Genome-Informed Design of a LAMP Assay for the Specific Detection of the Strain of 'Candidatus Phytoplasma asteris' Phytoplasma Occurring in Grapevines in South Africa.

Grapevine yellows is one of the most damaging phytoplasma-associated diseases worldwide. It is linked to several phytoplasma species, which can vary regionally due to phytoplasma and insect-vector diversity. Specific, rapid, and reliable detection of the grapevine yellows pathogen has an important role in phytoplasma control. The purpose of this study was to develop and validate a specific loop-mediated isothermal amplification (LAMP) assay for detection of a distinct strain of grapevine 'Candidatus Phytoplasma asteris' that is present in South Africa, through implementation of a genome-informed test design approach. Several freely available, user-friendly, web-based tools were coupled to design the specific LAMP assays. The criteria for selection of the assays were set for each step of the process, which resulted in four experimentally operative LAMP assays that targeted the ftsH/hflB gene region, specific to the aster yellows phytoplasma strain from South Africa. A real-time PCR was developed, targeting the same genetic region, to provide extensive validation of the LAMP assay. The validated molecular assays are highly specific to the targeted aster yellows phytoplasma strain from South Africa, with good sensitivity and reproducibility. We show a genome-informed molecular test design and an efficient validation approach for molecular tests if reference and sample materials are sparse and hard to obtain.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Differential Response of Grapevine to Infection with 'Candidatus Phytoplasma solani' in Early and Late Growing Season through Complex Regulation of mRNA and Small RNA Transcriptomes.

Bois noir is the most widespread phytoplasma grapevine disease in Europe. It is associated with 'Candidatus Phytoplasma solani', but molecular interactions between the causal pathogen and its host plant are not well understood. In this work, we combined the analysis of high-throughput RNA-Seq and sRNA-Seq data with interaction network analysis for finding new cross-talks among pathways involved in infection of grapevine cv. Zweigelt with 'Ca. P. solani' in early and late growing seasons. While the early growing season was very dynamic at the transcriptional level in asymptomatic grapevines, the regulation at the level of small RNAs was more pronounced later in the season when symptoms developed in infected grapevines. Most differentially expressed small RNAs were associated with biotic stress. Our study also exposes the less-studied role of hormones in disease development and shows that hormonal balance was already perturbed before symptoms development in infected grapevines. Analysis at the level of communities of genes and mRNA-microRNA interaction networks revealed several new genes (e.g., expansins and cryptdin) that have not been associated with phytoplasma pathogenicity previously. These novel actors may present a new reference framework for research and diagnostics of phytoplasma diseases of grapevine.

Determination of the Activity Signature of Key Carbohydrate Metabolism Enzymes in Phenolic-rich Grapevine Tissues.

Physiological studies in plants often require enzyme extraction from tissues containing high concentrations of phenols and polyphenols. Unless removed or neutralized, such compounds may hinder extraction, inactivate enzymes, and interfere with enzyme detection. The following protocol for activity assays for enzymes of primary carbohydrate metabolism, while based on our recently published one for quantitative measurement of activities using coupled spectrophotometric assays in a 96-well format, is tailored to the complexities of phenolic- and anthocyanin-rich extracts from grapevine leaf. As a case study we applied the protocol to grapevine leaf samples infected with plant pathogenic bacteria 'Candidatus Phytoplasma solani', known to alter carbohydrate metabolism in grapevine. The described adaptations may be useful for determination of metabolic fingerprints for physiological phenotyping of other plant species with inherently high levels of phenolic compounds.

Programmed cell death occurs asymmetrically during abscission in tomato.

