Entomopathogenic fungi associated to Prays oleae: isolation, characterization and selection for biological control

Tese de doutoramento em Ciências (área de especialiação em Biologia)Entomopathogenic fungi (EF) are nowadays considered one of the most promising alternatives to chemical pesticides. The use of fungal species or strains recovered from and adapted to a specific environment and host will increase the chances of success of biocontrol measures. In this work, we focused on one of the major pests affecting the olive culture, the lepidopteran Prays oleae Bern. A revision of available knowledge on entomopathogenic fungi is presented, focusing on their ecology and environmental factors that affected their occurrence, abundance and diversity. The infection mechanism is also addressed, as well as the enzymes and toxins related to both insect infection and death. The review on the compatibility of EF with chemical pesticides and other biological methods of pest control shows that different outcomes of those interactions are to be expected. Finally we address the current data about the use of EF for the control of olive pests. The first step was to ascertain which fungal entomopathogenic species were associated to P. oleae in an olive ecosystem. The undertaken survey, on larvae and pupae of the three annual generations (phyllophagous, antophagous and carpophagous) of P. oleae resulted in the identification of 43 species from 24 genera. Besides species best described as phytopathogenic, a large number of possible biocontrol agents, including antagonistic and entomopathogenic fungal species were found. Beauveria bassiana was the one found in higher abundance, especially in the phyllophagous generation. The internal transcribed spacer (ITS) region has been adopted as a barcode region for fungi, although many doubts still persist about the reliability of this marker. This work intends to evaluate and compare the efficiency of different molecular barcode markers (ITS, β-tubulin and RPB2), by amplifying these regions of fungal isolates retrieved from mycosed larvae and pupae of P. oleae. Although results showed higher performance of the ITS region, use of multiple barcode regions for fungal identification should be considered, as the use of β-tubulin and RBP2 increased the number of identified sequences. This multi-locus approach allowed the identification of fungal species associated with the death of larvae and pupae of P. oleae that present distinct roles in the ecosystem. The pathogenicity of four isolates of the major fungal taxa identified, B. bassiana, was evaluated on a lepidopteran pest of chestnut, Cydia splendana. The use of six concentrations of each fungal isolates showed a time- and concentration-dependent mortality of larvae, with mortalities ranging from 40 to 100%. Variations of virulence parameters were detected between isolates, both for median lethal concentration (LC50) and median lethal time (LT50). The results showed a high susceptibility of C. splendana to B. bassiana, indicating that EF can be useful to control this pest. After having determined the presence of EF in olive orchards, and their ability to infect insect pests, we decided to study some factors that can be responsible for the detected variation on their diversity and abundance in olive orchards. One of the studied factors was the impact that soil tillage has on EF. Larvae and pupae were sampled from olive orchards with or without soil tillage, and EF species associated to P. oleae were identified. A total of 120 isolates belonging to 8 EF species were found, being B. bassiana the most abundant one. Although no significant differences were observed between no-till and tilled orchards, higher occurrence, diversity and abundance was found in the former type of soil management. Four species were found exclusively on no-till orchards, indicating that the presence of natural vegetation creates more suitable conditions to EF, in olive orchards. The effect of the olive plant organs (leaves, flowers and fruits) on the manipulation of the entomopathogens B. bassiana and Paecilomyces formosa was studied under in vitro conditions. Both fungi are influenced by olive tree organs, but in different ways: Beauveria bassiana was more negatively affected by olives, while P. formosa was more negatively affected by leaves. Both volatile and diffusible compounds, as well as their interaction, are responsible for the observed effects. These results suggested that olive plant organ may be involved in the recruitment or maintain of specific fungal species which could partly explained the differences on fungal occurrence between P. oleae generations. Overall results showed that a pool of EF, for use as biocontrol agents, can be found in olive orchard, namely found in association to P. oleae, in high diversity and abundance. The most abundant species, B. bassiana, besides being isolated from P. oleae, also showed to be pathogenic to another lepidopteran pest, C. splendana. Of the factors that can interact with EF, different plant organs showed to have considerable influence on fungal behaviour. By other hand, different soil management practices didn‟t result in significant differences, although the presence of natural vegetation appears to be more suitable for EF. Combined information should give a significant thrust