Hyperspectral imaging reveals small-scale water gradients in apple leaves due to minimal cuticle perforation by Venturia inaequalis conidiophores.

Effects of Venturia inaequalis on water relations of apple leaves were studied under controlled conditions without limitation of water supply to elucidate their impact on the non-haustorial biotrophy of this pathogen. Leaf water relations, namely leaf water content and transpiration, were spatially resolved by hyperspectral imaging and thermography; non-imaging techniques-gravimetry, a pressure chamber, and porometry-were used for calibration and validation. Reduced stomatal transpiration 3-4 d after inoculation coincided with a transient increase of water potential. Perforation of the plant cuticle by protruding conidiophores subsequently increased cuticular transpiration even before visible symptoms occurred. With sufficient water supply, cuticular transpiration remained at elevated levels for several weeks. Infections did not affect the leaf water content before scab lesions became visible. Only hyperspectral imaging was suitable to demonstrate that a decreased leaf water content was strictly limited to sites of emerging conidiophores and that cuticle porosity increased with sporulation. Microscopy confirmed marginal cuticle injury; although perforated, it tightly surrounded the base of conidiophores throughout sporulation and restricted water loss. The role of sustained redirection of water flow to the pathogen's hyphae in the subcuticular space above epidermal cells, to facilitate the acquisition and uptake of nutrients by V. inaequalis, is discussed.

Isolation and characterization of the gene HvFAR1 encoding acyl-CoA reductase from the cer-za.227 mutant of barley (Hordeum vulgare) and analysis of the cuticular barrier functions.

The cuticle is a protective layer covering aerial plant organs. We studied the function of waxes for the establishment of the cuticular barrier in barley (Hordeum vulgare). The barley eceriferum mutants cer-za.227 and cer-ye.267 display reduced wax loads, but the genes affected, and the consequences of the wax changes for the barrier function remained unknown. Cuticular waxes and permeabilities were measured in cer-za.227 and cer-ye.267. The mutant loci were isolated by bulked segregant RNA sequencing. New cer-za alleles were generated by genome editing. The CER-ZA protein was characterized after expression in yeast and Arabidopsis cer4-3. Cer-za.227 carries a mutation in HORVU5Hr1G089230 encoding acyl-CoA reductase (FAR1). The cer-ye.267 mutation is located to HORVU4Hr1G063420 encoding ß-ketoacyl-CoA synthase (KAS1) and is allelic to cer-zh.54. The amounts of intracuticular waxes were strongly decreased in cer-ye.267. The cuticular water loss and permeability of cer-za.227 were similar to wild-type (WT), but were increased in cer-ye.267. Removal of epicuticular waxes revealed that intracuticular, but not epicuticular waxes are required to regulate cuticular transpiration. The differential decrease in intracuticular waxes between cer-za.227 and cer-ye.267, and the removal of epicuticular waxes indicate that the cuticular barrier function mostly depends on the presence of intracuticular waxes.

Hyperspectral Imaging in the UV Range Allows for Differentiation of Sugar Beet Diseases Based on Changes in Secondary Plant Metabolites.

Fungal infections trigger defense or signaling responses in plants, leading to various changes in plant metabolites. The changes in metabolites, for example chlorophyll or flavonoids, have long been detectable using time-consuming destructive analytical methods including high-performance liquid chromatography or photometric determination. Recent plant phenotyping studies have revealed that hyperspectral imaging (HSI) in the UV range can be used to link spectral changes with changes in plant metabolites. To compare established destructive analytical methods with new nondestructive hyperspectral measurements, the interaction between sugar beet leaves and the pathogens Cercospora beticola, which causes Cercospora leaf spot disease (CLS), and Uromyces betae, which causes sugar beet rust (BR), was investigated. With the help of destructive analyses, we showed that both diseases have different effects on chlorophylls, carotenoids, flavonoids, and several phenols. Nondestructive hyperspectral measurements in the UV range revealed different effects of CLS and BR on plant metabolites resulting in distinct reflectance patterns. Both diseases resulted in specific spectral changes that allowed differentiation between the two diseases. Machine learning algorithms enabled the differentiation between the symptom classes and recognition of the two sugar beet diseases. Feature importance analysis identified specific wavelengths important to the classification, highlighting the utility of the UV range. The study demonstrates that HSI in the UV range is a promising, nondestructive tool to investigate the influence of plant diseases on plant physiology and biochemistry.

