Complicações da utilização de equipamentos de proteção individual na pandemia de COVID-19 / Complications of personal protective equipment use during the COVID-19 pandemic / Complicaciones de la utilización de equipos de protección individual en la pandemia de COVID-19

Resumo Enquadramento: Em 2020, a COVID-19 é declarada pandemia. Os equipamentos de proteção individual (EPIs) surgem como medida eficaz para prevenir e controlar a transmissão, provocando diversas complicações nos profissionais. Objetivos: Identificar as complicações decorrentes da utilização de EPIs. Metodologia: Estudo transversal descritivo realizado em 118 profissionais de uma unidade local de saúde, responderam a um questionário online, através do convite, via email, do gestor do serviço. Parecer favorável da Comissão de Ética. Resultados: O respirador FPP2 (96,6%), a bata (79,6%) e o fato integral (89,3%) foram responsáveis pela maioria das complicações, predominou a hiperidrose relacionada com o uso de fato integral (95,3%), avental (93,1%) e bata (92,0%), as quedas associadas ao uso dos protetores dos sapatos (94,6%), a xerodermia com o uso de luvas (65,2%) e a dificuldade na comunicação verbal relacionada com o uso de máscaras. Conclusão: As complicações mais frequentes foram a hiperidrose, as quedas e a xerodermia, sugerindo-se o reforço de hidratação oral e cutânea bem como a limitação de utilização dos EPIs ao tempo absolutamente necessário.

Abstract Background: COVID-19 was declared a pandemic in 2020. Personal protective equipment (PPE) emerged as an effective measure to prevent and control transmission, resulting in several complications for health professionals. Objectives: To identify complications related to PPE use. Methodology: A descriptive cross-sectional study was carried out with 118 professionals from a local health unit who received an email invitation from the department manager to answer an online questionnaire. It received a favorable opinion from an Ethics Committee. Results: Most complications were associated with FPP2 respirators (96.6%), gowns (79.6%), and coveralls (89.3%), namely hyperhidrosis from wearing coveralls (95.3%), aprons (93.1%), and gowns (92.0%), falls from using shoe covers (94.6%), xeroderma from wearing gloves (65.2%), and oral communication difficulties due to mask use. Conclusion: The most frequent complications were hyperhidrosis, falls, and xeroderma. Oral and skin hydration is recommended. PPE should be worn for the minimum time possible.

Resumen Marco contextual: En 2020, el COVID-19 se declara pandemia. Los equipos de protección individual (EPI) surgen como medida eficaz para prevenir y controlar la transmisión, lo que provoca diversas complicaciones en los profesionales. Objetivos: Identificar las complicaciones derivadas del uso de EPI. Metodología: Estudio transversal, descriptivo, realizado en 118 profesionales de una unidad de salud local, que respondieron un cuestionario en línea a través de una invitación, vía correo electrónico, del responsable del servicio. Dictamen favorable del Comité de Ética. Resultados: El respirador FPP2 (96,6%), la bata (79,6%) y el traje general (89,3%) fueron los responsables de la mayoría de las complicaciones, la hiperhidrosis relacionada con el uso del traje general (95,3%), el delantal (93,1%) y la bata (92,0%), las caídas asociadas al uso de cubrezapatos (94,6%), la xerodermia con el uso de guantes (65,2%) y la dificultad en la comunicación verbal relacionada con el uso de mascarillas. Conclusión: Las complicaciones más frecuentes fueron hiperhidrosis, caídas y xerodermia, lo que sugiere reforzar la hidratación oral y cutánea, así como limitar el uso de EPI al tiempo absolutamente necesario.

Diploid Nuclei Occur throughout the Life Cycles of Pucciniales Fungi.

Within Eukaryotes, fungi are the typical representatives of haplontic life cycles. Basidiomycota fungi are dikaryotic in extensive parts of their life cycle, but diploid nuclei are known to form only in basidia. Among Basidiomycota, the Pucciniales are notorious for presenting the most complex life cycles, with high host specialization, and for their expanded genomes. Using cytogenomic (flow cytometry and cell sorting on propidium iodide-stained nuclei) and cytogenetic (FISH with rDNA probe) approaches, we report the widespread occurrence of replicating haploid and diploid nuclei (i.e., 1C, 2C and a small proportion of 4C nuclei) in diverse life cycle stages (pycnial, aecial, uredinial, and telial) of all 35 Pucciniales species analyzed, but not in sister taxa. These results suggest that the Pucciniales life cycle is distinct from any cycle known, i.e., neither haplontic, diplontic nor haplodiplontic, corroborating patchy and disregarded previous evidence. However, the biological basis and significance of this phenomenon remain undisclosed. IMPORTANCE Within Eukaryotes, fungi are the typical representatives of haplontic life cycles, contrasting with plants and animals. As such, fungi thus contain haploid nuclei throughout their life cycles, with sexual reproduction generating a single diploid cell upon karyogamy that immediately undergoes meiosis, thus resuming the haploid cycle. In this work, using cytogenetic and cytogenomic tools, we demonstrate that a vast group of fungi presents diploid nuclei throughout their life cycles, along with haploid nuclei, and that both types of nuclei replicate. Moreover, haploid nuclei are absent from urediniospores. The phenomenon appears to be transversal to the organisms in the order Pucciniales (rust fungi) and it does not occur in neighboring taxa, but a biological explanation or function for it remains elusive.

