AraDQ: an automated digital phenotyping software for quantifying disease symptoms of flood-inoculated Arabidopsis seedlings.

BACKGROUND: Plant scientists have largely relied on pathogen growth assays and/or transcript analysis of stress-responsive genes for quantification of disease severity and susceptibility. These methods are destructive to plants, labor-intensive, and time-consuming, thereby limiting their application in real-time, large-scale studies. Image-based plant phenotyping is an alternative approach that enables automated measurement of various symptoms. However, most of the currently available plant image analysis tools require specific hardware platform and vendor specific software packages, and thus, are not suited for researchers who are not primarily focused on plant phenotyping. In this study, we aimed to develop a digital phenotyping tool to enhance the speed, accuracy, and reliability of disease quantification in Arabidopsis. RESULTS: Here, we present the Arabidopsis Disease Quantification (AraDQ) image analysis tool for examination of flood-inoculated Arabidopsis seedlings grown on plates containing plant growth media. It is a cross-platform application program with a user-friendly graphical interface that contains highly accurate deep neural networks for object detection and segmentation. The only prerequisite is that the input image should contain a fixed-sized 24-color balance card placed next to the objects of interest on a white background to ensure reliable and reproducible results, regardless of the image acquisition method. The image processing pipeline automatically calculates 10 different colors and morphological parameters for individual seedlings in the given image, and disease-associated phenotypic changes can be easily assessed by comparing plant images captured before and after infection. We conducted two case studies involving bacterial and plant mutants with reduced virulence and disease resistance capabilities, respectively, and thereby demonstrated that AraDQ can capture subtle changes in plant color and morphology with a high level of sensitivity. CONCLUSIONS: AraDQ offers a simple, fast, and accurate approach for image-based quantification of plant disease symptoms using various parameters. Its fully automated pipeline neither requires prior image processing nor costly hardware setups, allowing easy implementation of the software by researchers interested in digital phenotyping of diseased plants.

A Novel Method for Quantifying Plant Morphological Characteristics Using Normal Vectors and Local Curvature Data via 3D Modelling-A Case Study in Leaf Lettuce.

Three-dimensional measurement is a high-throughput method that can record a large amount of information. Three-dimensional modelling of plants has the possibility to not only automate dimensional measurement, but to also enable visual assessment to be quantified, eliminating ambiguity in human judgment. In this study, we have developed new methods that could be used for the morphological analysis of plants from the information contained in 3D data. Specifically, we investigated characteristics that can be measured by scale (dimension) and/or visual assessment by humans. The latter is particularly novel in this paper. The characteristics that can be measured on a scale-related dimension were tested based on the bounding box, convex hull, column solid, and voxel. Furthermore, for characteristics that can be evaluated by visual assessment, we propose a new method using normal vectors and local curvature (LC) data. For these examinations, we used our highly accurate all-around 3D plant modelling system. The coefficient of determination between manual measurements and the scale-related methods were all above 0.9. Furthermore, the differences in LC calculated from the normal vector data allowed us to visualise and quantify the concavity and convexity of leaves. This technique revealed that there were differences in the time point at which leaf blistering began to develop among the varieties. The precise 3D model made it possible to perform quantitative measurements of lettuce size and morphological characteristics. In addition, the newly proposed LC-based analysis method made it possible to quantify the characteristics that rely on visual assessment. This research paper was able to demonstrate the following possibilities as outcomes: (1) the automation of conventional manual measurements, and (2) the elimination of variability caused by human subjectivity, thereby rendering evaluations by skilled experts unnecessary.

An Automated, Clip-Type, Small Internet of Things Camera-Based Tomato Flower and Fruit Monitoring and Harvest Prediction System.

Automated crop monitoring using image analysis is commonly used in horticulture. Image-processing technologies have been used in several studies to monitor growth, determine harvest time, and estimate yield. However, accurate monitoring of flowers and fruits in addition to tracking their movements is difficult because of their location on an individual plant among a cluster of plants. In this study, an automated clip-type Internet of Things (IoT) camera-based growth monitoring and harvest date prediction system was proposed and designed for tomato cultivation. Multiple clip-type IoT cameras were installed on trusses inside a greenhouse, and the growth of tomato flowers and fruits was monitored using deep learning-based blooming flower and immature fruit detection. In addition, the harvest date was calculated using these data and temperatures inside the greenhouse. Our system was tested over three months. Harvest dates measured using our system were comparable with the data manually recorded. These results suggest that the system could accurately detect anthesis, number of immature fruits, and predict the harvest date within an error range of ±2.03 days in tomato plants. This system can be used to support crop growth management in greenhouses.

Time-Series Growth Prediction Model Based on U-Net and Machine Learning in Arabidopsis.

