Apparatus and method for diagnosing environmental stress in plants

Abstract

The present invention relates to technology for improving a device for grasping and diagnosing an environmental stress state in plants.　This device for diagnosing environmental stress comprises a measurement light source 12, an inducing light source 14, and a transmitted light detector 18. The measurement light source 12 irradiates a plant sample S with first measurement light ML1 and second measurement light ML2. The inducing light source 14 irradiates the plant sample S with first photosynthesis-inducing light FR and second photosynthesis-inducing light AL. The transmitted light detector 18 detects first transmitted light TL1 and second transmitted light TL2.　A control unit 20 includes an analysis circuit 20a and a control circuit 20b, and the analysis circuit 20a calculates the light absorption difference between the first transmitted light TL1 and the second transmitted light TL2, then uses the light absorption difference in the calculation of an ROS marker (a reactive oxygen suppression index) for a plant serving as a value Y(ND) that is a state in which the P700 of an optical system I is oxidized in photosynthesis.　The analysis circuit 20a diagnoses the environmental stress state of the plant using the ROS marker.

Description

Related Applications

[0001] This application claims priority from Japanese Patent Application No. 2020-189008, filed November 12, 2020, which is incorporated herein by reference. [Technical Field]

[0002] The present invention relates to an improved technology for an apparatus for measuring photosynthetic activity, and in particular to an apparatus (environmental stress diagnostic apparatus) for grasping and diagnosing the environmental stress state in plants by measuring photosynthetic activity. [Background technology]

[0003] Chlorophyll fluorescence measurement has traditionally been used as a means of determining the photosynthetic activity of plants. This technique primarily detects the activity of photosystem II, the initial stage of photosynthesis. Chlorophyll fluorescence measurement monitors the small amount of light energy (chlorophyll fluorescence) emitted by chlorophyll, making it possible to quantitatively determine how many electrons are produced from water molecules in the photochemical reaction.

[0004] Plants are exposed to various environmental stresses on a daily basis, which inhibit photosynthesis and cause damage to plant growth by generating excess light energy, which in turn generates reactive oxygen species (ROS).

[0005] Specifically, the accumulation of ROS within plant cells causes the production of reactive nitrogen, lipid peroxides, and reactive carbonyls, which can lead to the damage of cellular functions and even death. For example, cucumber, a model plant of the Cucurbitaceae family and known as a cold-sensitive crop, suffers growth impairment due to ROS under cold stress. Therefore, temperature control is a significant cost, especially in greenhouse cultivation during the winter.

[0006] In other words, if ROS generation in plants, and ultimately environmental stress in plants, could be detected early, appropriate temperature control would be possible in the example of greenhouse cultivation of cucumbers mentioned above. Furthermore, early detection of environmental stress can be useful not only for reducing costs in plant cultivation and evaluating plant growth, but also for selecting stress-tolerant varieties. From this perspective, research into the early diagnosis of environmental stress in plants has been underway in recent years.

[0007] For example, Patent Document 1 discloses a technology related to a plant health diagnosis device that determines the smallest minimum point s (defined as S) that occurs after the maximum point p at which chlorophyll fluorescence intensity is at its maximum in a time-dependent change curve of chlorophyll fluorescence intensity, and the first maximum point m (defined as M) that occurs after the minimum point s, and compares the values ​​of S and M to diagnose the health of a plant. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 5881082 Summary of the Invention [Problem to be solved by the invention]

[0009] In the above-mentioned Patent Document 1, chlorophyll fluorescence measurement is used to perform analysis under predetermined conditions, enabling early detection of the health condition of plants. However, as mentioned above, chlorophyll fluorescence measurement is a technique that mainly detects the activity of photosystem II, which is the initial stage of photosynthesis. In other words, chlorophyll fluorescence measurement alone does not provide information on how electrons from photochemical reactions are used downstream of electron transport (photosystem I).

[0010] In other words, even if electrons are transferring oxygen to generate reactive oxygen species (ROS) in photosystem I, measuring chlorophyll fluorescence alone may result in this being detected as active photosystem II activity, and there is still room for improvement in order to accurately diagnose environmental stress.

[0011] The present invention has been made in consideration of the problems with the prior art described above, and its purpose is to realize an environmental stress diagnosis device and method for plants that can diagnose the environmental stress state of plants more accurately, earlier, and non-destructively than conventional methods, and that can be used outdoors. [Means for solving the problem]

[0012] In order to solve the above problems, the environmental stress diagnostic device according to the present invention comprises: An environmental stress diagnostic device for diagnosing an environmental stress state of a plant sample, comprising: a measurement light source that irradiates a plant sample with measurement light; a guidance light source that irradiates the plant sample with photosynthesis-inducing light; a sealed chamber that accommodates the plant sample and into which the measurement light and photosynthesis-inducing light can enter; a transmitted light detector that detects the measurement light that has passed through the plant sample as transmitted light; and a control unit that receives the transmitted light detected by the transmitted light detector as a measurement signal, the measurement light source irradiates the plant sample with two types of measurement light, a first measurement light and a second measurement light, which have different wavelengths; the induction light source irradiates the plant sample with two types of photosynthesis induction light, namely, first and second photosynthesis induction light, each having a different wavelength; the transmitted light detector detects the transmitted light of the first measurement light as first transmitted light and detects the transmitted light of the second measurement light as second transmitted light; the control unit includes a control circuit that controls the measurement light source and the guidance light source in accordance with the plant sample, and an analysis circuit that analyzes the detection result obtained by the transmitted light detector; the analysis circuit calculates a difference in light absorption between the first transmitted light and the second transmitted light, and calculates Y(ND), which is a state in which P700 of photosystem I is oxidized during photosynthesis, as an ROS marker that is an index of reactive oxygen suppression in plants, using the difference in light absorption; Furthermore, the analysis circuit is characterized in that it diagnoses the environmental stress state of the plant sample using the ROS marker.

[0013] In addition, in the environmental stress diagnostic device according to the present invention, the sealed chamber is provided with an oxygen concentration detector that measures the oxygen generation rate of the plant sample inside the sealed chamber; The analysis circuit is characterized by diagnosing the environmental stress state of the plant sample by utilizing the correlation between the ROS marker and the oxygen generation rate.

[0014] In addition, in the environmental stress diagnostic device according to the present invention, the analysis circuit creates a correlation analysis graph in which the correlation between the oxygen generation rate and the ROS marker in the plant sample is plotted with the oxygen generation rate on the horizontal axis and the ROS marker on the vertical axis; In the correlation analysis graph, when the region where plant samples not subjected to environmental stress are plotted is taken as the reference region in the correlation analysis graph, plant samples that are subjected to environmental stress and that show a normal defense response when subjected to the environmental stress are plotted in the defense response region, which is a region where the oxygen generation rate value is lower and the ROS marker value is higher than the reference region, and plant samples that are subjected to environmental stress and that do not show a normal defense response when subjected to the environmental stress are plotted in the damage region, which is a region where the oxygen generation rate value is lower and the ROS marker value is lower than the reference region, The analysis circuit is characterized in that it determines whether the plot position of the plant sample in the correlation analysis graph belongs to the reference region, defense response region, or damage region, and diagnoses the environmental stress state of the plant sample.

