Patent Application
20240344012
The present invention relates to a tomato pathogenic fungus detecting apparatus and a selective detecting method using the tomato pathogenic fungus detecting apparatus.
Phytopathogenic fungi have, as properties involving invasiveness into plants, features of forming an appressorium on a surface of a plant for attachment, and then searching for a pore, such as a stoma tissue, through which a hypha is extended into a plant body or secreting a plant cell wall degrading enzyme (a cellulase or a pectinase) from a hypha.
Making use of these features, for example, PTL 1 discloses a method for measuring an amount of a fungus using a microporous membrane support. NPL 1 discloses facts that a pseudohypha of Phytophthora sojae as one type of phytopathogenic oomycete grows downward as if attempting to go deep, rather than growing horizontally and that the pseudohypha penetrates a PET (polyethylene terephthalate) membrane having a pore of 3 μm.
Focusing on this property, the inventors of the present invention have already proposed a method for determining a phytopathogenic oomycete (PTL 2).
A target plant in the present invention, tomatoes are highly prone to disease caused by fungi, and pathogenic fungi that cause the disease are said to be dominated by three types of fungi, a tomato gray mold fungus (Botrytis cinerea, a tomato sooty mold fungus (Pseudocercospora fuligena), and a tomato leaf mold fungus (Passalora fulva). With regard to these pathogenic fungi, the gray mold fungus (Botrytis cinerea) is plurivorous and is infectious also to other plants, but the sooty mold fungus (Pseudocercospora fuligena) and the leaf mold fungus (Passalora fulva) present examples of infection only to tomatoes and are pathogenic fungi having high plant specificity. With regard to these pathogenic fungi that are said to have specificity to tomatoes, the inventors of the present invention have considered that it is necessary to detect the tomato pathogenic fungi in a stage where it is unclear what type of fungus is present on actual tomato leaves, that is, in a stage before pathogenesis, and have studied on this subject.
On the other hand, a pathogenic fungus selection technique using an artificial cell wall that is a basic selective fungus detection technique described in PTL 2 and used by the inventors of the present invention probably detects not only the tomato pathogenic fungi but also any phytopathogenic fungi. That is, if a fungus pathogenic to another plant is attached to a tomato leaf, the pathogenic fungus selection technique may possibly detect the fungus as a tomato pathogenic fungus. Tomato cultivation is mostly performed not by seeds but by seedlings, and a possibility cannot be ruled out of attaching a phytopathogenic fungus other than the tomato pathogenic fungi to a tomato seedling in a nursery garden, due to mixed cultivation with other plants and sharing of tools among a plurality of plants in a same facility. Similarly to the nursery garden described above, there is a possibility of attaching a fungus pathogenic to a plant other than tomatoes to a tomato seedling also in an actual cultivation site and a cultivation facility such as a vinyl greenhouse. If such attachment is left untreated, the phytopathogenic fungus other than the tomato pathogenic fungi possibly leads to presentation of a false positive in the pathogenic fungus selection technique using an artificial cell wall, to sometimes cause severe inconvenience in cultivation, such as useless chemical application or renewal of seedlings.
As a result of a research and investigation on this possibility of generating a false positive, the inventors of the present invention have actually encountered fungi that are other than the tomato pathogenic fungi and that lead to presentation of a false positive in a studying detecting method using an artificial cell wall. The fungi are four types of fungi, a Biscogniauxia genus fungus, a Penicillium genus fungus, a Phoma genus fungus, and a Trichoderma genus fungus, and a study on a method that does not detect these fungi has been required.
The present invention has been made in view of such actual circumstances, and an object of the present invention is to provide an apparatus and a method for selectively detecting a tomato pathogenic fungus.
As a result of an earnest study, the inventors of the present invention and others have found that a detecting apparatus configured as below can solve the above problem and further conducted the study based on the finding to complete the present invention.
That is, a tomato pathogenic fungus detecting apparatus related to one aspect of the present invention is characterized by including an artificial cell wall, a test sample solution inlet provided above the artificial cell wall, and a culture solution storage part provided under the artificial cell wall, wherein a culture solution contains a 15 mM to 30 mM buffer solution of a citrate salt in the culture solution storage part, and the culture solution has a pH of 5 to 5.5.
