JP2020141564A - Plant growth promoting agent and method for producing plant growth promoting agent - Google Patents
Plant growth promoting agent and method for producing plant growth promoting agent Download PDFInfo
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Abstract
Description
本発明は、家畜糞尿を原料とする植物生長促進剤及び植物生長促進剤の製造方法に関する。 The present invention relates to a plant growth promoter and a method for producing a plant growth promoter using livestock manure as a raw material.
家畜糞尿を原料とする堆肥は、地力を維持し且つ作物の生産性を向上させる有機質資材である。化学肥料の普及後は、作物の生産性向上には化学肥料が多用されてきた。しかしながら、近年の減農薬農法、減化学肥料農法又は有機農法の広がりにともない、堆肥の使用量も再び増加している。 Compost made from livestock manure is an organic material that maintains soil fertility and improves crop productivity. After the spread of chemical fertilizers, chemical fertilizers have been widely used to improve crop productivity. However, with the recent spread of pesticide-reduced farming methods, reduced chemical fertilizer farming methods, or organic farming methods, the amount of compost used has increased again.
牛糞、豚糞、鶏糞等の家畜糞を堆肥化する方法が広く知られている（例えば、特許文献１）。特許文献１の堆肥製造方法では、鶏糞に木片チップと微生物を混入した６０℃以上の混合物を、堆肥舎で６．５日間、その後発酵槽で２３．５日間発酵させる。得られた発酵物から、選別機で木片チップを回収した後、鶏糞堆肥を養生施設に送り、切返しをしながら６０℃以上で３．８日間発酵させて、鶏糞堆肥が完成する。 A method of composting livestock manure such as cow manure, pig manure, and chicken manure is widely known (for example, Patent Document 1). In the compost production method of Patent Document 1, a mixture of chicken manure mixed with wood chips and microorganisms at 60 ° C. or higher is fermented in a compost house for 6.5 days and then in a fermenter for 23.5 days. After collecting wood chips from the obtained fermented product with a sorter, the chicken manure compost is sent to a curing facility and fermented at 60 ° C. or higher for 3.8 days while turning back to complete the chicken manure compost.
このような堆肥を製造する際、堆肥の含水率を低くするため、***物から液体成分と固定成分とを分離することがある。この際に分離された液体成分についてはあまり利用されていなかった。 When producing such compost, the liquid component and the fixed component may be separated from the excrement in order to reduce the water content of the compost. The liquid component separated at this time was not used much.
そこで、本発明はこれらの点に鑑みてなされたものであり、家畜糞尿の液体成分を用いて植物の生長を促進することができる植物生長促進剤及び植物生長促進剤の製造方法を提供することを目的とする。 Therefore, the present invention has been made in view of these points, and provides a plant growth promoter and a method for producing a plant growth promoter capable of promoting plant growth using a liquid component of livestock manure. With the goal.
本発明は、以下の［１］〜［９］である。
［１］家畜の糞尿を原料とする植物生長促進剤であって、バクテロイデス門に含まれる微生物、クロロフレクサス門に含まれる微生物、ゲンマティモナス門に含まれる微生物、及びウェルコミクロビウム門に含まれる微生物からなる群のうち少なくともいずれか１つ以上を含む、植物生長促進剤。
［２］5′-CCTACGGGNGGCWGCAG-3′（配列番号１）で示される塩基配列（該塩基配列中、NはA、T、G又はCであり、WはA又はTである）を含む第１プライマーと5′-GACTACHVGGGTATCTAATCC-3′（配列番号２）で示される塩基配列（該塩基配列中、HはA、T又はCであり、VはA、C又はGである）を含む第２プライマーとを用いて、該植物生長促進剤中に含まれる微生物を検出したときに、バクテロイデス門に含まれる微生物、クロロフレクサス門に含まれる微生物、ゲンマティモナス門に含まれる微生物、及び、ウェルコミクロビウム門に含まれる微生物からなる群のうち少なくともいずれか１つ以上が検出される、［１］の植物生長促進剤。
［３］全炭素濃度が２，０００ｍｇ／ｋｇ以下である、［１］又は［２］の植物生長促進剤。
［４］ケルダール法で測定した全窒素濃度が５００ｍｇ／ｋｇ以下である、［１］〜［３］のいずれかの植物生長促進剤。
［５］全リン濃度が２，０００ｍｇ／ｋｇ以下である、［１］〜［４］のいずれかの植物生長促進剤。
［６］全カリウム濃度が５，０００ｍｇ／ｋｇ以下である、［１］〜［５］のいずれかの植物生長促進剤。
［７］前記植物生長促進剤は、液体、固形又はスラリーの態様である、［１］〜［６］のいずれかの植物生長促進剤。
［８］家畜の糞尿の液体成分を分離するステップと、5′-CCTACGGGNGGCWGCAG-3′（配列番号１）の塩基配列を含む第１プライマーと5′-GACTACHVGGGTATCTAATCC-3′（配列番号２）の塩基配列を含む第２プライマーとを用いて検出される微生物のうち、バクテロイデス門に含まれる微生物を１．５％以上、クロロフレクサス門に含まれる微生物を１．５％以上、ゲンマティモナス門に含まれる微生物を０．５％以上、及び、ウェルコミクロビウム門に含まれる微生物からなる群のうち少なくとも１種以上を０．５％以上含むようになるまで、前記液体成分を曝気させるステップとを含む、植物生長促進剤の製造方法。
［９］ 前記曝気させるステップにおいて、Ｆ［ｍ／ｓ］を空気流量、Ｖ［ｍ３］を処理液体積、ＨＬ［ｍ］を処理液深度とした場合に、
を満たす状態において前記液体成分を曝気させる、［８］の植物生長促進剤の製造方法。
The present invention is the following [1] to [9].
[1] A plant growth promoter made from livestock manure, which is contained in microorganisms contained in the phylum Bacteroidetes, microorganisms contained in the phylum Chloroflexi, microorganisms contained in the phylum Gemmatimonadetes, and phylum Verrucomicrobia. A plant growth promoter comprising at least one or more of the group consisting of microorganisms.
[2] A first containing the base sequence represented by 5'-CCTACGGGNGGCWGCAG-3'(SEQ ID NO: 1) (in the base sequence, N is A, T, G or C, and W is A or T). A second primer containing a primer and the base sequence shown by 5'-GACTACHVGGGTATCTAATCC-3'(SEQ ID NO: 2) (in the base sequence, H is A, T or C and V is A, C or G). When the microorganisms contained in the plant growth promoter were detected using, the microorganisms contained in the phylum Bacteroidetes, the microorganisms contained in the phylum Chloroflexi, the microorganisms contained in the phylum Gemmatimonadetes, and Verrucomicrobia. The plant growth promoter according to [1], wherein at least one or more of the group consisting of microorganisms contained in the phylum Verrucomicrobia is detected.
[3] The plant growth promoter according to [1] or [2], which has a total carbon concentration of 2,000 mg / kg or less.
[4] The plant growth promoter according to any one of [1] to [3], wherein the total nitrogen concentration measured by the Kjeldahl method is 500 mg / kg or less.
[5] The plant growth promoter according to any one of [1] to [4], which has a total phosphorus concentration of 2,000 mg / kg or less.
[6] The plant growth promoter according to any one of [1] to [5], which has a total potassium concentration of 5,000 mg / kg or less.
[7] The plant growth promoter according to any one of [1] to [6], wherein the plant growth promoter is a liquid, solid or slurry embodiment.
[8] The step of separating the liquid component of livestock manure, the first primer containing the base sequence of 5'-CCTACGGGNGGCWGCAG-3'(SEQ ID NO: 1), and the base of 5'-GACTACHVGGGTATCTAATCC-3'(SEQ ID NO: 2). Among the microorganisms detected using the second primer containing the sequence, 1.5% or more of the microorganisms contained in the phylum Bacteroidetes and 1.5% or more of the microorganisms contained in the phylum Chloroflexus are contained in the phylum Gemmatimonadetes. The step of aerating the liquid component until it contains 0.5% or more of the microorganisms and 0.5% or more of at least one of the group consisting of microorganisms contained in the Welcomicrobium phylum. A method for producing a plant growth promoter, including.
[9] In the aeration step, when F [m / s] is the air flow rate, V [m 3 ] is the treatment liquid volume, and HL [m] is the treatment liquid depth.
[8] The method for producing a plant growth promoter, which aerates the liquid component in a state satisfying the above conditions.
本発明の植物生長促進剤は、植物の生長を促進することができるという効果を奏する。そして、この植物の生長促進効果は、一般的に肥料に含まれるリンや窒素分の量が少なくても発揮される。 The plant growth promoter of the present invention has the effect of promoting the growth of plants. The growth promoting effect of this plant is generally exhibited even if the amount of phosphorus and nitrogen contained in the fertilizer is small.
［植物生長促進剤の成分］
本発明者らは、鋭意研究の結果、本実施の形態に係る家畜の糞尿の液体成分を原料とする植物生長促進剤が特定の微生物群を含むことにより、安定的な植物生長効果をもたらすことを見出した。家畜は、例えば、牛、豚、鶏、羊又は山羊であるが、これらの動物に限定されない。
[Ingredients of plant growth promoter]
As a result of diligent research, the present inventors have determined that the plant growth promoter using the liquid component of livestock manure according to the present embodiment contains a specific microbial group to bring about a stable plant growth effect. I found. Livestock are, for example, cows, pigs, chickens, sheep or goats, but are not limited to these animals.
