Tomato's Green Gold: Bioeconomy Potential of Residual Tomato Leaf Biomass as a Novel Source for the Secondary Metabolite Rutin.

At the end of the annual horticultural production cycle of greenhouse-grown crops, large quantities of residual biomass are discarded. Here, we propose a new value chain to utilize horticultural leaf biomass for the extraction of secondary metabolites. To increase the secondary metabolite content of leaves, greenhouse-grown crop plants were exposed to low-cost abiotic stress treatments after the last fruit harvest. As proof of concept, we evaluated the production of the flavonoid rutin in tomato plants subjected to nitrogen deficiency. In an interdisciplinary approach, we observed the steady accumulation of rutin in young plants under nitrogen deficiency, tested the applicability of nitrogen deficiency in a commercial-like greenhouse, developed a high efficiency extraction for rutin, and evaluated the acceptance of the proposed value chain by its key actors economically. On the basis of the positive interdisciplinary evaluation, we identified opportunities and challenges for the successful establishment of horticultural leaf biomass as a novel source for secondary metabolites.

Influence of the ethylene-related signal-inhibiting octapeptide NOP-1 on postharvest ripening and quality of 'Golden Delicious' apples.

BACKGROUND: Processes extending the shelf life of climacteric fruit play an important role in terms of a sustainable global food supply. In a previous study, a synthetic octapeptide (NOP-1) was shown to inhibit the interaction between ethylene receptor (ETR) and ethylene insensitive-2 (EIN2), and in consequence delay tomato ripening. We investigated for the first time the effect of NOP-1 on inhibiting the ripening of apples ('Golden Delicious') during postharvest. RESULTS: Using purified recombinant proteins from a bacterial expression system, we demonstrate here that EIN2 also interacts tightly (Kd = 136 ± 29 nmol L-1 ) with the corresponding apple ETR MdETR1. In line with previous binding studies on tomato ETRs, the ripening-delaying peptide NOP-1 clearly binds to the purified apple ETR. An NOP-1 solution (1000 µmol L-1 ) was applied with a brush or microdispenser and compared with apples treated with 1-methylcyclopropene (1-MCP) (SmartFresh™, Agrofresh) applied as gaseous treatment or untreated control fruits. NOP-1 inhibited colour development and chlorophyll degradation during shelf life. These effects were more pronounced with the brush application (surface film) than with microdroplets application (mimicking a sprayable formulation). NOP-1 did not alter ethylene release or respiration rate, whereas 1-MCP expectedly strongly suppressed both. There were no differences in quality parameters evaluated. CONCLUSION: Our study shows that NOP-1 binds to MdETR1 which results in delaying of ethylene-dependent ripening developments of skin colour and chlorophyll. Besides application methods, possible reasons for the weak effect of NOP-1 in comparison with previous tomato experiments could be different receptor affinity and penetration differences. © 2019 Society of Chemical Industry.

Agricultural adjuvants may impair leaf transpiration and photosynthetic activity.

Adjuvants such as surfactants are commonly incorporated into agrochemical formulations to enhance the biological efficiency of foliar sprays by improving the wetting behavior of the spray and/or the penetration of the active ingredients into the leaf tissues. Penetration accelerating adjuvants are known to increase the cuticular permeability and may alter the cuticular barrier to water loss. However, none or very little emphasis has been given to the impacts of adjuvants on crop water balance or drought tolerance, a very important factor affecting crop performance under water scarcity. Two model crops with strongly varying leaf traits, kohlrabi (Brassica oleracea) and apple (Malus domestica) seedlings were grown in controlled environments. Three adjuvants with varying solubility in the cuticle, i.e. octanol-water partition coefficients (logKow) were selected: rapeseed methyl ester (RME) and the surfactants alkyl polyglycoside (APG) and polyoxyethylated tallow amine (POEA). The higher the logKow of the adjuvant, the stronger was the increase of minimum epidermal conductance (gmin, an essential parameter describing plant drought tolerance). However, such effects depended on the physio-chemical properties of the leaf surface. In comparison to kohlrabi, the adjuvant effects on gmin of apple leaves were relatively weak. The increase of gmin was associated with a decrease in contact angle and with an alteration of the wax microstructure. Furthermore, POEA affected photochemical efficiency of kohlrabi leaves. Some adjuvants could have a temporal influence on transpirational water loss and gmin. At repeated applications, they might alter the effective water use and possibly reduce drought tolerance of some horticultural crops.

