Method for diagnosing sulfur deficiency in oryza genus plant

Abstract

To provide a method for diagnosing sulfur deficiency in Oryza genus plants.SOLUTION: Provided is a method for diagnosing sulfur deficiency in Oryza genus plants, comprising measuring the content of myo-inositol in the Oryza genus plant and / or measuring the ratio between glycine and alanine contents in the Oryza genus plant.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to methods for diagnosing sulfur deficiency in Oryza plants. [Background technology]

[0002] Sulfur deficiency, which has been reported in some areas of rice cultivation in recent years, is one of the conditions that inhibit the growth of rice plants. In recent years, it is said that the supply of sulfur to rice plants has decreased due to the continuous use of non-sulfate fertilizers to prevent autumn leaf fall caused by hydrogen sulfide in the soil of paddy fields, and the decrease in acid rain containing sulfur.

[0003] It is known that rice plants with sulfur deficiency show symptoms such as yellowing of leaves and a decrease in the number of stems. However, these symptoms are similar to those of nitrogen deficiency, which is also one of the conditions that inhibit the growth of rice plants, so it is often difficult to judge from the appearance. In fact, there have been reported cases where growth recovered after applying sulfur materials in fields where nitrogen fertilizer was applied after it was determined to be nitrogen deficiency based on the characteristics of the appearance and no improvement was observed in growth, and there are concerns about a decrease in yield due to such potential sulfur deficiency.

[0004] Regarding rice plants with sulfur deficiency, for example, Non-Patent Document 1 measures the sulfur and nitrogen contents in rice plants with sulfur deficiency and in the case where materials are applied to the rice plants (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Togo Tsuji, "Nutrition disorders and sulfur absorption characteristics of rice plants due to sulfur deficiency," Journal of the Japanese Society of Soil Science and Plant Nutrition, Vol. 71, No. 4, pp. 464-471, 2000. Summary of the Invention [Problem to be solved by the invention]

[0006] As one of the methods for diagnosing sulfur deficiency in rice plants including paddy rice, a method is used in which a sulfur material is actually applied to a part of a field suspected of sulfur deficiency based on characteristics such as appearance, and when the growth of the rice plant to which the material is applied is recovered, the rice plant in the field is judged to be in a sulfur-deficient state. However, it usually takes 7 to 14 days from the application of a sulfur material to a part of the field to determine from the appearance whether the growth of the plant in the applied area has recovered. Therefore, even if a sulfur material is applied to the field after diagnosing that the plant is in a sulfur-deficient state by this method, there is a time lag between when sulfur deficiency is suspected based on characteristics such as appearance and when the plant is diagnosed as being in a sulfur-deficient state. As a result, the rice plant will have to be cultivated in a sulfur-deficient state for a certain percentage of the tillering period, which is about several tens of days, during which the rice plant increases the number of stems, and there is a concern that this will cause a significant decrease in yield.

[0007] Another method for diagnosing sulfur deficiency in rice and other rice plants is to measure the amount of sulfur in paddy fields. More specifically, a method is known in which available sulfur content in the soil of a paddy field and / or sulfur concentration in irrigation water are measured to determine whether the paddy field is prone to sulfur deficiency. However, the amount of sulfur available for rice plants is limited, and some of the sulfur in the soil forms sulfides with metals and becomes unavailable to plants. Therefore, it is often impossible to explain (diagnose) the occurrence of sulfur deficiency in a paddy field solely from the available sulfur content in the paddy field soil.

[0008] On the other hand, if sulfur deficiency in rice plants could be diagnosed by taking a part or the whole of the plant as a sample and analyzing the sample, without relying on application tests or tests using soil samples, it may be possible to rapidly and directly analyze sulfur deficiency in rice plants.

[0009] The present disclosure aims to provide a method for diagnosing sulfur deficiency in Oryza plants. [Means for solving the problem]

[0010] The present inventors have discovered that sulfur deficiency in Oryza plants can be diagnosed by measuring the content of myo-inositol, a type of metabolite, and / or measuring the ratio of the contents of glycine and alanine, which are also metabolites, in Oryza plants, and have completed the present disclosure.

[0011] The present disclosure relates, for example, to the following: [1] A method for diagnosing sulfur deficiency in rice plants, comprising: Measuring the content of myo-inositol in the rice plant; and / or Measuring the ratio of glycine to alanine content in the rice plant; A method comprising: [2] The method according to [1], wherein the method comprises measuring a content of myo-inositol in an Oryza plant, and when the measured content of myo-inositol is lower than the content of myo-inositol in a control Oryza plant, the plant is diagnosed as being sulfur deficient. [3] The method according to [1], wherein the method comprises measuring a ratio of glycine to alanine content in an Oryza plant, and when the measured ratio of glycine to alanine content is greater than the ratio of glycine to alanine content in a control Oryza plant, the plant is diagnosed as being sulfur deficient. [4] The method according to [2] or [3], wherein the control rice plant is a rice plant that is not sulfur deficient. [5] The method according to any one of [1] to [3], wherein the rice plant is rice. Effect of the Invention

[0012] According to an embodiment of the present disclosure, sulfur deficiency in an Oryza plant can be diagnosed. According to an embodiment of the present disclosure, data for diagnosing sulfur deficiency in an Oryza plant can be obtained.

