Method for determining whether or not test sample contains exserohilum phytopathogenic fungus

The present invention provides a method for determining whether or not a test sample contains an Exserohilum phytopathogenic fungus. The method comprises: (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; (c) observing a back surface of the cellulose film; 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). The method further comprises a step of supplying a culture medium to the test sample before the step (b). The culture medium is a lactose casein hydrolysate agar medium containing carbendazim, captan, streptomycin sulfate, and neomycin sulfate. Alternatively, in the step (b), the test sample is left at rest while the back surface of the cellulose film is in contact with the culture medium.

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.

BACKGROUND

1. Technical Field

The present invention relates to a method for determining whether or not a test sample contains anExserohilumphytopathogenic fungus.

2. Description of the Related Art

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.FIG. 12shows a cross-sectional view of a microporous membrane supporting material used for the method disclosed therein. According to this method, the extended multiple pseudomycelia of a mold cell13cultured by a liquid culture or a mold cell13cultured on a microporous membrane1of a microporous membrane supporting material4are photographed and the shape, area and luminous intensity are recognized and analyzed by an image analytic means10. The number of the mold cells13can be counted by the culture for a short time. The microporous membrane1is interposed between a pressing ring2and a base3.NPL 1 discloses that pseudohyphae ofPhytophthora sojae, which is one of phytopathogenic oomycetes, penetrates the PET membrane having 3-micrometer pores.NPL2 discloses a semi-selective culture medium in which only anExserohilumgenus is grown.

CITATION LIST

Patent Literature

SUMMARY

An object of the present invention is to provide a method for determining whether or not a test sample contains anExserohilumphytopathogenic fungus.

The present invention provides a method for determining whether or not a test sample contains anExserohilumphytopathogenic 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 theExserohilumphytopathogenic 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:carbendazim,captan,streptomycin sulfate; andneomycin sulfate.

The present invention also provides a method for determining whether or not a test sample contains anExserohilumphytopathogenic 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 theExserohilumphytopathogenic 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:carbendazim;captan;streptomycin sulfate; andneomycin sulfate.

The present invention provides a method for determining whether or not a test sample contains anExserohilumphytopathogenic fungus.

DETAILED DESCRIPTION OF THE EMBODIMENT

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.

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 anExserohilumphytopathogenic 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 inFIG. 1, a container100is prepared. It is desirable that the container100comprises a flange102at the upper end thereof. The bottom surface of the container100is formed of a cellulose film104. It is desirable that the cellulose film104is supported by a substrate (not shown inFIG. 1). This will be described later.

As shown inFIG. 3, a test sample200is supplied to the inside of this container100. In this way, the test sample200is put on a front surface104aof the cellulose film104. When the test sample200contains a phytopathogenic fungus202, the phytopathogenic fungus202is put on the front surface104aof the cellulose film104, as shown inFIG. 4.

The test sample200is solid, liquid, or gaseous. It is desirable that the test sample200is solid or liquid. An example of the solid test sample200is soil or a crushed plant. Another example is an agricultural material such as vermiculite, rock wool or urethane. An example of the liquid test sample200is 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.

In the step (b), the test sample200is left at rest for a certain incubation time after the step (a). As one example, the incubation time is 24 hours.

As shown inFIG. 2, the cellulose film104may comprise a substrate170comprising a through hole172on at least one of the front surface104aand a back surface104bthereof. InFIG. 2, the cellulose film104comprises the substrate170on the front surface thereof. In other words, inFIG. 2, the substrate170comprises the cellulose film104on a back surface170bthereof. It is desirable that the through hole172has a diameter of more than 5 micrometers. More desirably, the through hole172has a diameter of not less than 8 micrometers. In case where the through hole172has a diameter of not more than 5 micrometers, the non-phytopathogenic fungus seldom reaches the inside of the through hole172. As a result, the non-phytopathogenic fungus fails to be brought into contact with the front surface of the cellulose film104. For more detail, see United States Patent Application Publications No. 2016/0355863 and No. 2016/0355864, both of which are incorporated herein by reference.

