Gnomoniopsis smithogilvyi, the causal agent of chestnut brown rot reported from Portugal

To ensure fruit quality and promote good storage practices, it is important to identify the fungi associated with chestnut (Castanea sativa) nut rots. In 2018, in the Trás-os-Montes region of Portugal, five orchards were selected and 300 fruits were collected from the ground by hand during the harvest period. In the laboratory, the fruits were observed visually and the number of decayed nuts recorded. Morphological symptoms were evaluated in diseased nuts and with the help of a scalpel, five sections (2 × 2 mm) of an infected cotyledon were cut for fungal isolation. Sections of the nuts were dipped in 70% (v/v) ethanol for 2 minutes and placed in 90 mm diameter Petri dishes containing potato dextrose agar (PDA; 39 g/l) to allow mycelial growth. Plates were incubated at 25 ±2ºC for seven days in the dark. Single colonies were transferred to new PDA plates to obtain purified isolates for subsequent identification. Identification of purified isolates was initially based on morphological characteristics and species identification obtained by molecular methods. DNA extraction was performed using the REDExtract-N-Amp™ Plant PCR Kit (Sigma-Aldrich, USA), and the ITS region amplified using the universal primers ITS1 and ITS4 (White et al., 1990). The amplified ITS region was sequencing by Stabvida Laboratories (Caparica, Portugal). Sequences were compared with published sequences in the GenBank databases using BLAST. Gnomoniopsis smithogilvyi was isolated from chestnut nuts which had cotyledons with a whitish-brown colour (Fig. 1). On PDA, the mycelium of G. smithogilvyi was woolly with a greyish colour and colonies developed in concentric circles (Fig. 2). This fungal species was found in 0 to 4.3% of fruits in the studied orchards (Table 1). Isolates Samil4.1, Samil6.1 and Brg-16 were preserved on agar slopes and kept at 4 ºC in the fungal collection of Instituto Politécnico de Bragança. The GenBank Accession Nos. of these sequences are MN956833, MN960397 and MN960408. G. smithogilvyi was identified in an industrial processing plant in 2017, 2018 and 2019 and the proportion of infected nuts were 8.0, 5.3 and 5.0% respectively. These values are in line with the study conducted by Driss (2019) on industrial samples from Portugal where it was observed 6.4% of the nuts were affected by G. smithogilvyi. This is the first detection of G. smithogilvyi on chestnut nuts in orchards in Portugal. This species has been reported as the main or among the major endophytes of chestnut tree (Visentin et al., 2012) and was isolated from rotten nuts from several countries in Europe and Asia, and in Australia (Lione et al., 2018). The disease symptoms in nuts are a colour alteration and texture degradation and sometimes the kernels are dehydrated (Lione et al., 2018). Several attacks can affect fruit production (Vannini et al., 2017).The authors are grateful to the Project BioChestnut - IPM - Implement effective control strategies against the chestnut and almond diseases under the Programme PDR2020-101-030959 for financial support.info:eu-repo/semantics/publishedVersio

O "Olho de Pavão" (Spilocaea oleagina) na região de Mirandela: distribuição, incidência, severidade e densidade de inóculo

Olive leaf-spot disease, studied by representative surveys, occurs on all olive groves of the region of Mirandela. Disease incidence and severity is higher on lower parts of canopy and at North and East directions. Fungus conidia are all ways present during the season with two main periods, November-December and April. Desfoliation by olive leaf-spot was in 1998 very important, with 80% of leaf-fall all two years twings. Por amostragem representativa da cultura da oliveira verificou-se que o "Olho de Pavão" está presente na generalidade dos olivais da região. A incidência da doença, avaliada par amostragem quinzenal, e superior na exposição Norte e Este e nas zonas mais baixas da copa. Os conídios do fungo encontram-se sempre presentes sendo a densidade de inoculo mais elevada nos meses de Novembro e Dezembro e ainda no mês de Abril. Nos olivais em estudo a desfoliação provocada pelo "Olho de Pavão" foi em média de 80% o que evidencia a importância desta doença na região