[0013] According to one embodiment of the present disclosure, sulfur deficiency in an Oryza plant can be diagnosed by using a part of the plant (e.g., a leaf blade) as a sample, which allows sulfur deficiency in the Oryza plant to be diagnosed simply and easily.

[0014] According to an embodiment of the present disclosure, sulfur deficiency in an Oryza plant can be diagnosed in a short time (for example, within one day). In addition, according to an embodiment of the present disclosure, sulfur deficiency in an Oryza plant can be diagnosed in a short time (for example, within one day) by using a part of the plant (for example, a leaf blade) as a sample. In this way, sulfur deficiency in an Oryza plant can be diagnosed while suppressing a decrease in yield caused by the time required for diagnosis.

[0015] According to an embodiment of the present disclosure, sulfur deficiency in rice plants can be diagnosed regardless of appearance. According to an embodiment of the present disclosure, sulfur deficiency and nitrogen deficiency can be distinguished in rice plants. Thus, sulfur deficiency can be diagnosed for rice plants that do not show symptoms of sulfur deficiency or in which nitrogen deficiency and sulfur deficiency cannot be distinguished from each other based on appearance. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 is a graph showing the amounts of metabolites in rice hydroponically grown under sulfur-deficient conditions in Example 1. [Diagram 2] FIG. 2 is a graph showing the sulfur and nitrogen concentrations in rice hydroponically grown under sulfur-deficient conditions in Example 2. [Diagram 3] FIG. 3 is a diagram showing the amount of metabolites in rice grown in a field where sulfur deficiency occurred in 2020, 2021, and 2022 in Example 3. [Figure 4]FIG. 4 is a diagram showing the amount of metabolites in rice grown in a field where sulfur deficiency occurred in 2023 in Example 3. [Diagram 5] FIG. 5 is a diagram showing the glycine / alanine ratio in rice hydroponically cultivated under sulfur-deficient conditions in Example 4. [Figure 6] FIG. 6 is a diagram showing the glycine / alanine ratio in rice grown in a field where sulfur deficiency occurred in 2020, 2021, and 2022 in Example 5. [Figure 7] FIG. 7 is a diagram showing the glycine / alanine ratio in rice grown in a field where sulfur deficiency occurred in 2023 in Example 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Although the present disclosure will be described below with reference to the accompanying drawings, the present disclosure is not limited to the following embodiments.

[0018] In this disclosure, unless otherwise specified, amino acids refer to amino acids in the L-form. For example, in this disclosure, the terms "alanine" and "L-alanine" refer to alanine in the L-form, and the term "D-alanine" refers to alanine in the D-form.

[0019] <Method for diagnosing sulfur deficiency in rice plants> One embodiment of the present disclosure is a method for diagnosing sulfur deficiency in a rice plant, comprising measuring the content of myo-inositol in the rice plant (myo-inositol measurement) and / or measuring the ratio of the content of glycine to alanine in the rice plant (glycine / alanine ratio measurement). That is, one embodiment of the present disclosure is a method for diagnosing sulfur deficiency in a rice plant, comprising myo-inositol measurement, another embodiment of the present disclosure is a method for diagnosing sulfur deficiency in a rice plant, comprising glycine / alanine ratio measurement, and yet another embodiment of the present disclosure is a method for diagnosing sulfur deficiency in a rice plant, comprising myo-inositol measurement and glycine / alanine ratio measurement. Another embodiment of the present disclosure may also be a method for obtaining data for diagnosing sulfur deficiency in a rice plant, comprising myo-inositol measurement and / or glycine / alanine ratio measurement.

[0020] <Rice plants> In this disclosure, unless otherwise limited, a "plant" includes a whole plant, a plant cell, a plant protoplast, a plant callus, or a plant part, such as an embryo, pollen, ovule, gamete, seed, leaf, flower, branch, fruit, stem, root, anther, etc.

[0021] In the present disclosure, "Oryza" refers to a plant belonging to the genus Oryza in the family Poaceae of the order Poales. As representative plants belonging to the Oryza genus, Oryza sativa and Oryza glaberrima are known, which are widely cultivated for food. The Oryza plant according to the present disclosure may be, for example, Oryza sativa or Oryza glaberrima, and in this case, Oryza sativa may be any of the japonica species (O. sativa subsp. japonica), the indica species (O. sativa subsp. indica), or the javanica species (O. sativa subsp. javanica), for example, the japonica species. In one embodiment, the Oryza plant according to the present disclosure may be paddy rice.

