JP5750806B2 - Evaluation method of white coat wilt control activity of soil - Google Patents

Description

  The present invention relates to a method for evaluating the activity of inhibiting white coat rot of soil, and an evaluation kit for the activity of inhibiting white coat rot of soil.

The white coat rot in fruit trees is caused by the white coat rot fungus belonging to the Ascomycota (R. necatrix, R. compacta out of more than 100 species belonging to the genus Rosellinia).
The white coat fungus is soil-borne and forms a white flora on the surface of fruit tree roots and a mycelium bundle under the tree. In particular, it shows symptoms of root rot. Therefore, it is not easy to observe the affected area of white coat feather disease, and when the symptom is confirmed on the ground part, the fruit tree itself often dies. Due to this feature, white coat rot has caused enormous damage to fruit tree production. Therefore, development of a reliable control method is desired from the production site of fruit trees.

Conventionally, methods for controlling diseases of filamentous fungi exhibiting phytopathogenicity have been carried out with chemical substances such as agricultural chemicals. However, because of the problem of causing environmental pollution by chemical substances due to the use of agricultural chemicals and the like, a control method using a living organism, which is a technology aiming at reducing the environmental load, has been attracting attention.
In particular, a biological control method using antagonistic microorganisms against white crest feather disease that originally survives in the soil is a particularly useful technique in terms of reducing the environmental burden. For example, bacteria such as Bacillus (see Patent Documents 1 to 3), filamentous fungi such as Trichoderma (see Patent Documents 4 and 5), mycelia of Enokitake (mushrooms) (Ref. 6 etc.)) have been reported to have an antagonistic action (lethal action, growth inhibitory action, etc.) against white coat feather disease.

Therefore, if it is possible to know in advance how much such antagonistic microorganisms are present in the cultivated soil at the cultivation site of fruit trees and horticultural plants, it is expected that the onset risk of white coat disease can be determined.
However, the conventional evaluation system for antagonism against white coat rot is focused on a specific microorganism, and whether the microorganism has an effect of inhibiting the growth of white coat rot or inhibiting the onset of plant infection. These were evaluated individually (see Patent Documents 1 to 6, etc.). That is, it was not a technique that could evaluate the inhibitory activity against white crest feather disease of the entire soil.

In addition, one example of a method for evaluating the deterrence of diseases that soil has has been reported (see Patent Document 7). In this method, the target soil pathogen is cultured on an agar medium, and (1) the target soil (specimen) or (2) the target soil sterilized (control) The degree of growth inhibition of pathogenic bacteria in soil is evaluated by comparing the degree of growth of pathogenic bacteria in (1) and (2).
However, this method is a technique that requires calculation of a numerical value called growth inhibition rate by comparison with a control. Therefore, based on the principle of the evaluation system, it was impossible to determine whether the growth of the white rot fungus has stopped or whether the white rot fungus has been killed.
In addition, when the method was applied to the evaluation against white coat fungus, there was a large variation in the calculated values, and a big problem was recognized in terms of detection sensitivity.
Furthermore, when carrying out the method, it is necessary to study conditions such as the determination of the mixing ratio of soil and sea sand, and in addition, facilities equipped with specialized equipment such as medium preparation and culture ( Laboratories etc.) were also required, and it was not recognized as a technology that could be implemented at production sites.

Japanese Patent Application Laid-Open No. 2002-284615 (white coat disease control agent containing thermophilic bacteria) Japanese Patent Application Laid-Open No. 2003-289854 (fungal control agent containing bacteria belonging to Bacillus mojavensis) JP 2005-206496 A (White Herb Feather Control Agent) Japanese Patent No. 3213112 (white coat feather control agent) JP 2006-199601 (Microbial material) JP 2009-292741 (Soil Disease Control Microbial Materials) Patent No. 3051920 Specification (Evaluation Method for Soil Disease Deterrence) JP 2007-129996 A (Treatment Method for White Crested Wing Tree)

Soil and Microorganisms Vol.65 No.2 (October 2011), p145

  In the present invention, there is provided a method that solves the above-mentioned problems and that can reliably (with extremely high sensitivity) evaluate the inhibitory activity against white feather disease of soil by a simple method that can be carried out even in the field of fruit trees. For the purpose.

  The present inventors performed (A) a process of killing only a part of the flora, and then (B) the dead sterilized flora and the viable flora Both were brought into contact with the soil to be evaluated and cultured, (C) the dead area on the piece of wood was measured, and (D) by evaluating, the inhibitory activity against white rot of the target soil was extremely simple and It was found that it can be evaluated with high sensitivity.

The greatest technical feature of the method of the present invention is that, in the soil contact of (C), dead cells of the flora specifically induce antagonistic microorganisms in the evaluation target soil and promote rapid growth. Thus, it is found that the sensitivity of the action (lethal action, growth inhibitory action) of antagonistic microorganisms contained in the soil against white coat fungus can be greatly amplified.
In the method of the present invention, if the killed area measured in (C) is larger than the area killed by the process (A) or does not change, the white area of the soil to be evaluated has. Immediate evaluation can be made if the inhibitory activity against antagonistic fungus (antagonism of antagonistic microorganisms) is strong.
Therefore, in the evaluation system of the present invention, it is possible in principle to evaluate the white coat feather inhibitory activity of the evaluation target soil without preparing a comparative control soil.

