<p>Oomycetes have long been considered as a separate class within the kingdom Fungi, but they are in fact closer to brown algae. They are currently classified in the Stramenopile eukaryotic kingdom, which includes heterokont algae and water molds. The major cell wall polysaccharides in Oomycetes are b-(1à3) and b-(1à6)-glucans, as well as cellulose, which has never been reported in any fungal species. Chitin - the major cell wall polysaccharide in fungi - occurs in minor amounts in the walls of some Oomycetes. Some Oomycete species are pathogens of great economical importance. For example, species of the genus <em>Phytophthora </em>are well studied plant pathogens that cause considerable economical losses in agriculture. Saprolegniosis, a fish disease caused by species from the genus <em>Saprolegnia</em>, is a major problem in the aquaculture industry and represents a threat to populations of salmonids in natural habitats. Currently, there are no chemicals available that are at the same time efficient Oomycete inhibitors, environmentally friendly and safe for human consumption of treated fishes. The biosynthesis of cellulose in Oomycetes is poorly understood, even though this biochemical pathway represents a potential target for new Oomycete inhibitors. In this work, cellulose biosynthesis was investigated in two selected Oomycetes, the plant pathogen <em>Phytophthora infestans</em> and the fish pathogen <em>Saprolegnia monoica</em>.</p><p> </p><p>A new Oomycete <em>CesA</em> gene family was identified. It contains four homologues designated as <em>CesA1, CesA2, CesA3</em> and <em>CesA4</em>. The gene products of <em>CesA1, 2</em> and <em>4 </em>contain Pleckstrin Homology domains located at the N-terminus. This represents a novel feature, unique to the Oomycete <em>CesA </em>genes. <em>CesA3</em> is the dominantly expressed <em>CesA </em>homologue in the mycelium of both <em>S. monoica</em> and <em>P. infestans</em>, while <em>CesA1</em> and<em> CesA2</em> are up-regulated in virulent life stages of <em>P. infestans</em>. <em>CesA4</em> was expressed only in minute amounts in all investigated types of cells. Gene silencing by RNA interference of the whole <em>CesA</em> gene family in <em>P. infestans</em> lead to decreased amounts of cellulose in the cell wall. The inhibitors of cellulose synthesis DCB and Congo Red had an up-regulating effect on <em>SmCesA</em> gene expression, which was accompanied by an increased b-glucan synthase activity <em>in vitro</em>. In addition, these inhibitors slowed down the growth of the mycelium from <em>S. monoica</em>. Zoospores from <em>P. infestans</em> treated with DCB were unable to infect potato leaves and showed aberrant cell wall morphologies similar to those obtained by silencing the <em>CesA</em> gene family.</p><p>Altogether these results show that at least some of the <em>CesA1-4</em> genes are involved in cellulose biosynthesis and that the synthesis of cellulose is crucial for infection of potato by <em>P. infestans</em>.</p><p> </p>

Vieni iš pastaruoju metu sparčiai plintančių augalų patogenų – Phytophthora genties patogenai. Šios genties patogenai pažeidžia vertingus vietinius ir iš svetimų kraštų kilusius augalus, augančius natūraliose augavietėse, miestų želdynuose ir medelynuose. Labai svarbu Phytophthora genties įvairių rūšių patogenų sukeliamas ligas kuo greičiau diagnozuoti, nustatyti šių grybų sąveiką su kitų rūšių patogeniniais organizmais bei pažeidžiamomis augalų rūšimis. Šio darbo tikslas – įvertinti juodalksnių (Alnus glutinosa (L.) Gaetrn.), augančių VDU Kauno botanikos sode, pažeistumą Phytophthora genties organizmais: iš infekuotų audinių išauginti ir morfologiškai apibūdinti grynas patogenų kultūras, identifikuoti patogenus naudojant ekspres testą ALERTTM bei išskirti DNR iš infekuotos medienos, lapų ir dirvožemio. Patogenais infekuoti ėminiai buvo auginami 12-oje Petri lėkštelių su morkų gabalėlių terpe ir 11-oje Petri lėkštelių su salyklo ekstrakto su chloramfenikoliu mitybine terpe. Greituoju testu buvo tikrinami 6 dirvožemio pavyzdžiai. Patogenų DNR buvo išskiriama iš infekuotos medienos, lapų ir dirvožemio. Phytophthora genties kolonijų augimo nepastebėta. Ištyrus dirvožemio ėminius buvo gauti teigiami testų rezultatai. Genominės DNR, išskirtos iš medienos bei lapų, koncentracija svyravo nuo 0,64 iki 22,66 ng/ml, o DNR, išskirtos iš dirvožemio, koncentracija svyravo nuo 5,78 iki 10,2 ng/ml. / Pathogens of Phytophthora genus, are very rapidly spreading plant