O gênero Burkholderia é composto por bactérias de elevada diversidade fisiológica e genética, sendo alvo para uma série de aplicações biotecnológicas. A linhagem Burkholderia seminalis TC3.4.2R3 foi capaz de controlar fungos e bactérias patogênicas, bem como produzir antibióticos. A análise de perfil fisiológico da planta hospedeira indica que não há a elicitação de respostas de defesa no controle dos sintomas da podridão mole por B. seminalis. Foi observado que o sucesso do controle depende do contato direto do agente de biocontrole e o patógeno B. gladioli, e que B. seminalis possui atividade antimicrobiana in vitro contra com a formação de um halo de inibição e redução de 40% da densidade de B. gladioli em co-cultura líquida com B. seminalis. A análise do mutante M01 defectivo no controle da podridão mole, que apresenta a região intergênica da patatina-ferritina nocauteada pelo transposon, não mostrou diferenças significativas comparado ao isolado selvagem para os mesmos ensaios. / The genus Burkholderia is composed of bacteria with high physiological and genetic diversity, and it is being goal for a number of biotechnological applications. The Burkholderia seminalis TC3.4.2R3 strain was able to control fungal and bacterial pathogens as well as produce antibiotics. The physiological profile analysis of the host plant indicates that there is no defense responses elicitation induced by B. seminalis involved in controlling symptoms of soft rot. It was observed that the success of control depends on the full contact of the biocontrol agent and the pathogen B. gladioli. B. seminalis has an antimicrobial activity in vitro showing a inhibition halo and reducing the density of B. gladioli in co-culture with B. seminalis by 40%. The M01 mutant, wich is defective in control of soft rot and showing a insertion of transposon in a intergenic region between patatin-ferritin, presented no significant differences compared to the wild strain for the same assays.

Orientadores: João Ernesto de Carvalho, Mary Ann Foglio / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T20:05:53Z (GMT). No. of bitstreams: 1 Gaspi_FernandaOliveiradeGaspari_D.pdf: 71779654 bytes, checksum: cfa74eb88bc93a25160ab6df284bff34 (MD5) Previous issue date: 2011 / Resumo: O conhecimento sobre as plantas, a descoberta de suas propriedades curativas têm sempre acompanhado a evolução do homem através dos tempos. A família Orchidaceae é a maior entre as angiospermas, são mais de 25 mil espécies já descritas. Entre elas está a Oncidium flexuosum Sims., uma importante espécie brasileira conhecida popularmente como bailarina e que possui grande valor ornamental. Porém, esta é a primeira pesquisa sobre as suas propriedades medicinais. Os objetivos deste trabalho foram avaliar as atividades antioxidante in vitro e in vivo, cicatrizante e antiulcerogênica dos extratos e das frações obtidas a partir das folhas de O. flexuosum e realizar as análises fitoquímicas e histoquímica preliminares. Para isto, foram utilizadas as análises da capacidade sequestradora do radical livre difenilhidrazil (DPPH), da atividade antiulcerogênica induzida por etanol, do reparo tecidual associado ou não a microcorrente, além das reações de caracterização e cromatografia de camada delgada. Também foram realizados testes histopatológicos para avaliar possíveis danos oxidativos. O extrato hidroalcoólico de O. flexuosum (EH) possui as atividades antioxidante in vitro diante do radical livre DPPH e in vivo, cicatrizante associado à microcorrente, estimulante da proliferação de fibroblastos in vitro e colágeno in vivo, e atividade antiulcerogênica. A fração terpeno (FrT) inibi o processo oxidativo causado pelo substâncias reativas do ácido tiobarbitúrico (TBARS). O extrato bruto enzimático (EBZ) e a FrT estimulam a proliferação de fibroblastos, porém com menor potência do que o EH. As análises fitoquímicas e histoquímica revelaram que as folhas de O. flexuosum possuem, entre os seus constituintes, as substâncias fenólicas, tais como flavonoides e taninos, e também cumarinas, triterpenos e esteróides. O EH não induziu danos renais nos animais, mas tem potencial hepatotóxico dose-tempo-dependente, causando inflamação e degeneração tecidual. Conclui-se