The present invention has been made based on these findings.
That is, the present invention according to [Claim 1] relates to a method for evaluating the white coat wing inhibitory activity of soil, comprising sequentially performing the steps described in (A) to (D) below.
(A) A step of killing a part of the bacterial flora of the wooden piece, all or a part of which is covered with the cultured flora of white coat fungus.
(B) The process which culture | cultivates for 7 to 120 days by making a part or all of the said killed flora and the surviving flora contact with soil.
(C) A step of measuring the length or area of the dead area of the flora of white coat fungus on the piece of wood.
(D) A step of evaluating the white coat feather inhibitory activity of the soil to be evaluated based on the measured value.
Further, in the present invention according to [Claim 2], the dead cells of the flora killed in the step (A) attracts antagonistic microorganisms against white rot fungus in the evaluation target soil, and the antagonists 2. The evaluation method according to claim 1, which has a property of promoting rapid growth of microorganisms.
In addition, the present invention according to [Claim 3] is characterized in that the white coat fungus cultivated in the step (A) is a non-pathogenic strain and / or a low pathogenic strain. It relates to the evaluation method described.
Further, the present invention according to [Claim 4] is any one of claims 1 to 3, wherein the treatment for killing a part of the flora in the step (A) is performed by contact with warm water of 35 ° C or higher. It is related with the evaluation method of crab.
Further, the present invention according to [Claim 5] relates to the evaluation method according to any one of Claims 1 to 4, wherein the wood piece is a long-axis wood piece.
Further, in the present invention according to [Claim 6], the treatment for killing a part of the flora in the step (A) is: a region of 5 mm or more from the tip of a long-axis-shaped piece of wood covered with the flora. 6. The evaluation method according to claim 5, which is a process for killing the bacterial flora.
Further, in the present invention according to [Claim 7], the soil contact treatment in the step (B) includes: the killed flora, and the border between the killed flora and the surviving flora 7. The evaluation method according to claim 5 or 6, which is a treatment for embedding living flora in an area of 5 mm or more.
Further, the present invention according to [Claim 8] has one or more of the constituent articles described in (a) below, and has a soil characterized by including the constituent articles described in (b) below. The present invention relates to an evaluation kit for white coat feather inhibitory activity.
(a) A container formed of glass, polyethylene, polyethylene terephthalate, polystyrene, or polypropylene and used in the step (A) and / or the step (B) according to any one of claims 1 to 7 and having an open top. .
(b) NITE P-, which is 3 to 40 cm long, and that all of the long-axis wood pieces, or a portion of 1 cm or more from the tip, is a non-pathogenic or low-pathogenic white coat fungus Long-axis wood pieces covered with 269 strains and / or FERM P-18142 strains.
Further, in the present invention according to (Claim 9), the container of the above (a) is a transparent or translucent container having an open top, and (b) 9. The evaluation kit according to claim 8, wherein the evaluation kit includes the component according to claim 8.
(c) a lid portion comprising: a plate-like object in which 2 to 100 pieces of long-axis wood pieces having the same length as described in (b) are fixed; When connected to the upper opening of the container according to (a), the lower tip of the long-axis wooden piece does not contact the bottom of the container (a), and the container of (a) A lid that can seal the inside.

-Usefulness in fruit tree cultivation site According to the present invention, it is a very simple method that can be carried out in the fruit tree cultivation site, and has extremely high sensitivity, and suppresses white herb wing disease activity (of antagonistic microorganisms) in the soil. Antagonism) can be evaluated.
Thereby, in this invention, before cultivation of a fruit tree, it becomes possible to determine the onset risk of the white coat feather disease of soil.

Further, in the method of the present invention, it is possible to directly evaluate outdoor soil by adopting a non-pathogenic strain as the white coat rot fungus cultured on a piece of wood.
Therefore, as a simplest aspect as the method of the present invention, the predetermined wood piece is pointed to the evaluation target soil, extracted after the lapse of a predetermined period, and the white sterilization disease of the evaluation target soil is observed only by visually observing the dead sterilized plexus area. It becomes possible to evaluate the inhibitory activity.

  Moreover, in this invention, it enables it to provide the evaluation kit which enables the said evaluation method to be implemented simply and reliably.

・ Prediction of hot water treatment effect and confirmation of sustainability “Hot water treatment” means that white rot fungus has a lethal temperature that is significantly lower than that of normal soil microorganisms. This refers to soil improvement technology that uses hot water drip treatment (see Patent Document 8, etc.). This technique is a technique that attracts attention as a technique that has a high effect of treating and controlling white crest feather disease and has a small environmental load.
The success or failure of warm water treatment is considered to depend largely on the strength (type, density, etc.) of the action of antagonistic microorganisms in the soil.
Therefore, by predicting the strength of the antagonism of microorganisms in the target soil (soil white coat disease inhibiting activity) in advance by the evaluation method of the present invention, the effect at the time of performing warm water treatment is predicted. Is possible.

In addition, in the hot water treatment, dead cells of white rot fungus killed by hot water attract antagonists and prevent the invasion of new white rot fungus into the soil after the hot water treatment. (See Non-Patent Document 1: Announcement subject to the provisions of Article 30 (1) of the Patent Act in this application).
Therefore, the sustainability of the hot water treatment effect is evaluated by evaluating the strength of the microbial antagonism of the soil after the hot water treatment is carried out by the evaluation method of the present invention (soil white coat disease inhibiting activity). It becomes possible.