Abscission occurs specifically in the abscission zone (AZ) tissue as a natural stage of plant development. Previously, we observed delay of tomato (Solanum lycopersicum) leaf abscission when the LX ribonuclease (LX) was inhibited. The known association between LX expression and programmed cell death (PCD) suggested involvement of PCD in abscission. In this study, hallmarks of PCD were identified in the tomato leaf and flower AZs during the late stage of abscission. These included loss of cell viability, altered nuclear morphology, DNA fragmentation, elevated levels of reactive oxygen species and enzymatic activities, and expression of PCD-associated genes. Overexpression of antiapoptotic proteins resulted in retarded abscission, indicating PCD requirement. PCD, LX, and nuclease gene expression were visualized primarily in the AZ distal tissue, demonstrating an asymmetry between the two AZ sides. Asymmetric expression was observed for genes associated with cell wall hydrolysis, leading to AZ, or associated with ethylene biosynthesis, which induces abscission. These results suggest that different abscission-related processes occur asymmetrically between the AZ proximal and distal sides. Taken together, our findings identify PCD as a key mechanism that occurs asymmetrically during normal progression of abscission and suggest an important role for LX in this PCD process.

Candidate pathogenicity factor/effector proteins of 'Candidatus Phytoplasma solani' modulate plant carbohydrate metabolism, accelerate the ascorbate-glutathione cycle, and induce autophagosomes.

The pathogenicity of intracellular plant pathogenic bacteria is associated with the action of pathogenicity factors/effectors, but their physiological roles for most phytoplasma species, including 'Candidiatus Phytoplasma solani' are unknown. Six putative pathogenicity factors/effectors from six different strains of 'Ca. P. solani' were selected by bioinformatic analysis. The way in which they manipulate the host cellular machinery was elucidated by analyzing Nicotiana benthamiana leaves after Agrobacterium-mediated transient transformation with the pathogenicity factor/effector constructs using confocal microscopy, pull-down, and co-immunoprecipitation, and enzyme assays. Candidate pathogenicity factors/effectors were shown to modulate plant carbohydrate metabolism and the ascorbate-glutathione cycle and to induce autophagosomes. PoStoSP06, PoStoSP13, and PoStoSP28 were localized in the nucleus and cytosol. The most active effector in the processes studied was PoStoSP06. PoStoSP18 was associated with an increase in phosphoglucomutase activity, whereas PoStoSP28, previously annotated as an antigenic membrane protein StAMP, specifically interacted with phosphoglucomutase. PoStoSP04 induced only the ascorbate-glutathione cycle along with other pathogenicity factors/effectors. Candidate pathogenicity factors/effectors were involved in reprogramming host carbohydrate metabolism in favor of phytoplasma own growth and infection. They were specifically associated with three distinct metabolic pathways leading to fructose-6-phosphate as an input substrate for glycolysis. The possible significance of autophagosome induction by PoStoSP28 is discussed.

Genome Size of Life Forms of Araceae-A New Piece in the C-Value Puzzle.

The genome size of an organism is an important trait that has predictive values applicable to various scientific fields, including ecology. The main source of plant C-values is the Plant DNA C-values database of the Royal Botanic Gardens Kew, which currently contains 12,273 estimates. However, it covers only 2.9% of known angiosperm species and has gaps in the life form and geographic distribution of plants. Only 4.5% of C-value estimates come from researchers in Central and South America. This study provides 41 new C-values for the aroid family (Araceae), collected in the Piedras Blancas National Park area in southern Costa Rica, including terrestrial, epiphytic and aquatic life forms. Data from our study are combined with C-value entries in the RBGK database for Araceae. The analysis reveals a wider range of C-values for terrestrial aroids, consistent with other terrestrial plants, a trend toward slightly lower C-values for epiphytic forms, which is more consistent for obligate epiphytes, and comparatively low C-values for aquatic aroids.

Geographical and Temporal Diversity of 'Candidatus Phytoplasma solani' in Wine-Growing Regions in Slovenia and Austria.

