Evaluation of the impact of herbicides on maize was done through multi- spectral and multi-modal imaging and multi-spectral fluorescence imaging combined with statistical methods. Spectra containing 13 wavelengths ranging from 375 nm to 940 nm were derived from multi-spectral images in transmission, reflection and scattering mode and fluorescence images obtained using high-pass filters (F450 nm, F500 nm, F550 nm, F600 nm, F650 nm) on control maize samples and maize samples treated with Herbextra herbicide were used. The appearance of the spectra allowed us to characterize the effect of the herbicide on the maize pigment concentration. The fluorescence images allowed us to track the fate of absorbed energy and through PLS-DA and SVM-DA to discriminate the two leaf categories with very low error rates for the test, i.e. 4.9% and 2% respectively. The results of this technique can be used in the context of precision agriculture.
Maize (Zea mays) is an annual tropical herb plant of the grass family. It is a highly prized cereal for its many qualities and applications [
Indeed the discovery of herbicides has been a relief for humanity in general but for the peasants in particular, allowing them to increase their area of exploitation. However, repeated use of herbicides has resulted in herbaceous species resistant to these products, leading to overdosage and to mixtures of products which do not often have the same active substance. Herbicides are biologically active and therefore intentionally toxic to target organisms. Because of their hazardous nature, the unintended contact of these substances with undesignated targets may cause serious problems for these targets [
Thus understanding the action of these products on photosynthetic constituents could help to combat the overdosage of these products. Several methods exist for monitoring the health of plants [
The experiment was carried out in the laboratory of instrumentation image and spectroscopy at the Institut National Polytechnique Houphouët-Boigny (INP- HB, Yamoussoukro, Cote d’Ivoire). In a greenhouse, seeds of corn were sown in pots containing a sterilized soil. Each pot contained three corn seeds. At the four-leaf stage, a portion of the maize plants were treated with HERBEXTRA (2, 4-D amine salt 720 g/l, SL), a selective herbicide acting on a large number of weeds. This herbicide is class III and harmful according to the FAO/WHO classification.
The treatment solution was prepared following the manufacturer’s instructions. Thus 8 ml of the 2,4-D amine salt 720 g/l were taken and mixed with 2 l of water in a high pressure sprayer. After homogenization of this mixture, the treatment is carried out on part of the young maize plants. The leaves of the plants treated with the herbicide and the leaves of the untreated plants (healthy leaves) are collected for measurements.
The multi-spectral and multimodal microscope is a microscope constructed from a commercial microscope (Brunel Metallurgical microscope, model SP80), in which all conventional sources were replaced by LEDs [
1) Hardware
The new motorized microscope consists of following components (
a) Camera
Indeed, the used enclosed digital Lumenera lt225M, camera has a pixel size of 5.5 × 5.5 μm. It has a high resolution 2/3 CMOSIS CMV2000 sensor with a fully
electronic global shutter. It uses the USB 3.0 technology with selectable 8 or 12-bit pixel data. That means it is very fast. The full resolution that can be reached is 1088 × 2048 with 2.2 MP. The maximum frame rate is 170 fps (frames per second). The camera has an exposure time range from 0 to 4 s.
b) Objective
In order to increase the size of viewed objects, we used a plan objective which is OLYMPUS NIKON NEOFLUAR 40/0.7. The use of this kind of objective is recommended when we need to see objects with more details is. But that objective undergoes chromatic aberration that makes the distance of found best-in- focus image related to a wavelength.
c) Laser and Data Acquisition Card (DAQ)
The sample is illuminated by lasers of wavelength 405 nm in transmission and 650 nm in reflection. Each wavelength can be used in transmission and also in reflection at any moment without modifying the system. A beam splitter is used between the objective and the camera. Its goal is to divide the beam put in reflection into two half parts: the first is obtained when the laser beam changes its direction after meeting the beam splitter so it interacts with the sample and the second half part is lost. Each output of lasers controllers meets optical densities whose goal is to reduce the power of used lasers. The lasers are controlled by a data acquisition card (DAQ) from National Instruments (USB 6008). Fibers are used in each mode to conduct the light from the source to the sample. Finally, a diffuser is used in order to homogenize the light coming from both lasers sources to the sample. The use of this component is helpful to reduce the speckle from lasers but move it can improve the level of speckle killing [
d) Servo-controllers
The moving of the sample in X, Y and Z directions is done using three motors from THORLABS. Those motors are controlled using TDC001 servo-controllers. They have a shaft-distance of 25 mm (0 to 25 mm) with a minimum step of 0.5 µm. Servo-controllers used are very compact footprints with 2.4 × 2.4 × 1.8. They can control motors from 12 to 15 v up to 2.5 w.
All components quoted above are combined together to build a very compact microscope equipped with two different modes: transmission and reflection. The built microscope is very flexible and easy to handle. It is also possible to use this system for fluorescence measurements. To achieve it, we put between the camera and the objective five high-pass-filters namely, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm.
B) Software
After that, easy software is designed to monitor the entire motorized microscope using MATLAB r2014a. With this software, it is possible to get the best-in- focus image, control the lasers and also the camera.
The images analyzed account for the spatial dimension and the spectral space represents the spectral dimension, i.e. a three dimensional space. From these microscopic images, we try to evaluate the action of the herbicide on the pigment concentration of maize leaves. In the image space, each object is characterized by a distribution of the intensity of the pixels. The objects are observed according to 13 wavelengths which are the variables. The average of the pixels is carried out according to each wavelength and a representation of the intensity as a function of the wavelengths is made subsequently to follow the behavior of each category of plant leaf.