Characterization of the Phytophthora cinnamomi lipase activity

Phyiophthora cínnamomi is soilborn pseudofungus, associated to the Oomycetes with ink disease of chestnut. Lipases (triacylglycerols hydrolases) are important enzymes in fat metabolism, catalyzing the breakdown of triacylglycerols to free fatty acids and g!ycerol. Owing to the very low so!ubility of their natural substrates, this hydrolysis is catalyzed at the interface between an insoluble substrate and the aqueous phase in which the enzyme is solubilized

Cancro do castanheiro: a luta cultural na redução do inóculo e manutenção sustentada dos soutos

Cryphonectria parasitica fungo Ascomiceta da ordem Diaphortales e família Valsaceae foi noticiado pela primeira vez em Portugal em 1989. Teve uma dispersão muito rápida em todas as regiões de castanheiro existindo focos com forte incidência da doença de maior ou menor dimensão em todos os locais onde foi introduzido. Este trabalho teve como objectivo avaliar a eficácia da extirpação dos cancros como meio de luta contra o cancro do castanheiro e estudar as características da população de C. parasitica. A extirpação dos cancros quando aplicada a sistemas de exploração de castanheiros para fruto revelou ser muito eficaz, necessitando no entanto de um acompanhamento continuado para ser eficiente. A população de C. parasitica evidenciou grande uniformidade nas características morfológicas relacionadas com a cor e crescimento dos isolamentos e maior variabilidade na produção de esporos, evidenciando ainda uniformidade em relação à sensibilidade ao cobre. Todos os isolamentos foram incluídos no mesmo grupo de compatibilidade vegetativa (VCG) e identificado como VCG 2-P o mais frequente na população de C. parasitica em Portugal

Sensitivity to Copper and Phosphite of Phytophthora Species Associated with Ink Diseases of Chestnut

Phytophthora cinnamomi (23 isolates) and P. cambivora (3 isolates), isolated from chestnut showing ink disease symptoms or from soil near dead roots of chestnut trees were tested to copper and phosphite sensitivity. Isolates were characterized using an amended agar assay. EC50 values (copper (or phosphite) concentration inhibiting mycelial growth of the pathogen by 50%) for each isolate were calculated by plotting probit percentage inhibition against log10 active ingredient (a.i.) concentration. EC50 values of copper ranged from 0.043×10-3 to 2.025×10-3 M in P. cinnamomi and 1.970×10-3 to 4.603×10-3 M in P. cambivora. EC50 values of phosphite ranged from 2.99 to 172.39 μg ml-1 in P. cinnamomi and 47.23 to 237.25 μg ml-1 in P. cambivora. This study may contribute to access the potential utility of these a.i. for managing ink disease of chestnut and to use in the future as benchmark for baseline sensitivity

Epidemiologia do cancro do castanheiro. Dinâmica da distribuição espacial de Cryphonectria parasitica (Murrill) Barr

Com este trabalho pretendeu-se conhecer as características biológicas da população patogénica, os mecanismos de multiplicação e dispersão espaço-temporal e o efeito da remoção do inóculo no desenvolvimento da doença, aspectos que foram estudados durante três anos consecutivos num souto de Parada-Bragança, localizado num dos focos iniciais do aparecimento do cancro em Portugal

Short and long term efficacy and prevalence of Cryphonectria parasitica hypovirulent strains released as biocontrol agents of chestnut blight

Chestnut blight, Cryphonectria parasitica (Murrill) Barr, was reported in Portugal in 1989. The disease rapidly spread within the chestnut region of the Trás-os-Montes (the Northeast of Portugal). Eradication and mechanical/burning exclusion failed to control the disease and natural hypovirulence is still absent or of very low incidence. The introduction of human-assisted therapeutic hypovirulence is therefore required to control the disease. We presente here the efficacy of field applications (short and long term) of a locally characterized hypovirulent strain (CHV1-RBB111), by the punch method in two chestnut orchards (Valpaços and Vinhais). The Valpaços disease incidence was 68% (65 out of 96 trees) and in Vinhais it was 46% (78 out of 169 trees). In both locations, diversity of vegetative incompatibility (vc type) was low and largely dominated (> 85%) by a single vc type (EU11). In Valpaços, all cankers of the orchard were treated (63 cankers) and in Vinhais 31 trees (cankers) were treated. One year after application, field efficacy was very high with 93.8% of healed cankers in Valpaços and 90.3% in Vinhais, these values increased to 100% and 93.5% (respectively) after four years. The persistence of the introduced CHV1-RBB 111 strain was high and present in almost all treated cankers and it was dominant both inwards and on the application site revealing an endophytic behavior. The non-C. parasitica microfungi were abundant with 14 different species. Biscogniauxia mediterranea (De Not.) Kuntze was the most abundant in both orchards. Treatment with the introduced hypovirulent strain (CHV1-RBB111) was found to be effective in controlling chestnut blight with regeneration of cortical tissues and recovery of treated trees.The authors are grateful to the Project BioChestnut -IPM- Implement effective control strategies against the chestnut and almond diseases under the Programme PDR2020- 101-030959 for financial support. This study was funded by the Project BioChestnut- IPM (PDR2020–101-030959).Data availabilityThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.info:eu-repo/semantics/publishedVersio

