JP2011140463A - Technique for controlling plant disease using waste mushroom bed of edible mushroom - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an agent for controlling plant diseases, which includes a waste mushroom bed of edible mushroom. <P>SOLUTION: The method for controlling plant diseases includes applying a waste mushroom bed of edible mushroom or its aqueous extract to plant bodies or cultivation soil. Lyophyllum decastes, Pleurotus eryngii, etc., are used as the edible mushrooms. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to a plant disease control technique. In detail, this invention relates to the disease control agent of the plant containing an edible mushroom waste fungus bed.

  Disease control of agricultural crops relies heavily on fungicides that directly kill pathogens. However, such a bactericide has a large impact on the environment, and development of a disease control technology that reduces the environmental load is strongly desired. As one of them, there is an interest in non-bactericidal resistance inducers that develop the resistance of plants and control diseases.

  When plants detect pathogenic bacteria to be infected, they develop resistance and protect themselves from external enemies. More than 80% of plant pathogenic fungi are filamentous fungi (fungi). In the infection of such fungi, the fungal cell walls are caused by chitin-degrading enzymes (chitinases) and glucan-degrading enzymes (β-1,3-glucanases) produced by plants. It has become clear that the components chitin and β-1,3-glucan are decomposed, and the various oligosaccharides that have been released are recognized as signal molecules (elicitors) by the plant and develop resistance. The plant-induced resistance includes local induced resistance that appears only in the part infected by pathogenic bacteria and whole body induced resistance that appears in the whole plant. Local induced resistance is temporarily expressed, but systemic induction It is known that resistance lasts for a long time (one month or more). Therefore, systemic induction resistance has attracted attention from the viewpoint of disease control. In recent years, in the field of plant infection physiology, research on the response mechanism of plants to elicitors has been energetically developed both at home and abroad, and analysis at the gene level is rapidly progressing (references such as Non-Patent Document 1). .

  While analysis of such plant infection mechanisms is progressing, crop disease control still depends heavily on fungicides. Many of the currently used bactericides are mainly selective bactericides from the viewpoint of safety, have a narrow antibacterial spectrum, and problems such as the emergence of resistant bacteria to the bactericides have arisen. On the other hand, disease control with plant systemic resistance inducers has no impact on the environment, is effective for a wide range of diseases, and there is no concern about the emergence of resistant bacteria, so it is expected that these problems can be solved at once. However, only a few compounds such as probenazole, a synthetic compound that is a rice blast control agent, have been put to practical use so far.

Kaku, H. et al .: Proceedings of the National Academy of Sciences of the USA, 103: 11086-11091, 2006

  The problem to be solved by the present invention was to develop a disease control technique of environmental load reduction type, in particular, a plant disease control technique using a new systemic resistance inducer.

  The inventors of the present invention have considered to use an elicitor of a fungal cell wall component that has been clarified from a study of plant infection mechanism analysis as a resistance inducer. Edible mushrooms are also a family of fungi, and plants, like pathogenic fungi, appear to develop resistance by recognizing mushroom cell wall components as elicitors. Many edible mushrooms are cultivated on the fungus bed, and the fungus bed after harvesting the mushrooms (waste fungus bed) is discarded in large quantities as garbage, but the waste fungus bed filled with mycelium is an abundant source of elicitor Is likely to be. Since there is no research on disease control using the waste fungus bed of such edible mushrooms, in this study, the disease control by the expression of systemic induced resistance of plants using the mycelium of the waste fungus bed of edible mushrooms. investigated. If this research is successful, it can be greatly expected to develop into two birds with one stone, such as the use of waste bacterial beds and the development of disease control agents.

  As a result of intensive studies based on such a concept, the present inventors have developed a systemic induced resistance of plants by using an edible mushroom waste fungus bed or an aqueous extract thereof, and have an effect on plant diseases. As a result, the present invention was completed.

