Soil microbiota improving agent, method for preparing soil microbiota improving agent, and method for improving soil microbiota

Abstract

To provide a soil microbiota improving agent, a method for preparing a soil microbiota improving agent, and a method for improving soil microbiota, employing a Lysobacter bacterium unused in conventional microbial pesticides or microbial formulations as a novel microbial resource.MEANS FOR SOLVING THE PROBLEM: The foregoing problem is solved by a soil microbiota improving agent comprising a Lysobacter bacterium or a bacterial flora comprising a Lysobacter bacterium.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a soil microflora improver, a method for preparing the soil microflora improver, and a method for improving soil microflora. [Background technology]

[0002] As an alternative to chemical pesticides, biological control technologies are being developed. Biological control is a method of controlling pathogenic microorganisms using microorganisms with functions such as antibiotics, parasitism, bacteriolysis, and competition. By utilizing the interactions between organisms in the ecosystem, biological control is possible that takes into consideration the environment, such as biodiversity, compared to chemical control that uses chemical pesticides.

[0003] The pesticides that can be used in organic farming are specified in the Organic JAS standard, and around 30 types of microbial pesticides, including biological control pesticides, are registered. In addition, there are many commercially available microbial preparations (preparations containing microorganisms such as bacteria and fungi that aim to manifest the functions of these microorganisms) that can be applied to farmland for the purpose of improving the soil of farmland.

[0004] Lysobacter bacteria are classified in the phylum Proteobacteria > class Gammaproteobacteria > order Xanthomonadales > family Xanthomonadaceae > genus Lysobacter. They are Gram-negative rods approximately 2 to 5 μm in length. Their biological control properties have recently attracted attention, and it has been revealed that they achieve "bacteriolysis" by secreting enzymes such as proteases and chitinases, and "antibiosis" by producing secondary metabolites that act as antibiotics (Non-Patent Document 1). Lysobacter bacteria are a relatively new genus whose classification was established relatively recently (Non-Patent Document 2). Only about 50 species have been identified, and only a few, such as L. enzymogenes, have been well studied (Non-Patent Document 3). Therefore, ongoing research is underway to identify new species and strains, as well as new enzymes and antibiotics (Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] G. Puopolo, J. Appl. Microbiol., 124, 15, (2018). [Non-patent document 2] Stackebrandt E., Int. J. Syst. Bacteriol., 38, 321, (1988) [Non-patent document 3] L. Lin, Front. Plant Sci., 14, 1116147, (2023) [Non-patent document 4] H. Hamamoto, Nat. Chem. Biol., 11, 127, (2015) [Non-Patent Document 5] Shigenobu Yoshida, Chemistry and Biology, 51, 541, (2013) [Non-patent document 6] Umi Etsuko, "Materials and Materials", 122,1, (2006) Summary of the Invention [Problem to be solved by the invention]

[0006] In organic farming, the pesticides that can be used are limited to natural or biological products as specified by the Organic JAS standards. This makes it more difficult to control crop diseases than in conventional farming, where a wide variety of chemical pesticides can be used, and has been a major factor hindering the expansion of organic farming.

[0007] The microorganisms used in microbial pesticides that meet the Organic JAS standard are only about 10 types of bacteria and fungi, such as the genera Bacillus, Pseudomonas, Talaromyces, and Trichoderma. Furthermore, most of these microbial pesticides target above-ground plant diseases, and the current situation is that there are almost no microbial pesticides that can be used against soil-borne diseases that are generally difficult to control and cause serious damage in the field (Non-Patent Document 5).

[0008] Microbial preparations that are applied to agricultural soil for the purpose of soil improvement have been commercialized, and preparations using lactic acid bacteria, koji mold, yeast, actinomycetes, etc. have been commercialized. However, these do not take into consideration compatibility with the soil environment or the prevention of inhibition of colonization by existing soil microbial flora. Furthermore, the microorganisms used are mainly microorganisms that are familiar from food processing, etc., and the colonization of agricultural soil by microorganisms and the resulting soil improvement effects of most of them have not been clear (Non-Patent Document 6).

[0009] An objective of the present invention is to provide a soil microflora improver that utilizes Lysobacter bacteria, which are not used in conventional microbial pesticides or microbial preparations, as a new microbial resource, a method for preparing a soil microflora improver, or a method for improving a soil microflora. [Means for solving the problem]

