A LAMP Protocol for the Detection of 'Candidatus Phytoplasma pyri', the Causal Agent of Pear Decline.

Phytoplasmas are cell-wall-less bacteria that cause diseases in approximately 1,000 plant species. 'Candidatus Phytoplasma pyri', the causal agent of pear decline, induces various symptoms on its hosts, leading to weakening and dieback of the plants, reduced fruit size and yield, and, consequently, considerable financial losses in all pear-growing areas. Fighting this disease requires a reliable and inexpensive method for pathogen detection in propagation material as well as plant stocks in orchards and breeding facilities. Here, we present a field-suitable detection protocol for 'Ca. P. pyri' based on loop-mediated isothermal amplification (LAMP) targeting the phytoplasmal 16S ribosomal DNA sequence. The combination of a simplified sample preparation method based on sodium hydroxide and colorimetric visualization of LAMP results enables a laboratory-independent pathogen detection. The detection limit is comparable with analysis by polymerase chain reaction; however, the pear decline LAMP detection method is superior in terms of ease of use, cost, and time effectiveness for obtaining results.

Erwinia amylovora affects the phenylpropanoid-flavonoid pathway in mature leaves of Pyrus communis cv. Conférence.

Flavonoids, which are synthesized by the phenylpropanoid-flavonoid pathway, not only contribute to fruit colour and photoprotection, they also may provide antimicrobial and structural components during interaction with micro-organisms. A possible response of this pathway was assessed in both mature and immature leaves of shoots of 2-year-old pear trees cv. Conférence, which were inoculated with the gram-negative bacterium Erwinia amylovora strain SGB 225/12, were mock-inoculated or were left untreated. The phenylpropanoid-flavonoid pathway was analysed by histological studies, by gene expression using RT-qPCR and by HPLC analyses of the metabolites at different time intervals after infection. Transcription patterns of two key genes anthocyanidin reductase (ANR) and chalcone synthase (CHS) related to the phenylpropanoid-flavonoid pathway showed differences between control, mock-inoculated and E. amylovora-inoculated mature leaves, with the strongest reaction 48 h after inoculation. The impact of E. amylovora was also visualised in histological sections, and confirmed by HPLC, as epicatechin -which is produced via ANR- augmented 72 h after inoculation in infected leaf tissue. Besides the effect of treatments, ontogenesis-related differences were found as well. The increase of certain key genes, the rise in epicatechin and the visualisation in several histological sections in this study suggest a non-negligible impact on the phenylpropanoid-flavonoid pathway in Pyrus communis due to inoculation with E. amylovora. In this study, we propose a potential role of this pathway in defence mechanisms, providing a detailed analysis of the response of this system attributable to inoculation with E. amylovora.

Loss of nuclear flavanols during drought periods in Taxus baccata.

Normally, needles of Taxus baccata during the growth period prominently stain blue for nuclear flavanols with the histochemical DMACA procedure. However, under excess heat and drought conditions, nuclear flavanols of current-year needles decline to zero. Nevertheless, greenish-yellow-coloured flavonols (quercetin derivatives) were still observed in nuclei. All of these yellow nuclei were in a silenced state and without mitosis. This link between drought and loss of nuclear flavanols was found in 3 years, 2003, 2007 and 2010. In 2007, exceptional drought occurred in early spring, interrupted by short rains. This, in turn, led to flushing of new sprouts, a characteristic feature in which nuclei were overloaded with flavanols. By the end of three drought periods, all nuclei developed blue-coloured nuclear flavanols. The flavanols seem to be associated with the histone proteins of chromatin. The oxidative degradation of catechin in Tris buffer (pH 8.0) containing MgCl2 was studied in the presence of the H4-core fragment TYTEHAKRKTVTAMD, modified according to the epigenetic histone code. The results show that catechin degradation can be significantly inhibited by the non-modified peptides and the methylated peptides (methylation at both lysine residues). The acetylated and formylated peptides do not show this behaviour. These observations indicate that flavanol association at chromosomes appears to be regulated by the epigenetic histone code.

Nondestructive application of laser-induced fluorescence spectroscopy for quantitative analyses of phenolic compounds in strawberry fruits (Fragaria x ananassa).