Hyperspectral mapping of the response of grapevine cultivars to Plasmopara viticola infection at the tissue scale.

Resistance of grapevine to Plasmopara viticola is associated with the hypersensitive reaction, accumulation of stilbenoids, and formation of callose depositions. Spectral characterization of infected leaf tissue of cvs 'Regent' and 'Solaris' with resistance genes Rpv 3-1 and Rpv 10 and Rpv 3-3, respectively, suggested that resistance is not dependent on large-scale necrotization of host tissue. Reactions of the resistant cultivars and a reference susceptible to P. viticola were studied using hyperspectral imaging (range 400-1000 nm) at the tissue level and microscopic techniques. Resistance of both cultivars was incomplete and allowed pathogen reproduction. Spectral vegetation indices characterized the host response to pathogen invasion; the vitality of infected and necrotic leaf tissue differed significantly. Resistance depended on local accumulation of polyphenols in response to haustorium formation and was more effective for cv. 'Solaris'. Although hypersensitive reaction of some cells prevented colonization of palisade parenchyma, resistance was not associated with extensive necrotization of tissue, and the biotrophic pathogen survived localized death of penetrated host cells. Hyperspectral imaging was suitable to characterize and differentiate the resistance reactions of grapevine cultivars by mapping of the cellular response to pathogen attack on the tissue level and yields useful information on host-pathogen interactions.

Pathogenesis of Plasmopara viticola Depending on Resistance Mediated by Rpv3_1, and Rpv10 and Rpv3_3, and by the Vitality of Leaf Tissue.

Grapevine cultivars vary in their resistance to Plasmopara viticola, causal agent of downy mildew. Genes from various Vitis species confer pathogen resistance (Rpv), resulting in reduced compatibility of the host-pathogen interaction and partial disease resistance that may become apparent at different stages of pathogenesis. This study describes the pathogenesis of P. viticola on the partially resistant cultivars Regent (Rpv3-1) and Solaris (Rpv3-3, Rpv10) as compared with the susceptible cultivar Mueller-Thurgau using various microscopic techniques, visual disease rating as well as qPCR. Host plant resistance had no effect on the initial steps of pathogenesis outside the host plant cells (zoospore attachment, formation of substomatal vesicle) and became detectable only after the formation of primary haustoria. The restricted compatibility resulted in reductions in haustorium size and in the number of secondary haustoria and was associated with callose depositions around haustoria and stomatal guard cells, collapsed mesophyll cells (hypersensitive reaction), and additional production of an amorphous substance in the intercellular space of cultivar Solaris. Resistance mechanisms reduced the efficiency of P. viticola haustoria and largely restricted tissue colonization to the spongy parenchyma; resistance of cultivar Solaris having thicker leaves was more effective than that of cultivar Regent. Despite of the effects of resistance genes, P. viticola was able to complete its life cycle by forming sporangiophores with sporangia through the stomata on both resistant cultivars indicating partial resistance. Differences in the pathogenesis on detached and attached grapevine leaves highlighted the impact of host tissue vitality on both resistance and susceptibility to the biotrophic pathogen.

Trichoderma species isolated from grapevine with tolerance towards common copper fungicides used in viticulture for plant protection.

BACKGROUND: Copper-containing fungicides are applied broadly in organic viticulture against downy mildew caused by Plasmopara viticola. Although long-term application of copper-based fungicides is associated with ecotoxic effects on the environment, their use in viticulture is required until sustainable alternatives are available. Trichoderma spp. might be a promising approach to fungicide reduction while promoting plant growth and development and displaying biocontrol activity. This study aims to examine the tolerance and compatibility of Trichoderma spp. to copper fungicides. This work contributes to the development of a spray application consisting of a copper-tolerant Trichoderma sp. combined with a downscaled copper fungicide rate against P. viticola. RESULTS: Trichoderma spp. isolated from grapevine wood in vineyards were identified and used for tolerance screening in various concentrations of copper fungicides. Copper hydroxide was identified as being highly compatible with Trichoderma. Two Trichoderma candidates, T. koningiopsis and T. harzianum, showed high copper tolerance in mycelial growth and germination tests, and were adapted to 2.85 g Cu L-1 of the selected fungicide. Microscopic investigations showed the attachment of copper compounds to fungal cell walls and copper uptake within the cytoplasm. In the case of high tolerance, large-scale copper uptake was prevented. CONCLUSION: Our findings identified two highly copper-tolerant Trichoderma isolates with natural adaptation to the vineyard ecosystem, which could be further tested as biostimulants and biocontrol agents, combined with a reduced fungicide rate for sustainable plant protection. © 2022 Society of Chemical Industry.