Thus, the present invention provides:
(1) An edible mushroom waste fungus bed or a plant disease control agent containing an aqueous extract thereof;
(2) The disease control agent according to (1), wherein the edible mushroom waste fungus bed or an aqueous extract thereof has been subjected to autoclave sterilization treatment;
(3) The disease control agent according to (1) or (2), wherein the waste fungus bed is a waste fungus bed of Hatakeshimeji;
(4) The disease control agent according to (1) or (2), wherein the waste fungus bed is a waste bed of eringi:
(5) The disease control agent according to (3) or (4), wherein coniferous sawdust is used for the fungus bed;
(6) The disease controlling agent according to any one of (1) to (5), wherein the disease is caused by fungal infection;
(7) The disease control agent according to any one of (1) to (6), which is mixed with soil;
(8) A disease control method for a plant, which comprises applying an edible mushroom waste fungus bed or an aqueous extract thereof to the plant.
I will provide a.

  The present invention develops systemic induced resistance to plant diseases and effectively controls plant diseases. Since the present invention does not use harmful drugs and enables effective use of a large amount of waste fungus beds that were difficult to treat in edible mushroom fungus bed cultivation, the development of a disease control agent that reduces the environmental load. It can contribute greatly.

FIG. 1 illustrates a method of treating waste cucumber floor water with cucumber and inoculating pathogens. FIG. 2 illustrates a method of waste bacterial bed treatment for cucumber seedlings. FIG. 3 is a photograph showing the disease-suppressing effect by the immersion treatment of fruit bodies and waste fungus floor water extract. T1 is treated with fruit body water extract, T2 is treated with waste bacterial bed water extract (no treatment = not autoclaved), T3 is treated with waste bacterial bed water extract (sterilization treatment = autoclave treatment), T4 is water It is the leaf processed with. FIG. 4 is a photograph showing the disease control effect of conidia suspension dripping inoculation of waste bacterial bed water extract treated leaves. FIG. 5 is a photograph showing suppression of lesion formation by treatment of sterilized waste fungi on nursery soil. FIG. 6 is a diagram showing the results of examining the antibacterial activity against anthrax of the waste bacterial bed water extract. FIG. 7 is a graph showing the results of examining the expression of disease resistance-related genes by treatment with waste bacterial water extract.

  The present invention provides a plant disease control agent comprising an edible mushroom waste fungus bed or an aqueous extract thereof. In the infection with pathogenic fungi, chitinase and β-1,3-glucanase induced by plants decompose chitin and β-1,3-glucan, which are fungal cell wall components, and the plants use various oligosaccharides as elicitors. Although a mechanism for recognizing and developing systemic induced resistance is known, the present invention is based on a novel idea of using a waste fungus bed or its aqueous extract as a source of fungal cell wall components. Furthermore, the present invention not only has an excellent disease control effect, but also can provide a disease control agent that is not toxic and harmless to edible mushroom waste fungi beds, and effective use of a large amount of waste fungi beds that have been in trouble with processing. And inducing the systemic resistance of the plant, it has a long-lasting effect and usually has an advantage that the effect lasts for more than one month.

  The waste microbial bed used in the present invention may be obtained by cultivating any mushroom as long as it is an edible mushroom. Examples of edible mushrooms include, but are not limited to, Hatake Shimeji, Beech Shimeji, Eringi, Shiitake, Maitake, Enokitake, Nameko, Mushroom, Agaricus, Oyster. Preferred edible mushrooms include Hatake shimeji and eringi.

  The waste fungus bed is a fungus bed after inoculating and cultivating and harvesting mushroom mycelia on a fungus bed that is a medium in which nutrient sources such as sawdust and rice bran are mixed. Methods for preparing and components of the fungus bed, methods for inoculating the fungus bed, and cultivation methods for mushrooms are known to those skilled in the art.

  The waste microbial bed or its aqueous extract may be autoclaved. Autoclave sterilization does not reduce the disease control effect. Rather, autoclaving is preferable because storability is enhanced and the disease control effect is prolonged.