que o EH possui atividade antioxidante, cicatrizante e antiulcerogênica, provavelmente devido à presença das substâncias fenólicas / Abstract: The knowledge about plants, the discovery of their healing properties has always followed the evolution of man through the ages. The Orchidaceae family is the largest among the angiosperms, they are more than 25,000 described species, among them is the Oncidium flexuosum Sims., an important Brazilian species popularly known as dancer and which has great ornamental value. However, this is the first survey of its medicinal properties. The objectives of this work were to investigate the antioxidant activity in vitro and in vivo, wound healing and antiulcerogenic activities of the extracts and fractions obtained from the leaves of Oncidium flexuosum and perform the preliminary phytochemical and histochemistry analysis. For this, analysis of free radical scavenging capacity with difenilhidrazil (DPPH) test, antiulcer activity induced by ethanol, tissue repair associated or not with microcurrent were made, other than the characterization reactions and thin layer chromatography. Histopathological tests were also performed to evaluate possible oxidative damage. The extract of Oncidium flexuosum (EH) has in vitro antioxidant activity against free radical DPPH and in vivo, cicatrizant associated with microcurrent, stimulating fibroblasts proliferation in vitro and collagen in vivo. The terpene fraction (FrT) inhibit the oxidative process caused by the thiobarbituric acid reactive substances (TBARS). The enzimatic crude extract (EBZ) and FrT stimulate proliferation of fibroblasts, but with less efficiency than the EH. The phytochemical and histochemistry analysis revealed that the leaves of Oncidium flexuosum plant species have among its constituents, the phenolic substances such as flavonoids and tannins, and also the coumarins, triterpenes and steroids. The EH was not induce kidney damage in animals, but has hepatotoxic potential dose-time-dependent, causing inflammation and tissue degeneration. It was concluded that the EH has antioxidant, cicatrizant and antiulcerogenic activities probably due to the presence of phenolic substances / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

O gênero Burkholderia é composto por bactérias de elevada diversidade fisiológica e genética, sendo alvo para uma série de aplicações biotecnológicas. A linhagem Burkholderia seminalis TC3.4.2R3 foi capaz de controlar fungos e bactérias patogênicas, bem como produzir antibióticos. A análise de perfil fisiológico da planta hospedeira indica que não há a elicitação de respostas de defesa no controle dos sintomas da podridão mole por B. seminalis. Foi observado que o sucesso do controle depende do contato direto do agente de biocontrole e o patógeno B. gladioli, e que B. seminalis possui atividade antimicrobiana in vitro contra com a formação de um halo de inibição e redução de 40% da densidade de B. gladioli em co-cultura líquida com B. seminalis. A análise do mutante M01 defectivo no controle da podridão mole, que apresenta a região intergênica da patatina-ferritina nocauteada pelo transposon, não mostrou diferenças significativas comparado ao isolado selvagem para os mesmos ensaios. / The genus Burkholderia is composed of bacteria with high physiological and genetic diversity, and it is being goal for a number of biotechnological applications. The Burkholderia seminalis TC3.4.2R3 strain was able to control fungal and bacterial pathogens as well as produce antibiotics. The physiological profile analysis of the host plant indicates that there is no defense responses elicitation induced by B. seminalis involved in controlling symptoms of soft rot. It was observed that the success of control depends on the full contact of the biocontrol agent and the pathogen B. gladioli. B. seminalis has an antimicrobial activity in vitro showing a inhibition halo and reducing the density of B. gladioli in co-culture with B. seminalis by 40%. The M01 mutant, wich is defective in control of soft rot and showing a insertion of transposon in a intergenic region between patatin-ferritin, presented no significant differences compared to the wild strain for the same assays.