pathogens, recently. Pathogens of this genus negatively effect valuable local plants and those, which are brought from foreign lands ang grow in natural habitats, urban green plantations and arboretums. It is very important to identify diseases, which are caused by this species or other Phytophthora‘s genus pathogens and to identify interaction of these fungi and other pathogenic organisms species, negatively effected plant species. The aim of the study is to assess the damage done by Phytophthora genus organisms to black alders (Alnus glutinosa), growing in VMU Kaunas botanical garden: to morphologically characterize pure culturs of pathogens and grow them from infected tussues, identify pathogens using Express ALERTTM test and extract DNA from infected wood, leaves and soil. Pathogen-infected samples were grown in 12 Petri dishes with carrot medium and in 11 Petri dishes with malt extract and chloramphenicol medium. High-speed test was used for 6 samples of the soil. Pathogen‘s DNA was extracted from infected wood, leaves and soil.Growth of Phytophthora genus polony was not observed. After the analysis of soil-positive test result was obtained. Concentration of genomic DNA from the wood and leaves was 0,64-22,66 ng/ml from leaves and 5,78-10,2 ng/ml from soil.

No description available.

Tree canopies form the most important interface between the earth and the atmosphere, but their role as a potential habitat for eukaryotic microorganisms has been severely understudied. This thesis addresses the hitherto insufficient characterisation of protist diversity and community composition in forest soils and the canopy region, while in particular focusing on the prominent and mostly plant pathogenic Oomycota. Accordingly, several microhabitat compartments were sampled on the ground and in the canopy with the Leipzig Canopy Crane in two seasons over two years, and additionally in a palaeotropical rainforest in Papua New Guinea. Taxon-specific primers were applied to characterise the oomycete diversity in a high-throughput metabarcoding approach. In four chapters, I assess the initial description of oomycete communities in the canopy, their distribution in the forest ecosystem via air dispersal, their seasonality and functional diversity including the quantification of plant pathogens, and their diversity in tropical canopies. These results contribute to a more comprehensive understanding of oomycetes and their pathogenic lineages, their diversity, ecology, distribution, and ecosystem functioning.

Oomycetes have long been considered as a separate class within the kingdom Fungi, but they are in fact closer to brown algae. They are currently classified in the Stramenopile eukaryotic kingdom, which includes heterokont algae and water molds. The major cell wall polysaccharides in Oomycetes are b-(1à3) and b-(1à6)-glucans, as well as cellulose, which has never been reported in any fungal species. Chitin - the major cell wall polysaccharide in fungi - occurs in minor amounts in the walls of some Oomycetes. Some Oomycete species are pathogens of great economical importance. For example, species of the genus Phytophthora are well studied plant pathogens that cause considerable economical losses in agriculture. Saprolegniosis, a fish disease caused by species from the genus Saprolegnia, is a major problem in the aquaculture industry and represents a threat to populations of salmonids in natural habitats. Currently, there are no chemicals available that are at the same time efficient Oomycete inhibitors, environmentally friendly and safe for human consumption of treated fishes. The biosynthesis of cellulose in Oomycetes is poorly understood, even though this biochemical pathway represents a potential target for new Oomycete inhibitors. In this work, cellulose biosynthesis was investigated in two selected Oomycetes, the plant pathogen Phytophthora infestans and the fish pathogen Saprolegnia monoica. A new Oomycete CesA gene family was identified. It contains four homologues designated as CesA1, CesA2, CesA3 and CesA4. The gene products of CesA1, 2 and 4 contain Pleckstrin Homology domains located at the N-terminus. This represents a novel feature, unique to the Oomycete CesA genes. CesA3 is the dominantly expressed CesA homologue in the mycelium of both S. monoica and P. infestans, while CesA1 and CesA2 are up-regulated in virulent life stages of P. infestans. CesA4 was expressed only in minute amounts in all investigated types of cells. Gene silencing by RNA interference of the whole CesA gene family in P. infestans lead to decreased amounts of cellulose in the cell wall. The inhibitors of cellulose synthesis DCB and Congo Red had an up-regulating effect on SmCesA gene expression, which was accompanied by an increased b-glucan synthase activity in vitro. In addition, these inhibitors slowed down the growth of the mycelium from S. monoica. Zoospores from P. infestans treated with DCB were unable to infect potato leaves and showed aberrant cell wall morphologies similar to those obtained by silencing the CesA gene family. Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that the synthesis of cellulose is crucial for infection of potato by P. infestans. / QC 20101110

Oomycetes (Stramenopiles, protists) are among the most severe plant pathogens, comprising species with a high economic and ecologic impact on forest ecosystems. Their diversity and community structures are well studied in terrestrial habitats, but tree canopies as huge and diverse habitats have been widely neglected. A recent study highlighted distinct oomycete communities in the canopy stratum compared to the ground region of three temperate deciduous trees (Quercus robur, Tilia cordata, Fraxinus excelsior). While the communities from the two strata were distinct when taking oomycete abundances into account, they were rather similar when only OTU presence/absence was considered. It remains, however, unknown if this homogeneity in the OTU presence also leads to a functional homogenisation among microhabitats within the two strata ground and canopy. In this study, we supplemented functional traits to oomycete communities in the tree microhabitats, which were determined over a time period of 2 years with a metabarcoding approach. Our results showed that even though most oomycetes occurred in all microhabitats, a strong discrepancy between the strata and correspondingly the distribution of oomycete lifestyles could be observed. This pattern was constant over several seasons. Obligate biotrophic species, exclusively feeding on living host tissue, dominated the canopy region, implying tree canopies to be a hitherto neglected reservoir for parasitic protists. OTUs assigned to the genus Hyaloperonospora—parasites highly specialised on hosts that were not sampled—could be determined in high abundances in the canopy and the surrounding air, challenging the strict host dependencies ruled for some oomycetes. Our findings further contribute to the understanding of oomycete ecosystem functioning in forest ecosystems

Some species of Oomycetes are well studied pathogens that cause considerable economical losses in the agriculture and aquaculture industries. Currently, there are no chemicals available that are environmentally friendly and at the same time efficient Oomycete inhibitors. The cell wall of Oomycetes consists of b-(1à3) and b-(1à6)-glucans, cellulose and in some species minute amounts of chitin. The biosynthesis of cellulose and chitin in Oomycetes is poorly understood. However, cell wall synthesis represents a potential target for new Oomycete inhibitors. In this work, cellulose and chitin synthase genes and gene products were analyzed in the plant pathogen Phytophthora infestans and in the fish pathogen Saprolegnia monoica.   A new Oomycete CesA gene family was identified, containing four subclasses of genes designated as CesA1 to 4. The gene products of CesA1, 2 and 4 contain pleckstrin homology (PH) domains located at the N-terminus, which is unique to the Oomycete CesAs. Our results show that the SmCesA2 PH domain binds to phosphoinositides, F-actin and microtubules in vitro and can co-localize with F-actin in vivo. Functional characterization of the CesA genes by gene silencing in P. infestans led to decreased cellulose content in the cell wall. The cellulose synthase inhibitors DCB and Congo Red inhibited the growth of the mycelium of S. monoica and had an up-regulating effect on SmCesA gene expression. Zoospores from P. infestans treated with DCB were unable to infect potato leaves. In addition, two full-length chitin synthase genes (Chs) were analyzed from S. monoica.  Expression of SmChs2 in yeast yielded an active recombinant protein. The biochemical characterization of the in vitro product of SmChs2 confirmed that the protein is responsible for chitin formation. The chitin synthase inhibitor nikkomycin Z inhibited the SmChs2 both in vivo and in vitro.   Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that synthesis of cellulose is crucial for infection of potato by P. infestans. The PH domain is involved in the interaction of CesA with the cytoskeleton. In addition, we firmly demonstrate that the SmChs2 gene encodes a catalytically active chitin synthase. / QC 20110531

