Patent Grant
10526636
This application claims the benefit of foreign priority of Japanese patent application 2017-010910 filed on Jan. 25, 2017, the content of which is incorporated herein by reference.
The present invention relates to a method for determining whether or not a test sample contains a phytopathogenic fungus.
PTL1 discloses a method for counting the number of mold cells in a specimen by the culture for a short time and capable of accurately counting the cell number.
NPL1 discloses that pseudohyphae of Phytophthora sojae, which is one of phytopathogenic oomycetes, penetrates the PET membrane having 3-micrometer pores.
An object of the present invention is to provide a method for selectively determining whether or not a test sample contains at least one phytopathogenic fungus selected from the group consisting of Gibberella fujikuroi, Fusarium avenaceum, and Glomerella tucumanensis.
The present invention provides a method for determining whether or not a test sample contains at least one phytopathogenic fungus selected from the group consisting of Gibberella fujikuroi, Fusarium avenaceum, and Glomerella tucumanensis, the method comprising:
(a) putting the test sample on a front surface of a cellulose film;
wherein
the cellulose film has a thickness of not less than 0.5 micrometers and not more than 2 micrometers; and
the cellulose film has no through hole;
(b) leaving the test sample at rest for a predetermined time after the step (a);
(c) observing a back surface of the cellulose film after the step (b); and
(d) determining that the test sample contains the at least one phytopathogenic fungus selected from the group consisting of Gibberella fujikuroi, Fusarium avenaceum, and Glomerella tucumanensis, if a fungus which has penetrated the cellulose film is found on the back surface of the film in the step (c).
The present invention provides a method for selectively determining whether or not a test sample contains at least one phytopathogenic fungus selected from the group consisting of Gibberella fujikuroi, Fusarium avenaceum, and Glomerella tucumanensis.
First, a fungus will be described. Fungi are roughly divided into a phytopathogenic fungus and a non-phytopathogenic fungus. For example, the phytopathogenic fungus is a Gibberella genus, a Fusarium genus, or a Glomerella genus. An example of the phytopathogenic fungus is Gibberella fujikuroi, Fusarium avenaceum, or Glomerella tucumanensis. These phytopathogenic fungi cause root rot disease, blast, anthrax, or gray mold. These three phytopathogenic fungi kill the plant, especially, corn. An example of the non-phytopathogenic fungus is Pythium catenulatum, Pythium torulosum or Pythium inflatum.
The term “phytopathogenic” means to have pathogenicity to plants. The term “non-phytopathogenic” means not to have pathogenicity to plants. Even if a fungus has pathogenicity, however, if the fungus has no pathogenicity to plants, the fungus is non-phytopathogenic. In other words, if a fungus does not have adverse effects on plants, the fungus is non-phytopathogenic. The prefix “non-” included in the term “non-phytopathogenic” does not modify “phyto”. The prefix “non-” modifies “pathogenic”.
Hereinafter, the embodiment of the present invention will be described in more detail with reference to the drawings.
(Step (a))
In the step (a), a test sample is put on a front surface of a cellulose film having a thickness of not less than 0.5 micrometers and not more than 2 micrometers. The importance of the thickness of the cellulose film will be described later.
In particular, as shown in
As shown in
The test sample 200 is solid, liquid, or gaseous. It is desirable that the test sample 200 is solid or liquid. An example of the solid test sample 200 is soil or a crushed plant. Another example is an agricultural material such as vermiculite, rock wool or urethane. An example of the liquid test sample 200 is agricultural water, a solution used for hydroponic culture, a liquid used for washing a plant, a liquid extracted from a plant, a liquid used for washing an agricultural material, or a liquid used for washing clothing or shoes of a worker.
(Step (b))
In the step (b), the test sample 200 is left at rest for a certain incubation time after the step (a). An example of the incubation time is 24 hours. Hereinafter, the importance of the thickness of the cellulose film 104 will be described.
In the step (b), various fungi contained in the test sample 200 are grown. As demonstrated in the experiments which will be described later, if the following requirement (I) is satisfied, the at least one phytopathogenic fungus 202 selected from the group consisting of Gibberella fujikuroi, Fusarium avenaceum, and Glomerella tucumanensis grows up so as to penetrate the cellulose film 104, as shown in
Requirement (I): The cellulose film 104 has a thickness of not less than 0.5 micrometers and not more than 2 micrometers.
