Extending Hyperspectral Imaging for Plant Phenotyping to the UV-Range

Previous plant phenotyping studies have focused on the visible (VIS, 400–700 nm), near-infrared (NIR, 700–1000 nm) and short-wave infrared (SWIR, 1000–2500 nm) range. The ultraviolet range (UV, 200–380 nm) has not yet been used in plant phenotyping even though a number of plant molecules like flavones and phenol feature absorption maxima in this range. In this study an imaging UV line scanner in the range of 250–430 nm is introduced to investigate crop plants for plant phenotyping. Observing plants in the UV-range can provide information about important changes of plant substances. To record reliable and reproducible time series results, measurement conditions were defined that exclude phototoxic effects of UV-illumination in the plant tissue. The measurement quality of the UV-camera has been assessed by comparing it to a non-imaging UV-spectrometer by measuring six different plant-based substances. Given the findings of these preliminary studies, an experiment has been defined and performed monitoring the stress response of barley leaves to salt stress. The aim was to visualize the effects of abiotic stress within the UV-range to provide new insights into the stress response of plants. Our study demonstrated the first use of a hyperspectral sensor in the UV-range for stress detection in plant phenotyping

In-Field Detection of Yellow Rust in Wheat on the Ground Canopy and UAV Scale

The application of hyperspectral imaging technology for plant disease detection in the field is still challenging. Existing equipment and analysis algorithms are adapted to highly controlled environmental conditions in the laboratory. However, only real time information from the field scale is able to guide plant protection measures and to optimize the use of resources. At the field scale, many parameters such as the optimal measurement distance, informative feature sets, and suitable algorithms have not been investigated. In this study, the hyperspectral detection and quantification of yellow rust in wheat was evaluated using two measurement platforms: a ground-based vehicle and an unmanned aerial vehicle (UAV). Different disease development stages and disease severities were provided in a plot-based field experiment. Measurements were performed weekly during the vegetation period. Data analysis was performed by three prediction algorithms with a focus on the selection of optimal feature sets. In this context, the across-scale application of optimized feature sets, an approach of information transfer between scales, was also evaluated. Relevant aspects for an on-line disease assessment in the field integrating affordable sensor technology, sensor spatial resolution, compact analysis models, and fast evaluation have been outlined and reflected upon. For the first time, a hyperspectral imaging observation experiment of a plant disease was comparatively performed at two scales, ground canopy and UAV

Evaluation of the benefits of combined reflection and transmission hyperspectral imaging data through disease detection and quantification in plant–pathogen interactions

Previous studies investigating the performance of transmission and reflection datasets for disease detection showed inconsistent results. Within the studies, the performance of transmission imaging varied significantly for the detection of biotroph and necrotrophy plant pathogens, while reflection imaging showed excellent results in both studies. The current study explores the hypothesis that the disparity between these results might be correlated with the different interactions of the respective pathogens with the host plants and the way light interacts with the plant tissue. Pyrenophora teres f. teres and Puccinia hordei—the causative agents of net blotch and brown rust in barley—have been investigated with focus on early-stage detection and quantification (disease severity) of symptoms. Datasets of hyperspectral imaging time-series measurements were analysed through application of multiple data analysis methods (support vector machines; principal component analysis with following distance classifier; spectral decomposition) in order to compare the performance of both datasets for the detection of disease symptoms. It could be shown that transmittance-based brown rust detection (e.g. 12% disease severity) is outperformed by reflectance-based detection (e.g. 36% disease severity) regardless of the algorithm. However, both the detection and quantification of brown rust through transmittance were more accurate than those of powdery mildew in earlier studies. Transmittance and reflectance performed similar for the detection of net blotch disease during the experiments (~ 1% disease severity for reflection and transmission). Each data analysis method outperformed manual rating in terms of disease detection (e.g. 15% disease severity according to manual rating and 36% through support vector machines for rust reflection data). Except for the application of a distance classifier on net blotch transmittance data, it could be shown that pixels, which were classified as symptomatic through the data analysis methods while estimated to represent healthy tissue during manual rating, correlate with areas at the edges of manually detected symptoms. The results of this study support the hypothesis that transmission imaging results are highly correlated with the type of plant–pathogen interaction of the respective pathogens, offering new insights into the nature of transmission-based hyperspectral imaging and its application range