Yield prediction for crops is essential information for food security. A high-throughput phenotyping platform (HTPP) generates the data of the complete life cycle of a plant. However, the data are rarely used for yield prediction because of the lack of quality image analysis methods, yield data associated with HTPP, and the time-series analysis method for yield prediction. To overcome limitations, this study employed multiple deep learning (DL) networks to extract high-quality HTTP data, establish an association between HTTP data and the yield performance of crops, and select essential time intervals using machine learning (ML). The images of Arabidopsis were taken 12 times under environmentally controlled HTPP over 23 days after sowing (DAS). First, the features from images were extracted using DL network U-Net with SE-ResXt101 encoder and divided into early (15-21 DAS) and late (∼21-23 DAS) pre-flowering developmental stages using the physiological characteristics of the Arabidopsis plant. Second, the late pre-flowering stage at 23 DAS can be predicted using the ML algorithm XGBoost, based only on a portion of the early pre-flowering stage (17-21 DAS). This was confirmed using an additional biological experiment (P < 0.01). Finally, the projected area (PA) was estimated into fresh weight (FW), and the correlation coefficient between FW and predicted FW was calculated as 0.85. This was the first study that analyzed time-series data to predict the FW of related but different developmental stages and predict the PA. The results of this study were informative and enabled the understanding of the FW of Arabidopsis or yield of leafy plants and total biomass consumed in vertical farming. Moreover, this study highlighted the reduction of time-series data for examining interesting traits and future application of time-series analysis in various HTPPs.

TheLNet270v1 - A Novel Deep-Network Architecture for the Automatic Classification of Thermal Images for Greenhouse Plants.

The real challenge for separating leaf pixels from background pixels in thermal images is associated with various factors such as the amount of emitted and reflected thermal radiation from the targeted plant, absorption of reflected radiation by the humidity of the greenhouse, and the outside environment. We proposed TheLNet270v1 (thermal leaf network with 270 layers version 1) to recover the leaf canopy from its background in real time with higher accuracy than previous systems. The proposed network had an accuracy of 91% (mean boundary F1 score or BF score) to distinguish canopy pixels from background pixels and then segment the image into two classes: leaf and background. We evaluated the classification (segment) performance by using more than 13,766 images and obtained 95.75% training and 95.23% validation accuracies without overfitting issues. This research aimed to develop a deep learning technique for the automatic segmentation of thermal images to continuously monitor the canopy surface temperature inside a greenhouse.

Image-Based Machine Learning Characterizes Root Nodule in Soybean Exposed to Silicon.

Silicon promotes nodule formation in legume roots which is crucial for nitrogen fixation. However, it is very time-consuming and laborious to count the number of nodules and to measure nodule size manually, which led nodule characterization not to be study as much as other agronomical characters. Thus, the current study incorporated various techniques including machine learning to determine the number and size of root nodules and identify various root phenotypes from root images that may be associated with nodule formation with and without silicon treatment. Among those techniques, the machine learning for characterizing nodule is the first attempt, which enabled us to find high correlations among root phenotypes including root length, number of forks, and average link angles, and nodule characters such as number of nodules and nodule size with silicon treatments. The methods here could greatly accelerate further investigation such as delineating the optimal concentration of silicon for nodule formation.

High-Throughput Phenotyping (HTP) Data Reveal Dosage Effect at Growth Stages in Arabidopsis thaliana Irradiated by Gamma Rays.

The effects of radiation dosages on plant species are quantitatively presented as the lethal dose or the dose required for growth reduction in mutation breeding. However, lethal dose and growth reduction fail to provide dynamic growth behavior information such as growth rate after irradiation. Irradiated seeds of Arabidopsis were grown in an environmentally controlled high-throughput phenotyping (HTP) platform to capture growth images that were analyzed with machine learning algorithms. Analysis of digital phenotyping data revealed unique growth patterns following treatments below LD50 value at 641 Gy. Plants treated with 100-Gy gamma irradiation showed almost identical growth pattern compared with wild type; the hormesis effect was observed >21 days after sowing. In 200 Gy-treated plants, a uniform growth pattern but smaller rosette areas than the wild type were seen (p < 0.05). The shift between vegetative and reproductive stages was not retarded by irradiation at 200 and 300 Gy although growth inhibition was detected under the same irradiation dose. Results were validated using 200 and 300 Gy doses with HTP in a separate study. To our knowledge, this is the first study to apply a HTP platform to measure and analyze the dosage effect of radiation in plants. The method enabled an in-depth analysis of growth patterns, which could not be detected previously due to a lack of time-series data. This information will improve our knowledge about the effects of radiation in model plant species and crops.

An automated, high-throughput plant phenotyping system using machine learning-based plant segmentation and image analysis.

A high-throughput plant phenotyping system automatically observes and grows many plant samples. Many plant sample images are acquired by the system to determine the characteristics of the plants (populations). Stable image acquisition and processing is very important to accurately determine the characteristics. However, hardware for acquiring plant images rapidly and stably, while minimizing plant stress, is lacking. Moreover, most software cannot adequately handle large-scale plant imaging. To address these problems, we developed a new, automated, high-throughput plant phenotyping system using simple and robust hardware, and an automated plant-imaging-analysis pipeline consisting of machine-learning-based plant segmentation. Our hardware acquires images reliably and quickly and minimizes plant stress. Furthermore, the images are processed automatically. In particular, large-scale plant-image datasets can be segmented precisely using a classifier developed using a superpixel-based machine-learning algorithm (Random Forest), and variations in plant parameters (such as area) over time can be assessed using the segmented images. We performed comparative evaluations to identify an appropriate learning algorithm for our proposed system, and tested three robust learning algorithms. We developed not only an automatic analysis pipeline but also a convenient means of plant-growth analysis that provides a learning data interface and visualization of plant growth trends. Thus, our system allows end-users such as plant biologists to analyze plant growth via large-scale plant image data easily.