Laser-induced fluorescence spectroscopy (LIFS) was nondestructively applied on strawberries (EX = 337 nm, EM = 400-820 nm) to test the feasibility of quantitatively determining native phenolic compounds in strawberries. Eighteen phenolic compounds were identified in fruit skin by UV and MS spectroscopy and quantitatively determined by use of rp-HPLC for separation and diode-array or chemical reaction detection. Partial least-squares calibration models were built for single phenolic compounds by means of nondestructively recorded fluorescence spectra in the blue-green wavelength range using different data preprocessing methods. The direct orthogonal signal correction resulted in r (2) = 0.99 and rmsep < 8% for p-coumaroyl-glucose, and r (2) = 0.99 and rmsep < 24% for cinnamoyl-glucose. In comparison, the correction of the fluorescence spectral data with simultaneously recorded reflectance spectra did not further improve the calibration models. Results show the potential of LIFS for a rapid and nondestructive assessment of contents of p-coumaroyl-glucose and cinnamoyl-glucose in strawberry fruits.

A dynamical model of environmental effects on allocation to carbon-based secondary compounds in juvenile trees.

BACKGROUND AND AIMS: Patterns and variations in concentration of carbon-based secondary compounds in plant tissues have been explained by means of different complementary and, in some cases, contradictory plant defence hypotheses for more than 20 years. These hypotheses are conceptual models which consider environmental impacts on plant internal demands. In the present study, a mathematical model is presented, which converts and integrates the concepts of the 'Growth-Differentiation Balance' hypothesis and the 'Protein Competition' model into a dynamic plant growth model, that was tested with concentration data of polyphenols in leaves of juvenile apple, beech and spruce trees. The modelling approach is part of the plant growth model PLATHO that considers simultaneously different environmental impacts on the most important physiological processes of plants. METHODS: The modelling approach for plant internal resource allocation is based on a priority scheme assuming that growth processes have priority over allocation to secondary compounds and that growth-related metabolism is more strongly affected by nitrogen deficiency than defence-related secondary metabolism. KEY RESULTS: It is shown that the model can reproduce the effect of nitrogen fertilization on allocation patterns in apple trees and the effects of elevated CO(2) and competition in juvenile beech and spruce trees. The analysis of model behaviour reveals that large fluctuations in plant internal availability of carbon and nitrogen are possible within a single vegetation period. Furthermore, the model displays a non-linear allocation behaviour to carbon-based secondary compounds. CONCLUSIONS: The simulation results corroborate the underlying assumptions of the presented modelling approach for resource partitioning between growth-related primary metabolism and defence-related secondary metabolism. Thus, the dynamical modelling approach, which considers variable source and sink strengths of plant internal resources within different phenological growth stages, presents a successful translation of existing concepts into a dynamic mathematical model.

The influence of polyphenol rich apple pomace or red-wine pomace diet on the gut morphology in weaning piglets.

Alternative food ingredients, e.g. secondary plant compounds, are discussed to have beneficial effects and improve gut health. In this study, the effect of three different diets - normal piglets starter without additives, with apple pomace or with red-wine pomace - on the intestinal morphology was investigated from 3 days prior to weaning to 4 weeks post-weaning. At five time points, six piglets from each treatment group were slaughtered; at first time point only six animals from control group were slaughtered. Villus height, crypt depth and breadth of villi and crypts were determined in the jejunum, ileum and colon in 78 piglets. Additionally, the area of the Peyer's patches in the ileum was measured. In jejunum (p < 0.01) and ileum (p < 0.001) the villus length in the control group decreased after weaning but increased over the entire feeding experiment (p < 0.001). In the two-pomace groups, no decrease was measured after weaning. In jejunum, an increase in villi breadth occurred, 73% in the control group and approximately 10% in both treatment groups. A 35% increase was found in the ileum in all groups. Peyer's patches area increased approximately 21% in the control group over 26 days of treatment, whereas in other groups no significant differences were found. Different polyphenol rich pomaces have diverse effects in the gastrointestinal tract. Red-wine pomace has an inhibitory effect on the jejunum villi growth, whereas apple and red-wine pomace have stimulating effect on crypt size in piglet colon. Apple and red-wine pomace can reduce the GALT activation via the Peyer's patches in the ileum. In conclusion, the flavanoids rich feeding regimen showed positive effects on villi morphology, GALT activation and can improve pig health.