  The disease for which the plant disease control agent of the present invention is effective is a disease caused by fungal infection, and the kind thereof is not particularly limited. Examples of plant diseases for which the disease control agent of the present invention is effective include anthrax, powdery mildew, downy mildew and the like in the case of cucumber. There is no particular limitation on the plant for which the plant disease control agent of the present invention is effective.

  The form of the plant disease control agent of the present invention is not particularly limited, and the waste microbial bed may be used as it is, or the waste microbial bed may be appropriately processed and used. For example, the waste microbial bed may be dried to form powder or granules. When applying the waste fungus bed to the plant disease control agent of the present invention, the mycelium may or may not be crushed. Alternatively, the plant disease control agent of the present invention may be a water extract of a waste fungus bed, or a product obtained by appropriately processing a water extract of a waste fungus bed. For example, the water extract of the waste microbial bed may be filtered, or the water extract of the waste microbial bed may be concentrated or dried. That is, the plant disease control agent of the present invention may be a solid (for example, a waste bacterial bed as it is, a ground product of a waste bacterial bed, a powder, a granule, a dried product of a water extract of a waste bacterial bed), a liquid (for example, water Extract, concentrate of water extract, etc.), or can be appropriately processed into various forms such as semi-solid and paste. Methods for obtaining these forms are well known to those skilled in the art. Furthermore, excipients, carriers, diluents, or inducers (spreading agents) known to those skilled in the art may be appropriately added to the plant disease control agent of the present invention.

  The plant disease control agent of the present invention can be applied to plants by various methods. For example, when the plant disease control agent of the present invention is in liquid form, it may be sprayed, sprayed or applied directly to the plant, or sprayed or mixed into the soil. When the plant disease control agent of the present invention is in the form of a concentrated solution, it may be diluted with water and sprayed onto plants. For example, when the plant disease control agent of the present invention is solid, it may be mixed with soil. For example, the waste microbial bed can be used as it is, or an autoclaved waste microbial bed can be mixed with soil. When the plant disease control agent of the present invention is in the form of a dried product, a spreading agent may be added, suspended in water and sprayed onto the plant. When the plant disease control agent of the present invention is mixed with soil, the soil may be a seedling soil or a field soil. The plant disease control agent of the present invention may be mixed with soil and used as a seedling soil form (for example, bagging), or may be grown in a cell tray or pot. The method of applying the plant disease control agent of the present invention to plants is not limited to the above.

  You may mix the disease control agent of another plant with the disease control agent of the plant of this invention. Alternatively, the plant disease control agent of the present invention may be used in combination with other plant disease control agents.

  The application amount of the plant disease control agent of the present invention to a plant can be appropriately determined by a person skilled in the art while considering the effect in consideration of the target plant, the target disease, other treatments on the plant, etc. Similarly, the number of times of application to plants can be appropriately determined by those skilled in the art. Also, the application time of the plant disease control agent of the present invention is not particularly limited, and may be any time until the plant grows from a seed state to a mature plant body.

  Furthermore, the present invention provides a disease control method for plants, which comprises applying an edible mushroom waste fungus bed or an aqueous extract thereof to plants.

  The present invention will be described in more detail and specifically with reference to the following examples. However, the examples are merely illustrative and should not be construed as limiting the scope of the present invention.

I. Materials and experimental methods Test waste bed The waste bed of Lyophyllum decastes (TMIC30940), which was developed as a good edible mushroom through joint research between the Fungus Research Institute of Japan Mushroom Center and Tottori Prefecture, was used. In some experiments, waste beds of Pleurotus ostreatus (TMIC35101), Pleurotus eryngii (TMIC35100), and Hericium erinaceum (TMIC35102) were also used.