As the causal agent of the grapevine yellows disease Bois noir, 'Candidatus Phytoplasma solani' has a major economic impact on grapevines. To improve the control of Bois noir, it is critical to understand the very complex epidemiological cycles that involve the multiple "Ca. P. solani" host plants and insect vectors, of which Hyalesthes obsoletus is the most important. In the present study, multiple genotyping of the tuf, secY, stamp, and vmp1 genes was performed. This involved archived grapevine samples that were collected during an official survey of grapevine yellows throughout the wine-growing regions of Slovenia (from 2003 to 2016), plus samples from Austrian grapevines, stinging nettle, field bindweed, and insect samples (collected from 2012 to 2019). The data show that the tuf-b2 type of the tuf gene has been present in eastern Slovenia since at least 2003. The hypotheses that the occurrence of the haplotypes varies due to the geographical position of Slovenia on the Italian-Slovenian Karst divide and that the haplotypes are similar between Slovenian and Austrian Styria were confirmed. The data also show haplotype changes for host plants and H. obsoletus associated with 'Ca. P. solani,' which might be linked to new epidemiological cycles of this phytoplasma that involve not just new plant sources and new insect vectors, but also climate and land-use changes.

Spatial and temporal profiles of cytokinin biosynthesis and accumulation in developing caryopses of maize.

BACKGROUND AND AIMS: Cytokinins are a major group of plant hormones and are associated with various developmental processes. Developing caryopses of maize have high levels of cytokinins, but little is known about their spatial and temporal distribution. The localization and quantification of cytokinins was investigated in maize (Zea mays) caryopsis from 0 to 28 d after pollination together with the expression and localization of isopentenyltransferase ZmIPT1 involved in cytokinin biosynthesis and ZmCNGT, the gene putatively involved in N9-glucosylation. METHODS: Biochemical, cellular and molecular approaches resolved the overall cytokinin profiles, and several gene expression assays were used for two critical genes to assess cytokinin cell-specific biosynthesis and conversion to the biologically inactive form. Cytokinins were immunolocalized for the first time in maize caryopses. KEY RESULTS: During the period 0-28 d after pollination (DAP): (1) large quantities of cytokinins were detected in the maternal pedicel region relative to the filial tissues during the early stages after fertilization; (2) unpollinated ovules did not accumulate cytokinins; (3) the maternal nucellar region showed little or no cytokinin signal; (4) the highest cytokinin concentrations in filial endosperm and embryo were detected at 12 DAP, predominantly zeatin riboside and zeatin-9-glucoside, respectively; and (5) a strong cytokinin immuno-signal was detected in specific cell types in the pedicel, endosperm and embryo. CONCLUSIONS: The cytokinins of developing maize caryopsis may originate from both local syntheses as well as by transport. High levels of fertilization-dependent cytokinins in the pedicel suggest filial control on metabolism in the maternal tissue; they may also trigger developmental programmed cell death in the pedicel.

New Cross-Talks between Pathways Involved in Grapevine Infection with 'Candidatus Phytoplasma solani' Revealed by Temporal Network Modelling.

Understanding temporal biological phenomena is a challenging task that can be approached using network analysis. Here, we explored whether network reconstruction can be used to better understand the temporal dynamics of bois noir, which is associated with 'Candidatus Phytoplasma solani', and is one of the most widespread phytoplasma diseases of grapevine in Europe. We proposed a methodology that explores the temporal network dynamics at the community level, i.e., densely connected subnetworks. The methodology offers both insights into the functional dynamics via enrichment analysis at the community level, and analyses of the community dissipation, as a measure that accounts for community degradation. We validated this methodology with cases on experimental temporal expression data of uninfected grapevines and grapevines infected with 'Ca. P. solani'. These data confirm some known gene communities involved in this infection. They also reveal several new gene communities and their potential regulatory networks that have not been linked to 'Ca. P. solani' to date. To confirm the capabilities of the proposed method, selected predictions were empirically evaluated.

A panel of real-time PCR assays for specific detection of three phytoplasmas from the apple proliferation group.