[0010] Item 1. A soil microflora improver comprising Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria. Item 2. The soil microbiota improver according to Item 1, which is applied to improve the soil microbiota of agricultural land to be improved so that it has a biological control effect against agricultural crop diseases. Item 3. The soil microbiome improver according to Item 1, further comprising a sterilized or sterilized carrier for allowing the Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria to establish an ecological niche in the soil of the agricultural land to be improved. Item 4. The soil microflora improver according to Item 1, further comprising an assimilable organic substance necessary for the survival and / or proliferation of the Lysobacter bacteria or a bacterial flora containing the Lysobacter bacteria. Item 5. The soil microbiome improver according to Item 2, characterized in that the farmland to be improved is at least one farmland selected from conventional farmland, organic farmland that has or has not been certified by the Organic JAS, special cultivation farmland, and environmental conservation farmland. Item 6. A soil microbiome improver according to Item 1, characterized in that it uses Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria recovered from at least one recovery soil selected from the soil of agricultural land selected from conventional agricultural land where the presence of Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria has been confirmed, organic agricultural land with or without JAS organic certification, special cultivation agricultural land, and environmental conservation agricultural land, and soil in the vicinity of the agricultural land. Item 7. The soil microbiota improver according to Item 6, wherein the soil for recovery is soil that has not been affected by agricultural crop diseases in the two to six years immediately preceding soil collection. Item 8. The soil microbiome improver according to Item 4, wherein the assimilable substance is at least one organic substance selected from the group consisting of (A) and (B) below: (A) at least one compound selected from the group consisting of proteins, amino acids, chitin, and N-acetyl-D-glucosamine; or (B) an organic fertilizer containing at least one natural organic compound selected from the group consisting of proteins, amino acids, chitin, and N-acetyl-D-glucosamine. Item 9. The soil microbiome improver according to Item 1, wherein the bacterial flora containing the Lysobacter bacteria is added as a soil bacterial flora solution obtained by recovering bacterial flora containing the Lysobacter bacteria that were indigenous to recovery soil. Item 10. The soil microbiome improver according to Item 1, wherein the bacterial flora containing Lysobacter bacteria is added as a composition rich in Lysobacter bacteria, which is prepared by recovering bacterial flora containing Lysobacter bacteria that have been indigenous to recovery soil and culturing the bacterial flora using assimilable substances. Item 11. The soil microbiome improver according to Item 1, wherein the bacterial flora containing the Lysobacter bacteria is added as soil that has been acclimated by mixing or spraying assimilable substances into recovery soil in which the bacterial flora containing the Lysobacter bacteria is native. Item 12. The soil microbiome improver according to Item 1, wherein the bacterial flora containing bacteria of the genus Lysobacter is added as a composition rich in bacteria of the genus Lysobacter obtained by culturing bacterial flora containing bacteria of the genus Lysobacter recovered from soil indigenously grown by mixing or spraying assimilable substances into the soil and acclimating the soil. Item 13. The soil microbiome improver according to Item 1, wherein the bacterial flora containing the Lysobacter bacteria is added as a composition rich in Lysobacter bacteria, which is obtained by culturing a concentrate obtained by concentrating the Lysobacter bacteria from a soil bacterial flora solution using multiple types of membrane filters with different pore sizes. Item 14. The soil microflora improver according to Item 12, wherein the content of Lysobacter bacteria in the bacterial flora contained in the composition with a high content of Lysobacter bacteria is 3.7% or more. Item 15. The soil microflora improver according to Item 10, wherein the bacterial flora contained in the composition with a high content of Lysobacter bacteria includes Pseudomonas bacteria as bacteria other than Lysobacter bacteria. Item 16. The soil microbiome improver according to Item 10, wherein the bacterial flora contained in the composition rich in Lysobacter bacteria contains 3% or more Lysobacter bacteria of unspecified species, or contains 16 or more different sequences in the V3-V4 region of 16S rRNA. Item 17. The soil microbiome improver according to Item 3, wherein the carrier comprises one or more of at least one porous body selected from the group consisting of soil from which Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria has been collected, biochar, and vermiculite; at least one adsorbent selected from the group consisting of bentonite and zeolite; and at least one mineral selected from the group consisting of mountain soil, mudstone, and shale. Item 18. The soil microbiome improver according to Item 17, wherein the biochar is powdered charcoal or shaped charcoal produced from unused plant biomass materials such as rice straw, rice husks, grass clippings, and waste wood, and wherein one or more types of unused plant biomass materials are used in combination. Item 19. A method for preparing a soil microbiome improver, comprising: preparing a soil sample collected from recovery soil for recovering Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria; and preparing a soil microbiome improver containing Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria, wherein the recovery soil is soil confirmed to contain Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria. Item 20. The method according to Item 19, wherein the preparation comprises mixing or spraying a nutrient-reducing substance into the soil for recovery to acclimate it, and preparing a portion of the acclimated soil as a soil sample. Item 21. A method in which preparing a soil microbiome improver comprises mixing a soil solution obtained by dispersing soil collected from recovery soil in an aqueous liquid, a soil bacterial flora solution that is the supernatant of the soil solution, a concentrated solution of the soil bacterial flora solution, or a composition rich in Lysobacter bacteria obtained by culturing the soil solution, the soil bacterial flora solution, or the concentrated solution, with a carrier and / or assimilable substances, wherein the carrier is used to enable the Lysobacter bacteria or the Lysobacter bacterial flora to obtain an ecological niche in the target soil. Item 22. A method for improving soil microflora, comprising applying the soil microflora improver according to Item 1 to the soil to be improved. [Effects of the Invention]

[0011] A new soil microbiota improver, a method for preparing a soil microbiota improver, or a method for improving soil microbiota can be provided. [Brief explanation of the drawings]

[0012] [Figure 1] This paper outlines methods for improving soil microflora. [Figure 2] 1 shows the change in the content of Lysobacter bacteria in the bacterial flora of the culture medium. DETAILED DESCRIPTION OF THE INVENTION

[0013] First, an example of the method for improving soil microflora of the present invention will be described with reference to FIG.

[0014] FIG. 1 shows a method for improving the microbiome of soil to be improved by carrying out Step I for preparing a soil microbiome improver and Step II for amending the soil with the prepared soil microbiome improver. In one embodiment, step I comprises:

[0015] Step Ia of preparing Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria recovered from recovery soil for recovering Lysobacter bacteria; Step Ib of culturing the prepared Lysobacter bacteria or bacterial flora containing Lysobacter bacteria in the presence of an assimilable substance; Step Ic: preparing a soil microflora amendment using the culture solution obtained in Step Ib Includes: Here, step Ib is an optional step.

