JP2013059299A - Marker for deciding water sufficiency degree of plant and method for deciding water sufficiency degree using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop a marker for deciding the water sufficiency degree, reflecting the water sufficiency-insufficiency state in a plant without being affected by an environmental factor, or the like, and to provide a method for deciding the water sufficiency-insufficiency state in a plant with a marker for deciding the water sufficiency degree.SOLUTION: A metabolite whose quantity is changed in dependency on the water sufficiency degree in a plant is used as a marker for deciding the water sufficiency degree to accurately decide the water sufficiency degree in the plant on the basis of the accumulation quantity of the marker in the plant.

Description

  The present invention relates to a marker for determining the degree of water sufficiency in a plant and a method for determining the degree of water sufficiency using the marker.

  In order to grow the target plant efficiently in a short period of time in agriculture and forestry, it is extremely important not only to supply the necessary nutrients when necessary, but also to provide an appropriate amount of water.

  In areas with relatively heavy rainfall throughout the year and areas with irrigation facilities such as Japan, hydration of plants is not a major problem. However, in areas with low rainfall, such as dry land, and areas where only the dry season is rainy, it is absolutely essential for the efficient production of agricultural and forestry products to accurately manage the moisture content of plants.

  In addition, when cultivating native wilderness and forests as planting sites, it is important in terms of production that the land contains the nutrients and moisture necessary for the growth of the target plant species to be planted. It becomes a problem. However, in the case of nutrients, improvement can be made relatively easily by fertilization even if the shortage is found after development. However, in the case of moisture, unless there is a sufficient amount of water source in the vicinity, a large-scale irrigation facility will be required, and it will be very expensive to improve the shortage. Therefore, in selecting a new planting site, it is very important to accurately determine the degree of soil moisture sufficiency in the candidate site in advance.

  Conventionally, the determination of the moisture content in the soil for the management of water sufficiency and the selection of planting sites in the agricultural and forestry fields has been performed by measuring the moisture content of the soil (Non-patent Document 1). However, the moisture content measured by this method is the value of the surface layer part of tens of centimeters at most from the soil surface, and the moisture content of the deep soil approximately 50 cm to 2 m below the ground surface mainly used by trees. Is difficult to measure by ordinary methods. Therefore, even if the moisture content of the surface layer of the soil shows an appropriate value in terms of measured values due to transient rainfall or irrigation, it is often possible that the soil has not been sufficiently submerged in the deep layer. In such a case, despite the fact that the plant is actually deficient in water, the producer misjudged that the water content is sufficient from the measured values in the soil surface area and took appropriate measures such as irrigation. Problems that don't do can arise. Therefore, it can be said that the said method is unsuitable for the plant which roots to the soil deep region like woody. All of the conventional methods are indirect inspection methods based on the moisture content of the soil. In other words, if the amount of water in the soil is sufficient, it is a method for determining that the water sufficiency of the plant growing there is also sufficient, and is a direct measurement of the substantial water sufficiency of the plant is not. Therefore, it is hard to say that it is a universally usable method.

  However, conventionally, a method for directly or objectively measuring or judging the substantial water sufficiency of plants has not been known. If there is a technique that can evaluate the degree of water sufficiency in a plant, the growth of the plant can be predicted more accurately. For this purpose, it is necessary to accurately grasp the water sufficiency state of the plant and perform appropriate irrigation according to the result.

Japan Soil Association Foundation Soil, water quality and plant analysis method for soil function monitoring survey

  An object of the present invention is to develop and provide a marker for determining the degree of water sufficiency that reflects the state of water sufficiency / insufficiency in plants without being affected by environmental factors.

  Moreover, it is providing the method of determining the sufficiency and insufficient state of the water | moisture content in a plant using the said marker for water sufficiency degree determination.

  In order to solve the above-mentioned problems, the present inventors have focused on a plant metabolite and found a metabolite that changes quantitatively depending on the water sufficiency in the plant. In addition, based on the accumulated amount of the metabolite in the plant, it became clear that the water sufficiency / insufficient state in the plant can be accurately determined. This invention is based on the said knowledge, ie, provides the following.

（２）前記植物が木本類である、（１）に記載のマーカー。
（３）前記木本類がユーカリプタス属である、（２）に記載のマーカー。
（４）植物における水分の充足度を判定する方法であって、被検植物の全部又は一部から代謝産物を含む抽出物を抽出する抽出工程、前記抽出工程で得られた抽出物中に含まれる（１）に記載の少なくとも一の水分充足度判定用マーカーの蓄積量を測定する測定工程、前記測定工程で得られた水分充足度判定用マーカーの蓄積量に基づいて、前記被検植物における水分の充足度を判定する判定工程
を含む、前記方法。
（５）前記判定工程において、水分の充足度を被検植物と対照植物のそれぞれから得られる水分充足度判定用マーカーの蓄積量の比較によって判定する、（４）に記載の判定方法。
（６）前記対象植物が水分不足状態にある、（５）に記載の判定方法。
（７）水分充足度判定用マーカーが表１におけるマーカーNo. 1〜3、6、8及び9の場合、当該マーカーの被検植物における蓄積量が対照植物における蓄積量よりも統計学的に有意に小さいときに被検植物における水分が充足状態にあると判定する、（６）に記載の判定方法。
（８）水分充足度判定用マーカーが表１におけるマーカーNo. 4、5、7、10及び11の場合、当該マーカーの被検植物における蓄積量が対照植物における蓄積量よりも統計学的に有意に大きいときに被検植物における水分が充足状態にあると判定する、（６）に記載の判定方法。
（９）前記対象植物が水分充足状態にある、（５）に記載の判定方法。
（１０）水分充足度判定用マーカーが表１におけるマーカーNo. 1〜3、6、8及び9の場合、当該マーカーの被検植物における蓄積量が対照植物における蓄積量よりも統計学的に有意に大きいときに被検植物における水分が不足状態にあると判定する、（９）に記載の判定方法。
（１１）水分充足度判定用マーカーが表１におけるマーカーNo. 4、5、7、10及び11の場合、当該マーカーの被検植物における蓄積量が対照植物における蓄積量よりも統計学的に有意に小さいときに被検植物における水分が不足状態にあると判定する、（９）に記載の判定方法。
（１２）前記植物が木本類である、（５）〜（１１）のいずれかに記載の判定方法。
（１３）前記木本類がユーカリプタス属である、（１２）に記載の判定方法。
（１４）（４）〜（１３）のいずれかに記載の判定方法を用いて、被検地における土中水分量を推定する土中水分量推定方法。
（１５）（１４）で土中水分量推定方法を用いて、対象植物の植栽候補地を選定する植栽候補地選定方法。
（１６）同一植物において、（４）〜（１３）のいずれかに記載の水分充足度判定方法によって得られた水分の充足度に関する経時的な二以上の判定結果に基づいて、その植物における生長状態を判定する、植物の生長状態判定方法。
（１７）二以上の経時的な判定結果が、いずれも充足状態にあるという結果であった場合には、その植物における生長状態は良好であると判定し、不足から充足に移行していることを示す場合には、生長状態が改善傾向にあると判定し、充足から不足に移行していることを示す場合には、生長状態が悪化傾向にあると判定し、又はいずれも不足していることを示す場合には、生長状態が悪いと判定する、（１６）に記載の判定方法。
（１８）（１６）又は（１７）に記載の植物の生長状態判定方法による判定結果に基づいて、その植物における水分の潅水量を決定する潅水量決定方法。 (1) It is a metabolite of a plant, and in high-performance liquid chromatography tandem mass spectrometry, when a temporal continuous concentration gradient of acetonitrile in liquid chromatography is formed at 0% 3% to 5 minutes 56%, the following The water content of any one of the plants indicated by marker Nos. 1 to 5 separated by the retention times shown in Table 1 and having the mass-to-charge ratio of the precursor ions and product ions in the mass spectrometry indicated by the values shown in Table 1 below Satisfaction degree marker.