forward on research on EF for olive pest control, as more knowledge is needed, for the understanding of the interaction EF- P. oleae- olive tree.Os fungos entomopatogénicos (FE) são, hoje em dia, uma das mais promissoras alternativas aos pesticidas químicos. O uso de espécies ou estirpes fúngicas isoladas e adaptadas a um determinado ecossistema e hospedeiro resultará num aumento das probabilidades de sucesso de métodos de luta biológica. Este trabalho focou-se numa das principais pragas que afecta a cultura da oliveira, o lepidóptero Prays oleae Bern. É apresentada uma revisão sobre o conhecimento disponível sobre os FE, incidindo sobre a sua ecologia, e factores que afectam a sua ocorrência, abundância e diversidade. O mecanismo de infecção destes fungos é também abordado, assim como enzimas e toxinas responsáveis pela infecção e morte do hospedeiro. A compatibilidade de EF com meios de controlo de pragas, quer químicos, quer de luta biológica é também apresentada. Finalmente, apresentam-se os dados disponíveis sobre o uso de FE contra pragas da oliveira. O primeiro passo foi verificar as espécies de FE encontradas associadas a P. oleae no ecossistema olival. A pesquisa efectuada, em larvas e pupas das três gerações anuais (filófaga, antófaga e carpófaga) de P. oleae resultou na identificação de 43 espécies. Para além de espécies descritas como fitopatogénicas, foi encontrado um grande número de possíveis agentes de luta biológica, incluindo espécies de fungos antagonistas e entomopatogénicas. Beauveria bassiana foi a espécie encontrada em maior abundância, principalmente na geração filófaga. A região espaçadora interna transcrita (internal transcribed spacer - ITS) foi recentemente definida como a região padrão -barcode- para os fungos, apesar de ainda subsistirem dúvidas sobre a sua confiabilidade. Este trabalho teve como objectivo avaliar e comparar a eficiência de diferentes regiões barcode (ITS, β-tubulin e RPB2), amplificando-os de isolados fúngicos obtidos de larvas e pupas de P. oleae. Apesar de os resultados mostrarem maior eficiência da região ITS, o uso de múltiplas regiões deve ser considerado, uma vez que aumenta o número de sequências identificadas. Esta abordagem multi-regiões permitiu a identificação de fungos associados a P. oleae, com distintas funções no ecossistema. A patogenicidade de quatro isolados da principal espécie identificada, B. bassiana, foi avaliada em Cydia splendana, um lepidóptero praga da cultura da castanha. A utilização de seis concentrações de cada isolado mostrou uma mortalidade de larvas dependente do tempo e da concentração, com valores compreendidos entre 40 a 100% de mortalidade. Variações nos parâmetros de virulência foram detectados entre isolados, quer na concentração letal média (LC50), quer no tempo letal médio (LT50). Os resultados mostraram uma alta susceptibilidade de C. splendana à infecção por B. bassiana, indicando que os FE podem ser úteis para o controlo desta praga. Após ter sido determinada a presença de FE em olivais, e a sua capacidade de infectar insectos, foram estudados alguns factores que poderão influenciar a sua diversidade e abundância nos olivais. Um desses factores estudados foi o impacto da mobilização do solo nos FE. Foram recolhidas larvas e pupas da praga em olivais com e sem mobilização de solo, identificando-se as espécies isoladas. No total, 120 isolados, pertencentes a 8 espécies de FE foram identificados, sendo B. bassiana a mais abundante. Apesar de não terem sido detectadas diferenças significativas entre olivais com e sem mobilização solo, foi observada maior ocorrência, diversidade e abundância em solos sem mobilização. Quatro espécies de FE foram encontradas exclusivamente em olivais sem mobilização, indicando que a vegetação natural pode criar condições mais favoráveis aos FE. O efeito dos órgãos de oliveira (folhas, flores e frutos) nos FE B. bassiana e Paecilomyces formosa foi estudado em condições in vitro. Ambas as espécies foram influenciadas pelos órgãos da oliveira, ainda que de forma distinta. Beauveria bassiana foi negativamente mais afectada pelos frutos, sendo que P. formosa foi negativamente mais afectada pelas folhas. Compostos voláteis, difusíveis, e a sua interacção são responsáveis pelos efeitos observados. Estes resultados sugerem que os órgãos da oliveira podem estar envolvidos no recrutamento ou manutenção de FE específicos, o que pode explicar, parcialmente, as diferenças na ocorrência de FE entre gerações de P. oleae. Os resultados globais revelaram a existência de um conjunto de FE no olival, em associação com P. oleae e em grande abundância e diversidade, que podem ser explorados como agentes de luta biológica. A espécie mais abundante, B. bassiana, além de isolada de P. oleae, também se mostrou patogénica em outra praga, C. splendana. Entre os factores que podem interagir com FE, os diferentes órgãos da planta mostraram ter uma influência considerável sobre o comportamento dos fungos. Por outro lado, os diferentes sistemas de maneio do solo não causaram diferenças significativas, embora a presença de vegetação natural pareça beneficiar os FE. A informação resultante deste trabalho deverá dar um impulso significativo à pesquisa sobre FE para a luta contra pragas de oliveira, uma vez que mais informação é necessária, para a compreensão da interacção FE- P. oleae- oliveira

