Apparatus and method for diagnosing environmental stress in plants

Abstract

The present invention relates to improved technology for a device that ascertains and diagnoses the state of environmental stress in plants.　A control circuit 20b controls a measurement light source 12 such that a second measurement light ML2 is higher powered than first measurement light ML1 and such that the first measurement light ML1 and the second measurement light ML2 have opposite phase square waves, and further controls the measurement light source 12 such that the first measurement light ML1 and the second measurement light ML2 are output synchronously, thereby forming the first measurement light ML1 and the second measurement light ML2 into a single pseudo synthesized square wave measurement light ML3 of 5-30 kHz.　A transmitted light detector 18 detects, as synthesized square wave transmitted light TL, the synthesized square wave measurement light ML3 that has passed through a plant sample S.　An analysis circuit 20a uses the synthesized square wave transmitted light TL to calculate a light absorption difference, uses the light absorption difference to calculate, as a ROS marker, Y(ND) which is the oxidation state of P700, and uses the ROS marker to diagnose the state of environmental stress of a plant.

Description

Related Applications

[0001] This application claims priority from Japanese Patent Application No. 2020-189009, 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 the generation of ROS 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 for reducing costs in plant cultivation, evaluating plant growth, and selecting stress-resistant varieties. Furthermore, early detection of environmental stress can also lead to early detection of mineral nutrient deficiencies in plants. 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 induction light; a sealed chamber that accommodates the plant sample and into which the measurement light and photosynthesis induction 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 outputs two types of measurement light, a first measurement light and a second measurement light, which have different wavelengths; The guidance light source outputs two types of photosynthesis guidance light, a first photosynthesis guidance light and a second photosynthesis guidance light, which have different wavelengths; the control unit includes an analysis circuit that analyzes the detection result obtained by the transmitted light detector, and a control circuit that controls the measurement light source and the guidance light source in accordance with the plant sample; the control circuit adjusts and controls the first and second measurement beams to have different output amplitudes and controls the measurement light source so that the first and second measurement beams become rectangular waves with opposite phases; the control circuit controls the measurement light source to synchronously output the first measurement light and the second measurement light, and forms the first measurement light and the second measurement light into a single pseudo-synthesized rectangular wave measurement light of 5 kHz to 30 kHz containing a DC component; the transmitted light detector detects the composite square-wave measurement light that has passed through the plant sample as composite square-wave transmitted light; the analysis circuit calculates a difference in light absorption between the first measurement light and the second measurement light transmitted through the plant sample using the composite rectangular wave transmitted light, and calculates Y(ND), which indicates 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, using the light absorption difference; The analysis circuit is characterized by diagnosing 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 environmental stress diagnosis device is provided with a communication unit for connecting to a network, and the environmental stress diagnosis device is connected to a communication terminal via the communication unit; the communication terminal is used to operate the environmental stress diagnostic device, and displays the ROS markers and the environmental stress diagnostic results as measurement results; The communication terminal is connected to a data server on which environmental stress diagnostic data is stored via a network, and compares the environmental stress diagnostic data with the ROS marker to diagnose the environmental stress state of the plant sample.

[0014] In addition, in the environmental stress diagnostic device according to the present invention, the control circuit synchronizes the first measurement light and the second measurement light output from the measurement light source by PWM control; the control circuit compares the timing of the falling edges of the rectangular waves of the first measurement light and the second measurement light with a reference signal waveform as a command frequency; The control circuit is characterized in that, when the timing of the falling edges of the first measurement light and the second measurement light is shifted due to the output from the measurement light source, the control circuit adjusts the timing of the falling edges in 0.25 μs units to maintain synchronization.

[0015] 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.

[0016] 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 (photosynthetic rate).

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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 the photosynthetic rate, Y(NPQ) as the light energy unavailable for photosynthesis, Y(NO) as the basic heat dissipation capacity in photosystem II, and 1-pL as the plastoquinone reduction rate from the chlorophyll fluorescence detection result (under saturated CO2 conditions) obtained by the fluorescence detector, The analysis circuit uses the optical absorption difference to calculate Y(I), which is the ground state of P700, and Y(NA), which is the state in which P700 absorbs optical energy; The analysis circuit is characterized by diagnosing a deficiency state of inorganic nutrients in the plant sample using all or any of Y(II), Y(NPQ), Y(NO), 1-pL, Y(I), Y(NA), and the ROS marker Y(ND).

[0021] In addition, in the environmental stress diagnostic device according to the present invention, the analysis circuit creates a sample diagnostic graph in which the time course is represented in a circular form and values ​​obtained by dividing Y(I), Y(ND), and Y(NA) by Y(II) are plotted; The analysis circuit is characterized by diagnosing a deficiency of all or any of the essential nutrients N, P, K, S, Mg, Ca, B, Zn, Mo, Cu, Fe, and Mn in the plant sample by comparing a basic diagnostic graph showing a plant in which inorganic nutrients have been controlled with the sample diagnostic graph.

[0022] In addition, in the environmental stress diagnostic device according to the present invention, The sealed chamber is characterized in that it is provided with a temperature control unit for controlling the temperature of the plant sample located inside the sealed chamber.

[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, and adjusting the first measurement light and the second measurement light output from the measurement light source and the first photosynthesis induction light and the second photosynthesis induction light output from the induction light source by a control circuit; controlling the measurement light source by the control circuit so that the second measurement light has a higher output than the first measurement light and the first and second measurement lights are square waves of opposite phases, and controlling the measurement light source by the control circuit so that the first and second measurement lights are output synchronously, forming the first and second measurement lights into a single pseudo-synthetic square wave measurement light of 5 kHz to 30 kHz containing a DC component, and irradiating the plant sample with the synthetic square wave measurement light together with the first photosynthesis induction light and the second photosynthesis induction light; detecting the composite square wave measurement light transmitted through the plant sample as composite square wave transmitted light of one frequency using a transmitted light detector; a step of calculating an optical absorption difference between the first measurement light and the second measurement light transmitted through the plant sample using the composite rectangular wave transmitted light by an analysis circuit, and calculating 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, by the analysis circuit using the optical absorption difference; and diagnosing the environmental stress state of the plant sample using the ROS marker. [Effects of the Invention]

[0024] According to the present invention, the control circuit controls the measurement light source to output the second measurement light slightly higher than the first measurement light and to output the first and second measurement lights as square waves with opposite phases. Furthermore, the control circuit controls the measurement light source to output the first and second measurement lights synchronously, thereby forming the first and second measurement lights into a single pseudo-synthetic square-wave measurement light of 5 kHz to 30 kHz containing a DC component. The ROS marker is calculated using the synthetic square-wave transmitted light transmitted through the plant sample, and the obtained ROS marker is used to perform environmental stress diagnosis. This provides an environmental stress diagnosis device that can diagnose environmental stress in plants more accurately and earlier than conventional chlorophyll fluorescence measurement.