[0022] <Sulfur deficiency> Sulfur deficiency is one of the conditions that inhibits the growth of rice plants. Sulfur-deficient rice plants show symptoms such as yellowing of leaves and a decrease in the number of stems. There is also concern about a decrease in yield due to potential sulfur deficiency, such as no visible symptoms or symptoms that are visible but are misdiagnosed as nitrogen deficiency.

[0023] The growth of a rice plant suffering from sulfur deficiency is restored by application of a sulfur material such as calcium sulfate or magnesium sulfate fertilizer. In other words, in one embodiment of the present disclosure, a rice plant whose growth is restored when a sulfur material is applied to the rice plant is a rice plant suffering from sulfur deficiency. In these cases, "growth is restored" may mean, for example, that yellowing of leaves and / or a decrease in the number of stems, which are external characteristics of sulfur deficiency, are improved or eliminated by application of a sulfur material, or that a rice plant to which a sulfur material is applied shows good growth (e.g., a larger number of stems and / or a smaller area of ​​yellowed leaves) compared to the same rice plant to which a sulfur material is not applied. In these cases, whether or not "growth has recovered" may be determined, for example, 5 to 30 days after application of the sulfur material; as a specific example, it may be determined 7, 10, 12, or 14 days later.

[0024] In one embodiment, the method for diagnosing sulfur deficiency in an Oryza plant according to the present disclosure may also be a method for predicting (or measuring or diagnosing) a growth recovery effect (improvement effect) of a sulfur material in an Oryza plant. That is, one embodiment of the present disclosure may be a method for predicting a growth recovery effect of a sulfur material in an Oryza plant, the method including myo-inositol measurement and / or glycine / alanine ratio measurement.

[0025] The "sulfur material" in the present disclosure is not particularly limited as long as it is a material that can guarantee soluble sulfur (a material that can guarantee that the fertilizer contains 1% or more of sulfur extracted with dilute hydrochloric acid, for example, a material that can supply sulfur in a form that can be utilized (absorbed) by plants of the genus Oryza), and examples thereof include ammonium sulfate, coated nitrogen fertilizer, superphosphate, triple superphosphate, coated phosphate fertilizer, processed phosphate fertilizer, potassium sulfate, potassium sulfate magnesium, coated potassium fertilizer, processed poultry manure fertilizer, dried bacterial cell fertilizer, coated compound fertilizer, calcium sulfate, magnesium sulfate fertilizer, processed magnesium fertilizer, coated magnesium fertilizer, manganese sulfate fertilizer, processed manganese fertilizer, and processed boron fertilizer, as well as chemical fertilizers, blended fertilizers, and designated mixed fertilizers that use these as raw materials, and specific components include materials (fertilizers) containing calcium sulfate, magnesium sulfate, potassium sulfate, ammonium sulfate, etc.

[0026] A rice plant diagnosed as being in a sulfur-deficient state by the method for diagnosing sulfur deficiency in a rice plant according to the present disclosure may have its growth restored by application of a sulfur material. The method for applying such a sulfur material is not particularly limited as long as it is a method that restores the growth of the rice plant, and examples of the method include full-layer fertilization method, side stripe fertilization method, and full-amount seedling box fertilization method (seedling box fertilization method).

[0027] <Diagnosis of sulfur deficiency> In the method for diagnosing sulfur deficiency according to the present disclosure, sulfur deficiency in an Oryza plant is diagnosed by measuring the content of myo-inositol in the Oryza plant (myo-inositol measurement) and / or measuring the ratio of the content of glycine to alanine in the Oryza plant (glycine / alanine ratio measurement).

[0028] In the present disclosure, myo-inositol measurement and glycine / alanine ratio measurement in rice plants are usually performed by collecting a part of an organ of the rice plant and measuring the myo-inositol content or the glycine / alanine content ratio in the part. In the present disclosure, the part of an organ of the rice plant used for myo-inositol measurement or glycine / alanine ratio measurement is not particularly limited as long as it can diagnose sulfur deficiency, and may be, for example, an embryo, pollen, ovule, gamete, seed, leaf, flower, branch, fruit, stem, root, anther, etc., preferably an embryo, pollen, ovule, gamete, seed, leaf, flower, branch, fruit, stem, or anther, more preferably a leaf or stem, and even more preferably a leaf. In addition, when a leaf is used, the part of the leaf is not particularly limited, and may be, for example, a leaf blade, a leaf sheath, a leaf auricle, or a leaf ligule, and a specific example may be a leaf blade.

