CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O Cajueiro (Anacardium occidentale L.) à uma planta com uma grande importÃncia social e econÃmica no Nordeste do Brasil. O bagaÃo do pedÃnculo do caju à uma das maiores fontes de resÃduos (90-94%) produzidos pela indÃstria cajueira. Neste estudo, foram preparadas cinzas do bagaÃo e submetidas à anÃlise da composiÃÃo e a testes de atividade antifÃngica in vitro contra espÃcies de Fusarium. Esta anÃlise indicou uma cristalinidade em torno de 73%, correspondendo Ãs seguintes fases solÃveis: carbonato Ãcido de potÃssio - KHCO3 (39,54%), sulfato de potÃssio - K2SO4 (24,87%), e estruvita-K - MgKPO4 â 6H2O (8,59%). As fases amorfas (cerca de 27%) foram identificadas como a fraÃÃo insolÃvel de cinzas. A soluÃÃo apresentou alta atividade antifÃngica contra F. oxysporum, F. moniliforme e F. lateritium. Sua aÃÃo foi maior do que o Cercobin (tiofanato metÃlico), indicando uma possÃvel utilizaÃÃo como um agente antifÃngico nÃo tÃxico. / Cashew (Anacardium occidentale L.) is a plant with a highly social and economic importance in Northeast Brazil. Cashew peduncle bagasse is one of the greatest sources of residues (90â94%) produced by the cashew agronomic industry. In this study, we prepared cashew peduncle bagasse ashes and submitted them to compositional analysis and tests for antifungal activity in vitro against Fusarium species. This analysis indicated a crystallinity around 73%, corresponding to the following soluble phases: acid potassium carbonate- KHCO3 (39.54%), potassium sulfate - K2SO4 (24.87%), and struvite-K - MgKPO4Â6H2O (8.59%). The amorphous phases (around 27%) were identified as the insoluble fraction of ashes. The solution showed high antifungal activity against F. oxysporum, F. moniliforme and F. lateritium. This activity of this product was greater than that of Cercobin (thiophanate-methyl), indicating a possible use of this material as a non-toxic antifungal agent.

Essa tese teve como objetivo avaliar todos os casos de candidose sistêmica por Candida albicans identificadas através de kit comercial ID 32C® (bioMérieux), diagnosticados no Laboratório de Micologia da Santa Casa de Misericórdia de Porto Alegre/RS, durante o período de 1999 a 2009, buscando identificar a prevalência de C. dubliniensis, bem como avaliar os fatores de virulência e diferença de perfil de suscetibilidade antifúngica entre os isolados clínicos. Foi realizado um levantamento clínico-epidemiológico dos casos incluídos no estudo, avaliando sexo, idade, manifestações clínicas, evolução, região proveniente do paciente, doença de base, condições predisponentes, utilização de corticóides e antibióticos e resposta ao tratamento recebido. Para a diferenciação das duas espécies utilizou-se testes fenotípicos (arranjo dos clamidosporos, teste de termotolerância, formação do tubo germinativo, crescimento em meio hipertônico e niger), molecular (espectrometria de massa) e genotípico (reação em cadeia da polimerase - PCR). Em adição, foi avaliada a eficácia do método de conservação das leveduras estocadas a -20ºC e comparamos quatro substratos (soro fresco, soro congelado, ágar e caldo Mueller-Hinton) para a prova do tubo germinativo. Determinou-se a produção da fosfolipase e proteinase em isolados incluídos no estudo. A atividade in vitro dos antifúngicos fluconazol, anfotericina B e anidulafungina frente aos isolados estudados foi determinada através da concentração inibitória mínima (CIM), a concentração fungicida mínima (CFM) e ponto de corte epidemiológico (ECV). Os casos de candidemia por C. albicans diagnosticados durante 10 anos ocorreram com maior frequência em pacientes adultos com presença de cateteres. Observamos que houve maior chance de ocorrência desta em pacientes oncológicos. O percentual de alta nos pacientes foi baixo. O método utilizado para a conservação de leveduras nesse estudo apresentou taxa de 70% de viabilidade. O ágar e o caldo Mueller-Hinton demonstraram sensibilidade de 90% e especificidade de 100%. Os isolados de C. albicans provenientes de hemocultivos apresentaram produção de fosfolipase em 78% e proteinase em 97% dos isolados. A espécie C. dubliniensis não foi identificada em isolados de hemocultivos, sendo todos os casos de candidemia por C. albicans. Os testes microcultivo em ágar fubá, espectrometria de massa, caldo niger e caldo hipertônico concordaram com o teste genotípico. Os isolados de C. albicans apresentaram maior suscetibilidade a anidulafungina, entretanto, os menores valores obtidos em 90% dos isolados (CIM90) foi pela anfotericina B. E através do ECV, os isolados poderiam ser resistentes ao fluconazol, demonstrando a importância da associação desses dois parâmetros. / The aim this tesis was to evaluate systemic candidiasis cases by Candida albicans through ID 32C® (bioMérieux), at Mycology Laboratory of the Santa Casa de Porto Alegre/RS, during 1999 to 2009, seeking to identify the C. dubliniensis prevalence, as well as evaluating the virulence factors and antifungal