[0015] In addition, in the environmental stress diagnostic device according to the present invention, The sealed chamber is provided with all or any of a temperature sensor, a humidity sensor, and an air pressure sensor as an environmental sensor; The analysis circuit corrects the oxygen generation rate detected by the oxygen concentration detector based on the detection result obtained by the environment sensor.

[0016] In addition, in the environmental stress diagnostic device according to the present invention, The oxygen concentration detector is a galvanic cell type oxygen concentration detector.

[0017] In addition, in the environmental stress diagnostic device according to the present invention, The environmental stress diagnostic device is provided with a fluorescence detector that detects chlorophyll fluorescence from the plant sample, the analysis circuit calculates Y(II) as a photosynthetic rate from the chlorophyll fluorescence detection result obtained by the fluorescence detector; The analysis circuit is characterized in that it diagnoses the environmental stress state of the plant sample by utilizing the correlation between the ROS marker and the Y(II).

[0018] In addition, in the environmental stress diagnostic device according to the present invention, the correlation between the ROS marker and the Y(II) is an inverse correlation in which a decrease in the Y(II) increases the ROS marker; The method is characterized in that the inverse correlation is utilized to diagnose drought stress or salt stress in plants.

[0019] In addition, in the environmental stress diagnostic device according to the present invention, the analysis circuit creates a correlation analysis graph in which the correlation between Y(II) and the ROS marker in the plant sample is plotted with the Y(II) on the horizontal axis and the ROS marker on the vertical axis; In the correlation analysis graph, when the region where plant samples not subjected to environmental stress are plotted is taken as the reference region in the correlation analysis graph, plant samples that are subjected to environmental stress and that show a normal defense response when subjected to the environmental stress are plotted in the defense response region, which is a region where the Y(II) value is lower and the ROS marker value is higher than the reference region, and plant samples that are subjected to environmental stress and that do not show a normal defense response when subjected to the environmental stress are plotted in the damage region, which is a region where the Y(II) value is lower and the ROS marker value is lower than the reference region, The analysis circuit is characterized in that it determines whether the plot position of the plant sample in the correlation analysis graph belongs to the reference region, defense response region, or damage region, and diagnoses the environmental stress state of the plant sample.

[0020] In addition, in the environmental stress diagnostic device according to the present invention, the guidance light source steadily irradiates the first photosynthesis induction light as continuous irradiation, and after the steady-state irradiation, pulses it as irradiation with a higher output than the steady-state irradiation without a rest period, and then steadily irradiates the second photosynthesis induction light with a rest period, and after the steady-state irradiation, pulses it without a rest period; The pulse irradiation is characterized in that the irradiation time is 1 ms to 300 ms.

[0021] In addition, in the environmental stress diagnostic device according to the present invention, The sealed chamber is characterized by being provided with an exhaled air introduction port for introducing exhaled air from the outside and an air output port for replacing the air inside the sealed chamber.

[0022] In addition, in the environmental stress diagnostic device according to the present invention, The environmental stress diagnosis device is characterized in that it is operated using a communication terminal and the results of the environmental stress diagnosis are displayed on the communication terminal.

[0023] The method for diagnosing environmental stress in plants according to the present invention comprises: a step of placing a plant sample in a sealed chamber, irradiating the plant sample with first and second measurement lights from a measurement light source, and irradiating the plant sample with first and second photosynthetic induction lights from a stimulation light source; detecting the first measurement light and the second measurement light transmitted through the plant sample as first transmitted light and second transmitted light, respectively, with a transmitted light detector; calculating a difference in light absorption between the first transmitted light and the second transmitted light using an analysis circuit, and using the difference in light absorption to calculate Y(ND), which indicates the state in which P700 of photosystem I is oxidized during photosynthesis, as an ROS marker, which is an index of reactive oxygen suppression in plants; and diagnosing the environmental stress state of the plant using the ROS marker. [Effects of the Invention]

[0024] According to the present invention, an environmental stress diagnostic device can be provided that can diagnose the environmental stress state of plants more accurately and earlier than conventional chlorophyll fluorescence measurements, by detecting transmitted light through a plant sample with a transmitted light detector and calculating ROS markers with an analysis circuit, and then performing environmental stress diagnosis using these ROS markers.

[0025] As a result, the ROS markers (and the correlation between the ROS markers and the oxygen generation rate) measured (and calculated) by the environmental stress diagnostic device according to the present invention can also be used as selection markers for varieties tolerant to low-temperature stress, for example. [Brief explanation of the drawings]

[0026] [Figure 1] 1 shows a schematic configuration diagram of an environmental stress diagnosis device according to an embodiment of the present invention. [Figure 2] 1 shows a schematic image diagram of an environmental stress diagnosis device according to an embodiment of the present invention. [Figure 3] 1A and 1B are diagrams illustrating a schematic operation of a guidance light source according to an embodiment of the present invention. [Figure 4]FIG. 1 shows a schematic image of the change in P700 absorbance obtained by irradiation with photosynthesis-inducing light in this embodiment. [Figure 5] 1A and 1B are schematic explanatory diagrams of an exhalation intake port and an air output port according to an embodiment of the present invention. [Figure 6] 1A and 1B are schematic diagrams illustrating an exhalation intake port and an air output port according to an embodiment of the present invention. [Figure 7] 1 shows an example of an environmental stress correlation according to an embodiment of the present invention. [Figure 8] FIG. 1 shows a schematic diagram of the induction principle of the ROS marker (Y(ND)). [Figure 9] 1 shows an example of the relationship between the ROS marker and the oxygen generation rate in this embodiment. [Figure 10] FIG. 1 shows an image diagram of the correlation between ROS markers and oxygen generation rate in this embodiment. [Figure 11] A comparative image (correlation analysis graph) of cold-tolerant and cold-sensitive varieties in terms of the correlation between ROS markers and oxygen generation rate is shown. [Figure 12] A comparative example of the varietal differences between cold-tolerant and cold-sensitive varieties is shown. [Figure 13] FIG. 10 is a schematic diagram showing the configuration of an environmental stress diagnosis device according to a modified example of the present invention. [Figure 14] This shows the change in soil moisture content over time during the drought stress experiment. [Figure 15] This shows the state of the plant (tomato) after drying treatment. [Figure 16] The relationship between soil moisture content and Y(ND) and Y(II) is shown. [Figure 17] The relationship between the number of days elapsed and Y(ND) and Y(II) is shown. [Figure 18] The correlation between Y(ND) and Y(II) in the drought stress experiment is shown. [Figure 19] This shows the state of plants (tomatoes) over the number of days that have passed in a salt stress experiment. [Figure 20] The relationship between the number of days elapsed at each salt concentration and Y(ND) and Y(II) is shown. [Figure 21] The correlation between Y(ND) and Y(II) in pots with controlled NaCl concentrations and pots treated with NaCl is shown. [Figure 22] The graph shows the change in Y(ND) / Y(II) over time as the number of days elapsed after salt stress treatment increases. [Figure 23] This shows an image (correlation analysis graph) comparing cold-tolerant and cold-sensitive varieties in the correlation between ROS markers and photosynthetic rate. [Figure 24] Examples of measurements of Y(ND), Y(I), Y(NA), and V(O2) are shown below. DETAILED DESCRIPTION OF THE INVENTION

[0027] The environmental stress diagnostic device of the present invention will be described below with reference to the drawings, but the present invention is not limited to the following examples as long as it does not depart from the spirit of the present invention.