Limitation of mineral supply as tool for the induction of secondary metabolites accumulation in tomato leaves.

Agricultural residues are natural sources for secondary metabolites as high value ingredients for industrial uses. The present work aims to exploit the accumulation potential of rutin and solanesol in tomato leaves following nitrogen and general mineral deficiency in a commercial-like greenhouse. Physiological responses of tomato plants were monitored non-destructively with a multiparametric fluorescence sensor, and biochemical parameters were determined by means of HPLC analysis. Nitrogen and general mineral limitation led to an accumulation of rutin in young tomato leaves while solanesol concentration was higher in mature leaves. In young leaves, the fluorescence indices SFR_R and NBI_G showed lower values compared to control plants for both stress treatments. On the contrary, FLAV and ANTH_RG values increased during the experiment, but no differences could be recorded in mature leaves. However, correlation analysis indicates, that the FLAV index is not a reliable tool to estimate the concentration of rutin and solanesol tomato leaves. To monitor fruit yield/quality as primary objective of tomato production, fruits showing symptoms of blossom end rot (BER) were counted before and after stress treatments. BER was determined more frequently for plants grown under a general mineral deficiency, concluding that a practical applicability at the end of fruit production is advisable. Our results indicate that by-products from Solanaceae plants are promising resources for valuable bioactive leaf compounds. To achieve the highest concentrations, the seasonal variation, the optimal environmental conditions, the concentrations in different plant organs and varieties as well as different production systems are of high interest for commercial implementation.

Suitability of fluorescence indices for the estimation of fruit maturity compounds in tomato fruits.

BACKGROUND: We examined the suitability of chlorophyll fluorescence-based indices to monitor and predict concentrations of fruit maturity compounds during tomato ripening under different growing conditions in the greenhouse. The aim of this study was to evaluate the effects of chlorophyll concentration changes on fluorescence-based indices and to exploit the relation between fluorescence and reflectance indices with the corresponding maturity compounds determined analytically. RESULTS: Fruits grown under water deficit matured faster than control fruits as recorded with fluorescence-based indices. The SFR_R index correlated well with the analytical determination of chlorophyll content, whereas the single-signal FRF_G correlated with lycopene content even if the sensor was unable to differentiate precisely between maturity stages 2 to 4. Neither the FLAV index nor the FLAV_UV index was suitable for flavonoid prediction in tomato fruits. Compared with fluorescence indices, the relation between the reflection index and pigment concentrations was lower for chlorophyll and higher for lycopene. CONCLUSION: Chlorophyll and lycopene content in tomato fruits can be estimated by means of fluorescence indices during the pre-harvest phase. Since the chlorophyll decrease during tomato ripening is the driving force affecting all fluorescence signals, the methods are not reliable for estimation of other maturity compounds in tomato fruits. © 2018 Society of Chemical Industry.

Novel Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for Ripening Control.