susceptibility profile difference of among isolates. The clinical and epidemiological survey was made through gender, age, clinical manifestations, evolution, patient's region, underlying disease, predisposing conditions, steroids and antibiotics use, and response to treatment. The phenotypic tests (tthermotolerance, germ tube, hypertonic and Niger medium), molecular (mass spectrometry) and genotypic (polymerase chain reaction – PCR) was used for two species identification. We also assessed if the mantainance of C. albicans stored at - 20ºC in a freezer with sterile distilled water was usefull.The four substrate (fresh and frozen serum, agar and broth Mueller-Hinton®) were used for germ tube formation and the phospholipase and proteinase activity were evaluated. The in vitro activity of fluconazole, amphotericin B and anidulafungin were compared through the minimum inhibitory concentration (MIC), the minimum fungicidal concentration (MFC) and epidemiological cutoff value (ECV). The candidemia cases by C. albicans for ten years occurred more frequently in adult and catheters use. We observed the more chance this occurrence in cancer patients. The survival percentage was low. The used method in the study for yeast stored had 70% of viability. The agar and broth Mueller-Hinton were 90% sensitivity and 100% specificity. The boodstream isolates of C. albicans produce virulence factors, such the germ tube production and hydrolytic enzymes (78% of phospholipase and 97% of protease) production. The C. dubliniensis was not identified in bloodstream isolates, thus all candidemia cases were by C. albicans. The mass spectrometry, cornmeal agar, Niger and hypertonic broth agreed with genotypic test. The isolates exhibited more susceptibility to anidulafungin, and 90% of them (MIC90) exhibited the lowest values against amphotericin B. Based on ECV and Pfaller classification, isolates could be resistant to fluconazole, demonstrating the importance of the combination of these parameters.

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-05-08T02:43:19Z No. of bitstreams: 1 127110045.pdf: 1527824 bytes, checksum: 960729a0a23069d5d40ec997cc3034b8 (MD5) / Made available in DSpace on 2015-05-08T02:43:19Z (GMT). No. of bitstreams: 1 127110045.pdf: 1527824 bytes, checksum: 960729a0a23069d5d40ec997cc3034b8 (MD5) Previous issue date: 2014-07-17 / O gênero Cuphea, popularmente conhecido no Brasil por “sete-sangrias”, tem seu uso medicinal reconhecido devido aos efeitos diurético, hipotensor e cardioprotetor. No sul do Brasil, em região característica do bioma Pampa, foi encontrada a espécie Cuphea glutinosa Cham. & Schltdl. Embora o uso popular, esta espécie é pouco descrita na literatura. O presente trabalho tem como objetivos o estudo da composição química dos extratos de C. glutinosa e a avaliação das atividades antifúngica e anti-hipertensiva. O material vegetal foi coletado na cidade de Uruguaiana (RS, Brasil), identificado e depositado em herbário. Após secagem e trituração do material vegetal, foram obtidos os extratos hidroetanólicos através de maceração exaustiva com etanol 40% (v/v) para folhas e etanol 70% (v/v) para raízes. Para a infusão, utilizou-se água a 80oC. As análises cromatográficas foram realizadas em equipamento cromatógrafo a líquido Prominence Shimadzu, em técnica por CLAE e CLUE. Utilizou-se sistema de fase reversa, eluição por gradiente com fase móvel composta por acetonitrila:metanol (4:1) e ácido fórmico 0,1% pH 3,0, coluna C18 analítica e fast, e detecção por UV-DAD e ESI-MS. Os teores de polifenóis totais e de flavonóides foram determinados por método colorimétrico, seguindo metodologia padronizada. A atividade antifúngica in vitro foi realizada utilizando o método de microdiluição em caldo, determinando-se a CIM, in-vitro, contra diferentes isolados clínicos. Para avaliação do potencial anti-hipertensivo in vivo, foram realizadas medições da pressão sanguínea pelo método de monitoramento hemodinâmico invasivo, através da inserção de cateter na artéria carótida. Os resultados de teor de fenólicos totais indicaram predominância destes componentes em extratos obtidos de folhas e por maceração, conforme os valores obtidos: 1,8501 mg EAG/mL (folhas) e 0,8467 mg EAG/mL (raízes) para infusão, e 3,7284 mgEAG/mL (folhas) e 2,6266 mg EAG/mL (raízes) para maceração. Quanto ao teor de flavonóides, os resultados quantitativos foram: 7,0959 mg/g (folhas) e 0,5664 mg/g (raízes) para a infusão, e 7,9511 mg/g (folhas) e 0,5994 mg/g (raízes) para maceração. Na análise cromatográfica, os extratos obtidos das folhas de C. glutinosa apresentaram picos cromatográficos bem separados, em perfil reprodutível. Na determinação por CLUE-MS, os dados de íon molecular e fragmentos de massa indicaram a composição predominante em flavonóides, sugerindo-se os componentes quercetina-3-O-glicosídeo, quercetina-3- arabinosídeo, quercetina-3-glicuronídeo, isoramnetina e quercetina-5-O-β-glicopiranosídeo. Para o potencial antifúngico, os extratos das folhas e raízes