[0028] FIG. 1 shows a schematic diagram of an environmental stress diagnosis device according to an embodiment of the present invention. The environmental stress diagnosis device 10 according to this embodiment is primarily for use outdoors. That is, the environmental stress diagnosis device 10 is a portable device that can be taken to a field environment and is brought to the site to directly measure plants (living leaves) and diagnose environmental stress. The environmental stress diagnosis device 10 according to this embodiment can be powered by a 12V power battery.

[0029] In this embodiment, for example, an operation display unit is provided in a part of the housing of the environmental stress diagnostic device 10, and the operation display unit can be used to operate the device and display diagnostic results (see FIG. 2(a)).

[0030] Furthermore, the environmental stress diagnosis device 10 may not be provided with an operation display unit (see FIG. 2(b)), and the device may be operated and the diagnosis results displayed using a communication terminal (mobile terminal) such as a smartphone or tablet. In this case, communication between the communication terminal and the environmental stress diagnosis device 10 can be performed wirelessly (in this case, the environmental stress diagnosis device is provided with a communication unit).

[0031] Since the environmental stress diagnosis device 10 according to this embodiment is a portable device, it actually requires batteries and other electrical components, but in Figure 1, components other than the main components required for environmental stress diagnosis are not shown.

[0032] The environmental stress diagnosis device 10 shown in Figure 1 is composed of a measurement light source 12 that irradiates a plant sample S with measurement light ML, an induction light source 14 that irradiates the plant sample S with photosynthesis-inducing light PL, a sealed chamber 16 that contains the plant sample S and into which the measurement light ML and photosynthesis-inducing light PL can enter, a transmitted light detector 18 that detects the measurement light ML (ML1, ML2) that has passed through the plant sample S as transmitted light TL (TL1, TL2), and a control unit 20 that receives the transmitted light TL detected by the transmitted light detector 18 as a measurement signal (electrical signal).

[0033] Furthermore, the sealed chamber 16 is provided with an oxygen concentration detector 22 that measures the oxygen generation rate (also called the change in oxygen concentration) inside the sealed chamber 16, and an environmental sensor 24 that grasps the environmental conditions inside the sealed chamber 16.

[0034] The environmental stress diagnostic device 10 of this embodiment can simultaneously measure ROS markers as an index of reactive oxygen inhibition and oxygen generation rate as photosynthetic activity on a plant sample S (living plant leaves) contained in a sealed chamber 16.

[0035] The measurement light source 12 irradiates the plant sample S with two types of measurement light, a first measurement light ML1 and a second measurement light ML2, which have different wavelengths. In this embodiment, the first measurement light ML1 and the second measurement light ML2 are used to perform two-wavelength light absorption difference measurement. The measurement light source 12 is configured to include, for example, two types of LEDs.

[0036] That is, as will be described in detail later, the P700 to be measured exists in three states: P700(Y(ND)) in the ground state, P700(Y(NA)) in the excited state, and P700+(Y(ND)) in the oxidized state. Therefore, to estimate the amount of Y(ND), a wavelength region that varies depending on Y(ND) is selected as the first measuring light ML1, and a wavelength region that is common to the three states and does not depend on Y(ND) is selected as the second measuring light ML2, thereby obtaining the difference between the first measuring light ML1 and the second measuring light ML2.

[0037] In this embodiment, the wavelength of the first measuring light ML1 is 810 nm or 830 nm, and the wavelength of the second measuring light ML2 is 880 nm or 910 nm. The wavelengths of the first measuring light ML1 and the second measuring light ML2 can be changed as appropriate depending on the type of plant sample S and the measurement.

[0038] In the two-wavelength light absorption difference measurement in this embodiment, a two-wavelength light absorption difference waveform can be obtained by subtracting waveform data of the first transmitted light TL1 obtained when the first measuring light ML1 passes through the plant sample S from waveform data of the second transmitted light TL2 obtained when the second measuring light ML2 passes through the plant sample S. In this embodiment, the ROS marker can be calculated using this two-wavelength light absorption difference waveform.

[0039] Furthermore, when the plant sample S becomes photosynthetically active, various parameters such as ROS markers change over time. At this time, when measuring light absorption (detecting light transmitted through the plant sample S), the background of the plant sample S also changes over time.

[0040] Therefore, in this embodiment, the background in the measurement can also be corrected by performing the two-wavelength light absorption difference measurement. That is, the two-wavelength absorption difference measurement in this embodiment cancels the change in the background over time, thereby realizing accurate measurement.

[0041] The second measuring light ML2 in this embodiment has a wavelength of 880 nm or 910 nm, which causes almost no absorption change and is close to the wavelength of the first measuring light ML1 (810 nm or 830 nm).

[0042] Guidance light source 14 irradiates plant sample S with two different wavelengths: first photosynthesis inducing light FR and second photosynthesis inducing light AL. Guidance light source 14 is configured, for example, with two types of LEDs. In this embodiment, the wavelength of the first photosynthesis inducing light FR is 740 nm, and the wavelength of the second photosynthesis inducing light AL is 640 nm. The wavelengths of the first photosynthesis inducing light FR and the second photosynthesis inducing light AL can be changed as appropriate depending on the type of plant sample S and the measurement. For example, the wavelength of the second photosynthesis inducing light AL can be changed as appropriate within the range of 400 nm to 700 nm.

[0043] In this embodiment, a filter can be provided between the LED for outputting the first photosynthesis induction light FR and the sealed chamber 16. By providing a filter, interference between the measurement light ML and the first photosynthesis induction light FR can be suppressed. Furthermore, in this embodiment, an optical filter that blocks the first photosynthesis induction light FR and AL can be provided between the sealed chamber 16 and the transmitted light detector 18 (in front of the transmitted light detector 18).

[0044] The first photosynthesis induction light FR and the second photosynthesis induction light AL are irradiated onto the plant sample S by combining continuous irradiation (called steady-state irradiation) and pulsed irradiation (called pulse irradiation, SP part in Figure 3) which is higher power irradiation than the steady-state irradiation, as shown in Figure 3.

[0045] Specifically, the guidance light source 14 steadily irradiates the first photosynthesis induction light FR as continuous irradiation, and after the steady irradiation, it pulses it as irradiation with a higher output than the steady irradiation without a rest period, and then steadily irradiates the second photosynthesis induction light AL with a rest period, and after the steady irradiation, it pulses it without a rest period.

[0046] Thereafter, only the second photosynthesis inducing light AL is irradiated onto the plant sample S, as shown in Figure 3. Irradiation with the first photosynthesis inducing light FR can also be performed, for example, at the end of the actual measurement. In this embodiment, steady irradiation is performed for approximately 5 to 60 seconds with both the first photosynthesis inducing light FR and the second photosynthesis inducing light AL. The steady irradiation time may be longer than 60 seconds depending on the type of plant sample S being measured.

[0047] In this embodiment, the first photosynthesis inducing light FR may be irradiated (steady irradiation and pulse irradiation) two or more times. By irradiating the first photosynthesis inducing light FR two or more times, the idling state of the plant sample S, which will be described later, can be more stably obtained.

