JP2018157816A - Fusarium bacteria-antagonistic bacteria growth promoting material and production method therefor, and method for inhibiting growth of fusarium bacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a material useful for inhibiting the growth of Fusarium bacteria in the soil and a production method therefor, and a method for inhibiting Fusarium disease using the material.SOLUTION: A material according to the present invention contains one or more compound selected from the group consisting of adenosine, 5'-S-methyl-5'-thioadenosine, and γ-glutamyl-S-allylcysteine, and is applied to the soil to promote the growth of the bacteria that antagonize Fusarium bacteria, thereby inhibiting the growth of Fusarium bacteria. A Fusarium bacteria-antagonistic bacteria growth promoting material according to the present invention can be extracted from Allium roots. The material according to the present invention can be applied to the soil to inhibit Fusarium disease.SELECTED DRAWING: Figure 21

Description

  The present invention relates to a substance that promotes the growth of bacteria that antagonize Fusarium bacteria and a method for producing the same. Further, the present invention relates to a method for inhibiting the growth of Fusarium bacteria using an antagonistic bacterial growth promoting substance of Fusarium bacteria.

  Fusarium is one type of filamentous fungus that infects crops and causes continuous cropping problems. Among Fusarium fungi, those with pathogenicity are those that enter more than 100 species of plants, mainly enter the roots and proliferate in the conduit, and inhibit water flow and cause symptoms such as leaf yellowing and wilting. Causes and sometimes dies. Fusarium fungi form highly durable thick-walled spores and can live in the soil for many years. Therefore, once Fusarium fungus grows once and a lesion originated from it grows, it becomes a host for a long time. Crop cultivation is no longer possible. In the agricultural field, the use of resistant varieties, cultivation using grafts to resistant rootstock, and soil fumigation have been carried out as countermeasures against lesions caused by Fusarium bacteria. Has not reached.

  Below, what has pathogenicity among Fusarium bacteria, for example, Fusarium oxysporum (Fusarium oxysporum) is also only called Fusarium bacteria. Various crop lesions caused by Fusarium bacteria are also referred to as Fusarium disease.

  Studies are underway on biocontrol methods that eliminate Fusarium bacteria without using disinfectants and other chemicals. The general strategy of biocontrol methods for soil diseases is to introduce a large amount of antagonistic microorganisms, especially bacteria called antagonistic bacteria, into the soil, and to suppress the occurrence of soil diseases by controlling the activity, growth or infection of plant bodies. It is to try to reduce. As a method for controlling Fusarium disease using antagonistic microorganisms, Patent Document 1 discloses a technique using a non-pathogenic Fusarium bacterium. Patent Document 2 discloses a technique using a bacterium belonging to the genus Colimonas. Patent Document 3 discloses a technique using Rhodopseudomonas bacteria and Bacillus bacteria. Patent Document 4 discloses a technique using Pseudomonas bacteria. Patent Document 5 discloses a technique using Streptomyces actinomycetes. However, due to soil factors such as pH, organic matter content, heavy metals, etc., weather conditions, survival competition with indigenous microorganisms, antagonistic microorganisms are unable to settle in the soil at the place where they are put in, and the expected disease control effect is achieved. There are many things that cannot be demonstrated.

  Development of technology to create soil that is unlikely to cause soil disease is being promoted by artificially activating the activity and growth of indigenous antagonistic microorganisms that inhabit each land. Hereinafter, the characteristic that disease is less likely to occur is referred to as disease prevention, and such soil is referred to as disease inhibition soil. Patent Document 6 and Non-Patent Documents 1 and 2 promote the growth of chitin and chitosan-degrading microorganisms by introducing into the soil a material containing crab and shrimp shells, crab shell-derived chitin, chitosan, and degradation products thereof. , Methods to reduce the number of pathogenic fungi containing chitin as the main component of the cell wall, prevent the occurrence of soil-borne fungal diseases such as Fusarium disease, and reduce the occurrence of continuous cropping disorders . However, in order to obtain a sufficient disease-suppressing effect with the materials disclosed in Patent Document 6 and Non-Patent Documents 1 and 2, a large amount of application to soil was required. On the other hand, chitin, chitosan and decomposition products thereof have recently been used for pharmaceuticals, health foods, etc., and their prices have risen, making it difficult to use them as soil conditioners.

  In some areas of China and Japan, mixed leeks and onion rotations are carried out on cucurbitaceae plants to control fusarium disease, and certain effects are observed. FIG. 16 shows the morbidity of each cucumber when cucumber and leek are mixed in the soil where Fusarium oxysporum is proliferating, when cucumber and leek are mixed, and when only cucumber is cultivated. It has been experimentally confirmed that leek mixed planting and rotation cultivation are effective in preventing Fusarium disease in various crops such as tomatoes and spinach. However, the mechanism by which Fusarium disease is prevented by cultivation of the genus Allium has not yet been elucidated. In addition, it is often not possible to plant or rotate a genus of genus in terms of the cropping system, cropping time, harvesting method, quality impact, etc. of crops that want to prevent Fusarium disease. It was.

  However, mixed planting of leeks and leek is effective not only for plants of the family Cucurbitaceae but also for diseases caused by Fusarium fungi on other plants. Spinach wilt is also a disease caused by Fusarium fungi, but as shown in FIG. 17, when spinach is mixed with leeks and leek, there is an effect of reducing the incidence of Fusarium disease. The mixed planting evaluation method shown in FIG. 17 is carried out by transplanting leek or leek seedlings to the soil in a pot mixed with spinach wilt fungus, seeding 10 spinach seeds around the seedling, and investigating the disease until 20 days after sowing. It is the result of calculating AUDPC (area under disease progression curve: a value representing the degree of disease development). It has been considered that effective measures against Fusarium disease can be taken if the components contained in the genus Allium can be identified and used.

Japanese Patent Laid-Open No. 06-256126 International Publication No. 2013/038575 JP2015-39359A JP 2010-229052 A JP 2003-277213 A JP 07-258636 A

Buxton et al. (1965) "Effect of solument with chitin on pear wilt caused by Fusarium oxysporum f. Sp. Psi." Ann. Biol. 55: 83-88 Komada et al. (1965) “Ecological study of yellow radish yellow rot (I) Relationship between pathogenic bacteria and other microorganisms in the soil and biological control by addition of chitin.” Soil and microorganisms 7: 41-48. Ashley et al. (1998) "Effects of chitin and chitosan on the incident and severity of Fusarium yellows of celery." Plant Dis. 82: 322-328.