apresentaram atividade in vitro contra Candida parapsilosis, Candida tropicalis e Trichosporon asahii, com CIM variando na faixa de 1,9-62,5 μg/mL. Nos testes hemodinâmicos realizados, os extratos das folhas não apresentaram efeito significativo sobre a pressão arterial. A identificação dos componentes em C. glutinosa, derivados de quercetina, torna promissora novas investigações a fim de aprofundar o conhecimento a respeito desta espécie, em especial na busca de respostas para a relatada ação anti-hipertensiva. / The Cuphea genus, popularly known in Brazil as "sete-sangrias", is used traditionally due the diuretic, hypotensive and cardioprotective effects. In southern Brazil, in characteristic region of Pampa biome, it was found the species Cuphea glutinosa Cham. & Schltdl. Although used popularly, this species is few reported in the literature. The present work aimed to study the chemical composition of extracts from C. glutinosa and to evaluate the antifungal and anti -hypertensive activities. The plant material was collected in the city of Uruguaiana (RS, Brazil), identified and deposited in a herbarium. After dryness and milling, the hydroethanolic extracts were obtained through exhaustive maceration using ethanol 40% (v/v) for leaves and ethanol 70% (v/v) for roots. The infusions were prepared using water at 80 °C. The chromatographic analyses were performed in liquid chromatography Prominence Shimadzu, for HPLC and UPLC assays. The method was conducted using reverse phase system, gradient elution with mobile phase composed by acetonitrile:methanol (4:1) and formic acid 0.1% pH 3.0, C18 analytical and fast column, and detection by UV-DAD and MS. The polyphenols and flavonoids contents were determined by colorimetric method. The in vitro antifungal activity was conducted by using the broth microdilution method, determining the MIC against different clinical isolates. For evaluation of in vivo anti-hypertensive potential, the blood pressure was measured by the method of invasive hemodynamic monitoring, through of insertion the catheter into the carotid artery. The results of phenolic content indicated the high concentration of these compounds in leaves extracts obtained by maceration: 1.8501 mgEAG/mL (leaves) and 0.8467 mgEAG/mL (roots) for infusion, and 3.7284 mgEAG/mL (leaves) and 2.6266 mgEAG/mL (roots) for maceration. For flavonoids, the contents were: 7.0959 mg/g (leaves) and 0.5664 mg/g (roots) for infusion, and 7.9511 mg/g (leaves) and 0.5994mg/g (roots) for maceration. In the chromatographic analyses, the leaf extracts from C. glutinosa presented chromatographic peaks well separated and reproducible. In the determination by UPLC-MS, the molecular ion and mass fragments indicated the predominant composition in flavonoids, suggesting the compounds quercetin-3- O-glucoside, quercetin-3-arabinoside, quercetin-3-glucuronide, isorhamnetin and quercetin-5- O-β-glucopiranoside. For the antifungal potential, the leaf and roots extracts presented activity against Candida parapsilosis, Candida tropicalis e Trichosporon asahii, with MIC values ranging 1,9-62,5 μg/mL. In the hemodynamic tests performed, the leaves extracts did not present significant effect in the arterial pressure, although a tendency in pressure reduction could be observed. The identification of quercetin derivatives in C. glutinosa becomes promisor further investigations about this species, mainly in respect to the anti-hypertensive action.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-04-07T12:58:36Z No. of bitstreams: 1 HEMERSON SILVA DA ROSA.pdf: 1232470 bytes, checksum: b64c04e3acdc1a67c9bd8559b68fb261 (MD5) / Made available in DSpace on 2016-04-07T12:58:36Z (GMT). No. of bitstreams: 1 HEMERSON SILVA DA ROSA.pdf: 1232470 bytes, checksum: b64c04e3acdc1a67c9bd8559b68fb261 (MD5) Previous issue date: 2013-01-24 / Sida tuberculata (Malvaceae), conhecida popularmente como “guanxuma”, é uma espécie vegetal de porte herbáceo, bem representada na região sul do Brasil. Na cultura popular é utilizada para tratamento de diversas enfermidades, em especial àquelas relacionadas ao diabetes e ao colesterol elevado. Para algumas espécies, existem relatos de eventual potencial antimicrobiano. Considerando a ausência de estudos sobre esta planta, o presente trabalho investigou a composição química dos extratos brutos de S. tuberculata e avaliou seu potencial antifúngico in vitro. Após a coleta e identificação, o material vegetal foi submetido aos processos de secagem e trituração. Submeteu-se a extração a frio por percolação, utilizando-se como solvente solução hidroetanólica a 40% para folhas e 70% para raízes. Para fins de comparação foram feitas extrações aquosas por infusão. Na sequência, foram determinados os teores de fenólicos totais e flavonóides totais. Posteriormente, as amostras foram analisadas através de método por CLAE-UV, com seleção das condições cromatográficas de melhor eficiência de separação. Sendo estas estabelecidas, efetuou-se análise por LC-MS em modo ESI