[0048] In this embodiment, the light amount (photon flux density) of the first photosynthesis-inducing light FR by steady irradiation is 30 μmol m -2 s -1 ~70 μmol m -2 s -1 The light intensity (photon flux density) of the second photosynthesis-inducing light AL is approximately 100 μmol m -2 s -1 ~22000 μmol m -2 s -1 That's about it.

[0049] Figure 4 shows a schematic image of the change in P700 absorbance obtained by irradiation with photosynthesis-inducing light in this embodiment. As shown in Figure 4, steady irradiation with the first photosynthesis-inducing light FR is performed to put photosystem I in the plant sample S into a steady operating state (idling state) (since the inside of the sealed chamber 16 is a dark place, this triggers the photosynthetic cycle in the plant sample S). Pulse irradiation with the first photosynthesis-inducing light FR is performed to grasp the total amount of P700 in photosystem I (the total amount of Y(I), Y(NA), and Y(ND), which represent each state of P700, as described below).

[0050] In this embodiment, by irradiating the plant sample S with pulses of the first photosynthesis-inducing light FR (740 nm), it is possible to activate only photosystem I without activating photosystem II (or to activate photosystem I faster than photosystem II).

[0051] Specifically, steady-state irradiation of FR causes photosystem I in plant sample S to enter a steady-state operating state. At this time, pulsed irradiation of FR causes P700 to enter a fully oxidized state. In other words, P700 enters a fully oxidized state before the photosynthetic cycle in photosystem I begins to operate, and by measuring this state, the total amount of P700 in photosystem I can be determined.

[0052] In addition, if the wavelength of the first photosynthesis-inducing light FR is 700 nm or longer, only photosystem I can be activated. In this manner, in this embodiment, the total amount of P700 can be appropriately determined by activating only photosystem I with pulsed irradiation of FR.

[0053] Steady irradiation of the second photosynthesis-inducing light AL (reaction light irradiation in Figure 4) is performed to idle the photosynthetic cycle (a photosynthetic cycle that operates both photosystem II and photosystem I) in the plant sample S and to grasp Y(ND), the state in which P700 is oxidized. In other words, the second photosynthesis-inducing light AL plays the role of pseudo-sunlight irradiation.

[0054] The pulsed irradiation of the second photosynthesis-inducing light AL is used to measure the reduced, ground state P700(Y(I)). Furthermore, by calculating the difference between the total amount of P700 measured by pulsed irradiation of FR and Y(I) + Y(ND) measured by AL irradiation, it is possible to measure P700*(Y(NA)), which is the state in which light energy is absorbed.

[0055] The irradiation time of the pulse irradiation SP is preferably 1 ms to 300 ms, more preferably 50 ms to 250 ms, and even more preferably 200 ms. For example, the light amount (photon flux density) of the pulse irradiation is 5000 μmol m -2 s -1 ~15,000 μmol m -2 s -1 The environmental stress diagnostic device 10 according to this embodiment can accurately measure the oxidation state of P700 in photosystem I (ROS marker) by using this characteristic stimulating light irradiation technique.

[0056] A plant sample S to be measured is accommodated in the sealed chamber 16. In this embodiment, a living leaf cut into pieces of approximately 16 mm square can be used as the plant sample S (the plant sample S is measured non-destructively). Although not shown in FIG. 1, the sealed chamber 16 is provided with a light-guiding window at a position that allows the measurement light ML (ML1, ML2) from the measurement light source 12 and the photosynthetic induction light PL (FR, AL) from the induction light source 14 to enter the interior of the chamber.

[0057] In this embodiment, a light guide can also be provided between the measurement light source 12 and the stimulation light source 14 and the sealed chamber 16. By providing a light guide, the measurement light ML and the photosynthetic stimulation light AL are irradiated along the same optical path, and uniform irradiation of the measurement sample S can be achieved.

[0058] Similarly, a light guide can be provided between the sealed chamber 16 and the transmitted light detector 18. By providing a light guide at this position, the transmitted light TL can be detected efficiently. Specifically, by providing a light guide, the transmitted light detector 18 can detect 20% to 30% more transmitted light TL than conventionally.

[0059] In this embodiment, the volume of the sealed chamber 16 is preferably about 2 ml to 20 ml, more preferably 5 ml to 10 ml, and particularly preferably 8 ml. By setting the sealed chamber 16 to such a volume, measurements can be performed for about 20 to 30 minutes from a saturated CO2 state without depleting the CO2.

[0060] Here, saturated CO2 state in this specification means a state in which the carbon dioxide concentration is about 1% to 4%. In this embodiment, the carbon dioxide concentration inside the sealed chamber 16 is set to about 1% to 2% and oxygen concentration measurement (or oxygen generation rate measurement) is performed. Furthermore, although the sealed chamber 16 in this embodiment has a cylindrical shape, it may have other shapes, such as a rectangular prism or a hemisphere.

[0061] Furthermore, in this embodiment, an exhaled breath introduction port 30a for introducing a person's exhaled breath (breathing) can be provided in the sealed chamber 16 (or connected to the sealed chamber 16 from outside the device), as shown in Figures 5 and 6(a), for example.

[0062] By introducing human breath into the sealed chamber 16 through the breath introduction port 30a, a saturated CO2 state can be easily created inside the sealed chamber 16 (FIG. 6(b)). Creating this saturated CO2 state makes it possible to measure maximum photosynthetic capacity during measurement (especially outdoor measurement). In other words, this embodiment allows for maximum photosynthetic capacity evaluation, enabling highly accurate oxygen concentration measurements that are easy to compare and evaluate and have high reproducibility, independent of the state of the stoma.

[0063] In this embodiment, carbon dioxide can also be generated in the sealed chamber 16 by, for example, putting a sodium bicarbonate solution into the sealed chamber 16. For example, in this embodiment, oxygen concentration can also be measured by soaking a cloth or felt in the sodium bicarbonate solution and putting the cloth or felt into the sealed chamber 16.

[0064] On the other hand, if the breath introduction port 30a is closed without breath being introduced, the sealed chamber 16 can be kept sealed, which results in a low CO2 state, making it possible to measure the minimum photosynthetic capacity.

[0065] 5 and 6(a), an air output port 30b can also be provided in the sealed chamber 16. In this embodiment, by providing this air output port 30b, the air inside the sealed chamber 16 can be easily replaced even when the plant sample S is contained inside the sealed chamber 16. The positions at which the breath introduction port 30a and the air output port 30b are provided are not particularly limited.

[0066] The oxygen concentration detector 22 provided in the sealed chamber 16 measures the photosynthetic activity of the plant sample S inside the sealed chamber as the oxygen generation rate. The oxygen concentration detector 22 is preferably, for example, a galvanic cell type oxygen concentration detector.

[0067] In this embodiment, by using a galvanic battery type (also called oxygen electrode type) oxygen concentration detector 22, a power source for the oxygen concentration detector 22 is not required when used outdoors, and improved maintainability can also be expected.

[0068] Furthermore, because the galvanic cell-type oxygen concentration detector detects oxygen as a voltage, there is no detection limit based on concentration, and it is possible to set a higher CO2 concentration (approximately 40,000 ppm). If the CO2 concentration can be set at a high level, CO2 can be quickly supplied to the inside of the leaf (inside the plant sample S) regardless of the open / closed state of the stomata, enabling rapid measurement of steady-state photosynthetic activity.