本発明の植物生長促進剤は、バクテロイデス門に含まれる微生物、クロロフレクサス門に含まれる微生物、ゲンマティモナス門に含まれる微生物、及びウェルコミクロビウム門に含まれる微生物からなる群のうち少なくともいずれか１つ以上の微生物を含む。 The plant growth promoter of the present invention is at least one of a group consisting of microorganisms contained in the phylum Bacteroidetes, microorganisms contained in the phylum Chloroflexi, microorganisms contained in the phylum Gemmatimonadetes, and microorganisms contained in the phylum Verrucomicrobia. Contains one or more microorganisms.
［バクテロイデス門］
バクテロイデス門は、グラム陰性細菌のグループであり、腸内細菌として知られている微生物が含まれる。下位分類（綱）として、バクテロイデス綱、フラボバクテリア綱、スフィンゴバクテリア綱、キティノファガ綱、キトファガ綱、サプロスピラ綱を含む。バクテロイデス門に含まれる微生物としては、例えば、Bacteroides plebeius、Bacteroides fragilisが挙げられる。
本発明の植物生長促進剤中、バクテロイデス門の微生物の含有割合は、好ましくは１．５％以上（後述する方法により本発明の植物生長促進剤から検出される微生物全体に対する割合。以下、微生物の含有割合については同様である。）であり、より好ましくは、５〜１２％である。
[Bacteroides Gate]
The phylum Bacteroides is a group of Gram-negative bacteria that include microorganisms known as gut flora. Subclasses (Classes) include Bacteroidia, Flavobacteriia, Sphingobacteriia, Kitinofaga, Chitofaga, and Saprospira. Examples of microorganisms contained in the phylum Bacteroides include Bacteroides plebeius and Bacteroides fragilis.
The content ratio of the microorganisms of the phylum Bacteroides in the plant growth promoter of the present invention is preferably 1.5% or more (the ratio of the microorganisms to the total microorganisms detected from the plant growth promoter of the present invention by the method described later. The content ratio is the same.), More preferably 5 to 12%.
［クロロフレクサス門］
クロロフレクサス門は、緑色滑走細菌門とも呼ばれ、下位分類（綱）として、クロロフレクサス綱、アナエロリネア綱、アルデンティカテナ綱、カルディリネア綱、クテドノバクテル綱、テルモフレクスス綱、テルモミクロビウム綱を含む。クロロフレクサス門に含まれる微生物としては、例えば、Thermomicrobium roseum、Sphaerobacter thermophilus、Caldilinea aerophila、Caldilinea tarbellica、Ktedonobacter、Thermosporothrix、Thermogemmatispora onikobensis、Thermogemmatispora foliorumが挙げられる。
本発明の植物生長促進剤中、クロロフレクサス門の微生物の含有割合は、好ましくは１．５％以上であり、より好ましくは、２〜１５％である。
[Chloroflexi Gate]
The phylum Chloroflexi, also called the phylum Chloroflexi, includes the subclasses (Classes) Chloroflexi, Anaerolinea, Ardenticatena, Cardilinea, Cutedonovactel, Thermoflexus, and Thermomicrobium. Examples of microorganisms contained in the phylum Chloroflexi include Thermomicrobium roseum, Sphaerobacter thermophilus, Caldilinea aerophila, Caldilinea tarbellica, Ktedonobacter, Thermosporothrix, Thermogemmatispora onikobensis, and Thermogemmatispora foliorum.
In the plant growth promoter of the present invention, the content ratio of microorganisms of the phylum Chloroflexi is preferably 1.5% or more, more preferably 2 to 15%.
［ゲンマティモナス門］
ゲンマティモナス門は、グラム陰性細菌の門である。下位分類（綱）として、ゲンマティモナス綱、ロンギミクロビウム綱が含まれる。ゲンマティモナス門に含まれる微生物としては、例えば、Gemmatimonas aurantiaca、Gemmatimonas phototrophica、Longimicrobium terrae、Gemmatirosa kalamazoonesisが挙げられる。
本発明の植物生長促進剤中、ゲンマティモナス門の微生物の含有割合は、好ましくは１．５％以上であり、より好ましくは、２％〜５％である。
[Gemmatimonadetes]
The phylum Gemmatimonadetes is the phylum of Gram-negative bacteria. Subclasses (Classes) include Gemmatimonadetes and Longimicrobium. Examples of microorganisms contained in the phylum Gemmatimonas include Gemmatimonas aurantiaca, Gemmatimonas phototrophica, Longimicrobium terrae, and Gemmatirosa kalamazoonesis.
In the plant growth promoter of the present invention, the content ratio of the microorganism of Gemmatimonadetes is preferably 1.5% or more, more preferably 2% to 5%.
［ウェルコミクロビウム門］
ウェルコミクロビウム門は、グラム陰性細菌の門である。下位分類（綱）として、ウェルコミクロビウム綱、オピトゥトゥス綱を含む。ウェルコミクロビウム門に含まれる微生物としては、例えば、Verrucomicrobium Haloferula、Verrucomicrobium Luteolibacter、 Verrucomicrobium Persicirhabdus、Verrucomicrobium Prosthecobacter、Verrucomicrobium Roseibacillus、Verrucomicrobium Roseimicrobium、Prosthecobacter dejongeii、Chthoniobacter flavus、Ellin514株、Puniceicoccus Cerasicoccusが挙げられる。
本発明の植物生長促進剤中、ウェルコミクロビウム門の微生物の含有割合は、好ましくは１．５％以上であり、より好ましくは、２％〜５％である。
[Verrucomicrobia]
The phylum Verrucomicrobia is the phylum of Gram-negative bacteria. Subclasses (classes) include Verrucomicrobiae and Opitutus. Examples of microorganisms contained in the phylum Verrucomicrobia include Verrucomicrobium Haloferula, Verrucomicrobium Luteolibacter, Verrucomicrobium Persicirhabdus, Verrucomicrobium Prosthecobacter, Verrucomicrobium Roseibacillus, Verrucomicrobium Roseimicrobium, Prosthecobacter dejonge.
In the plant growth promoter of the present invention, the content ratio of the microorganism of the phylum Verrucomicrobia is preferably 1.5% or more, more preferably 2% to 5%.
本発明の植物生長促進剤がこのような微生物を含むことにより、植物生長促進剤に含有されていた有機物が消費されていることに加えて、植物生長促進剤の原料である家畜の糞尿の液体成分に元々含まれていた植物発芽阻害物質が微生物により分解されていると推測される。その結果、本発明の植物生長促進剤は、安定した植物生長効果をもたらすことができると考えられる。 Since the plant growth promoter of the present invention contains such microorganisms, the organic matter contained in the plant growth promoter is consumed, and in addition, the liquid of livestock manure which is the raw material of the plant growth promoter It is presumed that the plant germination inhibitor originally contained in the components is decomposed by microorganisms. As a result, it is considered that the plant growth promoter of the present invention can bring about a stable plant growth effect.
本発明の植物生長促進剤は、さらに、アシドバクテリア門に含まれる微生物、クロロビウム門に属する微生物、及びプロテオバクテリア門に属する微生物からなる群から選ばれる一種以上の微生物を１．５％以上含むことが好ましい。 The plant growth promoter of the present invention further contains 1.5% or more of one or more microorganisms selected from the group consisting of microorganisms belonging to the phylum Acidobacteria, microorganisms belonging to the phylum Chlorobia, and microorganisms belonging to the phylum Proteobacteria. Is preferable.
［リアルタイムＰＣＲとシーケンスによる微生物の検出］
植物生長促進剤中において１６Ｓ ｒＲＮＡをターゲットとする第１プライマー及び第２プライマーを用いたリアルタイムＰＣＲを行うことにより植物生長促進剤に含まれる微生物群を特定することができる。リアルタイムＰＣＲでは、ＰＣＲ増幅産物の増加をリアルタイムに解析することにより、鋳型ＤＮＡを定量する。１６Ｓ ｒＲＮＡは、どの微生物においても配列の相動性が高いことから微生物の定量の指標として用いることができる。
[Detection of microorganisms by real-time PCR and sequencing]
The group of microorganisms contained in the plant growth promoter can be identified by performing real-time PCR using the first primer and the second primer targeting 16S rRNA in the plant growth promoter. In real-time PCR, template DNA is quantified by analyzing the increase in PCR amplification products in real time. 16S rRNA can be used as an index for quantification of microorganisms because the sequence compatibility is high in any microorganism.
第１プライマー及び第２プライマーは、リアルタイムＰＣＲにおいてＤＮＡポリメラーゼによる鋳型ＤＮＡの複製の開始起点となるオリゴヌクレオチドである。第１プライマーとして、配列番号１：
5′-CCTACGGGNGGCWGCAG-3′（配列番号１）
で示される塩基配列を含むＤＮＡ分子を用い、第２プライマーとして、配列番号２：
5′-GACTACHVGGGTATCTAATCC-3′（配列番号２）
で示される塩基配列を含むＤＮＡ分子を用いることができる。
The first and second primers are oligonucleotides that serve as the starting point for replication of the template DNA by DNA polymerase in real-time PCR. As the first primer, SEQ ID NO: 1:
5'-CCTACGGGNGGCWGCAG-3' (SEQ ID NO: 1)
Using a DNA molecule containing the base sequence shown by, as a second primer, SEQ ID NO: 2:
5'-GACTACHVGGGTATCTAATCC-3' (SEQ ID NO: 2)
A DNA molecule containing the base sequence shown by can be used.