  This invention is made | formed in view of the said problem of a prior art, Comprising: The provision of the technique which suppresses the onset by a Fusarium disease is made into the problem which should be solved. Specifically, the provision of a substance that promotes the growth of bacteria that antagonize Fusarium bacteria in the soil and suppresses the growth of Fusarium bacteria, that is, the provision of an antagonistic bacterial growth promoting substance of Fusarium, and a method for producing the same, and The present invention has been made as a problem to be solved by providing a method for suppressing Fusarium disease using an antagonistic bacterial growth promoting substance of Fusarium bacteria.

  The antagonistic bacterial growth promoting substance of Fusarium according to the present invention comprises one or more compounds selected from the group consisting of adenosine, 5′-S-methyl-5′-thioadenosine, and γ-glutamyl-S-allylcysteine. It is characterized by containing.

  The fusarium antagonistic bacterial growth promoting substance according to the present invention is a composition extracted from the root of the genus Allium.

  The present invention provides a method for inhibiting crop lesions caused by Fusarium fungi. In the method of the present invention, one or more compounds selected from the group consisting of adenosine, 5′-S-methyl-5′-thioadenosine, and γ-glutamyl-S-allylcysteine are used as a nursery for raising seedlings or plant seedlings. It is characterized by inhibiting Fusarium disease by applying to the soil of the field where the plants are planted.

  The method for inhibiting Fusarium disease of the present invention comprises a step of freezing the roots of the genus Allium, a step of grinding the roots of the frozen leeks, and suspending the suspended roots of the ground leeks in sterile water. Obtaining a liquid, filtering the suspension to obtain a filtrate, and subjecting the filtrate to chromatography to adenosine, 5′-S-methyl-5′-thioadenosine, and γ-glutamyl-S-allyl. A step of fractionating one or more compounds selected from the group consisting of cysteine, a step of applying the compound to a nursery for raising seedlings or a field soil for planting plant seedlings, and a step of growing antagonistic bacteria against Fusarium And a step of suppressing the growth of Fusarium bacteria.

  The present invention also provides a method for producing an antagonistic bacterial growth promoting substance of Fusarium. The method of the present invention includes a step of freezing the roots of the genus Allium, a step of grinding the frozen leeks root, and a step of suspending the ground roots of the leeks in sterile water to obtain a suspension. Filtering the suspension to obtain a filtrate and chromatographing the filtrate from the group consisting of adenosine, 5′-S-methyl-5′-thioadenosine, and γ-glutamyl-S-allylcysteine. And a step of fractionating one or more selected compounds.

  The inventor paid attention to the fact that mixed planting and rotation of the genus Allium, which is a traditional farming method, is effective in suppressing Fusarium disease, and analyzed the mechanism. As a result, it was confirmed that antagonistic bacteria of Fusarium bacteria grew in the soil by substances released from the genus Allium into the soil, and the antagonistic bacteria inhibited the growth of Fusarium bacteria and infection of plant bodies. The antagonistic bacterial growth promoting substance of Fusarium is one or more compounds selected from the group consisting of adenosine, 5′-S-methyl-5′-thioadenosine, and γ-glutamyl-S-allylcysteine. We identified and established a method for producing an antagonistic bacterial growth promoting substance of Fusarium from the roots of Allium. Furthermore, by adding the substance of the present invention to soil, it was confirmed that the occurrence of Fusarium disease was suppressed, and the present invention was made.

  The antagonistic bacterium of Fusarium in the present invention refers to a bacterium that directly or indirectly inhibits the action of Fusarium. In other words, it has antibacterial activity against Fusarium bacteria and suppresses the growth of Fusarium bacteria, as well as, for example, activates plant immunity and inhibits Fusarium bacteria's activities in tissues to prevent disease. It refers to bacteria in general.

  The antagonistic bacterial growth promoting substance of Fusarium bacterium of the present invention, when applied to soil, causes the antagonistic bacteria to accumulate and grow in the soil in about one week, and the antagonistic bacterium propagates Fusarium bacteria or infects plants. By inhibiting, Fusarium disease can be suppressed.

  The Fusarium antagonizing bacterial growth promoting substance of the present invention can prevent Fusarium disease even if it is applied before or after the Fusarium bacterium spreads in the soil. That is, it has both a preventive effect on Fusarium disease and a recovery effect.

  The antagonistic bacterial growth promoting substance of Fusarium according to the present invention has an action of activating an indigenous antagonistic microorganism that inhabits each land to promote growth. For this reason, even if the physicochemical properties of soil such as pH, organic matter content, heavy metals, etc., weather conditions, and biological properties such as indigenous microorganisms differ, the effect can be stably expressed.

  According to the present invention, it is possible to prevent Fusarium disease and recover from Fusarium disease even for crops for which resistant varieties and effective rootstocks have not been developed, and crops for which mixed planting or rotation with the Allium genus could not be used It becomes.

  According to the present invention, disease control soil can be obtained without using chemicals such as soil fumigation disinfectant. Moreover, when the antagonistic bacterial growth promoting substance of Fusarium of the present invention is applied once to soil, the effect continues for about 4 weeks or more.

  The Fusarium antagonizing bacterial growth promoting substance of the present invention can be easily and inexpensively produced from an onion genus extract.

