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The primary objectives of this research are to determine effective biological and alternative control strategies of insect and disease pests in order to reduce harsh chemical use during greenhouse crop production and transport s. This research includes two separate studies: 1) testing the practical viability of rearing and storing four species of entomopathogenic nematode (EPN), Steinernema feltiae, Steinernema carpocapsae, Heterorhabditis bacteriophora, Heterorhabditis indica; and, 2) the efficacy of UV-C radiation applied, pre-transport, as a preventative disease control strategy against Botrytis cinerea. A study was conducted testing EPN infectious juvenile (IJ) rearing production counts and IJ viability after a six-day storage period. When all four species are compared, S. feltiae had a greater number of infectious juveniles emerge from the wax moth cadavers and S. carpocasae had the least. All four species survived the six day storage period but EPN infectious juvenile counts were significantly different among species. Our second study tested the efficacy of UV-C radiation as an alternative control to traditional fungicides to deactivate B. cinerea in vitro and to determine plant tolerance to UV-C. The crops tested were poinsettia (Euphorbia pulcherrima) and primula (Primula vulgaris). All the UV-C doses, 1.0, 2.8, 3.7 or 4 W/m2, significantly decreased B. cinerea conidial germination in vitro and resulted in zero percent damage on poinsettia bracts. However, all UV-C doses during both replications caused minor damage, 15% or less, to primula flowers. / Master of Science in Life Sciences / Entomopathogenic nematodes (EPN) shows promise in being non-chemical and environmentally friendly solution for greenhouse pest and disease control. These can also be referred to as Biological Controls (Biocontrols). Entomopathogenic nematodes are used widely to control multiple greenhouse plant pests which include both Lycoriella spp., Fungus Gnats, and Frankliniella spp., Western Flower Thrips. However, there are challenges with EPN viability and storage from the manufacture to the greenhouse producer. We studied four EPN species, Steinernema feltiae, Steinernema carpocapsae, Heterorhabditis bacteriophora, Heterorhabditis indica, which were reared and stored to determine differences in production viability between species. Results show that the EPN species do not respond the same to storage and produce different amounts of infectious juveniles during rearing when conditions are the same. Separate from, but just as concerning as greenhouses plant pests are plant diseases. Ultraviolet radiation in the C spectra is known to be germicidal due to its narrow wavelengths. Because of this, UV-C has been shown to deactivate many different plant pathogens on contact and is being considered as a possible Biocontrol alternative to harsh traditional fungicides and bactericides. One disease that is known to contribute to the highest volume of annual crop losses is Botrytis cinerea. Botrytis cinerea is a plant disease that impacts floricultural crops to vegetables during propagation through the production supply chain to shipping and storage. We evaluated UV-C radiation at different doses, to determine if it could be used to replace a traditional fungicide before plants are shipped to reduce B. cinerea infection during transport. We found that UV-C successfully deactivated B. cinerea in vitro, but the viability of the application to plant tissue before transport has yet to be proven successful as a practical method of reducing B. cinerea during transport.