[0069] For example, when detecting oxygen concentration in a typical gas exchange measurement, a constant flow of air must be continuously flowing into the sealed chamber 16. This means that air is constantly being sprayed onto the plant sample S. In other words, for plants that are sensitive to dryness, such as aquatic plants, continuous spraying of air during gas exchange measurement can cause moisture loss, which can damage the plant sample S.

[0070] On the other hand, the galvanic cell type oxygen concentration detector 22 can maintain the inside of the sealed chamber 16 in a sealed and highly humid state, so that the plant sample S does not lose moisture during the oxygen concentration measurement.

[0071] The environmental sensor 24 is provided to measure the environmental state (environmental information) inside the sealed chamber 16. For example, the environmental sensor 24 may be any or all of a temperature sensor, a humidity sensor, and an air pressure sensor. Furthermore, the environmental sensor 24 is not limited to a temperature sensor, a humidity sensor, and an air pressure sensor, and may be a sensor for measuring other environmental parameters.

[0072] As described above, the environmental stress diagnosis device 10 according to this embodiment is primarily intended for use outdoors. Naturally, in outdoor field locations, the conditions of temperature, humidity, air pressure, and the like will vary depending on the region and environment. In this embodiment, when used outdoors in a different environment, data correction (correction processing) of the oxygen evolution rate of the plant sample S inside the sealed chamber 16 can be performed by detecting the temperature, humidity, and air pressure inside the sealed chamber 16.

[0073] Specifically, since the oxygen concentration changes due to fluctuations in humidity, temperature, and air pressure over time, the data on the oxygen generation rate of the plant sample S inside the sealed chamber 16 can be corrected using a correction coefficient γ that corrects for this.

[0074] [Number 1] γ=P(t) / P(t0)×[{(-4×10 -7 )×T 2 (t) - (2×10 -6 )×T(t)}×H(t)+1] H(t): Humidity sensor value at measurement time t [%] T(t): Temperature sensor value at measurement time t [℃] P(t): Humidity sensor value at measurement time t [hPa] t0: Time at calibration

[0075] In this embodiment, by using this correction coefficient γ, the oxygen concentration O2 can be calculated by the following formula.

[0076] [Number 2] O2[μmol O2] = K(t0)×Vs(t)×γ(t) Vs(t): Voltage signal of the oxygen sensor at measurement time t [V] K(t0): Conversion coefficient between oxygen concentration and voltage signal during calibration

[0077] Furthermore, the oxygen generation rate V(O2) can be calculated using the following formula:

[0078] [Number 3] V(O2) [μmol O2 / m 2 s] = 10 4 / A × K(t0)× d / dt {Vs(t)×γ(t)} A: Leaf area [cm 2 ]

[0079] The transmitted light detector 18 detects the first measuring light ML1 and the second measuring light ML2 that have passed through the plant sample S as transmitted light TL. Specifically, the transmitted light of the first measuring light ML1 is detected as the first transmitted light TL1, and the transmitted light of the second measuring light ML2 is detected as the second transmitted light TL2. The transmitted light detector 18 of this embodiment can be, for example, a PIN photodiode (generally, two PIN photodiodes are required to detect TL1 and TL2, respectively). In this embodiment, TL1 and TL2 can also be detected by a single PIN photodiode.

[0080] The control unit 20 has an analysis circuit 20a that analyzes the detection results obtained by the transmitted light detector 18 and the oxygen concentration detector 22, and a control circuit 20b that controls the measurement light source 12 and the guidance light source 14 in accordance with the plant sample S. For example, a microprocessor or an FPGA can be used for the analysis circuit 20a and the control circuit 20b. The analysis circuit 20b performs data analysis and environmental stress diagnosis of the plant, which are characteristic of this embodiment.

[0081] Next, the flow of measurement (and environmental stress diagnosis) of the plant sample S by the environmental stress diagnosis device 10 according to this embodiment will be described. The first measurement light ML1 and the second measurement light ML2 output from the measurement light source 12 reach the plant sample S located inside the sealed chamber 16. At this time, the intensities of the measurement light ML (ML1, ML2) are automatically adjusted by the control unit 20 (control circuit 20b) according to the type of plant sample S so that the signal intensities are the same (the measurement light ML is automatically adjusted so that appropriate transmission measurement can be performed).

[0082] At the start of the measurement, the plant sample S is not irradiated with photosynthesis-inducing light PL (FR, AL), and the measurement is performed in the dark. Note that in this embodiment, the measurement in the dark can be omitted. Thereafter, the first photosynthesis-inducing light FR and the second photosynthesis-inducing light AL are irradiated onto the plant sample S together with the first measurement light ML1 and the second measurement light ML2.

[0083] The plant sample S is irradiated with photosynthesis-inducing light PL (FR, AL) to enter a state of photosynthetic activity (photochemical reaction). The first measurement light ML1 and second measurement light ML2 irradiated onto the plant sample S are transmitted through the plant sample S, and the first transmitted light TL1 and second transmitted light TL2 transmitted through the plant sample S are detected by the transmitted light detector 18. The detected first transmitted light TL1 and second transmitted light TL2 are sent to the analysis circuit 20a of the control unit 20 as measurement signals (electrical signals).

[0084] At this time, the oxygen concentration detector 22 detects the oxygen generation rate of the plant sample S inside the sealed chamber 16, and the obtained detection result is sent to the analysis circuit 20a. Similarly, the environmental sensor 24 detects environmental information (temperature, humidity, air pressure, etc.) inside the sealed chamber 16, and the obtained detection result is sent to the analysis circuit 20a.

[0085] The analysis circuit 20a performs analysis based on the detection results. Specifically, the analysis circuit 20a calculates the difference in light absorption between the first transmitted light TL1 and the second transmitted light TL2 (two-wavelength absorption difference measurement). Then, the analysis circuit 20a uses this difference in light absorption to calculate Y(ND), which is the oxidized state of P700 in photosystem I during photosynthesis, as an ROS marker, which is an index of reactive oxygen suppression in plants.

[0086] The oxygen generation rate obtained by the oxygen concentration detector 22 is corrected based on the environmental information obtained by the environmental sensor 24. This correction process for the oxygen generation rate is particularly effective when measuring outdoors where various environmental conditions are expected.

[0087] The analysis circuit 20a then analyzes the correlation between the ROS marker and the oxygen generation rate (also referred to as a correlation analysis result or a correlation analysis graph), and performs environmental stress diagnosis in the plant using the obtained correlation analysis result.

[0088] The results of this correlation analysis are compared with environmental stress correlation data (also called an oxidative damage diagnostic manual) stored in advance in the analysis circuit 20a as shown in Fig. 7, for example, to enable accurate and early diagnosis of the environmental stress state of the plant (whether or not the plant is experiencing environmental stress, the degree of environmental stress, etc.) The environmental stress diagnosis in this embodiment is generally carried out according to the flow outlined above.

[0089] ROS markers (reactive oxygen suppression index) Here, the ROS marker in this embodiment will be described. The environmental stress diagnostic device 10 according to this embodiment utilizes the correlation between the ROS marker and the oxygen evolution rate (photosynthetic activity) to diagnose environmental stress in plants earlier and more accurately than conventional methods. That is, while conventionally environmental stress in plants has been diagnosed based solely on the photosynthetic activity in photosystem II, this embodiment performs diagnosis by analyzing the state of P700 in photosystem I in addition to photosystem II (or the oxygen evolution rate).