FIG. 1 is a flowchart showing an example of a method for producing an antagonistic bacterial growth promoting substance of Fusarium according to the present invention. FIG. 2 is a flowchart showing an example of a method for inhibiting Fusarium disease by the Fusarium antagonistic bacterial growth promoting substance of the present invention. FIG. 3: is a figure which shows the evaluation result of the inhibitory effect of the cucumber vine split disease of the cultivation soil of the genus Allium, and the soil which added the extract of the genus Allium of this invention. FIG. 4 is a diagram showing the relationship between the treatment temperature and the disease-suppressing effect when the soil cultivated with the genus Allium is heat-treated. FIG. 5 is a diagram showing the evaluation results of the heat resistance of the active ingredients of the leek root extract. FIG. 6 is a diagram showing the evaluation results of the severity of cucumber vine split disease when a leek root extract is applied to sterilized soil. FIG. 7: is a figure which shows the evaluation result of the onset degree of a cucumber vine split disease when the heat processing of a soil is performed after providing the extract of a leek root of this invention. FIG. 8 is a diagram showing the effect of the leek root extract of the present invention on soils having different biological and physicochemical properties. FIG. 9 is a diagram showing changes in the number of bacteria when Fusarium fungus is cultured in a medium containing a soil component to which an extract of the genus Allium of the present invention is added. FIG. 10 is a diagram showing a period until the onion root extract of the present invention induces the deterrence of cucumber vine split disease. FIG. 11A is a diagram showing the effect of inhibiting the growth of Fusarium fungus when the leek root extract of the present invention is applied to the soil on which cucumber vine split fungus has grown. FIG. 11B is a diagram showing the effect of inhibiting the growth of Fusarium fungi when the extract of the leek root of the present invention is applied to the soil on which the cucumber vine split fungus has grown, and cucumber is planted. FIG. 12 is a diagram showing the effect of reducing the degree of illness by applying the extract of the leek root of the present invention to the soil on which cucumber vine split fungus has grown. FIG. 13: is a figure which shows the induction effect of the disease suppression property of each division which fractionated the extract of the leek root of this invention with the organic solvent. FIG. 14 is a diagram showing the results of verifying the heat resistance of the antibacterial substance contained in the leek root extract of the present invention. FIG. 15 is a diagram showing a cluster analysis result of a bacterial community in the soil caused by addition of a plant root extract. FIG. 16 is a graph showing the effect of suppressing Fusarium disease when mixed genus Allium and cucumber are planted. FIG. 17 is a graph showing the effect of suppressing Fusarium disease when mixed onion and spinach are planted together. FIG. 18 is a graph showing changes in soil microorganisms when the soil on which the leeks and onions are grown is heat-treated. FIG. 19 is a graph showing a change in the effect of inhibiting the growth of Fusarium bacteria when heat treatment or antibiotic treatment is applied to the onion cultivation soil. FIG. 20 is a graph showing changes in the number of microorganisms in the cultivated soil when the onion cultivated soil is treated with antibiotics. FIG. 21 is a graph showing the inhibitory effect on the degree of occurrence of Fusarium disease in soil to which an antagonistic bacterial growth promoting substance according to the present invention is added. FIG. 22 is a graph showing the incidence of Fusarium disease when the antagonistic bacterial growth promoting substance according to the present invention is added to sterilized soil. FIG. 23 is a graph showing the growth inhibitory effect of Fusarium bacteria when the antagonistic bacterial growth promoting substance according to the present invention is added directly to the Fusarium culture medium. FIG. 24 is a graph showing the effect of inhibiting the growth of Fusarium bacteria in the soil to which the antagonistic bacterial growth promoting substance according to the present invention is added. FIG. 25 is a graph comparing the incidence of Fusarium disease between adenosine, which is one of antagonistic bacterial growth promoters, and other ribonucleosides. FIG. 26 is a graph showing the relationship between the concentration and the inhibitory effect of Fusarium disease in 5′-S-methyl-5′-thioadenosine and γ-glutamyl-S-allylcysteine, which are antagonistic bacterial growth promoters. FIG. 27 is a graph showing the effect of inhibiting the occurrence of Fusarium disease for each antagonistic bacterial group.

  Adenosine, 5′-S-methyl-5′-thioadenosine, and γ-glutamyl-S-allylcysteine, which are contained in the Fusarium antagonistic bacterial growth promoter of the present invention, control the Fusarium disease and its characteristics. Will be described with reference to the drawings. In the present embodiment, mainly Fusarium oxysporum EF. SP. Using Cucamerinum (Fusarium oxysporum f. Sp. Cucumerinum, hereinafter also referred to as cucumber vine split fungus), the pathogenesis inhibiting effect of Fusarium disease is evaluated.

  FIG. 21 shows a cucumber vine using soil to which adenosine, 5′-S-methyl-5′-thioadenosine and γ-glutamyl-S-allylcysteine, which are three types of antagonistic bacterial growth promoters of the present invention, are added. The result of having evaluated the disease prevention effect of split disease is shown.

  In order to quantitatively evaluate the effect, 6 kg of soil collected from the field was prepared, and each substance was applied at the concentrations shown below. Adenosine and 5'-S-methyl-5'-thioadenosine were applied to 6 kg of soil by dissolving 0.1 g in 1 liter of distilled water. γ-Glutamyl-S-allylcysteine was obtained by dissolving 1.34 g in 1 liter of distilled water and applying it to 6 kg of soil.

As a comparative example,
(1) additive-free soil that does not give anything,
(2) Prepare 1 liter of filtrate from 200 g of green onion root, and further dilute about 3 times by adding 650-700 ml of sterile water to 300-350 ml of filtrate. 1 liter added. That is, to which 6 Kg of soil was added with an extract extracted from about 66 g of green onion,
(3) A solution obtained by dissolving 0.1 g of adenine in 1 liter of distilled water and giving it to 6 kg of soil,
(4) What gave 0.1 kg of tyramine dissolved in 1 liter of distilled water to 6 kg of soil,
(5) A solution obtained by dissolving 0.1 g of 2-deoxyadenosine in 1 liter of distilled water and giving it to 6 kg of soil;
5 types of soil were prepared.

  The disease severity shown on the vertical axis in FIG. 21 is the result of evaluating the disease severity after cultivating each soil at 25 ° C. for 1 week, adding cucumber vine split fungi and planting cucumber seedlings. The disease severity here means that among all the young seedlings evaluated, the index of cotyledon wilting is index 1, yellowing or browning of canal and yellowing of hypocotyl is index 2, and the entire strain is severely wilting or It is a value calculated by the following formula so that complete death is indexed 3 and each lesion is weighted.

Disease severity = (3A + 2B + C) / 3N × 100
Here, A is the number of seedlings with index 3, B is the number of seedlings with index 2, C is the number of seedlings with index 1, and N is the number of all seedlings evaluated.

  As shown in FIG. 21, the disease severity of the non-added soil was 62%, whereas the disease severity of the soil to which the extract of the genus Allium was added was 24%. Was low. The severity of the soil to which the substance of the present invention was added was 24% for adenosine, 38% for 5′-S-methyl-5′-thioadenosine, and 18% for γ-glutamyl-S-allylcysteine, which was significantly The result was low. In addition, the adenine, tyramine, and 2-deoxyadenosine of the comparative examples all had a disease severity exceeding 50%, and no significant difference in disease severity was found between the additive-free soil.