Spatial Referencing of Hyperspectral Images for Tracing of Plant Disease Symptoms

The characterization of plant disease symptoms by hyperspectral imaging is often limited by the missing ability to investigate early, still invisible states. Automatically tracing the symptom position on the leaf back in time could be a promising approach to overcome this limitation. Therefore we present a method to spatially reference time series of close range hyperspectral images. Based on reference points, a robust method is presented to derive a suitable transformation model for each observation within a time series experiment. A non-linear 2D polynomial transformation model has been selected to cope with the specific structure and growth processes of wheat leaves. The potential of the method is outlined by an improved labeling procedure for very early symptoms and by extracting spectral characteristics of single symptoms represented by Vegetation Indices over time. The characteristics are extracted for brown rust and septoria tritici blotch on wheat, based on time series observations using a VISNIR (400⁻1000 nm) hyperspectral camera

Low-Cost 3D Systems: Suitable Tools for Plant Phenotyping

Over the last few years, 3D imaging of plant geometry has become of significant importance for phenotyping and plant breeding. Several sensing techniques, like 3D reconstruction from multiple images and laser scanning, are the methods of choice in different research projects. The use of RGBcameras for 3D reconstruction requires a significant amount of post-processing, whereas in this context, laser scanning needs huge investment costs. The aim of the present study is a comparison between two current 3D imaging low-cost systems and a high precision close-up laser scanner as a reference method. As low-cost systems, the David laser scanning system and the Microsoft Kinect Device were used. The 3D measuring accuracy of both low-cost sensors was estimated based on the deviations of test specimens. Parameters extracted from the volumetric shape of sugar beet taproots, the leaves of sugar beets and the shape of wheat ears were evaluated. These parameters are compared regarding accuracy and correlation to reference measurements. The evaluation scenarios were chosen with respect to recorded plant parameters in current phenotyping projects. In the present study, low-cost 3D imaging devices have been shown to be highly reliable for the demands of plant phenotyping, with the potential to be implemented in automated application procedures, while saving acquisition costs. Our study confirms that a carefully selected low-cost senso

Assessment of Fusarium Infection and Mycotoxin Contamination of Wheat Kernels and Flour Using Hyperspectral Imaging

Fusarium head blight (FHB) epidemics in wheat and contamination with Fusarium mycotoxins has become an increasing problem over the last decades. This prompted the need for non-invasive and non-destructive techniques to screen cereal grains for Fusarium infection, which is usually accompanied by mycotoxin contamination. This study tested the potential of hyperspectral imaging to monitor the infection of wheat kernels and flour with three Fusarium species. Kernels of two wheat varieties inoculated at anthesis with F. graminearum, F. culmorum, and F. poae were investigated. Hyperspectral images of kernels and flour were taken in the visible-near infrared (VIS-NIR) (400–1000 nm) and short-wave infrared (SWIR) (1000–2500 nm) ranges. The fungal DNA and mycotoxin contents were quantified. Spectral reflectance of Fusarium-damaged kernels (FDK) was significantly higher than non-inoculated ones. In contrast, spectral reflectance of flour from non-inoculated kernels was higher than that of FDK in the VIS and lower in the NIR and SWIR ranges. Spectral reflectance of kernels was positively correlated with fungal DNA and deoxynivalenol (DON) contents. In the case of the flour, this correlation exceeded r = −0.80 in the VIS range. Remarkable peaks of correlation appeared at 1193, 1231, 1446 to 1465, and 1742 to 2500 nm in the SWIR range

Quantitative assessment of disease severity and rating of barley cultivars based on hyperspectral imaging in a non-invasive, automated phenotyping platform

