Etienne Belin

High throughput quantitative phenotyping of plant resistance using chlorophyll fluorescence image analysis

BackgroundIn order to select for quantitative plant resistance to pathogens, high throughput approaches that can precisely quantify disease severity are needed. Automation and use of calibrated image analysis should provide more accurate, objective and faster analyses than visual assessments. In contrast to conventional visible imaging, chlorophyll fluorescence imaging is not sensitive to environmental light variations and provides single-channel images prone to a segmentation analysis by simple thresholding approaches. Among the various parameters used in chlorophyll fluorescence imaging, the maximum quantum yield of photosystem II photochemistry (Fv/Fm) is well adapted to phenotyping disease severity. Fv/Fm is an indicator of plant stress that displays a robust contrast between infected and healthy tissues. In the present paper, we aimed at the segmentation of Fv/Fm images to quantify disease severity.ResultsBased on the Fv/Fm values of each pixel of the image, a thresholding approach was developed to delimit diseased areas. A first step consisted in setting up thresholds to reproduce visual observations by trained raters of symptoms caused by Xanthomonas fuscans subsp. fuscans (Xff) CFBP4834-R on Phaseolus vulgaris cv. Flavert. In order to develop a thresholding approach valuable on any cultivars or species, a second step was based on modeling pixel-wise Fv/Fm-distributions as mixtures of Gaussian distributions. Such a modeling may discriminate various stages of the symptom development but over-weights artifacts that can occur on mock-inoculated samples. Therefore, we developed a thresholding approach based on the probability of misclassification of a healthy pixel. Then, a clustering step is performed on the diseased areas to discriminate between various stages of alteration of plant tissues. Notably, the use of chlorophyll fluorescence imaging could detect pre-symptomatic area. The interest of this image analysis procedure for assessing the levels of quantitative resistance is illustrated with the quantitation of disease severity on five commercial varieties of bean inoculated with Xff CFBP4834-R.ConclusionsIn this paper, we describe an image analysis procedure for quantifying the leaf area impacted by the pathogen. In a perspective of high throughput phenotyping, the procedure was automated with the software R downloadable at http://www.r-project.org/. The R script is available at http://lisa.univ-angers.fr/PHENOTIC/telechargements.html.

Multiscale imaging of plants: current approaches and challenges

We review a set of recent multiscale imaging techniques, producing high-resolution images of interest for plant sciences. These techniques are promising because they match the multiscale structure of plants. However, the use of such high-resolution images is challenging in the perspective of their application to high-throughput phenotyping on large populations of plants, because of the memory cost for their data storage and the computational cost for their processing to extract information. We discuss how this renews the interest for multiscale image processing tools such as wavelets, fractals and recent variants to analyse such high-resolution images.

Predicting sensorial attribute scores of ornamental plants assessed in 3D through rotation on video by image analysis

A method to construct morphometrical descriptors from rotating plants is proposed.Rotating virtual plants stimuli are appropriate for sensory profile experiments.Sensory attributes and morphometrical descriptors present strong relationships.Sensory attributes are efficiently predicted with few morphometrical descriptors. The visual appearance of a plant is tightly linked to its 3D architecture, and can be characterized by means of sensorial experiments. Providing a method to manage image features to predict objective visual traits of real or in silico ornamental plants seen and assessed in rotation, could be a valuable tool to take into account the 3D of the plants in order to reach faster, more faithful and more reproducible hedonic-free characterizations. The present study aims to present a simple approach to manage image data from rotating plant videos in order to predict some visual characteristics as beforehand determined through a non-hedonic sensory evaluation. It is proposed to implement plant morphometrical descriptors using common descriptive statistics computed from 2D features measured along the plant rotation with the aim to integrate the plant 3D. As a preliminary study to evaluate the potential of the proposed approach, the present experiment used virtual plants. First, a sensory profile on 20 virtual rose bushes videos for which 12 plant morphology-related sensory attributes were developed is presented. In parallel, 2D features from the video frames have been extracted considering an 8?-rotation interval and their discriminant power have been checked. Results showed that each sensory attributes presented at least one strong and significant linear relationship with a specific morphometrical descriptor (Pearsons correlation coefficient ?0.8, p-values<0.001). A stepwise predictor selection procedure to design ordinary least squares (OLS) regression models allowed quite good modeling of the sensory attributes with no more than four morphometrical descriptors (adjusted R2?0.9). Regression on components and penalized models presented also good to acceptable fit, but model cross-validation (CV) and model complexity confirmed the relevance of the OLS models and their selected morphometrical descriptors (R2-CV?0.9 and root mean square error of prediction <0.7) and strengthened the pertinence of transposing this image data management for experiments with real plants considering also their color characteristics thus achieving a proof of the concept.