  This experiment confirmed that the effect of inhibiting Fusarium disease can be obtained by adding adenosine, 5′-S-methyl-5′-thioadenosine and γ-glutamyl-S-allylcysteine to the soil. It was.

  In order to confirm the suppression mechanism of Fusarium disease by adenosine according to the present invention, 5′-S-methyl-5′-thioadenosine, and γ-glutamyl-S-allylcysteine, the same as when applied to soil in a field Each substance was applied to the sterilized soil at a concentration of 1, cultivated at 25 ° C. for 1 week, cucumber vine split fungi were added, cucumber seedlings were planted, and the disease severity was evaluated. The results are shown in FIG. In any of the substances of the present invention, when cucumber vine split fungus was added to sterilized soil and cultured, no reduction in disease severity was observed, and no clear effect of suppressing Fusarium disease was obtained. From this result, it was confirmed that the substance according to the present invention itself has no effect of suppressing Fusarium disease.

  Furthermore, adenosine, 5′-S-methyl-5′-thioadenosine, and γ-glutamyl-S-allylcysteine were each added to the cucumber vine split fungus inoculated on the PSB medium at 25 ° C. The results of measurement as the number of cucumber vine split fungi (GFP strain density) when cultured for 30 hours are shown in FIG. There was no significant difference in the number of cucumber vine split bacteria between the case where the culture was carried out in a medium not added with the substance and the case where the culture was carried out in a medium containing the substance of the present invention. It was confirmed that the substance of the present invention itself does not have the effect of inhibiting the growth of Fusarium bacteria.

  Next, the following evaluation was performed in order to evaluate the antagonistic property with respect to the Fusarium microbe of the soil which added the substance of this invention. An aqueous solution of adenosine 0.1 g / L, an aqueous solution of 5′-S-methyl-5′-thioadenosine 0.1 g / L, and an aqueous solution of γ-glutamyl-S-allylcysteine 1.34 g / L were prepared. Each aqueous solution was added to the soil in the field and cultured for 1 week. These aqueous solutions were further diluted 1000 times to prepare samples. Each sample was added to PSB medium inoculated with cucumber vine split fungus and cultured at 25 ° C. for 16 hours. At this time, the result of having measured the strain density of the cucumber vine split disease bacteria is shown in FIG. The strain density when cultured in an additive-free medium is 6.09 (Log10 bud cells / mL), whereas the strain density when adenosine is added is 5.84, and 5′-S-methyl- When 5′-thioadenosine was added, it was 5.82, and when γ-glutamyl-S-allylcysteine was added, it was 5.50. By adding the substance of the present invention, the number of cucumber vine split bacteria was significantly increased. Had decreased. From this result, it was confirmed that when the substance of the present invention was added to non-sterilized soil, the effect of inhibiting the growth of Fusarium bacteria was obtained.

  From the above results, the substance according to the present invention does not directly act on Fusarium bacteria, but the bacteria having the action of inhibiting the growth of Fusarium bacteria proliferate, thereby obtaining the effect of inhibiting Fusarium disease infection. It was predicted that Therefore, the bacteria that promote the growth by the substance of the present invention and suppress Fusarium disease were identified.

  In the genus Allium, the dominant bacteria group that grows around the roots of the onion and onion is isolated, and the cucumber vine cracking is performed using the soil that has been cultured for 16 days after adding each fungus group and cucumber vinegar. The severity of the disease was evaluated. As a result, the inhibitory effect of cucumber vine split disease as shown in FIG. 27 was confirmed in the three fungal groups of Flavobacterium, Burkholderia and Pseudomonas isolated from the surroundings of the roots of the onion and onion. The relative disease severity (%) shown in FIG. 27 is the disease severity of the soil inoculated with the strain relative to the disease severity of the soil not inoculated with the strain.

  The median relative morbidity of cucumber vine split disease in the soil to which Flavobacterium derived from leeks was added was 47%, and the median relative morbidity of Flavobacterium added to soil derived from onions was 50%. The median relative disease severity of onion-derived Burkholderia-added soil was 47%, and the median relative disease severity of onion-derived Burkholderia-added soil was 37.5%. The median relative disease severity of the onion-derived Pseudomonas-added soil was 75%, and the median relative disease severity of the onion-derived Pseudomonas-added soil was 67%. Thus, it was confirmed that the three fungal groups of Flavobacterium, Burkholderia, and Pseudomonas have an effect as an antagonistic bacterium of Fusarium bacteria and can suppress Fusarium disease.

  Table 1 below shows the results of confirming that the substance according to the present invention grows Fusarium antagonistic bacteria. Table 1 shows an aqueous solution of adenosine 0.1 g / L, an aqueous solution of 5′-S-methyl-5′-thioadenosine 0.1 g / L, and an aqueous solution of γ-glutamyl-S-allylcysteine 1.34 g / L. The values of the population density (cfu / dry soil) of Flavobacterium and Pseudomonas or Burkholderia, which are antagonistic bacteria of Fusarium, are shown when each field is added to the field soil and cultured for 1 week. .

  As shown in Table 1, in all of adenosine, 5′-S-methyl-5′-thioadenosine and γ-glutamyl-S-allylcysteine according to the present invention, Flavobacterium and Pseudomonas or Burkholderia which are antagonistic bacteria of Fusarium bacteria The effect of increasing the population density of the sea was confirmed.

  Since adenosine is a kind of ribonucleoside, it was confirmed whether other ribonucleosides also have an effect of promoting the growth of Fusarium antagonistic bacteria. The result is shown in FIG.

  A solution of 0.1 g of adenosine, guanosine, ribothymidine, uridine, and cytidine dissolved in 1 liter of distilled water is applied to 6 kg of soil, cultured at 25 ° C. for 1 week, and then added with cucumber vine split fungus. Then, seedlings of cucumber were planted and the severity was evaluated. As shown in FIG. 25, the disease severity of adenosine was 24%, while the disease severity of other ribonucleosides was 50% or more, which was significant in suppressing the disease severity of cucumber vine cracking disease. The effect was not confirmed.