配列番号１で示される塩基配列中、ＮはＡ（アデニン）、Ｔ（チミン）、Ｇ（グアニン）又はＣ（シトシン）であり、ＷはＡ又はＴである。配列番号２で示される塩基配列中、ＨはＡ、Ｔ又はＣであり、ＶはＡ、Ｃ又はＧである。 In the nucleotide sequence shown in SEQ ID NO: 1, N is A (adenine), T (thymine), G (guanine) or C (cytosine), and W is A or T. In the base sequence shown in SEQ ID NO: 2, H is A, T or C, and V is A, C or G.
第１プライマーは、配列番号１で示される塩基配列の５’側又は３’側に追加的な塩基配列を含んでもよい。このようなプライマーの例としては、５’側に追加的な塩基配列を含む配列番号３：
5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′のＤＮＡ分子が挙げられる。同様に、第２プライマーは、配列番号２で示される塩基配列の５’側又は３’側に追加的な塩基配列を含んでもよい。このようなプライマーの例としては、５’側に追加的な塩基配列を含む配列番号４：
5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′のＤＮＡ分子が挙げられる。
The first primer may contain an additional base sequence on the 5'side or 3'side of the base sequence shown in SEQ ID NO: 1. An example of such a primer is SEQ ID NO: 3: which contains an additional base sequence on the 5'side.
The DNA molecule of 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3' can be mentioned. Similarly, the second primer may contain an additional base sequence on the 5'side or 3'side of the base sequence shown in SEQ ID NO: 2. An example of such a primer is SEQ ID NO: 4: which contains an additional base sequence on the 5'side.
The DNA molecule of 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3' can be mentioned.
リアルタイムＰＣＲにより増幅されたＤＮＡ分子の配列を解読することにより、微生物群の種をそれぞれ特定することができる。解読されたＤＮＡ配列と既知の種のＤＮＡ配列との相同性が９７％以上である場合に、解読されたＤＮＡ配列がこの既知の種由来の１６Ｓ ｒＲＮＡの鋳型ＤＮＡ配列又は鋳型ＤＮＡの相補鎖のＤＮＡ配列であると判定することができる。 By sequencing the DNA molecules amplified by real-time PCR, the species of each microbial community can be identified. When the homology between the decoded DNA sequence and the DNA sequence of a known species is 97% or more, the decoded DNA sequence is the template DNA sequence of 16S rRNA derived from this known species or the complementary strand of the template DNA. It can be determined that it is a DNA sequence.
図１は、本発明の植物生長促進剤の一例（以下「植物生長促進剤Ａ」という。）中の微生物をリアルタイムＰＣＲで定量した際の解析結果を示す。図１には、リアルタイムＰＣＲにより検出された代表的な微生物のリード数を門ごとに示す。リード数は、リアルタイムＰＣＲにより増幅されたＤＮＡ分子の塩基配列を解読した結果、図１に示す門に属する微生物由来であると判定されたＤＮＡ分子の数である。図１の全リード数に対する割合は、塩基配列を解読したＤＮＡ分子の総数（全リード数）に対するリード数の割合の百分率である。なお、図１に示す結果を得るための解析方法の詳細については後述する。 FIG. 1 shows the analysis results when the microorganisms in an example of the plant growth promoter of the present invention (hereinafter referred to as “plant growth promoter A”) were quantified by real-time PCR. FIG. 1 shows the number of reads of typical microorganisms detected by real-time PCR for each phyla. The number of reads is the number of DNA molecules determined to be derived from the microorganism belonging to the phylum shown in FIG. 1 as a result of decoding the base sequence of the DNA molecule amplified by real-time PCR. The ratio to the total number of reads in FIG. 1 is a percentage of the ratio of the number of reads to the total number of DNA molecules whose nucleotide sequences have been decoded (total number of reads). The details of the analysis method for obtaining the result shown in FIG. 1 will be described later.
植物生長促進剤ＡのリアルタイムＰＣＲによる解析結果では、図１に示すように、全リード数に対するそれぞれの門に属する微生物のリード数の割合は、アシドバクテリア門に含まれる微生物が１４．８８％、バクテロイデス門に含まれる微生物が６．８０４％、クロロビウム門に含まれる微生物が８．６１９％、クロロフレクサス門に含まれる微生物が８．９５２％、ゲンマティモナス門に含まれる微生物が３．１２％、未培養門ＯＤ１に含まれる微生物が２．９２９％、プロテオバクテリア門に含まれる微生物が３６．７９２％、ウェルコミクロビウム門に含まれる微生物が２．８３％であった。 According to the results of real-time PCR analysis of the plant growth promoter A, as shown in FIG. 1, the ratio of the number of leads of microorganisms belonging to each phylum to the total number of reads was 14.88% for the microorganisms contained in the phylum Acidobacteria. 6.804% of the microorganisms contained in the phylum Bacteroides, 8.619% of the microorganisms contained in the phylum Chlorobium, 8.952% of the microorganisms contained in the phylum Chlorofexus, and 3.12% of the microorganisms contained in the phylum Genmatimonas. , 2.929% of the microorganisms contained in the uncultured phylum OD1, 36.792% of the microorganisms contained in the Proteobacterium phylum, and 2.83% of the microorganisms contained in the Welcomicrobium phylum.
植物生長促進剤Ａに含まれる微生物の割合は、植物生長促進剤Ａの製造場所の地理的条件等により多少ばらつくことがあるが、図１に示す下限値は、このばらつきによらずに検出可能な微生物の割合の下限値である。この下限値は、図１に示す全リード数に対する割合と同様に、それぞれの門に含まれる微生物が植物生長促進剤ＡのリアルタイムＰＣＲにおいて検出されるリード数の全リード数に対する百分率を示す。図１の下限値の欄に示すように、植物生長促進剤Ａは、アシドバクテリア門に含まれる微生物を３．５％以上、バクテロイデス門に含まれる微生物を１．５％以上、クロロビウム門に含まれる微生物を１．５％以上、クロロフレクサス門に含まれる微生物を１．５％以上、ゲンマティモナス門に含まれる微生物を０．５％以上、未培養門ＯＤ１に含まれる微生物を０．５％以上、プロテオバクテリア門に含まれる微生物を８％以上、ウェルコミクロビウム門に含まれる微生物を０．５％以上少なくとも含む。 The proportion of microorganisms contained in the plant growth promoter A may vary slightly depending on the geographical conditions of the place where the plant growth promoter A is produced, but the lower limit shown in FIG. 1 can be detected regardless of this variation. It is the lower limit of the proportion of various microorganisms. This lower limit indicates the percentage of the total number of reads that the microorganisms contained in each phylum are detected in the real-time PCR of the plant growth promoter A, similar to the ratio to the total number of reads shown in FIG. As shown in the lower limit column of FIG. 1, the plant growth promoter A contains 3.5% or more of microorganisms contained in the phylum Acidobacteria, 1.5% or more of microorganisms contained in the phylum Bacteroidetes, and phylum Chlorobium. 1.5% or more of the microorganisms contained in the phylum Chloroflexus, 1.5% or more of the microorganisms contained in the phylum Proteobacteria, 0.5% or more of the microorganisms contained in the uncultivated phylum OD1 % Or more, 8% or more of microorganisms contained in the phylum Proteobacteria, and 0.5% or more of microorganisms contained in the phylum Welcomicrobium.
［炭素分］
本発明の植物生長促進剤は、家畜の糞尿由来であり、炭素分（有機物）を含有していてもよい。ただし、本発明の植物生長促進剤は、炭素分が少ない方が好ましく、好ましくは、全炭素濃度が２，０００ｍｇ／ｋｇ以下である。このように、植物生長促進剤における炭素の濃度が、原料の糞尿に含有される炭素の濃度よりも低いので、植物に植物生長促進剤を与えた場合に、土壌中の有機物の濃度が高くなることを抑制することができる。このため、本発明の植物生長促進剤は、土壌中の酸素の欠乏や植物の根腐れを引き起こすことを抑制することができる。
[Carbon]
The plant growth promoter of the present invention is derived from livestock manure and may contain carbon (organic matter). However, the plant growth promoter of the present invention preferably has a low carbon content, and preferably has a total carbon concentration of 2,000 mg / kg or less. In this way, the concentration of carbon in the plant growth promoter is lower than the concentration of carbon contained in the manure of the raw material, so that when the plant is given the plant growth promoter, the concentration of organic matter in the soil becomes high. Can be suppressed. Therefore, the plant growth promoter of the present invention can suppress the cause of oxygen deficiency in soil and root rot of plants.
本発明の植物生長促進剤は、窒素分を含有していてもよいが、窒素分が少なくとも植物生長促進効果を発揮することができる。本発明の植物生長促進剤は、好ましくは、全窒素濃度が５００ｍｇ／ｋｇ以下である。この全窒素濃度は、一般的な肥料に含まれる全窒素濃度と比較すると少ない。 The plant growth-promoting agent of the present invention may contain nitrogen, but the nitrogen content can at least exert a plant growth-promoting effect. The plant growth promoter of the present invention preferably has a total nitrogen concentration of 500 mg / kg or less. This total nitrogen concentration is lower than the total nitrogen concentration contained in general fertilizers.