A Melanin-Deficient Isolate of Venturia inaequalis Reveals Various Roles of Melanin in Pathogen Life Cycle and Fitness.

Venturia inaequalis is the ascomycetous pathogen causing apple scabs and forms dark-pigmented spores and partially melanised infection structures. Although melanin is considered to be essential for the infection of host tissue, a spontaneously occurring melanin-deficient mutant was isolated from an abaxial side of an apple leaf and can be cultivated in vitro as well as in vivo. The morphology and development of the melanin-deficient-isolate SW01 on leaves of susceptible apple plants were compared to that of the corresponding wild-type isolate HS1. White conidia of SW01 were often wrinkled when dry and significantly increased their volume in suspension. Germination and formation of germtubes and appressoria were not impaired; however, the lack of melanisation of the appressorial ring structure at the interface with the plant cuticle significantly reduced the infection success of SW01. The colonisation of leaf tissue by non-melanised subcuticular hyphae was not affected until the initiation of conidiogenesis. Non-melanised conidiophores penetrated the plant cuticle from inside less successfully than the wild type, and the release of white conidia from less solid conidiophores above the cuticle was less frequent. Melanin in the outer cell wall of V. inaequalis was not required for the survival of conidia under ambient temperature or at -20 °C storage conditions, however, promoted the tolerance of the pathogen to copper and synthetic fungicides affecting the stability and function of the fungal cell wall, plasma membrane, respiration (QoIs) and enzyme secretion, but had no effect on the sensitivity to sulphur and SDHIs. The roles of melanin in different steps of the V. inaequalis life cycle and the epidemiology of apple scabs are discussed.

A Hyperspectral Library of Foliar Diseases of Wheat.

This work established a hyperspectral library of important foliar diseases of wheat induced by different fungal pathogens, representing a time series from infection to symptom appearance for the purpose of detecting spectral changes. The data were generated under controlled conditions at the leaf scale. The transition from healthy to diseased leaf tissue was assessed, and spectral shifts were identified and used in combination with histological investigations to define developmental stages in pathogenesis for each disease. The spectral signatures of each plant disease that indicate a specific developmental stage during pathogenesis, defined as turning points, were combined into a spectral library. Machine learning analysis methods were applied and compared to test the potential of this library to detect and quantify foliar diseases in hyperspectral images. All evaluated classifiers had high accuracy (≤99%) for the detection and identification of both biotrophic and necrotrophic fungi. The potential of applying spectral analysis methods in combination with a spectral library for the detection and identification of plant diseases is demonstrated. Further evaluation and development of these algorithms should contribute to a robust detection and identification system for plant diseases at different developmental stages and the promotion and development of site-specific management techniques for plant diseases under field conditions.

Fungicidal Efficacy of Drying Plant Oils in Green Beans against Bean Rust (Uromyces appendiculatus).

As biorationals, plant oils offer numerous advantages such as being natural products, with low ecotoxicological side effects, and high biodegradability. In particular, drying glyceride plant oils, which are rich in unsaturated fatty acids, might be promising candidates for a more sustainable approach in the discussion about plant protection and the environment. Based on this, we tested the protective and curative efficacy of an oil-in-water-emulsion preparation using drying plant oils (linseed oil, tung oil) and a semi-drying plant oil (rapeseed oil) separately and in different mixtures. Plant oils were tested in greenhouse experiments (in vivo) on green beans (Phaseolus vulgaris L.) against bean rust (Uromyces appendiculatus). We observed that a 2% oil concentration showed no or very low phytotoxic effects on green beans. Both tested drying oils showed a protective control ranging from 53-100% for linseed oil and 32-100% for tung oil. Longer time intervals of 6 days before inoculation (6dbi) were less effective than shorter intervals of 2dbi. Curative efficacies were lower with a maximum of 51% for both oils when applied 4 days past inoculation (4dpi) with the fungus. Furthermore, the results showed no systemic effects. These results underline the potential of drying plant oils as biorationals in sustainable plant protection strategies.