The story of coffee: legend and truth.

When we think about coffee, exotic tropical countries such as Colombia, Brazil, and Ethiopia first come to mind. However, the crucial contribution of Portugal and its scientists to each cup of coffee we drink remains either poorly known or overlooked.

Subcuticular−Intracellular Hemibiotrophy of Colletotrichum lupini in Lupinus mutabilis

Anthracnose caused by Colletotrichum lupini is the most important disease affecting lupin cultivation worldwide. Lupinus mutabilis has been widely studied due to its high protein and oil content. However, it has proved to be sensitive to anthracnose, which limits the expansion of its cultivation. In this work, we seek to unveil the strategy that is used by C. lupini to infect and colonize L. mutabilis tissues using light and transmission electron microscopy (TEM). On petioles, pathogen penetration occurred from melanized appressoria, subcuticular intramural hyphae were seen 2 days after inoculation (dai), and the adjacent host cells remained intact. The switch to necrotrophy was observed 3 dai. At this time, the hyphae extended their colonization to the epidermal, cortex, and vascular cells. Wall degradation was more evident in the epidermal cells. TEM observations also revealed a loss of plasma membrane integrity and different levels of cytoplasm disorganization in the infected epidermal cells and in those of the first layers of the cortex. The disintegration of organelles occurred and was particularly visible in the chloroplasts. The necrotrophic phase culminated with the development of acervuli 6 dai. C. lupini used the same infection strategy on stems, but there was a delay in the penetration of host tissues and the appearance of the first symptoms.

Complicações da utilização de equipamentos de proteção individual no atendimento a doentes por COVID-19 / Complications of personal protective equipment use in COVID-19 patient care

No final de 2019 surge, na china, um coronavírus altamente contagioso, que rapidamente se propagou pelos 5 continentes, tendo a OMS, a 11 de março de 2020, declarado a COVID-19 como uma pandemia. Sem tratamento eficaz, os Equipamentos de Proteção Individual (EPI) surgem como a medida mais eficaz de prevenir e controlar a transmissão. São vários os relatos e os estudos que surgem sobre as dificuldades e as complicações que os profissionais de saúde experienciam com a utilização prolongada destes. Assim, este estudo tem como objetivo analisar as complicações decorrentes da utilização dos EPI, por parte dos colaboradores dos serviços de Medicina Intensiva, Serviço de Urgência e Serviço de Medicina B Poente, de uma Unidade Local de Saúde do Norte de Portugal, no atendimento a doentes por COVID-19. Metodologia: Desenvolveu-se um estudo transversal analítico, em profissionais dos serviços de Medicina Intensiva, Serviço de Urgência e Serviço de Medicina B Poente, de uma Unidade Local de Saúde do Norte de Portugal, no atendimento a doentes por COVID- 19. Foram definidos como critérios de inclusão: prestarem cuidados a doentes com COVID- 19; utilizarem EPI, Instrumento de Recolha de Dados totalmente preenchido e disponibilizarem-se a participar no estudo. Obtivemos, assim, uma amostra de 28 médicos, 60 enfermeiros e 30 assistentes operacionais dos referidos serviços. Os participantes foram convidados, através do gestor de serviço e por mail, a responder a um questionário online no Google Docs., constituído por duas partes: a primeira com questões sobre a caraterização sociodemográfica e profissional e a segunda com questões relativas aos EPI e suas complicações. O estudo obteve o perecer favorável da Comissão de Ética n.º 25/2021. Os procedimentos de análise e tratamento de dados foram realizados no programa informático SPSS® através da estatística descritiva para todas as variáveis e de testes não paramétricos para análise inferencial, com valor de significância de 0,05. Resultados: Os principais resultados mostraram maior predomínio, do sexo feminino (72,0%); média de idade de 40±38,5 anos, com bacharelato/licenciatura (61,9%); casados ou em união de facto (61,9%); enfermeiros (50,8%), com tempo de serviço médio de 12,2±12 anos e a trabalhar no SU (52,5%). As luvas descartáveis (51,7%), a máscara cirúrgica (51,7%) e a máscara FFP2 (60,2%) foram os EPI utilizados por mais de 4 horas. Foram identificadas maiores dificuldades para o uso do macacão (71,2%), da máscara FFP2 (52,5%), dos óculos e da viseira (62,7%). A maior parte dos profissionais recorreu a medidas de prevenção e de tratamento das complicações decorrentes do uso dos EPI (89,8%) e foram identificadas complicações em quase todos os EPI decorrentes do seu uso (50,8%). Os EPI que registaram mais complicações na utilização foram a FFP2 (96,6%), a bata e macacão (89,3%) e os óculos (83,9%). O suor excessivo foi a complicação mais identificada por uso de macacão (95,3%), seguido do avental (93,1%) e bata (92,0%). As quedas por uso de cobre botas (94,6%) a pele seca por uso de luvas (65,2%) e a complicação na expressão oral por uso das máscaras em geral. Encontramos diferenças, estatisticamente significativas, entre as complicações e o sexo, a idade, o tempo de serviço, as habilitações literárias, a classe profissional, o tempo de utilização > a 2 horas e o serviço onde trabalha (p<0,05). Conclusão: Os EPI que registaram maiores complicações foram o macacão, a máscara FFP2 e os óculos e a viseira. As complicações mais identificadas foram o suor excessivo, as quedas, pele seca e dificuldade na expressão oral. As mulheres, os profissionais mais velhos, com mais tempo de serviço, os enfermeiros, os bacharéis/licenciados os que utilizaram mais de 2 horas os EPI e o SU e SMI registaram maior ordem de médias de complicações. Sugerimos a utilização de medidas preventivas para minimizar as complicações como reforço na hidratação, uso de apósitos de proteção, a redução do número de horas de utilização, sempre que possível e nos grupos mais identificados. A realização de outros estudos com amostras maiores, prospetivos e outras variáveis que permitam uma análise mais generalizada.

