artificial cell wall, and a culture solution storage unit provided below the artificial cell wall, wherein the test sample solution contains 50 to 70mM citrate buffer in the test sample solution application unit, and the test sample solution has a pH of 5 to 5.5.

With such a configuration, it is possible to provide a device and a method for selectively detecting tomato pathogenic fungi in a simple and safe manner.

Further, in the detection device, it is preferable that the artificial cell wall has a through hole having a pore diameter of 2 to 7 μm, and includes at least a substrate having 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 is 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 is considered to more reliably achieve the above-described effects.

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

Preferably, the detection device does not detect any fungi of the genera biscoccobacillus (biscogranhia), 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 further exhibited.

More preferably, the tomato nonpathogenic fungus is xylaria marinus (bisgonococcus maritima), Penicillium olgensis (Penicillium olsonii), Phoma polycephalum (Phoma multiratata) or Trichoderma asperellum (Trichoderma asperellum).

The method for detecting a tomato pathogenic fungus according to a further aspect of the present invention is characterized by comprising the step of selectively detecting a tomato pathogenic fungus using the above-described detection device.

The present invention will be described more specifically with reference to examples, but the scope of the present invention is not limited to these examples.