(2) The marker according to (1), wherein the plant is woody.
(3) The marker according to (2), wherein the woody species belongs to the genus Eucalyptus.
(4) A method for determining the degree of water sufficiency in a plant, the extraction step for extracting an extract containing a metabolite from all or part of the test plant, included in the extract obtained in the extraction step Measurement step of measuring the accumulated amount of at least one moisture sufficiency determination marker according to (1), based on the accumulated amount of the moisture sufficiency determination marker obtained in the measurement step, in the test plant The said method including the determination process which determines the sufficiency of a water | moisture content.
(5) The determination method according to (4), wherein, in the determination step, the degree of water sufficiency is determined by comparing the accumulated amount of the water sufficiency determination markers obtained from each of the test plant and the control plant.
(6) The determination method according to (5), wherein the target plant is in a moisture-deficient state.
(7) When the marker for determining the degree of water sufficiency is the marker Nos. 1 to 3, 6, 8, and 9 in Table 1, the accumulated amount of the marker in the test plant is statistically more significant than the accumulated amount in the control plant (6) The determination method as described in (6) which determines with the water | moisture content in a to-be-tested plant being in a sufficient state when it is small.
(8) When the water sufficiency determination marker is marker No. 4, 5, 7, 10 or 11 in Table 1, the accumulated amount of the marker in the test plant is statistically more significant than the accumulated amount in the control plant (6) The determination method as described in (6) which determines with the water | moisture content in a to-be-tested plant being in a sufficient state when it is large.
(9) The determination method according to (5), wherein the target plant is in a water-sufficient state.
(10) When the marker for determining water sufficiency is the marker Nos. 1 to 3, 6, 8, and 9 in Table 1, the accumulated amount of the marker in the test plant is statistically more significant than the accumulated amount in the control plant. (9) The determination method as described in (9) which determines with the water | moisture content in a test plant being in a shortage state when it is large.
(11) When the marker for determining the degree of water sufficiency is marker No. 4, 5, 7, 10 and 11 in Table 1, the accumulated amount of the marker in the test plant is statistically more significant than the accumulated amount in the control plant The determination method according to (9), wherein it is determined that the moisture in the test plant is in a deficient state when the water is small.
(12) The determination method according to any one of (5) to (11), wherein the plant is a tree.
(13) The determination method according to (12), wherein the woody species belongs to the genus Eucalyptus.
(14) A soil moisture content estimation method for estimating the soil moisture content in the test site using the determination method according to any one of (4) to (13).
(15) A planting site selection method for selecting a planting site for the target plant using the soil moisture content estimation method in (14).
(16) In the same plant, the growth in the plant based on two or more determination results with respect to the water sufficiency over time obtained by the water sufficiency determination method according to any one of (4) to (13) A method for determining a growth state of a plant, which determines a state.
(17) If two or more determination results over time are the results that both are in a satisfactory state, it is determined that the growth state in the plant is good, and the state has shifted from insufficient to sufficient In the case where the growth state is indicated, it is determined that the growth state is in an improvement trend, and in the case where it is indicated that the growth state is shifted from the satisfaction to the shortage, it is determined that the growth state is in a deterioration tendency, or both are insufficient. In the case of indicating this, the determination method according to (16), wherein the growth state is determined to be bad.
(18) An irrigation amount determination method for determining an irrigation amount of water in the plant based on the determination result by the growth state determination method of the plant according to (16) or (17).

  According to the plant water sufficiency determination marker of the present invention, it is possible to provide a marker that can directly and accurately determine the water sufficiency state of a plant without being influenced by other factors.

  According to the water content determination method of a plant of the present invention, by using the marker for determining the water content of a plant of the present invention, it is possible to accurately determine the water content state in a test plant without being influenced by other factors. can do.

  According to the soil moisture content estimation method of the present invention, it is possible to estimate the moisture content of soil in the test site, in particular, deep soil, through the moisture content of the plant, and based on the results, Candidate sites for planting can be selected.

It is the figure which showed the tree height as a growth amount of Eucalyptus genus hybrids (camaldrensis x deglupta) of planting 3 months in a water deficient district and a water sufficiency zone. It can be seen that in the water-deficient group, the growth of the plant is significantly inhibited as compared with the water-sufficient group. It is the figure which showed the tree height as the amount of growth of the Eucalyptus genus hybrid clone (Camaldrensis * deglutta) cultivated for 3 months changing nutrient conditions and water conditions. Each processing section (processing sections 1 to 4) in the figure corresponds to the processing section described in Table 4. That is, treatment section 1 is a treatment section where both fertilization amount and moisture amount are sufficient, and treatment section 2 is filled with fertilization amount, but treatment section where treatment amount is insufficient and treatment section 3 has a fertilization amount. The treatment section where the amount of moisture is insufficient, but the treatment section 4 is a treatment section where both the fertilizer application amount and the moisture amount are insufficient.

1. Marker for determining water sufficiency The first embodiment of the present invention is a marker for determining water sufficiency of a plant (hereinafter simply referred to as “marker” in the present specification). The marker of the present invention is a plant-derived metabolite that changes quantitatively depending on the degree of water content in the plant. Specifically, it refers to substances indicated by marker Nos. 1 to 11 in Table 1 above.

  As used herein, “plant” refers to a moss plant, a fern plant, and a seed plant. Preferably it is a seed plant. In the case of a seed plant, a gymnosperm or angiosperm, a herb or a tree is not questioned. Preferred are woods that mainly use moisture in the deep soil. More preferably, it is an angiosperm, more preferably a myrtaceae plant, and still more preferably a plant of the genus Eucalyptus. For example, Eucalyptus Camaldrensis, Eucalyptus Deglupta, Eucalyptus Grandis, Eucalyptus Eurofila, Eucalyptus Perita, Eucalyptus Globras, Eucalyptus Brassiana, Eucalyptus Teleticontis or their hybrids, such as Eucalyptus Camaldrens Hybrids of cis and eucalyptus deglupta (hereinafter referred to as “camaldrensis x degrupta”), hybrids of eucalyptus camaldrensis and eucalyptus eurofila (hereinafter referred to as “camaldrensis x eurofila”), eucalyptus Examples include hybrids of Perita and Eucalyptus brassana, or hybrids of Eucalyptus perita and Eucalyptus camaldrensis. As used herein, “deep soil” refers to soil having a depth of 30 cm to 2.5 m, 40 cm to 2.3 m, or 50 cm to 2.0 m from the ground surface.

  In the present specification, “degree of water sufficiency in a plant” refers to the degree of whether or not water has reached an amount necessary for the growth of the plant. For example, when the moisture content in a plant is low, it means that the amount of water in the plant has not reached the amount necessary for growth, that is, the plant is deficient or deficient. Further, when the degree of moisture content in a plant is high, it means that the amount of moisture in the plant has reached the amount necessary for growth, that is, the plant is in a sufficient state of moisture. In the present specification, the criterion for determining whether the degree of water satisfaction is high or low is determined based on the accumulated amount of the marker of the present invention. Since this will be described in detail in the determination process of the moisture sufficiency determination method described later, the description thereof is omitted here.