It is a schematic diagram of the evaluation kit which enables the evaluation of the white coat feather inhibitory activity which the soil of this invention has. (A): A simplified kit. (B): Precision measurement kit. FIG. 3 is a photographic image obtained by photographing the state at each processing stage in the method for evaluating the white coat feather inhibitory activity of soil in Example 1. (1): Culture treatment of white coat fungus on toothpick. (2): Partial extinction treatment of white rot fungus at the toothpick tip. (3): Contact treatment with test soil. (4): Measurement of the flora dead area of white coat fungus. The upper and lower plant boxes in (1), (3), and (4) were sealed with rose films. (A): In the measurement of Example 1 (4), it is a schematic diagram showing the vicinity of the borderline (black line) between the flora dead area and the survival area of white coat fungus on the toothpick. (B): Photographic image of the vicinity of the boundary (black line) between the microbiota killed area and the survival area after soil contact treatment in Test Zone 1-1. FIG. 3 is a photographic image obtained by enlarging a tip of a toothpick with a stereomicroscope in the measurement of Example 1 (4). (A): Test section 1-1 (partially killed + antagonistic microorganisms in the test soil). (B): Test section 1-2 (partially killed + no antagonistic microorganisms in the test soil). (C): Test area 1-3 (partially killed + antagonistic microorganisms in the test soil). In Example 2, the soil around the root of white coat rot (test area 2-1) or healthy soil between pear trees (test area 2-2) was tested, and the flora dead area of white coat fungus ( mm). FIG. 6 is a photographic image obtained by photographing the equipment used in Example 3 and the state of a processing stage. (A): Pear branch chip (seed fungus) obtained by cultivating white rot fungus (non-pathogenic NITE P-269 strain). (B): Culture treatment of white crest wing fungus on disposable chopsticks. (C): Contact treatment with test soil. In the measurement of Example 3 (4), it is the photograph image figure which imaged the whole chopsticks after soil contact processing. (A): Test area 3-1 (soil around diseased roots). (B): Test area 3-2 (healthy soil between pear trees). (C): Test area 3-3 (soil obtained by heat-treating the healthy soil at 55 ° C. for 3 days).

Hereinafter, the present invention will be described in detail.
The present invention relates to a method for evaluating white crest rot suppression activity of soil. Specifically, for the piece of wood covered with the flora of white rot fungus, (A) perform a treatment to kill only a part of the flora, then (B) evaluate both the dead sterilization flora and the viable flora The present invention relates to a method for evaluating the suppressive activity against white feather disease of a target soil by (C) measuring the dead area on a piece of wood and (D) evaluating by cultivating it in contact with the target soil.

[Wood pieces covered with bacterial flora]
In the evaluation method of the present invention, 'a piece of wood covered with white coat fungus flora' is used.
Here, the wooden piece may be any shape such as a long axis, plate, or sphere, but it is preferable to use a long axis (bar, column, skewer, long plate, etc.). .
Any material may be used, and examples thereof include birch and aspen. Moreover, the aggregate which hardened sawdust etc. can also be used.
It should be noted that it is not suitable to contain a lot of antibacterial substances, so it is desirable to check whether it can be cultured before use.
Further, it is desirable that the surface condition and material of the wood pieces be uniform for all the wood used for the same evaluation. If the surface unevenness and material are not uniform, the increase or decrease in the microbiota killed area may not be measured accurately, which is undesirable.

The length in the case of using a long-axis-shaped piece of wood may be a length that can visually confirm the increase or decrease of the microbiota killed area. Specifically, it is desirable to use a material of 3 cm or more, preferably 5 cm or more, particularly preferably 10 cm or more.
Further, the upper limit of the length may be any length suitable for operability, for example, 40 cm or less, preferably 30 cm or less, more preferably 25 cm or less.
Specific examples of such wooden pieces include toothpicks, disposable chopsticks, skewers for skewers, bamboo balls, and the like.

In order to prepare a piece of wood covered with the white rot fungus flora, sterilization (for example, high-temperature pressure treatment, boiling treatment, etc.) on the piece of wood, white seed rot (see Fig. 6 (A)) It is possible to incubate by inoculating and leaving at room temperature (specifically 10 to 30 ° C.) for 2 to 90 days, particularly 3 to 60 days, and further 4 to 40 days.
The culture can also be performed using a plastic bag, a coverable bucket, a clear case, a plastic case, etc., in a general household.

It is desirable to culture the flora on the piece of wood until the entire surface of the piece of wood (the whole) is covered with the flora, but even if a part of the surface is covered with the flora, If an area that can be confirmed is covered, it can be used in the present invention.
For example, in the case of a long-axis wood piece, at least 1 cm or more from the tip, preferably 2 cm or more, further 3 cm or more, further 4 cm or more, further 5 cm or more, further 6 cm or more, further 8 cm or more, further 10 cm or more In addition, a piece of wood covered with 15 cm or more can be mentioned.

In addition, as the white rot fungus used for the cultivation, any strain can be used as long as it belongs to Rosellinia necatrix or Rosellinia compact and has a normal growth ability. Preferably, non-pathogenic and / or low pathogenic strains are used.
Specific examples of non-pathogenic strains include NITE P-269 strain (W450 strain). Specific examples of low pathogenic strains include FERM P-18142 strain (W370 strain).
It is more desirable to use a non-pathogenic strain in view of leakage of white crest wing fungus to the outdoors.

The piece of wood covered with the white coat fungus flora can be prepared by starting the culture before the time when the evaluation method of the present invention is carried out (at least two days before).
In addition, by preparing the cultivated wood pieces and storing them at a low temperature (for example, 0 to 4 ° C.), it is possible to use them for about 3 months (preferably 2 months) in a timely manner.

[Partially killed]
The present invention is characterized by performing a process of killing a part of the bacterial flora on the wood pieces covered with the white coat fungus bacterial flora prepared above.
Here, the partial killing process refers to a process of killing only a part of the flora on the piece of wood, and not killing the whole.
If this step is not performed, the soil contact treatment in the next step does not attract and promote the growth of antagonistic microorganisms and cannot be evaluated with high sensitivity.
In addition, if the entire bacterial flora is killed, the evaluation of the present invention cannot be performed naturally.

As a means for performing a partial killing treatment, any means is adopted as long as it is not a treatment (for example, chemical treatment such as hypochlorous acid NA treatment) that prevents the attraction and growth of antagonistic microorganisms in the next step of soil contact treatment. Specifically, heat treatment can be employed.
Here, examples of the heat treatment include contact treatment with hot water, contact treatment with a solid phase such as a hot plate or a heat block, and treatment for directly inoculating the flora with a flame such as a burner or an alcohol lamp.
In the present invention, the one containing moisture in the killed dead cells is from the standpoint of promoting the growth of antagonistic microorganisms in the soil contact treatment in the next step (in terms of detection sensitivity of the measured value). Is preferred.
Therefore, when the dead cells after the heat treatment are in a dry state, it is preferable to perform a treatment including moisture.