[0016] In addition, in one embodiment, step I includes: Step Ia' of preparing soil (acclimatized soil) in which Lysobacter bacteria or bacterial flora containing Lysobacter bacteria have been acclimated by mixing or spraying a nutrient-reducing substance into recovery soil for recovering Lysobacter bacteria or bacterial flora containing Lysobacter bacteria; Step Ib': preparing a soil microbiota amendment using the conditioned soil obtained in step Ia'; Includes: Step II is Step IIa, mixing the soil microbiome amendment into the soil to be amended; Step IIb: Establishing Lysobacter in the amended soil Includes:

[0017] When Lysobacter bacteria are established in the soil to be improved, the enzymes and antibiotics produced by the Lysobacter bacteria can exert a control function against pathogenic microorganisms.

[0018] 1. Soil microflora improver and method for preparing the soil microflora improver An embodiment of the present invention relates to a soil microbiota improver and a method for preparing the soil microbiota improver. Preferably, the soil microbiota improver is applied to improve the soil microbiota of a target agricultural land (hereinafter also referred to as "target agricultural land") so as to have a biological control effect against agricultural crop diseases. In this specification, farmland may be an open field, a greenhouse, or an indoor field.

[0019] It is intended that the bacterial flora containing Lysobacter bacteria may also contain other indigenous bacteria in addition to Lysobacter bacteria. Examples of indigenous bacteria other than Lysobacter include Pseudomonas bacteria. The use of indigenous bacteria or bacterial flora is expected to prevent mismatches between Lysobacter bacteria and the soil of the target agricultural land. This method corresponds to the fertility management method stipulated in Article 4 of the Organic JAS standard, which only utilizes the functions of organisms (e.g., microorganisms) that inhabit the target agricultural land or its surrounding areas to improve and maintain and enhance soil.

[0020] The farmland to be improved is not particularly limited as long as it is farmland. Examples of farmland to be improved include conventional farmland, organic farmland with or without organic JAS certification, special cultivation farmland, and environmental conservation farmland. The farmland to be improved is preferably farmland where the use of chemical pesticides and chemical fertilizers is restricted, such as organic farmland, special cultivation farmland, and environmental conservation farmland. The soil microflora improver is prepared by the following method 1 or method 2.

[0021] 1-1. Method 1 The soil microflora improver includes bacteria of the genus Lysobacter or a bacterial flora containing bacteria of the genus Lysobacter.

[0022] (1) Step Ia This step includes preparing Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria, recovered from recovery soil for recovering Lysobacter bacteria. Here, a microbial flora containing Lysobacter bacteria is obtained from the recovery soil. In this specification, the identification of Lysobacter bacteria is performed using sequence information of the 16S ribosomal RNA (rRNA) gene encoded in the bacterial genome, and therefore, this is referred to as a bacterial flora containing Lysobacter bacteria. Therefore, this specification does not exclude the recovery of a microbial flora containing Lysobacter bacteria from recovery soil.

[0023] Preparation includes recovering Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria by the method described below, as well as obtaining (including purchasing) Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria recovered by the method described below.

[0024] For example, the bacterial flora containing Lysobacter bacteria contained in the soil microbiome improver is recovered from the recovery soil as the initial bacterial flora. The recovery soil is preferably soil in which the presence of Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria has been confirmed. The presence of Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria can be confirmed by bacterial culture of Lysobacter bacteria and other bacteria constituting the bacterial flora contained in a soil sample obtained by collecting a portion of the recovery soil. Alternatively, in addition to or instead of bacterial culture, confirmation can be achieved by detecting and / or analyzing the DNA of the bacteria contained in the soil sample. DNA detection can be performed, for example, by PCR. DNA analysis can include, for example, analyzing the sequence of the 16S ribosomal RNA (rRNA) gene encoded in the bacterial genome by sequencing or the like.

[0025] Examples of the soil for recovery include soil from farmland selected from conventional farmland, organic farmland with or without organic JAS certification, special cultivation farmland, and environmental conservation farmland, and / or soil in the vicinity of the farmland. The soil for recovery is preferably soil that has not been affected by agricultural crop diseases in the last 2 to 6 years, preferably the last 5 years, before the soil collection.

[0026] The recovery soil may be collected after about 1 to 5 weeks, preferably about 3 weeks, have passed since the utilizable substance described below was mixed or sprayed into the recovery soil. This step is referred to as pre-recovery acclimation in this specification. By applying the utilizable substance to the recovery soil in advance, the content of Lysobacter bacteria in the soil solution described below can be increased. For example, about 0.5 g to 10 g of the utilizable substance can be applied per 1 kg of recovery soil. Alternatively, about 100 kg to 1,000 kg of the utilizable substance can be applied per 1 tan of farmland (field). The bacterial flora containing Lysobacter bacteria recovered from soil that has been acclimated before recovery is sometimes referred to as the post-acclimation bacterial flora.

[0027] The recovery soil may be soil from agricultural land to be improved. That is, the Lysobacter bacteria or bacterial flora containing Lysobacter bacteria contained in the soil microbiome improver may be Lysobacter bacteria or bacterial flora containing Lysobacter bacteria that are indigenous to the soil to be improved.