O aumento da resistência microbiana devido a fatores como uso excessivo e ineficiente de antibióticos convencionais acarreta a necessidade da busca por novos compostos bioativos que atuem por mecanismos de ação diferentes aos fármacos já conhecidos. Na agricultura, o uso intensivo de pesticidas para o combate de microrganismos que comprometem principalmente a parte alimentícia também traz diversos problemas relacionados à resistência antimicrobiana e a riscos ambientais, oriundos do acúmulo dessas substâncias no solo. Dentro deste aspecto, o pseudofungo Pythium aphanidermatum, da classe dos oomicetos, destaca-se por ser uma espécie agressiva e altamente resistente a fungicidas comuns, apodrecendo raízes e frutos de cultivos de tomate, beterraba, pepino, pimentão, etc. A própolis verde, constituída em sua grande parte por material resinoso coletado e processado pela abelha da espécie Apis mellifera tem sido utilizada na medicina tradicional devido ao seu amplo espectro de ações preventivas e tratamentos de doenças, possuindo propriedades anti-inflamatórias, antimicrobianas, anticancerígenas e antioxidantes, tornando-se um produto de grande interesse na busca de novos compostos bioativos. Dentro destes aspectos apresentados, neste trabalho investigamos a ação da própolis verde contra o fitopatógeno P. aphanidermatum e identificamos através da técnica de cromatografia e bioensaios que a Artepillin C (3,5-diprenil-4-ácido-hidroxicinâmico), majoritária na própolis verde, foi o principal composto nesta ação. Os efeitos terapêuticos desta molécula tem sido foco de muitos estudos, porém ainda não há evidência em sua interação com agregados anfifílicos que mimetizam membranas celulares. O caráter anfifílico do composto, elevado pela presença dos grupos prenilados ligados ao ácido cinâmico, favoreceram a sua inserção nas membranas modelo, principalmente em seu estado agregado. Estas conclusões puderam ser inferidas devido às alterações nas propriedades das bicamadas lipídicas na presença da Artepillin C, podendo causar, especificamente para o caso de fitopatógenos como o P. aphanidermatum, perdas funcionais das proteínas de membranas, liberação de eletrólitos intracelulares e desintegração citoplasmática dos micélios e esporos. Ainda, as diferentes composições lipídicas nas vesículas influenciam no modo de interação do composto e consequentes alterações em suas estruturas, principalmente na presença do colesterol, que auxilia na manutenção da permeabilidade da bicamada lipídica, que pode contribuir para a integridade do conteúdo citoplasmático da célula. / The increase in the microbial resistance due to the excessive and inefficient use of conventional antibiotics brings the necessity to search new bioactive compounds which play their mechanism of action differently from the known drugs. In the agriculture, the intensive use of pesticide for the combat of microorganisms which undermine mainly the food portion also brings several issues related to the antimicrobial resistance and environment risks, originated from the high amount of these substances on the soil. In this aspect, the fungus-like Pythium aphanidermatum microorganism, from class Oomycete, stands out for being an aggressive species and highly resistant to common fungicides, rotting roots and fruits of tomato, beet, cucumber, pepper, etc. Green propolis, constituted by resinous material collected and processed by bees of the species Apis mellifera, has been used in the traditional medicine due its wide spectrum of preventive actions and diseases treatments, promoting anti-inflammatory, antimicrobial, anticancer and antioxidant properties, becoming a product of interest for investigation in the research of new bioactive compounds. Under all the aspects showed so far, in this work we investigated the action of the green propolis against the phytopathogen P. aphanidermatum and identified through chromatography and bioassays that Artepillin C (3,5-diprenyl-4-hydroxycinnamic acid), majority in the green propolis, was the main compound in this action. The therapeutic effects of this molecule have been the focus of several studies, but, so far there is no evidence for its interaction with amphiphilic aggregates that mimic cell membranes. The amphiphilic character of the