Barcoding of entomopathogenic fungi from olive tree pests : prospects and limitations

From all the barcoding initiatives in progress, fungal barcode is probably the one where more difficulties have been encountered. While for plants and animais the barcode regions were easy to define, for fungi the choice was not so straightforward. The internal transcribed spacer (ITS) region was one of the proposed DNA regions for barcoding fungi. This is an extensively used region, for molecular systematic and identification of species, being probably the most widely sequenced DNA region of fungi. This is due to the simplicity of the amplification, related to the multicopy nature of the rDNA; the possibility of using universal primers; and the high levei of sequence variation that occurs even between closely related species. Furthermore, a significant number of identified sequences for comparison are available in the GenBank database. Although the ITS region of rDNA was chosen for some groups of fungi, the use of this region presents very limited application for others, especially for Ascomycetes. As some of the most important entomopathogenic fungi are Ascomycetes, belonging to genera Beauveria, Cordyceps, Isaria, Lecanicillium and Paecilomyces, the use of the ITS region for barcoding purpose are being complemented with other regions. This work, based on the identification of fungal entomopathogens isolated directly from cadavers of one of the major pests in olive graves, the olive moth iPrevs oleae Bern.), intends to illustrate the application of the ITS region to identify these fungal species. The use of this region proved to be useful for the identification of most of the entomopathogenic fungi found in dead larvae and pupae of P. oleae. However, the use ot ITS region for barcode purposes did not allow the identification of several isolates, proving the requiremerít of using a second barcoding region, to enable full fungal identification.This work has been supported by FCT (PTDC/AGR-AAM/02600/2008)

Identification of fungi isolated from the olive moth (Prays oleae Bern.) based on ITS region

Fundação para a Ciência e Tecnologia (FCT) - Project PTDC/AGR-AAM/102600/200

Fungal diversity among different annual generations of Prays oleae

Comunicação em painelThe olives and olive oil production are important agricultural activities in Portugal. In the region of Trás-os-Montes, olive orchards are strongly affected by Prays oleae Bern., which is responsible for high losses in the olive production, as much as 40% of the expected yield. This lepidopteran presents three generations per year (phyllophagous, antophagous and carpophagous) that damage the olive tree in different organs (leaves, flowers and fruits, respectively). In an attempt to identify fungi that might cause the death of olive moths, larvae and pupae of those three annual generations were collected and surveyed for natural fungal infection. After their isolation, the fungal agents were molecularly identified by sequencing the amplified internal transcribed spacer (ITS) region of rDNA. In the present work, the diversity of fungal species associated to P. oleae, in several olive orchards located in Trás-os-Montes region, is discussed. The diversity and abundance of fungal species differed between all three generations. Higher diversity was found in the carpophagous generation, followed by the antophagous and phyllophagous generations. The use of already adapted fungal species to control one of the major pests of olive groves will increase the success of a future biocontrol strategy. In this context, the identification of fungi associated to P. oleae from olive orchards provided a pool of potential biocontrol agents. In this work, Beauveria bassiana proved to be the most promising fungus to be used as biocontrol agent against olive moth, being strongly associated to the phyllophagous generation. Other fungal species presenting entomopathogenic, antagonistic and phytopathogenic characteristics were also found.This work has been supported by FCT (PTDC/AGR-AAM/102600/2008)

Evaluation of the anti-inflammatory, antispasmodic and healing effects of walnut leaves Juglans regia L. aqueous extract.

As a part of the valorisation of medicinal plants of the Algerian flora, we have undertaken a study of plant extract (Juglans regia L.) growing in the Blida region, Algeria.The main objective of this work is to evaluate the in vivo pharmacological effects, namely the anti-inflammatory, antispasmodic and healing effects of common walnuts’s leaves aqueous extracts and ointment. The Carrageenan-induced paw edema test is used for screening of anti-inflammatory activity on 4 batches of mice (control batch, treated batch with Diclofenac® , treated batches with the aqueous extract at 5 and 10%), the analgesic effect was conducted using Acetic acid-induced endogenous spasm test, and the healing effect was conducted on rabbits. The anti-inflammatory test performed on laboratory mice, revealed a remarkable anti-inflammatory effect of the aqueous extract comparing with chosen standard pharmaceutical molecule (Diclofenac®), with a reduction percentage of 25 and 31.72 % for the aqueous extract and Diclofenac® respectively. The results for the analgesic effect was revealed to be significant comparing with those obtained with the reference product Spasfon. Finally, the healing effect was performed on rabbits, revealing high healing power comparing with those obtained with pharmaceutical healing paste ‘MADICASSOL” used as a reference. The ointment prepared using walnut leaves has shown more effectiveness with delay of healing less than a week compared to the ointment of reference which took almost 10 days for total healing effect. The obtained results confirm some information obtained during the ethnobotanical survey in a previous study; and that Juglans regia L. is endowed with an anti-inflammatory, antispasmodic and healing properties. 