positivo. Para os ensaios da atividade antifúngica foram utilizados os protocolos de microdiluição em ágar para determinação da concentração inibitória mínima (CIM) e concentração fungicida mínima (CFM). Também foi aplicada a metodologia para avaliação do potencial de remoção de biofilme em Cateter Venoso Central (CVC). Os resultados obtidos demonstraram um maior teor de fenólicos e flavonóides totais nos extratos das folhas. As análises por LC-UV-MS permitiram a identificação e proposição de cinco compostos, entre ecdisteróides, flavonóides e alcalóides. Nos ensaios de atividade antifúngica os extratos aquosos apresentaram atividade contra linhagens de Candida krusei, com valores de CIM variando entre 3.9 - 62.5 μg/ml para folhas e 1.95 - 31.25 μg/ml para raízes. No teste de remoção de biofilme, os extratos aquosos das folhas demonstraram um maior potencial de remoção. Os dados de composição química obtidos, nas variantes de diferentes partes da planta, bem como a atividade antimicrobiana detectada, geram expectativas quanto a novos estudos de exploração do potencial biológico de S. tuberculata. / Sida tuberculata (Malvaceae), popularly known as "guanxuma", is an herbaceous plant species present in southern Brazil. In popular culture, it is used for the treatment of several diseases, such as those related to diabetes and high cholesterol level. For some species of Sida, there are also reports about the antimicrobial potential. Considering the lack of studies at this species, this study proposed an investigation about the chemical composition of Sida tuberculata extracts and their in vitro antifungal activity. After collection and identification, the plant material was submitted to dryness and powdered. Then, it was submitted to extraction by percolation using hydroethanolic solution at 40% and 70% as solvent, to leaves and roots respectively. For comparison, aqueous extracts were obtained by infusion. The total phenolic and flavonoid contents of extracts were determined. The samples were also evaluated by HPLC-UV, testing the chromatographic conditions that promote the better separation efficiency. After, for the identification procedure, the analysis by LC-ESI-MS in positive mode was conducted using previously established conditions. For the antifungal activity assay, the agar microdilution protocols were used for determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). In addition, the extracts were tested for potential biofilm removal in Central Venous Catheter (CVC). The results demonstrated a higher concentration of total phenolic and flavonoids compounds in the leaves extracts. LC-MS analysis allowed the identification of five components, between ecdysteroids, flavonoids and alkaloids. In the assay of antifungal activity, the aqueous extract had activity against Candida krusei strains, with the MIC values varying between 3.9 - 62.5 μg/ml for leaves and 1.95 - 31.25 μg/ml for roots. In the CVC biofilm removal testing, the aqueous leaves extracts presented a greater potential. The chemical composition data obtained in this work, considering the different parts of plant, as well as the antimicrobial activity detected, bring perspectives of exploring this species concerning its biological potential.

Increased systemic infections and growing resistance of Candida species in immunosuppressed people have prompted research for additional treatment options. The purpose of this quantitative study was to investigate the potential of lemongrass, oregano, and thyme essential oils tested individually, combined, and combined with the antifungal agents fluconazole and caspofungin to kill Candida albicans isolates in a controlled laboratory setting. This study was grounded on the theoretical concepts of the epidemiologic triangle model. The experimental data collected were used to investigate risk factors related to age, gender, race, and comorbidities. Kill rates of lemongrass, oregano, and thyme essential oils individually and combined, kill rates of fluconazole, caspofungin, and the kill rates when the antifungals were each combined with the 3 essential oils were compared using 117 isolates recovered from bloodstream infections between January 2009 through August 1, 2017. The data collected were analyzed using 2-way repeated ANOVAS. According to study results, there were statistically significant increases in kill rates when the isolates were exposed to any of the combinations of essential oils tested. Using binomial and multinomial regression to analyze age, gender, race, and comorbidities resulted in the age group 25-34, kidney failure, and solid organ tumor cancer all being statistically significantly associated with an increased risk for Candida albicans bloodstream infections, and multiple organ failure negatively associated with the risk. Health care practitioners can use the results of this study to reduce the number of patients becoming infected with life-threatening yeast infections, which could reduce the costs associated with infections.