If the above requirement (I) is satisfied, a non-phytopathogenic fungus seldom penetrates the cellulose film 104. For this reason, the non-phytopathogenic fungus seldom appears on the back surface 104b of the cellulose film 104. In this way, the phytopathogenic fungus 202 appears on the back surface 104b selectively. In other words, the phytopathogenic fungus 202 appears outside of the container 100 selectively.
In case where the cellulose film 104 has a thickness of more than 2 micrometers, not only the non-phytopathogenic fungus but also the phytopathogenic fungus hardly penetrates the cellulose film 104 within the predetermined hours. Therefore, in case where the cellulose film 104 has a thickness of more than 2 micrometers, the selectivity is lost. In case where the cellulose film 104 has a thickness of less than 0.5 micrometers, not only the phytopathogenic fungus but also the non-phytopathogenic fungus penetrates the cellulose film 104 within the predetermined hours. Therefore, the selectivity is lost, in case where the cellulose film 104 has a thickness of less than 0.5 micrometers.
As shown in
Needless to say, as long as the cellulose film 104 is stretched taut, the substrate 170 is not required. In other words, if it is difficult to stretch the cellulose film 104 taut, the substrate 170 for supporting the cellulose film 104 is used. Unlike the substrate 170, note that the cellulose film 104 has no through hole.
A reference number 170a indicates the front surface of the substrate 170. As shown in
A culture medium may be supplied to the test sample 200 to accelerate the incubation of the fungus. In particular, a culture medium may be supplied to the inside of the container 100 containing the test sample 200. It is desirable that the culture medium is liquid. The culture medium may be supplied in the step (b). Alternatively, the culture medium may be supplied prior to the step (b). In other words, the culture medium may be supplied in the step (a). The culture medium may be supplied to the inside of the container 100 prior to the step (a).
Alternatively, after the first container 100 is stacked on the second container 300, the liquid culture medium 302 may be supplied between the back surface 104b of the cellulose film 104 and the bottom surface of the second container 300.
In place of the liquid culture medium 302, a viscous solid culture medium may also be used. As shown in
(Step (c))
In the step (c), the back surface 104b of the cellulose film 104 is observed after the step (b). It is desirable that the back surface 104b is observed using an optical microscope.
The phytopathogenic fungus 202 appears on the back surface 104b of the cellulose film 104, as described in the step (b). On the other hand, the non-phytopathogenic fungus does not appear on the back surface 104b of the cellulose film 104. In this way, in the present invention, the phytopathogenic fungus 202 appears on the back surface 104b of the cellulose film 104 selectively.
In other words, the phytopathogenic fungus 202 penetrates the cellulose film 104, whereas the non-phytopathogenic fungus does not penetrate the cellulose film 104. For this reason, the non-phytopathogenic fungus does not appear on the back surface 104b of the cellulose film 104. In this way, the phytopathogenic fungus 202 appears on the back surface 104b selectively. In other words, the phytopathogenic fungus 202 appears outside of the first container 100 selectively.
In the step (c), it is observed whether or not the phytopathogenic fungus 202 appears on the back surface 104b of the cellulose film 104.
In particular, the back surface 104b of the cellulose film 104 is observed as below.
As shown in
The liquid culture medium 302 and the solid culture medium 304 are removed from the second container 300. Then, a fluorescent agent having fungus combining ability is added to the inside of the second container 300. Hereinafter, such a fluorescent agent is referred to as “fungus fluorescent agent”. The reference number of the fungus fluorescent agent is 402. Then, as shown in
A part of the phytopathogenic fungus 202 which has appeared on the back surface 104b of the cellulose film 104 is dyed with the fungus fluorescent agent 402. Since the first container 100 is separated from the second container 300 by the cellulose film 104, the fungus fluorescent agent 402 does not spread into the first container 100. For this reason, the non-phytopathogenic fungus contained in the first container 100 is not dyed with the fungus fluorescent agent 402.