[0028] Lysobacter bacteria or bacterial flora containing Lysobacter bacteria can be recovered, for example, by collecting a portion of the soil sample from the soil to be recovered and dispersing the collected soil sample in a buffer solution such as PBS or an aqueous liquid such as water to obtain a soil solution. For example, the soil solution can be centrifuged at approximately 500 G to recover the supernatant, or by removing the sediment, to recover a supernatant containing Lysobacter bacteria or bacterial flora containing Lysobacter bacteria. In this specification, the supernatant is also referred to as a soil bacterial flora solution.

[0029] The soil bacterial flora solution containing Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria may be further concentrated. The soil bacterial flora solution can be concentrated, for example, by fractionation using a membrane filter or by centrifugation. The fractionation treatment of the soil bacterial flora solution is preferably carried out using multiple types of membrane filters with different pore sizes. The membrane filter is preferably a metal thin film filter in which pores are formed to a certain degree uniformly in one membrane filter.

[0030] The shape of the pores of the membrane filter can be approximately square, square, approximately circular, circular, or the like. The membrane filter can be produced by electroforming.

[0031] Examples of membrane filters with different pore sizes include metal mesh devices (manufactured by Murata Manufacturing Co., Ltd.) with precisely controlled filter structures (pore pitch, pore size, membrane thickness) and absolute size filters (manufactured by Optonix Precision Co., Ltd.).

[0032] The membrane filters with different pore sizes have a pore size that is smaller in the downstream filter than in the upstream filter, and the pore size can be in the range of 0.5 to 15 μm.

[0033] A concentrated solution of soil bacterial flora can be obtained by recovering the Lysobacter bacteria or bacterial flora containing Lysobacter bacteria trapped on the membrane filter.

[0034] Concentration of the soil bacterial flora solution by centrifugation can be carried out, for example, by centrifuging the soil bacterial flora solution at about 1,700 g to 6,000 g for about 3 to 90 minutes and removing the supernatant.

[0035] (2) Step Ib This step involves culturing the Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria prepared in step Ia in the presence of an assimilable substance.

[0036] The starting sample for culturing Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria is a soil sample, a soil bacterial flora solution, a concentrated solution, or the like. By mixing the starting sample with a nutrient-rich substance (described below) and culturing the mixture, preferably in a liquid medium, the Lysobacter bacteria are grown, yielding a composition rich in Lysobacter bacteria, which contains a high proportion of Lysobacter bacteria. The culturing is preferably carried out at room temperature (approximately 20°C to 28°C) for approximately 3 to 14 days, preferably 8 days.

[0037] The composition enriched with Lysobacter bacteria may be a liquid medium containing Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria after culture, or a suspension of Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria recovered from a liquid medium after culture and suspended in the aqueous liquid. Alternatively, the composition enriched with Lysobacter bacteria may be a sediment containing Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria recovered from a liquid medium by centrifugation. The sediment may be a dry product obtained by lyophilization. The Lysobacter bacteria used to obtain the composition enriched with Lysobacter bacteria may be isolated from a soil solution, a soil bacterial flora solution, or a concentrated solution, or may be isolated and then cultured.

[0038] When the composition rich in Lysobacter bacteria is a bacterial flora containing Lysobacter bacteria, the bacterial flora rich in Lysobacter bacteria may contain, for example, 0.5% to 30%, preferably 3% to 15%, and more preferably 3.7% to 10% of Lysobacter bacteria. The bacterial flora containing Lysobacter bacteria contained in the composition rich in Lysobacter bacteria may contain 1% or more, preferably 3% or more, of Lysobacter bacteria whose bacterial species have not been identified. Alternatively, the bacterial flora may contain 16 or more, preferably 16 to 60 or more, different sequences as determined by sequence analysis of the V3-V4 region of 16S rRNA.

[0039] (3) Step Ic This step is a process for preparing a soil microflora amendment.

[0040] To prepare the soil microbiota improver, the soil sample, soil microbiota solution, or concentrate containing the Lysobacter bacteria or the bacterial flora containing Lysobacter bacteria prepared in step Ia; or the Lysobacter bacteria-rich composition obtained in step Ib, is mixed with a carrier.

[0041] The carrier is used to allow Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria to establish an ecological niche in the soil of the agricultural land to be improved. "Establishing an ecological niche" refers to achieving soil establishment of Lysobacter bacteria, which is normally difficult. "Establishing an ecological niche" refers to, for example, increasing the amount of Lysobacter bacteria by approximately 2 to 5,000 times after application of the soil microbiome improver compared to the amount of Lysobacter bacteria in the soil before application. The carrier is preferably sterilized or aseptically treated.

[0042] Examples of the carrier include one or more types selected from the group consisting of at least one porous body selected from the group consisting of biochar and vermiculite; at least one adsorbent selected from the group consisting of bentonite and zeolite; and at least one mineral selected from the group consisting of mountain soil, mudstone, and shale.

[0043] The biochar may be made from unused plant biomass materials such as rice husks, rice straw, grass clippings, and waste wood, or a mixture thereof. The charcoal may be in the form of powdered charcoal, briquettes, or the like.

[0044] The average particle size of the carrier is approximately 10 μm to 10 mm. More specifically, the average particle size for powdered biochar is approximately 10 μm to 700 μm. The average particle size for pelleted biochar is approximately 3 mm to 10 mm. The average particle size for vermiculite, bentonite, zeolite, mountain soil, mudstone, and shale is approximately 300 μm to 8 mm.