[0029] The measurement of myo-inositol and the measurement of the glycine / alanine ratio in a part of an organ of a plant of the genus Oryza can be performed by extracting metabolites containing myo-inositol, alanine, and glycine from the part of an organ of a plant of the genus Oryza, and measuring the content of myo-inositol, alanine, and / or glycine in the extract. The method for extracting metabolites from a part of an organ of a plant of the genus Oryza is not particularly limited as long as it is a method capable of extracting myo-inositol and / or alanine and glycine, and can be performed according to a method commonly used by those skilled in the art. Examples of such extraction methods include a method in which a part of an organ of a plant of the genus Oryza is freeze-dried as necessary, then crushed, and an aqueous solution (e.g., a mixed solution of water, methanol, and chloroform) is added to the resulting crushed material and incubated to elute metabolites into the solution, and then insoluble components are removed by centrifugation, filtration, or the like. In one embodiment, the extraction of metabolites from a part of an organ of a plant of the genus Oryza does not need to include treatment with an enzyme having peptidase activity.

[0030] In the present disclosure, measuring the content of metabolites such as myo-inositol, alanine, and glycine may be to measure, as the content of the metabolite, for example, the content per unit mass (e.g., mass or amount of substance) of a plant organ or the concentration in the organ (e.g., molar concentration, molality, or mass percentage concentration), or it may be to obtain a parameter serving as an index of the content of the metabolite. However, from the viewpoint of measurement simplicity, in a preferred embodiment, it may be to obtain a parameter serving as an index of the content of the metabolite. In other words, the term "content" of the metabolite used in the present disclosure is a concept including both the content of the metabolite and the value (magnitude) of the parameter serving as an index of the content of the metabolite. In a preferred embodiment, the "content" of the metabolite may be the value (magnitude) of the parameter serving as an index of the content of the metabolite. Examples of such a parameter serving as an index of the content include peak intensity or peak area in mass spectrometry or optical measurement. Further, the measurement method capable of obtaining such a parameter is not particularly limited, but may be, for example, GC-MS analysis, LC-MS analysis, LC-MS / MS analysis, or measurement of absorbance or fluorescence intensity in HPLC, or may be signal intensity in, for example, a differential refractive index detector (RID), an evaporative light scattering detector (ELSD), an electrochemical detector (ECD), or a charged particle detector (CAD).

[0031] <Measurement of myo-inositol> A method for diagnosing sulfur deficiency according to the present disclosure includes, in one embodiment, measuring the content of myo-inositol. Myo-inositol is a type of cyclitol and is a compound with a CAS registration number of 87-89-8, which is the compound number defined by the American Chemical Society. As a metabolite in plants, it is widely present in a free or bound state in plants and is known to play many roles, such as forming membrane components and forming phytic acid (inositol hexaphosphate, IP6) to store phosphate. On the other hand, it has not been known until now that the amount of myo-inositol in gramineous plants varies in response to sulfur deficiency.

[0032] In one embodiment of the method for diagnosing sulfur deficiency according to the present disclosure, a rice plant may be diagnosed as being in a sulfur-deficient state when the content of myo-inositol in the rice plant is low. In one embodiment, a rice plant may be diagnosed as being in a sulfur-deficient state when the content of myo-inositol in the rice plant is lower than the content of myo-inositol in a control rice plant. The control rice plant may be, for example, a rice plant that is not in a sulfur-deficient state, and a specific example of the control rice plant may be a rice plant that is not in a sulfur-deficient state and is of the same variety as the rice plant diagnosed as being in a sulfur deficiency state. In addition, the control rice plant may be, for example, a rice plant that is resistant to sulfur deficiency, and the sulfur-deficiency resistant rice plant may be, for example, a rice plant of the same variety as the rice plant diagnosed as being in a sulfur deficiency state into which a gene for resistance to sulfur deficiency has been introduced, or may be, for example, a rice plant that is known not to be in a sulfur-deficient state or not to be in a sulfur-deficient state even when cultivated in a sulfur-deficient field.

[0033] In one embodiment of the present disclosure, the myo-inositol content in an Oryza plant is less than the myo-inositol content in a control Oryza plant may mean, for example, that the myo-inositol content measured in a diagnosed Oryza plant is less than the average or median of the myo-inositol contents in multiple control Oryza plants, and more specifically may be, for example, less than 1.00 times, 0.95 times or less, 0.90 times or less, 0.85 times or less, 0.80 times or less, 0.75 times or less, 0.70 times or less, or 0.50 times or less of the average or median. Furthermore, for example, the myo-inositol content in an Oryza plant being lower than the myo-inositol content in a control Oryza plant may mean, for example, that the myo-inositol content measured in a plurality of samples taken from the Oryza plant to be diagnosed is significantly lower than the myo-inositol content measured in a plurality of samples taken from one or more control Oryza plants. In this case, significantly lower may mean, for example, that the average myo-inositol content is lower than the average myo-inositol content in the control Oryza plants and that the p-value in a Dunnett's test between the two is less than 0.05, less than 0.01, or less than 0.001.