The present invention is capable of providing an apparatus and a method that are capable of selectively detecting a tomato pathogenic fungus simply and safely. The present invention enables presence of a tomato pathogenic fungus to be detected in a stage before pathogenesis caused by the fungus and enables presentation of a false positive attributed to a phytopathogenic fungus other than tomato pathogenic fungi to be avoided in the detection, so that the present invention is very useful for industrial use.
Hereinafter, an embodiment according to the present invention is specifically described. The present invention, however, is not limited to this embodiment.
Tomato pathogenic fungus detecting apparatus 1 according to the present embodiment is characterized by including, as illustrated in
Test sample solution inlet 3 is a vessel into which a test sample solution is charged, and the vessel desirably includes a flange on an upper end of the vessel. A bottom surface of test sample solution inlet 3 is formed of artificial cell wall 2.
Artificial cell wall 2 preferably includes, as illustrated in
Through hole 22 penetrates from a front-end surface to a back-end surface of substrate 21, and the through hole preferably has a hole diameter of 2 μm to 7 μm (sectional area of 4.5 μm2 to 38.5 μm2). The through hole having a hole diameter in the above range enables a target pathogenic fungus to be selectively detected more securely.
Further, in order to selectively detect a target pathogenic fungus more securely, a thickness of cellulose membrane 23 is also preferably adjusted. Specifically, cellulose membrane 23 preferably has a thickness of 0.5 μm to 2 μm.
It is considered that in artificial cell wall 2 of the present embodiment, adjusting the hole diameter of through hole 22 in substrate 21 and the membrane thickness of cellulose membrane 23 in the above range does not allow many of tomato non-pathogenic fungi to penetrate cellulose membrane 23, so that a part of the tomato non-pathogenic fungi can be excluded in this stage. On the other hand, a tomato pathogenic fungus targeted in the present embodiment selectively appears on the back surface of the substrate.
A thickness of substrate 21 is not particularly limited, but is preferably about 5 μm to 150 μm as one example.
As illustrated in
In the present embodiment, the test sample solution is mainly a solution (fungus collection solution) containing a fungus attached to a tomato leaf, and is not particularly limited as long as the test sample solution is a liquid probably containing a target pathogenic fungus. The test sample solution is, for example, a liquid having been used to wash a tomato leaf or a liquid in which a tomato leaf has been immersed, and examples include water, saline, surfactant-blended water (Tween80 0.01 to 0.1%).
The tomato pathogenic fungus targeted by the detecting apparatus of the present embodiment is preferably at least one selected from a tomato gray mold fungus (Botrytis cinerea), a tomato sooty mold fungus (Pseudocercospora fuligena), or a tomato leaf mold fungus (Passalora fulva).
The detecting apparatus of the present embodiment preferably does not detect fungi that are sometimes present on tomato leaves but are tomato non-pathogenic fungi, e.g., a Biscogniauxia genus fungus, a Penicillium genus fungus, a Phoma genus fungus, and a Trichoderma genus fungus. More specifically, the tomato non-pathogenic fungi are Biscogniauxia maritima, Penicillium olsonii, Phoma multirostrata, and Trichoderma asperellum.
In the present specification, the term “tomato pathogenic” means being pathogenic to tomatoes. The term “tomato non-pathogenic” means being non-pathogenic to tomatoes. A fungus that is pathogenic but is not pathogenic to tomatoes is “tomato non-phytopathogenic”. In other words, a fungus that does not adversely affect tomatoes is “tomato non-pathogenic”. The prefix “non-” included in the term “tomato non-pathogenic” does not modify the “tomato”, but modifies the “pathogenic”.
In the detecting apparatus of the present embodiment, culture solution 5 is put in culture solution storage part 4 provided under artificial cell wall 2. Culture solution 5 is not particularly limited as long as the culture solution is capable of culturing a fungus, and a general culture medium or culture solution is usable. For example, general culture media for culturing a fungus, i.e., a potato dextrose culture medium, Sabouraud dextrose culture medium, and the like are usable. In order to accelerate the culture of a fungus, a culture solution may be added not only to culture solution storage part 4 but also to the test sample solution.
In the present embodiment, it is important that culture solution 5 has a pH of 5 to 5.5 and culture solution 5 contains a 15 mM to 30 mM buffer solution of a citrate salt. These configurations enable obstructive fungi (tomato non-pathogenic fungi) that lead to a false positive in pathogenic fungus detection to be excluded and thus enable a target tomato pathogenic fungus to be selectively detected.