  As used herein, the term “metabolite” refers to all substances produced in plants by catabolic metabolism represented by respiration or anabolic metabolism represented by photosynthesis. Examples include proteins (including enzymes) and low molecular weight compounds (including plant hormones, polyphenols, sugars, amino acids, and nucleotides). Water solubility and fat solubility do not matter.

  Among the above metabolites, the marker for determination of water sufficiency according to the present invention is a high-performance liquid chromatograph tandem mass spectrometry with a time continuous gradient of acetonitrile in liquid chromatography from 0 min 3% to 5 min 56%. When formed, it is separated by the retention time shown in Table 1, and is a substance indicated by marker Nos. 1 to 11 in which the mass-to-charge ratio of precursor ions and product ions in mass spectrometry is specified by the values shown in Table 1. . More specifically, the substance is specified under the measurement conditions of Example 2 described later.

  “High-performance liquid chromatograph tandem mass spectrometry” (hereinafter abbreviated as “LC-MS / MS analysis”) is liquid chromatography (hereinafter abbreviated as “LC”). (MS: Mass Spectrometry) using a device in which two mass spectrometers are connected in series (Kachlickiet al) (Kachlickiet al). J Mass Spectrom. (2008) 43 (5): 572-586). In the LC-MS / MS system, a sample separated by LC at a specified retention time is ionized into precursor ions by the first MS (Q1) that can pass only ions having a specific mass-to-charge ratio, and then Q2 Metabolites are decomposed by high voltage and product ions are generated. In the subsequent second MS (Q3), only product ions having a specific mass-to-charge ratio pass and are detected by the detector. Therefore, even for metabolites whose chemical structures have not been identified, the sample separation conditions in LC and the mass-to-charge ratio of ions that can pass before and after decomposition are set in advance in the sample. The metabolite contained in can be identified.

  In the present invention, “a continuous concentration gradient of acetonitrile” refers to a continuous concentration gradient of acetonitrile, which is an LC solvent, within a predetermined time. Specifically, it means a concentration gradient in which the concentration at 0 minute is 3%, the concentration at 5 minutes is 56%, and the concentration with respect to time in the meantime forms a continuous gradient.

  As described above, each of the moisture sufficiency determination markers according to the present invention uses LC-MS / MS analysis to distribute a sample in a column together with acetonitrile that forms the temporal continuous concentration gradient. When the metabolites separated at the respective retention times described above are analyzed with a mass spectrometer, the precursor ions and the product ions are identified as substances having the mass-to-charge ratio shown in Table 1, the presence thereof is confirmed, and further quantification is performed. can do.

  In addition, as for the marker of this embodiment shown by marker No. 4, 5, 7, 10 and 11 of Table 1, as shown in Example 1 mentioned later, as for all, the accumulation | storage amount in a plant is accompanying the increase in a moisture content. A metabolite that has the property of increasing the amount of expression, synthesis, or secretion or release in a test plant when the water is full.

  On the other hand, as for marker of this embodiment shown by marker No. 1-3, 6, 8, and 9 of Table 1, as shown in Example 1 mentioned later, as for all, the accumulation | storage amount in a plant accompanies the reduction | decrease of a moisture content. A metabolite having a property that increases the amount of expression, synthesis, or secretion or release in the test plant due to lack or lack of water.

2. Water sufficiency determination method The 2nd Embodiment of this invention is the water sufficiency determination method in a plant. The method of the present invention includes an extraction step, a measurement step, and a determination step as essential steps. Hereinafter, each step will be specifically described.

2-1. Extraction Step The “extraction step” is a step of extracting an extract containing a metabolite from all or part of the test plant.

  In the present specification, the “test plant” refers to a plant that is subjected to the water content determination method of the present invention in order to determine the water content, and has grown or grown under any environment. It is a plant.

  In this specification, “the whole test plant” means all parts constituting the plant body of the test plant. The “part of the test plant” means an organ (for example, root, stem, leaf, flower, spore or seed) constituting the plant of the test plant, or a form constituting the organ. It refers to a tissue that is a group of cells that have been differentiated objectively and / or functionally, or cells that constitute the tissue. The part may use any part of the test plant, but is preferably a leaf part. This includes the marker for determining the degree of water sufficiency according to the first embodiment because it is easily available, has a relatively small load on the test plant, and contains the most metabolites among the plants. This is because there is a high possibility that In the present specification, in order to determine a more accurate water sufficiency level of the test plant, it is desirable to avoid sampling of the planted test plant in the field at least during or immediately after the rain. .

  In the present specification, the “extract” refers to a plant extract including a plurality of metabolites, usually a plant extract. As described above, since the metabolite of the present invention may be water-soluble or fat-soluble, the solvent used for extraction of the extract may be an aqueous solution or an organic solvent (for example, lower alcohol, ether, chloroform, ethyl acetate, Xylene, etc.).

  The method for extracting the extract from the test plant is not particularly limited as long as it is a method capable of extracting a metabolite from the test plant. For example, when obtaining a water-soluble metabolite, the plant body is crushed as necessary, then watered, soaked and / or squeezed for a predetermined period, and then filtered to collect the filtrate or centrifuged. What is necessary is just to collect | recover subsequent supernatants. In addition, when obtaining a fat-soluble metabolite, the plant body is pulverized as necessary, then immersed in an organic solvent such as methanol for a predetermined period, and then filtered to collect the filtrate or centrifuged. The supernatant may be collected. For the details of these extraction methods, techniques known in the art can be used. For example, a method described in a literature name (Iijima et al., The Plant Journal (2008) 54,949-962, Suzuki et al., Phytochemistry (2008) 69, 99-111) may be referred to.

  In the water sufficiency determination method of the present invention, in the determination step described later, when determining the water sufficiency level by comparing the amount of accumulated marker obtained from each of the test plant and the control plant, It is desirable to extract the extract from the control plant as well as the test plant. In this case, it is assumed that the control plant is the same species or the same hybrid as the test plant and is in a deficient state deficient in water or deficient in deficient water. The marker of the present invention includes other conditions other than the amount of water, such as growth weather conditions (for example, temperature, sunshine duration, humidity), soil conditions, temporal conditions (for example, growth period, growth period) and the plant. These conditions do not necessarily have to be the same for the test and control plants, as they are not affected or hardly affected by the health condition, but in order to obtain more accurate results, It is preferred that each condition other than the amount is the same as much as possible. Moreover, when extracting an extract from a part of individual | organism | solid, it is preferable that the part uses the same part between a test plant and a control plant. For example, when the extract is extracted from the leaves of the test plant, the extract of the control plant is preferably extracted from the leaves as well.

2-2. Measurement step The “measurement step” is a step of measuring the amount of accumulated marker contained in the extract obtained in the extraction step.

  The marker to be measured in this step is at least one of the 11 markers described in the first embodiment. Any one or more of the 11 markers may be arbitrarily selected. The use of a plurality of markers of 2 or more and 11 or less is also preferable for improving the reliability of the determination result in the determination method of the present invention.

  In this specification, the “accumulated amount” is the amount of a specific marker selected in the extract obtained in the extraction step. This amount may be a relative amount such as concentration, ionic strength, absorbance or fluorescence intensity, or may be an absolute amount such as the weight or volume of a marker included in a given amount of extract. .