本発明の植物生長促進剤は、リン分を含有していてもよいが、リン分が少なくとも植物生長促進効果を発揮することができる。本発明の植物生長促進剤は、好ましくは、全リン濃度が２，０００ｍｇ／ｋｇ以下である。この全リン濃度は、一般的な肥料に含まれる全リン濃度と比較すると少ない。 The plant growth promoter of the present invention may contain phosphorus, but the phosphorus can at least exert a plant growth promoting effect. The plant growth promoter of the present invention preferably has a total phosphorus concentration of 2,000 mg / kg or less. This total phosphorus concentration is lower than the total phosphorus concentration contained in general fertilizers.
本発明の植物生長促進剤は、カリウム（塩であってもよい）を含有していてもよいが、カリウムが少なくとも植物生長促進効果を発揮することができる。本発明の植物生長促進剤は、好ましくは、カリウム濃度が５，０００ｍｇ／ｋｇ以下である。このカリウム濃度は、一般的な肥料に含まれる全リン濃度と比較すると少ない。 The plant growth-promoting agent of the present invention may contain potassium (which may be a salt), but potassium can at least exert a plant growth-promoting effect. The plant growth promoter of the present invention preferably has a potassium concentration of 5,000 mg / kg or less. This potassium concentration is low compared to the total phosphorus concentration contained in general fertilizers.
本発明の植物生長促進剤は、剤型は限定されず、液体、固形又はスラリー等のいずれであってもよいが、液体であることが好ましい。
また、本発明の植物生長促進剤は、剤型に応じた副成分、例えば、希釈剤、安定剤、増粘剤、造粒剤などを含んでいてもよい。
The dosage form of the plant growth promoter of the present invention is not limited and may be liquid, solid, slurry or the like, but it is preferably liquid.
In addition, the plant growth promoter of the present invention may contain auxiliary components according to the dosage form, for example, a diluent, a stabilizer, a thickener, a granulator and the like.
本発明の植物生長促進剤の好適な態様として、全窒素濃度が５００ｍｇ／ｋｇ以下であり、全リン濃度が２，０００ｍｇ／ｋｇ以下であり、全カリウム濃度が５，０００ｍｇ／ｋｇ以下であり、アンモニア性窒素の濃度が２００ｍｇ／Ｌ以下であり、水溶性リン酸の五酸化リン酸換算の濃度が５質量％以下であり、且つ、水溶性カリウムの二酸化カリウム換算の濃度が５質量％以下である植物生長促進剤が挙げられる。本発明の植物生長促進剤における窒素、リン及び水溶性カリウムの濃度は、一般的な肥料における窒素、リン及び水溶性カリウムの濃度より低い。本発明の植物生長促進剤は、窒素等の濃度が低いにもかかわらず、後述の実施例において確認されているように植物生長促進効果がある。このことから、植物生長促進剤には上記微生物に起因する植物ホルモン様物質が含まれていると推定される。 As a preferred embodiment of the plant growth promoter of the present invention, the total nitrogen concentration is 500 mg / kg or less, the total phosphorus concentration is 2,000 mg / kg or less, and the total potassium concentration is 5,000 mg / kg or less. When the concentration of ammoniacal nitrogen is 200 mg / L or less, the concentration of water-soluble phosphoric acid in terms of phosphoric acid pentoxide is 5% by mass or less, and the concentration of water-soluble potassium in terms of potassium dioxide is 5% by mass or less. Some plant growth promoters are mentioned. The concentration of nitrogen, phosphorus and water-soluble potassium in the plant growth promoter of the present invention is lower than the concentration of nitrogen, phosphorus and water-soluble potassium in general fertilizers. Although the plant growth promoter of the present invention has a low concentration of nitrogen and the like, it has a plant growth promoting effect as confirmed in Examples described later. From this, it is presumed that the plant growth promoter contains a plant hormone-like substance caused by the above-mentioned microorganism.
［植物生長促進剤の好適な製造方法の例］
本発明の植物生長促進剤は、例えば、家畜、好ましくは牛や豚の糞尿を数日間以上曝気させることにより得ることができる。糞尿を曝気させることにより、上記した微生物が増殖し、本発明の植物生長促進剤が得られる。
曝気させる期間は、好ましくは、１ヶ月以上であり、より好ましくは、１０ヶ月以上であり、さらに好ましくは１２ヶ月以上であり、特に好ましくは、１３ヶ月〜１８ヶ月である。
[Example of suitable production method of plant growth promoter]
The plant growth promoter of the present invention can be obtained, for example, by aerating livestock, preferably cow or pig manure, for several days or longer. By aeration of manure, the above-mentioned microorganisms proliferate, and the plant growth promoter of the present invention is obtained.
The period of aeration is preferably 1 month or longer, more preferably 10 months or longer, still more preferably 12 months or longer, and particularly preferably 13 to 18 months.
［植物生長促進剤の好適な製造方法］
本発明の植物生長促進剤の好適な製造方法を説明する。本発明の植物生長促進剤は、好ましくは、以下の（１）及び（２）のステップを含む方法により製造される。
（１）家畜の糞尿の液体成分を分離するステップ
（２）液体成分を曝気させるステップ
（１）のステップでは、家畜の糞尿を蓄積し、蓄積した糞尿の液体成分を分離する。例えば、家畜の糞尿の上澄み液を液体成分として分離する。
[Preferable manufacturing method of plant growth promoter]
A suitable method for producing the plant growth promoter of the present invention will be described. The plant growth promoter of the present invention is preferably produced by a method including the following steps (1) and (2).
(1) Step of separating the liquid component of livestock manure (2) Step of aerating the liquid component In the step of (1), livestock manure is accumulated and the accumulated liquid component of manure is separated. For example, the supernatant of livestock manure is separated as a liquid component.
（２）のステップでは、家畜の糞尿の液体成分を曝気処理槽において曝気する。曝気処理槽においては、糞尿の液体成分中へ空気を送り込み、送り込んだ空気に液体を触れさせることにより、糞尿の液体成分を曝気させることができる。曝気処理槽のサイズに特に制限は設けないが、例えば、１Ｌ〜１００ｋＬの間である。曝気処理槽のサイズは、望ましくは１０Ｌ〜１０ｋＬ、更に望ましくは５００Ｌ〜５ｋＬである。処理液の供給形態は制限されるものではないが、曝気処理槽に連続的又は半連続的に家畜の糞尿又はその液体成分が供給されることが望ましい。 In step (2), the liquid component of livestock manure is aerated in an aeration treatment tank. In the aeration treatment tank, the liquid component of manure can be aerated by sending air into the liquid component of manure and bringing the liquid into contact with the sent air. The size of the aeration treatment tank is not particularly limited, but is, for example, between 1 L and 100 kL. The size of the aeration treatment tank is preferably 10 L to 10 kL, more preferably 500 L to 5 kL. The form of supply of the treatment liquid is not limited, but it is desirable that livestock manure or a liquid component thereof is continuously or semi-continuously supplied to the aeration treatment tank.
図２は、植物生長促進剤Ａを製造するための製造装置１００の一例を示す。製造装置１００は、糞尿貯め１０と、供給部２０と、第１曝気処理槽３０ａ〜第４曝気処理槽３０ｄと、コンプレッサ４０ａ〜コンプレッサ４０ｄと、回収部５０とを備える。糞尿貯め１０は、牛舎中の牛の糞尿を貯える。供給部２０は、糞尿貯め１０と第１曝気処理槽３０ａとの間に接続されたパイプを介して、糞尿貯め１０に貯えられた糞尿をポンプにより第１曝気処理槽３０ａへ供給する。供給部２０は、自動的且つ断続的に糞尿を供給し、例えば、１ヵ月に１回のペースで糞尿を供給する。 FIG. 2 shows an example of a manufacturing apparatus 100 for manufacturing a plant growth promoter A. The manufacturing apparatus 100 includes a manure storage 10, a supply unit 20, a first aeration treatment tank 30a to a fourth aeration treatment tank 30d, a compressor 40a to a compressor 40d, and a recovery unit 50. The manure reservoir 10 stores manure from cows in the barn. The supply unit 20 pumps the manure stored in the manure storage 10 to the first aeration treatment tank 30a via a pipe connected between the manure storage 10 and the first aeration treatment tank 30a. The supply unit 20 automatically and intermittently supplies manure, for example, manure once a month.
第１曝気処理槽３０ａ〜第４曝気処理槽３０ｄは、槽内に貯えている家畜の糞尿の液体成分を曝気させる。図２は、製造装置１００が４つの槽を有する例を示すが、製造装置１００は１槽のみを有してもよく、直列に配置された２〜８槽を有してもよい。第１曝気処理槽３０ａのサイズは、一例としては縦３ｍ、横３ｍ、深さ２．５ｍであり、第２曝気処理槽３０ｂ〜第４曝気処理槽３０ｄは、第１曝気処理槽３０ａと同じサイズであってもよい。 The first aeration treatment tanks 30a to the fourth aeration treatment tanks 30d aerate the liquid components of livestock manure stored in the tanks. FIG. 2 shows an example in which the manufacturing apparatus 100 has four tanks, but the manufacturing apparatus 100 may have only one tank, or may have 2 to 8 tanks arranged in series. The size of the first aeration treatment tank 30a is, for example, 3 m in length, 3 m in width, and 2.5 m in depth, and the second aeration treatment tank 30b to the fourth aeration treatment tank 30d are the same as the first aeration treatment tank 30a. It may be the size.