[0090] Figure 8 shows a schematic diagram of the induction principle of the ROS marker (Y(ND)) according to this embodiment. When plants are exposed to environmental stress, CO2 fixation through photosynthesis is suppressed. Recent research has revealed that the generation of reactive oxygen species (ROS) can be avoided by appropriately suppressing the electron supply from photosystem II (also known as PSII).

[0091] In plants, P700 (the chlorophyll that is the reaction center of photosystem I) can take three states: P700(Y(I)), which is reduced and in the ground state; P700*(Y(NA)), which is in a state where it is absorbing light energy (excited state); and P700+(Y(ND)), which is in a state where it has released light energy and is oxidized; the relationship between these states is Y(I)+Y(NA)+Y(ND)=1.

[0092] When P700 is in the Y(I) or Y(NA) state, which has an electron, the risk of ROS generation increases. On the other hand, when P700 is in the Y(ND) state, which has no electron, ROS generation is suppressed. In other words, the abundance ratio of Y(ND) can be used to determine whether ROS generation is suppressed in plants, that is, to non-destructively and early understand the environmental stress state in plants.

[0093] Specifically, when electrons are supplied from photosystem II, P700 in photosystem I (also called PSI) is oxidized to P700+. By detecting this P700+ (Y(ND)), the risk of ROS generation due to reduced photosynthesis can be detected early. In this embodiment, this (Y(ND)) is called an ROS marker as an indicator of reactive oxygen suppression.

[0094] Correlation between ROS markers and oxygen evolution rate Next, the correlation between the ROS marker (Y(ND)) and the oxygen generation rate (photosynthetic activity) according to this embodiment will be described. As described above, the environmental stress diagnostic device 10 according to this embodiment includes the transmitted light detector 18 and the oxygen concentration detector 22, and calculates the correlation between the ROS marker and the oxygen generation rate using the analysis circuit 20a, thereby enabling environmental stress diagnostics in plants to be performed earlier and more accurately than before.

[0095] Figure 9 shows an example of the relationship between ROS markers and the oxygen evolution rate V(O2). Figure 9 shows the results of measuring and analyzing plant samples in three fields: Field A (where plants (wheat) under environmental stress were grown; A in Figure 9), Field B (where plants (wheat) were grown without environmental stress; B in Figure 9), and Field C (where plants (wheat) were grown without environmental stress; C in Figure 9).

[0096] As shown in the figure, the value of Y(ND) / V(O2) is large in field A, where plants under environmental stress are being grown. On the other hand, this value is smaller in fields B and C, where plants not under environmental stress are being grown, than in field A. In this way, by understanding the relationship between the ROS marker (Y(ND)) and the oxygen evolution rate (V(O2)), it is possible to accurately diagnose the environmental stress state of plants.

[0097] 10 shows an image of the correlation between the ROS marker and the oxygen generation rate in this embodiment. A, B, and C in FIG. 10 represent A (field A), B (field B), and C (field C) in FIG. 9. The line in the center of FIG. 10 is a discriminant line for determining whether or not the plant is experiencing environmental stress.

[0098] As shown in the figure, the measurement results for field A (where plants under environmental stress were grown) are concentrated above the discriminant line (diagnosed as being under environmental stress), while the measurement results for fields B and C are concentrated below the discriminant line (diagnosed as not being under environmental stress). In other words, Figure 10 shows that by utilizing the correlation between ROS markers and oxygen generation rate, it is possible to obtain highly accurate environmental stress diagnosis results.

[0099] Figure 11 shows a comparative image (correlation analysis graph) of cold-tolerant and cold-sensitive varieties in terms of the correlation between ROS markers and oxygen generation rate. Figure 11 shows the state of cold-tolerant and cold-sensitive varieties after cold treatment. Here, cold treatment in this specification refers to a state in which a plant is exposed to a low temperature (0°C to 10°C) for 1 to 5 hours. Figure 11 also shows the state 5 hours after this cold treatment.

[0100] As shown in Figure 11, cold-tolerant varieties have a normal defense response when subjected to environmental stress. Specifically, cold-tolerant varieties show a decrease in the oxygen production rate (V(O2)) when subjected to environmental stress, while at the same time showing an increase in the ROS marker Y(ND).

[0101] On the other hand, in cold-sensitive varieties, the oxygen evolution rate and ROS marker values ​​decrease when exposed to environmental stress. This suggests damage caused by reactive oxygen species (ROS). Furthermore, this is not limited to cold-sensitive varieties; plants that are exposed to environmental stress for long periods of time and are no longer able to mount normal defense responses will also show results similar to those of the cold-sensitive varieties in Figure 11.

[0102] In other words, in the correlation analysis graph shown in Figure 11, if the area in which plant samples that have not been subjected to environmental stress are plotted is taken as the reference area in the correlation measurement graph, plant samples that have been subjected to environmental stress and that show normal defense responses when subjected to that environmental stress (low-temperature tolerant varieties) will be plotted in an area (called the defense response area) where the oxygen generation rate value is lower than the reference area and the ROS marker value is higher.

[0103] On the other hand, plant samples that are subjected to environmental stress and do not exhibit normal defense responses even when exposed to said environmental stress (low-temperature sensitive varieties or plants that have been exposed to environmental stress for a long period of time and are no longer able to exhibit normal defense responses) are plotted in an area (called the damage area) where the oxygen generation rate values ​​are lower than the reference area and the ROS marker values ​​are low.

[0104] The analysis circuit 20a of this embodiment can diagnose the environmental stress state of the plant sample S by determining whether the plot position of the plant sample S belongs to (or is close to) the reference region, defense response region, or damage region in the correlation analysis graph.

[0105] For example, as shown in Figure 12, when plants are returned to the optimum temperature after low temperature treatment, the growth of the cold-sensitive variety (top row) is clearly delayed compared to the cold-tolerant variety (bottom row). In other words, by detecting and evaluating ROS markers under a low-temperature stress environment, it is possible to compare not only the degree of cold damage but also the differences in varietal tolerance to cold damage.

[0106] Thus, according to the present invention, the oxygen concentration detector 22 is used in conjunction with the transmitted light detector 18 to simultaneously measure the transmitted light (ROS markers are calculated by the analysis circuit 20a) and oxygen generation rate of the plant sample S contained inside the sealed chamber 16, and the analysis circuit 20a is used to perform environmental stress diagnosis using the correlation between the ROS markers and the oxygen generation rate, thereby providing an environmental stress diagnosis device 10 that can diagnose the environmental stress state of plants more accurately and earlier than conventional chlorophyll fluorescence measurement.

[0107] Furthermore, in this embodiment, an environmental stress diagnostic device for plants has been described, but by performing the same steps as in this device, for example, it is possible to diagnose environmental stress earlier and more accurately than before.

[0108] Specifically, the method first places a plant sample in a sealed chamber, irradiates the plant sample with first and second measurement lights from a measurement light source, and irradiates the plant sample with first and second photosynthesis induction lights from a guidance light source, then detects the first and second measurement lights that have passed through the plant sample as first and second transmitted lights, respectively, with a transmitted light detector, and detects the oxygen evolution rate of the plant sample in the sealed chamber with an oxygen concentration detector.