Ethylene signaling is decisive for many plant developmental processes. Among these, control of senescence, abscission and fruit ripening are of fundamental relevance for global agriculture. Consequently, detailed knowledge of the signaling network along with the molecular processes of signal perception and transfer are expected to have high impact on future food production and agriculture. Recent advances in ethylene research have demonstrated that signaling of the plant hormone critically depends on the interaction of the ethylene receptor family with the NRAMP-like membrane protein ETHYLENE INSENSITIVE 2 (EIN2) at the ER membrane, phosphorylation-dependent proteolytic processing of ER-localized EIN2 and subsequent translocation of the cleaved EIN2 C-terminal polypeptide (EIN2-CEND) to the nucleus. EIN2 nuclear transport, but also interaction with the receptors sensing the ethylene signal, both, depend on a nuclear localization signal (NLS) located at the EIN2 C-terminus. Loss of the tight interaction between receptors and EIN2 affects ethylene signaling and impairs plant ethylene responses. Synthetic peptides derived from the NLS sequence interfere with the EIN2-receptor interaction and have utility in controlling plant ethylene responses such as ripening. Here, we report that a synthetic peptide (NOP-1) corresponding to the NLS motif of Arabidopsis EIN2 (aa 1262-1269) efficiently binds to tomato ethylene receptors LeETR4 and NR and delays ripening in the post-harvest phase when applied to the surface of sampled green fruits pre-harvest. In particular, degradation of chlorophylls was delayed by several days, as monitored by optical sensors and confirmed by analytical methods. Similarly, accumulation of ß-carotene and lycopene in the fruit pulp after NOP-1 application was delayed, without having impact on the total pigment concentration in the completely ripe fruits. Likewise, the peptide had no negative effects on fruit quality. Our molecular and phenotypic studies reveal that peptide biologicals could contribute to the development of a novel family of ripening inhibitors and innovative ripening control in climacteric fruit.

Acclimations to light quality on plant and leaf level affect the vulnerability of pepper (Capsicum annuum L.) to water deficit.

We investigated the influence of light quality on the vulnerability of pepper plants to water deficit. For this purpose plants were cultivated either under compact fluorescence lamps (CFL) or light-emitting diodes (LED) providing similar photon fluence rates (95 µmol m(-2) s(-1)) but distinct light quality. CFL emit a wide-band spectrum with dominant peaks in the green and red spectral region, whereas LEDs offer narrow band spectra with dominant peaks at blue (445 nm) and red (665 nm) regions. After one-week acclimation to light conditions plants were exposed to water deficit by withholding irrigation; this period was followed by a one-week regeneration period and a second water deficit cycle. In general, plants grown under CFL suffered more from water deficit than plants grown under LED modules, as indicated by the impairment of the photosynthetic efficiency of PSII, resulting in less biomass accumulation compared to respective control plants. As affected by water shortage, plants grown under CFL had a stronger decrease in the electron transport rate (ETR) and more pronounced increase in heat dissipation (NPQ). The higher amount of blue light suppressed plant growth and biomass formation, and consequently reduced the water demand of plants grown under LEDs. Moreover, pepper plants exposed to high blue light underwent adjustments at chloroplast level (e.g., higher Chl a/Chl b ratio), increasing the photosynthetic performance under the LED spectrum. Differently than expected, stomatal conductance was comparable for water-deficit and control plants in both light conditions during the stress and recovery phases, indicating only minor adjustments at the stomatal level. Our results highlight the potential of the target-use of light quality to induce structural and functional acclimations improving plant performance under stress situations.

Concentration of hinokinin, phenolic acids and flavonols in leaves and stems of Hydrocotyle leucocephala is differently influenced by PAR and ecologically relevant UV-B level.