Validação de potenciais biomarcadores proteómicos na Síndrome da Apneia Obstrutiva do Sono

Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015A Síndrome da Apneia Obstrutiva do Sono (SAOS) é um distúrbio respiratório muito prevalente na população em geral, com graves repercussões na saúde e na qualidade de vida dos doentes. A Proteómica tem vindo a ser aplicada no estudo da SAOS na pesquisa de novos biomarcadores de diagnóstico, prognóstico, monitorização e/ou alvo terapêutico para esta doença. Estudos preliminares realizados no Laboratório de Proteómica do INSARJ revelaram a existência de proteínas diferencialmente moduladas em glóbulos vermelhos (GVs) de doentes com SAOS comparados com roncadores simples. Algumas destas proteínas, como a catalase (CAT) e a peroxiredoxina 2 (Prx2), estão envolvidas em processos antioxidantes, e este mecanismo parece estar significativamente comprometido nos doentes com SAOS. O principal objectivo deste trabalho foi a validação da CAT e da Prx2 por técnicas ortogonais à Proteómica, como o western blotting (WB) e/ou por estudos de cinética enzimática em amostras de GVs de doentes com SAOS, antes e após seis meses de tratamento CPAP (Continuous Positive Airway Pressure), e em indivíduos roncadores simples como controlos. Dois tipos de amostras de GVs foram avaliadas por doente, amostra recolhida no final da tarde antes do exame de polissonografia – PSG (denominadas de “Noite”) e amostra recolhida na manhã seguinte ao exame (denominadas de “Manhã”). Após tratamento CPAP, apenas as amostras de GVs “Manhã” foram preparadas e, portanto, consideradas no estudo. O estudo da CAT, por cinética enzimática, revelou que a actividade da CAT é significativamente menor nos GVs “Manhã” em comparação com as amostras “Noite”, sejam de doentes SAOS ou de controlos (Anova p<0,05). Contudo, esta diminuição da actividade da CAT durante a manhã em relação à noite estava significativamente (T Student, p<0,05) mais acentuada nos doentes com SAOS do que nos controlos. Após seis meses de tratamento com CPAP, observou-se um aumento muito significativo da actividade da CAT nos GVs “Manhã”. Surpreendentemente, constatou-se por WB, não haver diferenças quantitativas e significativas dos níveis de expressão da CAT entre os grupos de doentes/condições analisados, e tão pouco havia, nos doentes SAOS, antes e após o tratamento CPAP. Estes resultados sugerem que a modulação da atividade da CAT nos GVs está essencialmente associada a modificações pós-traducionais da proteína, o que corrobora com os resultados obtidos pela Proteómica, que identificou oito proteoformas para a CAT nestas células. A avaliação do estado redox/oligomérico da Prx2 nos GVs por WB em SDS-PAGE não reduzido, utilizando anticorpos específicos para a Prx2 e para formas hiperoxidadas da Prx (PrxSO2/3), revelou que as formas monoméricas e diméricas da Prx2 estavam significativamente hiperoxidadas nos GVs “Manhã” ou “Noite” de doentes com SAOS. O tratamento CPAP diminui esta hiperoxidação nos monómeros e dímeros da Prx2. Contudo, as formas multiméricas da Prx2 hiperoxidadas, descritas com actividade chaperone/transdução de sinal associadas à protecção celular, surgiram quase que exclusivamente nos doentes SAOS após CPAP. A forma dissulfídica oxidada/hiperoxidada da Prx2 está associada a inativação da sua actividade peroxidática, e por conseguinte, a estado de stresse oxidativo. A conversão entre as diferentes funções da Prx2, ou seja, entre a actividade peroxidática antioxidante e a atividade chaperone/transdução de sinal, é regulada pelo seu estado redox/oligomérico na célula. Em resumo, os resultados deste trabalho validaram os resultados anteriormente obtidos por Proteómica, indicando nos GVs de doentes com SAOS, existência de desregulação no seu sistema redox-homeostase envolvendo a CAT e a