The marine surface-associated bacterium Pseudoaltermonas tunicata, produces a range of compounds that inhibit fouling organisms, including invertebrate larvae, bacteria, algal spores and fungi. In addition to these antifouling compounds P. tunicata cells produce both a yellow and a purple pigment. The aim of this study was to further characterise the antifouling activities, their regulation and relationship with pigmentation, and the ecological significance of P. tunicata and related organisms. It was discovered that the anti-algal compound was extracellular, heat sensitive, polar and between 3 and 10 kDa in size. The anti-fungal compound was found to be the yellow pigment and active against a wide range of fungal and yeast isolates. Chemical analysis suggests that this compound consists of a carbon ring bound to a fatty-acid side chain. Genetic analysis supports the chemical data for the active compound as a mutant in a gene encoding for a long-chain fatty-acid CoA ligase was deficient for anti-fungal activity. To address the regulation of antifouling compounds and their relationship to pigmentation transposon mutagenesis of P. tunicata was performed. Mutants lacking the yellow pigment displayed a reduced ability to inhibit fouling organisms. Further analysis of these mutants identified genes involved with the synthesis and regulation of synthesis of pigment and antifouling compounds. One of these mutants was disrupted in a gene (wmpR) with similarity to the transcriptional regulators ToxR from Vibrio cholerae and CadC from Escherichia coli. Analysis of global protein expression using two-dimensional gel electrophoresis showed that WmpR is essential for the expression of at least fifteen proteins important for the synthesis of fouling inhibitors. The ecological significance of antifouling bacteria was addressed by assessing the antifouling capabilities of a collection of bacteria isolated from different marine surfaces. Overall, isolates from living surfaces displayed more antifouling traits then strains isolated from non-living surfaces. Five dark-pigmented strains originating from the alga Ulva lactuca were further studied. Phylogenetic and phenotypic analysis revealed that they were all members of the genus Pseudoalteromonas and were closely related to P. tunicata. Two strains represented a novel species within the genus and were taxonomically defined as P. ulvae sp. nov.

Els avenços que han experimentat tots els camps de la medicina en els darrers 20 anys han influït de forma notable en els tipus de malalts que són atesos als centres hospitalaris, sobre tot en hospitals de tercer nivell. L'aplicació de noves tecnologies o actituds terapèutiques corn el trasplantament de medúl.la òssia o d'òrgan sòlid i la utilització d'agents quimioteràpics han esdevingut cada cop més freqüents. A més a més, factors com la millora en l'atenció dels malalts ingressats en unitats de cures intensives, la nutrició parenteral, l'hemodiàlisi o els antibiòtics d'ampli espectre han contribuït de forma clara i favorable al tractament dels pacients en situacions crítiques. Això però, té una contrapartida negativa que es tradueix en la presència d'un major nombre de malalts hospitalitzats amb un compromís immunitari evident o amb patologies de base molt greus. Considerats corn un conjunt, aquests tipus de pacients constitueixen una població altament susceptible a patir una infeccin nosocomial que pot estar causada per diversos microorganismes entre els que cal incloure els fongs. Les infeccions fúngiques en aquests malalts són sovint severes, ràpidament progressives i generalment, hi ha certes dificultats per arribar al seu diagnòstic o realitzar un tractament adequat. L'increment experimentat en el nombre d'infeccions nosocomials fúngiques ha comportat, de forma paral.lela, un augment en el nombre de comunicacions científiques que descriuen diferents brots epidèmics nosocomia1s causats per fongs (Fridkin and Jarvis, 1996). És obvi que el focus d'origen o el mecanisme de transmissió pot ser molt diferent d'un brot a l'altre segons les característiques de l'agent implicat. A més a més, per determinar la causa d'un brot epidèmic concret i poder adoptar les mesures de control més adients per aturar-lo, és fonamental conèixer la fisiopatologia del fong implicat. Però, tal com ha passat amb les infeccions bacterianes, a mesura que aprofundim en l'epidemiologia d'una determinada infecció fúngica, i per extensió, la dels brots epidèmics, es fa cada cop més evident la necessitat d'utilitzar una determinada metodologia que ens ajudi a avaluar les troballes obtingudes. L'aplicació de mètodes moleculars per compendie millor l'epidemiologia de les infeccions fúngiques i la consegüent tipificació dels fongs implicats ha experimentat un impuls notable en els darrers 10 anys. Tot i els diversos mètodes existents, no hi ha però, un mètode considerat "standard" i l'elecció d'un o altre dependrà de les preguntes plantejades i les possibilitats de cada centre (Soll, 2000). Per tot allò comentat anteriorment, és obvi que la millora en el pronòstic de les infeccions fúngiques, sobre tot en malalts amb immunosupressió, és un objectiu que cal assolir el més aviat possible. Aquest objectiu es pot abordar des de diferents punts de vista. Així, en la pràctica diària hi ha dues opcions que semblen clares: avançar en les tècniques de diagnòstic precoç d'aquestes infeccions i la introducció en el tractament de nous antifúngics que millorin l'activitat, tolerància i seguretat dels antifúngics actuals. Hi ha però altres possibilitats que també cal estudiar per aconseguir l'objectiu que hem comentat. Algunes són necessàries com a pas previ a les opcions anteriors i altres simplement les complementen En aquesta tesi hem centrat el nostre treball en un aspecte més bàsic i inicial com és la valoració de l'activitat in vitro de nous antifúngics sobre fongs patògens prevalents amb la finalitat d'esbrinar les seves possibilitats com a fàrmacs potencialment útils en el tractament de les infeccions fúngiques. A més a més, hi ha un segon aspecte que també ha estat objecte d'anàlisi com és l'aplicació de la sonda semirepetitiva Ca3 a l'estudi de les infeccions nosocomials per “Candida albicans”.