With an excitation source at 405 nm, we used five high-pass filters for each sample. Since the number of filters is greater than 3, we face a problem of multidimensional statistics. Fluorescence is the remission of part of the energy absorbed by chlorophyll molecules that initiate photosynthesis reactions. It is therefore in competition with the energy used in the mechanisms of regulation of photosynthesis. We monitor the intensity of the fluorescence as a function of the filters in order to understand the fate of the light intensity absorbed. Since the intensity of fluorescence is different at the two groups of leaves and also according to the filters, a model for the distinction of the leaves is required. Partial Least Squares Discriminant Analysis (PLS-DA), a linear classification method [
Data set | Healthy leaves | Leaves with herbextra | Total |
---|---|---|---|
Train (calibration set) | 82 | 80 | 162 |
Test | 41 | 40 | 81 |
Total | 123 | 120 | 243 |
proposed by these two statistical methods, parameters such as sensitivity, specificity, error ratio and accuracy are proposed by Ballabio et al. and Shrestha et al. These are defined as follows:
where TP (true positive) is the number of samples correctly classified in their respective group, FN is false negative samples, TN is the true negative and FP is false positive. Sensitivity is the ability of the model to correctly identify samples group, whereas the specificity is the capacity to reject the samples of others group.
We imaged 40 leaves of maize (20 healthy leaves and 20 leaves treated with Herbextra) with multi-spectral microscope I. The microscopic images allow us to distinguish the modes of transmission, reflection and scattering (
The difference between these two types of leaves is observed at the biochemical level. Indeed, whatever the mode, one sees a structural disorganization and deformation of the elements inside the leaves treated with the herbicides. This
structural difference of the healthy and treated leaves is revealed by the optical properties across the spectrum of these leaves (
The multi-functional microscope II allowed us to acquire fluorescence images of maize leaves with a set of filters F450, F500, F550, F600, F650 (
1) PLS-DA
Before establishing the model some parameters must be determined. This is
the case for the number of latent variables (LV). In our work we use four LV because the error is relatively small (0.018) (
In
3) SVM-DA
Since our problem in this study is not linearly separable, we used the Gaussian
PLS-DA | SVM-DA | |||||||
---|---|---|---|---|---|---|---|---|
Data set | Error rate (er) | Accuracy | Sensitivity (Sn) | Specificity (Sp) | Error rate (er) | Accuracy | Sensitivity (Sn) | Specificity (Sp) |
Train | 3% | 97% | 1.00 | 0.94 | 0% | 100% | 1.00 | 1.00 |
Validation | 2.4% | 97.6% | 1.00 | 0.95 | 1.8% | 98% | 1.00 | 0.96 |
Test | 4.9% | 95% | 1.00 | 0.91 | 2% | 98% | 0.95 | 1.00 |
function as a kernel function for data separation. The parameters of this function are chosen optimally, i.e. the choice is based on the minimization of the misclassification error. Thus the kernel width is maintained at 0.96 and the cost or constraint factor is set to 100. The model contains 44 support vectors used to determine the separation lines of the two groups. The model is very stable in the classification of the two groups of samples with relatively low error percentages (
The spectral signatures of our samples are all the same in each mode. However, in terms of intensity, in transmission the maize leaf with the herbicide transmits more light than the healthy corn leaf and that over the entire spectrum of the visible (400 nm - 700 nm). The interaction between the molecules of herbicide and the biochemical constituents of leaf generated the modification of the form of these constituents, creating a deformation of these at the microscopic level. These modifications show that plants produce some compounds to fight again external elements. This struggle for life is the base of deformation and change in optical properties. So this high transmission therefore demonstrates a weakness of the photosynthetic apparatus in the leaves with the herbicide linked to a low concentration of chlorophyll (Chl). Some studies show that stress can lead to a decrease in chlorophyll [
Healthy corn leaves diffuse less light than leaves treated with the herbicide at
590 nm and between 700 nm - 940 nm bound to the biochemical content of these leaves. The diffusion of the leaves is related to the variation of refractive index at the level of the leaf (air, water, etc.) [
Also, after the excitation phase of the leaf any non-transmitted or reflected energy is absorbed. Plants use this absorbed energy to make their organic matter through photosynthesis. The intensities of the fluorescence images show here that much of the energy absorbed by leaves treated with the herbicide is emitted in the form of fluorescence (chlorophyll fluorescence). Plant resistance responses to exterior attack commonly involve the accumulation of specific compounds with either signaling or antimicrobial properties. The latter can include structural modifications [
Our approach in this work allowed us to see that the combination of multi- spectral and multimodal imaging and fluorescence imaging is an effective asset in the diagnosis of the use of herbicides on plants. These two methods allowed us to see on the one hand the spectral behavior of the plants under treatment of herbextra and the effect of the latter on the amount of chlorophyll in the plants through the transmission, reflection and diffusion spectra. On the other hand, the fluorescence imaging allowed us to follow the fate of the energy absorbed by the plant and the rate of transmission of this energy to the reaction center. This transmission rate could thus be an important asset in the diagnosis of a stress situation in the plant. The models show the potential of VSM-DA and PLS-DA in data discrimination. One of the perspectives of this work will be to study the impact of water stress on herbicide treated plants due to the disturbance of rainfall due to climate change.
We would like to thank the International Science Program (ISP) and TWAS for financing the project.
Kouakou, A.K., Soro, A.P., Taky, A.K., Patrice, K. and Zoueu, J.T. (2017) Multi-Spectral and Fluorescence Imaging in Prevention of Overdose of Herbicides: The Case of Maize. Spectral Analysis Reviews, 5, 11-24. https://doi.org/10.4236/sar.2017.52002