This application claims the benefit of foreign priority of Japanese patent application 2017-010911 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 an Exserohilum 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.
An object of the present invention is to provide a method for determining whether or not a test sample contains an Exserohilum phytopathogenic fungus.
The present invention provides a method for determining whether or not a test sample contains an Exserohilum phytopathogenic fungus, the method comprising:
(a) putting the test sample on a front surface of a cellulose film having 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 Exserohilum phytopathogenic fungus, if a fungus which has penetrated the cellulose film is found on the back surface of the cellulose film in the step (c);
wherein
the method further comprises
a step of supplying a culture medium to the test sample before the step (b); and
the culture medium is a lactose casein hydrolysate agar medium containing:
The present invention also provides a method for determining whether or not a test sample contains an Exserohilum phytopathogenic fungus, the method comprising:
(a) putting the test sample on a front surface of a cellulose film having 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 Exserohilum phytopathogenic fungus, if a fungus which has penetrated the cellulose film is found on the back surface of the cellulose film in the step (c);
wherein
in the step (b), the test sample is left at rest while the back surface of the cellulose film is in contact with a culture medium; and
the culture medium is a lactose casein hydrolysate agar medium containing:
The present invention provides a method for determining whether or not a test sample contains an Exserohilum phytopathogenic fungus.
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 more than 14.5 micrometers. As one example, the cellulose film has a thickness of not less than 0.04 micrometers and not more than 14.5 micrometers. It would be difficult to form a cellulose film having a thickness of less than 0.04 micrometers. On the other hand, it takes too long a time for an Exserohilum phytopathogenic fungus to penetrate a cellulose film having a thickness of more than 14.5 micrometers. Therefore, the cellulose film having such a thickness is not impracticable.
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). As one example, the incubation time is 24 hours.
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.
A reference number 170a indicates the front surface of the substrate 170. As shown in
In the present embodiment, a culture medium is supplied to the test sample 200. As disclosed in Roberto Luis De Rossi et. al., the culture medium is a lactose casein hydrolysate agar medium containing carbendazim, captan, streptomycin sulfate; and neomycin sulfate. By using the culture medium, only an Exserohilum phytopathogenic fungus penetrates the cellulose film 104 to selectively appear on the back surface of the cellulose film 104 in the step (d). In other words, phytopathogenic fungi other than an Exserohilum phytopathogenic fungus do not appear on the back surface of the cellulose film 104 in the step (d).
In particular, a culture medium is 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 is supplied in the step (b). Alternatively, the culture medium is supplied prior to the step (b). In other words, the culture medium is 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.
Only an Exserohilum phytopathogenic fungus 202 penetrates the cellulose film 104 to appear on the back surface 104b of the cellulose film 104, as described in the step (b). On the other hand, phytopathogenic fungi other than an Exserohilum phytopathogenic fungus do not appear on the back surface 104b of the cellulose film 104. In this way, in the present invention, only the Exserohilum phytopathogenic fungus 202 appears on the back surface 104b of the cellulose film 104 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, whether or not the phytopathogenic fungus 202 appears on 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 an Exserohilum phytopathogenic fungus, 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 an Exserohilum phytopathogenic fungus, 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 Exserohium Turcicum)
Exserohium turcicum, one of phytopathogenic fungi, was inoculated on a V-8 agar culture medium. Then, the culture medium was left at rest at a temperature of 25 degrees Celsius for one week. Exserohium turcicum was given by a Professor, Dr. Shim, who belongs to Department of Plant Pathology and Microbiology, Texas A&M University.
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 Exserohium turcicum were released in the water disposed on the 12-well plate. In this way, an aqueous solution containing Exserohium turcicum was provided. Hereinafter, this aqueous solution is referred to as “phytopathogenic fungus aqueous solution”.
(Preparation of Culture Medium)
The present inventors added the following four reagents to a lactose casein hydrolysate agar medium in accordance with the disclosure of NPL2 to prepare a liquid culture medium 302.
The thus-prepared liquid culture medium 302 was supplied into the second container 300.
(Experiment 1)
The experiment 1 is composed of inventive examples 1A-1G.
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 7%. 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 PIEP 12R 48). The polyethylene terephthalate film served as the substrate 170. The polyethylene terephthalate film randomly had a plurality of through holes 172. In this way, the cellulose film 104 having a thickness of 14.5 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 30 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 Exserohium turcicum 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. As a result, the mean value of the number of the hyphae of Exserohium turcicum which appeared on the back surface 104b was 35.5.
(Experiment 2)
In the experiment 2, an experiment similar to the experiment 1 was conducted, except for using Gibberella fujikuroi in place of Exserohium turcicum. The experiment 2 is composed of comparative examples 2A-2G. Similarly to the Exserohium turcicum, Gibberella fujikuroi is one of phytopathogenic fungi.
(Experiment 3)
In the experiment 3, an experiment similar to the experiment 1 was conducted, except for using Fusarium avenaceum in place of Exserohium turcicum. The experiment 3 is composed of comparative examples 3A-3G. Similarly to the Exserohium turcicum, Fusarium avenaceum is one of phytopathogenic fungi.
(Experiment 4)
In the experiment 4, an experiment similar to the experiment 1 was conducted, except for using Glomerella tucumanensis in place of Exserohium turcicum. The experiment 4 is composed of comparative examples 4A-4G. Similarly to the Exserohium turcicum, Glomerella tucumanensis is one of phytopathogenic fungi.
The following Table 1-Table 4 show the number of the hyphae which penetrated the cellulose film 104 in the experiment 1-the experiment 4.
Exserohium turcicum
Gibberella fujikuroi
Fusarium avenaceum
Glomerella
tucumanensis
As is clear from Table 1-Table 4, only Exserohium turcicum appeared on the back surface 104b of the cellulose film 104. On the other hand, phytopathogenic fungi other than Exserohium turcicum did not appeared on the back surface 104b of the cellulose film 104.
The present invention can be used to determine easily whether or not a test sample such as agricultural water or soil contains an Exserohium phytopathogenic fungus.
Number | Date | Country | Kind |
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2017-010911 | Jan 2017 | JP | national |
Number | Name | Date | Kind |
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20160355863 | Uriu | Dec 2016 | A1 |
20160355864 | Uriu | Dec 2016 | A1 |
Number | Date | Country |
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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. |
Roberto Luis De Rossi et al., “Semi-selective culture medium for Exserohilum turcicum isolation from corn seeds”, Summa Phytopathol., Botucatu, vol. 40, No. 2, pp. 163-167, Jun. 9, 2014. |
Number | Date | Country | |
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20180208961 A1 | Jul 2018 | US |