Therefore, it is desirable to employ a contact treatment with warm water as the most suitable means for the heat treatment in the present invention. This is because the dead cells contain water.
In addition, hot water treatment can be performed by dipping or spraying, but by adopting the dipping mode in particular, it is possible to kill only a specific range of flora from the tip of the long axis piece of wood with a pinpoint. It becomes.

The temperature of the heat treatment (warm water treatment) can be 35 ° C or higher, but in order to surely kill it in a short time, 40 ° C or higher, 45 ° C or higher, 50 ° C or higher, Examples include 60 ° C. or higher, particularly 70 ° C. or higher.
For example, in the case of immersion in warm water at 45 ° C., the flora of white coat fungus on the immersed part can be killed by treatment for 30 minutes, and in the case of immersion in warm water at 70 ° C. for 20 seconds.

Note that the flora area to be killed by the treatment (microbiota killed area) is an area at least 5 mm from the tip of the long-axis piece of wood covered with the flora (the increase or decrease of the dead area can be determined visually) Territory), it is necessary to kill.
Preferably, it is desirable to kill 1 to 15 cm, particularly 2 to 10 cm, more preferably 3 to 8 cm from the tip in order to determine the increase or decrease of the killed area.

In addition, it is desirable that the partial killing process is performed so that the bacterial flora in an area of 5 mm or more from the boundary between the killed flora and the viable flora survives.
Preferably, it is desirable for the area to survive 1 to 15 cm, particularly 2 to 10 cm, and more preferably 3 to 8 cm in order to determine the increase or decrease of the dead area.

[Soil contact treatment]
The present invention is a technique characterized by performing a treatment in which both the bacterial flora killed by the above steps and the viable flora are brought into contact with the test soil to be evaluated and cultured.
In the soil contact treatment, the dead microorganisms killed in the above treatment specifically attract the antagonistic microorganisms in the soil, and the attracted antagonistic microorganisms rapidly feed on the wood pieces using the dead bacteria as food. This process is performed.
By this treatment, in the present invention, it becomes possible to greatly amplify and detect the sensitivity of the action (lethal action, growth inhibitory action) of the antagonistic microorganism contained in the soil against the white rot fungus.
Note that the soil contact treatment can be performed using a bucket, a plastic bag, a clear case, a plastic case, a water tank, a flower pot, a planter, or the like.

Here, the antagonistic microorganism refers to all microorganisms having a lethal action and a growth inhibitory action against white rot fungus.
Examples include bacteria such as Bacillus, filamentous fungi such as Trichoderma, and fungi of enokitake (mushrooms).

The aspect of the test soil contact in the treatment is carried out by bringing the killed flora and part or all of the surviving flora into contact with the soil.
Specifically, in addition to (i) the entire area of the flora killed area formed on the long-axis piece, (ii) the viable flora of a predetermined area from the boundary between the killed flora and the viable flora Can be mentioned (see, for example, FIG. 2 (3), FIG. 6 (C)).
Moreover, the said soil contact is specifically performed by insertion in a buried soil or soil.

In addition, it is desirable that the treatment is performed by bringing a region of at least 5 mm from the boundary between the killed flora and the viable flora (a region where the increase / decrease of the dead region can be visually determined) into contact with soil.
In particular, when the area is 1 to 15 cm, particularly 2 to 10 cm, and further 3 to 8 cm, it is preferable to determine the increase or decrease of the dead area.

The period for soil contact may be a period in which the action of antagonistic microorganisms in the soil can be detected, and examples include 7 to 120 days, preferably 14 to 90 days, and further 21 to 60 days. .
Moreover, as the temperature of the said process, it is desirable to carry out at room temperature (specifically 10-30 degreeC, Preferably 20-28 degreeC). In addition, since white rot fungus has a low lethal temperature and is vulnerable to high temperatures, it is desirable to carry out at 33 ° C or lower.

[Measurement of microbiota killed area]
In the method of the present invention, it is necessary to collect a piece of wood after performing the above-mentioned soil contact treatment, and to measure the flora dead area of white coat fungus.
The microbiota killed area in the wood pieces after the soil contact treatment is observed by increasing or decreasing the area depending on the strength of the action (antagonistic power) of the antagonistic microorganisms contained in the test soil.
When there are many antagonistic microorganisms in the test soil, the antagonistic microorganisms have a high lethal action and growth inhibitory action (antagonism) against white coat fungus. Therefore, due to the decrease in the viable flora of white coat fungus, the microbiota killed area is enlarged and observed.
On the other hand, when there are few antagonistic microorganisms in a test soil, the lethal action and growth inhibitory action (antagonism power) with respect to the white rot fungus of an antagonistic microorganism are low. Therefore, the microbiota killed area is observed to be decreased due to the growth of the flora of white coat fungus.

Examples of the measurement target include the area of the microbiota killed area on the piece of wood, the distance from one end of the piece of wood, and the like.
Specifically, in the case of a long-axis piece of wood, it is preferable to measure the distance from “the tip of the part where the partial killing process has been performed” to “the boundary line between the killed area and the living area” (FIG. 3). Reference 33).

Here, the boundary between the flora dead area and the viable flora area can be directly observed with a visual, loupe, or stereomicroscope after removing the soil on the surface of the wood chip.
Here, the boundary can be determined by washing away the bacterial flora on the piece of wood with water or the like, and using the black line deposited on the piece of wood as an index.
In addition, the said black line is a melanin component secreted for protection against antagonistic microorganisms by white coat fungus in the soil contact treatment, and is formed at a portion in contact with the antagonistic microorganisms. Moreover, it is a line | wire which can be observed clearly visually.
Therefore, in consideration of implementation at the fruit tree cultivation site, it is recognized that the mode using the black line as an index is simple and suitable.