Culture solution 5 having a pH of less than 5 or more than 5.5 may possibly make it impossible to completely exclude the tomato non-pathogenic fungi that obstruct the tomato pathogenic fungus detection. Culture solution 5 that contains the buffer solution having a concentration of the citrate salt of less than 15 mM may possibly make it impossible to completely exclude the tomato non-pathogenic fungi that obstruct the tomato pathogenic fungus detection. On the other hand, the culture solution that contains the buffer solution having a concentration of the citrate salt of more than 50 mM may possibly also exclude a part or all of targeted tomato pathogenic fungi.
The citrate salt is not particularly limited, but is preferably a monovalent citrate salt, and is preferably sodium citrate, potassium citrate, or the like more specifically. Further, the test sample solution normally preferably has an EC (electric conductivity) of about 2 mS/cm to 4 mS/cm.
The detecting apparatus of the present embodiment detects presence or absence of a tomato pathogenic fungus in a sample by observing, after a lapse of a certain culture period, whether or not the tomato pathogenic fungus has appeared on the back surface of cellulose membrane 23 of artificial cell wall 2. An observation means is not particularly limited, and optical observation can be conducted with microscope 6 by disposing microscope 6 under artificial cell wall 2 as illustrated in
The culture period of a fungus is not particularly limited and is preferably not less than 72 hours. A culture temperature is preferably set at about 20° C. to 28° C.
The present invention further encompasses a tomato pathogenic fungus detecting method including selectively detecting a tomato pathogenic fungus using the detecting apparatus described above.
The tomato pathogenic fungus detecting method of the present embodiment is not particularly limited in terms of steps other than using the detecting apparatus described above, and includes the steps of, for example: charging a test sample solution into test sample solution inlet 3 of the detecting apparatus; leaving the test sample solution to stand still in the detecting apparatus (culturing); observing a back surface of artificial cell wall 2 (cellulose membrane 23) in the detecting apparatus after the leaving; and determining that the test sample solution contains a tomato pathogenic fungus when the fungus is observed on the back surface of cellulose membrane 23.
The present specification discloses various forms of techniques as described above, from among which main techniques are summarized as follows.
A tomato pathogenic fungus detecting apparatus according to one aspect of the present invention is characterized by including an artificial cell wall, a test sample solution inlet provided above the artificial cell wall, and a culture solution storage part provided under the artificial cell wall, wherein a culture solution contains a 15 mM to 30 mM buffer solution of a citrate salt in the culture solution storage part, and the culture solution has a pH of 5 to 5.5.
These configurations enable provision of an apparatus and a method that are capable of selectively detecting a tomato pathogenic fungus simply and safely.
In the detecting apparatus, it is preferable that the artificial cell wall includes at least a substrate that has a through hole with a hole diameter of 2 μm to 7 μm and has a thickness of 5 μm to 150 μm, and a cellulose membrane that is provided on one surface of the substrate and has a thickness of 0.5 μm to 2 μm. These configurations are considered to enable the effects described above to be more securely obtained.
In the detecting apparatus, the citrate salt is preferably at least one selected from sodium citrate or potassium citrate. These configurations are considered to enable the effects described above to be more securely obtained.
In the detecting apparatus, a tomato pathogenic fungus to be a detection target is preferably at least one selected from a tomato gray mold fungus (Botrytis cinerea), a tomato sooty mold fungus (Pseudocercospora fuligena), or a tomato leaf mold fungus (Passalora fulva). This setting is considered to enable the effects described above to be more exhibited.
The detecting apparatus preferably does not detect fungi that are sometimes present on tomato leaves but are tomato non-pathogenic fungi, namely a Biscogniauxia genus fungus, a Penicillium genus fungus, a Phoma genus fungus, and a Trichoderma genus fungus. This setting is considered to enable the effects described above to be more exhibited.
The tomato non-pathogenic fungi are more preferably Biscogniauxia maritima, Penicillium olsonii, Phoma multirostrata, and Trichoderma asperellum.
A tomato pathogenic fungus detecting method according to another aspect of the present invention is characterized by selectively detecting a tomato pathogenic fungus using the detecting apparatus.
Hereinafter, the present invention is described further specifically by way of an example. A scope of the present invention, however, is not limited to this example.