We examined the effects of ambient, non-stressing ultraviolet (UV)-B (280-315nm) level combined with different intensities of photosynthetic active radiation (PAR, 400-700nm) on the accumulation of the lignan (-)-hinokinin, in leaves and stems of Hydrocotyle leucocephala. Plants were exposed in sun simulators under almost natural irradiance and climatic conditions to one of four light regimes, i.e. two PAR intensities (906 and 516µmolm(-2)s(-1)) including or excluding UV-B radiation (0 and 0.4Wm(-2)). Besides hinokinin, we identified three chlorogenic acid isomers, one other phenolic acid, 12 quercetin, and five kaempferol derivatives in the H. leucocephala extracts. Hinokinin was most abundant in the stems, and its accumulation was slightly enhanced under UV-B exposure. We therefore assume that hinokinin contributes to cell wall stabilization and consequently to a higher resistance of the plant to environmental factors. Quercetin derivatives increasingly accumulated under UV-B and high PAR exposure at the expense of kaempferols and chlorogenic acids, which was apparently related to its ability to scavenge reactive oxygen species. In general, the concentration of the constituents depended on the plant organ, the leaf age, the light regimes, and the duration of exposure. The distribution pattern of the compounds within the examined organs was not influenced by the treatments. Based on the chemical composition of the extracts a principal component analysis (PCA) enabled a clear separation of the plant organs and harvesting dates. Younger leaves mostly contained higher phenylpropanoid concentrations than older leaves. Nevertheless, more pronounced effects of the light regimes were detected in older leaves. As assessed, in many cases the individual compounds responded differently to the PAR/UV-B combinations, even within the same phenylpropanoid class. Since this is the first report on the influence of light conditions on the accumulation of lignans in herbaceous plants, it opens many perspectives for a more precise elucidation of all involved biochemical and molecular processes.

Proximal sensing of plant-pathogen interactions in spring barley with three fluorescence techniques.

In the last years fluorescence spectroscopy has come to be viewed as an essential approach in key research fields of applied plant sciences. However, the quantity and particularly the quality of information produced by different equipment might vary considerably. In this study we investigate the potential of three optical devices for the proximal sensing of plant-pathogen interactions in four genotypes of spring barley. For this purpose, the fluorescence lifetime, the image-resolved multispectral fluorescence and selected indices of a portable multiparametric fluorescence device were recorded at 3, 6, and 9 days after inoculation (dai) from healthy leaves as well as from leaves inoculated with powdery mildew (Blumeria graminis) or leaf rust (Puccinia hordei). Genotype-specific responses to pathogen infections were revealed already at 3 dai by higher fluorescence mean lifetimes in the spectral range from 410 to 560 nm in the less susceptible varieties. Noticeable pathogen-induced modifications were also revealed by the 'Blue-to-Far-Red Fluorescence Ratio' and the 'Simple Fluorescence Ratio'. Particularly in the susceptible varieties the differences became more evident in the time-course of the experiment i.e., following the pathogen development. The relevance of the blue and green fluorescence to exploit the plant-pathogen interaction was demonstrated by the multispectral fluorescence imaging system. As shown, mildewed leaves were characterized by exceptionally high blue fluorescence, contrasting the values observed in rust inoculated leaves. Further, we confirm that the intensity of green fluorescence depends on the pathogen infection and the stage of disease development; this information might allow a differentiation of both diseases. Moreover, our results demonstrate that the detection area might influence the quality of the information, although it had a minor impact only in the current study. Finally, we highlight the relevance of different excitation-emission channels to better understand and evaluate plant-physiological alterations due to pathogen infections.

Ecologically relevant UV-B dose combined with high PAR intensity distinctly affect plant growth and accumulation of secondary metabolites in leaves of Centella asiatica L. Urban.

We investigated the effects of environmentally relevant dose of ultraviolet (UV)-B and photosynthetic active radiation (PAR) on saponin accumulation in leaves on the example of Centella asiatica L. Urban. For this purpose, plants were exposed to one of four light regimes i.e., two PAR intensities with or without UV-B radiation. The experiment was conducted in technically complex sun simulators under almost natural irradiance and climatic conditions. As observed, UV-B radiation increased herb and leaf production as well as the content of epidermal flavonols, which was monitored by non-destructive fluorescence measurements. Specific fluorescence indices also indicate an increase in the content of anthocyanins under high PAR; this increase was likewise observed for the saponin concentrations. In contrast, UV-B radiation had no distinct effects on saponin and sapogenin concentrations. Our findings suggest that besides flavonoids, also saponins were accumulated under high PAR protecting the plant from oxidative damage. Furthermore, glycosylation of sapogenins seems to be important either for the protective function and/or for compartmentalization of the compounds. Moreover, our study revealed that younger leaves contain higher amounts of saponins, while in older leaves the sapogenins were the most abundant constituents. Concluding, our results proof that ambient dose of UV-B and high PAR intensity distinctly affect the accumulation of flavonoids and saponins, enabling the plant tissue to adapt to the light conditions.