Waxflower (Chamelaucium spp. and hybrids) is the singlemost important Australian export cut-flower. The major problem in waxflower trading is flower abscission after harvest. While several factors are involved, ethylene production resulting from preharvest infection with the fungus Botrytis cinerea is the most important cause. The general objectives of this study were to investigate the biology of Botrytis infecting waxflower flowers and potential elicitation of host defence against this pathogen. Effects of anti-ethylene and S-carvone treatments on Botrytis-induced flower abscission were also evaluated. Infection of flowers by Botrytis was studied on two waxflower cvs. Mullering Brook and My Sweet Sixteen using light and electron microscopy. Conidial germination and protoappressorial formation occurred within 8 h post-inoculation (hpi). Infection of most floral organs, including petals, anthers and filaments, stigma, and hypanthium, was within 24 hpi. Infection cushions on stamen bases were formed at 36 hpi by saprophytic hyphae that originated from anthers. This infection route probably gives rise to the typical tan-coloured Botrytis symptoms that appear to radiate from this part of the flower. Subcuticular hyphae were present at very high density near stamen bases. They evidently resulted at multiple penetrations from single infection cushions. Flower abscission occurred at 72 hpi. At this time, floral tube tissues remained uninfected. This temporal pattern infers the possible transmission of a signal (e.g. ethylene) upon Botrytis infection (6–36 hpi) that intiates a defence response of shedding infected flowers (72 hpi). Susceptibility of waxflower before and after harvest to B. cinerea under various environmental conditions (laboratory, greenhouse, and field) was investigated. Flowers, either on plants or on cut stems showed similar susceptibility to B. cinerea and abscised under cool temperatures (~20 ºC) and high humidity (>95% RH) conditions following infection. Compared to cv. Mullering Brook, cv. My Sweet Sixteen was somewhat more resistant to B. cinerea infection under field conditions. Constitutive and inducible antifungal compounds in waxflower flower tissues were screened in cvs. CWA Pink, Stephan’s Delight, Mullering Brook and My Sweet Sixteen using thin layer chromatography bioassays with isolates of B. cinerea and Alternaria alternata (pathogenic) and Cladosporium cladosporioides (non-pathogenic). Common inhibition zone observed at Rf 0.28–0.38, 0.46–0.56 and 0.67–0.76 contained phenolic compounds. There were at least five (cv. Mullering Brook) and one (cv. My Sweet Sixteen) inducible antifungal phenolic compounds as judged by increases in inhibition area as a result of B. cinerea infection and methyl jasmonate treatment. The total areas of B. cinerea- and MeJA-induced inhibition zones were approximately 2.0- and 2.5-folds greater, respectively, than zones from control flowers. Preharvest sprays of three different known host plant defence elicitors, methyl jasmonate (MeJA), benzothiadiazole (BTH), and silicon (Si), were applied to waxflower cvs. Mullering Brook and My Sweet Sixteen plants. BTH or Si sprays generally had no significant effect on postharvest Botrytis severity on either cultivar. MeJA sprays did not reduce B. cinerea on cv. Mullering Brook. MeJA slightly suppressed B. cinerea on cv. My Sweet Sixteen at 500 and 750 µM. Overall, field applications of these host plant defence elicitor chemicals as spray treatments had little effect on vase life, water uptake and relative fresh weight of the cut sprigs. Moreover, they did not appreciably suppress B. cinerea or associated postharvest floral abscission. The efficacy of combined elicitor treatments and combined pre- and postharvest MeJA treatments were assessed. Preharvest foliar applications of MeJA (1000 µM; 2 or 4 times), MeJA (1000 µM) combined with BTH (150 mg/L), and MeJA combined with Si (1500 mg SiO2/L) generally did not suppress postharvest B. cinerea development and flower abscission from harvested sprigs. A pre- plus post-harvest 1000 µM MeJA spray treatment consistently but only slightly suppressed B. cinerea infection on flowers from both pot- and field-grown plants. Pre- and post-harvest MeJA treatments reduced B. cinerea development, but increased flower abscission. Combined MeJA and anti-ethylene treatments were then screened for potential to suppress B. cinerea while preventing flower abscission. However, the combined MeJA and 1-MCP treatment reduced neither Botrytis disease nor flower abscission on sprigs from pot- and field-grown plants. The combined MeJA and STS treatment reduced disease severity for up to 6 days on sprigs harvested from pot-grown plants but tended to increase Botrytis severity on sprigs from field-grown plants 6 days after inoculation. Antifungal effects of the essential oil S-carvone against B. cinerea germination and mycelial growth were demonstrated in vitro. Inhibition increased with increasing S-carvone concentrations from 0.64 mM to 5.08 mM. However, in planta, S-carvone concentrations in this range did not affect either Botrytis disease levels or flower abscission on cut waxflower flowers.