  The marker described in the first embodiment used in the method of the present embodiment has an unidentified chemical structure, but can be identified by LC-MS / MS analysis as described in the first embodiment. The accumulated amount contained therein can be measured by using at least LC-MS / MS analysis. For example, in order to measure the accumulated amount of marker No. 1 contained in the extract, acetonitrile in LC is 3% at 0 minute, 56% at 5 minutes, The substance that flowed with the extract into the column so as to form a continuous concentration gradient with respect to time, separated in 0.43 minutes, the mass-to-charge ratio of precursor ions in Q1 to 175.2, and the mass of product ions in Q3 If marker No. 1 is present in the extract, it can be specifically detected and quantified by the detector by passing it through a tandem mass spectrometer having a charge ratio of 116.0.

  However, the method for measuring the accumulated amount of the marker contained in the extract is not particularly limited as long as it is a method capable of detecting and quantifying a specific metabolite from the extract other than the above method. For example, detection and quantification can be performed using mass spectrometry, a method applying an antigen-antibody reaction, electrophoresis and the like.

  In addition to the LC-MS / MS analysis method described above, mass spectrometry includes high-performance liquid chromatography mass spectrometry (LC-MS), gas chromatography mass spectrometry (GC-MS), gas chromatograph tandem mass spectrometry (GC-MS). MS / MS), capillary electrophoresis mass spectrometry (CE-MS) and ICP mass spectrometry (ICP-MS).

  The antigen-antibody reaction is applied using an antibody that specifically recognizes each marker, ELISA (Enzyme-Linked ImmunoSorbent Assay) method, surface plasmon resonance (SPR) method, or quartz crystal microbalance (QCM) The law can be used.

  When the metabolite to be analyzed is a protein, for example, two-dimensional electrophoresis can be used as the electrophoresis.

  Any of the above analysis methods are known in the art, and may be performed according to these methods. For example, literature names (Yoko Iijima et al., The Plant Journal (2008) 54,949-962, Masami Hirai et al. Proc Natl Acad Sci USA (2004) 101 (27) 10205-10210, Shigeru Sato et al., The Plant Journal (2004) 40 (1) 151-163, Motoyuki Shimizu et al., Proteomics (2005) 5,3919-3931).

2-3. Determination Step The “determination step” is a step of determining whether the moisture content in the test plant is high or low based on the accumulated amount of the marker obtained in the measurement step. “Based on the amount of accumulated marker” means that the amount of accumulated marker is used to determine the degree of water sufficiency.

  The criteria for determining the degree of water sufficiency are not particularly limited as long as the accumulated amount of the marker is used. For example, the value set when selecting the marker described in the first embodiment or the obtained value may be used as a reference value as a reference value, or the accumulated amount of marker in a control plant may be used as a reference value.

  Here, the “reference value” is an ionic strength value in LC-MS analysis obtained or set when a marker shown in Table 1 is selected as a moisture sufficiency determination marker from a number of metabolites.

  As described in Example 1 described later, the marker described in the first embodiment was cultivated in the water-sufficiency zone and the water-deficient zone and was obtained from individuals in both zones, as described in Example 1 below. When the accumulated amount of metabolites is compared, as shown in Table 2 of Example 1, the accumulated amount in the water-sufficiency individual has an ionic strength of 1.5 times or more, or 1 / 1.5 times or less. Is selected as a marker for determining the degree of water satisfaction. Therefore, based on the properties of each of the 11 markers shown in Tables 1 and 2, the reference value is 1.5 times or 1 / 1.5 times the ionic strength in the moisture-deficient section. More specific reference values for each marker are shown below.

  Markers No. 4, 5, 7, 10, and 11 shown in Table 2 are metabolites that have the property that their accumulated amount in plants increases as the amount of water increases. As shown in Table 2, the accumulated amount of these markers in the water-deficient individuals is ionic strength, 0.07 ± 0.06 for marker No. 4, 0.74 ± 0.24 for marker No. 5, 0.28 for marker No. 7. Since ± 0.08, 0.47 ± 0.16 for marker No. 10 and 0.05 ± 0.03 for marker No. 11, a value corresponding to 1.5 times the ionic strength, specifically 0.11 for marker No. 4. The reference value can be ± 0.06, 1.11 ± 0.24 for marker No. 5, 0.42 ± 0.08 for marker No. 7, 0.71 ± 0.16 for marker No. 10, and 0.08 ± 0.03 for marker No. 11. Therefore, in marker Nos. 4, 5, 7, 10, and 11, as a determination method, when the accumulated amount obtained in the test plant is equal to or higher than the reference value in ionic strength, the test plant It can be determined that the moisture sufficiency in is high, and conversely if it is below the reference value, it can be determined that the water sufficiency in the test plant is low.

  On the other hand, marker Nos. 1 to 3, 6, 8, and 9 shown in Table 2 are metabolites that have the property that the amount of accumulation in plants increases as the amount of water decreases. As shown in Table 2, the accumulated amount of these markers in the water-deficient individuals is ionic strength, 0.24 ± 0.07 for marker No.1, 0.37 ± 0.20 for marker No.2, 0.36 for marker No.3. ± 0.08, 0.34 ± 0.06 for marker No. 6, 2.51 ± 0.79 for marker No. 8, and 0.67 ± 0.08 for marker No. 9, a value corresponding to 1 / 1.5 times the ionic strength of each, Specifically, the marker No. 1 is 0.16 ± 0.07, the marker No. 2 is 0.25 ± 0.20, the marker No. 3 is 0.24 ± 0.08, the marker No. 6 is 0.23 ± 0.06, the marker No. 8 is 1.67 ± 0.79, For marker No. 9, 0.45 ± 0.08 can be used as a reference value. Therefore, in the marker Nos. 1 to 3, 6, 8, and 9, as the determination method, when the accumulated amount obtained in the test plant is not more than the reference value in terms of ionic strength, the test plant It can be determined that the moisture sufficiency in the plant is high, and conversely, if the moisture content exceeds the reference value, it can be determined that the moisture sufficiency in the test plant is low.

  In addition, the accumulated amount in the water-sufficiency zone of markers No. 4, 5, 7, 10, and 11 is ionic strength, 0.26 ± 0.21, 1.21 ± 0.22, 0.49 ± 0.11, 1.81 ± 0.62, and 0.12 ± 0.01, respectively. Therefore, this value may be a stricter reference value. In this case, if the accumulated amount of the marker obtained in the test plant is equal to or higher than the reference value in terms of ionic strength, it is determined that the moisture content in the test plant is high, and conversely the reference When the value is less than the value, it can be determined that the degree of moisture content in the test plant is low.

  Accumulated amounts in the water sufficiency section of markers No. 1 to 3, 6, 8 and 9 are ionic strength, 0.00 ± 0.00, 0.24 ± 0.06, 0.04 ± 0.02, 0.24 ± 0.03, 0.28 ± 0.03 and 0.50 ±, respectively. Since it was 0.05, this value may be a stricter reference value. In this case, if the accumulated amount of the marker obtained in the test plant is not more than the reference value in terms of ionic strength, it is determined that the degree of water sufficiency in the test plant is high, and conversely the reference When exceeding a value, it can also be determined that the degree of water sufficiency in the test plant is low.

  When using the above reference values in the determination process, the accumulated amount of markers analyzed by LC-MS analysis may cause slight errors between samples. It is desirable to standardize by dividing by the accumulated amount of the substance (eg (-) Epicatechin).