[0025] As a result, the ROS markers (or the correlation between the ROS markers and the oxygen evolution rate, etc.) measured (and calculated) by the environmental stress diagnostic device according to the present invention can be used, for example, as selection markers for varieties tolerant to low-temperature stress. Furthermore, the ROS markers (or the correlation between the ROS markers and the oxygen evolution rate, as well as chlorophyll fluorescence parameters) can also be used to diagnose mineral nutrient deficiencies in plants (diagnosis of mineral nutrient stress). [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 P700 absorption change 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] 10 shows a modified example of the environmental stress diagnosis device according to the embodiment of the present invention. [Figure 8] 1 shows an example of an environmental stress correlation according to an embodiment of the present invention. [Figure 9] FIG. 1 shows an image of the correlation between ROS markers and photosynthetic rate measured in an embodiment of the present invention. [Figure 10] 1 shows a schematic image diagram of an environmental stress diagnosis using a data server in this embodiment. [Figure 11] 1 shows an example of a display screen of a mobile terminal according to the present embodiment. [Figure 12] 1A and 1B are schematic explanatory diagrams of measurement light irradiation according to an embodiment of the present invention. [Figure 13] 10A and 10B are schematic diagrams illustrating the duty ratio of a synthetic rectangular wave measurement light according to the present embodiment. [Figure 14] 1 shows a schematic image diagram of a synthetic rectangular wave transmitted light according to an embodiment of the present invention. [Figure 15] 1 is a schematic explanatory diagram of a synchronization control according to an embodiment of the present invention; [Figure 16] FIG. 1 shows a schematic diagram of the induction principle of the ROS marker (Y(ND)). [Figure 17] 1 shows an example of the relationship between the ROS marker and the oxygen generation rate in this embodiment. [Figure 18] FIG. 1 shows an image diagram of the correlation between ROS markers and oxygen generation rate in this embodiment. [Figure 19] Examples of measurements of Y(ND), Y(I), Y(NA), and V(O2) are shown below. [Figure 20] A comparative image of a plant with normal mineral nutrients (control) and a plant with a mineral nutrient deficiency (free) is shown. [Figure 21] A comparative image of a plant with normal mineral nutrients (control) and a plant with a mineral nutrient deficiency (free) is shown. [Figure 22] FIG. 1 shows a schematic configuration diagram of an environmental stress diagnostic device 10 according to this embodiment when measuring chlorophyll fluorescence. [Figure 23] A schematic diagram of each parameter in chlorophyll fluorescence detection is shown. [Figure 24] A schematic diagram of each parameter in photosystem I is shown. [Figure 25] An example of a measurement result graph is shown below, in which the passage of time is represented as a circle and Y(I), Y(ND), Y(NA), and Y(II) are plotted. [Figure 26] An example of a measurement result graph showing values ​​obtained by dividing Y(I), Y(ND), and Y(NA) by Y(II) is shown below. [Figure 27] An example of a measurement result graph is shown, in which the time course is represented as a circle and Y(I), Y(ND), Y(NA), Y(II), Y(NO), Y(NPQ), and 1-qL are plotted. [Figure 28] An example of a measurement result graph showing values ​​obtained by dividing Y(ND), Y(NA), Y(NPQ), and 1-qL by Y(II) is shown below. [Figure 29] An example of a measurement result graph is shown below, where the magnitude of each value of Y(I), Y(ND), Y(NA), Y(II), Y(NO), Y(NPQ), and 1-qL is represented as a circle. [Figure 30] 1 shows an example of a Y(ND)-Y(II) diagnostic plot according to this embodiment. [Figure 31] This shows a schematic image of the growth of sunflowers two months after sowing. [Figure 32] 10A and 10B are schematic diagrams of diagnostic images based on sample diagnostic graphs in this embodiment. [Figure 33] FIG. 1 shows a schematic configuration diagram of an environmental stress diagnosis device according to an embodiment of the present invention, in which a temperature adjustment unit is added. [Figure 34] An example of a measurement result using the temperature control unit in this embodiment will be shown. 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 intended for use primarily 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 5V to 20V power battery (for example, 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 a battery 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 diagnostic 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 photodetector 18 that detects the measurement light ML that has passed through the plant sample S as transmitted light, and a control unit 20 that receives the transmitted light detected by the photodetector 18 as a measurement signal (electrical signal).

[0033] The environmental stress diagnostic device 10 according to this embodiment measures ROS markers as indicators of reactive oxygen suppression by irradiating a plant sample S (a living plant leaf) housed in a sealed chamber 16 with characteristic measurement light.

[0034] The measurement light source 12 outputs 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 characteristic measurement light irradiation and two-wavelength light absorption difference measurement. Specifically, as shown in FIG. 1, the first measurement light ML1 and the second measurement light ML2 are combined into one measurement light ML3 (referred to as a combined rectangular wave measurement light; details will be described later) which is irradiated onto the plant sample S.

[0035] The measurement light source 12 includes, for example, two types of LEDs. In this embodiment, the wavelength of the first measurement light ML1 is 810 nm or 830 nm, and the wavelength of the second measurement light ML2 is 880 nm or 910 nm. The wavelengths of the first measurement light ML1 and the second measurement light ML2 can be changed as appropriate depending on the type of plant sample S and the measurement.

[0036] Here, in the two-wavelength light absorption difference measurement in this embodiment, the two-wavelength light absorption difference waveform can be obtained by subtracting the waveform data of the transmitted light TL obtained when the first measurement light ML1 passes through the plant sample S from the waveform data of the transmitted light TL obtained when the second measurement light ML2 passes through the plant sample S.

[0037] 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 changes significantly 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 significantly 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.

[0038] In this embodiment, a single measurement light ML3 obtained by combining ML1 and ML2 can be used to obtain a two-wavelength optical absorption difference waveform, which can be used to calculate ROS markers, etc.

[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 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 has a small 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 includes, for example, two types of LEDs. In this embodiment, the wavelength of first photosynthesis inducing light FR is 740 nm, and the wavelength of second photosynthesis inducing light AL is 640 nm. The wavelengths of first photosynthesis inducing light FR and 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 second photosynthesis inducing light AL can be changed as appropriate within the range of 400 nm to less than 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 photodetector 18 (in front of the photodetector 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. Note that irradiation with the first photosynthesis inducing light FR can also be performed, for example, at the end of the irradiation of the irradiating light. In this embodiment, the steady irradiation of both the first photosynthesis inducing light FR and the second photosynthesis inducing light AL is performed for approximately 5 to 60 seconds. Note that 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 ~2000 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 of photosystem I 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, 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, photosystem I in plant sample S is in a steady state of operation due to steady irradiation of FR. At this time, pulsed irradiation of FR causes P700 to become fully oxidized. In other words, P700 becomes fully oxidized before the photosynthetic cycle in photosystem I begins operating, 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 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 second pulse of 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 FR pulse irradiation 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 being 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. In addition, the light amount (photon flux density) of the AL pulse irradiation in this embodiment 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 contained in the sealed chamber 16. In this embodiment, fresh leaves cut into pieces of approximately 8 to 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 (ML3) 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 guidance light source 14 and the sealed chamber 16. By providing a light guide, the measurement light ML and the photosynthesis guidance light AL are irradiated along the same optical path, and uniform irradiation onto the measurement sample S (surface of S) can be achieved.