At the end of 2019, a highly contagious coronavirus appears in China, which quickly spread across the 5 continents, with the WHO on March 11, 2020 declaring COVID- 19 a pandemic. Without effective treatment, Personal Protective Equipment (PPE) emerges as the most effective measure to prevent and control transmission. There are several reports and studies that arise about the difficulties and complications that health professionals experience with the prolonged use of these. Thus, this study aims to analyze the complications resulting from the use of PPE by employees of intensive care services, emergency services and medicine service B West, of a Local Health Unit of northern Portugal, in the care of patients by COVID-19. Methodology: An analytical cross-sectional study was developed in professionals from intensive care services, emergency services and medicine service B West, of a Local Health Unit in northern Portugal, in the care of patients by COVID-19. Inclusion criteria were defined: to provide care to patients with COVID-19; use PPE, fully completed Data Collection Tool and make themselves available to participate in the study. Thus, we obtained a sample of 28 physicians, 60 nurses and 30 operational assistants of these services. The participants were invited, through the service manager and by mail, to answer an online questionnaire in Google Docs., consisting of two parts: the first with questions about the sociodemographic and professional characteristics and the second with questions related to PPE and its complications. The study obtained the favorable perishing of the Ethics Committee No. 25/2021. The procedures of data analysis and treatment were performed in the SPSS® computer program through descriptive statistics for all variables and nonparametric tests for inferential analysis, with a significance value of 0.05. Results: The main results showed a higher predominance of females (72.0%); mean age of 40±38.5 years, with bachelor's degree/bachelor's degree (61.9%); married or in de facto union (61.9%); nurses (50.8%), with an average length of service of 12.2±12 years and working in the SU (52.5%). Disposable gloves (51.7%), surgical mask (51.7%) and FFP2 mask (60.2%) were epi used for more than 4 hours. Greater difficulties were identified for the use of the overall (71.2%), the mask FFP2 (52.5%), the glasses and the visor (62.7%). Most professionals resorted to prevention and treatment measures for complications resulting from the use of PPE (89.8%) and complications were identified in almost all PPE stemming from its use (50.8%). The PPE that had the most complications in the use were FFP2 (96.6%), gown and overalls (89.3%) and glasses (83.9%). Excessive sweating was the most identified complication by wearing overalls (95.3%), followed by apron (93.1%) and gown (92.0%). Falls due to the use of copper boots (94.6%) dry skin by the use of gloves (65.2%) and the complication in oral expression by wearing masks in general. We found statistically significant differences between complications and gender, age, time of service, educational qualifications, professional class, time of use > to 2 hours and the service where she works (p<0.05). Conclusion: The PPE that had the greatest complications were the overalls, the FFP2 mask and the glasses and the visor. The most identified complications were excessive sweating, falls, dry skin and difficulty in oral expression. Women, older professionals, with longer service, nurses, bachelors/graduates, those who used PPE more than 2 hours, PPE and SU and IMS had a higher order of means of complications. We suggest the use of preventive measures to minimize complications such as strengthening hydration, use of protective props, reducing the number of hours of use, whenever possible and in the most identified groups. Other studies with larger samples, prospects and other variables that allow a more generalized analysis.