As shown in
(Step (d))
In the step (d), it is determined that the test sample contains at least one phytopathogenic fungus selected from the group consisting of Gibberella fujikuroi, Fusarium avenaceum, and Glomerella tucumanensis, if a fungus is found on the back surface 104b of the cellulose film 104 in the step (c). Needless to say, it is determined that the test sample does not contain at least one phytopathogenic fungus selected from the group consisting of Gibberella fujikuroi, Fusarium avenaceum, and Glomerella tucumanensis, if a fungus is not found on the back surface 104b of the cellulose film 104 in the step (c).
The present invention will be described in more detail with reference to the following examples.
(Incubation of Gibberella fujikuroi)
Gibberella fujikuroi, one of the phytopathogenic fungi, was inoculated on a potato dextrose agar culture medium. Then, the culture medium was left at rest at a temperature of 25 degrees Celsius for one week. Gibberella fujikuroi was given by the National Bio-Resource Center (National Institute of Genetics, Mishima-city, Shizuoka, Japan)
Then, a part including ends of hyphae was cut together with the culture medium at a size of 1 centimeter×1 centimeter. The cut part was immersed in pure water disposed on a 12-well plate. Each of the pure water has a volume of 1 milliliter.
The water contained in the 12-well plate was observed using an optical microscope. As a result, the present inventors confirmed that spores of Gibberella fujikuroi were released in the water disposed on the 12-well plate. In this way, an aqueous solution containing Gibberella fujikuroi was provided. Hereinafter, this aqueous solution is referred to as “phytopathogenic fungus aqueous solution”.
(Preparation of Culture Medium)
A potato dextrose culture medium having a volume of 650 microliters was added as the liquid culture medium 302 to the second container 300. In this way, the second container 300 containing the liquid culture medium 302 was prepared.
(Experiment 1)
The experiment 1 is composed of inventive examples 1A-1D, comparative examples 1E-1Z, and comparative examples 1AA-1AB.
The first container 100 shown in
First, cellulose (available from SIGMA-ALDRICH Co. LLC, trade name: Avicel PH-101) was dissolved in an ionic liquid to prepare a cellulose solution having a concentration of 2%. The ionic liquid was 1-butyl-3-methyl imidazolium chloride (available from SIGMA-ALDRICH Co. LLC).
The cellulose solution was warmed to 60 degrees Celsius. Then, the cellulose solution was applied by a spin coat method for thirty seconds at a rotation speed of 2,000 rpm onto a back surface of a container having a polyethylene terephthalate film on the bottom surface thereof (available from Merck KGaA, trade name: Millicell PISP 12R 48). The polyethylene terephthalate film served as the substrate 170. The polyethylene terephthalate film randomly had a plurality of through holes 172 each having a diameter of three micrometers. In this way, the cellulose film 104 having a thickness of 2.0 micrometers was formed on the back surface of the polyethylene terephthalate film.
The container was left at rest in ethanol at room temperature for 12 hours. In this way, 1-butyl-3-methyl imidazolium chloride was replaced with ethanol. In other words, 1-butyl-3-methyl imidazolium chloride was removed from the cellulose film 104.
Finally, the container was dried in a vacuum desiccator. In this way, the first container 100 shown in
Then, as shown in
The first container 100 was left at rest at a temperature of 25 degrees Celsius for 24 hours. In other words, in the inventive example 1A, the incubation time was 24 hours.
The number of the hyphae of Gibberella fujikuroi which appeared on the back surface 104b of the cellulose film 104 was counted visually with an optical microscope. The inventive example 1A was repeated fifteen times.
In the inventive example 1B, an experiment similar to the inventive example 1A was conducted, except that each of the through holes 172 had a diameter of 5 micrometers. The container having a bottom surface comprising the through holes each having a diameter of 5 micrometers was available from Merck KGaA as a trade name: Millicell PIMP 12R 48.
In the inventive example 1C, an experiment similar to the inventive example 1A was conducted, except that each of the through holes 172 had a diameter of 8 micrometers. The container having a bottom surface comprising the through holes each having a diameter of 8 micrometers was available from Merck KGaA as a trade name: Millicell PIEP 12R 48.
In the inventive example 1D, an experiment similar to the inventive example 1A was conducted, except that the cellulose solution had a concentration of 1.0%, except that the cellulose film 104 had a thickness of 0.5 micrometers, and that each of the through hole 172 had a diameter of 3 micrometers.