Modification of primary and secondary metabolism of potato plants by nitrogen application differentially affects resistance to Phytophthora infestans and Alternaria solani.

Potato plants ( SOLANUM TUBEROSUM L. cv. Indira) were grown at two levels of N supply in the greenhouse. Plants supplied with 0.8 g N per plant (high N variant) showed significantly increased biomass as compared to plants without additional N fertilisation (low N variant). C/N ratio was lower and protein content was higher in leaves of the high N variant. The concentration of chlorogenic acids and flavonols was significantly lower in leaves from the high N variant. Whereas resistance to ALTERNARIA SOLANI increased when plants were supplied with additional nitrogen, these plants were more susceptible to PHYTOPHTHORA INFESTANS. After infection with both pathogens, we found a strong induction of p-coumaroylnoradrenaline and p-coumaroyloctopamine, which are identified for the first time in potato leaves and are discussed as resistance factors of other solanaceous plants.

Flavanols in somatic cell division and male meiosis of tea (Camellia sinensis) anthers.

Young anthers excised from closed tea flower buds ( Camellia sinensis L.) were stained as fresh tissues with p-dimethylaminocinnamaldehyde reagent to localize flavanols associated with nuclei and chromosomes, apart from those flavanols stored in vacuoles. This staining reagent yields a blue colour for flavanols. In the nonsporogenic somatic cells of developing anthers, flavanols were found to be attached to chromosomes at all mitotic stages. Male meiosis started at a bud size of about 3.5 mm in diameter in pollen mother cells which displayed generally more or less pronounced blue nuclei and cytoplasm. The meiotic divisions from prophase I to telophase II were characterized by blue stained nuclei and chromosomes, but within the cytoplasm there was, if any, a random and very poor reaction for flavanols. Metaphase and telophase of meiotic divisions showed maximally condensed chromosomes staining dark blue. Early in telophase II, the cytoplasm was again stained blue; this faded at late tetrad stage. Flavanols of young mitotic and older non-mitotic anthers were determined using high pressure liquid chromatography--chemical reaction detection (HPLC-CRD). Catechin, epicatechin, B2, and epigallocatechin were minor compounds, whereas epicatechin gallate and epigallocatechin gallate were found in higher amounts. The major flavanol compound of the anthers, epicatechin gallate, exhibited a significant affinity to histone sulphate, as shown by UV-VIS spectroscopic titration.

Significance of flavonoids in plant resistance and enhancement of their biosynthesis.

The roles of flavonoids in plant defence against pathogens, herbivores, and environmental stress are reviewed and their significant contribution to plant resistance is discussed. The induction of flavonoids is of particular interest for gathering evidence of their roles. Tools are mentioned which may enhance flavonoid biosynthesis and accumulation. These include metabolic engineering and UV light. The induction of defence-related flavonoids is modified by other determining factors and competition between growth and secondary metabolism may exist. In an evolutionary context, stress-related oxidative pressure may have been a major trigger for the distribution and abundance of flavonoids. UV protection is one of their most significant, or even the most significant, functional role for flavonoids. The multi-functionality of these compounds, however, often complicates the interpretation of experimental results but, overall, it supports the importance of flavonoids.

The plant's capacity in regulating resource demand.