<p>Syftet med detta projekt var att undersöka om det är möjligt att använda en fungicid vid odling av celler från Oncidium crispum, för att slippa kontaminering av mykorrhizasvamp. Kontaminering från denna sorts svamp har i tidigare försök varit ett stort promlem. I undersökningen användes fungiciden Nystain. Ett första försök gjordes för att testa om Nystatin hade effekt på den aktuella svampen. På näringsmedium fick hyfer från mykorrhizasvampen växa mot ett filterpapper indränkt i Nystatinlösning med olika koncentrationer. Som kontroll användes filterpapper indränkta i vatten och 20 % etanol. Efter 72 h mättes hur nära svampen växt pappret eller om den växt över pappret. Det kunde konstateras att Nystatin vid den starkaste koncentrationen hindrade svampens framväxt. Där stannade svampen innan pappret i större utsträckning än hos de lägre koncentrationerna. Av första försöket drogs därför slutsatsen att Nystatin hämmar mykorrhizasvampen. I ett andra försök doppades explantat från Oncidium crispum i en lösning av Nystatin i olika koncentrationer, som kontroll användes vatten och 20 % etanol. Explantaten lades på MS-medium med hormonerna auxin och cytokinin. Vid den starkaste koncentrationen av Nystatin hämmades tillväxten av svamphyfer fullständigt. Däremot kunde ingen kallustillväxt urskiljas hos något av explantaten. Som kontroll för att försäkra sig om att Nystainet inte påverkade växtmaterialet negativt gjordes andra delen av försöket även med Stapelia grandiflora, ett växtmaterial som är känt för att lätt kunna bilda kallus. Kallustillväxten var fullständig hos kontrollen med vatten, hos de övriga lösningarna var kallustillväxten tyvärr mycket begränsad.</p> / <p>The purpose of this project was to examine if it’s possible to use a fungicide when growing cells from Oncidium crispum, to avoid the contamination of mycorrhizafungus. The contamination from this kind of fungus has been a big problem in earlier experiments. The fungicide used in the experiment was Nystatin. In a first experiment the effect of Nystatin against the mycorrhizafungus was tested. On a nutrient medium hyphae from the mycorrhizafungus were growing towards a paper disc soaked with a solution of Nystatin in different concentration. Paper discs soaked with water and 20 % ethanol were used as a reference. After 72 h it was measured how close the hyphae has got to the paper disc or if it had grown over the paper. It was established that the strongest concentration of Nystatin inhibited the growth of hyphae, in this concentration it stopped growing before it came to the paper disc in a greater extent then by the other concentrations. Therefore the conclusion was drawn, that Nystatin inhibits growth of the mycorrhizafungus. In a second experiment explants from Oncidium crispum were dipped in a solution of Nystatin in different concentrations, water and 20 % ethanol was used as a reference. The explants were placed on a MS-medium with the hormones auxin and cytokinin. By the strongest concentration of Nystatin there was no growth of hyphae. Callus formation couldn’t be distinguished by any of the explants. To make sure that Nystatin did not have a bad influence on the plant material the second part of the experiment was also performed on Stapelia grandiflora, a plant material that is known for its ability to form callus. In the reference with water the callus formation was complete, but in the other solutions the callus formation was very limited.</p>

Syftet med detta projekt var att undersöka om det är möjligt att använda en fungicid vid odling av celler från Oncidium crispum, för att slippa kontaminering av mykorrhizasvamp. Kontaminering från denna sorts svamp har i tidigare försök varit ett stort promlem. I undersökningen användes fungiciden Nystain. Ett första försök gjordes för att testa om Nystatin hade effekt på den aktuella svampen. På näringsmedium fick hyfer från mykorrhizasvampen växa mot ett filterpapper indränkt i Nystatinlösning med olika koncentrationer. Som kontroll användes filterpapper indränkta i vatten och 20 % etanol. Efter 72 h mättes hur nära svampen växt pappret eller om den växt över pappret. Det kunde konstateras att Nystatin vid den starkaste koncentrationen hindrade svampens framväxt. Där stannade svampen innan pappret i större utsträckning än hos de lägre koncentrationerna. Av första försöket drogs därför slutsatsen att Nystatin hämmar mykorrhizasvampen. I ett andra försök doppades explantat från Oncidium crispum i en lösning av Nystatin i olika koncentrationer, som kontroll användes vatten och 20 % etanol. Explantaten lades på MS-medium med hormonerna auxin och cytokinin. Vid den starkaste koncentrationen av Nystatin hämmades tillväxten av svamphyfer fullständigt. Däremot kunde ingen kallustillväxt urskiljas hos något av explantaten. Som kontroll för att försäkra sig om att Nystainet inte påverkade växtmaterialet negativt gjordes andra delen av försöket även med Stapelia grandiflora, ett växtmaterial som är känt för att lätt kunna bilda kallus. Kallustillväxten var fullständig hos kontrollen med vatten, hos de övriga lösningarna var kallustillväxten tyvärr mycket begränsad. / The purpose of this project was to examine if it’s possible to use a fungicide when growing cells from Oncidium crispum, to avoid the contamination of mycorrhizafungus. The contamination from this kind of fungus has been a big problem in earlier experiments. The fungicide used in the experiment was Nystatin. In a first experiment the effect of Nystatin against the mycorrhizafungus was tested. On a nutrient medium hyphae from the mycorrhizafungus were growing towards a paper disc soaked with a solution of Nystatin in different concentration. Paper discs soaked with water and 20 % ethanol were used as a reference. After 72 h it was measured how close the hyphae has got to the paper disc or if it had grown over the paper. It was established that the strongest concentration of Nystatin inhibited the growth of hyphae, in this concentration it stopped growing before it came to the paper disc in a greater extent then by the other concentrations. Therefore the conclusion was drawn, that Nystatin inhibits growth of the mycorrhizafungus. In a second experiment explants from Oncidium crispum were dipped in a solution of Nystatin in different concentrations, water and 20 % ethanol was used as a reference. The explants were placed on a MS-medium with the hormones auxin and cytokinin. By the strongest concentration of Nystatin there was no growth of hyphae. Callus formation couldn’t be distinguished by any of the explants. To make sure that Nystatin did not have a bad influence on the plant material the second part of the experiment was also performed on Stapelia grandiflora, a plant material that is known for its ability to form callus. In the reference with water the callus formation was complete, but in the other solutions the callus formation was very limited.