Centelloside accumulation in leaves of Centella asiatica is determined by resource partitioning between primary and secondary metabolism while influenced by supply levels of either nitrogen, phosphorus or potassium.

In the present study we aimed to investigate the relevance of either N, P or K supply for herb and leaf yield and for centelloside concentrations in Centella asiatica L. Urban leaves. In this regard, we elucidated the causal relationship between assimilation rate, leaf N, P and K concentrations, herb and leaf production, and centelloside accumulation. The experiments were conducted consecutively in a greenhouse where C. asiatica was grown in hydroponic culture and fertigated with nutrient solutions at either 0, 30, 60, 100 or 150% of the N, P or K amount in a standard Hoagland solution. In general, the increase in N, P or K supply enhanced assimilation rate and herb and leaf yield. However, exceeding specific thresholds, the high availability of one single nutrient caused lower leaf N concentrations and a decline in assimilation rate and plant growth. Irrespective of N, P and K supply, the leaf centelloside concentrations were negatively associated with herb and leaf yield, which is in accordance with the assumptions of the carbon/nutrient balance and the growth differentiation balance hypotheses. Moreover, we found strong negative correlations between saponins and leaf N concentrations, while the respective sapogenins were negatively correlated with K concentrations. Using C. asiatica as model system, our experiments reveal for the first time that the accumulation of saponins and sapogenins is affected by resource allocation between primary and secondary metabolism and that besides carbon, also nutrient availability is relevant for the regulation of the centelloside synthesis. Finally, our results highlight the huge potential of optimized and carefully controlled mineral nutrition of medicinal plants for steering the bio-production of high-quality natural products.

Presymptomatic detection of powdery mildew infection in winter wheat cultivars by laser-induced fluorescence.

The sensor-based monitoring of diseases under controlled conditions establishes an objective tool that allows a better understanding of the pathogen-plant interactions in different situations. The purpose of our work was to implement the presymptomatic detection of powdery mildew on wheat leaves shortly after fungus inoculation by spectral and time-resolved laser-induced fluorescence spectroscopy. In the general scope of plant phenotyping, we hypothesized that it is possible to discriminate between wheat genotypes that are either resistant or susceptible to powdery mildew. According to our results, the presymptomatic detection of powdery mildew on wheat leaves was accomplished, irrespective of genotype, as early as one day after inoculation using the fluorescence amplitude ratio F451:F522. Similarly, the ratios F451:F522, F522:F687, and F522:F736 of the half-bandwidth are also appropriate parameters. Furthermore, in the spectral range between 410 nm and 620 nm, the mean lifetime was significantly longer in inoculated leaves than it was in control leaves. Finally, the short-term (10-12 hour) increase of the fluorescence mean lifetime at 530 nm and 560 nm following the inoculation suggests that the speed of the plant reaction might be associated to its resistance to the pathogen. Based on this information, we conclude that determinations of ultraviolet, laser-induced fluorescence intensity and lifetime are suitable approaches to presymptomatically detect powdery mildew on wheat leaves one day after inoculation.

Effects of relative humidity and substrate on the spatial association between glyphosate and ethoxylated seed oil adjuvants in the dried deposits of sessile droplets.

