A combined microscopy approach to study plant-phytoplasma interaction using Arabidopsis thaliana.

Phytoplasmas, obligate parasites of plants and phloem-feeding insects, belong to Mollicutes (Lee et al., 2004) and are associated with several hundreds of diseases affecting over one thousand plant species, including many economically important crops (Marcone, 2014). There is no effective curative strategy available so far, so the sole ways to limit the infection outbreaks are the use of insecticides and the removal of symptomatic plants (Bertaccini et al., 2014). Even if not all infections are necessarily deleterious, symptoms in infected plants suggest heavy disorders of phloem functions and growth-regulator balancing (Lee et al., 2000). Upon their discovery (Doi et al., 1967), the study of phytoplasmas has been hindered by the extreme difficulty to culture them in vitro, due to their lack of fundamental metabolic pathways (Bai et al., 2006). Moreover, the study in natural plant hosts is often limited by environmental conditions, long plant life cycle and poor knowledge of host-plant biology. Therefore, in the last decade some authors suggested to use Arabidopsis thaliana as model plant for studying phytoplasma-plant interactions. This choice was supported by the correspondence between the macroscopic symptoms developed in infected A. thaliana and those observed in natural host plants (Bressan and Purcell, 2005; Hoshi et al., 2009; Cettul and Firrao, 2011; MacLean et al., 2011). Nevertheless, morphological and ultrastructural modifications occurring in infected A. thaliana tissues have never been described in detail. In this work, we adopted a combined microscopy approach to verify if this plant is a reliable model for the study of phytoplasma-plant interactions at microscopical level. Using DAPI and fluorescence microscopy (FM), phytoplasma presence and localization were demonstrated in every infected plant. Transmission electron microscopy (TEM) observations confirmed phytoplasma massive presence into the sieve elements (SEs) (Figure 1). Phytoplasma appeared well preserved, with typical pleomorphic shape, free-floating and dividing in the lumen or adhered to SE membrane, probably connecting to the host (Marcone et al., 2014; Buxa et al., 2015). Phytoplasmas also established relationships with sieve element reticulum (SER). Pathogen presence, probably linked to nutrient uptake (Celli et al., 2015; Musetti et al., 2016), caused SER hyperproliferation, as observed in many other plant-phytoplasma interaction (Rudzinska-Langwald and Kaminska, 2001; Buxa et al., 2015) (Figure 1). Pathogen spread was documented by the passage through sieve pores. As remarked above, phytoplasma presence affected host plant development (Lee et al., 2000). In infected A. thaliana plants, light microscopy (LM) evidenced a profound disturbance in phloem morphology at histological level, mainly consisting in collapse, necrosis and hyperplasia of the phloem components. The relationship between necrosis and hyperplasia could be explained as a plant response to the impaired phloem functionality (Oshima et al., 2001) or due to pathogen effectors (Bai et al., 2009; Sugio et al., 2011). At ultrastructural level, as previously observed in other phytoplasma hosts (Musetti et al., 2000; 2013; Kaminska et al., 2001; Santi et al., 2013), phloem components showed plasmolysis or were collapsed or necrotized. Even in vital SEs, abnormalities of cell membrane profile and cell wall thickness were visible. TEM observations showed two typical plant responses to phytoplasma infection: phloem-protein agglutination and callose deposition at the sieve plates, which limited sieve-pore diameter (Figure 1). These phenomena have been interpreted as a plant reaction to physically limit pathogen spread (Lherminier et al., 2003; Gamalero et al., 2010; Luna et al., 2011; Musetti et al., 2010; 2013). Phloem functionality experiments using CFDA and confocal laser scanner microscopy (CLSM) suggested that sieve-pore obstruction leads to phloem impairment (Figure 2 A, C). This phenomenon is also associated to the accumulation of photo-assimilates, visible as chloroplast starch deposits under LM and TEM (Figure 2 B, D), as previously reported in other host plants (Maust et al., 2003; Junqueira et al., 2004; Musetti et al., 2013). This study proved that phloem tissue of infected A. thaliana presented the main morphological and ultrastructural response to phytoplasma infection as reported in natural hosts. Moreover, analyses carried on A. thaliana were not affected by troubles linked to low phytoplasma titre and uneven distribution, typical of woody plants. Therefore, we can state that A. thaliana revealed a reliable model plant for phytoplasma-plant interactions, concerning both macroscopic symptoms and morphological and ultrastructural changes

Orthology-Based Estimate of the Contribution of Horizontal Gene Transfer from Distantly Related Bacteria to the Intraspecific Diversity and Differentiation of Xylella fastidiosa