[0058] Similarly, a light guide can be provided, for example, between the sealed chamber 16 and the photodetector 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 approximately 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 approximately 20 to 30 minutes from a saturated CO2 state without depleting the CO2. In this specification, a saturated CO2 state refers to a state in which the carbon dioxide concentration is approximately 1% to 4%. In this embodiment, measurements are performed with the carbon dioxide concentration inside the sealed chamber 16 set to approximately 1% to 4%. Furthermore, although the sealed chamber 16 in this embodiment is cylindrical, it may have other shapes, such as a rectangular prism or a hemisphere.

[0060] 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.

[0061] 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 (particularly outdoor measurement). In other words, this embodiment allows for maximum photosynthetic capacity evaluation, making it possible to perform highly accurate measurements (such as ROS marker measurements and oxygen concentration measurements, which will be described later) that are easy to compare and evaluate and have high reproducibility, independent of the state of the stoma.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] The transmitted light detector 18 detects the composite square-wave measurement light ML3 (ML1 and ML2) that has passed through the plant sample S as composite square-wave transmitted light TL. The transmitted light detector 18 according to this embodiment can be, for example, a PIN photodiode.

[0066] The control unit 20 has an analysis circuit 20a that analyzes the detection results obtained by the transmitted light detector 18, 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 20a performs data analysis and environmental stress diagnosis of the plant, which are characteristic of this embodiment.

[0067] Next, a 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.

[0068] At this time, in this embodiment, the first measurement light ML1 and the second measurement light ML2 output from the measurement light source 12 are irradiated onto the plant sample S as a single pseudo measurement light (synthetic rectangular wave measurement light ML3) under characteristic control by the control circuit 20b. Furthermore, the intensities of the measurement lights ML (ML1, ML2) output from the measurement light source 12 are automatically adjusted by the control unit 20 (control circuit 20b) to match 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).

[0069] 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, this measurement in the dark can be omitted. Thereafter, the plant sample S is irradiated with the first photosynthesis-inducing light FR and the second photosynthesis-inducing light AL together with the composite rectangular wave measurement light ML3 (ML1 and ML2).

[0070] The plant sample S is irradiated with photosynthesis-inducing light PL (FR, AL) and enters a state of photosynthetic activity (photochemical reaction). The composite square-wave measurement light ML3 irradiated onto the plant sample S passes through the plant sample S, and the composite square-wave transmitted light TL that has passed through the plant sample S is detected by the transmitted light detector 18. The detected composite square-wave transmitted light TL is sent to the analysis circuit 20a of the control unit 20 as a measurement signal (electrical signal).

[0071] The analysis circuit 20a performs analysis based on the detection results. The analysis circuit 20a calculates the optical absorption difference between the first measurement light ML1 and the second measurement light ML2 transmitted through the plant sample S using the combined rectangular wave transmitted light TL (two-wavelength absorption difference measurement).

[0072] The analysis circuit 20a then uses this light absorption difference 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.The analysis circuit 20a then uses the ROS marker to diagnose environmental stress in plants.By using this ROS marker, environmental stress can be diagnosed earlier and more accurately than before.

[0073] Variations Next, a modified example of the environmental stress diagnostic device according to this embodiment will be described. Fig. 7 shows a modified example of the environmental stress diagnostic device according to this embodiment. In Fig. 7, components common to the environmental stress diagnostic device 10 shown in Fig. 1 will be described using the same reference numerals. As shown in the figure, the sealed chamber 16 of the environmental stress diagnostic device 10 according to this embodiment is provided with an oxygen concentration detector 22 that measures the oxygen generation rate (also called oxygen concentration change) of the plant sample S inside the sealed chamber 16, and an environmental sensor 24 that monitors the environmental conditions inside the sealed chamber 16.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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. In the present invention, oxygen measurements are also performed without light irradiation, and the dark respiration rate (oxygen consumption) is observed. In the analysis of this embodiment, the overall photosynthetic rate (photosynthetic activity) is determined by adding the rate of change in oxygen concentration (apparent photosynthetic rate) and the dark respiration rate when irradiated with photosynthesis-inducing light. Furthermore, when looking at photosynthetic capacity from chlorophyll fluorescence, Y(II) is used, but the value when CO2 is saturated is used because Y(II) when CO2 is not saturated does not accurately represent photosynthetic capacity. Also, when converting photosynthetic activity from the oxygen concentration change rate (photosynthetic activity) into Y(II) and evaluating it using a Y(ND)-Y(II) graph or Y(ND) / Y(II), the dark respiration rate is also included in the oxygen concentration change rate (photosynthetic activity). The advantage of converting the oxygen concentration change rate (photosynthetic activity) into Y(II) is that the dimension can be unified with Y(ND). The oxygen evolution rate V(O2) to Y(II) is calculated using the following formula: Y(II) = 4 / (α × PFD) × {V'(t) + |V' O (t)|} = 4 / (α × PFD) × {V(O2)} α: constant 0.42~0.48 PFD: Photon Flux Density: Photon Flux Density: Photosynthesis-inducing light AL [μmol / m 2 / s] V'(t): Apparent oxygen evolution rate (apparent photosynthetic rate) V'0(t): Dark respiration rate (value at steady state before light irradiation: value minus oxygen sensor consumption rate)

[0078] On the other hand, in the galvanic cell type oxygen concentration detector 22, the inside of the sealed chamber 16 can be sealed and water-soaked felt can be placed inside the chamber, so that a highly humid state can be maintained, and moisture in the plant sample S will not be lost during oxygen concentration measurement.

[0079] 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.

[0080] As described above, the environmental stress diagnosis device 10 according to this embodiment is primarily intended for use outdoors. Naturally, in outdoor field settings, the conditions of temperature, humidity, air pressure, and the like vary depending on the region and environment. When used outdoors in a different environment, the environmental stress diagnosis device 10 shown in FIG. 7 can detect the temperature, humidity, and air pressure inside the sealed chamber 16 to perform data correction (correction processing) on ​​the oxygen evolution rate of the plant sample S inside the sealed chamber 16.

[0081] 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.

[0082] [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): Pressure sensor value at measurement time t [hPa] t0: Time at calibration

[0083] In this embodiment, by using this correction coefficient γ, the oxygen concentration O2 can be calculated by the following formula.