A Histopathological Study Reveals New Insights Into Responses of Chestnut (Castanea spp.) to Root Infection by Phytophthora cinnamomi.

The European chestnut (Castanea sativa) is threatened by the hemibiotrophic oomycete Phytophthora cinnamomi, the causal agent of ink disease. Chestnut species have different susceptibility levels to P. cinnamomi, with the Asian species (C. crenata; C. mollissima) exhibiting the highest level of resistance. A histological approach was used to study the responses exhibited by susceptible and resistant chestnut genotypes by characterizing the early stages of P. cinnamomi infection and the cellular responses it induces in roots. C. sativa (susceptible) and C. crenata (resistant) plantlets were inoculated with a P. cinnamomi virulent isolate with a zoospore suspension or by direct contact with mycelia agar pieces. Root samples were collected at 0.5, 3.5, 24, 48, and 72 h after inoculation (hai) for microscopic observations. Penetration was observed in both species at 0.5 and 3.5 hai with mycelium and zoospore inoculations, respectively. In both inoculation methods, following penetration into the rhizodermis, P. cinnamomi hyphae grew inter- and intracellularly through the cortex and into the vascular cylinder. C. crenata cells displayed a delay in the pattern of infection by having fewer cell layers colonized compared with C. sativa. At 72 hai, the collapse of the first layers of C. sativa cortical cells was observed, indicating the beginning of necrotrophy. C. crenata was able to respond more efficiently to P. cinnamomi than C. sativa by restricting the pathogen's growth area through the early activation of resistance responses such as callose deposition around some intracellular hyphae, hypersensitive response-like cell death, cell wall thickening, and accumulation of phenolic-like compounds.

Pathological, Morphological, Cytogenomic, Biochemical and Molecular Data Support the Distinction between Colletotrichum cigarro comb. et stat. nov. and Colletotrichum kahawae.

The genus Colletotrichum has witnessed tremendous variations over the years in the number of species recognized, ranging from 11 to several hundreds. Host-specific fungal species, once the rule, are now the exception, with polyphagous behavior regarded as normal in this genus. The species Colletotrichum kahawae was created to accommodate the pathogens that have the unique ability to infect green developing coffee berries causing the devastating Coffee Berry Disease in Africa, but its close phylogenetic relationship to a polyphagous group of fungi in the C. gloeosporioides species complex led some researchers to regard these pathogens as members of a wider species. In this work we combine pathological, morphological, cytogenomic, biochemical, and molecular data of a comprehensive set of phylogenetically-related isolates to show that the Coffee Berry Disease pathogen forms a separate species, C. kahawae, and also to assign the closely related fungi, previously in C. kahawae subsp. cigarro, to a new species, C. cigarro comb. et stat. nov. This taxonomic clarification provides an opportunity to link phylogeny and functional biology, and additionally enables a much-needed tool for plant pathology and agronomy, associating exclusively C. kahawae to the Coffee Berry Disease pathogen.

Violência no namoro: estudo com adolescentes de uma Escola Secundária de Bragança / Dating violence: a study with adolescents from a Secondary School in Bragança

A violência no namoro tem vindo a ser assumida como um fenómeno preocupante. No sentido de analisar a violência no namoro, comportamentos e atitudes que legitimam essa violência em adolescentes, foi conduzido um estudo descritivo, analítico, quantitativo e transversal com 180 alunos de ambos os sexos, do Agrupamento de Escolas Abade de Baçal, em Bragança, com idades compreendidas entre os 15 e os 18 anos (M=16,13; Dp=0,893). Os dados foram recolhidos através de um inquérito por questionário constituído por três partes: questionário sociodemográfico, Inventário de Conflitos nas Relações de Namoro entre Adolescentes (CADRI) e Escala de Atitudes acerca da Violência no Namoro (EAVN). Os principais resultados descritivos e inferenciais mostraram uma maior presença da violência emocional e verbal (M=3,03; DP=3,003), embora os adolescentes consideram fazer uso de estratégias de resolução de conflitos no namoro não abusivas. As diversas formas de violência estudadas mostraram-se influenciadas pelo sexo (p≤0,01), idade (p≤0,01) e ano de escolaridade (p≤0,01). Foram encontradas relações positivas significativas entre o consumo de drogas (p≤0,05) e de álcool (p≤0,01), o envolvimento em comportamentos de risco (p≤0,01), vitimização prévia no namoro (p≤0,01) e conhecimento de violência no namoro no grupo de pares (p≤0,01) com os comportamentos de violência. Os resultados mostraram também atitudes de legitimação da violência masculina e feminina, influenciada pelo sexo (p≤0,01), consumo de tabaco (p≤0,05) e de outras drogas (p≤0,05), envolvimento em comportamentos de risco (p≤0,01), vitimização prévia no namoro (p≤0,01) e o conhecimento da violência no namoro no grupo de pares (p≤0,01). A partir dos resultados foi possível identificarem-se modelos explicados das estratégias de resolução de conflitos abusivas, não abusivas e comportamentos violentos, bem como da legitimação das atitudes de violência física, psicológica e sexual masculina e das atitudes de violência psicológica e sexual feminina. Perante os resultados encontrados, investigações futuras devem ser conduzidas com o intuito de averiguar a realidade da violência no namoro nos adolescentes, como ponto de partida para intervenções que promovam a mudança de comportamentos e de legitimação de atitudes de violência.