  Furthermore, when the accumulated amount of the marker in the control plant is used as a criterion, the accumulated amount in the extract of the test plant is compared with the accumulated amount in the extract of the control plant with respect to the marker of the present invention. What is necessary is just to determine the sufficiency of the water | moisture content in a test plant based on. The term “control plant” as used herein refers to a plant that is the same species or hybrid as the test plant and has the same level of growth, and is a water-satisfied individual to which moisture has been applied in a minimum amount, or a minimum amount required. And water deficient individuals whose water content is limited to be less than.

  When the accumulated amount of the marker in the control plant is used as a criterion, it is preferable that the extraction step and the measurement step are performed under the same conditions at the same time in the control plant and the test plant. Alternatively, the extraction process and the measurement process are performed on the control plant under various conditions, and the accumulated amount of the obtained marker is stored in a database in advance. The accumulated amount of the control plant marker that matches as much as possible may be selected and used.

  When determining the water sufficiency of the test plant using the accumulated amount of the marker in the control plant as a determination criterion, the specific determination method is not limited.

  For example, if the control plant is a water deficient individual, it is verified whether there is a statistically significant quantitative difference in the amount of accumulation of the marker in the test plant and the control plant. It can also be determined that the moisture in the test plant is in a sufficient state. As used herein, “statistically significant” means that there is a significant difference when the difference in the amount of accumulated marker is statistically processed. As a test method for statistical processing, a known test method capable of determining the presence or absence of significance may be appropriately used. For example, Student's t test or multiple comparison test can be used. Specifically, for example, the risk rate (significance level) is less than 5%, 1%, or 0.1%. Here, the markers No. 1 to 3, 6, 8, and 9 of the present invention are in inverse proportion to the amount of water because the amount of accumulation in the plant increases as the amount of water decreases. As a specific example of the significant quantitative difference, the marker accumulation amount in the test plant is ionic strength, which is 1 / 1.5 times or less, preferably 1/2 times or less, more preferably 1 / 2.5 times that of the control plant. The case where it has the following ionic strength is mentioned. In addition, markers No. 4, 5, 7, 10 and 11 of the present invention are proportional to the amount of water because the amount of accumulation in the plant increases as the amount of water increases, and are statistically significant. As a specific example of the quantitative difference, the marker accumulation amount in the test plant has an ionic strength that is 1.5 times or more, preferably 2 times or more, more preferably 2.5 times or more of that in the control plant. Is mentioned.

  On the other hand, if the control plant is a water-sufficient individual, it is verified whether there is a statistically significant quantitative difference in the amount of the marker accumulated in the test plant and the control plant. It can also be determined that the moisture in the test plant is in a deficient state. Since markers No. 1 to 3, 6, 8, and 9 of the present invention are in inverse proportion to the amount of water, as a specific example of a statistically significant quantitative difference, the amount of marker accumulation in the test plant is The ionic strength may be 1 / 1.5 times or more, preferably 1/2 times or more, more preferably 1 / 2.5 times or more that of the control plant. Further, since marker Nos. 4, 5, 7, 10 and 11 of the present invention are proportional to the amount of water, specific examples of statistically significant quantitative differences include marker accumulation in the test plant. Examples include the case where the amount has an ionic strength that is 1.5 times or less, preferably 2 times or less, more preferably 2.5 times or less that of the control plant.

  According to this method, the water sufficiency in the test plant can be accurately determined without being influenced by other factors.

  According to this method, the amount of water that can be substantially used by the plant in the planting site where the plant is planted can be determined from the water sufficiency of the plant.

3. Soil moisture content estimation method The third embodiment of the present invention is a soil moisture content estimation method. This method is a method for estimating the moisture content in the soil in the test site from the determination result using the method for determining the degree of water satisfaction of the second embodiment.

  The water sufficiency determination method of the second embodiment is a method for determining a substantial water sufficiency in a plant, but the test plant grows indirectly by using the result obtained by the method. It is possible to estimate the amount of moisture in the soil of the test site that is or has grown.

  In the present specification, the “test site” refers to a land that is a target of the soil moisture content estimation method of the present invention. It may be a plantation site where plants are planted and / or managed by human hands such as farms, plantations, and pastures, or land with little or no human hands such as forests or wilderness.

  In this specification, “the amount of moisture in the soil” refers to the amount of moisture contained in the soil below the surface of the earth or the moisture content of the soil. However, this method merely estimates whether or not the soil contains the moisture necessary for the growth of the test plant, and the amount of moisture or water content as an absolute value such as volume or weight. It does not measure or calculate relative values such as rate.

  The test plant used in the present method is not limited as long as it is a plant growing on the test site, but woody trees, especially the roots from the surface to a depth of 30 cm to 2.5 m, preferably 40 cm to 2.3 m, more preferably Is preferably a medium-high tree extending from 50 cm to 2.0 m. This is because the effectiveness of the present invention that can estimate the soil moisture content in the deep region, which was difficult to measure by the conventional method, is more exhibited. Moreover, even if it is a plant species which roots in the same deep layer area, it is preferable that it is a plant species with a high water-absorbing property, that is, a water-requiring requirement. This is because it is possible to more accurately estimate whether or not there is an amount of water in the soil that is sufficient for demand for plant species with high water requirements.

  The method includes an extraction process, a measurement process, a determination process, and an estimation process. Among these, the extraction process, the measurement process, and the determination process are the same as the moisture sufficiency determination method of the second embodiment, and thus the description thereof is omitted here.

  The “estimation step” estimates the moisture content in the soil of the test site where the test plant provided for the method grew based on the result of the determination step, that is, the result of the water sufficiency determination method of the second embodiment. It is a process. In the estimation method, when the determination in the determination process is a result that the water sufficiency level of the test plant is in a satisfactory state, the water content in the soil of the test site where the test plant grew is large. Can be estimated. In particular, if the depth of the soil where the root of the test plant is mainly stretched can be estimated from the plant height and tree height, the moisture content in the soil estimated in this step is the depth of the soil in the soil. It can be estimated as the amount of water. In general, Eucalyptus takes almost the same form, but for example, Camaldrensis × Deglupta is known to have roots mainly in the range of 30 cm to 2 m underground when the tree height is 20 m. Therefore, as a result of determining the degree of water sufficiency from a part of the above-mentioned height of camaldrensis planted in a predetermined plantation, the amount of water is determined to be satisfactory. Estimated that the amount of moisture in the soil 30 cm to 2 m underground of the plantation where Drensis x Degrupta was planted is sufficient, and conversely, if it is determined that the amount of moisture is insufficient, It can be estimated that the amount is small.

  According to this method, it is possible to indirectly estimate the moisture content of the deep underground region, which has been difficult in the past, based on the moisture sufficiency of the plants growing on the land.

4). Planting site selection method The fourth embodiment of the present invention is a planting site selection method. This method is a method of selecting a planting site of the target plant using the soil moisture content estimation method of the third embodiment.

  When cultivating a native wilderness or a forest as a planting site, knowing in advance whether or not the land is suitable for planting the target plant is important in selecting a planting candidate site. An important element. However, as described above, even if it is determined that the land is not suitable for planting the target plant after the candidate planting site has been determined, if the inappropriate cause is a lack of nutrients, fertilization is performed. It is possible to improve to suitable land by improving the soil. However, if the cause is inappropriate, if there is not enough water source in the vicinity, it will take a huge cost to improve after the candidate planting site is decided. It is preferable that the appropriateness can be grasped.

  In this method, from the result of the soil moisture content estimation method of the third embodiment, it is determined whether the planting candidate site is suitable for planting the target plant in terms of moisture content, and an appropriate candidate site is selected. Is the method.