2. Test plants and pathogenic fungi For the disease control test, the cucumber (Cucumis sativus) cultivar “Natsu Suzumi” and the cucumber anthracnose fungus (Colletotrichum orbiculare) C-14 strain (all agricultural) Used from the farming and technical center. The cucumber seeds were sown on the seedling raising soil in the cell tray, and the germinated seedlings were transplanted to the seedling soil in a pot (diameter 9 cm) and cultivated in a nursery room at 25 ° C., 14 hours light period, 10 hours dark period. On the other hand, when anthrax is cultivated and stored on a potato broth agar medium (PDA medium) to obtain spores (conidia) for inoculation, the anthracnose fungus is on a PDA medium (PDY medium) containing 0.5% yeast extract. And cultured at 25 ° C. for 7 to 12 days. The conidia formed on the medium are suspended in distilled water (DW), washed several times by centrifugation (800 xg, 5 minutes), adjusted to a conidia concentration of 5 x 10 ⁵ cells / ml, and immediately used for experiments. did.

3. Preparation of water extract from waste bacteria bed 500ml of distilled water was added to 200g of waste bacteria bed just after harvesting, and ground with a homogenizer (1,500rpm, 2 minutes), and the ground liquid was filtered with gauze The supernatant fraction (water extract) is obtained by centrifugation (800 × g, 5 minutes), and a part of the water extract is further autoclaved (sterilized) (121 ° C., 30 minutes) (see FIG. 1). And stored at −80 ° C. until use. In use, the water extract was thawed at room temperature. For comparison, a water extract (50 g fruit body / 250 ml distilled water) was obtained from the fruit body (mushroom) of Hatake shimeji mushroom by the same operation as the waste fungus bed.

4). Disease control test by waste fungus bed water extract and waste fungus bed treatment When treating water extract with cucumber, spray treatment or immersion treatment on the first leaf of potted cucumber seedling with fully developed second leaf (10 seconds) (see FIG. 1). In addition, DW was processed as a control. The treated cucumber seedlings were again placed in a nursery room at 25 ° C., 14 hours light period 10 hours dark period, and after 1 week, conidia suspension of anthrax was sprayed on the whole cucumber seedlings or inoculated dropwise onto cucumber leaves (10 μl of 30 μl). (See Fig. 1). As a control, DW was used. The cucumber seedling was placed in a growth cabinet at 90 ° C. and dark at 20 ° C. After 24 hours, the cucumber seedling was taken out and placed in a nursery room. One week later, the number of lesions was measured by spray inoculation, and the area of lesions was measured by drop inoculation.

  On the other hand, the waste fungus bed of Hatake Shimeji and the sterilized (121 ° C., 30 minutes) waste fungus bed were mixed with the cucumber seedling soil so that the nursery soil: waste fungus bed was 2: 1 (v / v) ( (See FIG. 2). Seed the cucumber seeds on the waste tray mixed seedling soil or seedling soil in the cell tray, and after 1 week, transplant the germinated seedlings to the waste fungus bed mixed seedling soil or seedling soil in a pot (diameter 9 cm), 25 ° C, 14 hours light period 10 It put in the nursery room of the time dark period, and was cultivated for 2-3 weeks (refer FIG. 2). The first and second leaves of cucumber seedlings were inoculated dropwise with conidia suspension of anthrax fungus (30 μl at 10 spots / leaf). DW was used as a control. The cucumber seedling was placed in a growth cabinet at 90 ° C. and dark at 20 ° C. After 24 hours, the cucumber seedling was taken out and placed in a nursery room. One week later, the lesion area was measured.

5. Antibacterial activity test of waste bacteria bed water extract Since the disease control effect of waste bacteria bed water extract treatment may be caused by the antibacterial activity against pathogenic bacteria of the water extract, Viables were suspended (5 × 10 ⁵ cells / ml), dropped onto a slide glass, and allowed to stand in a dark room at 25 ° C. for 24 hours, and then the spore germination rate and germ tube length were measured. In addition, an anthrax bacterial cell disc (5 mm in diameter) was planted on a PDY medium containing a sterilized water extract and a PDY medium as a control, and the diameter of mycelia grown after a certain period of time was measured in the dark at 25 ° C.