In the inventive example 1E, an experiment similar to the inventive example 1A was conducted, except that the cellulose film 104 had a thickness of 0.5 micrometers, and that each of the through holes 172 had a diameter of 5 micrometer.
In the inventive example 1F, an experiment similar to the inventive example 1A was conducted, except that the cellulose film 104 had a thickness of 0.5 micrometers, and that each of the through holes 172 had a diameter of 8 micrometers.
In the comparative example 1G, an experiment similar to the inventive example 1A was conducted, except that the cellulose film 104 had a thickness of 4.4 micrometers (namely, the cellulose solution had a concentration of 4.0%) and that each of the through holes 172 had a diameter of 1 micrometer.
In the comparative example 1H, an experiment similar to the inventive example 1A was conducted, except that the cellulose film 104 had a thickness of 4.4 micrometers (namely, the cellulose solution had a concentration of 4.0%) and that each of the through holes 172 had a diameter of 3 micrometers.
In the comparative example 1I, an experiment similar to the inventive example 1A was conducted, except that the cellulose film 104 had a thickness of 4.4 micrometers (namely, the cellulose solution had a concentration of 4.0%) and that each of the through holes 172 had a diameter of 5 micrometers.
In the comparative example 1J, an experiment similar to the inventive example 1A was conducted, except that the cellulose film 104 had a thickness of 4.4 micrometers (namely, the cellulose solution had a concentration of 4.0%) and that each of the through holes 172 had a diameter of 8 micrometers.
In the comparative example 1K, an experiment similar to the inventive example 1A was conducted, except that the cellulose film 104 had a thickness of 3.7 micrometers (namely, the cellulose solution had a concentration of 3.0%) and that each of the through holes 172 had a diameter of 1 micrometer.
In the comparative example 1L, an experiment similar to the inventive example 1A was conducted, except that the cellulose film 104 had a thickness of 3.7 micrometers (namely, the cellulose solution had a concentration of 3.0%) and that each of the through holes 172 had a diameter of 3 micrometers.
In the comparative example 1M, an experiment similar to the inventive example 1A was conducted, except that the cellulose film 104 had a thickness of 3.7 micrometers (namely, the cellulose solution had a concentration of 3.0%) and that each of the through holes 172 had a diameter of 5 micrometers.
In the comparative example 1N, an experiment similar to the inventive example 1A was conducted, except that the cellulose film 104 had a thickness of 3.7 micrometers (namely, the cellulose solution had a concentration of 3.0%) and that each of the through holes 172 had a diameter of 8 micrometers.
In the comparative examples 1O-1Z and 1AA-1AB, an experiment similar to the inventive example 1A was conducted in accordance with the disclosure shown in the following Table 1.
(Experiment 2)
In the experiment 2, a non-phytopathogenic fungus aqueous solution containing spores of Saccharomyces cerevisiae was used in place of the phytopathogenic fungus aqueous solution containing spores of Gibberella fujikuroi. Unlike Gibberella fujikuroi, Saccharomyces cerevisiae is one kind of non-phytopathogenic fungus. A non-phytopathogenic fungus aqueous solution containing spores of Saccharomyces cerevisiae was prepared similarly to the case of the phytopathogenic fungus aqueous solution containing spores of Gibberella fujikuroi. The experiment 2 is composed of comparative examples 2A-2Z and comparative examples 2AA-2AB.
In the comparative examples 2A-2Z and 2AA-2AB, an experiment similar to the inventive example 1A was conducted in accordance with the disclosure shown in the following Table 2.
(Experiment 3)
In the experiment 3, a phytopathogenic fungus aqueous solution containing spores of Fusarium avenaceum was used in place of the phytopathogenic fungus aqueous solution containing spores of Gibberella fujikuroi. Similarly to Gibberella fujikuroi, Fusarium avenaceum is one kind of phytopathogenic fungus. A phytopathogenic fungus aqueous solution containing spores of Fusarium avenaceum was prepared similarly to the case of the phytopathogenic fungus aqueous solution containing spores of Gibberella fujikuroi. The experiment 3 is composed of inventive examples 3A-3F, comparative examples 3G-3Z and comparative examples 3AA-3AB.