  In this specification, “planting candidate site” refers to a candidate site that can become a planting site in the future if the conditions are satisfied in the wilderness or forest. In the present specification, the “target plant” refers to a plant planted in a planting place for the purpose of cultivation. Herbs and woods are not limited, but woods, particularly medium and high trees, are preferred for the same reason as in the soil moisture content estimation method.

  This method is a plant that grows naturally in the planting candidate site, or has already been planted in the planting candidate site before the target plant, and has a tree height similar to that of the target plant, preferably the same family, Preferably, a plant belonging to the same subfamily, more preferably the same genus, more preferably the same species is used as the test plant. When the soil moisture content estimation method of the third embodiment is carried out and it is estimated from the estimation result that the soil moisture content of the planting candidate site where the test plant grew was small, the planting candidate site is Excluded from the candidate as an inappropriate planting area. On the other hand, if it is estimated that the amount of moisture in the soil of the candidate planting site is large, the candidate planting site is selected as a suitable planting site.

  According to the planting site selection method of the present invention, before selecting as a planting site, it is possible to determine whether the candidate site is suitable for planting the target plant in terms of moisture content in the soil. It becomes. As a result, it becomes possible to select an inappropriate planting site and avoid purchases at the stage of selecting a planting candidate site, which eliminates unnecessary investment and leads to cost reduction.

5. Plant Growth State Determination Method The fifth embodiment of the present invention is a plant growth state determination method.
As described in Example 1 to be described later, even if the same-type plants cultivated in the water-sufficiency zone and the water-sufficiency zone have a clear difference in growth according to the amount of irrigation even if the nutritional state and the like are the same. That is, the degree of water content in a plant greatly affects the growth of the plant. This method determines the growth state of the same plant based on a plurality of results over time of the water sufficiency determination method of the second embodiment, and predicts the future growth of the plant from the determination result. Is the method. Specifically, the growth state in the plant is determined on the basis of two or more determination results regarding the water satisfaction obtained from the same plant over time by the water satisfaction determination method of the second embodiment. And

  “Plant growth state” includes the growth rate of plants, the growth of plants within a predetermined period, or the transition of growth.

  “Two or more determination results regarding water sufficiency obtained over time from the same plant” means determination of water sufficiency according to the second embodiment performed on samples collected from the same plant at two or more different times Each judgment result by the method is said. The period between each sample may be determined according to the plant type, growth environment, etc., and is not particularly limited, but if the period is too short, there is a possibility that an accurate growth state cannot be determined. For example, it is preferable to leave it for 3 days or more, preferably 1 week or more, more preferably 1 month or more, or 3 months or more.

  What is necessary is just to perform the method of determining the water sufficiency in a test plant according to the method as described in the said 2nd Embodiment.

Specific examples of the method for determining the growth state of the plant based on two or more determination results obtained over time include the following methods.
(1) If two or more determination results over time are the results that both are in a satisfactory state, it is determined that the growth state of the plant is good. In this case, if the state can be maintained, it can be predicted that the future growth of the plant is high.
(2) When two or more determination results over time have shifted from a deficient state to a full state, it is determined that the growth state of the plant is in an improving trend. In this case, if the improved state can be maintained, it can be predicted that the future growth of the plant is high.
(3) If two or more determination results over time have shifted from the satisfactory state to the insufficient state, it is determined that the growth state tends to deteriorate. In this case, if appropriate measures such as irrigation are not taken, the plant can be predicted to grow worse in the future.
(4) If two or more determination results with time are both in a shortage state, it is determined that the growth state is bad. In this case, if appropriate measures such as quick irrigation are not taken, it can be predicted that the plant will grow worse and possibly wither in some cases.

  How much water should be irrigated may be appropriately determined based on the determination result obtained by the method for determining the degree of water satisfaction according to the second embodiment. For example, if it is determined that the water content of the test plant is insufficient, and if water has already been irrigated to the land where the test plant has already been planted, the amount of irrigation is small Therefore, water should be irrigated in a larger amount. If rainwater has been left and the land being planted has not been watered before, it should be actively watered.

  Furthermore, if it is determined that the water content of the test plant is in a sufficient state, and the land on which the test plant has already been planted has been irrigated, the irrigation amount is maintained. Then, it is only necessary to continue irrigation after that, and if it is not irrigated after leaving rainwater, there will be no change such as shifting to the dry season etc. or extremely sunny days without rain. As long as it is left alone.

  According to this method, it is possible to determine whether or not to give water to the land on which the test plant grows and how much water should be irrigated. It also makes it possible to maintain or improve the growth state of the plant by avoiding or eliminating the deficiency or deficiency state of the plant. As a result, plant production and production efficiency can be increased.

<Example 1: Selection of marker for diagnosis of water sufficiency>
(material)
Two test plots (water-sufficiency zone and water-deficient zone) 10 eucalyptus hybrid clones (camaldolensis x deglupta) were cultivated in a greenhouse with a soil volume of 13 L in a greenhouse for 3 months. The cultivation conditions in both test plots are all the same, except that 1 L of water is irrigated every day in the water-sufficiency zone and 1 L is irrigated once every 3 days in the water-deficient zone.

  In both test groups, as shown in FIG. 1, a difference in growth according to the amount of irrigation was clearly recognized. Therefore, the individuals in these two test sections were used as materials for selecting a marker for diagnosing water sufficiency.

(Method)
1. Leaf Sampling For each Eucalyptus individual in the above-mentioned water-sufficiency group and water-deficient group, leaves were cut out from the petiole portion with scissors. The main vein portion of the collected leaves was removed, and the rest was dried at 50 ° C. overnight.

2. Extraction of Extract Each individual of about 30 mg in dry weight was placed in a ceflock tube. A zirconia ball (Tosoh) having a diameter of 5 mm was added to the tube, and the leaf sample was pulverized with Tissue Lyser II (Qiagen) at 25 Hz for 30 seconds. Next, add 1 mL of extract (water: methanol: chloroform = 1: 2.5: 1 (v: v: v), 5 mg / mL (-) containing Epicatechin as an internal standard substance) to the tube, and add Tissue Lyser II. And extracted by shaking for 120 seconds at 25 Hz. The obtained extract was centrifuged at 12000 × g for 10 minutes, 0.4 mL of water was added to about 0.9 mL of the supernatant, and centrifuged at 12000 × g for 10 minutes, and the upper layer (aqueous methanol layer) and the lower layer (chloroform) Layer). Subsequently, the upper layer was filtered using Millex-LG (Millipore), and the filtrate was recovered.

3. LC-MS analysis 10 μL of the obtained filtrate was subjected to LC-MS analysis under the following conditions. About the specific analysis method, the instruction manual attached to each equipment used was followed.