Xylella fastidiosa is a xylem-limited bacterium phylogenetically related to the xanthomonads, with an unusually large and diversified range of plant hosts. To ascertain the origin of its peculiarities, its pan-genome was scanned to identify the genes that are not coherent with its phylogenetic position within the order Xanthomonadales. The results of the analysis revealed that a large fraction of the genes of the Xylella pan-genome have no ortholog or close paralog in the order Xanthomonadales. For a significant part of the genes, the closest homologue was found in bacteria belonging to distantly related taxonomic groups, most frequently in the Betaproteobacteria. Other species, such as Xanthomonas vasicola and Xanthomonas albilineans which were investigated for comparison, did not show a similar genetic contribution from distant branches of the prokaryotic tree of life. This finding indicates that the process of acquisition of DNA from the environment is still a relevant component of Xylella fastidiosa evolution. Although the ability of Xylella fastidiosa strains to recombine among themselves is well known, the results of the pan-genome analyses stressed the additional relevance of environmental DNA in shaping their genomes, with potential consequences on their phytopathological features

Epifluorescence microscopy imaging of phytoplasmas in embedded leaf tissues using DAPI and SYTO13 fluorochromes

The use of DNA-specific dyes, i. e. DAPI, is extensively reported for phytoplasma detection in fresh plant materials. However, fluorescence-based microscopy and imaging of fresh tissues often evidences technical limitations which are more significant in infected tissues, because phenolic and other defense-related compounds accumulate in the cell wall and in the vacuole making difficult sample preparation. In this paper we describe a method based on the use of epifluorescence microscopy and the DNA probes DAPI and SYTO13\uae for phytoplasma visualization in resin-embedded plant tissues. The method allows detection of phytoplasmas and it is recommended for tissues that are recalcitrant to conventional imaging

Exploring the Equity Impact of Current Digital Health Design Practices:Protocol for a Scoping Review

BACKGROUND: The field of digital health has grown rapidly in part due to digital health tools’ potential to reduce health inequities. However, such potential has not always been realized. The design approaches used in digital health are one of the known aspects that have an impact on health equity. OBJECTIVE: The aim of our scoping review will be to understand how design approaches in digital health have an impact on health equity. METHODS: A scoping review of studies that describe how design practices for digital health have an impact on health equity will be carried out. The scoping review will follow the methodologies laid out by Arksey and O’Malley, the Joanna Briggs Institute, and the PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews) checklist. The PubMed, Embase, Web of Science, and ACM Digital Library databases will be searched for peer-reviewed papers. The ProQuest Dissertations and Theses and Global Index Medicus databases will be searched for gray literature. The results will be screened against our inclusion and exclusion criteria. Subsequently, the data extracted from the included studies will be analyzed. RESULTS: As of March 2022, a preliminary search of the peer-reviewed databases has yielded over 4900 studies, and more are anticipated when gray literature databases are searched. We expect that after duplicates are removed and screening is completed, a much smaller number of studies will meet all of our inclusion criteria. CONCLUSIONS: Although there has been much discussion about the importance of design for lowering barriers to digital health participation, the evidence base demonstrating its impacts on health equity is less obvious. We hope that our findings will contribute to a better understanding of the impact that design in digital health has on health equity and that these findings will translate into action that leads to stronger, more equitable health care systems

A história africana nos livros didáticos do ensino fundamental de Montividiu (Goiás – Brasil)

Resumo: O propósito do estudo ora apresentado é mostrar como a história africana é retratada nos livros didáticos do 6º ano de 2015, na única escola urbana da rede municipal de ensino fundamental de Montividiu-GO. Verificou-se que, embora as obras estejam de acordo com a Lei 10.639/03, a história africana tem pouco destaque, aparecendo fragmentada e desconexa da história eurocêntrica que norteia o material didático. Através de uma pesquisa bibliográfica utilizando como universo os vinte livros que constam no Guia de Livros Didáticos PNLD 2014 selecionou-se, como espaço amostral, apenas seis em virtude destes terem sido recebidos para análise dos professores de História da Escola Municipal Armando Gomes da Fonseca. Palavras-chave: Livro didático. Lei 10.639/03. História africana

A IMPORTÂNCIA DA RECREAÇÃO PARA O ENSINO DO ATLETISMO NA ESCOLA: UM RELATO DE EXPERIÊNCIA

A Educação Física é de suma importância para o desenvolvimento dos alunos, abrangendo as dimensões conceitual, dimensional procedimental e atitudinal, preparando assim, o aluno para a sociedade. O objetivo deste trabalho é relatar os acontecimentos do Estágio Curricular Multidisciplinar I, que foi realizado em uma escola municipal do interior do município de Alpestre-RS. No estágio foi realizado atividades de atletismo que proporcionaram aos alunos um desenvolvimento integral, por meio de vivências que exploraram as habilidades motoras básicas como correr, saltar, pular, arremessar de forma simples e prazerosa, respeitando a individualidade de cada um e estimulando o desenvolvimento motor, cognitivo, afetivo e social. A recreação é importantíssima na fase escolar das crianças, pois desenvolve a integração com o grupo, a locomoção, o raciocínio, a psicomotricidade, o espírito esportivo, a resistência física e a psicológica, a criação de estratégias, a motivação, a criatividade, a comunicação, o espírito de liderança, as noções de tempo e espaço, entre outros. Os métodos utilizados nas aulas foram prioritariamente os métodos parcial, recreativo e global. O Estágio proporcionou a vivencia da docência, estar efetivamente de frente a uma turma, aplicando nas aulas os conhecimentos aprendidos na universidade, vivenciando a função do professor e as dificuldades que são encontradas no decorrer do ano letivo