  As a result of confirming the concentration of 5′-S-methyl-5′-thioadenosine and γ-glutamyl-S-allylcysteine, the inhibitory effect of the disease is expressed among the antagonistic bacterial growth promoting substances of Fusarium of the present invention. Is shown in FIG. About 5'-S-methyl-5'-thioadenosine and γ-glutamyl-S-allyl cysteine, 0.1 g, 0.5 g and 1 g, respectively, were dissolved in 1 liter of distilled water. Prepared aqueous solutions were applied to 6 kg of soil. After cultivating this soil at 25 ° C. for 1 week, cucumber vine split fungi were added, cucumber seedlings were planted, and the disease severity after growing for 12 hours and 21 days was evaluated.

  5'-S-methyl-5'-thioadenosine has a disease severity of 28% when applied with an aqueous solution of 0.1 g / L, and 5% by Dunnett test compared to soil to which nothing has been added. A significant difference of less than was confirmed. γ-Glutamyl-S-allylcysteine has a disease severity of 18% when a 1 g / L aqueous solution is applied, and a Dunnett test confirms a significant difference of less than 1% compared to soil to which nothing has been added. It was done. From this result, it was confirmed that 5'-S-methyl-5'-thioadenosine was effective in a smaller amount than γ-glutamyl-S-allylcysteine.

  The concentration at which the fusarium antagonistic bacterial growth promoting substance of the present invention is used can be appropriately adjusted depending on the physicochemical properties and biological properties of the soil.

  When adenosine, 5′-S-methyl-5′-thioadenosine and γ-glutamyl-S-allylcysteine according to the present invention are applied to soil, the growth of Flavobacterium and Pseudomonas or Burkholderia, which are antagonistic bacteria of Fusarium, It is an antagonistic bacterial growth promoting substance of Fusarium that promotes These substances can be easily and inexpensively manufactured using, for example, the roots, stems, and leaves of the genus Allium. An example of the method for producing an antagonistic bacterial growth promoting substance of Fusarium according to the present invention is shown in FIG. This production method includes a step of freezing the roots of the genus Allium (step S1), a step of grinding the roots of the frozen leeks (step S2), and suspending the ground roots of the ground leeks in sterilized water. It includes a step of obtaining a suspension (step S3), a step of filtering the suspension to obtain a filtrate (step S4), and a step of fractionating substances by chromatography (step S5). . In addition, the manufacturing method of the antagonistic bacterial growth promoting substance of Fusarium according to the present invention is not limited to the method illustrated in FIG. The method described in FIG. 1 is an exemplification, and any method such as synthesis can be adopted as long as it is a method capable of obtaining these substances, not limited to extraction from natural products.

  FIG. 2 shows a flowchart of an example of a method for inhibiting Fusarium disease using a growth promoting substance of an antagonistic bacterium of Fusarium according to the present invention. The method of the present invention includes a step of freezing the roots of the genus Allium (step S11), a step of grinding the roots of the frozen leeks (step S12), and suspending the ground roots of the leeks in sterilized water. A step of obtaining a suspension (step S13), a step of filtering the suspension to obtain a filtrate (step S14), a step of fractionating a substance by chromatography (step S15), and a liquid containing the substance Including a step (step S16) of imparting to the soil, a step of growing antagonistic bacteria against Fusarium bacteria (Step S17), and a step of suppressing the growth of Fusarium bacteria (Step S18). The method for suppressing Fusarium disease described in FIG. 2 is also an example, and the method for obtaining a substance to be applied to this method is not limited to the method for extracting from the root of the genus Allium. In order to suppress Fusarium disease, it is sufficient that the antagonistic bacterial growth promoting substance of Fusarium bacterium according to the present invention can be introduced, and Steps S11 to S15 can be replaced with a method for producing a substance such as synthesis.

  The antagonistic bacterial growth promoting substance of Fusarium according to the present invention is adenosine, 5′-S-methyl-5′-thioadenosine, γ-glutamyl-S-allylcysteine, and a crude extract of the genus Allium containing these substances. By using this, it is possible to easily obtain an equivalent effect of growing an antagonistic bacterium of Fusarium bacteria.

  The “genus onion” described below includes leek, onion, leek and garlic.

  The method for producing the onion genus extract is the same as steps S1 to S4 in FIG. That is, a step of freezing leek roots (step S1), a step of grinding frozen leek roots (step S2), and a suspension of ground leek roots suspended in sterile water And a step of filtering the suspension to obtain a filtrate (step S4).

  In the method for producing an onion genus extract of the present invention, before performing chromatography, the filtrate obtained by filtering the suspension is subjected to liquid-liquid distribution in the order of hexane, ether, and ethyl acetate, and then each fraction is separated. A step of concentrating and drying with an evaporator can be provided.

  The soil improvement method using the extract of the genus Allium can apply the processes from steps S11 to S14 and steps S16 to S18 in FIG. As shown in the flowchart, the step of freezing the roots of the genus Allium (step S11), the step of grinding the roots of the frozen leeks (step S12), and the ground roots of the ground leeks are suspended in sterile water. A step of obtaining a suspension (step S13), a step of filtering the suspension to obtain a filtrate (step S14), a step of applying the filtrate to soil (step S16), and a growth of antagonistic bacteria against Fusarium bacteria And a step of suppressing the growth of Fusarium bacteria (step S18).

  In the method for improving soil with the root extract of the present invention, the filtrate obtained in step S14 is liquid-liquid distributed in the order of hexane, ether, and ethyl acetate, and then each fraction is concentrated and dried with an evaporator. This fraction can be applied to the soil.

  Below, the result of having evaluated the characteristic which grows the antagonistic bacteria of Fusarium bacteria of the crude extract of Allium is shown. In the following evaluation, among Fusarium oxysporum, Fusarium oxysporum EF. SP. The characteristics of the extract of the present invention and the effect of the soil improvement method using the extract will be described by giving an example in which the disease inhibitory effect of Cusarium num (Fusarium oxysporum f. Sp. Cucumerinum) was evaluated.

  An extract of the genus Allium is prepared by preparing a 1 liter filtrate from 200 g of the root, and further diluting it about 3 times by adding 650 to 700 ml of sterilized water to 300 to 350 ml of the filtrate. 1 liter is applied to 6 kg of soil. This means that an extract extracted from about 66 g of green onion was applied to 6 kg of soil.