台灣蘭花業者過去憑藉著優異的育種技術以及豐富的種源，在全球蘭花市場上佔有一席之地。由於蘭花經濟價值高，近年已吸引許多國外業者投入栽植與生產，目前包括荷蘭、美國、日本、韓國、泰國、加拿大以及中國大陸等國家的業者都開始切入蘭花市場。就蘭花的銷售而言，影響蘭花價值的因子除了有商品定位形象外，隨著建築風格的改變、空間配置的調整，新的消費需求將決定未來的蘭花價值。 美國蘭花市場是台灣第二大外銷市場，台灣蘭花外銷美國的外銷量在過去3年中平均每年有超過60%成長（外銷值平均則是每年有超過38%的成長），因此美國蘭花市場的變化將可牽動國內蘭花業者的獲利空間。 本研究目的在於分析美國蘭花市場的供應鏈的現況以及批發市場現況、並調查美國政府對於蘭花進口的相關法規制度以及台灣蘭花輸美現況，進以剖析美國蘭花市場以及找尋台灣蘭花在美國市場的商業機會。 經調查發現：美國蘭花市場主要是盆花市場，在1999年至2007年間，美國蘭花盆花市場的批發值（約1.2億美元/每年）約為蘭花切花 批發市場的10 – 16倍；以進出口的比例來看，美國蘭花盆花的進口價值為出口價值的6倍以上，因此美國屬於蘭花盆花淨輸入國；美國蘭花盆花的主要輸入源為：台灣、泰國、荷蘭、加拿大、中國與韓國；其中過去9年中，美國每年自中國、韓國、加拿大以及泰國進口的蘭花價值或是重量的複合年成長率都超越台灣；因此雖然目前台灣仍是美國的蘭花的最主要進口源，但是未來在開創美國市場時，除了應注意荷蘭業者的動態外，亦需注意中國、韓國、加拿大以及泰國的蘭花業者狀態。 美國蘭花切花進口量在2004年達最大以後即開始下滑，雖然進口量下滑但是進口總值提升，足見進口單價明顯提升。 就美國的蘭花進口法規來看，美國農業部認為絕大部分的貿易用蘭花切花都是來自於人工繁殖的植株，因此不屬於CITES的限制範圍；除非有明顯證據證實是採集自野外，否則，在檢查後，蘭花切花的商業性運輸將會被海關放行；另外，美國政府主要是依CITES的規定來執行蘭花盆花進口的查核，而目前CITES中關於人工繁殖的雜交蕙蘭（Cymbidium）、石斛蘭（Dendrobium）（血色石斛蘭（Dendrobium cruentum）除外）、蝴蝶蘭（Phalaenopsis）以及萬代蘭（Vanda）的蘭花有相同的標準，蝴蝶蘭並不是唯一的豁免種類。 就美國國內交易市場現況來看，荷蘭業者以及加拿大業者已將花卉公開拍賣市場機制引進美國，並會透過快遞業者進行蘭花遞送。 總體而言，台灣蘭花盆花以及蘭花切花在美國市場都有發展機會；建議未來的發展方向可包含：1. 依據不同地理區位市場的氣候環境與人口組成，透過育種技術來提供適地適境並符合市場期待的蘭種，並透過主張植物品種權及/或專利權及/或透過營業秘密的方式掌握育種優勢；2. 開始植根美國當地，直接引進台灣的育種與育苗技術進入美國境內（或是鄰近國家）進行蘭花的生產；3. 充分掌握國際與美國國內法規變化、定期調查美國市場需求變動，並善用美國多元化通路，不疏漏美國蘭花切花市場，也不輕忽蝴蝶蘭以外的蘭花盆花市場。 / Taiwan is a major worldwide supplier of orchids due to its outstanding seeding and breeding techniques and the natural environment. In the recent years, the lucrative economic value of orchids has prompted many industries from other countries, such as Holland, United States, Japan, Korea, Thailand, Canada and China to invest in orchid productions. In addition to the merchandise image of orchids that affects their price, demands resulting from the changing architecture styles and interior designs further determine the future value of orchids. The US orchid market is the second biggest place for orchids exported from Taiwan. Over the past three years, the average annual growth rate of orchid export weights from Taiwan to the US is over 60%, while the orchid export value on average has increased by over 38%. Therefore, the role and the possible commercial opportunities of orchids from Taiwan in the US market require further attention from the orchid farmers and the government in Taiwan. The aim of this study is to analyze the possible commercial opportunity of orchid from Taiwan in the US market by understanding the current supply chain and wholesale value of the US orchid market, ascertaining the relevant import and export laws and regulations, and investigating the current state of orchids from Taiwan in the US orchid market. According to previous investigations, it is realized that the potted orchid is the major type purchased in the US. Between 1999 and 2007, the wholesale value of potted orchids is around 120 million dollars per year, which is about 10-16 times more than that of the cut orchid. Furthermore, the US is a net import country of orchids, where the import orchid value is 6 times more than the export orchid value. Taiwan, Thailand, Holland, Canada, China and Korea are the main sources from which orchids are imported