Thesis (PhD (Botany and Zology))--University of Stellenbosch, 2006. / The well-known, and often phytopathogenic, ophiostomatoid fungi are represented in South Africa by the two phylogenetically distantly related genera Ophiostoma (Ophiostomatales) and Gondwanamyces (Microascales). They are commonly associated with the fruiting structures (infructescences) of serotinous members of the African endemic plant genus Protea. The species O. splendens, O. africanum, O. protearum, G. proteae and G. capensis have been collected from various Protea spp. in South Africa where, like other ophiostomatoid fungi, they are thought to be transported by arthropod vectors. The present study set out to identify the vector organisms of Protea-associated members of mainly Ophiostoma species, using both molecular and direct isolation methods. A polymerase chain reaction (PCR) and taxon specific primers for the two Protea-associated ophiostomatoid genera were developed. Implementation of these newly developed methods revealed the presence of Ophiostoma and Gondwanamyces DNA on three insect species. They included a beetle (Genuchus hottentottus), a bug (Oxycarenus maculates) and a psocopteran species. It was, however, curious that the frequency of these insects that tested positive for ophiostomatoid DNA was very low, despite the fact that ophiostomatoid fungi are known to colonise more than 50% of Protea infructescences. Subsequent direct isolation methods revealed the presence of reproductive propagules of Ophiostoma spp. on four Protea-associated mite species (Oodinychus sp., two Tarsonemus spp. and Proctolaelaps vandenbergi). These mites are numerous within Protea infructescences and Ophiostoma spp. were isolated from a high frequency of these individuals. The Oodinychus sp. mite was found to vector most of the Protea-associated Ophiostoma species. It was thus postulated that the mites (in particular the Oodinychus sp.) act as primary vectors of the Protea-associated Ophiostoma species. The association between Oodinychus mites collected from P. repens and O. splendens proved to be mutualistic. Mites feeding on this fungus showed significantly higher population growth than mites feeding on any of the other fungal species tested. The short- and long-distance dispersal methods of these mites were also investigated. Firstly the ability of mites to move from drying infructescences to moist and sheltered areas such as provided by intact infructescences on the same plant was investigated experimentally. Significantly more mites were found to actively disperse from drying infructescences to artificially manufactured infructescences containing moistened filter paper shreds than to artificially manufactured infructescences containing dry filter paper shreds. The frequent fires associated with the habitat of these mites would, however, require movement over larger areas than what would be possible through self-dispersal. Dispersal of mites via air currents was thus investigated using sticky traps, but no Ophiostoma-vectoring mites were captured in this way. Self-dispersal aided by air currents could thus be ruled out, and our investigations shifted to vectored dispersal. Numerous insects emerging from Ophiostoma-containing P. repens and P. neriifolia infructescences were collected using specially designed emergence cages. Scanning electron microscopy and stereo-microscopy revealed that all three Ophiostoma-vectoring mite genera were phoretic on the beetle G. hottentottus. Tarsonemus spp. and P. vandenbergi were also phoretic on the beetles Trichostetha fascicularis and T. capensis associated with P. repens and P. neriifolia flowers. Mites collected from the surface of these beetles were found to vector reproductive propagules of various Ophiostoma spp. This thus seems to be the only method of long-distance dispersal of these mites and subsequently also the Protea-associated Ophiostoma species. Molecular phylogenetic reconstruction based on large subunit, ITS and beta-tubulin DNA sequence data suggests a polyphyletic origin for the Protea-associated members of Ophiostoma, which proposes multiple invasions of this unusual niche by these fungi. These studies also revealed the presence of four new species of Ophiostoma associated with Protea spp. The new species O. palmiculminatum, O. phasma, O. gemellus and Sporothrix variecibatus were thus described. Ophiostoma palmiculminatum is associated with P. repens infructescences and the Oodinychus mites collected from them. Ophiostoma phasma was collected from various Protea and mite species. Ophiostoma gemellus and Sporothrix variecibatus were initially only isolated from mites, but have subsequently also been isolated from Protea spp. The present study clarifies many aspects pertaining to the phylogeny and ecology of the interesting members of Ophiostoma associated with Protea hosts. As such this study will form the platform for further studies on the co-evolution of these insect / mite / fungi / plant associations.