  In FIG. 3, the result of the suppression effect of the cucumber vine split disease of the cultivation soil of the genus Allium and the soil which added the extract of the genus Allium is shown. The disease severity shown on the left side of FIG. 3 is obtained by adding cucumber, tomato, leek, onion and leek root extract to the same non-sterile soil and culturing at 25 ° C. for 1 month, It is the result of planting cucumber seedlings with the addition of the disease fungus and evaluating the disease severity. The morbidity shown on the right side of FIG. 3 is that the cucumber, tomato, onion, onion and leek are cultivated in the same non-sterile soil, and after removing the cultivated plants, cucumber vine split fungus is added and It is the result of planting young seedlings and evaluating the disease severity.

  In soils cultivated with leeks, onions, and leek, and soils added with an extract of the genus leeks, pathogenesis was significantly suppressed compared to soils without any added soil and cultivated cucumbers and tomatoes. The Moreover, the soil which added the extract of the genus leek genus has the disease inhibitory effect equivalent to the soil which cultivated leek, onion, and leek.

  FIG. 4 is a graph showing the relationship between the treatment temperature and the disease-suppressing effect when the soil cultivated with the genus Allium is heat-treated. As the genus Allium, soil cultivated with onion, onion, leek and garlic was used. For comparison, soil that was not cultivated and soil that cultivated cucumber were used. The above six kinds of soils were each heat-treated at 40 ° C., 60 ° C., and 80 ° C. for 30 minutes. Moreover, it heat-processed for 60 minutes at the temperature of 121 degreeC. To each soil after the heat treatment, cucumber vine split fungi were added and cucumber seedlings were planted, and the severity of the disease was evaluated.

  The soil in which cucumber was cultivated showed a high disease severity equivalent to the soil in which nothing was cultivated, regardless of the presence or absence of temperature treatment. On the other hand, the soil which cultivated leek and onion was confirmed to have a very high disease control effect until heating at 40 ° C. However, when the heat treatment at a temperature of 60 ° C. or higher was performed, the effect of suppressing the disease was hardly observed. Soil cultivated with leek and garlic was found to have a significantly higher disease control effect than cucumber cultivated soil until heating at 40 ° C. The inhibitory effect was almost not recognized. From the above, the disease-suppressing effect is 1) a substance that is inactivated at 60 ° C. or higher supplied from the roots of Allium, or 2) a heat of 60 ° C. or higher that is propagated by a substance supplied from the roots of Allium. One of the weak soil microorganisms has the effect of suppressing the disease.

  FIG. 5 is a graph showing an evaluation of the heat resistance of the onion root extract-containing material. The evaluation method is as follows. The leek root extract produced by the production method shown in FIG. 2 is heated at 40 ° C., 50 ° C., 60 ° C., 70 ° C., 80 ° C., and 121 ° C. for 30 minutes, respectively, and is not sterilized. And cultured at 25 ° C. for 1 month. Then, cucumber vine split fungi were added, and cucumber seedlings were planted to evaluate the disease severity. For comparison, an extract of a leek root that was not heat-treated was applied to evaluate the severity of the disease.

  The extract that was subjected to the heat treatment at 40 ° C. had reduced cucumber vine split disease pathogenesis. On the other hand, even in the case of an extract subjected to heat treatment at 50 ° C., 60 ° C., and 70 ° C., a certain disease prevention effect is recognized, and the disease inhibition property is further reduced to 80 ° C. or more. This was the case where the heat treatment was performed. From this, it is clear that the disease-inhibiting substance contained in the roots of leeks consists of at least two kinds of substances that are inactivated at less than 40 ° C. and substances that have heat resistance up to around 70 ° C. It was.

  FIG. 6 is a graph showing an evaluation of the severity of cucumber vine split disease when an onion root extract is applied to sterilized soil. The evaluation method is as follows. The leek root extract produced by the production method shown in FIG. 2 was applied to sterilized soil, cultured at 25 ° C. for 1 month, added with cucumber vine split fungus, planted with cucumber seedlings, and evaluated for disease severity. . In addition, sterilized soil that had been cultivated at 25 ° C for 1 month with an extract of a leek root was further heat-treated at four stages of 40 ° C, 60 ° C, 80 ° C, and 121 ° C for 30 minutes, respectively. Cucumber seedlings were planted with the addition of the disease fungus and the severity of the disease was evaluated. As a comparative evaluation, sterilized soil was cultured at 25 ° C. for one month, cucumber vine split fungi were added, cucumber seedlings were grown, and the disease severity was evaluated.

  When leek root extract was applied to sterilized soil, a mild inhibitory effect on cucumber vine split disease was observed, but almost no cucumber vine split disease inhibitory effect was observed when heated at 40 ° C or higher. From this, it was found that the suppression of cucumber vine split disease is due to the effect of soil microorganisms that are propagated by the substance supplied from the root of the genus Allium, rather than the effect of the substance itself contained in the extract of the onion root.

  FIG. 7 is a graph showing an evaluation of the degree of occurrence of cucumber vine split disease when an onion root extract is applied to non-sterilized soil and the soil is heat-treated. The evaluation method is as follows. The leek root extract produced by the production method shown in FIG. 2 was applied to non-sterilized soil and cultured at 25 ° C. for 1 month. Thereafter, the soil was heat-treated at 40 ° C. and 60 ° C. for 30 minutes, respectively. Moreover, it heat-processed for 60 minutes at the temperature of 121 degreeC. To each soil after the heat treatment, cucumber vine split fungi were added and cucumber seedlings were planted, and the severity of the disease was evaluated. As a comparative evaluation, after adding an onion root extract to non-sterilized soil and non-sterilized soil and culturing at 25 ° C. for one month, cucumbers were grown without heat treatment, and the disease severity was evaluated.

  When leek root extract is applied to soil that has not been sterilized, the effect of suppressing cucumber vine split disease is remarkable with heat treatment at 40 ° C or lower, but the effect of suppressing cucumber vine split disease with heat treatment at 60 ° C or higher. No effect was observed. From these results, it was found that the suppression of cucumber vine split disease is greatly affected by soil microorganisms that are propagated by substances supplied from the roots of the genus Allium, and the soil microorganisms are vulnerable to heat.