(Culture of Botrytis cinerea)
Botrytis cinerea, which is one of tomato pathogenic fungi and a pathogenic fungus of tomato gray mold disease was inoculated into a potato dextrose agar culture medium (Difco™ Potato Dextrose Agar). Next, the culture medium was left to stand still at a temperature of 25 degrees Celsius for one week. Botrytis cinerea was given by associate professor Shimizu belonging to Faculty of Applied Biological Sciences, Gifu University. Thereafter, the Botrytis cinerea-cultured potato dextrose agar culture medium in which hyphae sufficiently grew was left under irradiation with black light for not less than four days and left in a room-temperature environment for not less than two weeks to promote spore formation. Several ml of sterile pure water was dropped to the treated Botrytis cinerea-cultured potato dextrose agar culture medium, and surfaces of the hyphae were rubbed with a platinum loop, an ink brush, or the like to give a crushed hypha and spore mixed suspension.
(Culture of Pseudocercospora fuligena)
Pseudocercospora fuligena, which is one of tomato pathogenic fungi and a pathogenic fungus of tomato sooty mold disease was inoculated into a potato dextrose agar culture medium. Next, the culture medium was left to stand still at a temperature of 28 degrees Celsius for one week. Pseudocercospora fuligena was gotten from The Genetic Resources Center, NARO (the National Agriculture and Food Research Organization) (MAFF No. 306728). Thereafter, hyphae of Pseudocercospora fuligena were transplanted from the potato dextrose agar culture medium to a burdock powder agar culture medium, and further left to stand still at a temperature of 28 degrees Celsius for one to two weeks. After sufficiently growing again, the hyphae was subjected to mechanical stress such as rubbing surfaces of the hyphae with a platinum loop, an ink brush, or the like, thereafter left under irradiation with black light for not less than four days, and then left in a room-temperature environment for not less than two weeks to promote spore formation again. Several ml of sterile pure water was dropped to the treated Pseudocercospora fuligena-cultured burdock powder agar culture medium, and surfaces of the hyphae were rubbed with a platinum loop, an ink brush, or the like to give a crushed hypha and spore mixed suspension.
(Culture of Passalora fulva)
Passalora fulva, which is one of tomato pathogenic fungi and a pathogenic fungus of tomato leaf mold disease was inoculated into a potato dextrose agar culture medium. Next, the culture medium was left to stand still at a temperature of 23 degrees Celsius for one to two weeks. Passalora fulva was gotten from The Genetic Resources Center, NARO (the National Agriculture and Food Research Organization) (MAFF No. 726744). Thereafter, several ml of sterile pure water was dropped to the Passalora fulva-cultured potato dextrose agar culture medium in which hyphae sufficiently grew, and surfaces of the hyphae were rubbed with a platinum loop, an ink brush, or the like to give a crushed hypha and spore mixed suspension.
(Culture of Biscogniauxia maritima, Penicillium olsonii, Phoma multirostrata, and Trichoderma asperellum)
Biscogniauxia maritima, Penicillium olsonii, Phoma multirostrata, and Trichoderma asperellum that were not tomato pathogenic fungi but were present on tomato leaves were collected from the tomato leaves, separated, and then inoculated into a potato dextrose agar culture medium. Separation sources, tomatoes were collected from a plurality of locations. A separation method was as follows. Collected several tomato leaves were charged into a clear resin vessel or a resin bag together with a fungus collection solution that consists of saline containing 0.1% of surfactant Tween 80 (SIGMA-ALDRICH), stirred for one minute to transfer fungi attached to the leaves to the fungus collection solution. The fungus collection solution was diluted and applied to a potato dextrose agar culture medium containing 100 mg/L of streptomycin sulfate (Wako Pure Chemical Industries, Ltd.) by a plate agar smear method. Then, fungi that emerged in culture at 25 degrees Celsius for several days were separated from a fungus colony. Identification of the fungi was commissioned to Japan Food Research Laboratories (general incorporated foundation), Tama Laboratory. After the isolation, Biscogniauxia maritima, Penicillium olsonii, Phoma multirostrata, and Trichoderma asperellum that were inoculated into potato dextrose agar culture media were left to stand still at a temperature of 25 degrees Celsius for one week. Thereafter, several ml of sterile pure water was dropped to the potato dextrose agar culture media for culturing these four types of fungi in which hyphae sufficiently grew or spores were sufficiently formed, and surfaces of the hyphae were rubbed with a platinum loop, an ink brush, or the like to give a crushed hypha and spore mixed suspension.
The artificial cell wall in the detecting apparatus was prepared as follows.