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Les hydrocarbures aromatiques polycyliques (HAPs) figurent parmi les polluants organiques persistants majeurs des sols pollués et présentent une toxicité avérée vis-à-vis de l'homme et des écosystèmes. Parmi les méthodes de remédiation des sols pollués par les HAPs, la phytoremédiation assistée par les champignons mycorhiziens à arbuscules (CMA), pourrait représenter une alternative innovante, écologique et économique. L'utilisation des mycorhizes comme outil de phytoremédiation des sols pollués présente plusieurs avantages dont une meilleure tolérance à la toxicité des HAPs, une meilleure nutrition hydrique et minérale ainsi qu'une meilleure dissipation des HAPs. De rares études ont décrit l'impact des HAPs sur le développement des CMA en lien avec une péroxydation lipidique et une perturbation des teneurs en lipides du CMA, mais ni les cibles d'action de ces polluants au niveau du métabolisme lipidique, ni le rôle de ces modifications dans sa tolérance aux HAPs et dans leur dissipation n'ont été étudiés. C'est pourquoi, le premier objectif de ce travail vise tout d'abord à comprendre l'impact des HAPs sur le métabolisme lipidique. Le radiomarquage par l'acétate [1-¹⁴C] a permis de montrer une perturbation de la biosynthèse des lipides membranaires du CMA extra-racinaire. D'autre part, nos résultats montrent que les HAPs affectent la nutrition phosphatée. Par ailleurs, la capacité des mycorhizes à dégrader et à bioaccumuler le benzo[a]pyrène est démontrée. Enfin, l'implication du métabolisme des lipides de réserve (les triacylglycérols) du mycélium extra-racinaire dans la régénération des membranes altérées, la lutte contre le stress oxydant induit par les HAPs et dans leur métabolisation/bioaccumulation est discutée. / Polycyclic aromatic hydrocarbons (PAHs) are among the major persistent organic pollutant frequently found in the polluted soils and are harmful for human health and its environment. To clean-up the PAHs polluted soils, phytoremediation assisted by arbuscular mycorrhizal fungi (AMF) could represent an innovative, ecological and cost-effective alternative. The use of mycorrhizas, as phytoremediation tool, has several advantages including increased tolerance to the pollutant toxicity, improved water and mineral nutrition as well as a better pollutant dissipation. Few studies have described the impact of PAHs on the AMF development related with lipid peroxidation and total lipid content disturbance. However, so far neither the target action of these pollutants on the metabolism, nor the role of these lipid changes in PAH tolerance and in their dissipation have been studied. Therefore, the present work aims firstly to improve our understanding of the PAHs impact on the CMA lipid metabolism. Thanks to radiolabeling experiments with [1-¹⁴C] acetate, our results showed a disruption of the membrane lipid biosynthesis pathways in the AMF extraradical mycelium, grown in the presence of PAHs. Secondly, it was highlighted that the PAHs affectef the phosphate nutrition. Finally, the mycorrhizas abilities to degrade and to bioaccumulate the benzo[a]pyrene, were pointed out. The involvement of extraradical mycelium storage lipid (triacyglycerols) metabolism in the membrane regeneration, the fight against the PAH induced-oxidative stress and the PAH metabolism/bioaccumulation is discussed.

Em cana-de-açúcar, a colonização do caule por fungos oportunistas, como Fusarium verticillioides e Colletotrichum falcatum, está diretamente ligada ao ataque da lagarta Diatraea saccharalis (Lepidoptera: Crambidae). Duas proteínas, SUGARWIN1 e SUGARWIN2 são produzidas em cana-de-açúcar, em resposta ao dano mecânico e ao ataque de D. saccharalis, porém estas proteínas não afetam o inseto, e sim ocasionam alterações fisiológicas e morfológicas em F. verticillioides e C. falcatum, ocasionando a morte destes fungos por apoptose. Dietas artificiais suplementadas com estes fungos oportunistas ocasionaram o ganho de peso da D. saccharalis. Esses dados indicam uma interação mais íntima entre o inseto e estes patógenos de cana, sendo que, neste estudo procuramos identificar relações simbióticas entre os indivíduos, analisando se a forma de transmissão desses fungos é mediado pela D. saccharalis. Os resultados mostraram a presença do F. verticillioides em todas as fases de desenvolvimento