In the flora dead area of the white rot fungus after the soil contact treatment, the antagonistic microorganisms attracted by the dead fungus of the white rot fungus grow and colonies and flora are formed.
When a soil having no antagonistic microorganisms is used, it is natural that colonies and flora of antagonistic microorganisms are not formed.

  In addition, in order to reduce the variation in evaluation, more than 3 pieces of wood (3 pieces in the case of long axis), preferably 4 pieces (4 pieces) or more, and further 5 pieces (5 pieces) to one test soil It is desirable to measure using the above.

[Evaluation of white coat wilt control activity of soil]
In the present invention, it is possible to evaluate the white rot prevention activity of the test soil based on the measured values of the above-mentioned microbiota killed area. In principle, the evaluation can be performed without comparison with the measured value of the control soil.
For example, (i) when the microbiota killed area after the soil contact treatment is 'expanded' compared to the area before the treatment, the action of the entire antagonistic microorganism contained in the soil is high. Accordingly, in this case, it can be evaluated that the soil white feather disease inhibitory activity of the soil is “high”.
In addition, (ii) when the microbiota killed area after the soil contact treatment and the area before the treatment are the same, the growth of white rot fungus is suppressed by the action of the antagonistic microorganisms contained in the soil as a whole State. That is, in this case as well, it can be evaluated that the soil white coat disease inhibitory activity of the soil is 'high'.
On the other hand, (iii) when the microbiota killed area after the soil contact treatment is 'decreased' compared to the area before the treatment, the action of the entire antagonistic microorganism contained in the soil is low. Therefore, in this case, it can be evaluated that the soil white coat disease inhibitory activity of the soil is 'low'.

Further, in the present invention, a more detailed degree of activity is evaluated by comparing with each test soil or control soil (for example, soil etc. artificially killing antagonistic microorganisms by heat treatment etc.). It becomes possible.
In this case, even if the killed area is decreased by the measurement, the relative strength of the activity can be evaluated by comparing the degree of decrease.

Evaluation by the method of the present invention can be suitably used particularly in soil evaluation in the cultivation of fruit trees and flower buds.
Specifically, apple, pear, grape, loquat, fig, kiwi fruit, peach, plum, sweet cherry, apricot, plum, oyster, citrus, chestnut, mulberry, cha, cherry, oak, oak, poplar, maple, camellia, This is an effective technique in that it can evaluate the soil when cultivating azaleas, roses, chrysanthemum, moto, peony, and the like.
In addition, as the deterrent activity of the soil that can be evaluated by the method of the present invention, any disease suppressive activity can be evaluated as long as it is a plant disease (white crest rot) caused by bacteria belonging to Rosellinia necatrix or Rosellinia compact. It is.

[Evaluation Kit]
The evaluation method in the present invention is a technique that can be performed using general instruments that are generally provided by farmers or the like, but in order to perform a simpler and more accurate evaluation, the following configuration is used. An embodiment with an evaluation kit containing an article is preferred.

・ Simple measurement version As a kit for performing the above-described evaluation of the present invention simply, as an aspect consisting of a minimum number of constituent articles, (a) a container having an open top and (b) covered with white coat fungus flora Mention may be made of a kit comprising a long wooden piece. For example, one embodiment of the simplified version kit is shown in FIG.

Here, the “upper opening container (reference numeral 2 in FIG. 1)” of (a) is specifically a container formed of glass, polyethylene, polyethylene terephthalate, polystyrene, or polypropylene, preferably transparent. Or a translucent container can be mentioned.
The said container is a container which makes it possible to perform the said partial killing process by putting hot water in a container. Moreover, it becomes possible to perform a soil contact process as it is by putting test soil after discarding hot water.
Moreover, it can also be set as the container used exclusively by each said process by packing the said container in multiple numbers with a kit.

In addition, as (b) 'long-axis wood pieces covered with white coat fungus flora (reference numerals 3 and 4 in FIG. 1)', the length is 3 to 40 cm, and the long-axis shape The whole piece of wood or the part of 1 cm or more from the tip indicates a long-axis piece of wood covered with the flora of white rot fungus.
Here, with respect to suitable aspects such as the length and material of the piece of wood (reference numeral 3 in FIG. 1), the degree of coverage of the bacterial flora, etc., the aspect described in the above paragraph can be adopted.
Also, the white coat rot fungus (reference numeral 4 in FIG. 1) refers to NITE P-269 and / or FERM P-18142, which is a non-pathogenic or low pathogenic white coat rot. is there.
In addition, in order to reduce the variation in evaluation, it is desirable to use three (preferably more) pieces of wood (b) for each sample soil. It is desirable to pack many pieces of wood.

Precision measurement versionAs another aspect of the evaluation kit of the present invention, instead of the above-mentioned (b) wood pieces, (c) a lid portion comprising a long-axis wood piece covered with white coat fungus flora, The aspect which packs can be mentioned. For example, one embodiment of the precision measurement plate kit is shown in FIG.

Here, as the “lid portion (symbol 6 in FIG. 1)” of (c), long-axis wood pieces having the same length as described in (b) above are used; A lid having a fixed plate-like object (reference numeral 5 in FIG. 1); and when connected to the upper opening of the container according to (a), The lower tip indicates a lid that can seal the inside of the container of (a) without contacting the bottom of the container (a).
In this aspect, a plurality of pieces of wood having the same length are attached to the lid. Therefore, in all the long-axis-shaped pieces of wood that have been mounted, it is extremely easy to align the areas that are partly killed and the areas that are brought into contact with the soil into the same length. Therefore, this aspect is an aspect suitable for multi-sample processing.