Regulation of resource allocation in plants is the key to integrate understanding of metabolism and resource flux across the whole plant. The challenge is to understand trade-offs as plants balance allocation between different and conflicting demands, e.g., for staying competitive with neighbours and ensuring defence against parasites. Related hypothesis evaluation can, however, produce equivocal results. Overcoming deficits in understanding underlying mechanisms is achieved through integrated experimentation and modelling the various spatio-temporal scaling levels, from genetic control and cell metabolism towards resource flux at the stand level. An integrated, interdisciplinary research concept on herbaceous and woody plants and its outcome to date are used, while drawing attention to currently available knowledge. This assessment is based on resource allocation as driven through plant-pathogen and plant-mycorrhizosphere interaction, as well as competition with neighbouring plants in stands, conceiving such biotic interactions as a "unity" in the control of allocation. Biotic interaction may diminish or foster effects of abiotic stress on allocation, as changes in allocation do not necessarily result from metabolic re-adjustment but may obey allometric rules during ontogeny. Focus is required on host-pathogen interaction under variable resource supply and disturbance, including effects of competition and mycorrhization. Cost/benefit relationships in balancing resource investments versus gains turned out to be fundamental in quantifying competitiveness when related to the space, which is subject to competitive resource exploitation. A space-related view of defence as a form of prevention of decline in competitiveness may promote conversion of resource turnover across the different kinds of biotic interaction, given their capacity in jointly controlling whole plant resource allocation.

Growth-promoting nitrogen nutrition affects flavonoid biosynthesis in young apple (Malus domestica Borkh.) leaves.

Enhanced shoot growth and a decrease in flavonoid concentration in apple trees grown under high nitrogen (N) supply was observed in previous studies, along with increasing scab susceptibility of cultivar "Golden Delicious" after high N nutrition. Several hypotheses have suggested that there is a trade-off between primary and secondary metabolism because of competition for common substrates, but nothing is known about regulation at the enzyme level. In this study, a set of experiments was performed to elucidate the effect of N nutrition on the activities of key enzymes involved in flavonoid biosynthesis (phenylalanine ammonia-lyase [PAL], chalcone synthase/chalcone isomerase [CHS/CHI}, flavanone 3-hydroxylase [FHT], flavonol synthase [FLS], dihydroflavonol 4-reductase [DFR]) and the accumulation of different groups of phenylpropanoids. The inhibition of flavonoid accumulation by high N nutrition could be confirmed, but the influence of N supply on the flavonoid enzymes CHS/CHI, FHT, DFR, and FLS was not evident. However, PAL activity seems to be downregulated, thus forming a bottleneck resulting in a generally decreased flavonoid accumulation. Furthermore, the response of the scab-resistant cultivar "Rewena" to high N nutrition was not as strong as that of the susceptible cultivar "Golden Delicious".

Flavonoid biosynthesis in the tea plant Camellia sinensis: properties of enzymes of the prominent epicatechin and catechin pathways.

Leaves of tea (Camellia sinensis L.) contain extraordinary large amounts of (-)-epigallocatechin, (-)-epicatechin, (+)-gallocatechin, and (+)-catechin and derivatives of these compounds that show positive effects on human health. The health-promoting effects of flavan 3-ols, especially those of green tea, are of scientific and public interest. Furthermore, they play a crucial role in defense against pathogens of tea. Therefore, biosynthesis of these flavonoid compounds was investigated. The anthocyanidin reductase enzyme recently described from Arabidopsis and Medicago was shown to be present in tea with very high activity and produces epicatechin as well as epigallocatechin from the respective anthocyanidins, thus explaining the very high contents of these compounds. A strong combined dihydroflavonol 4-reductase/leucoanthocyanidin 4-reductase activity was demonstrated and catalyzes the key steps in catechin and gallocatechin formation. Together with the enzyme activities and substrate specificities of the preceding enzymatic reactions, the biosynthesis of the most prominent flavonoids of tea is elucidated.

Flavanol binding of nuclei from tree species.

Light microscopy was used to examine the nuclei of five tree species with respect to the presence of flavanols. Flavanols develop a blue colouration in the presence of a special p-dimethylaminocinnamaldehyde (DMACA) reagent that enables those nuclei loaded with flavanols to be recognized. Staining of the nuclei was most pronounced in both Tsuga canadensis and Taxus baccata, variable in Metasequoia glyptostroboides, faint in Coffea arabica and minimal in Prunus avium. HPLC analysis showed that the five species contained substantial amounts of different flavanols such as catechin, epicatechin and proanthocyanidins. Quantitatively, total flavanols were quite different among the species. The nuclei themselves, as studied in Tsuga seed wings, were found to contain mainly catechin, much lower amounts of epicatechin and traces of proanthocyanidins. Blue-coloured nuclei located centrally in small cells were often found to maximally occupy up to 90% of a cell's radius, and the surrounding small rim of cytoplasm was visibly free of flavanols. A survey of 34 gymnosperm and angiosperm species indicated that the first group has much higher nuclear binding capacities for flavanols than the second group.