[0034] <Glycine / alanine ratio measurement> In one embodiment, the method for diagnosing sulfur deficiency according to the present disclosure includes measuring the ratio of the content of glycine to alanine (glycine / alanine ratio). Glycine and alanine are α-amino acids that are ubiquitous in organisms, including plants. Glycine and alanine in organisms are produced by enzymatic degradation of proteins, as well as by biosynthesis. The glycine / alanine ratio according to the present disclosure means the ratio of the content of glycine and alanine that are present in a free state in an Oryza plant, and does not mean the ratio of the total amount of glycine, including glycine residues and alanine residues contained in peptides, proteins, etc., present in an Oryza plant, to the total amount of alanine.

[0035] The terms "glycine to alanine content ratio" and "glycine / alanine ratio" in the present disclosure refer to the glycine content normalized by the alanine content. In other words, the terms "glycine to alanine content ratio" and "glycine / alanine ratio" in the present disclosure refer to the value obtained by dividing the glycine content by the alanine content.

[0036] The glycine / alanine ratio according to the present disclosure may be a ratio of the content (e.g., mass or substance amount) per unit mass of a plant organ or a ratio of the concentration (e.g., volume molar concentration, mass molar concentration, or mass percent concentration) in the organ, or a ratio of a parameter that is an index of the content of a metabolite. Specific examples of the glycine / alanine ratio include the mass ratio, concentration ratio, and substance amount ratio of glycine and alanine contained in an extract from a part of a plant organ or the like. Other specific examples of the glycine / alanine ratio include the peak area ratio and signal intensity ratio of a signal derived from glycine and a signal derived from alanine when an extract from a part of a plant organ or the like is analyzed by GC-MS analysis, LC-MS analysis, LC-MS / MS analysis, or HPLC absorbance or fluorescence intensity measurement, etc., and the peak area in this case may be the peak area when the horizontal axis is the retention time and the vertical axis is the signal intensity.

[0037] In one embodiment of the method for diagnosing sulfur deficiency according to the present disclosure, a rice plant may be diagnosed as being sulfur deficient when the ratio of the contents of glycine and alanine (glycine / alanine ratio) is large. In one embodiment, a rice plant to be diagnosed may be diagnosed as being sulfur deficient when the glycine / alanine ratio in the rice plant is larger than the glycine / alanine ratio in a control rice plant. The control rice plant may be, for example, a rice plant that is not sulfur deficient, and a specific example of the control rice plant may be a rice plant that is not sulfur deficient and is of the same variety as the rice plant to be diagnosed as being sulfur deficient. In addition, the control rice plant may be, for example, a rice plant that is resistant to sulfur deficiency, and the sulfur deficiency resistant rice plant may be, for example, a rice plant of the same variety as the rice plant to be diagnosed as being sulfur deficiency into which a gene for resistance to sulfur deficiency has been introduced, or may be, for example, a rice plant that is known not to be or is unlikely to be sulfur deficient even when cultivated in a field with sulfur deficiency.

[0038] In one embodiment of the present disclosure, the glycine / alanine ratio in an Oryza plant being greater than the glycine / alanine ratio in a control Oryza plant may mean, for example, that the glycine / alanine ratio measured in a diagnosed Oryza plant is greater than the average or median of the glycine / alanine ratios in a plurality of control Oryza plants, and more specifically, may be, for example, more than 1.0 times, 1.2 times or more, 1.4 times or more, 1.6 times or more, 1.8 times or more, 2.0 times or more, 2.5 times or more, 3.0 times or more, 5.0 times or more, or 10 times or more of the average or median. Furthermore, for example, a glycine / alanine ratio in an Oryza plant that is greater than the glycine / alanine ratio in a control Oryza plant may mean, for example, that the glycine / alanine ratio measured in a plurality of samples taken from the Oryza plant to be diagnosed is significantly greater than the glycine / alanine ratio measured in a plurality of samples taken from one or more control Oryza plants. In this case, significantly greater may mean, for example, that the average glycine / alanine ratio is greater than the average glycine / alanine ratio in the control Oryza plants, and the p-value in a Dunnett's test between the two is less than 0.05, less than 0.01, or less than 0.001.

[0039] <Metabolite labeling> In the measurement of the content and / or the parameter that is an index of the content, myo-inositol, alanine and / or glycine may be labeled as necessary, and the labeling may be either pre-column derivatization or post-column derivatization. The labeling agent used for such labeling may be one that is commonly used by those skilled in the art. For example, when the measurement is performed by GC-MS, myo-inositol, alanine and / or glycine may be labeled with, for example, N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) or N,O-bis(trimethylsilyl)acetamide (BSA). Furthermore, for example, when measuring the absorbance or fluorescence intensity of myo-inositol, alanine, and / or glycine separated by HPLC, these may be labeled with a labeling agent having an absorber and / or a fluorescent wavelength in a wavelength region detectable by a photometer, such as o-phthalaldehyde, phenylisothiocyanate, or dansyl chloride.