Violence in dating has been taken as a worrying phenomenon. In order to analyze the violence in dating, behaviors and attitudes that legitimizes violence in adolescents, was conducted a descriptive, analytical, quantitative and transversal study, with 180 students of both sexes, attending the grouping of schools Abade de Baçal, in Bragança, aged between 15 and 18 years (M = 16.13; SD = 0.893). The data were collected through a survey consisting of three parts: socio-demographic questionnaire, Inventory of Conflicts Dating Relationships among Adolescents (CADRI) and Attitudes about Violence in Dating (EAVN). The main descriptive and inferential results showed more emotional and verbal violence (M=3.03; SD=3.003), although teenagers consider make use of non-abusive conflict resolution strategies in relationship. The various forms of violence studied were influenced by gender (p≤0.01), age (p≤0.01) and grade level (p≤0.01). Significant positive relationships were found between the consumption of drugs (p≤0.05) and alcohol (p≤0.01), engaging in risky behavior (p≤0.01), prior victimization in dating (p≤0.01) and awareness of dating violence in peer group (p≤0.01). The results also showed legitimate attitudes of male and female violence, influenced by sex (p≤0.01), consumption of tobacco (p≤0.05) and other drugs (p≤0.05), engaging in risky behavior (p≤0.01), prior victimization in dating (p≤0.01) and awareness of dating violence in peer group (p≤0.01). From the results it was possible to identify models which explained abusive and non-abusive conflict strategies resolution and violent behavior, as well as legitimate attitudes of male physical, psychological, and sexual violence, and female psychological and sexual violence. Attending these results, future investigations should be conducted in order to ascertain the reality of dating violence in adolescents, as a starting point for interventions that promote behavior change and legitimating of attitudes to violence.

A first insight into the involvement of phytohormones pathways in coffee resistance and susceptibility to Colletotrichum kahawae.

Understanding the molecular mechanisms underlying coffee-pathogen interactions are of key importance to aid disease resistance breeding efforts. In this work the expression of genes involved in salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) pathways were studied in hypocotyls of two coffee varieties challenged with the hemibiotrophic fungus Colletotrichum kahawae, the causal agent of Coffee Berry Disease. Based on a cytological analysis, key time-points of the infection process were selected and qPCR was used to evaluate the expression of phytohormones biosynthesis, reception and responsive-related genes. The resistance to C. kahawae was characterized by restricted fungal growth associated with early accumulation of phenolic compounds in the cell walls and cytoplasmic contents, and deployment of hypersensitive reaction. Similar responses were detected in the susceptible variety, but in a significantly lower percentage of infection sites and with no apparent effect on disease development. Gene expression analysis suggests a more relevant involvement of JA and ET phytohormones than SA in this pathosystem. An earlier and stronger activation of the JA pathway observed in the resistant variety, when compared with the susceptible one, seems to be responsible for the successful activation of defense responses and inhibition of fungal growth. For the ET pathway, the down or non-regulation of ET receptors in the resistant variety, together with a moderate expression of the responsive-related gene ERF1, indicates that this phytohormone may be related with other functions besides the resistance response. However, in the susceptible variety, the stronger activation of ERF1 gene at the beginning of the necrotrophic phase, suggests the involvement of ET in tissue senescence. As far as we know, this is the first attempt to unveil the role of phytohormones in coffee-C. kahawae interactions, thus contributing to deepen our understanding on the complex mechanisms of plant signaling and defense.

The coffee leaf rust pathogen Hemileia vastatrix: one and a half centuries around the tropics.