[0084] [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

[0085] Furthermore, the oxygen generation rate V(O2) can be calculated using the following formula:

[0086] [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]

[0087] 7 uses the composite rectangular-wave transmitted light TL to calculate the optical absorption difference between the first measurement light ML1 and the second measurement light ML2 transmitted through the plant sample S (two-wavelength absorption difference measurement).Then, using this optical absorption difference, the analysis circuit 20a calculates 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. In this embodiment, the irradiation intensity of the measurement light is adjusted by constant current control of the LED light source, and the drive current can be changed linearly. This embodiment is characterized in that the waveform after passing through the sample and the photodetector becomes a single composite square wave. By setting the sample and making the photodetection signals of the two measurement lights as similar as possible, the two spectral characteristics can be made the same. By irradiating the cells with light (photosynthesis-inducing light) in this state, the difference between the two types of light absorption can be calculated with high accuracy. Specifically, in two-wavelength absorption difference measurement, a sample is first set so that the signal intensities of the two wavelengths of measurement light are equivalent. However, in reality, there is a noise width, making it difficult to achieve the same signal intensity with a DC signal. In this embodiment, a single composite square wave is generated, and lock-in amplifier processing is performed in the signal processing. This lock-in amplifier processing calculates a signal intensity difference below the noise width, making it possible to reduce the signal strength of the two-wavelength measurement light, and canceling out the light absorption when passing through the sample or optical system and the spectral sensitivity characteristics of the photodetector. In lock-in amplifier processing, frequency locking will not occur unless there is a difference between the two signals, so a detectable difference is created, but the difference between the two signals is about 1 to 2%, which is distinguishable. This difference is usually below the noise level, so it does not affect the actual measurement. In this embodiment, the intensities of the measurement lights of the two wavelengths are adjusted as follows. First, the signal intensity of one of the measurement lights (here, ML2) is measured through the sample, and the current supplied to the ML2 light source (LED) is adjusted to achieve the desired signal intensity. Next, the other measurement light (ML1) is irradiated, the signal intensity is measured through the sample, and the current supplied to the ML1 light source (LED) is adjusted by lock-in amplifier processing so that the difference in signal intensity with ML2 is approximately 1 to 2% of the desired signal intensity. These are performed automatically after the sample is placed, and both ML1 and ML2 are adjusted to the desired signal strength and the difference in strength is 2% or less.

[0088] 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.

[0089] The analysis circuit 20a corrects the oxygen generation rate obtained by the oxygen concentration detector 22 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 a variety of environmental conditions are expected.

[0090] 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.

[0091] The results of this correlation analysis are compared with environmental stress correlation data (also called an oxidative damage diagnostic manual) that is pre-stored in the analysis circuit 20a, as shown in Figure 8, for example, to enable accurate and early diagnosis of the environmental stress state of the plant (whether or not it is under environmental stress, the degree of environmental stress, etc.).

[0092] Furthermore, it is also possible to diagnose whether the stress load on the plant is increasing or decreasing based on the correlation between the measured ROS marker and photosynthetic activity (oxygen evolution rate) V(O2), as shown in Figure 9. It is also possible to calculate Y(II) from the oxygen evolution rate V(O2) and use the correlation between the ROS marker and Y(II) as the photosynthetic rate.

[0093] Furthermore, the environmental stress diagnostic device of this embodiment can also diagnose environmental stress on plants by using, for example, a data server on a network. Figure 10 shows a schematic image diagram of environmental stress diagnosis using a data server.

[0094] As shown in the figure, the environmental stress diagnosis device 10 according to this embodiment can be connected to the internet via a communication terminal such as a smartphone or tablet terminal. In this case, the environmental stress diagnosis device 10 is provided with a communication unit (not shown) for connecting to the network. The environmental stress diagnosis device 10 is connected to the communication terminal via this communication unit (wireless communication in FIG. 10). Note that the network connection can also be made via a wired connection.

[0095] 10, the environmental stress diagnosis device 10 is operated using a communication terminal. The communication terminal displays, for example, ROS markers as measurement results and environmental stress diagnosis results.

[0096] For example, as shown in the screen image of Fig. 11, in addition to the setting screen, the screen of the mobile terminal can also display a real-time measurement display, a diagnostic plot screen, etc. In addition, in this embodiment, the measurement results and environmental stress diagnostic results may be displayed using, for example, an app.

[0097] The communication terminal is connected via a network to a data server that stores, for example, environmental stress diagnostic data, etc. The data server stores past measurement data (ROS markers, other measurement results measured by the device, etc.), meteorological information, other growth information, etc. as a database (collectively referred to as environmental stress diagnostic data).

[0098] The environmental stress diagnostic device 10 (or the mobile terminal) can also diagnose the environmental stress state of the plant sample by comparing the environmental stress diagnostic data stored in the data server with the measurement results (ROS markers, etc.). It is also possible to perform classification prediction and environmental stress diagnosis using machine learning, for example.

[0099] In this way, the environmental stress diagnostic device of this embodiment can accurately and early diagnose environmental stress in a plant sample by using ROS markers regardless of the location of the plant (even outdoors). Environmental stress diagnosis in this embodiment is generally carried out according to the above-mentioned procedure.

[0100] Synthetic square wave measurement light Next, the synthetic rectangular wave measurement light will be described in detail. In this embodiment, measurement light irradiation using synthetic rectangular wave measurement light is performed to measure ROS markers with high accuracy. Fig. 12 shows a schematic explanatory diagram of measurement light irradiation according to this embodiment. Fig. 12 is a block diagram showing the operation (control) of the control circuit 20b in Fig. 1 (and Fig. 7).

[0101] The control circuit 20b performs constant current control using PWM control (ON / OFF switching control in PWM control) to form a composite rectangular wave measurement light. As shown in Fig. 12, the control circuit 20b has CH1 for controlling the first measurement light ML1 and CH2 for controlling the second measurement light ML2. CH1 and CH2 output digital signals as control commands.

[0102] The digital signals from the control circuits 20b (CH1 and CH2) are converted into predetermined current values ​​(analog signals) by the D / A converter 1 (DAC1) and the D / A converter 2 (DAC2), respectively, and the converted analog signals are input to the constant current driver 1 and the constant current driver 2.

[0103] At this time, the control circuit 20b outputs a PWM signal (on / off signal) for PWM control to the constant current driver 1 and the constant current driver 2. This PWM signal causes the constant current driver 1 and the constant current driver 2 to operate in accordance with the PWM signal. Specifically, the PWM signals are input to the constant current driver 1 and the constant current driver 2 in synchronization so that ML1 and ML2 are rectangular waves with opposite phases.

[0104] Furthermore, the control circuit 20b according to this embodiment controls the constant current driver 1 and the constant current driver 2 so that the second measurement light ML2 has a higher output power than the first measurement light ML1. The control circuit 20b can also control the constant current driver 1 and the constant current driver 2 so that the first measurement light ML1 has a higher output power than the second measurement light ML2. Based on this PWM signal, the constant current driver 1 causes the measurement light source 12 to output the first measurement light ML1, and the constant current driver 2 causes the measurement light source 12 to output the second measurement light ML2.