[0040] <Cultivation method and growth stage of rice plants to be diagnosed> The cultivation method of the rice plant to be diagnosed by the method for diagnosing sulfur deficiency according to the present disclosure is not particularly limited, and may be any of rice grown in paddy fields (paddy rice), rice grown hydroponically, and rice grown in fields (upland rice). However, from the viewpoint that sulfur deficiency is more likely to occur by continuous use of a sulfate-free fertilizer, in a preferred embodiment, rice grown in paddy fields (paddy rice) or rice grown hydroponically may be used, and in a more preferred embodiment, rice grown in paddy fields (paddy rice) may be used.

[0041] In the method for diagnosing sulfur deficiency according to the present disclosure, the growth stage of the rice plant at which the myo-inositol amount and / or the glycine / alanine ratio are measured is not particularly limited, but in one embodiment, it may be the tillering stage, for example, the effective tillering stage or a period of a predetermined number of days (1 day, 2 days, 3 days, 5 days, 7 days, 10 days, or 14 days) immediately before the maximum tillering stage. In the present disclosure, the tillering stage refers to a growth stage at which the number of stems increases. In the present disclosure, the effective tillering stage refers to a period during the tillering stage during which the number of stems increases rapidly, and more specifically, refers to a period from the start of tillering to the first time when the number of stems reaches the same number as the number of stems at the start of the panicle formation stage. The maximum tillering stage refers to a growth stage at which the number of stems per unit area is at its maximum. By diagnosing sulfur deficiency at the above-mentioned times, it is possible to understand the sulfur deficiency state of rice plants and apply sulfur materials appropriately, which can minimize the period of sulfur deficiency during the tillering stage when stem number is increasing rapidly and avoid a decrease in yield.

[0042] <Actions and Effects>

[0043] According to one embodiment of the present disclosure, sulfur deficiency in Oryza plants can be diagnosed.

[0044] According to an embodiment of the present disclosure, sulfur deficiency in an Oryza plant can be diagnosed by using a part of the plant, such as an organ, as a sample. In this way, sulfur deficiency in an Oryza plant can be easily diagnosed.

[0045] According to an embodiment of the present disclosure, sulfur deficiency in a rice plant can be diagnosed quickly and simply. Also, according to an embodiment of the present disclosure, sulfur deficiency in a rice plant can be diagnosed quickly and simply by using a part of a plant such as an organ as a sample. In this way, sulfur deficiency in a rice plant can be diagnosed while suppressing a decrease in yield caused by the time required for diagnosis (time lag).

[0046] According to an embodiment of the present disclosure, sulfur deficiency in rice plants can be diagnosed regardless of appearance. According to an embodiment of the present disclosure, sulfur deficiency and nitrogen deficiency can be distinguished in rice plants. Thus, sulfur deficiency can be diagnosed for rice plants that do not show symptoms of sulfur deficiency or in which nitrogen deficiency and sulfur deficiency cannot be distinguished from each other based on appearance.

[0047] <Other embodiments> Another embodiment of the present disclosure may be an Oryza plant obtained by cultivating an Oryza plant diagnosed by the method for diagnosing sulfur deficiency according to an embodiment of the present disclosure. EXAMPLES

[0048] The present disclosure will be described more specifically below using examples, but the present disclosure is not limited to the following examples.

[0049] Example 1: Metabolite amounts in rice grown hydroponically under sulfur-deficient conditions Over the two years of 2020 and 2021, rice was grown under non-sulfur-deficient conditions (normal nutrient solution, normal) or sulfur-deficient conditions (sulfur-deficient nutrient solution, S-deficient), and their metabolites were evaluated.

[0050] In 2020, cultivation was performed using normal culture solution (normal) or sulfur-deficient culture solution (S-deficient) shown in Table 1. These culture solutions were used by adding the missing components to Kimura's B solution, known as a culture solution used for hydroponic cultivation, and adjusting the pH to 5.0 to 5.5. In addition, in 2020, rice was cultivated using these culture solutions under four conditions: a sulfur-deficient condition area for the entire period (cultivated in sulfur-deficient culture solution for the entire period after transplanting), a sulfur-sufficient condition area (cultivated in normal culture solution for the entire period after transplanting), a sulfur-deficient condition area in the late growth period (cultivated in normal culture solution at the time of transplanting, and from about one week after the start of tillering, cultivated in sulfur-deficient culture solution), and a sulfur-deficient condition area in the early growth period (cultivated in sulfur-deficient culture solution at the time of transplanting, and from about one week after the start of tillering, cultivated in normal culture solution). Note that special grade reagents were used as reagents, and deionized water was used as the solvent. The SiO2·xH2O was prepared by passing an aqueous solution of sodium silicate through a cation exchange resin (Amberlite®, IR120B) to remove sodium ions. The chlorine concentration was 52 ppm in the normal culture medium and 65 ppm in the sulfur-deficient culture medium.