6). Expression analysis of induced resistance-related genes in waste fungus floor water extract and waste fungus bed treated leaves Cucumber seedling treated with waste fungus floor water extract and waste fungus bed before and 1 day after inoculation of anthracnose fungus RNA was extracted from leaves using a conventional method, and resistance-related gene expression was analyzed by real-time PCR. Resistance-related genes were examined for 6 genes already reported in cucumber (callose synthase gene, lignin peroxidase gene, chitinase gene, β-1,3-glucanase gene, PR-protein 1 gene and PAL gene).

II. Results and discussion Disease control effect of waste microbial bed water extract treatment on cucumber leaves First, examine whether the mycelium component of Hatake-shimeji shows disease control effect, and also consider the water extract treatment time for cucumber seedlings necessary for disease control Therefore, the water extract obtained from the fruit body of Hatake shimeji was sprayed or immersed in the whole cucumber seedling, and after a certain time, the conidia suspension of anthrax was sprayed on the whole cucumber seedling and one week later the leaf disease The number of spots was examined. Table 1 shows the results of the immersion treatment on the first cucumber seedling leaves. The disease-suppressing effect was observed 5 hours after the immersion in the water extract, and the suppressing effect was remarkable on the 4th day. After 7 days, the suppressing effect was almost 100%. In addition, when the water extract was sprayed over the entire cucumber seedling, almost the same result as the immersion treatment was obtained. Therefore, the disease suppression effect test by waste bacterial bed treatment was carried out one week after the water extract treatment.

Furthermore, in order to see if the disease control effect of the fruit body water extract treatment is also induced by the waste fungus bed water treatment, the fruit body water extract and the waste fungus bed water extract (sterilized) are added to the first leaf of the cucumber seedling. Treatment and non-treatment) were carried out, and after 1 week, the conidia suspension was spray-inoculated on the cucumber seedlings, and the number of lesions on the second leaf was examined. As a result, in any water extract treatment, the number of lesions is remarkably suppressed, the waste fungus floor water extract exhibits the same disease control effect as the fruit body water extract, It was clarified that the heat-stable and disease-suppressing effect was also observed in leaves other than the treatment (Table 2, FIG. 3).

Next, in order to examine an efficient conidial inoculation method for simply seeing the disease-suppressing effect, the whole cucumber seedling is sprayed or soaked in the first leaf, and conidia after one week. The suspension was inoculated dropwise into a cucumber seedling to examine the lesion area. As a result, both the waste bacterial bed water extract spray treatment and the first leaf immersion treatment showed a marked decrease in the lesion area due to the conidial suspension dripping (Table 3, FIG. 4). Therefore, in subsequent disease control experiments, the dripping inoculation method was used in which the amount of conidial suspension to be inoculated was small and the disease control test could be easily performed.

2. Disease control effect of waste microbial bed treatment on cucumber seedling soil Mix cucumber seedling soil with waste fungus bed or sterilization waste fungus bed to 2: 1 (v / v), and add cucumber seeds to the mixed soil in the cell tray. Then, cucumber seedlings were transplanted into the mixed soil in the pot, and the disease control effect on the first and second leaves of the cucumber seedlings was examined. As a result, in both the waste fungus bed and the sterilization waste fungus bed, the disease suppression effect was observed in the cucumber seedlings transplanted to the mixed soil in the pot (Table 4, FIG. 5). In particular, the suppression effect of cucumber seedlings cultivated in soil mixed with sterilized waste fungus bed was remarkable, and it became clear that killing the mycelium in the waste fungus bed by sterilization showed a higher effect. This is probably because sterilization released more elicitor from the mycelium in the waste fungus bed than without treatment. In addition, when the fungal bed before inoculation with Hatake-shimeji was used as a control, DW was used because the growth of the cucumber seedling was inhibited. Since the waste microbial bed after cultivation of Hatake shimeji does not show growth inhibition, it is considered that the growth inhibitory substance present in the microbial bed is decomposed by Hatake shimeji.