da D. saccharalis após contato com o fungo, ou seja, depois de se alimentarem em dieta suplementada por F. verticillioides no 4º instar, permaneceram infectadas pelo fungo ao longo de toda a fase pupal e adulta, em ambos os sexos. Além disso, o F. verticillioides foi transmitido para os descendentes de D. saccharalis, sendo que o fungo foi detectado nos ovos, ou seja, um caso original de transmissão vertical. Por meio de microscopia, também foi possível verificar a alta intensidade de F. verticillioides no interior do intestino de lagartas. Estes dados inferem em uma relação simbiótica entre F. verticillioides e D. saccharalis, onde o simbionte é transferido verticalmente para as gerações subsequentes. As respostas obtidas com o fungo C. falcatum diferiram daquelas obtidas com F. verticillioides, uma vez que não se detectou a presença do fungo a partir da fase pupal. Neste caso, a relação de simbiose entre o fungo e o inseto pode resultar em uma transmissão horizontal. Com este estudo foi possível identificar diferentes formas de transmissão por D. saccharalis para dois fungos envolvidos em podridão de colmo em cana-de-açúcar. Estes dados mudam a forma como é vista a transmissão de F. verticillioides por D. saccharalis em cana-de-açúcar, podendo influenciar a forma de manejo da podridão de Fusarium e da broca nos canaviais. / In sugarcane, stem colonization by opportunistic fungi, such as Fusarium verticillioides and Colletotrichum falcatum, is directly linked to the attack of Diatraea saccharalis (Lepidoptera: Crambidae) caterpillar. Two proteins, SUGARWIN1 and SUGARWIN2 are produced in sugarcane, in response to mechanical damage and attack of D. saccharalis, however these proteins do not affect the insect, but cause physiological and morphological changes in F. verticillioides and C. falcatum, causing the death of these fungi by apoptosis. Artificial diets supplemented with these opportunistic fungi caused the weight gain of D. saccharalis. These data indicate a more intimate interaction between the insect and the sugarcane pathogens. In this study, we sought to identify symbiotic relationship among individuals, analyzing whether the transmission of these fungi is mediated by D. saccharalis. The results showed the presence of F. verticillioides in all stages of D. saccharalis development after contact with the fungus, in the 4th instar. The caterpillars remained infect by the fungus throughout the pupal and adult phase, in both sexes. In addition, F. verticillioides was transmitted to D. saccharalis offspring, being detected in eggs, an original case of vertical transmission. Through the microscopy results, it was also possible to verify the high intensity of F. verticillioides inside the intestines of caterpillar. These data infer in a symbiotic relationship between F. verticillioides and D. saccharalis, where the symbiont is transferred vertically to the offspring. The responses obtained with C. falcatum differed from those obtained with F. verticillioides, since the presence of the fungus was not detected from the pupal phase. In this case, the symbiont relationship between fungus and insect can result in a horizontal transmission. With this study was possible to identify different forms of fungi transmission by D. saccharalis. These data change the way the transmission of F. verticillioides by D. saccharalis in sugarcane is viewed, and may influence the management of Fusarium rot and sugarcane borer attack in sugarcane.

Plants respond to insect and pathogen attack by inducing and accumulating a large set of defense proteins. Colonization of sugarcane stalk by opportunistic fungi, such as Fusarium verticillioides and Colletotrichum falcatum, usually occurs after Diatraea saccharalis (Lepidoptera: Cambridae) caterpillars attack increasing the damage caused by the borer. Two homologous of BARWIN protein were identified in sugarcane, SUGARWIN1 and SUGARWIN2. Their gene expression is induced in response to wound and Diatraea saccharalis damage. However, the recombinant SUGARWIN protein does not affect insect development; but promotes significant morphological and physiological changes in Fusarium verticillioides and Colletotrichum falcatum, which lead to fungal cell death via apoptosis, indicating that SUGARWINs may work as a first layer of defense against the fungi infection. In this study, we deepen our understanding of the role of SUGARWINs in plant defense and the molecular mechanisms by which these proteins affect fungi by elucidating their molecular targets. Our results show that SUGARWINs play an important role in plant defense against opportunistic pathogens. We demonstrated that SUGARWINs are induced by