TAXONOMY AND HISTORY: Hemileia vastatrix Berk. and Broome (Basidiomycota, Pucciniales) was described in 1869 in eastern Africa and Ceylon as the agent of coffee leaf rust and has spread to all coffee cultivation areas worldwide. Major disease outbreaks in Asia, Africa and America caused and continue to cause severe yield losses, making this the most important disease of Arabica coffee, a cash crop for many tropical and sub-tropical countries. LIFE CYCLE AND DISEASE SYMPTOMS: Hemileia vastatrix is a hemicyclic fungus with the urediniosporic life cycle as its most important (if not only) source of inoculum. Chlorotic spots are the first macroscopic symptoms, preceding the differentiation of suprastomatal, bouquet-shaped, orange-coloured uredinia. The disease can cause yield losses of up to 35% and have a polyetic epidemiological impact on subsequent years. DISEASE CONTROL: Although the use of fungicides is one of the preferred immediate control measures, the use of resistant cultivars is considered to be the most effective and durable disease control strategy. The discovery of 'Híbrido de Timor' provided sources of resistance that have been used in several breeding programmes and that have been proven to be effective and durable, as some have been in use for more than 30 years. GENETIC DIVERSITY AND MOLECULAR PATHOGENICITY: Although exhibiting limited genetic polymorphism, the very large genome of H. vastatrix (c. 797 Mbp) conceals great pathological diversity, with more than 50 physiological races. Gene expression studies have revealed a very precocious activation of signalling pathways and production of putative effectors, suggesting that the plant-fungus dialogue starts as early as at the germ tube stage, and have provided clues for the identification of avr genes.

Comparative Validation of Conventional and RNA-Seq Data-Derived Reference Genes for qPCR Expression Studies of Colletotrichum kahawae.

Colletotrichum kahawae is an emergent fungal pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa. Currently, the molecular mechanisms underlying the Coffea arabica-C. kahawae interaction are still poorly understood, as well as the differences in pathogen aggressiveness, which makes the development of functional studies for this pathosystem a crucial step. Quantitative real time PCR (qPCR) has been one of the most promising approaches to perform gene expression analyses. However, proper data normalization with suitable reference genes is an absolute requirement. In this study, a set of 8 candidate reference genes were selected based on two different approaches (literature and Illumina RNA-seq datasets) to assess the best normalization factor for qPCR expression analysis of C. kahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of C. kahawae bearing different aggressiveness patterns (Ang29, Ang67, Zim12 and Que2), at different stages of fungal development and key time points of the plant-fungus interaction process. Gene expression stability was assessed using the pairwise method incorporated in geNorm and the model-based method used by NormFinder software. For C. arabica-C. kahawae interaction samples, the best normalization factor included the combination of PP1, Act and ck34620 genes, while for C. kahawae samples the combination of PP1, Act and ck20430 revealed to be the most appropriate choice. These results suggest that RNA-seq analyses can provide alternative sources of reference genes in addition to classical reference genes. The analysis of expression profiles of bifunctional catalase-peroxidase (cat2) and trihydroxynaphthalene reductase (thr1) genes further enabled the validation of the selected reference genes. This study provides, for the first time, the tools required to conduct accurate qPCR studies in C. kahawae considering its aggressiveness pattern, developmental stage and host interaction.

A method for obtaining RNA from Hemileia vastatrix appressoria produced in planta, suitable for transcriptomic analyses.

Appressoria are the first infection structures developed by rust fungi and require specific topographic signals from the host for their differentiation. The ease in obtaining appressoria in vitro for these biotrophic fungi led to studies concerning gene expression and gene discovery at appressorial level, avoiding the need to distinguish plant and fungal transcripts. However, in some pathosystems, it was observed that gene expression in appressoria seems to be influenced by host-derived signals, suggesting that transcriptomic analyses performed from in planta differentiated appressoria would be potentially more informative than those from in vitro differentiated appressoria. Nevertheless analysing appressorial RNA obtained from in planta samples is often hampered by an excessive dilution of fungal RNA within plant RNA, besides uncertainty regarding the fungal or plant origin of RNA from highly conserved genes. To circumvent these difficulties, we have recovered Hemileia vastatrix appressoria from Arabica coffee leaf surface using a film of nitrocellulose dissolved in butyl and ethyl acetates (nail polish), and extracted fungal RNA from the polish peel. RNA thus obtained is of good quality and usable for cDNA synthesis and transcriptomic (quantitative PCR) studies. This method could provide the means to investigate specific host-induced appressoria-related fungal pathogenicity factors.

Proteomic analysis of apoplastic fluid of Coffea arabica leaves highlights novel biomarkers for resistance against Hemileia vastatrix.