With or Without You: Altered Plant Response to Boron-Deficiency in Hydroponically Grown Grapevines Infected by Grapevine Pinot Gris Virus Suggests a Relation Between Grapevine Leaf Mottling and Deformation Symptom Occurrence and Boron Plant Availability

Despite the increasing spread of Grapevine Leaf Mottling and Deformation (GLMD) worldwide, little is known about its etiology. After identification of grapevine Pinot gris virus (GPGV) as the presumptive causal agent of the disease in 2015, various publications have evaluated GPGV involvement in GLMD. Nevertheless, there are only partial clues to explain the presence of GPGV in both symptomatic and asymptomatic grapevines and the mechanisms that trigger symptom development, and so a consideration of new factors is required. Given the similarities between GLMD and boron (B)-deficiency symptoms in grapevine plants, we posited that GPGV interferes in B homeostasis. By using a hydroponic system to control B availability, we investigated the effects of different B supplies on grapevine phenotype and those of GPGV infection on B acquisition and translocation machinery, by means of microscopy, ionomic and gene expression analyses in both roots and leaves. The transcription of the genes regulating B homeostasis was unaffected by the presence of GPGV alone, but was severely altered in plants exposed to both GPGV infection and B-deficiency, allowing us to speculate that the capricious and patchy occurrence of GLMD symptoms in the field may not be related solely to GPGV, but to GPGV interference in plant responses to different B availabilities. This hypothesis found preliminary positive confirmations in analyses on field-grown plants

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues

Despite the increasing impact of Grapevine Pinot gris disease (GPG-disease) worldwide, etiology about this disorder is still uncertain. The presence of the putative causal agent, the Grapevine Pinot Gris Virus (GPGV), has been reported in symptomatic grapevines (presenting stunting, chlorotic mottling, and leaf deformation) as well as in symptom-free plants. Moreover, information on virus localization in grapevine tissues and virus-plant interactions at the cytological level is missing at all. Ultrastructural and cytochemical investigations were undertaken to detect virus particles and the associated cytopathic effects in field-grown grapevine showing different symptom severity. Asymptomatic greenhouse-grown grapevines, which tested negative for GPGV by real time RT-PCR, were sampled as controls. Multiplex real-time RT-PCR and ELISA tests excluded the presence of viruses included in the Italian certification program both in field-grown and greenhouse-grown grapevines. Conversely, evidence was found for ubiquitous presence of Grapevine Rupestris Stem Pitting-associated Virus (GRSPaV), Hop Stunt Viroid (HSVd), and Grapevine Yellow Speckle Viroid 1 (GYSVd-1) in both plant groups. Moreover, in every field-grown grapevine, GPGV was detected by real-time RT-PCR. Ultrastructural observations and immunogold labelling assays showed filamentous flexuous viruses in the bundle sheath cells, often located inside membrane-bound organelles. No cytological differences were observed among field-grown grapevine samples showing different symptom severity. GPGV localization and associated ultrastructural modifications are reported and discussed, in the perspective of assisting management and control of the disease. \ua9 2017 The Author(s

Filamentous sieve element proteins are able to limit phloem mass flow, but not phytoplasma spread

In Fabaceae, dispersion of forisomes\u2014highly ordered aggregates of sieve element proteins\u2014in response to phytoplasma infection was proposed to limit phloem mass flow and, hence, prevent pathogen spread. In this study, the involvement of filamentous sieve element proteins in the containment of phytoplasmas was investigated in non-Fabaceae plants. Healthy and infected Arabidopsis plants lacking one or two genes related to sieve element filament formation\u2014AtSEOR1 (At3g01680), AtSEOR2 (At3g01670), and AtPP2-A1 (At4g19840)\u2014were analysed. TEM images revealed that phytoplasma infection induces phloem protein filament formation in both the wild-type and mutant lines. This result suggests that, in contrast to previous hypotheses, sieve element filaments can be produced independently of AtSEOR1 and AtSEOR2 genes. Filament presence was accompanied by a compensatory overexpression of sieve element protein genes in infected mutant lines in comparison with wild-type lines. No correlation was found between phloem mass flow limitation and phytoplasma titre, which suggests that sieve element proteins are involved in defence mechanisms other than mechanical limitation of the pathogen