C. falcatum, and the induction of SUGARWINs can vary among sugarcane varieties. The sugarcane variety exhibiting the highest level of SUGARWIN induction exhibited a considerable reduction in C. falcatum infection. Furthermore, SUGARWIN1 exhibited ribonuclease and chitinase activity, whereas SUGARWIN2 exhibited only chitinase activity. This variable enzymatic specificity seems to be the result of divergent amino acid composition within the substrate-binding site. Additionally, plants attacked by insects and pathogens display profound physiological, morphological and chemical changes or adaptations, which may result in organism attraction or avoidance. In this study, we also aimed to understand the insect-fungi association in sugarcane and the role of fungal volatile compounds in this association. Our results have shown that D. saccharalis positively influences C. falcatum infection on sugarcane, inducing a fast growing when compared to C. falcatum treatment without D. saccharalis attack. In addition, both fungi, C. falcatum and F. verticillioides, have been shown a double effect on D. saccharalis caterpillar, they promoted a strong attraction for insects due volatile organic compound emission and positively influenced D. saccharalis feeding and weight gain in diets supplemented with fungi. Fungal volatile organic compounds from C. falcatum and F. verticillioides were identified and quantified; acoradiene and acorenol were specifically induced by the fungi. These data suggest a synergistic interaction, mediated by organic volatile compounds, between D. saccharalis and the fungi C. falcatum and F. verticillioides in sugarcane. / As plantas respondem ao ataque de insetos e patógenos induzindo e acumulando um grande conjunto de proteínas de defesa. A colonização do caule de cana por fungos oportunistas, como Fusarium verticillioides e Colletotrichum falcatum, geralmente ocorre após o ataque de lagartas de Diatraea saccharalis (Lepidoptera: Cambridae), resultando no aumento do dano causado pelo inseto. Dois homólogos da proteína BARWIN foram identificados em cana-de-açúcar, SUGARWIN1 e SUGARWIN2. A expressão desses genes é induzida em resposta ao ferimento mecânico e ao ataque de Diatraea saccharalis, entretanto, a proteína não afeta o desenvolvimento do inseto, mas promove alterações morfológicas e fisiológicas significativas em Fusarium verticillioides e Colletotrichum falcatum, causando a morte destes fungos por apoptose. Esses dados indicam que as SUGARWINs podem funcionar como uma defesa inicial contra a infecção fúngica. Neste estudo, aprofundamos nosso entendimento do papel das SUGARWINs na defesa de plantas e os mecanismos moleculares pelos quais essas proteínas afetam os fungos, elucidando seus alvos moleculares. Nossos resultados mostraram que as SUGARWINs desempenham um papel importante na defesa da planta contra patógenos oportunistas. Foi demonstrado que essas proteínas também são induzidas por C. falcatum em cana-de-açúcar, e sua indução pode variar entre as variedades de cana-de-açúcar. A variedade de cana-de-açúcar que apresentou o maior nível de indução de SUGARWINs apresentou uma redução considerável na infecção por C. falcatum. Além disso, SUGARWIN1 exibiu atividade de ribonuclease e quitinase, enquanto que SUGARWIN2 exibiu apenas atividade de quitinase. Esta especificidade enzimática parece ser o resultado da composição divergente de aminoácidos no sítio de ligação do substrato. Além disso, as plantas atacadas por insetos e patógenos exibem profundas alterações fisiológicas, morfológicas e químicas ou adaptações, que podem resultar em atração ou repelência do organismo, dessa forma, estudamos também a associação inseto-fungos na cana-de-açúcar, e o papel dos compostos voláteis fúngicos nessa associação. Nossos resultados mostraram que D. saccharalis influencia positivamente a infecção por C. falcatum em cana-de-açúcar, induzindo crescimento rápido do fungo quando comparado ao tratamento com C. falcatum sem ataque de D. saccharalis. Além disso, ambos os fungos, C. falcatum e F. verticillioides, mostraram um efeito duplo sobre lagartas de D. saccharalis, promovendo uma forte atração desses insetos devido à emissão de compostos orgânicos voláteis e influenciando positivamente a alimentação de D. saccharalis e ganho de peso em dietas suplementadas com fungos. Os compostos orgânicos voláteis fúngicos de C. falcatum e F. verticillioides foram identificados e quantificados; acoradieno e acorenol foram especificamente induzidos pelos fungos. Estes dados sugerem uma interação sinergistica, mediada por compostos orgânicos voláteis, entre D. saccharalis e os fungos C. falcatum e F. verticillioides em cana-de-açúcar.