Políticas públicas e desenvolvimento humano: fatores que impactam o IDH em municípios brasileiros

The human development approach considers that economic growth alone is not sufficient to explain progress, which results from a greater process of transformation. In this context, the Human Development Index – HDI has proved to be an important indicator to support the direction of public policies capable of promoting progress and increasing the quality of life of the populations. To contribute to this debate, the present research aims to investigate which factors explain the HDI of Brazilian municipalities. From the literature, socioeconomic variables with potential functional relationship with the HDI were identified. Through panel data analysis of 1,292 Brazilian municipalities, selected by geographical position, population representativeness and data availability, the three alternative econometric formulations were tested: Pooled Regression, Random Effects Model and Fixed Effects Model. The results of the regression indicated that the conditions of electricity, population size, location, income and sanitation proved to be explanatory factors for the HDI. From a theoretical point of view, the results complemented studies that relate socioeconomic variables to the HDI and, in a practical perspective, indicated ways for politicians and managers to promote public policies capable of improving human development in Brazilian municipalities.El desarrollo es una construcción política de la sociedad caracterizada por la mejora de la calidad de vida, el avance tecnológico, la mejora de las instituciones y la evolución de los indicadores sociales. En este contexto, el Índice de Desarrollo Humano - IDH ha demostrado ser un indicador importante para apoyar la dirección de políticas públicas capaces de aumentar la calidad de vida de las poblaciones. Por lo tanto, la presente investigación tiene como objetivo identificar los factores socioeconómicos que explican el IDH, a través del análisis de datos de panel de 1.292 municipios brasileños, en los años 1991, 2000 y 2010. Los resultados indicaron que las condiciones de energía eléctrica, ubicación, población, ingresos, saneamiento y violencia demostraron ser HDI explicativo. Las políticas públicas para mejorar estas condiciones tienden a promover mejoras en el IDH y en la calidad de vida de la sociedad.A abordagem do desenvolvimento humano considera que apenas o crescimento econômico não é suficiente para explicar o progresso, que decorre de um processo maior de transformação. Neste contexto, o Índice de Desenvolvimento Humano – IDH tem se mostrado um importante indicador para apoiar o direcionamento de políticas públicas capazes de promover o progresso e aumentar a qualidade de vida das populações. Para contribuir com este debate, a presente pesquisa tem como objetivo investigar quais fatores explicam o IDH de municípios brasileiros. A partir da literatura, foram identificadas variáveis socioeconômicas com potencial relação funcional com o IDH. Por meio de análise de dados em painel de 1.292 municípios brasileiros, selecionados por posição geográfica, representatividade populacional e pela disponibilidade de dados, foram testadas as três formulações econométricas alternativas: Pooled Regression, Modelo de Efeitos Aleatórios e Modelo de Efeitos Fixos. Os resultados da regressão indicaram que as condições de energia elétrica, tamanho populacional, localização, renda e saneamento se mostraram fatores explicativos do IDH. Do ponto de vista teórico, os resultados complementaram estudos que relacionam variáveis socioeconômicas ao IDH e, em perspectiva prática, indicou caminhos para que políticos e gestores possam promover políticas públicas capazes de melhorar o desenvolvimento humano em municípios brasileiros

Rheological properties of alkaline activated fly ash used in jet grouting applications

The application of alkaline activated fly ash to soil stabilisation has been recently studied, and although the structural behaviour was adequate, some concerns were raised regarding its apparent viscosity, which proved to be an important issue in jet grouting applications. Therefore, this paper deals with the experimental study of rheology of alkaline activated fly ash-based grouts, namely with: setting time of the freshly mixed grout; fluidity; capillary absorption; shrinkage and expansion behaviour; mass variation during the curing process and density. In order to establish some correlation points with structural performance, uniaxial compression strength (UCS) at 28 days curing was also determined. Six different grout compositions, defined based on Na2O (alkali)/ash and activator / ash ratios, were analysed. The activator was composed of sodium silicate and two different sodium hydroxide concentrations: 10.0 and 12.5 molal. Results show that the fluidity of the grouts correlate very well with UCS, with an increase of the former resulting in a decrease in the latter, which is a concern in jet grouting applications since this parameter controls the velocity of the grout at the nozzle and the soil/grout mixing capability. In terms of porosity, this type of material seems to be less porous than cement-based grouts. However, drying shrinkage values obtained were in general higher than usual for concrete, which is in accordance with results obtained by other researchers.Teixeira Duarte Engenharia e Construções S.A. e Fundação para a Ciência e Tecnologia (FCT