compound, enhanced by the presence of two prenylated groups bounded to the cinnamic acid, favors the insertion of the compound in the model membranes mainly in its aggregation state. These conclusions could be inferred due the alterations in the properties of the lipid bilayer in the presence of Artepillin C, that may cause, specifically in the case of phytopathogens like P. aphanidermatum, functional losses of membrane proteins, releasing of intracellular electrolytes and cytoplasmatic disintegration of mycelium and spores. Moreover, the difference of the lipid composition in the vesicles influence in the action of the compound and consequent alteration in their structures, mainly in the presence of cholesterol, that provides the maintenance of permeability of the lipid bilayer, contributing to the integrity of the cytoplasmic material of the cell.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-17T13:13:31Z No. of bitstreams: 1 Antonio Alves Pimenta Neto.pdf: 891078 bytes, checksum: 3c3c0666af1521e887fa16686d4d993c (MD5) / Made available in DSpace on 2017-02-17T13:13:31Z (GMT). No. of bitstreams: 1 Antonio Alves Pimenta Neto.pdf: 891078 bytes, checksum: 3c3c0666af1521e887fa16686d4d993c (MD5) Previous issue date: 2012-02-29 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / This study aimed to evaluate the effectiveness of extracts and vegetable oils in controlling diseases of Solanaceae caused by Phytophthora nicotianae and its effects on the isolates of the pathogen. The effect of means and methods of cultivation in the sporulation of the pathogen was first studied to enable the inoculations and tests with oils and plant extracts. The first study was to evaluate the influence of various liquid and solid culture media, obtained from plant host tissues and/or indicated in the literature for the cultivation of Phytophthora spp, as well as different light regimes: constant dark (D), 12h alternating light (LD), and constant light (L) and the hydrogen potential in growth and sporulation of P. nicotianae. The mycelial growth was obtained at 10 and 15 days of culture, through the quantification of biomass. The development of the colonies in the agar media was monitored through daily measurement of radial growth. The spore (zoospores/mL) was obtained in different ways in the 10th and 15th day in a Neubauer chamber, and the data transformed to √ x +1. In the agar media, (D) showed the largest growth increases, however means differed in all light regimes, especially those of cassava, eggplant, tomato and vegetable juice (V8), the first two with the largest increases and the last two with the lowest. The light regimes did not significantly affect growth in liquid media with 10 days of incubation. In some ways the presence of light is inversely proportional to the vegetative growth, but was an essential factor for sporulation, since it showed the presence of zoospores only (L) and (LD), except for the V8 medium, where it obtained the highest sporulation in the absence of light (D). Strains, media, and the presence of agar, promoted statistical difference in the number of zoospores/mL in (L) and (LD). More acids culture media induced more sporulation of P. nicotianae and less mycelial growth. The second study was divided in "in vitro" and "in vivo” bioassays aiming to analyse the antifungal and biocontrole effect of vegetable oils and extracts of Syzygium aromaticum and Cymbopogon nardus on fruit and tomato and eggplant seedlings inoculated with P. nicotianae. It was found that the products inhibited the germination of the mycelial growth and zoospores obtained from S. aromaticum at 0,5 μL/ ml and 10% concentrations CAE and EO, respectively. Treatments with C. nardus EO and CAE at 1,0 μL/ ml and 20% respectively, delayed progression of disease in fruit and seedlings compared to inoculated control. It can be inferred that the products obtained from S. aromaticum and C. nardus, have the potential to reduce the attack of this pathogen on tomato and eggplant. / O presente trabalho teve como objetivo avaliar a eficiência de extratos e óleos vegetais no controle de doenças causadas por Phytophthora nicotianae em tomateiro e berinjela, bem como seus efeitos sobre os isolados do patógeno. A esporulação do patógeno foi inicialmente estudado para viabilizar as inoculações e os testes com os óleos e extratos vegetais. O primeiro estudo consistiu em avaliar a influência de diferentes meios de cultura líquidos e agarisados, obtidos de órgão vegetais de hospedeiros