BACKGROUND: In recent years, several studies have shown the impact of adjuvants on the characteristics of herbicide deposits on leaf surfaces. Until now, most studies have addressed the distribution of active ingredients (AIs), whereas few experiments have focused on the location of the adjuvants. The objective of this study was a systematic examination of the particle distribution profile of both the AI (glyphosate, Gly) and the adjuvants after the application of sessile microdroplets on hydrophobic (Teflon) and hydrophilic (glass and aluminium) model surfaces. RESULTS: The association degree (AD) was surface dependent and specific for the tested adjuvants. In general, the rather hydrophobic adjuvant RSO 5 showed decreasing AD with Gly at increasing relative humidity (RH) levels. The rather hydrophilic RSO 60 adjuvant displayed higher AD between the compounds at a higher RH. A high concentration of the adjuvant reduced the AD for both of the RSO adjuvants evaluated. CONCLUSION: The combination of surface properties, the type of adjuvant and the relative humidity determines the degree of association between Gly and the adjuvants. The present results suggest that the interaction between the AI and an adjuvant determines whether spatial separation occurs, whereas physical processes (e.g. capillary particle movement, inward and outward Marangoni flows and the evaporation rate) are decisive for the extent of the separation. Coffee-ring structures were formed exclusively with the adjuvant+Gly mixtures, whereas Gly alone formed either one big deposit or several small islands distributed within the droplet footprint.

Use of blue-green and chlorophyll fluorescence measurements for differentiation between nitrogen deficiency and pathogen infection in winter wheat.

In recent years, several sensor-based approaches have been established to early detect single plant stresses, but the challenge of discriminating between simultaneously occurring stressors still remains. Earlier studies on wheat plants strongly affected by pathogens and nitrogen deficiency indicated that chlorophyll fluorescence might be suited to distinguish between the two stressors. Nevertheless, there is lack of information on the pre-symptomatic detection of synchronized occurrence of slight N-deficiency and the early stages of pathogen infection. The usefulness of the blue, green, and yellow fluorescence signals in this context has not yet been explored. We hypothesized that differentiation between wheat plants' physiological reaction due to N-deficiency and leaf rust (Puccinia triticina) as well as N-deficiency and powdery mildew (Blumeria graminis f. sp. tritici) might be accomplished by means of UV laser-induced fluorescence spectral measurements between 370 and 620nm in addition to chlorophyll fluorescence (640-800nm). Plants were provided with either a normal or a modified Hoagland nutrient solution in order to induce a slight N deficit. Pathogen inoculation was carried out on the second fully developed leaf. Four experimental groups were evaluated: (a) N-full-supply [N+]; (b) N-deficiency [N-]; (c) N-full-supply+pathogen [N+/LR] or [N+/PM]; (d) N-deficiency+pathogen [N-/LR] or [N-/PM]. The results revealed that, in addition to the amplitude ratio of R/FR fluorescence, B/G fluorescence also facilitated reliable and robust discrimination among the four experimental groups. The discrimination among the experimental groups was accomplished as early as one and two days after inoculation for powdery mildew and leaf rust infection, respectively. During the 3days evaluation period, the differences among the treatment groups became more evident. Moreover, several other amplitude ratios and half-bandwidth ratios proved to be suited to early detect fungal infection, irrespective of the nitrogen status of the plant.

UV-induced fluorescence spectra and lifetime determination for detection of leaf rust (Puccinia triticina) in susceptible and resistant wheat (Triticum aestivum) cultivars.