[0105] The first measurement light ML1 and the second measurement light ML2 from the measurement light source 12 are controlled as rectangular waves with opposite phases, forming a single pseudo rectangular wave (composite rectangular wave measurement light ML3) containing a DC component, as shown in FIG. 12. The frequency of the composite rectangular wave measurement light ML3 is preferably 5 kHz to 30 kHz, and more preferably 8 kHz to 20 kHz, to perform a good optical absorption difference measurement for obtaining the ROS marker. In this embodiment, the frequency of the composite rectangular wave measurement light ML3 is controlled to 10 kHz. The control circuit 20b can adjust and control the output amplitude of the first measurement light ML1 and the second measurement light ML2.

[0106] That is, the control circuit 20b according to this embodiment controls the measurement light source 12 so that the second measurement light ML2 has a higher output power than the first measurement light ML1 and the first measurement light ML1 and the second measurement light ML2 are rectangular waves of opposite phases. Furthermore, the control circuit 20b controls the measurement light source 12 to output the first measurement light ML1 and the second measurement light ML2 in synchronization with each other, thereby forming the first measurement light ML1 and the second measurement light ML2 into a single pseudo-synthesized rectangular wave measurement light ML3 of 5 kHz to 30 kHz containing a DC component.

[0107] In this embodiment, feedback control is performed while monitoring with the transmitted light detector 18 so that the output value of the first measuring light ML1 and the output value of the second measuring light ML2 are the same output value, and the detection signal of the second measuring light ML2 (or the first measuring light ML1) transmitted through the plant sample S is controlled to within 1 to 5% or 1 to 2% of the target set detection value, thereby achieving highly accurate measurements.

[0108] The control circuit 20b controls the measurement light source 12 so that the difference in output value (output amplitude) between the first measurement light ML1 and the second measurement light ML2 is within 1% to 5%, or 1% to 2% (this value is close to the actual noise level). However, if the difference in output value becomes 0 (the same output value), the lock-in amplifier signal processing described below becomes impossible. In other words, since it is not possible to obtain a composite rectangular wave measurement light ML3 containing an AC component, the output value of the first measurement light ML1 and the output value of the second measurement light ML2 are controlled so that there is a slight difference.

[0109] Specifically, in this embodiment, when the output value (current value or voltage value) of the first measuring light ML1 is 100%, the output value of the second measuring light ML2 is about 101% to 105%, or about 101% to 102%. By adjusting the output values ​​of the first measuring light ML1 and the second measuring light ML2 within this range, accurate measurement can be achieved.

[0110] Furthermore, the PWM control (PWM signal) by the control circuit 20b may have a phase difference of 90 degrees and a duty ratio of 50% for each of ML1 and ML2 (duty ratio 5:5), as shown in FIG. 13(a), or may have a phase difference of 90 degrees and a duty ratio of 80% for ML1 and 20% for ML2 (duty ratio 8:2), as shown in FIG. 13(b). The duty ratios of ML1 and ML2 are not limited to those shown in FIG. 10 and may be set appropriately depending on the measurement. In this embodiment, the phase difference is set to 90 degrees and the duty ratios of 50% for each of ML1 and ML2, taking into account the ease of waveform shaping technology and the convenience of analysis.

[0111] In addition, the degree of overlap of the two measurement beams can be adjusted by PWM control of the fall times of the two measurement beams, so the overlap time interval can be created by setting the duty ratio to 48:48 or 47:47.

[0112] The composite square-wave measurement light ML3 obtained by the first measurement light ML1 and the second measurement light ML2 is obtained as a pseudo-square wave having a DC component and an AC component. The composite square-wave measurement light ML3 transmitted through the plant sample S is detected as composite square-wave transmitted light TL by the transmitted light detector 18 (see FIG. 1) (see FIG. 14). As shown in FIG. 14, the composite square-wave transmitted light TL contains a DC component and an AC component. The AC component of this composite square-wave transmitted light TL corresponds to the two-wavelength optical absorption difference between the first measurement light ML1 and the second measurement light ML2 transmitted through the plant sample S.

[0113] That is, in this embodiment, it is possible to detect two types of transmitted light that have passed through the plant sample S with one transmitted light detector 18. Therefore, the stress diagnosis device 10 according to this embodiment can reduce the number of components compared to conventional devices, thereby realizing miniaturization.

[0114] The transmitted light detector 18 detects the composite rectangular wave transmitted light TL as a 10 kHz rectangular wave with a slight unevenness (AC component) on top of the DC component (FIG. 14). In this embodiment, for example, AC coupling can be used to remove the DC component and detect only the AC component. Then, by amplifying this AC component with an amplifier, a sufficient dynamic range can be ensured (even small signal changes can be sufficiently expanded and measured).

[0115] In addition, in this embodiment, by using such a composite rectangular wave measurement light ML3 (composite rectangular wave transmitted light TL), a noise reduction effect can be expected compared to the conventional method of detecting the two-wavelength light absorption difference using the first measurement light ML1 and the second measurement light ML2. Furthermore, even if the baseline changes irregularly in the measurements of ML1 and ML2, the difference in minute changes in the two-wavelength absorption can be calculated with a high S / N ratio by performing lock-in amplifier signal processing using the composite rectangular wave measurement light ML3.

[0116] Here, as described above, the second measuring light ML2 is made to have a higher output than the first measuring light ML1 (specifically, the output is made slightly higher in the range of about 1% to 5%), and the first measuring light ML1 and the second measuring light ML2 are synchronously controlled as square waves of opposite phases to obtain a composite square-wave measuring light ML3. However, in reality, very high-speed control of the light source output waveform (frequency 5 kHz to 30 kHz) is performed, and the LED control currents of the first measuring light ML1 and the second measuring light ML2 are variably adjusted depending on the difference in absorption of the sample to be measured.

[0117] Therefore, the settling time varies depending on the set current (LED control current), and the first measurement light ML1 and the second measurement light ML2 may overlap (the first measurement light ML1 and the second measurement light ML2 may become out of synchronization, or the composite waveform may become distorted). In this embodiment, the settling time (fall time) is set to several tens to several hundreds of nanoseconds. In the case of such high-speed control, even a slight deviation in synchronization makes it impossible to obtain the composite rectangular wave measurement light ML3.

[0118] Therefore, the control circuit 20b according to this embodiment not only controls the formation of the composite rectangular wave measurement light ML3 but also simultaneously controls the synchronization between the first measurement light ML1 and the second measurement light ML2 (synchronization control).

[0119] 15 is a schematic diagram illustrating synchronization control according to this embodiment. As shown in the figure, the control circuit 20b monitors the rising and falling timings of the rectangular waves in the first measurement light ML1 and the second measurement light ML2 and the reference signal as a command frequency when adjusting the measurement light source at the start of measurement. The control circuit 20b then compares the falling timings of the rectangular waves in ML1 and ML2 with the waveform of the reference signal.