In the inventive examples 3A-3F, the comparative examples 3G-3Z and 3AA-3AB, an experiment similar to the inventive example 1A was conducted in accordance with the disclosure shown in the following Table 3.
(Experiment 4)
In the experiment 4, a phytopathogenic fungus aqueous solution containing spores of Glomerella tucumanensis was used in place of the phytopathogenic fungus aqueous solution containing spores of Gibberella fujikuroi. Similarly to Gibberella fujikuroi, Glomerella tucumanensis is one kind of phytopathogenic fungus. A phytopathogenic fungus aqueous solution containing spores of Glomerella tucumanensis was prepared similarly to the case of the phytopathogenic fungus aqueous solution containing spores of Gibberella fujikuroi. The experiment 4 is composed of inventive examples 4A-4F, comparative examples 4G-4Z and comparative examples 4AA-4AB.
In the inventive examples 4A-4F, the comparative examples 4G-4Z and 4AA-4AB, an experiment similar to the inventive example 1A was conducted in accordance with the disclosure shown in the following Table 4.
(Experiment 5)
In the experiment 5, a non-phytopathogenic fungus aqueous solution containing spores of Penicillium chysogeum was used in place of the phytopathogenic fungus aqueous solution containing spores of Gibberella fujikuroi. Unlike Gibberella fujikuroi, Penicillium chysogeum is one kind of non-phytopathogenic fungus. A non-phytopathogenic fungus aqueous solution containing spores of Penicillium chysogeum was prepared similarly to the case of the phytopathogenic fungus aqueous solution containing spores of Gibberella fujikuroi. The experiment 5 is composed of comparative examples 5A-5Z and comparative examples 5AA-5AB.
In the comparative example 5A-5Z and 5AA-5AB, an experiment similar to the inventive example 1A was conducted in accordance with the disclosure shown in the following Table 5.
The following Table 1-Table 5 show the number of the hyphae which penetrated the cellulose film 104 in the experiments.
Gibberella fujikuroi
Saccharomyces
cerevisiae
Fusarium avenaceum
Glomerella
tucumanensis
Penicillium chysogeum
The comparative examples 1Q, 1U, 3Q, 3U, 4Q, and 4U were not conducted actually.
As is clear from Table 1-Table 5, when the following requirement (I) is satisfied, the corn phytopathogenic fungus appears on the back surface 104b of the cellulose film 104 selectively. In other words, the corn phytopathogenic fungus 202 appears outside of the container 100 selectively.
Requirement (I): The cellulose film 104 has a thickness of not less than 0.5 micrometers and not more than 2 micrometers.
As demonstrated in the inventive example 1A, the number of hyphae which penetrated cellulose film 104 is 7.2 at a minimum, as long as the requirement (I) is satisfied. On the other hand, as long as the requirement (I) is satisfied, the non-phytopathogenic fungus hardly penetrates the cellulose film 104. As demonstrated in the comparative example 5F, the number of hyphae which penetrated cellulose film 104 is 6.8 at a maximum, when the requirement (I) is satisfied.
The present invention can be used to determine easily whether or not a test sample such as agricultural water or soil contains at least one phytopathogenic fungus selected from the group consisting of Gibberella fujikuroi, Fusarium avenaceum, and Glomerella tucumanensis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017-010910 | Jan 2017 | JP | national |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20160355863 | Uriu | Dec 2016 | A1 |
| 20160355864 | Uriu | Dec 2016 | A1 |
| Number | Date | Country |
|---|---|---|
| 2005-287337 | Oct 2005 | JP |
| Entry |
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| Cao et al. Ind. Eng. Chem. Res., 2006, 45:4193-4199. |
| Candau et al. Applied and Environmental Microbiology, 1991, 57(11):3378-3382. |
| Paul F. Morris et al., “Chemotropic and Contact Responses of Phytophthora sojae Hyphae to Soybean Isoflavonoids and Artificial Substrates”, Plant Physiol. (1998) 117:1171-1178, Aug. 1, 1998. |
| Number | Date | Country | |
|---|---|---|---|
| 20180208960 A1 | Jul 2018 | US |