Phenoplant: a web resource for the exploration of large chlorophyll fluorescence image datasets

BackgroundImage analysis is increasingly used in plant phenotyping. Among the various imaging techniques that can be used in plant phenotyping, chlorophyll fluorescence imaging allows imaging of the impact of biotic or abiotic stresses on leaves. Numerous chlorophyll fluorescence parameters may be measured or calculated, but only a few can produce a contrast in a given condition. Therefore, automated procedures that help screening chlorophyll fluorescence image datasets are needed, especially in the perspective of high-throughput plant phenotyping.ResultsWe developed an automatic procedure aiming at facilitating the identification of chlorophyll fluorescence parameters impacted on leaves by a stress. First, for each chlorophyll fluorescence parameter, the procedure provides an overview of the data by automatically creating contact sheets of images and/or histograms. Such contact sheets enable a fast comparison of the impact on leaves of various treatments, or of the contrast dynamics during the experiments. Second, based on the global intensity of each chlorophyll fluorescence parameter, the procedure automatically produces radial plots and box plots allowing the user to identify chlorophyll fluorescence parameters that discriminate between treatments. Moreover, basic statistical analysis is automatically generated. Third, for each chlorophyll fluorescence parameter the procedure automatically performs a clustering analysis based on the histograms. This analysis clusters images of plants according to their health status. We applied this procedure to monitor the impact of the inoculation of the root parasitic plant Phelipanche ramosa on Arabidopsis thaliana ecotypes Col-0 and Ler.ConclusionsUsing this automatic procedure, we identified eight chlorophyll fluorescence parameters discriminating between the two ecotypes of A. thaliana, and five impacted by the infection of Arabidopsis thaliana by P. ramosa. More generally, this procedure may help to identify chlorophyll fluorescence parameters impacted by various types of stresses. We implemented this procedure at http://www.phenoplant.org freely accessible to users of the plant phenotyping community.

On the value of the Kullback---Leibler divergence for cost-effective spectral imaging of plants by optimal selection of wavebands

The practical value of a criterion based on statistical information theory is demonstrated for the selection of optimal wavelength and bandwidth of low-cost lighting systems in plant imaging applications. Kullback–Leibler divergence is applied to the problem of spectral band reduction from hyperspectral imaging. The results are illustrated on various plant imaging problems and show similar results to the one obtained with state-of-the-art criteria. A specific interest of the proposed approach is to offer the possibility to integrate technological constraints in the optimization of the spectral bands selected.

Computer vision under inactinic light for hypocotyl-radicle separation with a generic gravitropism-based criterion

Seedling heterotrophic growth monitoring is done by means of computer vision.A separation of radicle and hypocotyl is performed in inactinic light.This is obtained with a generic criterion based on gravitropism.This allows high-throughput phenotyping equipment for analysis of seeds quality. This article proposes a computer-vision based protocol, useful to contribute to high-throughput automated phenotyping of seedlings during elongation, the stage following germination. Radicle and hypocotyl are two essential organs which start to develop at this stage, with the hypocotyl growing towards the soil surface and the radicle exploring deeper layers for nutrient absorption. Early identification and measurement of these two organs are important to the characterization of the plant emergence and to the prognosis of the adult plant. In normal conditions, this growth process of radicle and hypocotyl takes place in the soil, in the dark. Identification and measurement of these two organs are therefore challenging, because they need to be achieved with no light that could alter normal growth conditions. We propose here an original protocol exploiting an inactinic green light, produced by a controlled LED source, coupled to a standard low-cost gray-level camera. On the resulting digital images, we devise a simple criterion based on gravitropism and amenable to direct computer implementation. The automated criterion, through comparison with the performance of human experts, is demonstrated to be efficient for the detection and separation of radicle and hypocotyl, and generic for various species of seedlings. Our protocol especially brings improvement in terms of cost reduction over the current method found in the recent literature which resorts to higher-cost passive thermal imaging to perform the same task in the dark, and that we also consider here for comparison. Our protocol connected to automation of image acquisition, can serve to improve high-throughput phenotyping equipments for analysis of seed quality and genetic variability.