Needless to say, as long as the cellulose film104is stretched taut, the substrate170is not required. In other words, if it is difficult to stretch the cellulose film104taut, the substrate170for supporting the cellulose film104is used.

A reference number170aindicates the front surface of the substrate170. As shown inFIG. 2, it is desirable that the substrate170has a plurality of through holes172. The thickness of the substrate170is not limited; however, as one example, it is desirable that the substrate170has a thickness of not less than 1 micrometer and not more than 500 micrometers. The cellulose film104is significantly thin. However, if the cellulose film104is arranged on the substrate170, it is easy to handle the cellulose film104. As shown inFIG. 2, note that the cellulose film104has no through hole.

In the present embodiment, a culture medium is supplied to the test sample200. 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 anExserohilumphytopathogenic fungus penetrates the cellulose film104to selectively appear on the back surface of the cellulose film104in the step (d). In other words, phytopathogenic fungi other than anExserohilumphytopathogenic fungus do not appear on the back surface of the cellulose film104in the step (d).

In particular, a culture medium is supplied to the inside of the container100containing the test sample200. 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 container100prior to the step (a).

FIG. 6shows another embodiment of the supply of the culture medium. As shown inFIG. 6, it is desirable that the back surface104bof the cellulose film104is in contact with a liquid culture medium302. First, a second container300having the liquid culture medium302therein is prepared. Hereinafter, the container100is referred to as “first container100” to distinguish it from the second container300. The first container100is stacked on the second container300in such a manner that the lower surface of the flange102is in contact with the upper end of the second container300. In other words, the first container100is supported by the upper end of the second container300. In this way, the liquid culture medium302is sandwiched between the back surface104bof the cellulose film104and the bottom surface of the second container300.

Alternatively, after the first container100is stacked on the second container300, the liquid culture medium302may be supplied between the back surface104bof the cellulose film104and the bottom surface of the second container300.

In place of the liquid culture medium302, a viscous solid culture medium may also be used. As shown inFIG. 6, both of a solid culture medium304and the liquid culture medium302may be used. In this case, the liquid culture medium302is sandwiched between the solid culture medium304and the cellulose film104. As shown inFIG. 5, the incubation of the phytopathogenic fungus202which has appeared on the back surface104bis accelerated by at least one of the liquid culture medium302and the solid culture medium304.

In the step (c), the back surface104bof the cellulose film104is observed after the step (b). It is desirable that the back surface104bis observed using an optical microscope.

Only anExserohilumphytopathogenic fungus202penetrates the cellulose film104to appear on the back surface104bof the cellulose film104, as described in the step (b). On the other hand, phytopathogenic fungi other than anExserohilumphytopathogenic fungus do not appear on the back surface104bof the cellulose film104. In this way, in the present invention, only theExserohilumphytopathogenic fungus202appears on the back surface104bof the cellulose film104selectively.

In the step (c), it is observed whether or not the phytopathogenic fungus202appears on the back surface104bof the cellulose film104.

In particular, whether or not the phytopathogenic fungus202appears on the back surface104bof the cellulose film104is observed as below.

As shown inFIG. 8, while the cellulose film104is irradiated with light emitted from a light source500arranged above the front surface104aof the cellulose film104, the phytopathogenic fungus202is observed optically using a microscope600arranged below the back surface104bof the cellulose film104.

The liquid culture medium302and the solid culture medium304are removed from the second container300. Then, a fluorescent agent having fungus combining ability is added to the inside of the second container300. Hereinafter, such a fluorescent agent is referred to as “fungus fluorescent agent”. The reference number of the fungus fluorescent agent is402. Then, as shown inFIG. 7, the first container100is stacked on the second container300having the fungus fluorescent agent402therein. Alternatively, the fungus fluorescent agent402may be supplied between the back surface104bof the cellulose film104and the bottom surface of the second container300after the first container100is stacked on the second container300.