O fungo Pleurotus ostreatus pertence ao grupo de basidiomicetos que degradam madeira. Este cogumelo cultivado em todo mundo apresenta grande rusticidade e produtividade, e pode ainda ser usado em processos de biorremediação e biopolpação. Devido a seu potencial biotecnológico, torna-se interessante a compreensão da interação deste com outros microrganismos. Neste sentido, recentemente foi observada a presença de bactérias associadas a P. ostreatus em culturas in vitro, que apresentavam grande pleomorfismo. A partir desta observação foram elaborados ensaios que visaram a confirmação da presença de bactérias. Para tanto, foi utilizada a estratégia do "Ciclo Completo de Análise do rRNA" (full-cycle rRNA analysis) empregada em microrganismos não cultiváveis ou de crescimento fastidioso, além do emprego de técnicas de microbiologia básica, e de estudos de ultraestrutura. Os estudos de microbiologia básica indicaram que se tratava de um microrganismo fastidioso e que se desenvolvia melhor na presença do fungo em sistema de co-cultivo em meios contendo Tween 80 ou Tween 20. Por sua vez, a análise de ultraestrutura demonstrou a presença de estruturas pleomórficas, tanto internamente como externamente à hifa. Em relação ao "Ciclo completo de Análise do rRNA" este se iniciou pela amplificação e seqüenciamento de parte do rDNA bacteriano, que revelou a proximidade desta bactéria com o Complexo Burkholderia cepacia (CBC). A partir desta seqüência, foi realizado um estudo de bioinformática que indicou sondas específicas para este grupo de bactérias. Completando o Ciclo completo de Análise do rRNA, foram realizados ensaios de hibridização in situ fluorescente (FISH) para a confirmar a relação entre as estruturas bacterianas e a seqüência obtida. Este método comprovou a presença das bactérias no interior das hifas de P. ostreatus. Este trabalho constitui o primeiro relato de bactérias pleomórficas pertencente ao complexo B. cepacia associados a P. ostreatus. / The fungus Pleurotus ostreatus, which belongs to white rot basidiomycete group, is a widely cultivated mushroom; this species has high productivity and rusticity, besides its use in biobleaching and bioremediation processes. This biotechnological potential justifies microbial interaction studies between this fungi and others microorganisms. In P. ostreatus mycelia, it has been observed pleomorphic bacteria growing on agar media. This research describes several assays to confirm bacterial presence in this sample. Therefore, the full-cycle rRNA analysis (described for unculturable or fastidious microorganism), ultrastructure and basic microbiology approaches were employed. Basic microbiology approaches indicated slow growing bacteria, which grown faster near to fungi colonies in solid media amended with Tween 80 or Tween 20 (co-culture system). Ultrastructure studies confirm the presence of intracellular and extracellular pleomorphic bacteria. The full-cycle rRNA analysis started with 16S rDNA amplification and sequencing. This approach demonstrated a relation between these bacteria with Burkholderia cepacia complex. By bioinformatics analysis was determinate which DNA probes can be use to identified this bacterial group. The last step for full-cycle rRNA analysis was applying fluorescent in situ hybridization (FISH). This technique confirmed the relationship between 16S rDNA bacterial sequence and bacterial forms. This is the first time that a pleomorphic bacteria from B. cepacia complex is found associated with P. ostreatus.

Foram estudadas as enzimas implicadas no metabolismo de cAMP, bem como as variações na concentração deste nucleotídeo cíclico e na atividade de adenilato ciclase durante o ciclo biológico de B. emersonii. Demonstrou-se que os zoósporos contêm enzimas específicas e distintas para a hidrólise de cAMP e cGMP. Existe apenas uma espécie molecular da cAMP fosfodiesterase, que hidrolisa cAMP a 5\'-AMP com um Km aparente de 2-4 µM; a presença de cGMP nas misturas de reação, não altera as propriedades cinéticas da enzima. A adenilato ciclase de B. emersonii é uma enzima particulada, provavelmente ligada à membrana plasmática do zoósporo, que exige especificamente Mn2+ para sua atividade. A enzima não é ativada por NaF, catecolaminas ou outros compostos de estrutura semelhante. O estudo das propriedades cinéticas da adenilato ciclase sugere um modelo simples no qual o verdadeiro substrato da enzima é o complexo MnATP2- e tanto ATP corno Mn2+ , nas suas formas livres, competem com o complexo pelo sítio catalítico da enzima, que apresenta uma afinidade maior pelas formas livres do que pelo complexo MnATP2-. A atividade especifica da adenilato ciclase, determinada durante o ciclo biológico do fungo, mostra-se elevada nos zoósporos, cai lentamente