In modern agriculture, the use of cultivars that are resistant against specific stresses, e.g. pathogen infections, is an integral component. Considering the great demand for a rapid and objective screening method for stress resistance of new cultivars, the question arises, whether time resolved fluorescence spectroscopy is suitable for such purposes. Amongst others, infected plants might accumulate specific compounds such as salicylic acid and phenylpropanoid compounds as key substances in plant disease resistance, whereas synthesis and accumulation may influence fluorescence parameters such as absolute intensity of single peaks, ratios between peaks and lifetime. Experiments were conducted in a controlled-environment cabinet cultivating four leaf rust susceptible and three leaf rust resistant genotypes. Fluorescence measurements were conducted using a compact fibre-optic fluorescence spectrometer with a nanosecond time-resolution. Results of experiments revealed that UV-induced measurements of spectral characteristics as well as determination of fluorescence lifetime are suited to detect leaf rust (Puccinia triticina) in wheat (Triticum aestivum L.) cultivars as early as 2 days after inoculation (dai). For this purpose several parameters such as the fluorescence (F) amplitude ratios F451/F522, F451/F687, F451/F736, F522/F687, F522/F736 as well as fluorescence mean lifetime especially at 470nm, might be used. Discrimination between resistant and susceptible cultivars to the leaf rust pathogen could be accomplished 3dai by using the ratio of fluorescence amplitude between the blue (F451nm) and red (F687nm) peak, and mean lifetime at 440, 500 and 530nm. Our results indicate that the combination of spectrally and time-resolved fluorescence could be an additional tool in plant breeding programs for an automatic and precise high-throughput system for evaluation of the pathogen resistance of new genotypes.

Ultraviolet-B and photosynthetically active radiation interactively affect yield and pattern of monoterpenes in leaves of peppermint (Mentha x piperita L.).

Solar radiation is a key environmental signal in regulation of plant secondary metabolism. Since metabolic responses to light and ultraviolet (UV) radiation exposure are known to depend on the ratio of spectral ranges (e.g., UV-B/PAR), we examined effects of different UV-B radiation (280-315 nm) and photosynthetically active radiation (PAR, 400-700 nm) levels and ratios on yield and pattern of monoterpenoid essential oil of peppermint. Experiments were performed in exposure chambers, technically equipped for realistic simulation of natural climate and radiation. The experimental design comprised four irradiation regimes created by the combination of two PAR levels including or excluding UV-B radiation. During flowering, the highest essential oil yield was achieved at high PAR (1150 micromol m(-2) s(-1)) and approximate ambient UV-B radiation (0.6 W m(-2)). Regarding the monoterpene pattern, low PAR (550 micromol m(-2) s(-1)) and the absence of UV-B radiation led to reduced menthol and increased menthone contents and thereby to a substantial decrease in oil quality. Essential oil yield could not be correlated with density or diameter of peltate glandular trichomes, the epidermal structures specialized on biosynthesis, and the accumulation of monoterpenes. The present results lead to the conclusion that production of high quality oils (fulfilling the requirements of the Pharmacopoeia Europaea) requires high levels of natural sunlight. In protected cultivation, the use of UV-B transmitting covering materials is therefore highly recommended.

Does the microclimate under hail nets influence micromorphological characteristics of apple leaves and cuticles?

A higher frequency of hail storms, possibly due to climate change, has led to increased installation of hail nets worldwide. The objective of the present work was to investigate potential effects of the microclimate under these hail nets on micromorphological characteristics of the leaves and adaxial leaf cuticles. Leaves of apple cultivars 'Pinova' and 'Fuji' grown on trees under white (highly translucent) or red-black (low transmittance) hail nets or on uncovered (control) trees were evaluated in June, August, September and October. The microclimate under the colored hail nets had no impact on leaf micromorphology, amount of cuticular wax, or leaf thickness. Similarly, no differences in thickness and permeability for calcium could be established between cuticles of leaves grown on trees under the two types of hail nets or uncovered trees. For all evaluated parameters, significant differences were detected between the two cultivars examined. In both cultivars, leaf wax synthesis followed a characteristic curve, increasing from the first to the second evaluation, and then decreasing continuously without affecting cuticular penetration of calcium. Overall, our results show that a reduction of the hail nets by 6-10% in both light and humidity was insufficient to influence the surface properties of apple leaves and permeability of cuticles. This may suggest that pest management strategies, i.e. formulation of agrochemicals, their application and dose, do not need to be adapted when used under hail nets. Overall, the present results indicate that the microclimatic changes brought about by colored hail nets are sufficient to enhance the vegetative growth and induce the 'shade avoidance syndrome', but do not appear to affect the leaf cuticular properties.