[0045] By using a carrier, the carrier becomes a habitat for Lysobacter bacteria to establish a niche (ecological position) in the soil, and it is expected that the existing soil microbial flora will be prevented from inhibiting the establishment of Lysobacter bacteria.

[0046] The soil microbiome improver may further contain an assimilable substance. The assimilable substance is organic matter necessary for the survival and / or proliferation of Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria. For example, the assimilable substance may include organic fertilizers such as rice bran, chicken manure, oil cake, bone meal, fish or meat meal, wood ash, tea leaves, rice straw, rice husks, wheat bran, cow manure, and compost (fermented food waste fertilizer); fermented products of the above organic fertilizers; and at least one component of an organic fertilizer selected from the group consisting of proteins, amino acids, sugars, phospholipids, and fats.

[0047] The utilizable substance is preferably at least one organic substance selected from the group consisting of the following (A) and (B): (A) at least one compound selected from the group consisting of proteins, amino acids, chitin, and N-acetyl-D-glucosamine; (B) An organic fertilizer containing at least one natural organic compound selected from the group consisting of proteins, amino acids, chitin, and N-acetyl-D-glucosamine. The compounds described in (A) above may include chemically synthesized compounds and compounds isolated and purified from natural materials. The organic fertilizers described in (B) above may include fermented fertilizers.

[0048] Preferably, the soil microbiome improver contains Lysobacter bacteria or Lysobacter bacteria, a carrier, and a bioavailable substance. When the soil microbiome improver is applied to the soil to be improved, the Lysobacter bacteria use the carrier in the soil microbiome improver as a foothold to establish an initial living space, and at the same time, begin to grow using the bioavailable substance in the soil microbiome improver as a nutrient source, ultimately achieving soil establishment. The bioavailable substance is then decomposed by soil microorganisms, including the Lysobacter bacteria, and ultimately supplies nutrients (e.g., nitrogen) necessary for crop growth.

[0049] Additionally, the soil microflora amendment may comprise a portion of the recovery soil.

[0050] Method 1 may include, prior to step Ia, investigating whether or not the recovery soil contains Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria.

[0051] 1-2. Method 2 In this embodiment, Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria is added to the soil microflora improver as recovery soil (in a state where it is contained in a part of the recovery soil).

[0052] As the soil microflora improver, a portion of the soil to be recovered containing Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria may be recovered as is, or may be recovered after being acclimated before recovery. In this section, the explanations for the recovery soil, carrier, assimilable substances, etc. are given in the above Method 1 by reference.

[0053] (1) Step Ia' This step includes preparing a soil sample collected from recovery soil for recovering Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria. The description of the soil sample is incorporated herein by reference in the above paragraph 1. Preparation includes collecting a soil sample from the recovery soil or receiving (including purchasing) a soil sample collected from the recovery soil.

[0054] Preferably, the preparation includes collecting a portion of the acclimated soil as a soil sample after pre-recovery acclimation, in which nutrient-reducing substances are mixed into or sprayed on the soil to be recovered, or preparing the soil sample by receiving (including purchasing) the soil sample. Pre-recovery acclimation can be carried out by mixing or spraying the assimilable substance into the soil to be recovered before recovery, and leaving it to stand for about 1 to 5 weeks, preferably about 3 weeks (watering as necessary).

[0055] (2) Step Ib' To prepare the soil microflora amendment, the acclimated soil is mixed with a carrier as needed. The soil microflora amendment may further contain assimilable substances.

[0056] Method 2 may include, prior to step Ia', investigating whether or not the recovery soil contains Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria.

[0057] 1-3. Formulation of soil microflora improver The formulation of the soil microflora improver is not limited and may be a liquid, powder, solid, or the like.

[0058] The soil microbiome improver may comprise a soil solution, a soil microbiome solution, or a concentrated soil microbiome solution containing the Lysobacter bacteria or a microbiome containing Lysobacter bacteria obtained in step Ia; or a Lysobacter bacteria-rich composition containing the Lysobacter bacteria obtained in step Ib. The content of the Lysobacter bacteria or the microbiome containing Lysobacter bacteria in the soil microbiome improver, converted into Lysobacter bacteria, can be approximately 0.1 to 20 parts by mass, preferably approximately 0.2 to 10 parts by mass, per 100 parts by mass of the soil microbiome improver. The content of the carrier can be approximately 10 to 90 parts by mass, per 100 parts by mass of the soil microbiome improver. The content of the assimilable substance can be approximately 2 to 50 parts by mass, per 100 parts by mass of the soil microbiome improver. When the soil microflora improver contains recovery soil, the content of the recovery soil can be about 1 to 30 parts by mass when the entire soil microflora improver is taken as 100 parts by mass.

[0059] 2. Methods for improving soil microflora One embodiment of the present invention relates to a method for improving soil microflora by applying the soil microflora improver prepared in 1 above. The method corresponds to step II shown in FIG. 1 and includes applying a soil microbiome amendment to the soil to be amended.

[0060] The soil microflora improver is used for improving 1 tan (approximately 998 m) of farmland. 2 It is preferable to apply about 100 kg to 1,000 kg per acre. Top dressing is preferably done once at the start of improvement, and preferably repeated once or twice every month. It is also preferable to apply it one month (if the improvement is applied once) to three months (if it is applied three times) before planting. The soil microflora improver may also be applied as a top dressing between planting and harvesting, or after harvesting.