Information-theoretic modeling of trichromacy coding of light spectrum

Trichromacy is the representation of a light spectrum by three scalar coordinates. Such representation is universally implemented by the human visual system and by RGB (Red Green Blue) cameras. We propose here an informational model for trichromacy. Based on a statistical analysis of the dynamics of individual photons, the model demonstrates a possibility for describing trichromacy as an information channel, for which the input–output mutual information can be computed to serve as a measure of performance. The capabilities and significance of the informational model are illustrated and motivated in various situations. The model especially enables an assessment of the influence of the spectral sensitivities of the three types of photodetectors realizing the trichromatic representation. It provides a criterion to optimize possibly adjustable parameters of the spectral sensitivities such as their center wavelength, spectral width or magnitude. The model shows, for instance, the usefulness of some overlap with smooth graded spectral sensitivities, as observed for instance in the human retina. The approach also, starting from hyperspectral images with high spectral resolution measured in the laboratory, can be used to devise low-cost trichromatic imaging systems optimized for observation of specific spectral signatures. This is illustrated with an example from plant science, and demonstrates a potential of application especially to life sciences. The approach particularizes connections between physics, biophysics and information theory.

Shape descriptors to characterize the shoot of entire plant from multiple side views of a motorized depth sensor

A low-cost depth camera recently introduced is synchronized with a specially devised low-cost motorized turntable. This results in a low-cost motorized depth sensor, able to provide a large number of registered side views, which is exploited here for the quantitative characterization of the shoots of entire plants. A set of four new shape descriptors of the shoots, constructed from the depth images on multiple side views of the shoots of plants, is proposed. The four descriptors quantify effective volume, multiscale organization, spatial symmetries and lacunarity of the plants. The four descriptors are here defined, validated on synthetic scenes with known properties, and then applied on nine different-looking real plants to illustrate their abilities for quantitative characterization and comparison. The resulting motorized depth sensor and associated image processing open new perspectives to various plant science applications including plant growth and architecture monitoring, plant response to stresses or the assessment of aesthetic parameters for ornamental plants.

Quantum image coding with a reference-frame-independent scheme

For binary images, or bit planes of non-binary images, we investigate the possibility of a quantum coding decodable by a receiver in the absence of reference frames shared with the emitter. Direct image coding with one qubit per pixel and non-aligned frames leads to decoding errors equivalent to a quantum bit-flip noise increasing with the misalignment. We show the feasibility of frame-invariant coding by using for each pixel a qubit pair prepared in one of two controlled entangled states. With just one common axis shared between the emitter and receiver, exact decoding for each pixel can be obtained by means of two two-outcome projective measurements operating separately on each qubit of the pair. With strictly no alignment information between the emitter and receiver, exact decoding can be obtained by means of a two-outcome projective measurement operating jointly on the qubit pair. In addition, the frame-invariant coding is shown much more resistant to quantum bit-flip noise compared to the direct non-invariant coding. For a cost per pixel of two (entangled) qubits instead of one, complete frame-invariant image coding and enhanced noise resistance are thus obtained.

Digital Image Processing with Quantum Approaches

We devise and analyze illustrative examples of image processing tasks that show the capability of specifically quantum properties to afford enhanced performance inaccessible with classical processing. The quantum approaches here essentially demonstrate and exploit the possibility of parallel processing stemming from superposition of quantum states. The results illustrate the rich potential, yet largely to be explored, of quantum information and computation for image and signal processing.