Next, the relationship between the storage period of the waste bacteria bed and the disease control effect was examined. The waste bacteria bed left in the room and the sterilized waste bed left in the room were taken out after a certain period of time and mixed with the seedling soil to examine the disease control effect. As a result, it was shown that the disease-suppressing effect disappeared in the waste bed left in the room for 2 weeks, but the inhibitory effect was maintained in the waste bed that was left in the room for 3 weeks, and it could be stored for a long time. (Table 5). Although the cause of the disappearance of the effect of the waste fungus bed left in the room is not clear, it is considered that the waste fungus bed has become dry and hard, so that the mixing with the seedling soil is insufficient.

Next, in order to see if there is a disease-inhibiting effect even in the waste fungi beds of edible mushrooms other than Hatake shimeji, we examined using sterilized waste fungi beds of oyster mushrooms, eringi and yamabushitake. When sterilized waste beds of oyster mushrooms and yamabushitake were mixed with the seedling soil, the growth inhibition of cucumber seedlings was observed, so the disease control effect was not investigated. The disease suppression effect more remarkable than the waste bacteria bed was shown (Table 6). Hatakeshimeji and eringi use coniferous sawdust in the fungus bed, whereas hiratake and Yamabushitake, which showed growth inhibition, use hardwood sawdust in the fungus bed, which is involved in the suppression of plant growth Is also possible.

3. Antibacterial Activity of Waste Bacteria Bed Water Extract The disease suppression effect of waste bed extract and sterilized extract may be due to antibacterial action against pathogenic bacteria, not induced resistance of plants. After suspending in the waste bacterial bed water extract, conidia germination and germ tube elongation were examined 24 hours later. As a result, no antibacterial activity was observed, and the water extract was more active in both conidia germination and germ tube elongation (Table 7, FIG. 6). This is probably because the aqueous extract contained nutrients in the fungus bed and affected the growth of anthrax. On the other hand, when a mycelium of anthrax (5 mm) was placed on a Czapek medium (9 cm petri dish) containing a sterilized water extract and mycelial growth was measured after one week, the mycelium growth was vigorous in any medium. No antibacterial substance was found in Table 7 (FIG. 7, FIG. 6). From these results, it is considered that the effect of the plant hindrance control agent of the present invention is not due to the antibacterial action against pathogenic bacteria, but due to the induction of plant resistance.

4). Expression analysis of disease resistance-related genes in cucumber seedlings treated with waste fungus floor water extract Cucumber is a cucumber to see if the whole body induced resistance of plants is involved in the disease control effect of waste fungus floor water extract treatment The expression of 6 types of genes that have been shown to be involved in systemic induced resistance was analyzed. As a result, the expression of chitinase gene was increased in the spray-treated leaves of the water extract, and the expression of chitinase gene and β-1,3-glucanase gene was increased in the second leaf obtained by immersing the water extract in the first leaf. Further remarkable expression was observed 1 day after suspension inoculation (FIG. 7). From these results, it was shown that the effect of the plant interference control agent of the present invention is exhibited by inducing systemic resistance of the plant.

  The present invention can be used in the agricultural field, the agricultural chemical field, and the like.

Claims (8)

  A plant disease control agent comprising an edible mushroom waste fungus bed or an aqueous extract thereof.   The disease control agent according to claim 1, wherein the edible mushroom waste fungus bed or its aqueous extract has been subjected to autoclave sterilization treatment.   The disease control agent according to claim 1 or 2, wherein the waste fungus bed is a waste fungus bed of Hatakeshimeji.   The disease control agent according to claim 1 or 2, wherein the waste fungus bed is a waste bed of eringi.   The disease control agent according to claim 3 or 4, wherein coniferous wood sawdust is used for the fungus bed.   The disease control agent according to any one of claims 1 to 5, wherein the disease is caused by fungal infection.   The disease control agent according to any one of claims 1 to 6, which is mixed with soil.   A plant disease control method comprising applying an edible mushroom waste fungus bed or an aqueous extract thereof to a plant.

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