We report here on the development of combination of assays for fast, reliable, specific and sensitive detection and discrimination of 'Candidatus Phytoplasma mali', 'Ca. P. prunorum' and 'Ca. P. pyri' from the 16Sr-X (apple proliferation - AP) group. These phytoplasmas are causal agents of diseases of fruit trees within the family Rosaceae, namely apple proliferation (AP), European stone fruit yellows (ESFY) and pear decline (PD). The designed panel of assays uses TaqMan minor groove binder probes (MGB). It comprises the same set of primers and specific probes for species-specific amplification within the 16S-23S rRNA intergenic spacer region, a set of primers and probes for amplification of the 16S ribosomal DNA region for the universal phytoplasma detection, and an additional set of primers and probe for 18S rRNA as an endogenous quality control of DNA extraction. The performance characteristics of the panel were evaluated. The advantages of new assays were shown in a comparative study with the conventional PCR, which proved their higher sensitivity combined with three-fold shorter time of testing process; and in comparison with two reported multiplex real-time PCR assays for detection of 'Ca. P. mali' or 'Ca. P. pyri'. New panel of assays were tested on the DNA samples of 'Ca. P. mali', 'Ca. P. prunorum', 'Ca. P. pyri', other phytoplasmas and other bacteria isolated from plant material. Additionally, 198 symptomatic and asymptomatic fruit tree field samples collecting during several growing seasons were tested with new assays as well. The results of this study indicate that the combination of three specific assays may be applied in routine phytoplasma surveys and in the certification programs.

Cytokinins and their function in developing seeds.

Cytokinins are a major group of plant hormones that control various processes in plant growth and development. Chemically they are N6-substituated adenine derivatives, including their respective ribotides, ribosides and glucosides. The inter-conversion between different cytokinins metabolites represents the transition between active, inactive, storage and transport forms. This process is in vivo rather dynamic and rapid. We have finally begun to understand the role of cytokinins in plant development through the identification of genes for the first plant enzyme in the biosynthetic pathway, isopentenyl transferases, less than 10 years ago, in addition to research of other enzymes and their corresponding genes that are involved in cytokinin metabolism and signal transduction. This review focuses on the recent findings on cytokinins with an emphasis on their role during the development of seedsfrom which the first natural cytokinin was isolated more that half a century ago.

Development and Validation of a New TaqMan Real-Time PCR for Detection of 'Candidatus Phytoplasma pruni'.

Phytoplasmas of the 16SrIII group are wide spread, and have a broad plant host range. Among these, 'Candidatus phytoplasma pruni' ('Ca. P. pruni'; phytoplasmas of 16SrIII subgroup A) can cause serious diseases in Prunus species and 'Ca. P. pruni'-related strains can infect other plant species, including grapevines. In this study, a new real-time PCR detection system was developed for 'Ca. P. pruni' using TaqMan chemistry. This test was designed to detect 'Ca. P. pruni', by amplifying the species-specific secY gene. In addition, a test to amplify the group-specific 16S rRNA gene region was also developed. The performances of both tests were evaluated. The test that amplifies the secY gene provided reliable and quick detection of 'Ca. P. pruni'. Using the newly developed and validated test, 'Ca. P. pruni' was not found in any of the 434 field samples collected from different plants species grown in different regions of Slovenia.

'Bois noir' phytoplasma induces significant reprogramming of the leaf transcriptome in the field grown grapevine.