第１曝気処理槽３０ａ〜第４曝気処理槽３０ｄは、糞尿を固体成分と液体成分とに分離する。第１曝気処理槽３０ａ〜第４曝気処理槽３０ｄは、槽内の糞尿の固体成分を沈降させる。一方、第１曝気処理槽３０ａからあふれた糞尿の液体成分は、第２曝気処理槽３０ｂへ移動する。液体成分が、第３曝気処理槽３０ｃ、第４曝気処理槽３０ｄへ順次移動し、それぞれの曝気処理層において糞尿の固体成分が沈降することにより、糞尿の固体成分がほぼ除去される。 The first aeration treatment tank 30a to the fourth aeration treatment tank 30d separate manure into a solid component and a liquid component. The first aeration treatment tank 30a to the fourth aeration treatment tank 30d settle the solid component of manure in the tank. On the other hand, the liquid component of manure overflowing from the first aeration treatment tank 30a moves to the second aeration treatment tank 30b. The liquid component sequentially moves to the third aeration treatment tank 30c and the fourth aeration treatment tank 30d, and the solid component of manure is settled in each aeration treatment layer, so that the solid component of manure is substantially removed.
コンプレッサ４０ａ〜コンプレッサ４０ｄは、空気を圧縮して、第１曝気処理槽３０ａ〜第４曝気処理槽３０ｄ内において糞尿の液体成分中に圧縮した空気を送り込むための装置である。第１曝気処理槽３０ａ〜第４曝気処理槽３０ｄでは、コンプレッサ４０ａ〜コンプレッサ４０ｄにより送り込まれた空気に液体成分が触れるので、液体成分を曝気させることができる。第１曝気処理槽３０ａ〜第４曝気処理槽３０ｄは、コンプレッサ４０ａ〜コンプレッサ４０ｄを用いて液体成分内の微生物の増殖に要する酸素を供給することにより、微生物の増殖を促進させることができる。微生物の増殖を促進させることで、糞尿の液体成分に含まれる植物発芽阻害成分の分解量を増やすことができる。 The compressors 40a to 40d are devices for compressing air and sending the compressed air into the liquid component of manure in the first aeration treatment tank 30a to the fourth aeration treatment tank 30d. In the first aeration treatment tanks 30a to the fourth aeration treatment tank 30d, since the liquid component comes into contact with the air sent by the compressors 40a to 40d, the liquid component can be aerated. The first aeration treatment tanks 30a to the fourth aeration treatment tanks 30d can promote the growth of microorganisms by supplying oxygen required for the growth of microorganisms in the liquid component by using the compressors 40a to 40d. By promoting the growth of microorganisms, the amount of decomposition of the plant germination inhibitor component contained in the liquid component of manure can be increased.
（２）のステップでは、Ｆ［ｍ／ｓ］を空気流量、Ｖ［ｍ３］を処理液体積、ＨＬ［ｍ］を処理液深度とした場合に、
曝気が行われている間の糞尿の液体成分の温度は、０℃〜４０℃の範囲である。曝気が行われている間の糞尿の液体成分のｐＨは、６．０〜８．０の間が好適である。 The temperature of the liquid component of manure during aeration is in the range of 0 ° C to 40 ° C. The pH of the liquid component of manure during aeration is preferably between 6.0 and 8.0.
曝気時間は、糞尿の液体成分の植物発芽阻害成分が十分に除去できるまでの時間であり、好ましくは、１ヶ月以上、さらに好ましくは１２カ月以上である。 The aeration time is the time until the plant germination inhibitory component of the liquid component of manure can be sufficiently removed, and is preferably 1 month or longer, more preferably 12 months or longer.
また、微生物による植物発芽阻害成分の除去を促進させるため、外部から何らかの従属栄養性好気微生物を添加してもよい。従属栄養性好気微生物は、例えば、酵母、乳酸菌等である。 Further, in order to promote the removal of the plant germination inhibitory component by the microorganism, some heterotrophic aerobic microorganism may be added from the outside. Heterotrophic aerobic microorganisms are, for example, yeast, lactic acid bacteria and the like.
図２の回収部５０は、例えばポンプを用いて、第４曝気処理槽３０ｄに接続されたパイプを介して、曝気された後の液体成分を植物生長促進剤として回収する。回収された植物生長促進剤では、（２）のステップの処理を実行することにより、含有する微生物が増殖しており、第１プライマー及び第２プライマーを用いて検出される微生物のうち、バクテロイデス門に含まれる微生物を１．５％以上、クロロフレクサス門に含まれる微生物を１．５％以上、ゲンマティモナス門に含まれる微生物を０．５％以上、又は、ウェルコミクロビウム門に含まれる微生物を０．５％以上含む。 The recovery unit 50 in FIG. 2 recovers the liquid component after aeration as a plant growth promoter through a pipe connected to the fourth aeration treatment tank 30d, for example, using a pump. In the recovered plant growth promoter, the microorganisms contained in the recovered plant growth promoter are grown by carrying out the treatment of step (2), and among the microorganisms detected using the first primer and the second primer, the phylum Bacteroidetes 1.5% or more of microorganisms contained in, 1.5% or more of microorganisms contained in Chloroflexi, 0.5% or more of microorganisms contained in Gemmatimonadetes, or Verrucomicrobia Contains 0.5% or more of microorganisms.
回収された植物生長促進剤は、そのままもしくは５０倍まで希釈して植物に使用することができ、植物生長促進剤単体、又は植物生長促進剤と無機肥料もしくは有機肥料とを混合して使用することにより、相乗的に植物生長促進効果が期待できる。 The recovered plant growth promoter can be used as it is or diluted up to 50 times for plants, and the plant growth promoter alone or a mixture of the plant growth promoter and an inorganic fertilizer or an organic fertilizer should be used. Therefore, a synergistic effect of promoting plant growth can be expected.
回収された植物生長促進剤は、（２）のステップにおいて微生物により液体成分に含まれる有機物がほぼ完全に消費されているので、植物に植物生長促進剤を加えた場合に、土壌中の有機物濃度が高くなることを抑制することができる。このため、高い有機物濃度に起因する酸素の欠乏や植物の根腐れを抑制することができる。また、これまで利用方法が乏しかった家畜糞尿の液体成分を植物生長促進剤として有効活用することができる。植物生長促進剤を使用する対象となる植物の栽培形態は特に限定されず、畑作、稲作及び水耕栽培において生長促進効果を示すことが確認されている。また、植物生長促進剤に有機肥料又は無機肥料以外の副成分を混合した状態で植物の生長促進のために用いてもよい。 In the recovered plant growth promoter, the organic matter contained in the liquid component is almost completely consumed by the microorganisms in the step (2). Therefore, when the plant growth promoter is added to the plant, the concentration of the organic matter in the soil Can be suppressed from becoming high. Therefore, oxygen deficiency and root rot of plants due to high organic matter concentration can be suppressed. In addition, the liquid component of livestock manure, which has been poorly used so far, can be effectively used as a plant growth promoter. The cultivation form of the target plant to which the plant growth promoter is used is not particularly limited, and it has been confirmed that it exhibits a growth promoting effect in upland farming, rice cultivation and hydroponics. Further, it may be used for promoting the growth of plants in a state where the plant growth promoter is mixed with auxiliary components other than organic fertilizer or inorganic fertilizer.
［実施例１］
［植物生長促進剤Ａの製造］
図２に示した製造装置１００を用いて植物生長促進剤Ａを製造した。植物生長促進剤Ａの原材料として牛舎中の牛の糞尿を用いた。図２の第４曝気処理槽３０ｄの貯留液から１ヶ月に１回約６トンの植物生長促進剤Ａを取り出した。第１曝気処理槽３０ａ〜第４曝気処理槽３０ｄの体積はそれぞれ２０ｋＬとした。曝気量は各曝気処理槽で約３．７５ｍ３／分とした。Ｆ［ｍ／ｓ］を空気流量、Ｖ［ｍ３］を処理液体積、ＨＬ［ｍ］を処理液深度とした場合に、以下の式（ａ）：
[Manufacturing of plant growth promoter A]
The plant growth promoter A was produced using the production apparatus 100 shown in FIG. Manure from cows in the barn was used as a raw material for the plant growth promoter A. Approximately 6 tons of plant growth promoter A was taken out from the reservoir of the fourth aeration treatment tank 30d in FIG. 2 once a month. The volumes of the first aeration treatment tank 30a to the fourth aeration treatment tank 30d were set to 20 kL, respectively. The amount of aeration was about 3.75 m 3 / min in each aeration treatment tank. When F [m / s] is the air flow rate, V [m 3 ] is the treatment liquid volume, and HL [m] is the treatment liquid depth, the following equation (a):
［植物生長促進剤Ａの組成分析］
簡易ＣＯＤメーターＨＣ−６０７を用いて化学的酸素要求量（ＣＯＤ）を測定した。水素イオン濃度ｐＨは、ＪＩＳ Ｋ０１０２ １７．１に従い、ガラス電極法を用いて測定した。全炭素濃度は、全有機炭素計（ＴＯＣ−Ｖ，ＳＳＭ−５０００Ａ，島津製作所）を用いて測定した。全窒素濃度の測定では、ケルダール法により変換されたアンモニア態窒素をインドフェノール青法で測定した。全リン濃度の測定では、植物生長促進剤Ａ中のリンにモリブデン酸アンモニウムを加えて錯体を形成させ、アスコルビン酸により還元させ、還元反応による生成物の７１０ｎｍの吸収を測定した。全カリウム濃度は、原子吸光法により測定した。無機窒素は試料４．０ｇを１．０ Ｍ ＫＣｌ ４０ｍＬに溶解させ、遠心分離後の上清を測定に用いた。
[Composition analysis of plant growth promoter A]
The chemical oxygen demand (COD) was measured using a simple COD meter HC-607. The hydrogen ion concentration pH was measured using the glass electrode method according to JIS K0102 17.1. The total carbon concentration was measured using a total organic carbon meter (TOC-V, SSM-5000A, Shimadzu Corporation). In the measurement of total nitrogen concentration, the ammonia nitrogen converted by the Kjeldahl method was measured by the indophenol blue method. In the measurement of the total phosphorus concentration, ammonium molybdate was added to phosphorus in the plant growth promoter A to form a complex, which was reduced by ascorbic acid, and the absorption of the product by the reduction reaction at 710 nm was measured. The total potassium concentration was measured by atomic absorption spectroscopy. For inorganic nitrogen, 4.0 g of the sample was dissolved in 40 mL of 1.0 M KCl, and the supernatant after centrifugation was used for the measurement.