durante a germinação e permanece baixa em todo o período de crescimento, só voltando a apresentar um aumento na atividade após a indução da esporulação. Quando este processo e induzido na presença de cicloheximida, a atividade permanece baixa, sugerindo que a enzima é sintetizada \"de novo\" nesta fase do ciclo evolutivo. A concentração intracelular de cAMP foi também determinada nas várias fases do ciclo biológico de B. emersonii. No zoósporo encontrou-se um valor médio de 33 pmoles cAMP/mg proteína. Durante a germinação, os níveis de cAMP aumentam, atingindo um máximo (~ 100 pmoles/mg proteína)quando a quase totalidade dos zoósporos se transformou em esferócitos. A partir daí observou-se um declínio gradual nos níveis de cAMP, que permanecem baixos durante toda a fase de crescimento, voltando a elevar-se na fase final da esporulação até alcançar o nível de zoósporo. O grande aumento na concentração intracelular de cAMP na fase de esferócitos é parcialmente explicado pela predominância da atividade de adenilato ciclase sobre a atividade de cAMP fosfodiesterase neste período; a possibilidade de uma ativação \"in vivo\" da adenilato ciclase, neste estágio do ciclo, não pode ser excluída. A queda nos níveis de cAMP que ocorre na passagem de esferócito a gérmen, numa fase onde a atividade de cAMP fosfodiesterase já e muito baixa, é devido principalmente a excreção deste nucleotídio cíclico para o meio extracelular. O grande aumento nos níveis de cAMP durante a transição de zoósporo a esferócito pode estar relacionado com a ativação metabólica ocorrendo nesta fase e pode também refletir uma característica de sistemas em diferenciação, isto é, a necessidade de altos níveis de cAMP para a transição entre dois estados celulares diferenciados. / The enzymes involved in the metabolism of cAMP have been studied, as well as the fluctuations in the concentration of this cyclic nucleotide and in the adenylate cyclase activity during the life cycle of B. emersonii. Zoospores were shown to contain independent specific enzymes involved in the hydrolysis of cAMP and cGMP. A single molecular species was found for the cAMP phosphodiesterase activity, which catalyses the hydrolysis of cAMP to 5\'-AMP. This enzyme displays normal Michaelis kinetics with an apparent Km of 2-4 µM; the addition of cGMP to the reaction mixtures does not modify the kinetic properties of the enzyme. Adenylate cyclase activity in B. emersonii is associated with particulate subcellular fractions, most probably bound to the zoospore plasma membrane. The activity requires Mn2+ and it is not activated by NaF, cathecolamines or other related compounds. The enzyme substrate is the MnATP2- complex and the kinetic data obtained studying the adenylate cyclase activity can be explained by a simple model where free ATP and Mn2+ compete with MnATP2- for the catalytic site of the enzyme, the affinity for MnATP2- being lower than for free Mn2+ and ATP. The specific activity of adenylate cyclase has been determined throughout the fungus life cycle. The enzyme activity is high in zoospores, falls slowly during germination remaining low at the growth phase and rising again during the later stage of sporulation. When this process is induced in the presence of cycloheximide, there is no increase in adenylate cyclase activity, suggesting that \"de novo\" synthesis of the enzyme occurs at this stage. Fluctuations in the intracellular levels of cAMP during the cell cycle of B. emersonii have also been shown. Zoospores contain an average concentration of 33 pmoles cAMP/mg protein. During germination, a significant increase in the cAMP levels is observed, reaching a maximum (ca. 100 pmoles/mg protein) when the majority of the zoospores have changed into round cells. From then on a gradual decline in the cAMP levels is observed. During the growth phase the cAMP contents of the cells remain low, increasing again late in the sporulation stage. The large increase in the intracellular concentration of cAMP in the round cell phase is partially explained by the predominance of adenylate cyclase activity over cAMP phosphodiesterase activity (during this stage); the possibility of an \"in vivo\" activation of the adenylate cyclase during this period, however, cannot be excluded. The decrease in the cAMP levels occurring during the passage of round cells to germlings, in a stage where cAMP phosphodiesterase activity is negligible, is mainly due to the excretion of this cyclic nucleotide to the extracellular medium. The rise in cAMP contents during encystment might be related to the activation of metabolism occurring in this phase and may also reflect a characteristic of differentiating systems, that is, high cAMP levels being necessary for a differentiative transition.