BACKGROUND: Phytoplasmas are bacteria without cell walls from the class Mollicutes. They are obligate intracellular plant pathogens which cause diseases in hundreds of economically important plants including the grapevine (Vitis vinifera). Knowledge of their biology and the mechanisms of their interactions with hosts is largely unknown because they are uncultivable and experimentally inaccessible in their hosts. We detail here the global transcriptional profiling in grapevine responses to phytoplasmas. The gene expression patterns were followed in leaf midribs of grapevine cv. 'Chardonnay' naturally infected with a phytoplasma from the stolbur group 16SrXII-A, which is associated with the grapevine yellows disease 'Bois noir'. RESULTS: We established an on field experimental system in a productive vineyard that allowed application of molecular tools in a plant natural environment. Global transcription profiles of infected samples were compared with the healthy ones using microarray datasets and metabolic pathway analysis software (MapMan). The two-year-long experiment revealed that plant genes involved in primary and secondary metabolic pathways were changed in response to infection and that these changes might support phytoplasma nutrition. A hypothesis that phytoplasmas interact with the plant carbohydrate metabolism was proven and some possibilities how the products of this pathway might be utilized by phytoplasmas are discussed. In addition, several photosynthetic genes were largely down-regulated in infected plants, whereas defense genes from the metabolic pathway leading to formation of flavonoids and some PR proteins were significantly induced. Few other genes involved in defense-signaling were differentially expressed in healthy and infected plants. A set of 17 selected genes from several differentially expressed pathways was additionally analyzed with quantitative real-time PCR and confirmed to be suitable for a reliable classification of infected plants and for the characterization of susceptibility features in the field conditions. CONCLUSION: This study revealed some fundamental aspects of grapevine interactions with the stolbur 'Bois noir' phytoplasma in particular and some plant interactions with phytoplasmas in general. In addition, the results of the study will likely have an impact on grape improvement by yielding marker genes that can be used in new diagnostic assays for phytoplasmas or by identifying candidate genes that contribute to the improved properties of grape.

A comparative study on the role of cytokinins in caryopsis development in the maize miniature1 seed mutant and its wild type.

We report here on a comparative developmental profile of plant hormone cytokinins in relation to cell size, cell number and endoreduplication in developing maize caryopsis of a cell wall invertase-deficient miniature1 (mn1) seed mutant and its wild type, Mn1, genotype. Both genotypes showed extremely high levels of total cytokinins during the very early stages of development, followed by a marked and genotype specific reduction. While the decrease of cytokinins in Mn1 was associated with their deactivation by 9-glucosylation, the absolute and the relative part of active cytokinin forms was higher in the mutant. During the exponential growth phase of endosperm between 6 d after pollination and 9 d after pollination, the mean cell doubling time, the absolute growth rate and the level of endoreduplication were similar in the two genotypes. However, the entire duration of growth was longer in Mn1 compared with mn1, resulting in a significantly higher cell number in the Mn1 endosperm. These data correlate with the previously reported peak levels of the Mn1-encoded cell wall invertase-2 (INCW2) at 12 d after pollination in the Mn1 endosperm. A model showing possible crosstalk among cytokinins, cell cycle and cell wall invertase as causal to increased cell number and sink strength of the Mn1 developing endosperm is discussed.

Plant Hormones in Phytoplasma Infected Plants.

Phytoplasmas are bacterial plant pathogens that need a plant host and an insect vector for their spread and survival. In plants, the physiological responses that phytoplasmas trigger result in symptom development through effects on hormonal, nutritional, and stress signaling pathways, and the interactions between these. In this review, recent advances on the involvement of plant hormones together with their known and deduced roles in plants infected with phytoplasmas are discussed. Several studies have directly, or in many cases indirectly, addressed plant hormone systems in phytoplasma-infected plants. These have provided accumulating evidence that phytoplasmas extensively affect plant hormone pathways. Phytoplasmas thus, with disturbing complex plant hormone networks, suppress plant immunity and modify plant structure, while optimizing their nutrient acquisition and facilitating their colonization of the plants, and their dissemination among plants by their insect vectors.

Oxidative stress in duckweed (Lemna minor L.) caused by short-term cadmium exposure.

The mechanisms of plant defence against cadmium toxicity have been studied by short-term exposure of Lemna minor L. (common duckweed) to concentrations of CdCl2 ranging from 0 to 500microM. High accumulation of cadmium was observed (12,320+/-2155microgg(-1) at 500microM CdCl2), which caused a gradual decrease of plant growth, increased lipid peroxidation, and weakened the entire antioxidative defence. Total glutathione concentration decreased significantly; however, the concentration of oxidized glutathione remained stable. The responses of four antioxidant enzymes showed that catalase was the most inhibited after CdCl2 exposure, ascorbate peroxidase and guaiacol peroxidase moderately, and glutathione reductase least. The total antioxidative potential revealed an induced antioxidative network at 0.1microM CdCl2 (137+/-13.2% of the control) and its reduction to only 47.4+/-4.0% of the control at higher cadmium concentrations. The possible application of the examined biomarkers in ecotoxicological research is discussed.