A proteomic analysis of the apoplastic fluid (APF) of coffee leaves was conducted to investigate the cellular processes associated with incompatible (resistant) and compatible (susceptible) Coffea arabica-Hemileia vastatrix interactions, during the 24-96 hai period. The APF proteins were extracted by leaf vacuum infiltration and protein profiles were obtained by 2-DE. The comparative analysis of the gels revealed 210 polypeptide spots whose volume changed in abundance between samples (control, resistant and susceptible) during the 24-96 hai period. The proteins identified were involved mainly in protein degradation, cell wall metabolism and stress/defense responses, most of them being hydrolases (around 70%), particularly sugar hydrolases and peptidases/proteases. The changes in the APF proteome along the infection process revealed two distinct phases of defense responses, an initial/basal one (24-48 hai) and a late/specific one (72-96 hai). Compared to susceptibility, resistance was associated with a higher number of proteins, which was more evident in the late/specific phase. Proteins involved in the resistance response were mainly, glycohydrolases of the cell wall, serine proteases and pathogen related-like proteins (PR-proteins), suggesting that some of these proteins could be putative candidates for resistant markers of coffee to H. vastatrix. Antibodies were produced against chitinase, pectin methylesterase, serine carboxypeptidase, reticuline oxidase and subtilase and by an immunodetection assay it was observed an increase of these proteins in the resistant sample. With this methodology we have identified proteins that are candidate markers of resistance and that will be useful in coffee breeding programs to assist in the selection of cultivars with resistance to H. vastatrix.

Flow cytometry reveals that the rust fungus, Uromyces bidentis (Pucciniales), possesses the largest fungal genome reported--2489 Mbp.

Among the Eukaryotes, Fungi have relatively small genomes (average of 44.2 Mbp across 1850 species). The order Pucciniales (Basidiomycota) has the largest average genome size among fungi (305 Mbp), and includes the two largest fungal genomes reported so far (Puccinia chrysanthemi and Gymnosporangium confusum, with 806.5 and 893.2 Mbp, respectively). In this work, flow cytometry was employed to determine the genome size of the Bidens pilosa rust pathogen, Uromyces bidentis. The results obtained revealed that U. bidentis presents a surprisingly large haploid genome size of 2489 Mbp. This value is almost three times larger than the previous largest fungal genome reported and over 50 times larger than the average fungal genome size. Microscopic examination of U. bidentis nuclei also showed that they are not as different in size from the B. pilosa nuclei when compared with the differences between other rusts and their host plants. This result further reinforces the position of the Pucciniales as the fungal group with the largest genomes, prompting studies addressing the role of repetitive elements and polyploidy in the evolution, pathological specialization and diversity of fungal species.

Genome size analyses of Pucciniales reveal the largest fungal genomes.

Rust fungi (Basidiomycota, Pucciniales) are biotrophic plant pathogens which exhibit diverse complexities in their life cycles and host ranges. The completion of genome sequencing of a few rust fungi has revealed the occurrence of large genomes. Sequencing efforts for other rust fungi have been hampered by uncertainty concerning their genome sizes. Flow cytometry was recently applied to estimate the genome size of a few rust fungi, and confirmed the occurrence of large genomes in this order (averaging 225.3 Mbp, while the average for Basidiomycota was 49.9 Mbp and was 37.7 Mbp for all fungi). In this work, we have used an innovative and simple approach to simultaneously isolate nuclei from the rust and its host plant in order to estimate the genome size of 30 rust species by flow cytometry. Genome sizes varied over 10-fold, from 70 to 893 Mbp, with an average genome size value of 380.2 Mbp. Compared to the genome sizes of over 1800 fungi, Gymnosporangium confusum possesses the largest fungal genome ever reported (893.2 Mbp). Moreover, even the smallest rust genome determined in this study is larger than the vast majority of fungal genomes (94%). The average genome size of the Pucciniales is now of 305.5 Mbp, while the average Basidiomycota genome size has shifted to 70.4 Mbp and the average for all fungi reached 44.2 Mbp. Despite the fact that no correlation could be drawn between the genome sizes, the phylogenomics or the life cycle of rust fungi, it is interesting to note that rusts with Fabaceae hosts present genomes clearly larger than those with Poaceae hosts. Although this study comprises only a small fraction of the more than 7000 rust species described, it seems already evident that the Pucciniales represent a group where genome size expansion could be a common characteristic. This is in sharp contrast to sister taxa, placing this order in a relevant position in fungal genomics research.

Overview of the functional virulent genome of the coffee leaf rust pathogen Hemileia vastatrix with an emphasis on early stages of infection.