[0051] [Table 1]

[0052] In 2021, rice was grown using the four media shown in Table 2 under four sulfur concentration conditions: 0 ppm (S0 area, S-free area), 0.087 ppm (0.01 times area), 0.87 ppm (0.1 times area), and 8.7 ppm (normal area, normal area). The culture solution used under these conditions was Kimura B solution, known as a culture solution used for hydroponic cultivation, with the missing components added and the pH adjusted to 5.0 to 5.5. Special grade reagents were used as reagents, and deionized water was used as solvent. SiO2·xH2O was prepared by passing a sodium silicate aqueous solution through a cation exchange resin (Amberlite (registered trademark), IR120B) to remove sodium ions. In addition, the chlorine concentration was 45 mg / L in the normal area, 63 mg / L in the 0.1x area, 65 mg / L in the 0.01x area, and 65 mg / L in the S-free area.

[0053] [Table 2]

[0054] The rice plants obtained in this way in 2020 or 2021 were sampled by collecting the top fully expanded leaves. The top fully expanded leaves collected were freeze-dried and then crushed to obtain freeze-dried samples.

[0055] 800μL of extraction solution (methanol:chloroform:water=3:1:1) was added to 5mg of freeze-dried sample, mixed, and incubated for 10 minutes to extract water-soluble metabolites. 170μL of water was further added to the mixture, mixed, and centrifuged at 14000rpm for 5 minutes at 4℃. The supernatant was collected and further centrifuged at 14000rpm for 7 minutes at 4℃. The supernatant was collected and dried by vacuum centrifugation to obtain the extracted sample. 60μL of 20mg / mL methoxyamine hydrochloride solution was added to the extracted sample, and the sample was dissolved by incubating at 35℃ for 90 minutes. 30μL of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was added thereto, incubated at 35℃ for 30 minutes to derivatize metabolites, and then centrifuged at 14000rpm for 5 minutes at room temperature, and the supernatant was used as the GC-MS analysis sample. For the analysis, a GCMS-QP2010 Ultra (Shimadzu Corporation) was used. In the following examples, the vertical axis of the GC-MS results is expressed as peak area.

[0056] The results are shown in Figure 1. The data in Figure 1 are shown as mean ± standard error (n = 3), and *, **, and *** in the figure indicate that the p-value in the Dunnett test was less than 0.05, less than 0.01, and less than 0.001, respectively. According to Figure 1, under the conditions of sulfur deficiency for the entire period (2020) and sulfur concentration 0 ppm (2021), the contents of glutamine, serine, and glycine were significantly higher among the metabolites, and the content ratio of glycine in particular was larger than that of the group without sulfur deficiency. In addition, under the conditions of sulfur deficiency for the entire period (2020) and sulfur concentration 0 ppm (2021), the content of myo-inositol was significantly lower. On the other hand, in alanine and tyrosine, no significant changes in the amount of metabolites were observed under the conditions of sulfur deficiency for the entire period (2020) and sulfur concentration 0 ppm (2021) as observed above.

[0057] <Example 2: Sulfur and nitrogen concentrations in rice grown hydroponically under sulfur-deficient conditions> The ratio of sulfur concentration to nitrogen concentration in the leaf blade of rice is known to be one of the parameters that can be used as an indicator for diagnosing sulfur deficiency in rice plants. Therefore, the sulfur and nitrogen concentrations were measured in the leaf blade of rice plants grown under conditions that were not sulfur deficient (normal nutrient solution, normal) or sulfur deficient (sulfur deficient nutrient solution, S deficient).

[0058] A sufficient amount of nitric acid was added to the frozen and crushed sample prepared in the same manner as in Example 1, and the mixture was heated to 90°C and incubated until decomposition. The mixture was then diluted with water to a nitric acid concentration of 3%, mixed, and allowed to stand overnight or longer. The supernatant was collected, and the solution was diluted 5 times with water to measure the sulfur concentration by ICP-MS (7700x ICP-MS, manufactured by Agilent). The nitrogen concentration of the weighed frozen and crushed sample was measured using a Sumigraph (NC-22F, manufactured by Sumika Chemical Analysis Center Co., Ltd.) using a dry combustion method.

[0059] The results are shown in Figure 2. The data in Figure 2 are shown as mean ± standard error (n = 3), and * and *** in the figure indicate that the p-values ​​in the Dunnett test were less than 0.05 and 0.001, respectively. Figure 2 suggests that the sulfur and nitrogen concentrations in the leaf blades show a similar dependency on the sulfur concentration in the culture solution, and that it may not be easy to diagnose sulfur deficiency using the ratio as an indicator.