Antioxidative responses of duckweed (Lemna minor L.) to short-term copper exposure.

GOAL, SCOPE AND BACKGROUND: Elevated concentrations of copper in the environment result in accumulation of the metal in plants and cause an increase in reactive oxidative species (ROS). The first response to elevated amounts of ROS is increased levels of enzymatic and non-enzymatic antioxidants that reduce oxidative stress. The aim of our study was to evaluate the early stages of antioxidative responses to the low copper concentrations usually present in moderately polluted environments. In addition, some other parameters were examined to evaluate the effect of copper on plants. METHODS: Duckweed (Lemna minor L.) was exposed to different concentrations of copper sulphate for up to 24 hours. Glutathione concentration and enzymatic activities of catalase, guaiacol peroxidase and glutathione reductase were measured spectrophotometrically. Additionally, delayed and prompt chlorophyll fluorescence was measured by luminometry and fluorometry, respectively. The accumulation of copper in plants exposed for 24 hours to various concentrations of copper sulphate was measured by flame atomic absorption spectrophotometry. RESULTS: The treatment of plants with copper sulphate resulted in an immediate decrease of the glutathione pool, which was replenished after 24 hours at CuSO4 concentrations lower than 2 microM. Higher CuSO4 concentrations caused a decrease of reduced glutathione. The responses of the antioxidant enzymes glutathione reductase, guaiacol peroxidase and catalase to CuSO4 differed during the first six hours of exposure, but their enzyme activities all increased after 24 hours of exposure. All these enzymes displayed biphasic activity curves with maximum values between 0.5 microM and 1 microM CuSO4. The response of guaiacol peroxidase was the most pronounced and statistically significantly specific and that of catalase the least. Delayed chlorophyll fluorescence decreased after exposure to 1 microM CuSO4, but no significant effect on maximum quantum yield of photosystem II (Fv/Fm) was observed. L. minor accumulated relatively high concentrations of copper. The accumulation rate was higher at lower concentrations of copper in the test medium (up to 2 microM CuSO4) than at concentrations above 2 microM CuSO4. DISCUSSION: One of the most pronounced antioxidative responses to copper exposure was modified levels of oxidized and reduced forms of glutathione. The decrease of the glutathione pool is most probably coupled with induced production of phytochelatins. Antioxidative enzymes showed the biphasic enzyme activity characteristic of stress response. Guaiacol peroxidase exhibited the greatest significant increase of activity, even at higher CuSO4 concentrations at which the activity of catalase and glutathione reductase dropped. The intensity of delayed chlorophyll fluorescence decreased, indicating reduced photosynthesis of plants under stress. All the measured parameters showed that plants respond to even low copper concentrations very soon after exposure. The accumulation rate of copper in duckweed tissues indicates that L. minor is an accumulator species. CONCLUSIONS: The synchronized and prompt inducibility of antioxidants indicates their involvement in a general plant defence strategy for coping with metal-induced oxidative stress. Glutathione concentration and guaiacol peroxidase activity were found to be the most sensitive of the early indicators of exposure to copper concentrations present in polluted water bodies. RECOMMENDATION AND PERSPECTIVES: The experimental design of the present study allowed us to compare the sensitivity of various methods and parameters for detecting plant responses to heavy metal-induced oxidative stress. The level of glutathione and the enzyme activities of guaiacol peroxidase and glutathione reductase could be used as a rapidly determined early warning system in toxicity studies.