Waxflower (Chamelaucium spp. and hybrids) is the singlemost important Australian export cut-flower. The major problem in waxflower trading is flower abscission after harvest. While several factors are involved, ethylene production resulting from preharvest infection with the fungus Botrytis cinerea is the most important cause. The general objectives of this study were to investigate the biology of Botrytis infecting waxflower flowers and potential elicitation of host defence against this pathogen. Effects of anti-ethylene and S-carvone treatments on Botrytis-induced flower abscission were also evaluated. Infection of flowers by Botrytis was studied on two waxflower cvs. Mullering Brook and My Sweet Sixteen using light and electron microscopy. Conidial germination and protoappressorial formation occurred within 8 h post-inoculation (hpi). Infection of most floral organs, including petals, anthers and filaments, stigma, and hypanthium, was within 24 hpi. Infection cushions on stamen bases were formed at 36 hpi by saprophytic hyphae that originated from anthers. This infection route probably gives rise to the typical tan-coloured Botrytis symptoms that appear to radiate from this part of the flower. Subcuticular hyphae were present at very high density near stamen bases. They evidently resulted at multiple penetrations from single infection cushions. Flower abscission occurred at 72 hpi. At this time, floral tube tissues remained uninfected. This temporal pattern infers the possible transmission of a signal (e.g. ethylene) upon Botrytis infection (6–36 hpi) that intiates a defence response of shedding infected flowers (72 hpi). Susceptibility of waxflower before and after harvest to B. cinerea under various environmental conditions (laboratory, greenhouse, and field) was investigated. Flowers, either on plants or on cut stems showed similar susceptibility to B. cinerea and abscised under cool temperatures (~20 ºC) and high humidity (>95% RH) conditions following infection. Compared to cv. Mullering Brook, cv. My Sweet Sixteen was somewhat more resistant to B. cinerea infection under field conditions. Constitutive and inducible antifungal compounds in waxflower flower tissues were screened in cvs. CWA Pink, Stephan’s Delight, Mullering Brook and My Sweet Sixteen using thin layer chromatography bioassays with isolates of B. cinerea and Alternaria alternata (pathogenic) and Cladosporium cladosporioides (non-pathogenic). Common inhibition zone observed at Rf 0.28–0.38, 0.46–0.56 and 0.67–0.76 contained phenolic compounds. There were at least five (cv. Mullering Brook) and one (cv. My Sweet Sixteen) inducible antifungal phenolic compounds as judged by increases in inhibition area as a result of B. cinerea infection and methyl jasmonate treatment. The total areas of B. cinerea- and MeJA-induced inhibition zones were approximately 2.0- and 2.5-folds greater, respectively, than zones from control flowers. Preharvest sprays of three different known host plant defence elicitors, methyl jasmonate (MeJA), benzothiadiazole (BTH), and silicon (Si), were applied to waxflower cvs. Mullering Brook and My Sweet Sixteen plants. BTH or Si sprays generally had no significant effect on postharvest Botrytis severity on either cultivar. MeJA sprays did not reduce B. cinerea on cv. Mullering Brook. MeJA slightly suppressed B. cinerea on cv. My Sweet Sixteen at 500 and 750 µM. Overall, field applications of these host plant defence elicitor chemicals as spray treatments had little effect on vase life, water uptake and relative fresh weight of the cut sprigs. Moreover, they did not appreciably suppress B. cinerea or associated postharvest floral abscission. The efficacy of combined elicitor treatments and combined pre- and postharvest MeJA treatments were assessed. Preharvest foliar applications of MeJA (1000 µM; 2 or 4 times), MeJA (1000 µM) combined with BTH (150 mg/L), and MeJA combined with Si (1500 mg SiO2/L) generally did not suppress postharvest B. cinerea development and flower abscission from harvested sprigs. A pre- plus post-harvest 1000 µM MeJA spray treatment consistently but only slightly suppressed B. cinerea infection on flowers from both pot- and field-grown plants. Pre- and post-harvest MeJA treatments reduced B. cinerea development, but increased flower abscission. Combined MeJA and anti-ethylene treatments were then screened for potential to suppress B. cinerea while preventing flower abscission. However, the combined MeJA and 1-MCP treatment reduced neither Botrytis disease nor flower abscission on sprigs from pot- and field-grown plants. The combined MeJA and STS treatment reduced disease severity for up to 6 days on sprigs harvested from pot-grown plants but tended to increase Botrytis severity on sprigs from field-grown plants 6 days after inoculation. Antifungal effects of the essential oil S-carvone against B. cinerea germination and mycelial growth were demonstrated in vitro. Inhibition increased with increasing S-carvone concentrations from 0.64 mM to 5.08 mM. However, in planta, S-carvone concentrations in this range did not affect either Botrytis disease levels or flower abscission on cut waxflower flowers.