Role of flavanols in yellowing beech trees of the Black Forest.

Beech leaves were sampled at the end of a prolonged hot dry period at a tree decline site in the Black Forest, Germany to investigate the potential role of flavanols in defense mechanisms against environmental stress. Green and yellowing leaves were harvested from the uppermost canopy of trees that were more than 200 years old and 30 m high. Yellowing leaves had a 7.4-fold higher concentration of total flavanols than green leaves. Green leaves contained flavanol inclusions, but during yellowing the inclusions disintegrated and the cells became filled with flavanols. Abscisic acid (ABA) stimulated the release of flavanols from intravacuolar inclusions of leaf petioles and flower pedicels. In addition, ABA caused flavanols to leach from the trichomes of beech galls. The antioxidative potential of leaf extracts, as estimated by indoleacetic acid (IAA) oxidation, was significantly higher in yellowing leaves than in green leaves. In vitro experiments revealed that (+)-catechin promoted growth of beech tissue.

Developmental changes in the phenol concentrations of 'golden delicious' apple fruits and leaves.

The phenolic content of apple fruit skin and leaves was determined at the developmental stage of each organ. Phenolic levels decreased on a dry weight basis during the seasonal development of fruits and leaves with respect to their ontogenesis but the single compounds did not behave uniformly. A shift in flavanol pools from monomeric to oligomeric structures during fruit growth indicated the biosynthetic tendency towards the formation of procyanidins at the end of the growing period. Among the procyanidins identified epicatechin-(4 beta,6)-epicatechin-(4 beta,6)-epicatechin has not been reported previously from apple.

Accumulation of flavanols in yellowing beech leaves from forest decline sites.

Beech leaves were sampled during two consecutive years from three sites characterized by forest decline. Both monomeric and oligomeric flavanols from green and yellowing leaves were determined quantitatively by reversed-phase, high-performance liquid chromatography (HPLC) combined with a chemical reaction detection technique (CRD). Yellowing leaves generally contained more than twice the quantity of flavanols than normal green leaves. The monomeric flavanols, epicatechin and (+)-catechin, comprised up to 80% of the total flavanol fraction. Histochemical staining with p-dimethylaminocinnamaldehyde was used to determine local deposition of flavanols, including insoluble oligomeric flavanols, in leaf tissues. The yellowing leaves stained intensely, whereas the green leaves stained lightly. Flavanol staining was strongest in the spongy parenchyma followed by the palisade cells. The upper epidermis stained more intensely than the lower epidermis. A 4-day treatment of small leaf pieces (5 x 5 mm) with 0.04 mM paraquat resulted in browning of the leaf pieces. The browning reaction was prevented when the paraquat treatment was carried out in the presence of 0.16 mM (+)-catechin, indicating an antioxidative property for this flavanol.

Localization and quantitative determination of catechins and proanthocyanidins in the phloem of elm and cherry.

The localization of catechins and proanthocyanidins in the phloem of elm (Ulmus minor Mill.) and cherry (Prunus avium L.) was determined histochemically by use of 4-dimethylaminocinnamaldehyde (DMACA) reagent. The two tree species showed a characteristic distribution of these phenols, which were most abundant in the phloem rays of cherry, but were largely confined to upright parenchyma cells of elm. Quantitative determination and qualitative separation of catechins and proanthocyanidins were performed using HPLC-CRD (chemical reaction detection), with 4-dimethylaminocinnamaldehyde as the staining reagent. Up to 14 different catechins and proanthocyanidins were found in both species.

Inhibition of growth and xylogenesis and promotion of vacuolation in prunus callus by the flavanone prunin.

Callus tissue of Prunus avium L. responded to supplied prunin (naringen in 7-glucoside) showing vaculoation and storage of oligomeric proanthocyanidins. In addition, prunin caused restricted callus initiation and/or less callus growth. When prunin was omitted from the medium numerous tracheids and more peroxidases were formed in the callus.