[0060] Example 3: Metabolite amounts in rice grown in a field Based on the results of Example 1, in paddy fields where sulfur deficiency is a concern, rice was grown under sulfur treatment under multiple conditions, and the amount of metabolites was measured. In field A in 2020, "Niji no Kirameki" was grown as the test variety, and sulfur was applied to the entire layer as calcium sulfate ("field calcium") at three levels: 0 kg / 10a, 40 kg / 10a, and 60 kg / 10a. In field B in 2020, "Yume Obako" was grown, and sulfur was applied to the seedling box as calcium sulfate at three levels: 0 g / box, 125 g / box, and 500 g / box. In field A in 2021, "Niji no Kirameki" was grown, and sulfur was applied to the entire layer as calcium sulfate at three levels: 0 kg / 10a, 40 kg / 10a, and 60 kg / 10a. In 2021, field C was used to grow Yumeobako, and sulfur was applied to the seedling boxes at three levels: 0g / box, 250g / box, and 500g / box. In 2022, field D was used to grow Akitakomachi, and sulfur was applied under three conditions: no treatment, full-layer application of 100kg / 10a, and box application of 500g / box. In 2023, field D was used to grow Miyamanishiki, and sulfur was applied to the seedling boxes at three levels: 40kg / 10a or 100kg / 10a as calcium sulfate, or 250g / box.

[0061] The rice plants thus obtained were sampled by collecting the top fully expanded leaves. The top fully expanded leaves were freeze-dried and then crushed to obtain freeze-dried samples. The amounts of metabolites in the freeze-dried samples were analyzed in the same manner as in Example 1.

[0062] The results for 2020, 2021 and 2022 are shown in Figure 3. The results for 2023 are shown in Figure 4. The data in Figures 3 and 4 are shown as mean ± standard error (n = 3), and *, ** and *** in the figures indicate that the p-value in the Dunnett test was less than 0.05, less than 0.01 and less than 0.001, respectively. According to Figures 3 and 4, for glycine, which is one of the metabolites, in all fields where the development of sulfur deficiency was a concern, the amount of glycine tended to increase when the amount of sulfur in the soil was low. On the other hand, for alanine, there was a tendency for the amount of alanine to fluctuate less depending on the amount of sulfur in the soil. Furthermore, for myo-inositol, which is one of the metabolites, in all fields, the amount of myo-inositol tended to decrease when the amount of sulfur in the soil was low. From these results, it became clear that sulfur deficiency in rice plants can be diagnosed by measuring the content of myo-inositol.

[0063] Example 4: Glycine / alanine ratio in rice grown under sulfur-deficient conditions Based on the results of Examples 1 and 3, it was examined whether sulfur deficiency in rice plants can be diagnosed using an index obtained by normalizing the glycine content with the alanine content (glycine / alanine ratio, Gly / Ala ratio).

[0064] FIG. 5 shows the results of the glycine / alanine ratio, which is the value obtained by dividing the glycine-derived peak area under each condition in the hydroponic cultivation of Example 1 by the alanine-derived peak area under each condition. The results of the glycine / alanine ratio, which is the value obtained by dividing the glycine-derived peak area under each condition in the cultivation at the on-site field in Example 3 by the alanine-derived peak area under each condition, are shown in FIG. 6 for the results in 2020, 2021, and 2022, and in FIG. 7 for the results in 2023. According to FIG. 5, FIG. 6, and FIG. 7, the glycine / alanine ratio of the rice plant cultivated under sulfur-deficient conditions was higher than that under non-sulfur-deficient conditions for hydroponic cultivation and cultivation at the on-site field. From this result, it became clear that sulfur deficiency in rice plants can be diagnosed by measuring the ratio of glycine and alanine content.

Claims

1. 1. A method for diagnosing sulfur deficiency in an Oryza plant, comprising: Measuring the content of myo-inositol in the rice plant, and / or Measuring the ratio of glycine to alanine content in the rice plant; A method comprising:

2. 2. The method of claim 1, wherein the method comprises measuring a content of myo-inositol in an Oryza plant, and the sulfur deficiency is diagnosed when the measured content of myo-inositol is less than the content of myo-inositol in a control Oryza plant.

3. 2. The method of claim 1, wherein the method comprises measuring a ratio of glycine to alanine content in an Oryza plant, and a sulfur deficiency is diagnosed when the measured ratio of glycine to alanine content is greater than the ratio of glycine to alanine content in a control Oryza plant.

4. The method according to claim 2 or 3, wherein the control Oryza plant is a rice plant that is not sulfur deficient.

5. The method according to any one of claims 1 to 3, wherein the rice plant is rice.

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