BackgroundPhenotyping is a bottleneck for the development of new plant cultivars. This study introduces a new hyperspectral phenotyping system, which combines the high throughput of canopy scale measurements with the advantages of high spatial resolution and a controlled measurement environment. Furthermore, the measured barley canopies were grown in large containers (called Mini-Plots), which allow plants to develop field-like phenotypes in greenhouse experiments, without being hindered by pot size.ResultsSix barley cultivars have been investigated via hyperspectral imaging up to 30 days after inoculation with powdery mildew. With a high spatial resolution and stable measurement conditions, it was possible to automatically quantify powdery mildew symptoms through a combination of Simplex Volume Maximization and Support Vector Machines. Detection was feasible as soon as the first symptoms were visible for the human eye during manual rating. An accurate assessment of the disease severity for all cultivars at each measurement day over the course of the experiment was realized. Furthermore, powdery mildew resistance based necrosis of one cultivar was detected as well.ConclusionThe hyperspectral phenotyping system combines the advantages of field based canopy level measurement systems (high throughput, automatization, low manual workload) with those of laboratory based leaf level measurement systems (high spatial resolution, controlled environment, stable conditions for time series measurements). This allows an accurate and objective disease severity assessment without the need for trained experts, who perform visual rating, as well as detection of disease symptoms in early stages. Therefore, it is a promising tool for plant resistance breeding

Discovering coherency of specific gene expression and optical reflectance properties of barley genotypes differing for resistance reactions against powdery mildew.

Hyperspectral imaging has proved its potential for evaluating complex plant-pathogen interactions. However, a closer link of the spectral signatures and genotypic characteristics remains elusive. Here, we show relation between gene expression profiles and specific wavebands from reflectance during three barley-powdery mildew interactions. Significant synergistic effects between the hyperspectral signal and the corresponding gene activities has been shown using the linear discriminant analysis (LDA). Combining the data sets of hyperspectral signatures and gene expression profiles allowed a more precise differentiation of the three investigated barley-Bgh interactions independent from the time after inoculation. This shows significant synergistic effects between the hyperspectral signal and the corresponding gene activities. To analyze this coherency between spectral reflectance and seven different gene expression profiles, relevant wavelength bands and reflectance intensities for each gene were computed using the Relief algorithm. Instancing, xylanase activity was indicated by relevant wavelengths around 710 nm, which are characterized by leaf and cell structures. HvRuBisCO activity underlines relevant wavebands in the green and red range, elucidating the coherency of RuBisCO to the photosynthesis apparatus and in the NIR range due to the influence of RuBisCO on barley leaf cell development. These findings provide the first insights to links between gene expression and spectral reflectance that can be used for an efficient non-invasive phenotyping of plant resistance and enables new insights into plant-pathogen interactions

Screening of Barley Resistance Against Powdery Mildew by Simultaneous High-Throughput Enzyme Activity Signature Profiling and Multispectral Imaging

Molecular marker analysis allow for a rapid and advanced pre-selection and resistance screenings in plant breeding processes. During the phenotyping process, optical sensors have proved their potential to determine and assess the function of the genotype of the breeding material. Thereby, biomarkers for specific disease resistance traits provide valuable information for calibrating optical sensor approaches during early plant-pathogen interactions. In this context, the combination of physiological, metabolic phenotyping and phenomic profiles could establish efficient identification and quantification of relevant genotypes within breeding processes. Experiments were conducted with near-isogenic lines of H. vulgare (susceptible, mildew locus o (mlo) and Mildew locus a (Mla) resistant). Multispectral imaging of barley plants was daily conducted 0–8 days after inoculation (dai) in a high-throughput facility with 10 wavelength bands from 400 to 1,000 nm. In parallel, the temporal dynamics of the activities of invertase isoenzymes, as key sink specific enzymes that irreversibly cleave the transport sugar sucrose into the hexose monomers, were profiled in a semi high-throughput approach. The activities of cell wall, cytosolic and vacuole invertase revealed specific dynamics of the activity signatures for susceptible genotypes and genotypes with mlo and Mla based resistances 0–120 hours after inoculation (hai). These patterns could be used to differentiate between interaction types and revealed an early influence of Blumeria graminis f.sp. hordei (Bgh) conidia on the specific invertase activity already 0.5 hai. During this early powdery mildew pathogenesis, the reflectance intensity increased in the blue bands and at 690 nm. The Mla resistant plants showed an increased reflectance at 680 and 710 nm and a decreased reflectance in the near infrared bands from 3 dai. Applying a Support Vector Machine classification as a supervised machine learning approach, the pixelwise identification and quantification of powdery mildew diseased barley tissue and hypersensitive response spots were established. This enables an automatic identification of the barley-powdery mildew interaction. The study established a proof-of-concept for plant resistance phenotyping with multispectral imaging in high-throughput. The combination of invertase analysis and multispectral imaging showed to be a complementing validation system. This will provide a deeper understanding of optical data and its implementation into disease resistance screening