[0109] Then, an analysis circuit calculates the difference in light absorption between the first transmitted light and the second transmitted light, and the analysis circuit uses this difference in light absorption to calculate Y(ND), which indicates the oxidized state of P700 in photosystem I during photosynthesis, as an ROS marker, which is an index of reactive oxygen suppression in plants.Finally, a step is performed in which the correlation between the ROS marker and the oxygen generation rate is used to diagnose the environmental stress state of the plant, allowing for earlier and more accurate environmental stress diagnosis than before.

[0110] The environmental stress diagnostic device 10 according to this embodiment primarily measures ROS markers and oxygen generation rate, but can also measure chlorophyll fluorescence, for example. By analyzing the chlorophyll fluorescence measurement results along with the correlation between the ROS markers and oxygen generation rate, it becomes possible to perform environmental stress diagnosis with even greater accuracy. When the environmental stress diagnostic device 10 according to this embodiment is equipped with a chlorophyll fluorescence measurement function, a 450 nm LED, for example, can be used as the light source.

[0111] Variations A modified example of the environmental stress diagnostic device according to this embodiment is shown in Figure 13. In this modified example, a fluorescence detector 40 that detects chlorophyll fluorescence is provided instead of the oxygen concentration detector 22 (and the environmental sensor 24) in Figure 1, and the photosynthetic rate, Y(II), is measured by the fluorescence detector 40.

[0112] Specifically, in this modification, the analysis circuit 20a shown in FIG. 13 calculates Y(II), which represents the photosynthetic rate, from the chlorophyll fluorescence detection results obtained by the fluorescence detector 40. In this case, the measurement is performed by setting the inside of the sealed chamber 16 in a saturated CO2 state. The analysis circuit 20a then diagnoses the environmental stress state of the plant sample S using the correlation between the ROS marker and Y(II). For example, the correlation between the ROS marker and Y(II) can be used to determine the drought stress or salt stress of the plant. Below, we will explain drought stress experiments and salt stress experiments using this modification.

[0113] Figures 14 to 18 show the results of a drought stress experiment using tomatoes as plant samples. The drought stress experiment was conducted under natural light in a glass greenhouse (e.g., at temperatures above 20°C to 30°C). Six pots were prepared, each with controlled soil moisture and six pots with dry soil moisture. Soil moisture content, ROS markers, and Y(II) were measured over time, and the results were compared between the control pots and the dry pots. Soil moisture content was measured using the Delta-T Device SM150KIT (SM150T (soil moisture sensor) + HH150 (display)).

[0114] As shown in Figure 14, the soil moisture content decreased with the drying treatment, but the plants (tomatoes) looked healthy until the 12th day, when the value reached approximately 20% (Dry in Figure 15). Then, as shown in Figure 15, on the 15th day, when the soil moisture content reached approximately 10%, rapid leaf wilting was observed.

[0115] Furthermore, as shown in Figure 16, in the dry pots, as the soil moisture content decreased, the ROS marker Y(ND) increased (Figure 16(a)), while the photosynthetic rate Y(II) decreased (Figure 16(b)). In other words, the ROS marker and Y(II) are in an inverse correlation relationship, where an increase in one causes a decrease in the other. Furthermore, in this inverse correlation relationship, the ROS marker Y(ND) increases as the photosynthetic rate Y(II) decreases, so the results in Figure 16 are thought to indicate the defense response in Figure 11.

[0116] In addition, as shown in Figures 17 and 18, in this drought stress experiment, in the dry pots (Dry), there was a point (around the 8th day) where the Y(ND) value rose significantly relative to the Y(II) value. This suggests that in this drought stress experiment, some kind of change in the water physiology response occurred around this timing of the 8th day (water or drought stress can be detected at a much earlier stage than when visible symptoms appear).

[0117] 19 to 22 show the results of a salt stress experiment using tomatoes as plant samples. Similar to the drought stress experiment described above, the salt stress experiment was carried out in a glass greenhouse (e.g., at 20 to 30°C or higher) under natural light. Four pots each were prepared to receive salt stress at NaCl concentrations of 50 mM, 100 mM, and 250 mM, and these were compared.

[0118] In this salt stress experiment, there was no significant effect on the appearance of the leaves in pots treated with NaCl concentrations of 50 mM and 100 mM, but some individuals showed slightly smaller growth on the 16th day compared to those in the control pots (not shown). As shown in Figure 19, plants at a NaCl concentration of 250 mM were healthy until the 9th day, but from around the 12th day, signs such as browning of the leaves were observed, and by the 16th day, significant damage was observed.

[0119] Furthermore, Figure 20(a) shows that in pots with a NaCl concentration of 250 mM, the ROS marker Y(ND) increases from the 8th day after salt stress treatment, and Figure 20(b) shows that the photosynthetic rate Y(II) decreases from the 8th day after salt stress treatment (salt stress can be detected at a much earlier stage than when visible symptoms appear).

[0120] Figure 21 shows the relationship between Y(ND) and Y(II) in pots with a controlled NaCl concentration and those treated with NaCl. As shown in the figure, there is an inverse correlation between the Y(ND) and Y(II) values. The inverse correlation here means that as one increases, the other decreases, as mentioned above. Furthermore, the higher the salt concentration, the greater the variability in the Y(ND) / Y(II) plots depending on the number of days of treatment.

[0121] Figure 22 shows the change in Y(ND) / Y(II) over time with respect to the number of days after salt stress treatment. As shown in the figure, by calculating the ratio of Y(ND) / Y(II), it is possible to detect changes in ROS markers in response to salt stress more clearly. That is, in the environmental stress diagnosis device according to this modification, the analysis circuit 20a calculates Y(ND) / Y(II), and the presence or absence of salt stress can be diagnosed by using this value of Y(ND) / Y(II).

[0122] The correlation between the ROS marker and Y(II) measured in this modified example is similar to the correlation between the ROS marker and V(O2) shown in Fig. 11. In other words, if the region in which plant samples not subjected to environmental stress are plotted is taken as the reference region in the correlation measurement graph as shown in Fig. 23, plant samples that are subjected to environmental stress and exhibit normal defense responses when subjected to the environmental stress (low-temperature tolerant varieties) will be plotted in a region (referred to as the defense response region) where the Y(II) value is lower and the ROS marker value is higher than the reference region.

[0123] On the other hand, plant samples that are subjected to environmental stress and do not exhibit normal defense responses even when exposed to said environmental stress (low-temperature sensitive varieties or plants that have been exposed to environmental stress for a long time and are no longer able to exhibit normal defense responses) will have lower Y(II) values ​​than the reference region and will be plotted in a region (called the damage region) where the ROS marker values ​​are low.

[0124] Furthermore, in this embodiment (and its variations), environmental stress in a plant can be diagnosed using, for example, the value of the ROS marker Y(ND), without relying on the correlation with V(O2) or Y(II) as described above. Specifically, for example, when the value of the ROS marker is 0.1 to 0.3 or less, more preferably 0.1 or 0.2 or less, it can be diagnosed as not being subjected to environmental stress.