アンモニア態窒素の濃度の測定は、アンモニア態窒素の抽出液１．０ｍＬにフェノールニトロプルシッド溶液４００μＬを混合し、次亜塩素酸ナトリウム溶液６００μＬを加えてから撹拌し、４５分後に６３５ｎｍの光の吸光度を分光光度計で測定することにより行った。 To measure the concentration of ammonia nitrogen, mix 400 μL of phenol nitropulside solution with 1.0 mL of ammonia nitrogen extract, add 600 μL of sodium hypochlorite solution, stir, and after 45 minutes, measure the light of 635 nm. This was done by measuring the absorbance with a spectrophotometer.
また、亜硝酸態窒素の濃度の測定では、まず、亜硝酸態窒素の１．０ｍＬの抽出液に１００μＬのスルファニルアミド溶液を加えてから撹拌し、３分間放置した。その後、ナフチルエチレンジアミン溶液１００μＬを加えてから撹拌し、２０分間放置した。その後、５４０ｎｍの光の吸光度を分光光度計で測定することにより亜硝酸態窒素の濃度を測定した。 In the measurement of the concentration of nitrite nitrogen, first, 100 μL of sulfanilamide solution was added to 1.0 mL of the extract of nitrite nitrogen, and the mixture was stirred and left for 3 minutes. Then, 100 μL of the naphthylethylenediamine solution was added, and the mixture was stirred and left for 20 minutes. Then, the concentration of nitrite nitrogen was measured by measuring the absorbance of light at 540 nm with a spectrophotometer.
硝酸態窒素の測定では、硝酸態窒素の抽出液２００μＬにブルシン・４−アミノベンゼンスルホン酸溶液１００μＬを加えた。その後、硫酸（水：濃硫酸＝３：２０）１．０ｍＬを加えてから撹拌し、冷暗所で１０分間反応させた。さらに水１．０ｍＬを加えた後に、４１０ｎｍの光の吸光度を分光光度計で測定した。 In the measurement of nitrate nitrogen, 100 μL of a brucine 4-aminobenzenesulfonic acid solution was added to 200 μL of the nitrate nitrogen extract. Then, 1.0 mL of sulfuric acid (water: concentrated sulfuric acid = 3:20) was added, and the mixture was stirred and reacted in a cool and dark place for 10 minutes. After further adding 1.0 mL of water, the absorbance of light at 410 nm was measured with a spectrophotometer.
植物生長促進剤ＡのｐＨは８．８２、化学的酸素要求量（ＣＯＤ）は７４９．３±３３．４ｍｇ／Ｌ（試行回数３回、±以下の数値は標準偏差）であった。全炭素濃度は１，１００ ｍｇ／ｋｇ、全窒素濃度は２９０ｍｇ−Ｎ／ｋｇ、全リン濃度は、２６ｍｇ／ｋｇ、全カリウム濃度は２，７００ｍｇ／ｋｇであった。水溶性硝酸態窒素濃度は３８０ｍｇ−Ｎ／ｋｇ、アンモニア性窒素は不検出、水溶性リン酸濃度は五酸化リン酸換算で６４ｍｇ−Ｐ２Ｏ５／ｋｇ、水溶性カリウム濃度は酸化カリウム換算で３，２５４ｍｇ−Ｋ２Ｏ／ｋｇであった。 The pH of the plant growth promoter A was 8.82, and the chemical oxygen demand (COD) was 749.3 ± 33.4 mg / L (three trials, values below ± are standard deviations). The total carbon concentration was 1,100 mg / kg, the total nitrogen concentration was 290 mg-N / kg, the total phosphorus concentration was 26 mg / kg, and the total potassium concentration was 2,700 mg / kg. Water-soluble nitrate nitrogen concentration is 380 mg-N / kg, ammoniacal nitrogen is not detected, water-soluble phosphoric acid concentration is 64 mg-P 2 O 5 / kg in terms of pentoxide, and water-soluble potassium concentration is in terms of potassium oxide. It was 3,254 mg-K 2 O / kg.
北海道施肥ガイド２０１５（北海道ホームページ、 http://www.pref.hokkaido.lg.jp/ns/shs/clean/sehiguide2015.htm、２０１９年１月２１日閲覧）によると、一般的な牛糞堆肥の全炭素量は７２，０００ｍｇ／ｋｇ、全窒素量は６，０００ｍｇ−Ｎ／ｋｇ、水溶性リン酸は４，０００ｍｇ−Ｐ２Ｏ５／ｋｇ、水溶性カリウムは５，０００ｍｇ−Ｋ２Ｏ／ｋｇ程度である。したがって、植物生長促進剤Ａの中に含まれる肥料成分は、一般的な牛糞堆肥に比べて極めて希薄であることがわかる。 According to the Hokkaido Fertilization Guide 2015 (Hokkaido homepage, http://www.pref.hokkaido.lg.jp/ns/shs/clean/sehiguide2015.htm, viewed January 21, 2019), all of the general cow dung compost Carbon content is 72,000 mg / kg, total nitrogen content is 6,000 mg-N / kg, water-soluble phosphoric acid is 4,000 mg-P 2 O 5 / kg, and water-soluble potassium is 5,000 mg-K 2 O / kg. Degree. Therefore, it can be seen that the fertilizer component contained in the plant growth promoter A is extremely dilute as compared with general cow dung compost.
［植物生長促進効果の検証（水耕栽培）］
ＬＥＤ光源付の栽培棚でトマト及び小松菜を栽培することにより、植物生長促進効果の確認試験を実施した。試験区では、水で１／１０希釈した植物生長促進剤Ａを吸収させた約１ｃｍ角のウレタンフォームに、種子が上を向いた状態で並べた。一方、比較のための対照区では、脱イオン水を入れたバットに、種子が上を向いた状態で並べた。１２時間ごとに光源の点灯状態と消灯状態とを切り替えた。温度の調節は、実験室のエアコンを用いて、２８℃に設定することにより行った。それぞれの試験区で２７検体の試験を実施し、発芽して生育した植物体の高さを測定し、平均高さ及び標準偏差を求めた。発芽しなかった植物体は実験結果の評価対象から除外した。
[Verification of plant growth promotion effect (hydroponics)]
A confirmation test of the plant growth promoting effect was carried out by cultivating tomatoes and Japanese mustard spinach on a cultivation shelf equipped with an LED light source. In the test plot, the seeds were arranged in a urethane foam of about 1 cm square absorbed with the plant growth promoter A diluted 1/10 with water with the seeds facing upward. On the other hand, in the control group for comparison, the seeds were arranged in a vat containing deionized water with the seeds facing upward. The lighting state and the extinguishing state of the light source were switched every 12 hours. The temperature was adjusted by setting it to 28 ° C. using a laboratory air conditioner. Twenty-seven samples were tested in each test plot, the height of the germinated and grown plants was measured, and the average height and standard deviation were determined. Plants that did not germinate were excluded from the evaluation of experimental results.
図３は、播種後１ヶ月の小松菜及びトマトを撮影した様子を示す写真である。対照区の水のみで生育したトマトならびに小松菜の地上部の高さは、それぞれ１．９８±０．１３ｃｍ、１．９３±４．３４ｃｍであったのに対し、試験区にて１／１０希釈した植物生長促進剤Ａで生育したものは、それぞれ７．５５±０．８６ｃｍ、５．０４±０．６２ｃｍであった。小松菜及びトマトは、試験区においては対照区と比べて、それぞれ３．８倍、２．６倍の高さまで生長した。したがって、製造した植物生長促進剤Ａは、小松菜及びトマトの両方において植物生長促進効果をもたらすことを確認できた。 FIG. 3 is a photograph showing a photograph of Japanese mustard spinach and tomatoes one month after sowing. The heights of the above-ground parts of tomatoes and Japanese mustard spinach grown only in the water of the control group were 1.98 ± 0.13 cm and 1.93 ± 4.34 cm, respectively, whereas they were diluted 1/10 in the test group. Those grown with the plant growth promoter A were 7.55 ± 0.86 cm and 5.04 ± 0.62 cm, respectively. Komatsuna and tomato grew to a height of 3.8 times and 2.6 times in the test group as compared with the control group, respectively. Therefore, it was confirmed that the produced plant growth-promoting agent A had a plant growth-promoting effect on both Japanese mustard spinach and tomato.