Hemileia vastatrix is the causal agent of coffee leaf rust, the most important disease of coffee Arabica. In this work, a 454-pyrosequencing transcriptome analysis of H. vastatrix germinating urediniospores (gU) and appressoria (Ap) was performed and compared to previously published in planta haustoria-rich (H) data. A total of 9234 transcripts were identified and annotated. Ca. 50% of these transcripts showed no significant homology to international databases. Only 784 sequences were shared by the three conditions, and 75% were exclusive of either gU (2146), Ap (1479) or H (3270). Relative transcript abundance and RT-qPCR analyses for a selection of genes indicated a particularly active metabolism, translational activity and production of new structures in the appressoria and intense signaling, transport, secretory activity and cellular multiplication in the germinating urediniospores, suggesting the onset of a plant-fungus dialogue as early as at the germ tube stage. Gene expression related to the production of carbohydrate-active enzymes and accumulation of glycerol in germinating urediniospores and appressoria suggests that combined lytic and physical mechanisms are involved in appressoria-mediated penetration. Besides contributing to the characterization of molecular processes leading to appressoria-mediated infection by rust fungi, these results point toward the identification of new H. vastatrix candidate virulence factors, with 516 genes predicted to encode secreted proteins.

454-pyrosequencing of Coffea arabica leaves infected by the rust fungus Hemileia vastatrix reveals in planta-expressed pathogen-secreted proteins and plant functions in a late compatible plant-rust interaction.

Coffee (Coffea arabica L.), one of the key export and cash crops in tropical and subtropical countries, suffers severe losses from the rust fungus Hemileia vastatrix. The transcriptome of H. vastatrix was analysed during a compatible interaction with coffee to obtain an exhaustive repertoire of the genes expressed during infection and to identify potential effector genes. Large-scale sequencing (454-GS-FLEX Titanium) of mixed coffee and rust cDNAs obtained from 21-day rust-infected leaves generated 352 146 sequences which assembled into 22 774 contigs. In the absence of any reference genomic sequences for Coffea or Hemileia, specific trinucleotide frequencies within expressed sequence tags (ESTs) and blast homology against a set of dicots and basidiomycete genomes were used to distinguish pathogen from plant sequences. About 30% (6763) of the contigs were assigned to H. vastatrix and 61% (13 951) to C. arabica. The majority (60%) of the rust sequences did not show homology to any genomic database, indicating that they were potential novel fungal genes. In silico analyses of the 6763 H. vastatrix contigs predicted 382 secreted proteins and identified homologues of the flax rust haustorially expressed secreted proteins (HESPs) and bean rust transferred protein 1 (RTP1). These rust candidate effectors showed conserved amino-acid domains and conserved patterns of cysteine positions suggestive of conserved functions during infection of host plants. Quantitative reverse transcription-polymerase chain reaction profiling of selected rust genes revealed dynamic expression patterns during the time course of infection of coffee leaves. This study provides the first valuable genomic resource for the agriculturally important plant pathogen H. vastatrix and the first comprehensive C. arabica EST dataset.

Validation of RT-qPCR reference genes for in planta expression studies in Hemileia vastatrix, the causal agent of coffee leaf rust.

Hemileia vastatrix is a biotrophic fungus, causing coffee leaf rust in all coffee growing countries, leading to serious social and economic problems. Gene expression studies may have a key role unravelling the transcriptomics of this pathogen during interaction with the plant host. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is currently the golden standard for gene expression analysis, although an accurate normalisation is essential for adequate conclusions. Reference genes are often used for this purpose, but the stability of their expression levels requires validation under experimental conditions. Moreover, pathogenic fungi undergo important biomass variations along their infection process in planta, which raises the need for an adequate method to further normalise the proportion of fungal cDNA in the total plant and fungus cDNA pool. In this work, the expression profiles of seven reference genes [glyceraldehyde-3-phosphate dehydrogenase (GADPH), elongation factor (EF-1), Beta tubulin (ß-tubulin), cytochrome c oxidase subunit III (Cyt III), cytochrome b (Cyt b), Hv00099, and 40S ribosomal protein (40S_Rib)] were analysed across 28 samples, obtained in vitro (germinated uredospores and appressoria) and in planta (post-penetration fungal growth phases). Gene stability was assessed using the statistical algorithms incorporated in geNorm and NormFinder tools. Cyt b, 40S_Rib, and Hv00099 were the most stable genes for the in vitro dataset, while 40S_Rib, GADPH, and Cyt III were the most stable in planta. For the combined datasets (in vitro and in planta), 40S_Rib, GADPH, and Hv00099 were selected as the most stable. Subsequent expression analysis for a gene encoding an alpha subunit of a heterotrimeric G-protein showed that the reference genes selected for the combined dataset do not differ significantly from those selected specifically for the in vitro and in planta datasets. Our study provides tools for correct validation of reference genes in obligate biotrophic plant pathogens, as well as the basis for RT-qPCR studies in H. vastatrix.