Innovation for Sustainability and Networking

Throughout human history, innovation has been the main factor in adapting humanity to its settings. On the basis of earlier practice, human creativity allows the finding of new, permanent ways to do things. their applications encourage new spaces, new necessities and new lifestyles. Innovation has been an element of human capacities from its earlier stages, but it has been recognized only recently as a clear device of social and economic change

Avaliação ultrassonográfica de variações anatômicas entre vasos femorais realizada por estudantes de medicina

Introduction: Central vein puncture is a medical procedure traditionally done following anatomical landmarks as a reference to successfully achieving the vessel. However, this traditional technique is commonly unsuccessful due to anatomical variations that may be found. Point of care ultrasonography (POC-US) is used to assist central catheterization by directly visualizing the vessel, increasing procedure security and minimizing risks. Objectives: Evaluate anatomical variations prevalence in femoral vessels, utilizing POC-US, done by medical students submitted to a short period of time training in ultrasonography. Methods: Five medical students, without previous experience on the use of ultrasonography, were submitted to an eight-hour theorical practical training in POC-US. The students evaluated one hundred femoral vessels of volunteers. Results: The right limb femoral vein was found more frequently in the medial position (43%) in comparison to the right limb femoral artery. On the left limb, the posteromedial position was the most found (45%). Conclusion: The insertion of a central catheter following the traditional technique is based on anatomical landmarks, and does not take into account existing anatomical variations. With a short period of training, POC-US is capable of qualifying professionals to acknowledge the real location of the vessel and avoid inadvertent punctures and complications.Introdução: A punção venosa central é um procedimento médico tradicionalmente realizado seguindo os marcadores anatômicos como referência para atingir o vaso, às cegas. No entanto, nem sempre o sucesso nessa técnica é alcançado, fato que se deve principalmente às variações anatômicas. A ultrassonografia point of care (US-POC) é utilizada para auxiliar a cateterização central por visualização direta do vaso, aumentando a segurança do procedimento. Objetivo: Avaliar a prevalência das variações anatômicas de vasos femorais através da utilização da US-POC por estudantes de medicina submetidos a curto período de treinamento. Materiais e Métodos: Cinco estudantes de medicina, sem experiência prévia em US-POC, foram submetidos a oito horas de treinamento teórico-prático. Foram avaliados os vasos femorais de cem voluntários. Resultados: A veia femoral direita foi encontrada mais frequentemente na posição medial (43%) em relação à artéria femoral direita. À esquerda, a posição posteromedial foi a mais observada (45%). Conclusão: A técnica tradicional de punção de acesso central se baseia em marcadores anatômicos e não leva em consideração as variações anatômicas existentes. Um treinamento de curto período para uso da US-POC é capaz de capacitar o profissional para reconhecer o posicionamento real dos vasos e evitar punções inadvertidas

A small TAT-TrkB peptide prevents BDNF receptor cleavage and restores synaptic physiology in Alzheimer's disease

Publisher Copyright: Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.In Alzheimer's disease (AD), amyloid β (Aβ)-triggered cleavage of TrkB-FL impairs brain-derived neurotrophic factor (BDNF) signaling, thereby compromising neuronal survival, differentiation, and synaptic transmission and plasticity. Using cerebrospinal fluid and postmortem human brain samples, we show that TrkB-FL cleavage occurs from the early stages of the disease and increases as a function of pathology severity. To explore the therapeutic potential of this disease mechanism, we designed small TAT-fused peptides and screened their ability to prevent TrkB-FL receptor cleavage. Among these, a TAT-TrkB peptide with a lysine-lysine linker prevented TrkB-FL cleavage both in vitro and in vivo and rescued synaptic deficits induced by oligomeric Aβ in hippocampal slices. Furthermore, this TAT-TrkB peptide improved the cognitive performance, ameliorated synaptic plasticity deficits and prevented Tau pathology progression in vivo in the 5XFAD mouse model of AD. No evidence of liver or kidney toxicity was found. We provide proof-of-concept evidence for the efficacy and safety of this therapeutic strategy and anticipate that this TAT-TrkB peptide has the potential to be a disease-modifying drug that can prevent and/or reverse cognitive deficits in patients with AD.publishersversionpublishe