[0120] Here, the control circuit 20b can synchronize the rising timing of the first measurement light ML1 and the second measurement light ML2. However, the falling timing of the first measurement light ML1 and the second measurement light ML2 may be shifted due to the output (output current, etc.) of the measurement light source 12 caused by the transmittance of the sample, etc. (In reality, the falling manner of the rectangular wave differs depending on the output value.) For example, due to the difference in the output value of the first measurement light ML1 and the second measurement light ML2, the first measurement light ML1 and the second measurement light ML2 may have different falling timings.

[0121] Therefore, the control circuit 20b according to this embodiment adjusts the falling timings of the first and second measurement lights ML1 and ML2 in 0.25 μs increments (−0.25 μs to +0.25 μs) to maintain synchronization when the falling timings are shifted. Specifically, the control circuit 20b compares the falling timings of the rectangular waves of the first and second measurement lights ML1 and ML2 with a reference signal waveform as a command frequency. When the falling timings of the first and second measurement lights ML1 and ML2 are shifted due to the output from the measurement light source 12, the control circuit 20b adjusts the falling timings in 0.25 μs increments to maintain synchronization.

[0122] The control circuit 20b synchronizes the first and second measurement beams ML1 and ML2 output from the measurement light source 12 by PWM control, delaying their rising timings by a half cycle. The falling timings of the first and second measurement beams ML1 and ML2 can be adjusted in increments of 0.25 μs to compensate for the difference in falling time resulting from the difference in measurement beam intensity (the degree of overlap between the two measurement beams).

[0123] In this manner, in this embodiment, a stable composite rectangular wave measuring light can be obtained by dealing with synchronization errors that inevitably occur in the control of the measuring light, thereby achieving highly accurate environmental stress diagnosis.

[0124] 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 (or the photosynthetic rate Y(II) calculated from V(O2)) to diagnose environmental stress in plants earlier and more accurately than in the past. That is, while environmental stress in plants has conventionally been diagnosed based only on the photosynthetic activity in photosystem II, in this embodiment, diagnosis is made by analyzing the state of P700 in photosystem I in addition to photosystem II (or the oxygen evolution rate).

[0125] FIG. 16 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 supply of electrons from photosystem II (also known as PSII).

[0126] In plants, P700 (the chlorophyll that is the reaction center of photosystem I) can take three states: P700(Y(I)), which is in the reduced ground state; P700*(Y(NA)), which is in the state where it absorbs light energy; and P700+(Y(ND)), which is in the state where it releases light energy and is oxidized. The relationship is Y(I)+Y(NA)+Y(ND)=1.

[0127] 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.

[0128] 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.

[0129] Correlation between ROS markers and oxygen evolution rate Next, the correlation between the ROS marker (Y(ND)) and the oxygen generation rate (photosynthetic rate) 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.

[0130] An example of the relationship between ROS markers and oxygen production rate is shown in Figure 17. Figure 17 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 17), Field B (where plants (wheat) were grown without environmental stress; B in Figure 17), and Field C (where plants (wheat) were grown without environmental stress; C in Figure 17).

[0131] 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.

[0132] Fig. 18 shows an image of the correlation between the ROS marker and the oxygen generation rate in this embodiment. A, B, and C in Fig. 18 represent A (field A), B (field B), and C (field C) in Fig. 17. The line in the center of Fig. 18 is a discriminant line for determining whether or not the plant is experiencing environmental stress.

[0133] 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 18 shows that by utilizing the correlation between ROS markers and oxygen generation rate, it is possible to obtain highly accurate environmental stress diagnosis results.

[0134] As described above, according to the present invention, the control circuit 20b forms the composite square wave measurement light ML3 (ML2 is made higher output than ML1 and ML1 and ML2 are synchronously controlled as square waves of opposite phases), and the oxygen concentration detector 22 is used in conjunction with the transmitted light detector 18 to simultaneously measure the composite square wave transmitted light TL (the ROS marker is calculated by the analysis circuit 20a) and the oxygen generation rate of the plant sample S contained inside the sealed chamber 16. Furthermore, by using the analysis circuit 20a to perform environmental stress diagnosis utilizing the correlation between the ROS marker and the oxygen generation rate, an environmental stress diagnosis device 10 can be obtained that can diagnose the environmental stress state of plants more accurately and earlier than conventional chlorophyll fluorescence measurement.

[0135] 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.

[0136] Specifically, the plant sample is first placed in a sealed chamber, and the control circuit adjusts the first and second measurement lights output from the measurement light source, as well as the first and second photosynthetic induction lights output from the induction light source.

[0137] Then, the control circuit controls the measurement light source so that the second measurement light has a higher output than the first measurement light and the first and second measurement lights become square waves of opposite phases, and the control circuit also controls the measurement light source so that the first and second measurement lights are output synchronously, forming the first and second measurement lights into a single pseudo-synthetic square-wave measurement light of 5 kHz to 30 kHz containing a DC component, and irradiating the plant sample with the synthetic square-wave measurement light together with the first photosynthesis induction light and the second photosynthesis induction light.

[0138] Then, a step of detecting the composite square-wave measurement light transmitted through the plant sample as composite square-wave transmitted light with a transmitted light detector is performed. Then, a step of calculating the optical absorption difference between the first measurement light and the second measurement light transmitted through the plant sample using the composite square-wave transmitted light with an analysis circuit, and a step of calculating 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, using the optical absorption difference with the analysis circuit is performed. Finally, a step of diagnosing the environmental stress state of the plant using the ROS marker is performed, enabling environmental stress diagnosis to be performed earlier and more accurately than before.

[0139] Furthermore, the environmental stress diagnosis device 10 according to this embodiment can also calculate Y(I) and Y(NA) in addition to Y(ND) and the oxygen generation rate V(O2), as shown in Figure 19. By combining these other parameters, optimal analysis (environmental stress diagnosis) can be performed.

[0140] Diagnosis of mineral nutrient stress As described above, in this embodiment, environmental stress is diagnosed using measurement information (mainly ROS markers) from photosystem I and the oxygen evolution rate (or photosynthetic rate). In addition, the present inventors have discovered that inorganic nutrient stress in plants can be diagnosed by using other measurement information from photosystem II in addition to the measurement information from photosystem I.

[0141] Figures 20 and 21 show comparative images of a plant with normal mineral nutrients (control) and a plant with a mineral nutrient deficiency (free). In this specification, mineral nutrients refer to elements essential for plants, excluding C, O, and H. Figure 20 shows a comparative image of N, P, K, S, Mg, and Ca, which are essential nutrients for plants. Figure 21 shows a comparative image of B, Zn, Mo, Cu, Fe, and Mn.

[0142] Figures 20 and 21 show a comparison of sunflower growth. Specifically, sunflowers were grown for two weeks, and then the concentrations of each mineral nutrient were changed. Then, one week later, the growth of each was compared. Below, the measurement results using fresh sunflower leaves as plant sample S are shown.

[0143] As shown in Figures 20 and 21, it can be seen that a lack of inorganic nutrients has a significant impact on plant growth. In this embodiment, by detecting various parameters of photosystem I and photosystem II, it is possible to detect a deficiency of inorganic nutrients at an early stage. Diagnosis of inorganic nutrient stress in plants is expected to reduce mismatches in the timing of top dressing, for example, and as a result, ensure stable plant harvests.