The number of long-axis wood pieces is preferably 3 or more in order to reduce variation in evaluation, but more preferably 4 or more, further 5 or more, and more preferably 9 or more. Is preferred.
The upper limit of the number is not particularly limited as long as the flora of adjacent pieces of wood do not come into contact with each other, and examples thereof include 100 or less, particularly 81 or less, and 64 or less.
In addition, it is desirable to arrange | position each wooden piece at equal intervals.

The material of the lid portion is not particularly limited, and specific examples include glass, polyethylene, polyethylene terephthalate, polystyrene, or polypropylene.
In addition, (a) it is possible to have a mode in which packing or an O-ring is bitten on the connecting portion with the container or covered with parafilm.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, the scope of the present invention is not limited by these.

[Example 1] "Method for evaluating the white crest rot suppression activity of soil"
In order to establish a method for evaluating the white crest rot suppression activity of soil, tests with different combinations of the following treatment conditions were conducted (test groups 1-1 to 1-3).

(1) “Cultivation of white rot fungus on toothpick”
25 holes (2.5mm in length x 5 in width) were drilled at 1cm intervals on the bottom of the plant box to create an "upper plant box" that was autoclaved.
In addition, a toothpick (birch material) that was autoclaved in distilled water was prepared.

  On the other hand, a PDA agar medium (50 ml) was separately prepared in a sterilized plant box to obtain a 'lower plant box'. Then, 4 mm square PDA agar medium (inoculation source) in which white coat fungus was cultured was placed on the medium at equal intervals, covered, and cultured at 23 ° C. for about 4 days. In addition, Roselinia necatrix W563 strain was used as a white coat fungus.

After cultivation, the lid of the lower plant box was opened in a clean bench, and the upper plant box was stacked by sterilization. Next, the toothpick (25 pieces) was inserted into each of the drill holes on the bottom surface of the plant box until the tip touches the flora of white coat fungus in the lower plant box.
And the connection part of the upper and lower plant boxes and the lid part of the upper plant box were sealed with parafilm to prevent drying and accidental separation of the plant box.

Thereafter, by culturing at 23 ° C. for 3 weeks, white rot fungus was cultured until the flora covered the entire toothpick. Fig. 2 (1) shows a photographic image of the state after culture.
In this embodiment, the upper plant box with a lid attached with a toothpick corresponds to the lid 6 in FIG. The lower plant box corresponds to the upper opening container 2.

(2) “Partial killing of white coat fungus”
After adding about 100 ml of sterilized distilled water to a new sterilized plant box, a plant box containing hot water at 45 ° C. was prepared by boiling water.

The plant box cultured in (1) above (only test sections 1-1 and 1-2) was collected, and the upper plant box was removed from the lower plant box in the clean bench. At this time, a toothpick covered with a flora of white coat fungus is inserted into (attached to) the bottom surface of the upper plant box.
The removed upper plant box was overlaid on a partially killed plant box containing warm water so that “about 1 cm from the tip” of the toothpick after culture was immersed in the warm water. Then, the lower plant box was bathed and the warm water was maintained at 45 ° C., and left for 30 minutes (partial killing of white coat fungus).

As a result of the treatment, the toothpick in the test sections 1-1 and 1-2 was in a state where the flora from the tip to 1 cm was killed and only the upper flora was alive.
After the treatment, the upper plant box was removed and the hot water in the killed plant box was discarded. The upper plant box was placed again and left to cool for about 10 minutes. A photographic image of this state is shown in FIG. 2 (2).
In the test section 1-3 where the treatment was not performed, it was still covered with the flora.

(3) “Contact treatment with test soil”
A plant box was prepared in which about 200 ml of each sample soil to be evaluated was placed in a new plant box. Each soil sample was collected in the field and then used to remove stones with a sieve (4 mm).
As soil, Trichoderma harzianum (filamentous fungus that is an antagonistic microorganism of white coat fungus) after autoclave sterilization is mixed uniformly (test groups 1-1 and 1-3), and soil that is just autoclaved (test group) 1-2) was prepared.

For all plant boxes in test areas 1-1 to 1-3, separate the upper plant box in a clean bench and place it on the plant box for soil contact treatment with each test soil. Was inserted into the soil.
Then, it was left in a 23 ° C. incubator for 1 month (soil contact treatment). A photographic image obtained by photographing the state of the processing is shown in FIG. 2 (3).

(4) “Measurement of the extinction area of white rot fungus”
A new sterilization plant box containing 2-3 sheets of sterile filter paper (diameter 5 cm) and 4-5 ml of sterile distilled water was prepared. Distilled water was added to prevent drying in the plant box.

The plant box that had undergone the above treatment (3) was recovered, and the upper plant box was removed. In addition, since the soil adhered to the toothpick surface, the surface soil was removed with tweezers or the like.
This was overlaid on a plant box containing filter paper and distilled water and allowed to stand at 23 ° C. for 2 days. A photographic image of the state of the processing is shown in FIG. 2 (4).

Then, remove the upper plant box again in the laboratory and observe the tip of the cultured toothpick (16 pieces located at the periphery) under a stereomicroscope. The length of the area where the white flake fungus flora was killed was measured from the tip (see reference numeral 32 in FIG. 3).
The measurement results are shown in Table 1. Note that the nine toothpicks in the center were not used for measurement due to large variations in growth conditions.
Further, microscopic images of the tips of toothpicks left for 5 days after measurement are shown in FIGS. 4 (A) to (C).