Spatially resolved quantification of agrochemicals on plant surfaces using energy dispersive X-ray microanalysis.

BACKGROUND: In the present study the principle of energy dispersive X-ray microanalysis (EDX), i.e. the detection of elements based on their characteristic X-rays, was used to localise and quantify organic and inorganic pesticides on enzymatically isolated fruit cuticles. Pesticides could be discriminated from the plant surface because of their distinctive elemental composition. RESULTS: Findings confirm the close relation between net intensity (NI) and area covered by the active ingredient (AI area). Using wide and narrow concentration ranges of glyphosate and glufosinate, respectively, results showed that quantification of AI requires the selection of appropriate regression equations while considering NI, peak-to-background (P/B) ratio, and AI area. The use of selected internal standards (ISs) such as Ca(NO(3))(2) improved the accuracy of the quantification slightly but led to the formation of particular, non-typical microstructured deposits. The suitability of SEM-EDX as a general technique to quantify pesticides was evaluated additionally on 14 agrochemicals applied at diluted or regular concentration. CONCLUSION: Among the pesticides tested, spatial localisation and quantification of AI amount could be done for inorganic copper and sulfur as well for the organic agrochemicals glyphosate, glufosinate, bromoxynil and mancozeb.

Surfactant-induced deposit structures in relation to the biological efficacy of glyphosate on easy- and difficult-to-wet weed species.

BACKGROUND: Typical active ingredient (AI) residue patterns are formed during droplet drying on plant surfaces owing to the interaction of spray solution characteristics and leaf micromorphology. Currently, comparatively little is known about the influence of AI deposit patterns within a spray droplet residue area on the penetration and biological efficacy of glyphosate. A scanning electron microscope with energy dispersive X-ray microanalysis has been used to characterise residue patterns and to quantify the area ultimately covered by glyphosate within the droplet spread area. RESULTS: The easy-to-wet weed species Stellaria media L. and Viola arvensis L., as well as the difficult-to-wet Chenopodium album L. and Setaria viridis L., differing in their surface micromorphology, have been used. Rapeseed oil ethoxylates (RSO 5 or RSO 60) were added to glyphosate solutions to provide different droplet spread areas. Addition of RSO 5 enhanced droplet spread area more than RSO 60, and both caused distinct glyphosate residue patterns. The biological efficacy of treatment solutions showed no significant correlation with the area ultimately covered by glyphosate. CONCLUSION: The results have implications on herbicide uptake models. This study shows that droplet spread area does not correspond to the area ultimately covered by glyphosate, and that the latter does not affect glyphosate phytotoxicity.

Retention and rainfastness of mancozeb as affected by physicochemical characteristics of adaxial apple leaf surface after enhanced UV-B radiation.

It is not clear so far whether alteration of leaf micromorphology and surface wax chemistry due to the impact of environmental factors, such as UV-B radiation, affects retention and rainfastness of applied pesticide solutions. In this study; UV-B treated and untreated adaxial leaf surfaces of apple seedlings (Malus domestica Borkh.) were characterized in terms of chemical composition, micromorphological fine structure, hydrophobicity, and wettability. Furthermore, the retention and rainfastness of applied fungicide mancozeb were studied. The samples were examined 0, 24 and 48 h after ultraviolet (UV)-B radiation (0.022 kW m(-2) for 150 min) The total wax mass, recovered from the adaxial leaf surface, amounted from 0.38 microg cm(-2) (control) up to 0.49 microg cm(-2) (24 h). Chemical composition of surface wax altered, whereas the contact angle of applied water droplets on leaf surface of UV-B treated plants did not change significantly compared to the control. The alteration of surface wax quantity and quality significantly affected retention of a.i.; it increased at a sampling time of 24 h after UV-B irradiation, whereas rainfastness of the fungicide spray solution was not significantly influenced.