Prx2. Este trabalho propõe ainda, a CAT e a Prx2, como fortes candidatos a biomarcadores de gravidade e/ou de monitorização do tratamento CPAP. Contudo, serão necessários mais estudos para consolidar este resultados e contribuir para uma melhor compreensão acerca do diagnóstico, monitorização e tratamento para a SAOS.The Obstructive Sleep Apnea (OSA) is a breathing disorder very prevalent in the general population, with serious effects on health and quality of life of the patients. The Proteomics has been applied to the study of the OSA, in order to identify new biomarkers of diagnosis, prognosis, monitoring and/or as therapeutic target for this disease. Preliminary studies at Proteomics Laboratory of INSARJ revealed the existence of proteins differentially modulated in red blood cells (RBCs) of OSA patients compared with simple snorers (controls). Some of these proteins such as catalase (CAT) and peroxiredoxin 2 (Prx2), are involved in antioxidants processes, which seem to be significantly compromised in OSA patients. The main objective of this work was the validation of these proteins by orthogonal technologies to Proteomics, such as western blotting (WB) and/or kinetic enzyme assay on RBCs samples from OSA patients, before and after six months of treatment CPAP (Continuous Positive Airway Pressure), and from simple snorers as controls. Two types of RBCs samples were evaluated per patient, sample taken late in the afternoon and before polysomnography (PSG) (denominated here "Night") and sample collected in the morning after the examination ("Morning"). After CPAP treatment, only "Morning" RBC samples were prepared and therefore considered in this study. The kinetic enzyme assay showed that CAT activity is significantly higher in RBC "Morning" compared to the "Night" samples, whatever were OSA or control (Anova p<0.05). However, this decrease in “Morning” CAT activity compared to “Night” was even greater in OSA RBC samples than in controls ones (Student's T p <0.05). After six months of CPAP treatment, there was a very significant increase in CAT activity in this "Morning" RBC OSA samples. Surprisingly, no significant differences were observed in the CAT expression level among the patient’s group/condition analysed. These results suggest that the modulation of CAT activity in RBCs is mainly associated with post-translational modifications, which supports the previous proteomics data identifying eight proteoforms for CAT in these cells. The evaluation of redox/oligomeric study of the Prx2 in RBCs by WB in SDS-PAGE, non-reduced, using antibodies specific for Prx2 and hyperoxidized forms of Prx (PrxSO2/3) demonstrated that the monomeric and dimeric form of Prx2 were significantly hyperoxidized in “Morning” or “Night” OSA RBCs. The CPAP treatment reduced this hyperoxidation observed on monomers and dimers of Prx2. However, hyperoxidized multimeric forms of Prx2, described with chaperone activity, appeared almost exclusively in OSA patients after CPAP. The disulfide oxidized/hiperoxidized forms of Prx2 are associated with the inactivation of its peroxidatic activity and therefore with the state of stress-oxidative. The conversion between the different functions of Prx2, i.e. between antioxidant peroxidatic activity and chaperone activity or transduction signaling, is regulated by its redox/oligomeric state in the cell. In summary, the results of this study validated the results previously obtained by proteomics, indicating that in OSA RBCs the redox- homeostasis system involving the CAT and Prx2 is dysregulated. This work also proposes CAT and Prx2 as a promising candidates biomarkers of severity and/or to monitor CPAP treatment. However, more studies will be needed to consolidate this results and contribute to a better understanding of OSA diagnosis and therapy monitoring