[0144] As described above, the environmental stress diagnostic device 10 according to this embodiment primarily measures ROS markers and oxygen generation rate, but can also measure chlorophyll fluorescence to achieve inorganic nutrition stress diagnosis. Figure 22 shows a schematic diagram of the environmental stress diagnostic device 10 according to this embodiment when measuring chlorophyll fluorescence. In Figure 22, components common to the environmental stress diagnostic device 10 shown in Figures 1 and 7 are described using the same reference numerals.

[0145] As shown in the figure, the environmental stress diagnostic device 10 is equipped with a transmitted light detector 18, an oxygen concentration detector 22 (and an environmental sensor 24), and a fluorescence detector 40 for measuring chlorophyll fluorescence. In this embodiment, a 450 nm LED, for example, can be used as a light source to measure chlorophyll fluorescence. In this embodiment, for example, a 450 nm LED can be added to the guidance light source 14.

[0146] In this way, by measuring chlorophyll fluorescence along with ROS markers and oxygen evolution rate and analyzing the obtained chlorophyll fluorescence detection results, it becomes possible to perform more specific environmental stress diagnosis, for example, diagnosis of inorganic nutrient stress in plants.

[0147] The analysis circuit 20a in Figure 22 can calculate Y(II) as the photosynthetic rate, Y(NPQ) as the light energy that cannot be used for photosynthesis, Y(NO) as the basic heat dissipation capacity in photosystem II, and 1-pL as the plastoquinone reduction rate using the chlorophyll fluorescence detection results obtained by the fluorescence detector 40 (Figure 23).

[0148] Furthermore, as described above, the analysis circuit 20a can use the light absorption difference to calculate Y(I), which is the ground state of P700, Y(NA), which is the state in which P700 is absorbing light energy, and Y(ND), which is an ROS marker (Figure 24).

[0149] Figure 25 shows an example of a measurement result graph (also called an RFM Original Plot) in which the passage of time is represented in a circle (pie chart) and Y(I), Y(ND), Y(NA), and Y(II) are plotted. In Figure 25, one revolution of the pie chart represents the passage of 10 minutes of time. In Figure 25, even when comparing the plot trajectories (trajectory shapes), it cannot be said that the changes in trajectory shapes due to differences in deficient elements are clearly evident.

[0150] On the other hand, Figure 26 shows an example of a measurement result graph (also called an RFM Diagnosis Plot) showing the values ​​obtained by dividing Y(I), Y(ND), and Y(NA) in Figure 25 by Y(II). Compared with Figure 25, Figure 26 shows the characteristics of the trajectory shape due to differences in deficient elements.

[0151] 26, it is possible to diagnose mineral nutrient stress in plants (diagnosis of mineral nutrient deficiency) by comparing the trajectory shapes of the basic diagnostic graph obtained from a plant in which mineral nutrients have been controlled with the sample diagnostic graph obtained from plant sample S. Furthermore, the comparison of the trajectory shapes in the basic diagnostic graph with the trajectory shapes in the sample diagnostic graph can also be performed by automatically recognizing trajectory shape patterns using, for example, AI machine learning.

[0152] Similarly, Figure 27 shows an example of a measurement result graph (also called a Diagnosis Plot) in which the passage of time is represented as a circle and Y(I), Y(ND), Y(NA), Y(II), Y(NO), Y(NPQ), and 1-qL are plotted. Also, Figure 28 shows an example of a measurement result graph (also called an Original Plot) in which Y(ND), Y(NA), Y(NPQ), and 1-qL are divided by Y(II). Comparing Figure 27 and Figure 28, it is difficult to see the difference in the trajectory shapes in Figure 27, whereas it is relatively easy to see the difference in the trajectory shapes due to the deficient elements in Figure 28.

[0153] 29 shows an example of a measurement result graph (also called a radar chart) in which the magnitude of each value of Y(I), Y(ND), Y(NA), Y(II), Y(NO), Y(NPQ), and 1-qL is represented in a circular form. The measurement results in FIG. 29 can also be used to diagnose inorganic nutrient stress according to this embodiment.

[0154] In other words, when the measurement results in Figures 25 to 29 are examined, the analysis circuit 20a of this embodiment can diagnose inorganic nutrient stress in plants (diagnose inorganic nutrient deficiency) by using all or any of Y(II), Y(NPQ), Y(NO), 1-pL, Y(I), Y(NA), and the ROS marker Y(ND).

[0155] Furthermore, the analysis circuit 20a creates a sample diagnostic graph in which the passage of time is represented in a circular form and the values ​​obtained by dividing Y(I), Y(ND), and Y(NA) by Y(II) are plotted, and by comparing the sample diagnostic graph with a basic diagnostic graph showing a plant in which inorganic nutrients have been controlled, it is possible to diagnose a deficiency state of all or any of the essential nutrients in the plant: N, P, K, S, Mg, Ca, B, Zn, Mo, Cu, Fe, and Mn.

[0156] For example, in the comparison of sunflower growth shown in this example, it can be seen that there are differences in the plot positions showing the correlation between the ROS marker and Y(II) in fields A to D, as shown in Figure 30. These differences in plot positions can be distinguished (classified) as area A, area B, area C, and area D in the correlation diagram of Figure 30. In this embodiment, plant growth can also be predicted by analyzing the areas of these plot positions.

[0157] Specifically, as shown in Figure 30, field C (area C) is a healthy field with good growth (an area with low ROS marker generation), and the growth prediction based on the results of this example is C>B>A>D (poor growth in areas where photosynthetic activity is decreasing and ROS marker generation tends to increase).

[0158] Figure 31 shows a schematic image of the growth of sunflowers two months after sowing. As shown in the figure, the growth order was C>B>A>D, similar to the growth prediction in Figure 30. In this way, by using the environmental stress diagnostic device according to this embodiment, it is possible to predict the growth status of plants from the correlation between ROS markers and photosynthetic activity (Y(II) and V(O2)).

[0159] Furthermore, as described above, the deficiency state of essential nutrients in plants can be predicted or diagnosed in more detail from the plot shape of sample diagnostic graphs (e.g., Figures 26 and 28) in which the time course is represented in a circular form and the values ​​obtained by dividing Y(I), Y(ND), and Y(NA) by Y(II) are plotted.

[0160] Specifically, as shown in Figure 32, there is a clear difference in the shape of the plot when a plant is in a state of deficiency in, for example, N or Mn compared to the shape of the plot when the plant is in a controlled state. In this way, by analyzing the shape of the plot of the sample diagnostic graph using the environmental stress diagnostic device according to this embodiment, it is possible to diagnose deficiencies of essential nutrients in plants at an early stage.

[0161] Furthermore, the environmental stress diagnosis device according to this embodiment can also be provided with a temperature control unit 42 in the sealed chamber 16 as shown in Fig. 33. In this embodiment, by providing this temperature control unit 42 in the sealed chamber 16, the internal leaf temperature (temperature of the plant sample S) can be controlled.