[0061] Prior to, simultaneously with, or after mixing or spraying the soil microbiota amendment, a carrier and / or a bioavailable substance may be applied to the area of ​​farmland to be improved with the soil microbiota amendment.

[0062] A method for improving soil microbiomes may involve applying the soil microbiome improver to a portion of the farmland to be improved, or to soil collected from the farmland to be improved and transferred to a container such as a planter, before applying the soil microbiome improver to the entire farmland to be improved, and then conducting a pre-application survey to determine whether Lysobacter bacteria or a Lysobacter bacterial flora have become established.

[0063] Pre-application surveys can be conducted by mixing or spraying a soil microbiome improver on a portion of the farmland to be improved, leaving it for a certain period of time, such as 1 to 8 weeks (and irrigating as necessary), then collecting a soil sample and conducting bacterial culture of the Lysobacter bacteria and other bacteria that make up the bacterial flora contained in the soil sample. Alternatively, in addition to or instead of bacterial culture, confirmation can be conducted by detecting and / or analyzing the DNA of the bacteria contained in the soil sample. Methods for DNA detection and / or analysis are as described above. [Example]

[0064] The present invention will be described in more detail below with reference to examples, but the present invention should not be construed as being limited to these examples.

[0065] 1. Example 1 Approximately 1 kg of soil was collected from organic open-field farmland in Koka City, Shiga Prefecture (A), organic greenhouse farmland in Koka City, Shiga Prefecture (B), organic open-field farmland in Omihachiman City, Shiga Prefecture (C), conventional open-field farmland in Aisho Town, Shiga Prefecture (D), conventional open-field farmland in Kuwana City, Mie Prefecture (E), and abandoned farmland in Koka City, Shiga Prefecture (F) and subjected to the following acclimation process. During the acclimation process, the collected soil was placed in flowerpots and fertilized with approximately 2 g of BSA (bovine serum albumin, protein) as organic fertilizer (assimilable substance). The pots were then placed in greenhouses and allowed to stand for 3 weeks, with regular watering and weed removal. The soil bacterial flora was then analyzed by next-generation sequencing (NGS) for amplicon analysis of the 16S rRNA V3-V4 region before and 3 weeks after fertilization. The results for Lysobacter bacteria are shown in Table 1.

[0066] [Table 1]

[0067] The analysis revealed that the increase in Lysobacter bacteria was most pronounced in farmland (A), which was organic open-field farmland that had experienced almost no crop disease over the past 50 years.

[0068] Next, a soil microbial flora improver was prepared using the soil from farmland (A) and applied to farmlands (A) to (F) to verify its effectiveness in improving the soil microbial flora. The soil microbial flora improver was prepared and applied three times in total, in different seasons (spring, summer, and autumn).

[0069] Using the procedure described above, we prepared acclimated soil from farmland (A) 3 weeks after BSA fertilization. Approximately 400 g of acclimated soil was suspended in PBS to obtain approximately 1 L of soil solution. After ultrasonic dispersion, the soil components were separated by centrifugation at 500 G, and the supernatant was used as the soil microbiota solution [solution (a)] to produce the soil microbiota conditioner.

[0070] Next, a soil bacterial flora solution was prepared using freshly collected soil from farmland (A) and PBS using the procedure described above. 1.5 mg of BSA was added to 175 mL of the prepared soil bacterial flora solution as a bioavailable substance, and the solution was cultured for 8 days with aeration. The culture solution after 8 days of culture was designated as a Lysobacter -enriched composition [solution (b)] for use in the production of a soil microbiome conditioner. During the culture period, the bacterial flora of the culture solution was analyzed by amplicon analysis on days 2, 4, 6, and 8. Figure 2 shows the change in the Lysobacter content of the bacterial flora in the culture solution.

[0071] The content of Lysobacter bacteria increased depending on the number of days of culture. This analysis revealed that in addition to Lysobacter bacteria, Pseudomonas bacteria were the dominant bacteria in this culture medium. Pseudomonas bacteria are known to contribute to the degradation of synthetic chemicals such as pesticides. Using the soil bacterial flora solution prepared from the newly collected soil of agricultural land (A), plate culture was performed on agar medium supplemented with BSA and various antibiotics, and Lysobacter bacteria were isolated and passaged (expanded). Approximately 20 samples of the obtained Lysobacter bacteria were suspended in PBS to create the Lysobacter bacteria solution [solution (c)] used to manufacture the soil microbiome improver.

[0072] Biochar (palm-derived carbonaceous material with a particle size of approximately 1–5 mm) was autoclaved to serve as a bacterial carrier. Eight grams of the sterilized biochar was mixed with 2 grams of BSA as an organic fertilizer (assimilable substance), and the resulting mixture was diluted to approximately 50 mL with solution (a), (b), or (c) to prepare soil microbiota conditioners (a)–(c). The resulting soil conditioners were applied to approximately 1 kg of soil from each of the farmland samples (A)–(F) collected in flowerpots. Amplicon analysis was used to compare the content of Lysobacter bacteria in the soil microbiota before and 3 weeks after application. The results are shown in Table 2. [Table 2]

[0073] Application of soil microbiota conditioners (a) to (c) resulted in the establishment of Lysobacter bacteria in farmland (A) to (F), and their content increased. The increase rate (content 3 weeks after application / content before application) was 124 times on average across all data, with a minimum of 7.4 times and a maximum of 830 times (the results for summer soil C were excluded because the data before application was 0).

