Experimental laboratory investigations of the laser-induced fluorescence spectra of watercress and lawn grass were conducted. The fluorescence spectra were excited by YAG:Nd laser emitting at 532 nm. It was established that the influence of stress caused by mechanical damage, overwatering, and soil pollution is manifested in changes of the spectra shapes. The mean values and confidence intervals for the ratio of two fluorescence maxima near 685 and 740 nm were estimated. It is presented that the fluorescence ratio could be considered a reliable characteristic of plant stress state.
Fluorescence analysis is a widely used high-sensitivity method that is applied in many scientific and technical fields. A viable application of the technique is the analysis of plant state [
Chlorophyll is the basic fluorescent component of green leaf in the red and far-red regions. The fluorescence spectrum of a green leaf at room temperature exhibits two maxima in the red band (680–690 nm) and in the far-red band (730–740 nm) [
There are wide experimental data on the fluorescence spectra of various plant species, both stressed and nonstressed, excited at wavelength ranges of 266–635 nm [
In this paper, experimental results of the analysis of fluorescence spectra variation of different samples of a plant species in both normal and stressed states are presented.
The fluorescence spectra were excited at a wavelength of 532 nm. It is common to use lasers with wavelengths at 337, 335, and 532 nm for fluorescence excitation in experimental research. The laser source used in this study was selected because of the advantages offered by the solid-state YAG:Nd laser at the wavelength of 532 nm (for remote sensing equipment development), in comparison with both the nitrogen gas laser at the wavelength of 337 nm and the solid-state YAG:Nd laser at the wavelength of 355 nm (the third harmonic of the YAG laser has lower pulse intensity than its second harmonic).
The laboratory configuration used to measure fluorescence spectra is shown in Figure
Laboratory configuration for laser-induced fluorescence experiments.
An EKSPLA NL210 solid-state YAG:Nd laser with diode pumping and frequency doubling was used as the source of fluorescence excitation. Laser light was transmitted by means of the optical system to the target plant located at a distance of 1 m from the optical system. The apparent diameter of the laser beam on the plant sample was approximately 25 mm. The laser spot has covered 15–20 plants. The fluorescent radiation of the plants was collected from the same spot size together with the reflected laser light by the optical system and directed into the optical fiber. The optical fiber was used to transmit light to the input of the polychromator. The reflected light from the laser beam was prevented from entering a polychromator by using an NF01-532U Semrock filter. Fluorescent radiation from 595 to 800 nm was detected. An M266 Solar LS polychromator was used as the spectral device and all transitions within the polychromator fully automated (i.e., the swapping of diffraction grids and optical filters and slit width selection).
The fluorescence spectrum was detected using a highly sensitive detector (Matrix-430k-ns Deltatekh) based on CCD array with an image intensifier. The image intensifier (generation II+, diameter 18 mm) has quantum efficiency 15% at the wavelength 550 nm. The image was transferred by the optical system from the image intensifier to the CCD. The image was converted into a digital array and transmitted to the computer. Special software developed with LabVIEW National Instruments was used to control the setup. The major specifications of the setup are presented in Table
Specifications of laboratory setup.
Specifications | Value |
---|---|
Laser pulse energy, mJ | 2.1 |
Laser wavelength, nm | 532 |
Laser pulse duration, ns | <7 |
Laser repetition rate, Hz | <500 |
Laser beam spread, mrad | <3 |
Spectral band of registration, nm | 595–800 |
Spectral resolution, nm | 6 |
Diameter of optical detection system, mm | 15 |
Distance to sample, m | 1 |
The experiment included equipment calibration as a preparatory step. The polychromator was calibrated by wavelength using a calibration light source based on a mercury-argon lamp (SL2 StellarNet Inc.) with a linear spectrum. The test was performed at the wavelength of 546.07 nm. Calibration of sensitivity of the registration system was performed using a light source based on halogen lamp (DH-2000-CAL Ocean Optics Inc.) with a continuous spectrum. Known spectrum of the lamp was acquired for sensitivity calculation.
The experimental research of laser-induced fluorescence spectra was performed using easy to keep fast-growing plant species, that is, salads, watercress, mustard, common borage, cucumbers, and lawn grass. The experimental measurements of fluorescence spectra of watercress (
The plants in normal state were grown in favorable condition for their development. The watercress plants have height of approximately 4 cm, and the lawn grass plants 8 cm.
By the leaf cutting of the watercress, the half of one leaf of each plant was dissected. The leaf laying was conducted using 7
The overwatering stress condition was implemented by placing the pot of the watercress sample in a watering can. The level of water in the watering can was always slightly below the level of soil in the plant pot; thus, it was not visually obvious that the root system of the plant sample was constantly in overwatered soil.