3-1. Liquid Chromatography (LC) Conditions • Equipment used: UFLC (Shimadzu Corporation)
・ Eluent A: 0.1% formic acid (Wako) in water rechromorub for water chromatography (Merck)
・ Eluent B: Hypergrade resolvosolve (Merck) for acetonitrile LC / MS with 0.1% formic acid (Wako)
・ Column: TSKgel ODS-100V 3.0x 750mm particle 3μm (Tosoh)
Guard column: TSKguardgel ODS-100V, particle 5μm (Tosoh)
・ Column temperature: 40 ℃
・ Flow rate: 0.5mL / min
・ Time continuous gradient (time schedule gradient) B%
0 minutes: 3% to 10 minutes: 97%

3-2. MS conditions-Equipment used: 3200QTRAP
・ Mass range: 100-1000
-Scan mode: Enhanced Mass Scan
・ Ionization method: ESI-Positive
・ Scanning speed: 4000 amu / sec
・ Analysis software: Analyst 1.4.2

3-3. MSMS conditions-Equipment used: 3200QTRAP
・ Mass range: 50-1000
-Scan mode: Enhanced Product Ion Scan
・ Ionization method: ESI-Positive
・ Scanning speed: 4000 amu / sec
・ Analysis software: Analyst 1.4.2

3-4. Alignment conditions-Metabolite data alignment: Marker View ^TM 1.2 Software
・ Elution time tolerance: 1 minute ・ Mass tolerance: 25 ppm
・ Intensity threshold: 10,000

4). Selection of markers for determining water sufficiency From the metabolites obtained in the LC-MS / MS analysis, select metabolites that show water sufficiency in terms of accumulated volume using Volcano Plot of GeneSpring MS (Agilent Technologies). did. Specifically, when the accumulated amount of metabolites was compared between individuals in the water-sufficiency group and those in the water-deficient group, the accumulated amount in the sufficient-sufficiency individual was ionic strength, and the metabolism of 1.5 times or more than that in the individual in the deficient group A product or a metabolite of 1 / 1.5 times or less was selected as a marker for determining the degree of water satisfaction.

  All metabolites are substances that vary in the plant depending on the amount of water, but the former is proportional to the amount of water, that is, the amount of accumulation in the plant increases as the amount of water increases. In contrast, the latter is a marker whose amount of accumulation in a plant body decreases as the amount of water increases, that is, a marker that is inversely related to the amount of water.

(result)
As a result of selection, 11 metabolites (markers No. 1 to 11) shown in Tables 2 and 3 were obtained. Table 2 shows the results of comparing the amount of accumulated metabolites between the water-deficient and water-sufficient individuals, and Table 3 shows how to identify each of the obtained markers by LC-MS / MS analysis. LC retention time, precursor ion and product ionic strength are shown.

  As shown in this table, markers No. 4, 5, 7, 10 and 11 are metabolites whose accumulated amount in the water-sufficiency zone is classified as 1.5 times or more of that in the water-deficient zone in terms of ionic strength. It became clear that. That is, the metabolites of markers No. 4, 5, 7, 10 and 11 of the present invention were markers having a property proportional to the amount of water.

  On the other hand, marker Nos. 1 to 3, 6, 8, and 9 are metabolites that are classified as less than 1 / 1.5 times the accumulated amount in individuals in the water-sufficiency zone with ionic strength and less in individuals in the water-deficient zone Became clear. That is, the metabolites of the markers 1 to 3, 6, 8, and 9 of the present invention were markers that have an inversely proportional relationship with the amount of water.

<Example 2: Determination of water sufficiency in test plants of different nutritional states using a water sufficiency determination method>
Verify whether the water sufficiency determination method of the present invention can determine the water sufficiency without being affected by the nutritional state, even if the amount of fertilization (ie, nutritional state) is different using a greenhouse cultivated individual did.

(material)
A total of 20 hybrid clones of the genus Eucalyptus cultivated in a greenhouse with a soil volume of 13 L in a greenhouse for 3 months (Camaldrensis x Deglupta) were used. At that time, four treatment zones shown in Table 4 below were provided.

(Method)
1. Sampling of leaves About the individual | organism | solid of each said test plot, the leaf was cut out from the petiole part with the scissors every week for the period of 2-6 weeks after planting. Therefore, the total number of samples is 100 because 20 individuals collect 5 leaves. In each sample, the main vein portion of the collected leaf was removed, and the rest was dried overnight at 50 ° C.

2. Extraction Extraction The extraction was performed according to the method described in Example 1.

3. LC-MS / MS analysis 10 μL of the obtained filtrate was subjected to LC-MS / MS analysis under the following analysis conditions. About the specific analysis method, the instruction manual attached to each equipment used was followed.

3-1. Liquid Chromatography (LC) Conditions • Equipment used: UFLC (Shimadzu Corporation)
・ Eluent A: 0.1% formic acid (Wako) containing water resolve for water chromatography
・ Eluent B: Hypergrade resolvosolve for acetonitrile LC / MS with 0.1% formic acid (Wako)
・ Column: TSKgel ODS-100V 3.0x 750mm particle 3μm (Tosoh)
Guard column: TSKguardgel ODS-100V, particle 5μm (Tosoh)
・ Column temperature: 40 ℃
・ Flow rate: 1.0 ml / min
・ Time continuous gradient (time schedule gradient) B%
0 minutes: 3% to 5 minutes: 56%

3-2. MS / MS conditions-Equipment used: 3200QTRAP
-Scan mode: Multiple Reaction Monitoring
・ Ionization method: ESI-Positive
・ Scanning speed: 4000 amu / sec
・ Analysis software: Analyst 1.4.2
・ Target Mass:
No.1: Q1: m / z: 175.2, Q3: m / z: 116.0 RT = 0.4
No.2: Q1: m / z: 188.0, Q3: m / z: 118.0 RT = 2.0
No.3: Q1: m / z: 511.1, Q3: m / z: 511.1 RT = 3.1
No.4: Q1: m / z: 769.2, Q3: m / z: 153.0 RT = 2.9
No.5: Q1: m / z: 104.2, Q3: m / z: 104.2 RT = 0.4
No.6: Q1: m / z: 501.1, Q3: m / z: 501.1 RT = 3.8
No.7: Q1: m / z: 639.1, Q3: m / z: 639.1 RT = 4.1
No.8: Q1: m / z: 291.1, Q3: m / z: 291.1 RT = 0.5
No.9: Q1: m / z: 533.2, Q3: m / z: 362.2 RT = 0.5
No.10: Q1: m / z: 812.3, Q3: m / z: 812.3 RT = 2.0
No.11: Q1: m / z: 421.1, Q3: m / z: 259.2 RT = 4.5
No.12: Q1: m / z: 291.2, Q3: m / z: 139.1 RT = 2.6
Here, No. 12 is an internal standard substance (-) Epicatechin.
Q1 and Q3 are mass-to-charge ratio data (m / z) of each marker, and RT is a retention time (min).

3-3. Alignment conditions-Metabolite data alignment: Marker View ^TM 1.2 Software
・ Elution time tolerance: 1 minute ・ Mass tolerance: 25 ppm
・ Intensity threshold: 1,000

3-4. Standardization of data In the case of a mass spectrometer, the accumulated amount of marker is slightly different for each analysis even for the same sample, so standardization is required. Here, the accumulation amount of various markers was standardized by dividing by the accumulation amount of the internal standard substance ((−) Epicatechin).

4. Discriminant analysis
Discriminant analysis was performed on the data acquired by LC-MS / MS using GeneSpring MS (Agilent Technology). Specifically, the accumulated amount data in the water sufficiency group and the water deficiency group of the water sufficiency degree diagnosis marker of Example 1 is set as a training data set for discriminant analysis, that is, a base text sample, This is compared with the accumulated amount of the marker for diagnosis of water sufficiency in the plant of this example (20 samples), and it is determined whether the accumulated amount of the test plant is closer to the water sufficiency group or the water deficient group of Example 1 did. The determination method was performed under the following conditions.