(5) Effectiveness of measurement results and evaluation
・ Effectiveness of the evaluation system From the comparison of the measured values of test sections 1-1 and 1-2, increase or decrease in the flora dead area (mm) of white rot fungus by the presence or absence of antagonistic microorganisms in the test soil It was shown that the behavior was quite different.
Specifically, when the soil containing antagonistic microorganisms (test section 1-1) was tested, the microbiota killed area (mm) was significantly larger than 1 cm from the tip (partially killed area) ( It was shown to expand to 3 cm from the tip (see Fig. 4 (A)). This is presumed that in the treatment of (3) above, the killed area was expanded due to the lethal action and the growth inhibitory action against the white rot fungus possessed by the antagonistic microorganisms in the soil.
On the other hand, when a soil (test group 1-2) in which no antagonistic microorganism was present was used, it was shown that the microbiota killed area (mm) had completely disappeared (FIG. 4B). This is considered to be due to the fact that the white rot fungus has grown again because there is no microorganism that prevents the growth of the white rot fungus in the soil by the treatment of (3) above.

From these results, by carrying out a series of treatments in this example, “the strength of the activity of antagonistic microorganisms against the white rot fungus of the test soil (that is, the strength of the white rot prevention activity possessed by the soil). ) ”Can be evaluated.
For example, if the killed area expands more than the partially killed area (1 cm from the tip in this example) and does not change from 1 cm, the activity of antagonistic microorganisms present in the test soil is extremely strong Can be evaluated.
On the other hand, even when the killed area is smaller than the partially killed area (1 cm from the tip in this example), the relative strength of the antagonistic microbial activity can be evaluated by comparison between the test soils.

・ About the effect of partial killing treatment Among the above series of treatments, when the partial killing treatment of white coat rot fungus by warm water (the treatment of (2) above) was not performed (test section 1-3: control), Despite the presence of antagonistic microorganisms in the soil, white feather fungus was hardly reduced (FIG. 4 (C)).
That is, it was shown that the detection sensitivity is extremely low when the partial killing process is not performed.

From this, it has been clarified that in the evaluation method of the present invention, it is essential to bring the dead cells of the white rot fungus dead by the partial killing treatment into contact with the antagonistic microorganisms in the soil.
In addition, the principle of the evaluation method of the present invention is that sensitivity of detecting antagonistic microbial activity in soil by specifically collecting antagonistic microorganisms in the toothpick as dead bait of white coat disease and promoting rapid growth. Was presumed to be greatly amplified.

・ About the black line In addition, since the boundary line between the microbiota killed area and the living area after soil contact treatment is observed as a black line (melanin component secreted by white coat fungus for defense against antagonistic microorganisms) It was shown that measurement can be performed even if the black line is used as an index (see reference numeral 33 in FIG. 3).

[Example 2] "Evaluation of white crest rot suppression activity of soil"
The evaluation method established in Example 1 was used to actually evaluate white coat wilt prevention activity in outdoor soil.

Except that the soil around the affected root (test zone 2-1) or the healthy soil between pear trees (test zone 2-2) was used as the test soil in the pear field where white coat disease occurred In the same manner as in the methods described in Examples 1 (1) to (4), the flora dead area (mm) of white rot fungus was measured.
In addition, the sampling of each soil sample was performed 3 times at different places, and the measurement was performed for each. The measurement results are shown in FIG.

As a result, when measured using the soil around the roots affected by white crest feathers (Test Zone 2-1), compared to the case of providing healthy soil between pear trees (Test Zone 2-2), It was shown that the flora killing area (mm) of white coat rot fungus of the potato was greatly increased.
This was presumed to be due to the fact that the density of antagonistic microorganisms against white crest wing fungus was greatly increased in the soil around diseased roots.
From this, it was shown that the soil around the roots affected by white coat rot has higher activity of antagonistic microorganisms against white coat rot fungi (that is, the activity to suppress white coat rot is high) compared to healthy soil. .

[Example 3] "Aspect using general-purpose instrument"
In order to make the technology easy to use even in the fruit tree production site, is it possible to carry out the method for evaluating white coat wing deterrent activity established in Example 1 using commercially available equipment that is commonly used by general farmers? It was investigated.

(1) “Cultivation of white coat fungus on disposable chopsticks”
30 split chopsticks (aspen, 20 cm long) divided into two were sterilized by boiling for 10-15 minutes using a pan containing boiling tap water. Then, the pan was left with the lid on, and the temperature of the hot water was reduced to 40 ° C or lower.
Using a new disposable chopstick, the “boiled and sterilized disposable chopstick” was taken out from the pan and immediately put into a commercially available transparent plastic bag (40 × 30 cm).

On the other hand, pear branch chip (see Fig. 6 (A)) in which white coat rot fungus (NITE P-269, which is non-pathogenic) is cultured, is taken in 50mL and broken down into pieces using a new disposable chopstick. I put it in. Then, the chopsticks that had been boiled and sterilized previously were sprinkled evenly.
The bag mouth was closed so that a lot of air could enter the plastic bag, and it was left in a 23 ° C. incubator for 1 month to cultivate white rot fungus until the flora covered the entire chopsticks. A photographic image of the state is shown in FIG. 6 (B).

(2) “Partial killing of white coat fungus”
By immersing 5 cm of one tip of the disposable chopsticks cultured with white crest wing fungus in 70 ° C. hot water for 20 seconds, the bacterial flora from the tip to 5 cm was killed.

(3) “Contact treatment with test soil”
The test soil was put in a commercially available bucket (5 L) to a depth of about 10 cm. Each sample soil was collected in the field, and then stones and the like were removed using a sieve (4 mm).
As soil, soil around diseased roots (Test Zone 3-1: soil with many antagonistic microorganisms), healthy soil between pear trees (Test Zone 3-2), and the healthy soil was heat treated at 55 ° C for 3 days Soil (test section 3-3: soil in which antagonistic microorganisms were artificially reduced) was prepared.
In these buckets, the disposable chopsticks treated in (2) were inserted to the bottom of the bucket, and 10 cm from the tip was inserted into the soil. A photographic image of the state is shown in FIG. 6 (C).
Then, it was covered so that the soil would not dry, and left in a dark place at about 20 ° C for 1 month.