The fluorescence spectra of different samples of watercress grown under normal conditions are shown in Figure
Fluorescence spectra of different watercress samples in normal state.
As it can be seen in Figure
The fluorescence spectra of different samples of watercress stressed by leaf laying mechanical damage are shown in Figure
Fluorescence spectra of different watercress samples in stressed state caused by leaf laying.
The fluorescence spectra of watercress in a stressed state caused by leaf laying fluctuate considerably (Figure
The differences between the fluorescence spectra of plants under normal and stressed conditions are illustrated clearly by averaging the measurements of the fluorescence spectra. Figure
Averaged fluorescence spectra of watercress samples in normal and stressed conditions: (1) normal state, (2) leaf laying stress, (3) leaf cutting stress, and (4) root system damage stress.
It is clearly illustrated in Figure
The laser-induced fluorescence spectra of watercress in stressed state caused by overwatering are comparable with those presented in Figures
Fluorescence spectra of different watercress samples in stressed state caused by overwatering during 24 days.
As it is clearly illustrated in Figure
Figure
Fluorescence spectra of different watercress samples in stressed state caused by overwatering: (1) normal state, (2) 11-day overwatering, (3) 17-day overwatering, (4) 24-day overwatering.
Plot 1 in Figure
The results presented in Figures
The ratio of fluorescence intensities in the 680–690 and 730–740 nm spectral bands is widely used in experimental research to characterize the fluorescence spectrum shape. Analysis of experimental data indicated that the ratio of fluorescence intensities near 685 and 735 nm can be used to characterize plant stress state.
Histograms of distribution of fluorescence intensities ratio (
Histograms of distribution of fluorescence ratio, for watercress in a normal state and in a stressed state caused by leaf laying: (1) histogram for normal state, (2) histogram approximation for normal state, (3) histogram for stressed state, and (4) histogram approximation for stressed state.
The mean value of the fluorescence ratio is 0.81 and the standard deviation is 0.05 for the watercress plants in a normal state. The mean value of the fluorescence ratio is 0.96 and the standard deviation is 0.11 for the watercress plants in a stressed state caused by leaf laying.
Histograms of distribution of the fluorescence intensities ratio at 685 and 740 nm for watercress in normal condition in 16 days after planting and in a stressed condition after 24 days of overwatering are shown in Figure
Histograms of distribution of fluorescence ratio, for watercress in a normal state and stressed state caused by overwatering for 24 days: (1) histogram for normal state, (2) histogram approximation for normal state, (3) histogram of stressed state, and (4) histogram approximation for stressed state.
The mean value of the fluorescence ratio is 0.81 and the standard deviation is 0.05 for watercress plants in a normal state. The mean value of the fluorescence ratio is 0.97 and the standard deviation is 0.07 for watercress in a stressed state caused by overwatering during 24 days.
As it is shown in Figures
The mean values (with 95% confidence intervals) of the experimental laser-induced fluorescence spectra of watercress under different stress conditions (leaf cutting, leaf laying, root system damage, and root system overwatering during 11, 17, and 24 days) are shown in Figure
Fluorescence ratio (
Columns 1, 3, 5, 7, 9, and 11 in Figure
The changes of fluorescence spectra for plants in stress conditions described above are typical not only for watercress but also for other plants in stress conditions caused by different impact. The effect of soil pollution on lawn grass is considered below.
The aggregated statistical results (mean values and 95% confidence intervals) of the experimental laser-induced fluorescence spectra of lawn grass under different stress conditions caused by soil pollution (copper sulfate, CuSO4, ferric sulfate, FeSO4, and sodium chloride, NaCl) are shown in Figure
Fluorescence ratio (
Columns 1, 3, 5, 7, and 9 in Figure
It is clearly illustrated in Figures
This means that fluorescence excitation at 532 nm wavelength and the ratio of fluorescence intensities in the red (685 nm) and far-red (740 nm) bands can be used as signatures of plant stress state caused by various factors.
By the processing of the experimental results of fluorescence spectra (induced by a 532 nm wavelength laser) of plants in normal and stressed states caused by mechanical damage, overwatering, and soil pollution the following conclusions can be postulated. The fluorescence spectra of different samples of a plant species revealed repeatability of the spectra shapes. Ratio The difference between the mean value of ratio
The experimental results obtained allow us to develop a remote laser system for detecting plant stress state. However, to ensure the reliability of the measurements, it is necessary to calculate the mean value of ratio
The authors declare that they have no competing interests.