-Judgment algorithm: K-neighbor method In the case of a class classification that is given a training case with a class label: find k cases in order from the one closest to the case to be classified. A method of classifying the k cases into the class with the largest number.
・ Marker: 6 types of markers for determining water sufficiency (Markers No.1, 3, 4, 8, 10, 11)
・ Training data set: Marker profile obtained from the water deficit group and the water sufficiency group in Example 1 ・ Test sample data: Marker profile obtained from each of the 20 samples in the four treatment groups of this example

(result)
The results are shown in FIG. Among the treatment groups, the test groups 1 and 3, that is, the treatment groups in which irrigation was carried out every day showed better growth. This result shows that plant growth is more dependent on irrigation than fertilizer.

  Moreover, when the moisture sufficiency determination method of the present invention was used to determine the degree of water sufficiency for each individual in the four treatment zones shown in Table 4 every 2 to 6 weeks after planting, as shown in Table 5 In 50 samples of water deficiency (treatment zones 2 and 4), there was a misjudgment (indicated by an asterisk in the table) that there was a mismatch in 5 samples. In the ward (processing wards 1 and 3), a correct judgment result that all 50 samples are in a satisfactory state is obtained, and the judgment method of the present invention has a correct answer rate that the judgment result and the actual condition match is 95% as a whole. The actual moisture could be judged with very high accuracy. Note that in Table 5, “−” indicates that the reliability of the result by the K-nearest neighbor method is low, and it was not possible to determine whether it is a sufficient or insufficient zone.

<Example 3: Afforestation site & diagnosis of water sufficiency for other tree species>
In a plantation site that is considered to be affected by a complex environmental factor, the water content level determination method of the present invention is used for a eucalyptus tree species sample different from those in Examples 1 and 2, and the water content is actually accurately measured. It was verified whether the degree could be judged.

(material)
A 0.8 year-old Eucalyptus hybrid planted in two test zones (test zone A and test zone B) in Laos plantation was used as a sample.

(Method)
1. Leaf Sampling In both the above test areas, the leaves of the individuals were cut from the petiole part with scissors twice in the dry season and the beginning of the rainy season. Each was used as a water-deficient sample and a water-satisfied sample in each test section. The main vein portion of the collected leaves was removed, and the rest was dried at 50 ° C. overnight.
・ Moisture deficient sample: dry season (monthly precipitation: 12mm)
・ Moisture satisfaction sample: Beginning of the rainy season (monthly precipitation: 98 mm)

2. Extraction Extraction The extraction was performed according to the method described in Example 2.

3. LC-MS / MS analysis The LC-MS / MS analysis was performed according to the method described in Example 2.

4). Judgment The metabolite data obtained from the water deficient sample and the water sufficiency sample at the test site A is used as a training data set for discriminant analysis, that is, a base text sample, and the sample at the test site B as the test sample (total) 145 specimens) were determined based on the profile of the two markers in Table 1. The determination method was performed under the following conditions.

-Judgment algorithm: K nearest neighbor method (p-value <0.2)
・ Marker: 2 types of markers for determining water sufficiency (Markers No. 3 and 6)
・ Training data set: Profile of markers obtained from water deficient sample (water deficient individual) and water sufficient sample (water deficient individual) at test site A in Laos plantation ・ Test sample data: Test site B in Laos plantation Marker profiles obtained from 66 dry season samples and 79 samples from the beginning of the rainy season

(result)
The results are shown in Table 6. When the moisture sufficiency of 66 samples in the dry season sample of the test site B was determined, all the markers showed a determination result that all the samples were in a deficient state. That is, 100% correct answer was obtained. On the other hand, when 79 samples were determined at the beginning of the rainy season, it was determined that 63 samples had sufficient water. In other words, 88.9% of correct answers were obtained.

  As described above, according to the water sufficiency determination method using the marker of the present invention, a high determination accuracy of 90% correct answer rate even for plantation site samples that are considered to be affected by complex environmental factors. It was proved that the water sufficiency of plants growing on the land can be judged.

  As described above, in this example, the plant species used in the experiment are different from those in Examples 1 and 2. The marker of the present invention is a metabolite isolated from the plant species described in Example 1. However, in this example, it was revealed that the marker was also effective for different plant species, and the generality of the marker was proved. .

Claims (18)

A metabolite of a plant, and in high performance liquid chromatography tandem mass spectrometry,
When a time continuous gradient of acetonitrile in liquid chromatography was formed at 0 min 3% to 5 min 56%, it was separated with the retention time shown in Table 1 below, and precursor ion and product ion in mass spectrometry A marker for determining the degree of water sufficiency of any one of the plants indicated by marker Nos. 1 to 11 identified by the values shown in Table 1 below.

  The marker according to claim 1, wherein the plant is woody.   The marker according to claim 2, wherein the woody species belongs to the genus Eucalyptus. A method for determining the sufficiency of moisture in a plant,
An extraction step for extracting an extract containing a metabolite from all or a part of the test plant;
A measurement step of measuring the accumulated amount of at least one moisture sufficiency determination marker according to claim 1 contained in the extract obtained in the extraction step,
The said method including the determination process of determining the sufficiency of the water | moisture content in the said test plant based on the accumulation amount of the marker for water sufficiency determination obtained at the said measurement process.   The determination method according to claim 4, wherein in the determination step, the degree of water sufficiency is determined by comparing the accumulated amounts of the water sufficiency determination markers obtained from each of the test plant and the control plant.   The determination method according to claim 5, wherein the target plant is in a moisture-deficient state.   When the marker for determining the degree of water sufficiency is marker No. 1 to 3, 6, 8 and 9 in Table 1, the accumulated amount of the marker in the test plant is statistically significantly smaller than the accumulated amount in the control plant The determination method according to claim 6, wherein the water in the test plant is determined to be in a sufficient state.   When the marker for water sufficiency determination is marker No. 4, 5, 7, 10 and 11 in Table 1, the accumulated amount of the marker in the test plant is statistically significantly larger than the accumulated amount in the control plant The determination method according to claim 6, wherein the water in the test plant is determined to be in a sufficient state.   The determination method according to claim 5, wherein the target plant is in a water-sufficient state.   When the marker for determining the degree of water sufficiency is marker No. 1 to 3, 6, 8 and 9 in Table 1, the accumulated amount of the marker in the test plant is statistically significantly larger than the accumulated amount in the control plant The determination method according to claim 9, wherein it is determined that water in the test plant is in a deficient state.   When the marker for water sufficiency determination is marker No. 4, 5, 7, 10 or 11 in Table 1, the accumulated amount of the marker in the test plant is statistically significantly smaller than the accumulated amount in the control plant The determination method according to claim 9, wherein it is determined that water in the test plant is in a deficient state.   The determination method according to claim 5, wherein the plant is a tree.   The determination method according to claim 12, wherein the woody species is Eucalyptus.   A soil moisture content estimation method for estimating the soil moisture content in the test site using the determination method according to any one of claims 4 to 13.   A planting site selection method for selecting a planting site for the target plant using the soil moisture content estimation method according to claim 14.   In the same plant, the growth state in the plant is determined based on two or more determination results over time regarding the moisture sufficiency obtained by the moisture sufficiency determination method according to any one of claims 4 to 13. A method for determining the growth state of a plant.   When two or more determination results over time are the results that both are in a satisfactory state, it is determined that the growth state in the plant is good and indicates that it has shifted from insufficient to full In the case of determining that the growth state is in an improving trend and indicating that the growth state has shifted from sufficient to insufficient, it is determined that the growth state is in a worsening tendency, or that both are insufficient The determination method according to claim 16, wherein in this case, it is determined that the growth state is bad.   The irrigation amount determination method which determines the irrigation amount of the water | moisture content in the plant based on the determination result by the growth state determination method of the plant of Claim 16 or 17.

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