  FIG. 8 shows the heating of the soil after applying the root extract of leeks to two types of non-sterile soils with different biological and physicochemical properties collected from a different location from the soil used in FIG. It is the graph which evaluated the morbidity degree of the cucumber vine split disease at the time of processing. Even when applied to different soils, the leek root extract showed a remarkable inhibitory effect on cucumber vine split disease. It was also the same that this effect was maintained by heat treatment at 40 ° C. or lower. Since the extract of the genus Allium of the present invention has an action of activating and promoting proliferation of indigenous antagonistic microorganisms that inhabit each land, the soil physics and chemistry such as pH, organic matter content, heavy metals, etc. It was confirmed that the effects can be expressed stably even if the biological properties of the microorganisms and indigenous microorganisms are different.

  FIG. 9 shows the change in the number of bacteria when a cucumber vine split fungus is cultured in a medium containing a soil component to which an extract of the genus Allium is added. The evaluation method is as follows. The leek root extract produced by the production method shown in FIG. 2 was applied to non-sterilized soil and cultured at 25 ° C. for 1 month. Thereafter, a diluted soil solution (hereinafter also referred to as a soil component) to be added to the medium was heat-treated at a temperature of 40 ° C. or 60 ° C. for 30 minutes. And after adding 10% of soil components to the liquid medium, cucumber vine split fungus was added, and this liquid medium was used as a sample. As another sample, a soil component added with an antibacterial antibiotic and shake-cultured for 1 hour was added to a liquid medium together with cucumber vine split fungus to prepare a sample. Each sample was subjected to shaking culture. As a comparative sample, a liquid medium to which cucumber vine split fungus was added was also prepared for sterilized water-added soil, and shake culture was performed. In addition, the cucumber vine split fungus was cultured with shaking in the medium alone. The number of spores (or bud cells) of cucumber vine split fungi after culturing of each sample was counted.

  In the liquid medium diluted with the soil to which the extract of the leek root was applied, the effect of inhibiting the growth of cucumber vine split fungus was obtained by heat treatment at 40 ° C. On the other hand, in the heat treatment at 60 ° C., the growth inhibitory effect of cucumber vine split fungus was lowered. Even when an antibacterial antibiotic was added, the growth inhibitory effect of cucumber vine split fungus decreased. From this, it was found that the bacteria influence the growth inhibitory effect of the pathogenic bacteria, and the bacteria accumulate and grow by the substance supplied from the root of the genus Allium, but are weak against heat.

  FIG. 10 shows a period until the onion root extract induces the deterrence of the onset of the cucumber vine split fungus. The evaluation method is as follows. The leek root extract produced by the production method shown in FIG. 2 is applied to non-sterilized soil, and each of 0 days, 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks is used at 25 ° C. Incubated with Then, the cucumber vine split fungus was added, the cucumber seedling was planted, and the disease severity was evaluated. As a result, it was confirmed that after 1 week of culturing, the disease-inhibiting property was remarkably increased and the effect was stably continued.

  FIG. 11A and FIG. 11B show the growth inhibitory effect of Fusarium fungus when the extract of the leek root of the present invention is added to soil in which cucumber vine split fungus has already grown and been contaminated. The evaluation method is as follows. The leek root extract produced by the production method shown in FIG. 2 was applied to the soil on which cucumber vine split fungi had grown and cultured at 25 ° C. for 1 month. Then, the result of having evaluated the number of the cucumber vine split bacteria in soil is shown to FIG. 11A. As a comparative example, a soil to which sterilized water was applied instead of the onion root extract was evaluated. As a result, the soil to which the extract of the leek root was applied had a significantly reduced number of fusarium bacteria compared to the soil to which sterilized water was applied. It was confirmed that the number of fungi was decreased by adding the extract of leeks root. Further, after extracting a leek root extract to soil contaminated with cucumber vine split fungi and culturing at 25 ° C. for one month, cucumber seedlings were planted, and then the number of cucumber vine split fungi was evaluated. As a comparative example of this test, cucumber seedlings were planted for the same period in soil contaminated with cucumber vine split bacteria cultivated with sterilized water, and the number of cucumber vine split fungi was evaluated. The result is shown in FIG. 11B. Compared to the soil with sterilized water, the soil with the leek root extract had a significantly reduced number of Fusarium fungi even after planting cucumbers, and the soil with Fusarium fungi already growing was reduced. Even so, it was confirmed that the addition of the onion root extract reduced the number of Fusarium fungi, and the effect persisted after planting of the crop.

  FIG. 12 shows the effect of decreasing the disease severity when an extract of a leek root is added to soil that has already been contaminated with cucumber vine split fungi. The evaluation method is as follows. The leek root extract produced by the production method shown in FIG. 2 was applied to the soil on which cucumber vine split fungi had grown and cultured at 25 ° C. for 1 month. Thereafter, seedlings of cucumber were planted and the severity was evaluated. As a comparative example, a soil to which sterilized water was applied instead of the onion root extract was evaluated. Compared with soil with sterilized water, the soil with leek root extract has a significantly reduced disease severity, even if the cucumber vine split fungus has already grown. It was also confirmed from the difference in the disease severity that the disease severity was reduced by adding the root extract.

  FIG. 13 shows the disease-inhibiting effect of each section obtained by fractionating an extract of leek root with an organic solvent. The evaluated sample was obtained by separating the filtrate of the onion root extract obtained in step S4 of FIG. 1 in the order of hexane, ether and ethyl acetate, and then concentrating and drying each fraction with an evaporator. is there. Each fraction was re-dissolved in water and applied to the soil, and the disease-inhibiting effect after culturing for 1 week and 4 weeks was confirmed. The fraction indicated by “emulsion layer” refers to an emulsion layer generated in the intermediate layer during extraction with hexane.

  As shown in FIG. 13, the emulsion layer and the hexane layer induce remarkable disease suppression within one week after the addition, and the effect continues even after four weeks. On the other hand, the other fractions also induce a certain degree of deterrence within one week of addition, but the deterrence was further enhanced after 4 weeks, especially for the soil with an added aqueous layer.

  The result of verifying the heat resistance of the antibacterial substance contained in the leek root extract is shown in FIG. In FIG. 14, samples obtained by heating the onion root extract at 40 ° C. and 60 ° C. and samples not subjected to the heat treatment were added to a liquid medium together with cucumber vine split fungus and cultured, and then cucumber vine split fungus was cultured. The result of measuring the amount of spore growth is shown. The comparative example is a cucumber root extract. The extract of the leek root which has not been heat-treated has an inhibitory effect on the growth of cucumber vine split fungi. It was confirmed by the verification results shown in FIG. 14 that the extract of leek root has an effect of suppressing the growth of Fusarium fungi.