Waxflower (Chamelaucium spp. and hybrids) is the singlemost important Australian export cut-flower. The major problem in waxflower trading is flower abscission after harvest. While several factors are involved, ethylene production resulting from preharvest infection with the fungus Botrytis cinerea is the most important cause. The general objectives of this study were to investigate the biology of Botrytis infecting waxflower flowers and potential elicitation of host defence against this pathogen. Effects of anti-ethylene and S-carvone treatments on Botrytis-induced flower abscission were also evaluated. Infection of flowers by Botrytis was studied on two waxflower cvs. Mullering Brook and My Sweet Sixteen using light and electron microscopy. Conidial germination and protoappressorial formation occurred within 8 h post-inoculation (hpi). Infection of most floral organs, including petals, anthers and filaments, stigma, and hypanthium, was within 24 hpi. Infection cushions on stamen bases were formed at 36 hpi by saprophytic hyphae that originated from anthers. This infection route probably gives rise to the typical tan-coloured Botrytis symptoms that appear to radiate from this part of the flower. Subcuticular hyphae were present at very high density near stamen bases. They evidently resulted at multiple penetrations from single infection cushions. Flower abscission occurred at 72 hpi. At this time, floral tube tissues remained uninfected. This temporal pattern infers the possible transmission of a signal (e.g. ethylene) upon Botrytis infection (6–36 hpi) that intiates a defence response of shedding infected flowers (72 hpi). Susceptibility of waxflower before and after harvest to B. cinerea under various environmental conditions (laboratory, greenhouse, and field) was investigated. Flowers, either on plants or on cut stems showed similar susceptibility to B. cinerea and abscised under cool temperatures (~20 ºC) and high humidity (>95% RH) conditions following infection. Compared to cv. Mullering Brook, cv. My Sweet Sixteen was somewhat more resistant to B. cinerea infection under field conditions. Constitutive and inducible antifungal compounds in waxflower flower tissues were screened in cvs. CWA Pink, Stephan’s Delight, Mullering Brook and My Sweet Sixteen using thin layer chromatography bioassays with isolates of B. cinerea and Alternaria alternata (pathogenic) and Cladosporium cladosporioides (non-pathogenic). Common inhibition zone observed at Rf 0.28–0.38, 0.46–0.56 and 0.67–0.76 contained phenolic compounds. There were at least five (cv. Mullering Brook) and one (cv. My Sweet Sixteen) inducible antifungal phenolic compounds as judged by increases in inhibition area as a result of B. cinerea infection and methyl jasmonate treatment. The total areas of B. cinerea- and MeJA-induced inhibition zones were approximately 2.0- and 2.5-folds greater, respectively, than zones from control flowers. Preharvest sprays of three different known host plant defence elicitors, methyl jasmonate (MeJA), benzothiadiazole (BTH), and silicon (Si), were applied to waxflower cvs. Mullering Brook and My Sweet Sixteen plants. BTH or Si sprays generally had no significant effect on postharvest Botrytis severity on either cultivar. MeJA sprays did not reduce B. cinerea on cv. Mullering Brook. MeJA slightly suppressed B. cinerea on cv. My Sweet Sixteen at 500 and 750 µM. Overall, field applications of these host plant defence elicitor chemicals as spray treatments had little effect on vase life, water uptake and relative fresh weight of the cut sprigs. Moreover, they did not appreciably suppress B. cinerea or associated postharvest floral abscission. The efficacy of combined elicitor treatments and combined pre- and postharvest MeJA treatments were assessed. Preharvest foliar applications of MeJA (1000 µM; 2 or 4 times), MeJA (1000 µM) combined with BTH (150 mg/L), and MeJA combined with Si (1500 mg SiO2/L) generally did not suppress postharvest B. cinerea development and flower abscission from harvested sprigs. A pre- plus post-harvest 1000 µM MeJA spray treatment consistently but only slightly suppressed B. cinerea infection on flowers from both pot- and field-grown plants. Pre- and post-harvest MeJA treatments reduced B. cinerea development, but increased flower abscission. Combined MeJA and anti-ethylene treatments were then screened for potential to suppress B. cinerea while preventing flower abscission. However, the combined MeJA and 1-MCP treatment reduced neither Botrytis disease nor flower abscission on sprigs from pot- and field-grown plants. The combined MeJA and STS treatment reduced disease severity for up to 6 days on sprigs harvested from pot-grown plants but tended to increase Botrytis severity on sprigs from field-grown plants 6 days after inoculation. Antifungal effects of the essential oil S-carvone against B. cinerea germination and mycelial growth were demonstrated in vitro. Inhibition increased with increasing S-carvone concentrations from 0.64 mM to 5.08 mM. However, in planta, S-carvone concentrations in this range did not affect either Botrytis disease levels or flower abscission on cut waxflower flowers.