e/ou indicados na literatura para o cultivo de Phytophthora spp; além de diferentes regimes de luminosidade: ausência de luz (E), alternância luminosa de 12h (LE), e luz constante (L); e potencial hidrogeniônico no crescimento e esporulação de P. nicotianae. Nos meios agarisados, (E) proporcionou os maiores crescimentos, entretanto em todos os regimes de luz os meios se diferenciaram, destacando-se dos demais os de mandioca, berinjela, tomate, e suco de vegetais (V8), os dois primeiros com os maiores crescimentos e os dois últimos com os menores. Os regimes de luz não influenciaram significativamente o crescimento nos meios líquidos com 10 dias de incubação. Em alguns meios a presença de luz foi inversamente proporcional ao crescimento vegetativo, mas foi um fator essencial para a esporulação, pois se verificou a presença de zoósporos somente em (L) e (LE), a exceção do meio V8, no qual se obteve a mais alta esporulação na ausência de luz (E). Em (L) e (LE), os isolados, meios e a presença ou não de ágar, promoveram diferenças estatísticas quanto ao número de zoósporos/mL. Verificou-se que meios mais ácidos proporcionam um menor crescimento, mas uma maior esporulação para P. nicotianae. O segundo estudo foi dividido em bioensaios “in vitro” e “in vivo”, analisado o efeito fungitóxico e biocontrolador de óleos e extratos vegetais de Syzygium aromaticum e Cymbopogon nardus em frutos e plântulas de tomateiro e berinjela inoculados com P. nicotianae. Constatou-se que os produtos que mais inibiram o crescimento micelial e a germinação dos zoósporos, foram obtidos de S. aromaticum, a partir das concentrações de 0,5 μL/mL e 10% do OE e EBA, respectivamente. Enquanto que o tratamento que mais retardou a evolução da doença em frutos e plântulas quando comparado com a testemunha inoculada, foi o OE e EBA de C. nardus nas concentrações de 1,0 μL/mL e 20%, respectivamente. Com isso podemos inferir que os produtos obtidos de S. aromaticum e C. nardus, têm potencial para reduzir o ataque deste patógeno em plantas de tomate e berinjela.

The interactions between Medicago truncatula and Phytophthora medicaginis were examined using skl, a mutant blocked in ethylene perception, and a range of wild accessions of this plant species. P. medicaginis infection of M. truncatula plants resulted in compatible responses, whereas the mutant genotype was found to be hyper-susceptible to the pathogen. Phytophthora reproduction and colonization rates of Medicago tissues supported this conclusion. Infection of skl with different pathogens reinforced this observation. Ethylene production in infected A17 and skl roots showed reduced ethylene evolution in the mutant and suggested that a positive feedback loop, known as autocatalytic ethylene production, amplified the ethylene signal. To complement the study, expression analyses of defense response genes in this interaction were studied by real time RTPCR of Phytophthora-infected and mock-infected roots. The genes analyzed were PAL, CHS, IFR, ACC oxidase, GST, and PR10. The sequences needed for the analysis were found through the scrutiny of the M. truncatula EST database employing phylogenetics and bio-informatics tools. In A17 all the genes studied were up-regulated, although the specific gene expression patterns differed. The comparison of gene expression between A17 and skl genotypes allowed the differentiation between ethylene-dependent and ethylene-independent responses. Discrete results showed that ACC oxidase homologues were downregulated in the ethylene perception mutant, corroborating the ethylene observations. However, the expression of genes involved in the phenylpropanoid metabolism was increased in skl relative to A17, suggestive of an antagonism between the ethylene perception pathway and the regulation of the phenylpropanoid pathway. This result implied that Medicago phytoalexins accumulate in the disease interaction, but raised questions about their role in resistance to Phytophthora infection. This study establishes a link between mechanisms that regulate symbiotic infection and the regulation of disease resistance to Oomycete pathogens, especially P. medicaginis. The results served to identify a series of Phytophthora-induced genes, which remain pathogen-responsive even in the absence of a functional ethylene perception pathway. While it is possible that the products of these genes are involved in resistance to P. medicaginis, the present results demonstrate that ethylene perception is required for resistance.