to the US. Over the last 9 years, the compound annual growth rates of both the quantity and the value of orchids imported from China, Korea, Canada and Thailand have exceeded those from Taiwan. Therefore, although Taiwan is still the major source where orchids in the US are imported from, the orchid industries from China, Korea, Canada and Thailand in addition to Holland should also be monitored. The peak of imported cut orchid quantity was in 2004 before starting to decline. Meanwhile, however, the imported cut orchid value increased. Therefore, the price for imported cut orchid thrived over the last few years. Based on Cut Flowers and Greenery Import Manual, Animal and Plant Health Inspection Service (APHIS)of United States Department of Agriculture considers that “the great majority of orchids encountered in the trade are from artificially propagated plants and therefore fall outside the scope of the CITES Regulations; therefore, INSPECT and RELEASE commercial shipments of cut orchids unless you have convincing proof that the orchids were collected in the wild; neither permits nor certificates are necessary for orchid blossoms from artificially propagated plants”. This indicates that the US government holds a relatively open attitude towards the international cut orchid trades. Moreover, based on CITES appendices valid since July 1, 2008, it is known that “artificially propagated hybrids of the following genera are not subject to the provisions of the Convention, if conditions, as indicated under a) and b), are met: Cymbidium, Dendrobium, Phalaenopsis and Vanda”. Therefore, Cymbidium, Dendrobium（except Dendrobium cruentum）, Phalaenopsis and Vanda are under the same protection level based on CITES. Moreover, companies from Holland and Canada have introduced an auction mechanism for orchids into the US orchid market, and the auctioned orchids are delivered by express. The orchid industry in Taiwan has the experience and ability to export a variety of orchids to various countries, and the US orchid market is relatively open to various orchids. Both potted and cut orchids from Taiwan show commercially competitive advantage in the US market, but new marketing strategies of orchids from Taiwan in the US market are required to be implemented prior to the development thereto. Suggestions for the orchid industry in Taiwan include the following. 1. to develop customised orchid plants that are most suitable for specific markets based on the natural environment and/or the demographic make-up, and to allow further development of these dominant species by applying protection under PVPA, plant patent and/or utility patent; 2. to directly introduce the seeding and breeding experiences and techniques from Taiwan to the US or nearby countries to shorten transportation time and to allow for mass production; 3. to regularly acquire information about the changes to the CITES, US import laws and/or regulations and trends in the orchid market, and to fully utilize the diverse orchid distribution routes and the resources from research institutes for the full development of cut and potted orchid markets.