[0125] Furthermore, the environmental stress diagnostic device 10 according to this embodiment calculates the ROS marker and oxygen evolution rate (or photosynthetic rate), but it is also possible to calculate Y(I) and Y(NA) along with Y(ND) and the oxygen evolution rate V(O2), as shown in Fig. 24. By combining these other parameters, optimal analysis (environmental stress diagnosis) can be performed. [Explanation of symbols]

[0126] 10 Environmental stress diagnostic device 12 Measurement light source 14 Guidance light source 16 Sealed Chamber 18 Transmitted light detector 20 Control Unit 20a analysis circuit 20b Control circuit 22 Oxygen concentration detector 24 Environmental Sensors 30a Exhalation port 30b Air output port 40 Fluorescence Detector ML measurement light ML1 1st measurement light ML2 Second measuring beam PL photosynthesis-induced light FR First photosynthesis-induced light AL second photosynthesis-induced light TL transmits light TL1 First Transmitted Light TL2 Second Transmitted Light

Claims

1. An environmental stress diagnostic device for diagnosing an environmental stress state of a plant sample, comprising: a measurement light source that irradiates a plant sample with measurement light; a guidance light source that irradiates the plant sample with photosynthesis-inducing light; a sealed chamber that accommodates the plant sample and into which the measurement light and photosynthesis-inducing light can enter; a transmitted light detector that detects the measurement light that has passed through the plant sample as transmitted light; and a control unit that receives the transmitted light detected by the transmitted light detector as a measurement signal, the measurement light source irradiates the plant sample with two types of measurement light, a first measurement light and a second measurement light, which have different wavelengths; the induction light source irradiates the plant sample with two types of photosynthesis induction light, a first type and a second type, which have different wavelengths; the transmitted light detector detects the transmitted light of the first measurement light as first transmitted light and detects the transmitted light of the second measurement light as second transmitted light; the control unit includes a control circuit that controls the measurement light source and the guidance light source in accordance with the plant sample, and an analysis circuit that analyzes the detection result obtained by the transmitted light detector; the analysis circuit calculates a difference in light absorption between the first transmitted light and the second transmitted light, and calculates Y(ND), which is a state in which P700 of photosystem I is oxidized during photosynthesis, as an ROS marker that is an index of active oxygen suppression in plants, using the difference in light absorption; The environmental stress diagnostic device is further characterized in that the analysis circuit utilizes the ROS marker to diagnose the environmental stress state of the plant sample.

2. The environmental stress diagnosis device according to claim 1, the sealed chamber is provided with an oxygen concentration detector that measures the oxygen generation rate of the plant sample inside the sealed chamber; The environmental stress diagnostic device is characterized in that the analysis circuit diagnoses the environmental stress state of the plant sample by utilizing the correlation between the ROS marker and the oxygen generation rate.

3. The environmental stress diagnosis device according to claim 2, the analysis circuit creates a correlation analysis graph in which the correlation between the oxygen generation rate and the ROS marker in the plant sample is plotted with the oxygen generation rate on the horizontal axis and the ROS marker on the vertical axis; In the correlation analysis graph, when the region where plant samples not subjected to environmental stress are plotted is taken as the reference region in the correlation analysis graph, plant samples that are subjected to environmental stress and that show a normal defense response when subjected to the environmental stress are plotted in the defense response region, which is a region where the oxygen generation rate value is lower and the ROS marker value is higher than the reference region, and plant samples that are subjected to environmental stress and that do not show a normal defense response when subjected to the environmental stress are plotted in the damage region, which is a region where the oxygen generation rate value is lower and the ROS marker value is lower than the reference region, The analysis circuit determines whether the plot position of the plant sample in the correlation analysis graph belongs to the reference region, the defense response region, or the damage region, and diagnoses the environmental stress state of the plant sample.

4. The environmental stress diagnosis device according to claim 2 or 3, The sealed chamber is provided with all or any of a temperature sensor, a humidity sensor, and an air pressure sensor as an environmental sensor; The environmental stress diagnostic device is characterized in that the analysis circuit corrects the oxygen generation rate detected by the oxygen concentration detector based on the detection result obtained by the environmental sensor.

5. The environmental stress diagnosis device according to any one of claims 2 to 4, The environmental stress diagnostic device is characterized in that the oxygen concentration detector is a galvanic cell type oxygen concentration detector.

6. The environmental stress diagnosis device according to claim 1, The environmental stress diagnostic device is provided with a fluorescence detector that detects chlorophyll fluorescence from the plant sample, the analysis circuit calculates Y(II) as a photosynthetic rate from the chlorophyll fluorescence detection result obtained by the fluorescence detector; The environmental stress diagnostic device is characterized in that the analysis circuit diagnoses the environmental stress state of the plant sample by utilizing the correlation between the ROS marker and the Y(II).

7. The environmental stress diagnosis device according to claim 6, the correlation between the ROS marker and the Y(II) is an inverse correlation in which a decrease in the Y(II) results in an increase in the ROS marker; An environmental stress diagnostic device that utilizes the inverse correlation to diagnose drought stress or salt stress in plants.

8. The environmental stress diagnosis device according to claim 6, the analysis circuit creates a correlation analysis graph in which the correlation between Y(II) and the ROS marker in the plant sample is plotted with the Y(II) on the horizontal axis and the ROS marker on the vertical axis; In the correlation analysis graph, when the region where plant samples not subjected to environmental stress are plotted is taken as the reference region in the correlation analysis graph, plant samples that are subjected to environmental stress and that show a normal defense response when subjected to the environmental stress are plotted in the defense response region, which is a region where the Y(II) value is lower and the ROS marker value is higher than the reference region, and plant samples that are subjected to environmental stress and that do not show a normal defense response when subjected to the environmental stress are plotted in the damage region, which is a region where the Y(II) value is lower and the ROS marker value is lower than the reference region, The analysis circuit determines whether the plot position of the plant sample in the correlation analysis graph belongs to the reference region, the defense response region, or the damage region, and diagnoses the environmental stress state of the plant sample.

9. 9. The environmental stress diagnosis device according to claim 1, the guidance light source steadily irradiates the first photosynthesis induction light as continuous irradiation, and after the steady-state irradiation, pulses the first photosynthesis induction light at a higher output than the steady-state irradiation without a pause, and then steadily irradiates the second photosynthesis induction light with a pause, and after the steady-state irradiation, pulses the second photosynthesis induction light without a pause, The environmental stress diagnostic device is characterized in that the pulse irradiation time is 1 ms to 300 ms.

10. 10. The environmental stress diagnosis device according to claim 1, The environmental stress diagnostic device is characterized in that the sealed chamber is provided with an exhaled air introduction port for introducing exhaled air from the outside and an air output port for replacing the air inside the sealed chamber.

11. The environmental stress diagnosis device according to any one of claims 1 to 10, The environmental stress diagnosis device is characterized in that it is operated using a communication terminal and the results of the environmental stress diagnosis are displayed on the communication terminal.

12. A method for diagnosing environmental stress in plants, comprising: a step of placing a plant sample in a sealed chamber, irradiating the plant sample with first and second measurement lights from a measurement light source, and irradiating the plant sample with first and second photosynthesis induction lights from a stimulation light source; detecting the first measurement light and the second measurement light transmitted through the plant sample as first transmitted light and second transmitted light, respectively, with a transmitted light detector; calculating a difference in light absorption between the first transmitted light and the second transmitted light using an analysis circuit, and using the difference in light absorption to calculate Y(ND), which is a state in which P700 of photosystem I is oxidized during photosynthesis, as an ROS marker, which is an index of reactive oxygen suppression in plants; and diagnosing an environmental stress state of the plant using the ROS marker.

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