Observation of plant–pathogen interaction by simultaneous hyperspectral imaging reflection and transmission measurements

Hyperspectral imaging sensors are valuable tools for plant disease detection and plant phenotyping. Reflectance properties are influenced by plant pathogens and resistance responses, but changes of transmission characteristics of plants are less described. In this study we used simultaneously recorded reflectance and transmittance imaging data of resistant and susceptible barley genotypes that were inoculated with Blumeria graminis f. sp. hordei to evaluate the added value of imaging transmission, reflection and absorption for characterisation of disease development. These datasets were statistically analysed using principal component analysis, and compared with visual and molecular disease estimation. Reflection measurement performed significantly better for early detection of powdery mildew infection, colonies could be detected 2 days before symptoms became visible in RGB images. Transmission data could be used to detect powdery mildew 2 days after symptoms becoming visible in reflection based RGB images. Additionally distinct transmission changes occurred at 580–650 nm for pixels containing disease symptoms. It could be shown that the additional information of the transmission data allows for a clearer spatial differentiation and localisation between powdery mildew symptoms and necrotic tissue on the leaf then purely reflectance based data. Thus the information of both measurement approaches are complementary: reflectance based measurements facilitate an early detection, and transmission measurements provide additional information to better understand and quantify the complex spatio-temporal dynamics of plant-pathogen interactions

Quantitative and qualitative phenotyping of disease resistance of crops by hyperspectral sensors: seamless interlocking of phytopathology, sensors, and machine learning is needed!

Determination and characterization of resistance reactions of crops against fungal pathogens are essential to select resistant genotypes. In plant breeding, phenotyping of genotypes is realized by time consuming and expensive visual plant ratings. During resistance reactions and during pathogenesis plants initiate different structural and biochemical defence mechanisms, which partly affect the optical properties of plant organs. Recently, intensive research has been conducted to develop innovative optical methods for an assessment of compatible and incompatible plant pathogen interaction. These approaches, combining classical phytopathology or microbiology with technology driven methods — such as sensors, robotics, machine learning, and artificial intelligence — are summarized by the term digital phenotyping. In contrast to common visual rating, detection and assessment methods, optical sensors in combination with advanced data analysis methods are able to retrieve pathogen induced changes in the physiology of susceptible or resistant plants non-invasively and objectively. Phenotyping disease resistance aims different tasks. In an early breeding step, a qualitative assessment and characterization of specific resistance action is aimed to link it, for example, to a genetic marker. Later, during greenhouse and field screening, the assessment of the level of susceptibility of different genotypes is relevant. Within this review, recent advances of digital phenotyping technologies for the detection of subtle resistance reactions and resistance breeding are highlighted and methodological requirements are critically discusse

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

Monitoring wound healing in a 3D wound model by hyperspectral imaging and efficient clustering.

Wound healing is a complex and dynamic process with different distinct and overlapping phases from homeostasis, inflammation and proliferation to remodelling. Monitoring the healing response of injured tissue is of high importance for basic research and clinical practice. In traditional application, biological markers characterize normal and abnormal wound healing. Understanding functional relationships of these biological processes is essential for developing new treatment strategies. However, most of the present techniques (in vitro or in vivo) include invasive microscopic or analytical tissue sampling. In the present study, a non-invasive alternative for monitoring processes during wound healing is introduced. Within this context, hyperspectral imaging (HSI) is an emerging and innovative non-invasive imaging technique with different opportunities in medical applications. HSI acquires the spectral reflectance of an object, depending on its biochemical and structural characteristics. An in-vitro 3-dimensional (3-D) wound model was established and incubated without and with acute and chronic wound fluid (AWF, CWF), respectively. Hyperspectral images of each individual specimen of this 3-D wound model were assessed at day 0/5/10 in vitro, and reflectance spectra were evaluated. For analysing the complex hyperspectral data, an efficient unsupervised approach for clustering massive hyperspectral data was designed, based on efficient hierarchical decomposition of spectral information according to archetypal data points. It represents, to the best of our knowledge, the first application of an advanced Data Mining approach in context of non-invasive analysis of wounds using hyperspectral imagery. By this, temporal and spatial pattern of hyperspectral clusters were determined within the tissue discs and among the different treatments. Results from non-invasive imaging were compared to the number of cells in the various clusters, assessed by Hematoxylin/Eosin (H/E) staining. It was possible to correlate cell quantity and spectral reflectance during wound closure in a 3-D wound model in vitro

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

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

Specim IQ: Evaluation of a New, Miniaturized Handheld Hyperspectral Camera and Its Application for Plant Phenotyping and Disease Detection

Hyperspectral imaging sensors are promising tools for monitoring crop plants or vegetation in different environments. Information on physiology, architecture or biochemistry of plants can be assessed non-invasively and on different scales. For instance, hyperspectral sensors are implemented for stress detection in plant phenotyping processes or in precision agriculture. Up to date, a variety of non-imaging and imaging hyperspectral sensors is available. The measuring process and the handling of most of these sensors is rather complex. Thus, during the last years the demand for sensors with easy user operability arose. The present study introduces the novel hyperspectral camera Specim IQ from Specim (Oulu, Finland). The Specim IQ is a handheld push broom system with integrated operating system and controls. Basic data handling and data analysis processes, such as pre-processing and classification routines are implemented within the camera software. This study provides an introduction into the measurement pipeline of the Specim IQ as well as a radiometric performance comparison with a well-established hyperspectral imager. Case studies for the detection of powdery mildew on barley at the canopy scale and the spectral characterization of Arabidopsis thaliana mutants grown under stressed and non-stressed conditions are presente

Automated and efficient interpretation of 3D wound models by non-invasive hyperspectral imaging <i>in vitro</i>.

<p>Experimental design and work flow showing the different steps for monitoring wound healing from hyperspectral imaging data to interpretation using an efficient approach for unsupervised classification of wound tissue based on hierarchical decomposition according to archetypal data points.</p

Representative spectra of 3-dimensional wound models.

<p>(a) Cluster representatives after computing of XHC for the hyperspectral image data for. The number of cluster was set to <i>k</i> = 7 that could reflect biological processes in this cell culture system. The corresponding signatures represented different regions of the tissues. The dotted line was produced by a part of the tissue covered with fluid resulting in overexposure during the measurement. (b-c) The quantification of pixel densities per cluster. The y-axis is shown in log scale. AWF and CWF induced no significant differences in pixel densities compared to the control situation, however, the dark blue cluster was absent after 10 days <i>in vitro</i> without any supplements.</p

Histological classification of hyperspectral cluster means in relation to wound healing processes.

<p>(a) H/E staining was performed to monitor the wound healing progress morphologically over a specific time period <i>in vitro</i>. The different stages were presented as false color zoomed images of the hyperspectral clusters for the wounds. (b) The quantification of the cell number in different regions of interests revealed the first time a correlation between spectral reflectance and cell quantity in the tissue. (c) Immunohistochemical investigation of CXCR4, a marker for migratory cells, determined no correlations to reflectance data. Additionally, the cells generating the characteristic hyperspectral signature did not correspond to Caspase 3-expressing apoptotic cells (not shown). Scale bar: 250 <i>μ</i>m.</p