Sensory assessment of Cercospora beticola sporulation for phenotyping the partial disease resistance of sugar beet genotypes.

BACKGROUND: Due to its high damaging potential, Cercospora leaf spot (CLS) caused by Cercospora beticola is a continuous threat to sugar beet production worldwide. Breeding for disease resistance is hampered by the quantitative nature of resistance which may result from differences in penetration, colonization, and sporulation of the pathogen on sugar beet genotypes. In particular, problems in the quantitative assessment of C. beticola sporulation have resulted in the common practice to assess field resistance late in the growth period as quantitative resistance parameter. Recently, hyperspectral sensors have shown potential to assess differences in CLS severity. Hyperspectral microscopy was used for the quantification of C. beticola sporulation on sugar beet leaves in order to characterize the host plant suitability / resistance of genotypes for decision-making in breeding for CLS resistance. RESULTS: Assays with attached and detached leaves demonstrated that vital plant tissue is essential for the full potential of genotypic mechanisms of disease resistance and susceptibility. Spectral information (400 to 900 nm, 160 wavebands) of CLSs recorded before and after induction of C. beticola sporulation allowed the identification of sporulating leaf spot sub-areas. A supervised classification and quantification of sporulation structures was possible, but the necessity of genotype-specific reference spectra restricts the general applicability of this approach. Fungal sporulation could be quantified independent of the host plant genotype by calculating the area under the difference reflection spectrum from hyperspectral imaging before and with sporulation. The overall relationship between sensor-based and visual quantification of C. beticola sporulation on five genotypes differing in CLS resistance was R2 = 0.81; count-based differences among genotypes could be reproduced spectrally. CONCLUSIONS: For the first time, hyperspectral imaging was successfully tested for the quantification of sporulation as a fungal activity depending on host plant suitability. The potential of this non-invasive and non-destructive approach for the quantification of fungal sporulation in other host-pathogen systems and for the phenotyping of crop traits complex as sporulation resistance is discussed.

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.

Surface wax esters contribute to drought tolerance in Arabidopsis.

Waxes are components of the cuticle covering the aerial organs of plants. Accumulation of waxes has previously been associated with protection against water loss, therefore contributing to drought tolerance. However, not much information is known about the function of individual wax components during water deficit. We studied the role of wax ester synthesis during drought. The wax ester load on Arabidopsis leaves and stems was increased during water deficiency. Expression of three genes, WSD1, WSD6 and WSD7 of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT or WSD) family was induced during drought, salt stress and abscisic acid treatment. WSD1 has previously been identified as the major wax ester synthase of stems. wsd1 mutants have shown reduced wax ester coverage on leaves and stems during normal or drought condition, while wax ester loads of wsd6, wsd7 and of the wsd6wsd7 double mutant were unchanged. The growth and relative water content of wsd1 plants were compromised during drought, while leaf water loss of wsd1 was increased. Enzyme assays with recombinant proteins expressed in insect cells revealed that WSD6 and WSD7 contain wax ester synthase activity, albeit with different substrate specificity compared with WSD1. WSD6 and WSD7 localize to the endoplasmic reticulum (ER)/Golgi. These results demonstrated that WSD1 is involved in the accumulation of wax esters during drought, while WSD6 and WSD7 might play other specific roles in wax ester metabolism during stress.

Ergot Alkaloids and their Hallucinogenic Potential in Morning Glories.

Naturally occurring and semisynthetic ergot alkaloids play a role in health care or as recreational drugs in Western and indigenous Mexican societies. Evidence is summarized that ergot alkaloids present in Central American Convolvulaceae like Turbina corymbosa, Ipomoea violacea, and Ipomoea asarifolia are colonized by different species of a newly described clavicipitaceous fungal genus named Periglandula. The fungi are associated with peltate glandular trichomes on the adaxial leaf surface of its host plants. The Periglandula fungi are not yet culturable in vitro but were demonstrated to have the capacity to synthesize ergot alkaloids. The alkaloids do not remain in the fungal mycelium but are translocated via the glandular trichomes into their plant host. Both fungi and host benefit from a symbiotic lifestyle. In evolutionary terms the alkaloid biosynthetic gene cluster in the Periglandula/Ipomoea symbiosis is likely to have a conserved (basic) structure while biosynthetic ergot gene clusters within the genera Claviceps and Epichloe were under ecological selection for alkaloid diversification.