Obstructive sleep apnea associated with Diabetes mellitus Type 2: a proteomic study

Background: We previously showed that Obstructive sleep apnea (OSA), a common public health concern causing deleterious cardiometabolic dysfunction, induced proteomic alterations in red blood cells (RBC) such as changes in the redox-oligomeric state of peroxiredoxin 2 (PRDX2)1-2. Herein, we aimed to investigate whether OSA patients with Type 2 Diabetes Mellitus before and after positive airway pressure (PAP) treatment present similar changes in the RBC antioxidant protein PRDX2 to better understand the molecular basic mechanisms associated with OSA and OSA outcomes. Methods: RBC samples from control snorers (n=22 being 3 diabetics) and OSA patients before and after six month of PAP-treatment (n=29 being 8 diabetics) were analysed by non-reducing western blot using antibody against PRDX2 or PRDXSO2/3 to measure the total and overoxidized levels of monomeric/dimeric/multimeric forms of PRDX2. Results: We confirmed previously data by showing that in OSA RBC the overoxidation on the monomeric forms of PRDX2 was higher compared to controls. After PAP treatment, this overoxidation decreased followed by an increase of multimeric-overoxidized forms of PRDX2 described to be associated with chaperone protective function. In contrast, the level of PRDX2 monomers in RBC diabetic OSA, although higher abundant its overoxidation level was much lower than those observed in OSA without comorbidity and did not significant change after treatment. Moreover, the level of PAP-induced PRDX2-overoxidized-multimers was also lower in these diabetic OSA patients. The level of overoxidized monomeric/dimeric forms of PRDX2 correlated negatively with levels of insulin / triglycerides and HbA1C, respectively. After PAP, the level of (overoxidized) PRDX2SO2/3 multimers correlated positively with adrenaline levels. Conclusions: The redox/oligomeric state of RBC PRDX2 that is regulated by overoxidation of the active cysteines was differentially modulated in diabetic OSA patients compared to OSA without this comorbidity. PAP-induced overoxidized oligo forms of PRDX2 that is associated with chaperone protective function showed decreased in OSA patients with diabetes. The clinical impact of these findings needs further investigation and validation.Project partially supported by Harvard Medical School-Portugal Program (HMSP-ICJ/0022/2011), ToxOmics - Centre for Toxicogenomics and Human Health (FCT-UID/BIM/00009/2013).info:eu-repo/semantics/publishedVersio

Characterization of chestnut bark fungal communities in healthy trees and blight recovered through natural or introduced hypovirulence

Various fungal species together with Cryphonectria parasitica (Murr.) Barr. have been isolated from chestnut tissues with blight symptoms. Microfungi remain in cankers during tissue healing, which occurs by transmitting hypovirulence by Cryphonectria hypovirus 1 (CHV1) compatible strains. However, studies focused on the diversity and ecology of the non-C. parasitica taxa on chestnut bark are lacking. This work evaluated the composition and richness of microfungi species associated with healthy chestnut trees, those with cankers healed by natural hypovirulence and those treated with hypovirulent strains (artificially introduced hypovirulence). Microfungi from diseased trees were isolated from six randomly selected points in the inner and external areas of the healed canker. In healthy trees, tissue samples were collected from 12 random locations on each tree’s trunk. Fungal species were identified based on morphological characteristics and ITS region sequencing using the universal primers ITS1 and ITS4. Four hundred thirty-one fungal isolates were obtained from which 38 operational taxonomic units (OTUs) were identified. The fungal communities varied from tree to tree and did not display similar patterns. The endophyte Biscogniauxia mediterranea and epiphyte Cytospora eucalypticola fungi were detected in all study locations and tree health conditions. Notably, C. parasitica (virulent and hypovirulent strains) was dominant in the inner area of healed cankers, accounting for 64.3% of the isolates, and the saprobe fungi Penicillium glabrum was dominant among non-C. parasitica microfungi species. Dissimilarity analyses showed low similarity between the microfungi communities found in the inner and external areas of the healed cankers. The study reveals the long-life span of C. parasitica in healed cankers and the therapeutic effect of natural and introduced hypovirulence.This study was funded by the Project BioChestnut PDR2020-101-030959info:eu-repo/semantics/publishedVersio