[0074] Antagonism tests against crop disease bacteria were conducted using soil from summer farmland E (colored in Table 2) before application (0.05% Lysobacter content) and soil from farmland A three weeks after application (12.7% Lysobacter content). Soil microflora solutions were prepared from both farmland soils using PBS solution using the same procedure described above. The solutions were then centrifuged at 11,000 G to separate bacteria and other microorganisms. The supernatant, presumably containing enzymes and antibiotics produced by Lysobacter, was used as the test solution. Representative examples of crop disease bacteria were selected: Ralstonia solanacearum (bacterial wilt disease) and Pythium oomycete (rhizome rot disease). Antagonism tests were conducted using the test solution with plate culture samples of these pathogens to confirm the presence or absence of biological control activity. As a result, it was confirmed that the test solution from farmland (E) had no antagonistic effect, while the test solution from farmland (A) had antagonistic effect on both pathogens.

[0075] In the above examples, the content of Lysobacter bacteria was reported for classifications with unknown bacterial species. However, the Lysobacter yangpyeongensis species was also detected. Including this, the content of Lysobacter bacteria in solutions (a) and (b) used to prepare the soil microbiome improver was approximately 0.7% higher on average. Furthermore, it was revealed that the Lysobacter bacteria (bacterial species unknown) in solutions (a) and (b) used to prepare the soil microbiome improver contained 16 to 60 different sequences based on the sequences of the V3-V4 region of 16S rRNA used in amplicon analysis.

[0076] 2. Example 2 A soil bacterial flora solution was prepared using soil from farmland (D) and PBS solution using the same procedure as in Example 1. 1.5 mg of BSA was added as an assimilable substance to 175 mL of this soil bacterial flora solution, and liquid culture was carried out for 8 days with aeration. The content of Lysobacter bacteria in the bacterial flora of the resulting culture solution was 9.6%. Biochar (derived from rice husks) was autoclaved to obtain sterilized biochar as a carrier for supporting bacteria. A soil microbiome conditioner was prepared by mixing 8 g of sterilized biochar with 2 g of either a Group A compound (as described in the Detailed Description of the Invention) selected from 1) BSA, 2) albumin (derived from egg white), 3) arginine, 4) cysteine, 5) chitin, and 6) N-acetyl-D-glucosamine, or a Group B organic fertilizer (as described in the Detailed Description of the Invention) selected from 7) fish meal, 8) rice bran, and 9) soybean meal, or a mixture of a Group A compound and a Group B organic fertilizer. The mixture was then diluted to approximately 50 mL with the culture medium. The prepared soil microbiome conditioner was applied and mixed with approximately 1 kg of soil collected in flowerpots from farmland (A) to (F). Amplicon analysis was used to compare the content of Lysobacter bacteria in the soil microbiome before and 3 weeks after application. The results are shown in Table 3.

[0077] [Table 3]

[0078] It was found that the application of Groups A and B, and soil microflora improvers using these as assimilable substances, resulted in the establishment of Lysobacter bacteria in farmland (A) to (F), and their content increased. Furthermore, the increase rate (content 3 weeks after application / content before application) was 89 times on average across all data, with a minimum of 6.9 times and a maximum of 500 times.

[0079] 3. Example 3 A soil bacterial flora solution was prepared using agricultural soil (A) and PBS solution using the same procedure described above. The soil bacterial flora solution was fractionated using metal membrane filters (Murata Manufacturing Co., Ltd.) with pore sizes of 10 μm, 5 μm, 3 μm, and 1 μm, in that order, and the fractionated bacterial flora was trapped on each filter. Amplicon analysis was performed on the soil bacterial flora in the solution before fractionation and on the fractionated bacterial flora isolated on each filter to determine the Lysobacter content in the bacterial flora. The results were 0.21% (pre-fractionation solution), 0.72% (10 μm filter), 9.9% (5 μm filter), 4.7% (3 μm filter), and 1.1% (1 μm filter). A soil microbiota solution was prepared by backwashing multiple 5 μm pore filters with PBS to recover and suspend the fractionated bacterial flora. 0.7 mg of BSA was added to 175 mL of this solution as a bioavailable substrate and cultured for 8 days with aeration. The resulting culture solution contained 32.1% Lysobacter bacteria. Biochar (palm-derived carbonaceous material with a particle size of approximately 1–5 mm) was autoclaved as a bacterial support. 8 g of sterilized biochar and 2 g of BSA were mixed with the culture solution to a final volume of approximately 50 mL. The resulting soil conditioner was applied and mixed with approximately 1 kg of soil collected from agricultural fields (A)–(F) in flowerpots. Amplicon analysis was used to compare the Lysobacter bacteria content in the soil microbiota before and 3 weeks after application. The results are shown in Table 4. [Table 4]

[0080] Application of a soil microbiota conditioner containing a fractionated bacterial flora enriched for Lysobacter bacteria resulted in the establishment of Lysobacter bacteria in farmland (A) to (F), and its content increased. The increase in content (= content 3 weeks after application / content before application) was 180-fold on average across all data, with a minimum of 23-fold and a maximum of 750-fold.

Claims

1. A soil microflora improver comprising Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria.

2. The soil microbiota improver according to claim 1, which is applied to improve the soil bacterial flora of agricultural land to be improved so that it has a biological control effect against agricultural crop diseases.

3. The soil microbiome improver according to claim 1, further comprising a sterilized or sterilized carrier for allowing the Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria to obtain an ecological niche in the soil of the agricultural land to be improved.