First, cellulose (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-methyl imidazolium chloride (manufactured by SIGMA-ALDRICH). The cellulose solution was heated to 60 degrees Celsius and next applied to a back surface of a vessel (Millipore, trade name: Millicell PISP 12R 48) including a polyethylene terephthalate film as a bottom surface by spin coating for 30 seconds at a rotation rate of 2000 rpm. The polyethylene terephthalate film functioned as substrate 21 of the artificial cell wall in
The vessel including the cellulose membrane formed on the back surface of the polyethylene terephthalate film as the bottom surface was left to stand still in ethanol for 12 hours at room temperature. Thus, 1-Butyl-3-methyl imidazolium chloride was replaced with ethanol and removed, and then the vessel was dried in a vacuum desiccator at the end. Thus, the artificial cell wall was obtained that was tested in the present example and comparative examples.
The vessel that was formed into the artificial cell wall and included the cellulose membrane on the back surface of the polyethylene terephthalate film (substrate) as the bottom surface was put on a culture medium vessel (culture solution storage part) to form a tomato pathogenic fungus detecting apparatus. As the culture medium vessel, a 24-well flat bottom culture plate (Corning Incorporated, trade name: 24 Well Cell Cluture Cluster Flat Bottom) was used, and a space between the culture medium vessel and the artificial cell wall-forming vessel was filled with 600 μL of a liquid culture medium (culture solution) so that the back surface of the artificial cell wall-forming vessel was in contact with the liquid culture medium. As the liquid culture medium, a diluted potato dextrose liquid culture medium (Difco™ Potato Dextrose Broth 2.4 g/L aqueous solution) was used.
The crushed hypha and spore mixed suspensions respectively containing 200 pieces of hyphae and spores of Botrytis cinerea, Pseudocercospora fuligena, Passalora fulva, Biscogniauxia maritima, Penicillium olsonii, Phoma multirostrata, and Trichoderma asperellum were separately added into the artificial cell wall-forming vessel, and sterile purified water was added to the vessel so that a total volume of each of the resultant crushed hypha and spore mixed suspensions and the sterile purified water became 200 μL. Thus, test sample solutions were obtained.
A sodium citrate buffer solution was added to the prepared culture solution in the detecting apparatus so that a concentration of the buffer solution was adjusted to 20 mM. After the addition of the sodium citrate buffer solution, the sodium citrate buffer solution-containing diluted potato dextrose liquid culture medium (culture solution) in the culture solution storage part had a pH of 5.3 and an EC of 3.1 mS/cm.
Then, the test sample solutions respectively containing the seven types of fungi were disposed in the detecting apparatus, which was left to stand still at a temperature of 25 degrees Celsius for 72 hours. Thereafter, a number of hyphae that penetrated the artificial cell wall and observed on the back surface of the artificial cell wall was counted by visual inspection via an optical microscope.
A test was performed similarly to in Example 1 except that no sodium citrate buffer solution was added to the culture solution and the diluted potato dextrose liquid culture medium was directly used.
A test was performed similarly to in Example 1 except that the sodium citrate buffer solution was added to the prepared culture solution in the detecting apparatus so that the concentration of the buffer solution was adjusted to 10 mM. The culture solution had a pH of 5.4 and an EC of 1.7 mS/cm.
A test was performed similarly to in Example 1 except that the sodium citrate buffer solution was added to the prepared culture solution in the detecting apparatus so that the concentration of the buffer solution was adjusted to 60 mM. The culture solution had a pH of 5.5 and an EC of 12 mS/cm.
In Comparative Example 1 (
Also in Comparative Example 2 (
In Comparative Example 3 (
In contrast,
A tomato pathogenic fungus detecting apparatus of the present disclosure is capable of selectively detecting a target tomato pathogenic fungus while excluding a tomato non-pathogenic fungus leading to a false positive. Therefore, the detecting apparatus of the present disclosure can be suitably utilized for removing a tomato pathogenic fungus that adversely affects tomatoes or for other purposes in technical fields such as agriculture involving tomatoes.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-098831 | May 2018 | JP | national |
This application is a Divisional Application of U.S. patent application Ser. No. 17/069,911, filed on Oct. 14, 2020, which is a Continuation Application of International Patent Application No. PCT/JP2019/015050, filed on Apr. 5, 2019, which claims the benefit of Japanese Patent Application No. 2018-098831, filed on May 23, 2018, the entire contents of each are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17069911 | Oct 2020 | US |
| Child | 18750849 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2019/015050 | Apr 2019 | WO |
| Child | 17069911 | US |