Spectral Patterns Reveal Early Resistance Reactions of Barley Against Blumeria graminis f. sp. hordei.

Differences in early plant-pathogen interactions are mainly characterized by using destructive methods. Optical sensors are advanced techniques for phenotyping host-pathogen interactions on different scales and for detecting subtle plant resistance responses against pathogens. A microscope with a hyperspectral camera was used to study interactions between Blumeria graminis f. sp. hordei and barley (Hordeum vulgare) genotypes with high susceptibility or resistance due to hypersensitive response (HR) and papilla formation. Qualitative and quantitative assessment of pathogen development was used to explain changes in hyperspectral signatures. Within 48 h after inoculation, genotype-specific changes in the green and red range (500 to 690 nm) and a blue shift of the red-edge inflection point were observed. Manual analysis indicated resistance-specific reflectance patterns from 1 to 3 days after inoculation. These changes could be linked to host plant modifications depending on individual host-pathogen interactions. Retrospective analysis of hyperspectral images revealed spectral characteristics of HR against B. graminis f. sp. hordei. For early HR detection, an advanced data mining approach localized HR spots before they became visible on the RGB images derived from hyperspectral imaging. The link among processes during pathogenesis and host resistance to changes in hyperspectral signatures provide evidence that sensor-based phenotyping is suitable to advance time-consuming and cost-expensive visual rating of plant disease resistances.

Maize leaf trichomes represent an entry point of infection for Fusarium species.

Fifteen day old maize seedlings were inoculated with Fusarium graminearum, Fusarium proliferatum, and Fusarium verticillioides. More than 90 % F. proliferatum and F. verticillioides conidia and 50 % of F. graminearum formed one germ tube whereas the other 50 % of F. graminearum conidia formed two to three germ tubes. The germ tubes of F. graminearum conidia were longer than those of F. proliferatum and F. verticillioides. The three species of Fusarium infected bi-cellular trichomes by adhering and growing along the trichomes or by attaching to the cap cell of the trichomes 48 h after inoculation. Hyphae penetrated into the trichomes at the base, the side or at the top of the cap cells. The hyphae colonized the cap cells and then spread to base cells. Prickle trichomes were infected 72 h after inoculation. The hyphae either wrapped around prickle trichomes or formed a mass of hyphae around the top of prickle trichomes or formed appressorium. Macro trichomes were infected by F. graminearum 7 d after inoculation. Following penetration, the fungus spread to adjacent epidermal cells and to the subcuticle. This investigation provides the first assessment of F. graminearum, F. proliferatum, and F. verticillioides infection via trichomes of maize leaves.

Histopathological assessment of the infection of maize leaves by Fusarium graminearum, F. proliferatum, and F. verticillioides.

Young maize plants were inoculated on unfolded mature leaves and on folded immature leaves with Fusarium graminearum, Fusarium proliferatum, and Fusarium verticillioides suspensions. Infection and symptom development of disease on these asymptomatic mature leaves and immature leaves were then documented. Subcuticular infection was found by the three Fusarium species on both symptomatic and symptomless leaves. The three Fusarium species penetrated the stomata of immature leaves by the formation of appressoria-like structures, infection cushions or by direct penetration. Infection by the three species of Fusarium via stomata is reported here for the first time. The superficial hyphae and re-emerging hyphae of the three species produced conidia. The macroconidia of F. graminearum produced secondary macroconidia and F. proliferatum formed microconidia inside the leaf tissues that sporulated through stomata and trichomes. The infection of maize leaves by the three species of Fusarium and their sporulation may contribute inoculum to cob and kernel infection.

Plant Phenotyping using Probabilistic Topic Models: Uncovering the Hyperspectral Language of Plants.