4. The soil microflora improver according to claim 1, comprising an assimilable organic substance necessary for the survival and / or proliferation of the Lysobacter bacteria or a bacterial flora containing the Lysobacter bacteria.

5. The soil microbiome improver described in claim 2, characterized in that the agricultural land to be improved is at least one agricultural land selected from conventional agricultural land, organic agricultural land that has or has not obtained organic JAS certification, special cultivation agricultural land, and environmental conservation agricultural land.

6. The soil microbiome improver according to claim 1, characterized in that it uses Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria recovered from soil of agricultural land selected from conventional agricultural land where the presence of Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria has been confirmed, organic agricultural land with or without organic JAS certification, special cultivation agricultural land, and environmental conservation agricultural land, and at least one recovery soil selected from soil in the vicinity of the agricultural land.

7. The soil microbiota improver according to claim 6, wherein the recovery soil is soil that has not been affected by agricultural crop diseases in the two to six years immediately preceding the soil collection.

8. The soil microbiome improver according to claim 4, wherein the assimilable substance is at least one organic substance selected from the group consisting of the following (A) and (B): (A) at least one compound selected from the group consisting of proteins, amino acids, chitin, and N-acetyl-D-glucosamine; (B) An organic fertilizer containing at least one natural organic compound selected from the group consisting of proteins, amino acids, chitin, and N-acetyl-D-glucosamine.

9. The soil microflora improver according to claim 1, wherein the bacterial flora containing the Lysobacter bacteria is added as a soil bacterial flora solution obtained by recovering a bacterial flora containing the Lysobacter bacteria that was indigenous to the recovery soil.

10. The soil microbiome improver according to claim 1, wherein the bacterial flora containing Lysobacter bacteria is added as a composition rich in Lysobacter bacteria, which is obtained by recovering bacterial flora containing Lysobacter bacteria that were native to the soil for recovery and culturing them using assimilable substances.

11. The soil microbiome improver according to claim 1, wherein the bacterial flora containing the Lysobacter bacteria is added as soil in which a nutrient-rich substance has been mixed or sprayed into recovery soil in which the bacterial flora containing the Lysobacter bacteria is native and acclimatized.

12. The soil microbiome improver according to claim 1, wherein the bacterial flora containing Lysobacter bacteria is added as a composition rich in Lysobacter bacteria obtained by culturing a bacterial flora containing Lysobacter bacteria recovered from soil in which a bacterial flora containing Lysobacter bacteria is native, the soil being acclimated by mixing or spraying a nutrient-rich substance into the soil.

13. The soil microbiome improver according to claim 1, wherein the bacterial flora containing the Lysobacter bacteria is added as a composition rich in Lysobacter bacteria, which is obtained by culturing a concentrated solution obtained by concentrating the Lysobacter bacteria from a soil bacterial flora solution using multiple types of membrane filters with different pore sizes.

14. The soil microflora improver according to claim 12, wherein the content of Lysobacter bacteria in the bacterial flora contained in the composition with a high content of Lysobacter bacteria is 3.7% or more.

15. The soil microflora improver according to claim 10, wherein the bacterial flora contained in the composition with a high content of Lysobacter bacteria includes Pseudomonas bacteria as bacteria other than Lysobacter bacteria.

16. The soil microbiome improver according to claim 10, wherein the bacterial flora contained in the composition with a high content of Lysobacter bacteria contains 3% or more of Lysobacter bacteria whose bacterial species has not been identified, or contains 16 or more different sequences in the V3-V4 region of 16S rRNA.

17. The soil microbiome improver according to claim 3, wherein the carrier comprises one or more of at least one porous body selected from the group consisting of soil, biochar, and vermiculite from which Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria has been collected; at least one adsorbent selected from the group consisting of bentonite and zeolite; and at least one mineral selected from the group consisting of mountain soil, mudstone, and shale.

18. The soil microbiome improver according to claim 17, characterized in that the biochar is powdered charcoal or shaped charcoal produced from unused plant biomass materials such as rice straw, rice husks, grass clippings, and waste wood, and that one type of unused plant biomass material or a mixture of multiple types of unused plant biomass materials is used.

19. Preparing a soil sample collected from the soil for recovery of Lysobacter bacteria or bacterial flora containing Lysobacter bacteria; and Preparing a soil microflora improver containing Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria; 1. A method for preparing a soil microbiome amendment, comprising: The recovery soil is soil in which the presence of Lysobacter bacteria or a bacterial flora containing Lysobacter bacteria has been confirmed. method.

20. 20. The method according to claim 19, wherein the preparation comprises acclimating the soil for recovery by mixing or spraying a nutrient-reducing substance into the soil, and preparing a portion of the acclimated soil as a soil sample.

21. The step of preparing the soil microbiota improver comprises mixing a soil solution obtained by dispersing soil collected from the soil for recovery in an aqueous liquid, a soil bacterial flora solution which is the supernatant of the soil solution, a concentrated solution of the soil bacterial flora solution, or a composition with a high content of Lysobacter bacteria obtained by culturing the soil solution, the soil bacterial flora solution, or the concentrated solution, with a carrier and / or an assimilable substance; The method, wherein the carrier is used to allow Lysobacter bacteria or a Lysobacter bacterial flora to obtain an ecological niche in the target soil.

22. A method for improving soil microflora, comprising applying the soil microflora improver according to claim 1 to the soil to be improved.

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