[0162] For example, as shown in Figure 34(a), when comparing the measurement results of sunflower and wheat, if measurements are taken at the same temperature (25°C), it is difficult to capture the characteristics of wheat because the photoinduced phenomenon moves more quickly than that of sunflower.

[0163] Therefore, as shown in Figure 34(b), by controlling the temperature during wheat measurements (17°C in Figure 34(b)), the movement of the analysis parameters can be slowed down, making it easier to capture their characteristics (by forcibly slowing down the rate of the oxygen reaction, making it possible to identify and diagnose nutritional deficiencies in wheat).

[0164] In this embodiment, environmental stress diagnosis in plants is performed using measurement information from photosystem II or photosystem I, but the measurement information and analysis results obtained by the environmental stress diagnosis device 10 of this embodiment can also be used for other purposes, such as plant growth diagnosis and breeding.

[0165] Furthermore, although the environmental stress diagnostic device according to this embodiment is designed to place a plant sample inside a sealed chamber, it is also possible to achieve non-destructive, time-dependent monitoring by, for example, sandwiching a leaf between the chambers and measuring (by measuring without cutting the leaf). In addition, by using multiple environmental stress diagnostic devices in this embodiment (including mobile terminals and data servers), it is also possible to simultaneously measure multiple samples. [Explanation of symbols]

[0166] 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 42 Temperature control unit ML measurement light ML1 1st measurement light ML2 Second measuring beam ML3 Synthetic square wave measurement light PL photosynthesis-induced light FR First photosynthesis-induced light AL second photosynthesis-induced light TL synthesized rectangular wave 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 outputs two types of measurement light, a first measurement light and a second measurement light, which have different wavelengths; The guidance light source outputs two types of photosynthesis guidance light, a first photosynthesis guidance light and a second photosynthesis guidance light, which have different wavelengths; the control unit includes an analysis circuit that analyzes the detection result obtained by the transmitted light detector, and a control circuit that controls the measurement light source and the guidance light source in accordance with the plant sample; the control circuit adjusts and controls the first and second measurement beams to have different output amplitudes and controls the measurement light source so that the first and second measurement beams become rectangular waves with opposite phases; the control circuit controls the measurement light source to synchronously output the first measurement light and the second measurement light, and forms the first measurement light and the second measurement light into a single pseudo-synthetic rectangular wave measurement light of 5 kHz to 30 kHz containing a DC component; the transmitted light detector detects the composite square-wave measurement light that has passed through the plant sample as composite square-wave transmitted light; the analysis circuit calculates a difference in light absorption between the first measurement light and the second measurement light transmitted through the plant sample using the composite rectangular wave transmitted light, and calculates 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 active oxygen suppression in plants, using the light absorption difference; The environmental stress diagnostic device is characterized in that the analysis circuit diagnoses the environmental stress state of the plant sample using the ROS marker.

2. The environmental stress diagnosis device according to claim 1, The environmental stress diagnosis device is provided with a communication unit for connecting to a network, and the environmental stress diagnosis device is connected to a communication terminal via the communication unit; the communication terminal is used to operate the environmental stress diagnosis device, and displays the ROS markers as measurement results and the environmental stress diagnosis results; The communication terminal is connected to a data server on a network in which environmental stress diagnostic data is stored, and compares the environmental stress diagnostic data with the ROS marker to diagnose the environmental stress state of the plant sample.

3. The environmental stress diagnosis device according to claim 1 or 2, the control circuit synchronizes the first measurement light and the second measurement light output from the measurement light source by PWM control; the control circuit compares the timing of the falling edges of the rectangular waves of the first measurement light and the second measurement light with a reference signal waveform as a command frequency; the control circuit adjusts the timing of the falling edges of the first measurement light and the second measurement light in increments of 0.25 μs to maintain synchronization when the timing of the falling edges of the first measurement light and the second measurement light is shifted due to the output from the measurement light source.

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

5. 5. 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.

6. The environmental stress diagnosis device according to claim 5, 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.

7. The environmental stress diagnostic device according to claim 5 or 6, The environmental stress diagnostic device is characterized in that the oxygen concentration detector is a galvanic cell type oxygen concentration detector.

8. The environmental stress diagnosis device according to any one of claims 1 to 7, 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.

9. 9. 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 the photosynthetic rate, Y(NPQ) as the light energy unavailable for photosynthesis, Y(NO) as the basic heat dissipation capacity in photosystem II, and 1-pL as the plastoquinone reduction rate from the chlorophyll fluorescence detection result obtained by the fluorescence detector; The analysis circuit uses the optical absorption difference to calculate Y(I), which is the ground state of P700, and Y(NA), which is the state in which P700 absorbs optical energy; The analysis circuit diagnoses the deficiency state of inorganic nutrients in the plant sample by using all or any of Y(II), Y(NPQ), Y(NO), 1-pL, Y(I), Y(NA), and Y(ND), which is an ROS marker.

10. The environmental stress diagnosis device according to claim 9, the analysis circuit creates a sample diagnostic graph in which the time course is represented in a circular form and values ​​obtained by dividing Y(I), Y(ND), and Y(NA) by Y(II) are plotted; The analysis circuit compares a basic diagnostic graph showing a plant in which inorganic nutrients have been controlled with the sample diagnostic graph to diagnose a deficiency of all or any of the essential nutrients N, P, K, S, Mg, Ca, B, Zn, Mo, Cu, Fe, and Mn in the plant sample.

11. The environmental stress diagnostic device according to claim 9 or 10, An environmental stress diagnostic device, characterized in that the sealed chamber is provided with a temperature control unit for controlling the temperature of the plant sample located inside the sealed chamber.

12. A method for diagnosing environmental stress in plants, comprising: a step of placing a plant sample in a sealed chamber, and adjusting the first measurement light and the second measurement light output from the measurement light source and the first photosynthesis induction light and the second photosynthesis induction light output from the induction light source by a control circuit; controlling the measurement light source by the control circuit so that the second measurement light has a higher output than the first measurement light and the first and second measurement lights are rectangular waves of opposite phases, and controlling the measurement light source by the control circuit so that the first and second measurement lights are output synchronously, forming the first and second measurement lights into a single pseudo-synthetic rectangular wave measurement light of 5 kHz to 30 kHz containing a DC component, and irradiating the plant sample with the synthetic rectangular wave measurement light together with the first photosynthesis induction light and the second photosynthesis induction light; detecting the composite square wave measurement light transmitted through the plant sample as composite square wave transmitted light of one frequency using a transmitted light detector; calculating an optical absorption difference between the first measurement light and the second measurement light transmitted through the plant sample using the composite rectangular wave transmitted light by an analysis circuit, and calculating 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 active oxygen suppression in plants, by the analysis circuit using the optical absorption difference; and diagnosing an environmental stress state of the plant using the ROS marker.

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