［実施例２］
［無機栄養源との併用効果］
実施例１と同様の条件で、試験区において、水で５００倍希釈したハイポネックス社製のハイポネックス原液（マニュアルの標準使用量。以下、無機栄養とも表記する）と１０倍希釈した植物生長促進剤Ａとを併用した状態で、トマト及び小松菜を栽培した。対照区において、水で５００倍希釈したハイポネックス原液のみを用いて植物生長促進剤Ａを加えない状態で、トマト及び小松菜を栽培した。
[Example 2]
[Effect of combined use with inorganic nutrient sources]
Under the same conditions as in Example 1, in the test plot, Hyponex stock solution (standard amount used in the manual; hereinafter also referred to as inorganic nutrition) diluted 500 times with water and plant growth promoter A diluted 10 times. Tomatoes and Komatsuna were cultivated in combination with. In the control group, tomatoes and Japanese mustard spinach were cultivated using only the Hyponex stock solution diluted 500 times with water and without adding the plant growth promoter A.
植物生長促進剤Ａを添加せずに無機栄養のみを用いた対照区では、小松菜及びトマトの生長後の高さがそれぞれ５．９７±２．４ｃｍ、４．７０±２．１ｃｍであったのに対し、植物生長促進剤Ａと無機栄養を併用した試験区では、それぞれ１０．２２±５．５８ｃｍ、４．９４±３．２８ｃｍであった。小松菜においては約４０％の生長促進効果が認められた。トマトを栽培する場合、無機栄養のみの場合と比べて、有意な生長促進効果は認められなかった。以上のように、実施例に記載した植物生長促進剤Ａは、市販の肥料と併用する場合に植物の生長促進の相乗効果を発揮することを確認した。 In the control group using only inorganic nutrients without adding the plant growth promoter A, the heights of Japanese mustard spinach and tomato after growth were 5.97 ± 2.4 cm and 4.70 ± 2.1 cm, respectively. On the other hand, in the test group in which the plant growth promoter A and the inorganic nutrient were used in combination, the heights were 10.22 ± 5.58 cm and 4.94 ± 3.28 cm, respectively. In Komatsuna, a growth promoting effect of about 40% was observed. When tomatoes were cultivated, no significant growth promoting effect was observed as compared with the case of using only inorganic nutrients. As described above, it was confirmed that the plant growth promoter A described in the examples exerts a synergistic effect of plant growth promotion when used in combination with a commercially available fertilizer.
［微生物群集解析］
実施例１に記載した方法で製造した植物生長促進剤Ａ（１ｍＬ）を遠心分離（２０，０００×ｇ、５分間）し、沈殿物を回収した。沈殿物を５６０μＬのＴＥ緩衝液（１０ｍＭ Ｔｒｉｓ緩衝液（ｐＨ８．０）、１ｍＭ ＥＤＴＡ）に溶解した後、３０μＬのＳＤＳ溶液、１０μＬのＰｒｏｔｅｉｎａｓｅＫ（１０ｍｇ／ｍＬ）を添加し、混ぜた後、３７℃で１時間保温した。その後、１００μＬの５Ｍ ＮａＣｌを添加し、よく混ぜた後、８０μＬのＣＴＡＢ／ＮａＣｌ溶液を加え、よく混ぜた。サンプルを６５℃で１０分間保温した。保温後、０．７ｍＬのクロロホルム／イソアミルアルコール（２４：１）を添加し、反転混合した。その後、遠心分離（２０，０００×ｇ、５分間）した。生じた上清を新しいサンプルチューブに移し、フェノールクロロホルム抽出、イソプロパノール沈殿により、ＤＮＡを調整した。
[Microbial community analysis]
The plant growth promoter A (1 mL) produced by the method described in Example 1 was centrifuged (20,000 × g, 5 minutes), and the precipitate was collected. The precipitate was dissolved in 560 μL TE buffer (10 mM Tris buffer (pH 8.0), 1 mM EDTA), then 30 μL SDS solution, 10 μL Proteinase K (10 mg / mL) was added, mixed, and then 37 ° C. I kept it warm for 1 hour. Then, 100 μL of 5M NaCl was added and mixed well, then 80 μL of CTAB / NaCl solution was added and mixed well. The sample was kept warm at 65 ° C. for 10 minutes. After heat retention, 0.7 mL of chloroform / isoamyl alcohol (24: 1) was added and inverted and mixed. Then, it was centrifuged (20,000 × g, 5 minutes). The resulting supernatant was transferred to a new sample tube and DNA was prepared by phenol-chloroform extraction and isopropanol precipitation.
分離したＤＮＡを鋳型として、5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′（配列番号４）の塩基配列からなる第１プライマー、及び、5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′（配列番号３）の塩基配列からなる第２プライマーを用いて、１６Ｓ ｒＲＮＡの部分配列を増幅した。具体的には、5 ng/μlのDNA2.5 μlにAmplion PCR Forward Primer （1 μM） 5 μl、Amplicon PCR Reverse Primer （1 μM） 5 μl、KAPA HiFi Hotstart ReadyMix （KAPA Biosystems） 12.5 μlを混合し、サーマルサイクラーT-100 （BioRad）を用いてＰＣＲ増幅した。サーマルサイクル反応は初期変性（９５℃、３分）の後、変性（９５℃、３０秒）、アニーリング（５５℃、３０秒）、伸長反応（７２℃、３０秒）を２５サイクル行い、７２℃、５分の伸長反応を行った。 Using the separated DNA as a template, the first primer consisting of the base sequence of 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3'(SEQ ID NO: 4) and the primer consisting of the base sequence of 5'-TCGTCGGCAGCGTCAGATGTGTATAAGACAGCCTACGGGNGGCWGCAG-3' (SEQ ID NO: 3) Was used to amplify a partial sequence of 16S rRNA. Specifically, 5 ng / μl of DNA 2.5 μl was mixed with 5 μl of Amplion PCR Forward Primer (1 μM), 5 μl of Amplifier PCR Reverse Primer (1 μM), and 12.5 μl of KAPA HiFi Hotstart ReadyMix (KAPA Biosystems). , PCR amplification using thermal cycler T-100 (BioRad). In the thermal cycle reaction, after initial denaturation (95 ° C., 3 minutes), denaturation (95 ° C., 30 seconds), annealing (55 ° C., 30 seconds), and extension reaction (72 ° C., 30 seconds) are performed for 25 cycles, and 72 ° C. The elongation reaction was carried out for 5 minutes.
アガロースゲル電気泳動を用いてＰＣＲ増幅反応を確認後、AMPure XPビーズを用いて、ＰＣＲ増幅産物の精製をした。ＰＣＲ増幅産物を遠心機でスピンダウンした後、ボルテックスでよく撹拌したAMpure XPビーズを20 μl加え、ピペッティングで１０回混合した。室温で５分静置後、マグネティックスタンドにセットした状態で２分静置した。その後、透明になった上清をピペッティングで取り除いた。８０％エタノールを200 μl加え、３０秒静置した。上清を再び取り除き、再度８０％エタノールを200 μl加えた。３０秒静置後、ピペッティングでエタノールを完全に取り除き、１０分風乾した。 After confirming the PCR amplification reaction using agarose gel electrophoresis, the PCR amplification product was purified using AM Pure XP beads. After spinning down the PCR amplification product in a centrifuge, 20 μl of vortex-stirred AMpure XP beads were added and mixed 10 times by pipetting. After allowing to stand at room temperature for 5 minutes, it was allowed to stand for 2 minutes while being set on a magnetic stand. Then, the clear supernatant was removed by pipetting. 200 μl of 80% ethanol was added, and the mixture was allowed to stand for 30 seconds. The supernatant was removed again and 200 μl of 80% ethanol was added again. After allowing to stand for 30 seconds, ethanol was completely removed by pipetting, and the mixture was air-dried for 10 minutes.
続いて、マグネティックスタンドからチューブを取り出し、10 mM Tris-HCl （pH 8.5）を52.5 μl加え、ピペッティングでよく撹拌した。室温で２分静置した後、マグネティックスタンドにセットし、さらに２分静置した。ＰＣＲ産物が含まれる上清を新しいチューブに移した。Qubit fluorometerを用いて、ＤＮＡ濃度を確認し、0.2 ng/μl以上であることを確認した。 Subsequently, the tube was removed from the magnetic stand, 52.5 μl of 10 mM Tris-HCl (pH 8.5) was added, and the mixture was well stirred by pipetting. After allowing to stand at room temperature for 2 minutes, it was set on a magnetic stand and allowed to stand for another 2 minutes. The supernatant containing the PCR product was transferred to a new tube. The DNA concentration was confirmed using a Qubit fluorometer, and it was confirmed that the DNA concentration was 0.2 ng / μl or more.