1-Aminocyclopropane-1-Carboxylate Oxidase Induction in Tomato Flower Pedicel Phloem and Abscission Related Processes Are Differentially Sensitive to Ethylene.

Ethylene has impact on several physiological plant processes, including abscission, during which plants shed both their vegetative and reproductive organs. Cell separation and programmed cell death are involved in abscission, and these have also been correlated with ethylene action. However, the detailed spatiotemporal pattern of the molecular events during abscission remains unknown. We examined the expression of two tomato ACO genes, LeACO1, and LeACO4 that encode the last enzyme in ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate oxidase (ACO), together with the expression of other abscission-associated genes involved in cell separation and programmed cell death, during a period of 0-12 h after abscission induction in the tomato flower pedicel abscission zone and nearby tissues. In addition, we determined their localization in specific cell layers of the flower pedicel abscission zone and nearby tissues obtained by laser microdissection before and 8 h after abscission induction. The expression of both ACO genes was localized to the vascular tissues in the pedicel. While LeACO4 was more uniformly expressed in all examined cell layers, the main expression site of LeACO1 was in cell layers just outside the abscission zone in its proximal and distal part. We showed that after abscission induction, ACO1 protein was synthesized in phloem companion cells, in which it was localized mainly in the cytoplasm. Samples were additionally treated with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene actions, and analyzed 8 h after abscission induction. Cell-layer-specific changes in gene expression were observed together with the specific localization and ethylene sensitivity of the hallmarks of cell separation and programmed cell death. While treatment with 1-MCP prevented separation of cells through inhibition of the expression of polygalacturonases, which are the key enzymes involved in degradation of the middle lamella, this had less impact on the occurrence of different kinds of membrane vesicles and abscission-related programmed cell death. In the flower pedicel abscission zone, the physical progressions of cell separation and programmed cell death are perpendicular to each other and start in the vascular tissues.

Metabolic Consequences of Infection of Grapevine (Vitis vinifera L.) cv. "Modra frankinja" with Flavescence Dorée Phytoplasma.

Flavescence dorée, caused by the quarantine phytoplasma FDp, represents the most devastating of the grapevine yellows diseases in Europe. In an integrated study we have explored the FDp-grapevine interaction in infected grapevines of cv. "Modra frankinja" under natural conditions in the vineyard. In FDp-infected leaf vein-enriched tissues, the seasonal transcriptional profiles of 14 genes selected from various metabolic pathways showed an FDp-specific plant response compared to other grapevine yellows and uncovered a new association of the SWEET17a vacuolar transporter of fructose with pathogens. Non-targeted metabolome analysis from leaf vein-enriched tissues identified 22 significantly changed compounds with increased levels during infection. Several metabolites corroborated the gene expression study. Detailed investigation of the dynamics of carbohydrate metabolism revealed significant accumulation of sucrose and starch in the mesophyll of FDp-infected leaves, as well as significant up-regulation of genes involved in their biosynthesis. In addition, infected leaves had high activities of ADP-glucose pyrophosphorylase and, more significantly, sucrose synthase. The data support the conclusion that FDp infection inhibits phloem transport, resulting in accumulation of carbohydrates and secondary metabolites that provoke a source-sink transition and defense response status.

Real-time PCR for specific detection of three phytoplasmas from the apple proliferation group.

In this chapter, we describe a real-time PCR detection system for fast, reliable, specific, and sensitive detection and discrimination of 'Candidatus Phytoplasma mali', 'Ca. P. prunorum', and 'Ca. P. pyri' from the 16SrX (apple proliferation-AP) group. These phytoplasmas are causal agents of fruit tree diseases within the Rosaceae family, namely apple proliferation, European stone fruit yellows, and pear decline. The assays use (hydrolysis) TaqMan(®) minor groove binder probes. The panel of assays comprises the same set of primers and specific probes for species-specific amplification, and an additional set of primers and probe for 18S rRNA as an endogenous quality control of DNA extraction. The assays described can be used in routine phytoplasma surveys and in certification programmes.