  The effect of suppressing the growth of Fusarium fungus by the extract of leeks shown in FIG. 14 is lost by heating the extract of leeks at 40 ° C. The temperature at which the effect is lost is lower than the result of verification using soil and cucumber seedlings. From this, the inhibitory effect of Fusarium disease by the extract of the genus Allium is supplied from the direct antibacterial effect by the heat-sensitive antibacterial substance contained in the extract of the Allium genus and the root of the Allium genus It was confirmed that both the effects of antagonistic bacteria that accumulate and proliferate with the substance were included.

Bacteria that increase characteristically when an extract of the genus Allium is added to the soil, a gram-negative bacterium with low heat resistance, and a high abundance ratio in any soil There are the following five types of bacteria.
Betaproteobacteria class Burkholderia gammaproteobacteria class Xanthomonas order Alphaproteobacteria class Sphingomonas order Alphaproteria class Rhizobium order Solibacterias class
Some or all of these bacteria function as antagonistic bacteria for Fusarium oxysporum.

  In Fig. 15, non-sterilized soil collected from the same field was added with leeks, leek, onion, cucumber, and tomato root extract and cultured for 1 month. Results of cluster analysis based on this are shown. The numbers attached to the end of each plant name in FIG. 15 indicate the number of repetitions using the same soil, and the length of the branched branches reflects the difference in similarity. From these results, it can be seen that the bacterial community composition formed by the addition of the extract differs depending on the type of plant, and that a similar bacterial community was formed in the soil added with the root extract of allium, and the disease control mechanism Was also found to be almost the same.

  As a result of the above analysis, the extract of the genus Allium of the present invention itself has an action effect of suppressing the growth of Fusarium oxysporum, and further activates and proliferates the antagonistic bacteria that inhabit the soil. It has been clarified that by promoting, the growth of Fusarium oxysporum is suppressed and the incidence of Fusarium disease is lowered.

  FIG. 18 is a graph showing changes in soil microorganisms when the soil on which the leeks and onions are grown is heat-treated. The heat treatment conditions are 30 minutes at temperatures of 40 ° C., 60 ° C., 80 ° C., and 121 ° C., respectively. Although no microorganisms are reduced by heat treatment at 40 ° C., Gram-negative bacteria and filamentous fungi are greatly reduced by treatment at 60 ° C. In particular, it was confirmed that the number of gram-negative bacteria decreased to below the detection limit.

  FIG. 19 is a graph showing changes in the effect of inhibiting the growth of pathogenic bacteria when heat treatment or antibiotic treatment is applied to the onion cultivation soil. In leek cultivation soil, there is a factor that suppresses spore growth of cucumber vine split fungi. The inhibitory factor is not inactivated by the heat treatment at 40 ° C., but is deactivated by the heat treatment at 60 ° C. or the addition of an antibacterial antibiotic, or the effect thereof is reduced. From this result, it was confirmed that heat-sensitive bacteria are involved in the action of suppressing spore growth of cucumber vine split disease. Furthermore, it has been clarified that accumulation and growth of such antagonistic bacteria having an inhibitory effect on the growth of pathogenic bacteria are the main factors related to the disease-inhibiting ability of the cultivated scallion.

  FIG. 20 is a graph showing changes in the number of microorganisms in the cultivated soil when the onion cultivated soil is treated with antibiotics. Leek cultivation soil is diluted 1000 times, antibacterial antibiotics are added to the diluted solution, and the number of bacteria and the number of filamentous fungi are counted after 1 hour of culture. As shown in FIG. 20, only the bacteria decrease with the addition of antibiotics, and the number of filamentous fungi hardly changes.

  From the above results, the cause of the pathogenic bacteria growth suppression effect brought about by the heat treatment and the addition of antibiotics was brought about by the decrease of antagonistic bacteria in the green onion culture soil. It was confirmed to be the leading role. That is, it has been clarified that the accumulation and growth of Gram-negative antagonistic bacteria are involved in the pathogenesis of soil suppression by the cultivation of Allium spp. It is clear that a similar mechanism works in the soil to which the root extract of the genus Allium is added, and the disease is suppressed to disease.

  Since the antagonistic bacterial growth promoting substance of Fusarium according to the present invention can suppress lesions of various crops caused by Fusarium, it can be used as a substitute for agricultural chemicals or a soil improver.

Claims (5)

  An antagonistic bacterial growth promoter for Fusarium, comprising one or more compounds selected from the group consisting of adenosine, 5'-S-methyl-5'-thioadenosine, and γ-glutamyl-S-allylcysteine.   The fusarium antagonistic bacterial growth promoting substance according to claim 1, which is a composition extracted from the root of the genus Allium.   Field soil for planting seedlings or plant seedlings for raising seedlings with one or more compounds selected from the group consisting of adenosine, 5′-S-methyl-5′-thioadenosine, and γ-glutamyl-S-allylcysteine To suppress Fusarium disease by applying to Freezing allium roots;
Grinding the frozen leeks root;
Suspending ground leek roots in sterile water to obtain a suspension;
Filtering the suspension to obtain a filtrate;
Fractionating one or more compounds selected from the group consisting of adenosine, 5′-S-methyl-5′-thioadenosine, and γ-glutamyl-S-allylcysteine by subjecting the filtrate to chromatography; ,
Applying the compound to a nursery for raising seedlings or soil in a field for planting plant seedlings;
Growing an antagonistic bacterium against Fusarium,
A step of inhibiting the growth of Fusarium bacteria,
The method for inhibiting Fusarium disease according to claim 3, further comprising: Freezing allium roots;
Grinding the frozen leeks root;
Suspending ground leek roots in sterile water to obtain a suspension;
Filtering the suspension to obtain a filtrate;
Fractionating one or more compounds selected from the group consisting of adenosine, 5′-S-methyl-5′-thioadenosine, and γ-glutamyl-S-allylcysteine by subjecting the filtrate to chromatography; ,
A method for producing an antagonistic bacterial growth promoting substance of Fusarium fungus characterized by comprising:

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