アダプタ及びインデックスを付加するため、２回目のＰＣＲを行った。精製済みのPCR産物5 μlに対してNextera XT Index Primer mix 10 μl、KAPA HiFi HotStart ReadyMix （2 x） 25 μl、RCR grade water 10 μlを混合し、サーマルサイクル反応を行った。サーマルサイクル反応は初期変性（９５℃、３分）の後、変性（９５℃、３０秒）、アニーリング（５５℃、３０秒）、伸長反応（７２℃、３０秒）を８サイクル行い、７２℃、５分の伸長反応を行った。その後、AMPure XPビーズを用いて、ＰＣＲ増幅産物の精製をした。更に、Qubit fluorometerを用いて、ＤＮＡ濃度を確認し、5 ng/μl以上であることを確認した。 A second PCR was performed to add the adapter and index. Nextera XT Index Primer mix 10 μl, KAPA HiFi HotStart ReadyMix (2 x) 25 μl, and RCR grade water 10 μl were mixed with 5 μl of the purified PCR product, and a thermal cycle reaction was carried out. In the thermal cycle reaction, after initial denaturation (95 ° C., 3 minutes), denaturation (95 ° C., 30 seconds), annealing (55 ° C., 30 seconds), and extension reaction (72 ° C., 30 seconds) are carried out for 8 cycles, and 72 ° C. The elongation reaction was carried out for 5 minutes. The PCR amplification product was then purified using AM Pure XP beads. Furthermore, the DNA concentration was confirmed using a Qubit fluorometer, and it was confirmed that the DNA concentration was 5 ng / μl or more.
Illumina社MiSeqシーケンサーを用いて、ペアエンドシークエンスした。得られたリードデータはクオリティーチェック、アダプタならびにインデックス配列を除去し、Qiimeプログラムを用いたクラスター解析に供した。９７％以上の相同性に基づくOperated Taxonomic Unit （OTU） を作成し、ＢＬＡＳＴ解析を用いて、系統学的な情報を付与した。計算過程でOTU代表配列データ、多様性解析データを得た。 Paired-end sequencing was performed using the Illumina MiSeq sequencer. The obtained read data was subjected to quality check, adapter and index sequence removal, and cluster analysis using the Qiime program. An Operated Taxonomic Unit (OTU) based on 97% or more homology was created and phylogenetic information was given using BLAST analysis. OTU representative sequence data and diversity analysis data were obtained during the calculation process.
ＯＴＵ代表配列データを門レベルでまとめた表を図１に示す。代表配列に含まれるリード数ならびに全リードに対する割合を示している。全リード数は４６，６０５リードであった。アシドバクテリア門に含まれる微生物の割合は１４．８８％、バクテロイデス門に含まれる微生物の割合は６．８０４％、クロロビウム門に含まれる微生物の割合は８．６１９％、クロロフレクサス門に含まれる微生物の割合は８．９５２％、ゲンマティモナス門に含まれる微生物は３．１２％、未培養門ＯＤ１に含まれる微生物は２．９２９％、プランクトミケス門に含まれる微生物は３．５２５％、プロテオバクテリア門に含まれる微生物は３６．７９２％、ウェルコミクロビウム門に含まれる微生物は２．８３％含まれていることを確認した。 A table summarizing the OTU representative sequence data at the phylum level is shown in FIG. The number of reads included in the representative sequence and the ratio to all reads are shown. The total number of leads was 46,605 leads. The proportion of microorganisms contained in the phylum Acidobacteria is 14.88%, the proportion of microorganisms contained in the phylum Bacteroides is 6.804%, the proportion of microorganisms contained in the phylum Chlorobium is 8.619%, and the proportion of microorganisms contained in the phylum Chloroflexus is The ratio of microorganisms is 8.952%, microorganisms contained in the phylum Genmatimonas are 3.12%, microorganisms contained in the uncultivated phylum OD1 are 2.929%, and microorganisms contained in the phylum Proteobacteria are 3.525%. It was confirmed that the microorganisms contained in the phylum Proteobacteria were 36.792% and the microorganisms contained in the phylum Welcomicrobium were 2.83%.
以上、実施形態を用いて本発明を説明したが、本発明の技術的範囲は上記実施の形態に記載の範囲には限定されず、その要旨の範囲内で種々の変形及び変更が可能である。例えば、装置の全部又は一部は、任意の単位で機能的又は物理的に分散・統合して構成することができる。また、複数の実施の形態の任意の組み合わせによって生じる新たな実施の形態も、本発明の実施の形態に含まれる。組み合わせによって生じる新たな実施の形態の効果は、もとの実施の形態の効果を併せ持つ。 Although the present invention has been described above using the embodiments, the technical scope of the present invention is not limited to the scope described in the above embodiments, and various modifications and changes can be made within the scope of the gist thereof. .. For example, all or a part of the device can be functionally or physically distributed / integrated in any unit. Also included in the embodiments of the present invention are new embodiments resulting from any combination of the plurality of embodiments. The effect of the new embodiment produced by the combination has the effect of the original embodiment.
本実施形態の植物生長促進剤は、植物の生長を促進するので、農作物の収穫量を増加させることができる。 Since the plant growth promoter of the present embodiment promotes the growth of plants, the yield of agricultural products can be increased.
１０ 糞尿貯め
２０ 供給部
３０ａ 第１曝気処理槽
３０ｂ 第２曝気処理槽
３０ｃ 第３曝気処理槽
３０ｄ 第４曝気処理槽
５０ 回収部
１００ 製造装置
10 Manure storage 20 Supply unit 30a 1st aeration treatment tank 30b 2nd aeration treatment tank 30c 3rd aeration treatment tank 30d 4th aeration treatment tank 50 Recovery unit 100 Manufacturing equipment
Claims (9)
植物生長促進剤。 A plant growth promoter made from livestock manure, from microorganisms contained in the phylum Bacteroidetes, microorganisms contained in the phylum Chloroflexi, microorganisms contained in the phylum Gemmatimonadetes, and microorganisms contained in the phylum Verrucomicrobia. Includes at least one or more of the group
Plant growth promoter.
請求項１に記載の植物生長促進剤。 A first primer containing the nucleotide sequence shown by 5'-CCTACGGGNGGCWGCAG-3'(SEQ ID NO: 1) (in the nucleotide sequence, N is A, T, G or C, and W is A or T) and 5 Using a second primer containing the base sequence shown by ′-GACTACHVGGGTATCTAATCC-3 ′ (SEQ ID NO: 2) (in the base sequence, H is A, T or C and V is A, C or G). When the microorganism contained in the plant growth promoter is detected, the microorganism contained in the phylum Bacteroidetes, the microorganism contained in the phylum Chloroflexi, the microorganism contained in the phylum Gemmatimonadetes, and the phylum Verrucomicrobia At least one or more of the group consisting of the contained microorganisms is detected.
The plant growth promoter according to claim 1.
請求項１又は２に記載の植物生長促進剤。 Total carbon concentration is 2,000 mg / kg or less,
The plant growth promoter according to claim 1 or 2.
請求項１〜３のいずれか一項に記載の植物生長促進剤。 The total nitrogen concentration measured by the Kjeldahl method is 500 mg / kg or less.
The plant growth promoter according to any one of claims 1 to 3.
請求項１〜４のいずれか一項に記載の植物生長促進剤。 Total phosphorus concentration is 2,000 mg / kg or less,
The plant growth promoter according to any one of claims 1 to 4.
請求項１〜５のいずれか一項に記載の植物生長促進剤。 Total potassium concentration is 5,000 mg / kg or less,
The plant growth promoter according to any one of claims 1 to 5.
請求項１〜６のいずれか一項に記載の植物生長促進剤。 The plant growth promoter is in the form of a liquid, solid or slurry.
The plant growth promoter according to any one of claims 1 to 6.
5′-CCTACGGGNGGCWGCAG-3′（配列番号１）の塩基配列を含む第１プライマーと5′-GACTACHVGGGTATCTAATCC-3′（配列番号２）の塩基配列を含む第２プライマーとを用いて検出される微生物のうち、バクテロイデス門に含まれる微生物を１．５％以上、クロロフレクサス門に含まれる微生物を１．５％以上、ゲンマティモナス門に含まれる微生物を０．５％以上、及び、ウェルコミクロビウム門に含まれる微生物からなる群のうち少なくとも１種以上を０．５％以上含むようになるまで、前記液体成分を曝気させるステップとを含む、
植物生長促進剤の製造方法。 Steps to separate the liquid components of livestock manure,
Microorganisms detected using the first primer containing the nucleotide sequence of 5'-CCTACGGGNGGCWGCAG-3'(SEQ ID NO: 1) and the second primer containing the nucleotide sequence of 5'-GACTACHVGGGTATCTAATCC-3'(SEQ ID NO: 2) Of these, 1.5% or more of the microorganisms contained in the phylum Bacteroidetes, 1.5% or more of the microorganisms contained in the phylum Chloroflexi, 0.5% or more of the microorganisms contained in the phylum Gemmatimonadetes, and Welcomicrobium. A step of exposing the liquid component to 0.5% or more of at least one of the group of microorganisms contained in the phylum is included.
A method for producing a plant growth promoter.
を満たす状態において前記液体成分を曝気させる、
請求項８に記載の植物生長促進剤の製造方法。 In the aeration step, when F [m / s] is the air flow rate, V [m 3 ] is the treatment liquid volume, and H L [m] is the treatment liquid depth.
The liquid component is aerated in a state satisfying the above conditions.
The method for producing a plant growth promoter according to claim 8.
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