Modern phenotyping and plant disease detection methods, based on optical sensors and information technology, provide promising approaches to plant research and precision farming. In particular, hyperspectral imaging have been found to reveal physiological and structural characteristics in plants and to allow for tracking physiological dynamics due to environmental effects. In this work, we present an approach to plant phenotyping that integrates non-invasive sensors, computer vision, as well as data mining techniques and allows for monitoring how plants respond to stress. To uncover latent hyperspectral characteristics of diseased plants reliably and in an easy-to-understand way, we "wordify" the hyperspectral images, i.e., we turn the images into a corpus of text documents. Then, we apply probabilistic topic models, a well-established natural language processing technique that identifies content and topics of documents. Based on recent regularized topic models, we demonstrate that one can track automatically the development of three foliar diseases of barley. We also present a visualization of the topics that provides plant scientists an intuitive tool for hyperspectral imaging. In short, our analysis and visualization of characteristic topics found during symptom development and disease progress reveal the hyperspectral language of plant diseases.

Improvement of Lesion Phenotyping in Cercospora beticola-Sugar Beet Interaction by Hyperspectral Imaging.

Cercospora leaf spot (CLS) caused by Cercospora beticola is the most destructive leaf disease of sugar beet and may cause high losses in yield and quality. Breeding and cultivation of disease-resistant varieties is an important strategy to control this economically relevant plant disease. Reliable and robust resistance parameters are required to promote breeding progress. CLS lesions on five different sugar beet genotypes incubated under controlled conditions were analyzed for phenotypic differences related to field resistance to C. beticola. Lesions of CLS were rated by classical quantitative and qualitative methods in combination with noninvasive hyperspectral imaging. Calculating the ratio of lesion center to lesion margin, four CLS phenotypes were identified that vary in size and spatial composition. Lesions could be differentiated into subareas based on their spectral characteristics in the range of 400 to 900 nm. Sugar beet genotypes with lower disease severity typically had lesions with smaller centers compared with highly susceptible genotypes. Accordingly, the number of conidia per diseased leaf area on resistant plants was lower. The assessment of lesion phenotypes by hyperspectral imaging with regard to sporulation may be an appropriate method to identify subtle differences in disease resistance. The spectral and spatial analysis of the lesions has the potential to improve the screening process in breeding for CLS resistance.

Hyperspectral imaging reveals the effect of sugar beet quantitative trait loci on Cercospora leaf spot resistance.

The quantitative resistance of sugar beet (Beta vulgaris L.) against Cercospora leaf spot (CLS) caused by Cercospora beticola (Sacc.) was characterised by hyperspectral imaging. Two closely related inbred lines, differing in two quantitative trait loci (QTL), which made a difference in disease severity of 1.1-1.7 on the standard scoring scale (1-9), were investigated under controlled conditions. The temporal and spatial development of CLS lesions on the two genotypes were monitored using a hyperspectral microscope. The lesion development on the QTL-carrying, resistant genotype was characterised by a fast and abrupt change in spectral reflectance, whereas it was slower and ultimately more severe on the genotype lacking the QTL. An efficient approach for clustering of hyperspectral signatures was adapted in order to reveal resistance characteristics automatically. The presented method allowed a fast and reliable differentiation of CLS dynamics and lesion composition providing a promising tool to improve resistance breeding by objective and precise plant phenotyping.

Osmotic adjustment of young sugar beets (Beta vulgaris) under progressive drought stress and subsequent rewatering assessed by metabolite analysis and infrared thermography.

The main objective of this work was to provide the chronology of physiological and metabolic alterations occurring under drought and demonstrate how these relate to a phenotypic approach (infrared thermal imaging, IRT). This should provide tools to tailor phenotyping approaches for drought tolerance and underlying metabolic alterations. In the present study, destructive analysis of growth and cell morphology, water status, osmotic adjustment, metabolic changes and membrane damage were combined with non-destructive determination of leaf temperature using infrared thermography (IRT) in 6-week-old sugar beets subjected to progressive drought stress and subsequent rewatering. Different methods were suitable for the characterisation of the dynamic development of distinct stress phases: although IRT allowed detection of initial impairment of transpiration within 1 day of drought stress, destructive methods allowed us to distinguish a phase of metabolic adjustment including redirection of carbon flow into protective mechanisms and a subsequent phase of membrane destabilisation and cellular damage. Only the combination of invasive and non-invasive methods allowed for the differentiation of the complete sequence of physiological changes induced by drought stress. This could be especially beneficial for the selection of phenotypes that are adapted to early drought. During rewatering, sugar beet shoots rapidly re-established water relations, but membrane damage and partial stomatal closure persisted longer, which could have an impact on subsequent stress events. During the onset of secondary growth, taproots required more time to recover the water status and to readjust primary metabolites than shoots.