(4) “Measurement of the extinction area of white rot fungus”
The disposable chopsticks were removed from the soil, and the soil attached to the disposable chopsticks was washed while being rubbed strongly in tap water.
Then, the position of a black line (border line between the flora killed area and the living area) formed 10 cm from the tip of the disposable chopsticks was observed (see reference numeral 33 in FIG. 7), and the distance from the tip was measured. The results are shown in Table 2 and FIG.

(5) Effectiveness of measurement results and evaluation
As a result, when the affected area soil (Study Zone 3-1: soil with many antagonistic microorganisms) was tested, the microbiota killed area was approximately less than the area that was initially partially killed (5 cm from the tip). An enlargement of 1.5 to 2.5 cm was observed (see FIG. 7 (A)).
In addition, when normal healthy soil (test section 3-2) was tested, the microbiota killed area may expand by about 0.1 to 0.5 cm from the area where it was first partially killed (5 cm from the tip). Observed (see FIG. 7 (B)).
On the other hand, when heat-treated soil (Test Section 3-3: Soil with artificially reduced antagonistic microorganisms) was used, the microbiota killed area was approximately less than the area initially killed (5 cm from the tip). A decrease of 3.0-5.0 cm was observed (see FIG. 7 (C)).

From this, even when using tools that are always available at farmers, such as disposable chopsticks, buckets, plastic bags, etc., as in the case of using the culture case of Example 1, the white soil rot prevention activity of the test soil is reduced. It was shown that it can be evaluated.
Moreover, it was shown that it can measure and evaluate more easily by using a black line as an index.

The present invention is widely disseminated as a useful technique for very simply evaluating the activity of inhibiting white rot of the target soil at production sites such as apple or pear orchards where white rot occurs. There is expected.
Moreover, when the technology is spread, it is expected that a simple evaluation kit will be developed and commercialized so that the present invention can be implemented more easily.

In addition, until now, technological development relating to hot water treatment has been promoted as a soil improvement technique for white crest feather disease, but the present invention makes it possible to predict the effect when hot water treatment is performed. Thereby, it became possible to ensure that the effect of the hot water treatment was obtained.
Moreover, since it became possible to confirm the effect after the hot water treatment, it became possible to grasp the persistence of the effect and prevent the recurrence of white coat feather disease.
Therefore, it is expected that the present invention contributes to the widespread use of warm water treatment (a technology that has a high effect of treating and controlling white crest feather disease and has a low environmental load) in fruit tree production sites.

1: Evaluation kit for white-white feather disease suppression activity of soil 2: Upper open container 3: Long-shaft-shaped piece of wood 4: White flora of white-white feather disease fungus 5: Plate-like material 6: Lid 30: Partially killed Later cultured toothpick 31: Cultured toothpick after soil contact treatment 32: Fungal extinction zone of white coat fungus 33: Boundary between the fungal dead zone and the survival zone (black line)
34: Viable flora of white coat fungus

Claims (9)

A method for evaluating the white coat wing inhibitory activity of soil, comprising sequentially performing the steps described in (A) to (D) below.
(A) A step of killing a part of the bacterial flora of the wooden piece, all or a part of which is covered with the cultured flora of white coat fungus.
(B) The process which culture | cultivates for 7 to 120 days by making a part or all of the said killed flora and the surviving flora contact with soil.
(C) A step of measuring the length or area of the dead area of the flora of white coat fungus on the piece of wood.
(D) A step of evaluating the white coat feather inhibitory activity of the soil to be evaluated based on the measured value.   The dead cells of the flora killed in the step (A) have the property of attracting antagonistic microorganisms against white coat fungus in the evaluation target soil and promoting rapid growth of the antagonistic microorganisms. The evaluation method according to claim 1.   3. The evaluation method according to claim 1, wherein the white coat fungus cultivated in the step (A) is a non-pathogenic strain and / or a low pathogenic strain.   The evaluation method according to any one of claims 1 to 3, wherein the treatment for killing a part of the flora in the step (A) is performed by contact with warm water of 35 ° C or higher.   The evaluation method according to claim 1, wherein the piece of wood is a long-axis piece of wood.   The process for killing a part of the flora in the step (A) is a process for killing; a flora in an area of 5 mm or more from the tip of a long-axis-shaped piece of wood covered with the flora; 5. The evaluation method according to 5.   The soil contact treatment in the step (B) is performed by: a living flora in an area of 5 mm or more from the boundary part of the killed flora and the dead flora and the surviving flora; 7. The evaluation method according to claim 5 or 6, which is a treatment for embedding the soil. A kit for evaluating the activity of inhibiting white coat rot of soil, comprising one or more of the constituent articles described in (a) below and the constituent article described in (b) below.
(a) A container formed of glass, polyethylene, polyethylene terephthalate, polystyrene, or polypropylene and used in the step (A) and / or the step (B) according to any one of claims 1 to 7 and having an open top. .
(b) NITE P-, which is 3 to 40 cm long, and that all of the long-axis wood pieces, or a portion of 1 cm or more from the tip, is a non-pathogenic or low-pathogenic white coat fungus Long-axis wood pieces covered with 269 strains and / or FERM P-18142 strains. The container of the above (a) is a transparent or translucent container having an open top, and includes the constituent article described in the following (c) instead of the wood piece of the above (b), The evaluation kit according to claim 8.
(c) a lid portion comprising: a plate-like object in which 2 to 100 pieces of long-axis wood pieces having the same length as described in (b) are fixed; When connected to the upper opening of the container according to (a), the lower tip of the long-axis wooden piece does not contact the bottom of the container (a), and the container of (a) A lid that can seal the inside.