A part of the phytopathogenic fungus202which has appeared on the back surface104bof the cellulose film104is dyed with the fungus fluorescent agent402. Since the first container100is separated from the second container300by the cellulose film104, the fungus fluorescent agent402does not spread into the first container100. For this reason, the non-phytopathogenic fungus contained in the first container100is not dyed with the fungus fluorescent agent402.

As shown inFIG. 9, the phytopathogenic fungus202dyed with the fungus fluorescent agent402is observed using the epifluorescence microscope600located under the back surface104bof the cellulose film104. Needless to say, the phytopathogenic fungus202may be observed without using the fungus fluorescent agent402.

In the step (d), it is determined that the test sample contains anExserohilumphytopathogenic fungus, if a fungus is found on the back surface104bof the cellulose film104in the step (c). Needless to say, it is determined that the test sample does not contain anExserohilumphytopathogenic fungus, if a fungus is not found on the back surface104bof the cellulose film104in the step (c).

EXAMPLES

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 turcicumwas 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 ofExserohium turcicumwere released in the water disposed on the 12-well plate. In this way, an aqueous solution containingExserohium turcicumwas 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 medium302.

The thus-prepared liquid culture medium302was supplied into the second container300.

The experiment 1 is composed of inventive examples 1A-1G.

The first container100shown inFIG. 1was prepared as below.

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 substrate170. The polyethylene terephthalate film randomly had a plurality of through holes172. In this way, the cellulose film104having 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 film104.

Finally, the container was dried in a vacuum desiccator. In this way, the first container100shown inFIG. 1was obtained. InFIG. 1, note that the polyethylene terephthalate film serving as the substrate170is not illustrated.

Then, as shown inFIG. 6, the first container100was stacked on the second container300. The back surface104bof the cellulose film104was in contact with the liquid culture medium302. Subsequently, water having a volume of 200 microliters was added to the inside of the first container100. Furthermore, the phytopathogenic fungus aqueous solution containing 1,000 spores ofExserohium turcicumwas added to the inside of the first container100.

The first container100was 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 ofExserohium turcicumwhich appeared on the back surface104bof the cellulose film104was 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 ofExserohium turcicumwhich appeared on the back surface104bwas 35.5.FIG. 10is a microscope photograph of the back surface104bof the cellulose film104in the inventive example 1A.

In the experiment 2, an experiment similar to the experiment 1 was conducted, except for usingGibberella fujikuroiin place ofExserohium turcicum. The experiment 2 is composed of comparative examples 2A-2G. Similarly to theExserohium turcicum, Gibberella fujikuroiis one of phytopathogenic fungi.

In the experiment 3, an experiment similar to the experiment 1 was conducted, except for usingFusarium avenaceumin place ofExserohium turcicum. The experiment 3 is composed of comparative examples 3A-3G. Similarly to theExserohium turcicum, Fusarium avenaceumis one of phytopathogenic fungi.

In the experiment 4, an experiment similar to the experiment 1 was conducted, except for usingGlomerella tucumanensisin place ofExserohium turcicum. The experiment 4 is composed of comparative examples 4A-4G. Similarly to theExserohium turcicum, Glomerella tucumanensisis one of phytopathogenic fungi.

The following Table 1-Table 4 show the number of the hyphae which penetrated the cellulose film104in the experiment 1-the experiment 4.

As is clear from Table 1-Table 4, onlyExserohium turcicumappeared on the back surface104bof the cellulose film104. On the other hand, phytopathogenic fungi other thanExserohium turcicumdid not appeared on the back surface104bof the cellulose film104.

INDUSTRIAL APPLICABILITY

The present invention can be used to determine easily whether or not a test sample such as agricultural water or soil contains anExserohiumphytopathogenic fungus.

REFERENCE SIGNS LIST