Os objetivos do presente trabalho foram: avaliar o processo de biodeterioração do lenho das árvores de tipuana na cidade de São Paulo e relacionar as alterações da sua estrutura anatômica e propriedades físico-mecânicas com o seu risco de queda. Nos passeios públicos de regiões selecionadas da cidade, 1109 árvores de tipuana foram caracterizadas quanto às condições de entorno, estado geral, variáveis dendrométricas, biodeterioração externa e interna e o risco de queda, estimado com modelo de cálculo estrutural desenvolvido pelo IPT. A biodeterioração e os mecanismos de resistência das árvores foram avaliados ao nível macroscópico e microscópico. As alterações nas propriedades do lenho de tipuana foram analisadas por densitometria de raios X e pelos ensaios físico-mecânicos (densidade de massa aparente; resistência à flexão) e aplicado o método de elementos finitos para análise da distribuição de tensões no lenho. As árvores de tipuana de elevada idade não apresentaram sinais de declínio, embora com estado geral considerado como crítico, pelo plantio em locais indequados, manejo inapropriado e/ou ocorrência de injúrias. O lenho foi deteriorado por fungos apodrecedores, cupins-subterrâneos e brocas-de-madeira, sendo os fungos e cupins os principais organismos xilófagos, afetando a região do cerne; a associção entre os fungos, os cupins, as variáveis dendrométricas e o risco de queda foram estatisticamente significantes ao nível de 5 %. O cupim-subterrâneo Coptotermes gestroi ocorreu em elevados níveis de infestação nas árvores, sendo considerado, no entanto, como um organismo-praga secundário, fortemente associado com lenho previamente apodrecido por Ganoderma sp.. Os fungos apodrecedores ocorrentes foram classificados como de podridão branca e mole, sendo a primeira mais comum no cerne das árvores. Como mecanismo de resistência à biodeterioração observou-se a compartimentalização do lenho, caracterizada pelo acúmulo de extrativos nas células. Os fungos xilófagos causaram reduções significativas na densidade de massa aparente, tensão de ruptura à flexão estática e módulo de elasticidade em estágios incipiente e intenso de apodrecimento do lenho. Ruptura do tronco e o risco de queda das árvores de tipuana relacionaram-se, principalmente, com a intensidade da deterioração do lenho e com a sua dendrometria. A alteração da distribuição das tensões no lenho foi analisada em relação ao estágio, tamanho e localização da deterioração no tronco das árvores de tipuana. / The objectives of the present work were to evaluate the process of biodeterioration in the wood of tipuana trees in the city of São Paulo and to relate the alterations in the anatomical structures and physical and mechanical properties with there falling risk. In the sidewalks of selected regions of the city, 1109 trees were characterized taking into consideration aspects as surroundings, general conditions, dendrometric variables, external and internal biodeterioration and falling risk estimated by using a structural model developed by IPT. The biodeterioration and the resistance mechanisms of the trees were evaluated at a macro and microscopic levels. The alterations on the wood properties were analysed by X-rays densitometry and by performing physical and mechanical tests (specific gravity; bending resistance); the tension distribution in the wood was analyze by finite-elements method. The old aged trees encountered, did not show any sign of decline, eventhough, the general aspect of their surroundings was critical by the inadequate places they were planted; inappropriate management and/or occurrence of injuries. The trees had their trunks deteriorated by xylophagous organisms, mainly fungi and subterranean-termites, and some wood borer beetle too, which affected the heartwood. The association between the presence of the first two organinsms, the dendrometric variables and the risk of falling, was statistically significant at the 5% level. The infestation of the trees by the subterranean-termite Coptotermes gestroi was at high levels, however, it was considered as a secundary plague-organism, because its attack was strongly associated with the previously decayed heartwood by Ganoderma sp.. The decaying fungi observed were classified as white rot and soft rot, being the white rot more common in the heartwood of the trees. As a resistance mechanism of the trees against biodeterioration, it was observed the compartimentalization of the wood characterized by the accumulation of extractives in the cells. Decaying fungi caused significant reductions in the following wood properties: specific gravity, rupture strength to bending and modulus of elasticity either in incipient or intense decay. Trees trunk rupture and falling risk were associated mainly to biodeterioration intensity and dendrometric variables. The alteration of tension distribution in the wood was analyzed in relation to the stage, size and position of the deterioration in the wood of the trees.