Three yeast antagonists (two strains of Cryptococcus laurentii and one of Candida sake) from orange trees reduced incidence of green mold by 80 to 95% when tested in wounded orange fruits inoculated with Penicillium digitatum and incubated at 7ºC for 30 days. The yeasts inhibited conidial germination of the pathogen, but did not kill the spores. Effectiveness of the three yeasts as antagonists was associated in part with their ability to rapidly colonize wound sites, despite low nutrient availability. Observations suggested that production of extracellular matrix by the yeasts may have facilitated rapid wound colonization. Germination of P. digitatum conidia was significantly inhibited when the pathogen and antagonists were in direct physical contact in a culture suspension. The results supported the view that competition for nutrients is also a mode of action of yeasts against P. digitatum.

Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human and animal infection have been already reported. This species is reported to be heterogenous. The taxonomy of yeast Cryptococcus laurentii was always highly ambiguous. The application of molecular biology and bioinformatic methods led to dividing of searched strains to two distinct phylogenetic groups, some varieties were recognized as species and the locution „Cryptococcus laurentii group“ was introduced. The taxonomy of this group is likely not definitive and with advancing knowledge will change. Our aim was the identification of individual species within this group based on matrix-assisted laser desorption/ionization – time of flight mass spectrometry (MALDI – TOF MS), which has recently been described as a rapid, reliable, cost-effective and powerful tool for analyzing of microorganisms, even on variety level. Generally, the yeasts of genus Cryptococcus form highly resistant polysaccharide capsules and produce large amount of extracellular polysaccharides, therefore belong to so called „difficult“ cases for biotyping. The experimental protocol has been optimized for MS analysis of this genus on the selected strains of Cr. neoformans, Cr. laurentii and Cr. magnus from the Culture Collection of Yeasts (CCY).Thirty-three strains, originally classified as Cryptococcus laurentii has been identified by chosen method. These strains were distributed into six different groups according their spectra similarities. It was selected at least one strain of each group, which was classified based on the sequence analysis of the D1/D2 domains of the LSU rRNA gene. This strain (with known sequence) became representative for its group. Type strain of Cryptococcus laurentii (CCY 17-3-2) belongs to the group I. Its MS spectrum of ribosomal proteins differ from mass spectra of all other biotyped species, even with strains identified as Cryptococcus laurentii was the similarity of the spectra low, which could be caused by identification of two different varieties. The group II is represented by Cryptococcus laurentii CCY 17-3-17. Except this strain, thirteen more strains belong to the group II. The group III represents Cryptococcus flavescens CCY 17-3-29. This group included 12 additional strains with almost identical mass spectra. Group IV included only one strain (CCY 17-3-13), which was identified as Cryptococcus carnescens based on gene sequence analysis. Similarly, one representative (CCY 17-3-5) has the group V. Strain CCY 17-3-5 was identified as Cryptococcus flavus. The last group VI of three members represents strain 17-3-35 identified as Bulleromyces albus. While Cr. laurentii and Cr. flavescens belong to phylogenetic group I and Cr. carnescens to the phylogenetic group II, four strains giving two types of different MS spectra and identified as Cr. flavus (1 strain) and Bulleromyces albus (3 strains) were excluded from „Cr. laurentii group.“

Orientador: Márcia Marinho / Resumo: A criptococose é uma doença sistêmica causada pela inalação de basidiosporos desidratados de espécies patogênicas do gênero Cryptococcus presentes no ambiente. Os complexos espécies mais associados à doença são os complexos de espécies Cryptococcus neoformans e C. gattii. O complexo de espécies C. neoformans está associado a casos de criptococose em pacientes imunossuprimidos, sendo comumente isolados do solo e de fezes de aves. O complexo de espécies de C. gattii, por sua vez, afeta principalmente indivíduos imunocompetentes e geralmente é isolado em espécies de árvores nativas ou exóticas em estado de biodegradação. Este estudo teve como objetivo realizar o mapeamento ambiental de espécies patogênicas do gênero Cryptococcus em amostras ambientais arbóreas em locais públicos da cidade de Birigui, São Paulo. Para a coleta das amostras, foram selecionadas regiões com maior trânsito neste município. Após processamento, foram realizados testes morfo-fisiológicos, bioquímicos, teste de assimilação de carbono e nitrogênio e PCR URA5 e RFLP com dupla digestão enzimática para caracterização do complexo de espécies/espécie de Cryptococcus. Entre fevereiro de 2018 e março de 2019, foram coletadas 100 amostras de diferentes árvores em locais arborizados públicos. A porcentagem de colonização das árvores por C. gattii VGII foi de 5%, por C. neoformans VNIV de 1% e foram encontrados 6% de C. laurentii e 3% de C. albidus. Estes resultados sugerem a existência de micro-focos e diversidade ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cryptococcosis is a systemic disease caused by the inhalation of dehydrated basidiospores of pathogenic species of Cryptococcus genus present in the environment. The two species complex associated with the disease are the Cryptococcus neoformans and C. gattii species complex. C. neoformans species complex are associated with cases of cryptococcosis in immunosuppressed patients, being commonly isolated from the soil and birds feces. C. gattii species complex affects mainly immunocompetent individuals and are often isolated in native or exotic tree species in some state of biodegradation. This study aimed to perform the environmental mapping of pathogenic species of the genus Cryptococcus in tree samples of public places of the city of Birigui, São Paulo. For collection, the samples were collected from regions with greater traffic in this city. After processing, morphophysiological, biochemical, carbon and nitrogen assimilation and URA5 PCR and RFLP with double enzymatic digestion tests were performed to characterize the Cryptococcus species/species complex. Between february 2018 and march 2019 was collected a total of 100 samples from different trees in public arborized locations. The percentage of tree colonization by the C. gattii VGII was 5%, for C. neoformans VNIV was 1% and was found 6% of C. laurentii and 3% of C. albidus species. These results obtained suggests that there are a micro-foci and diversity of pathogenic Cryptococcus species in some arboreal environment loca... (Complete abstract click electronic access below) / Mestre

Cryptococcus laurentii and Cryptococcus flavescens are nonfermenting yeasts forming extracellular polysaccharide capsule. Both species are mainly saprofytic but Cr. laurentii is also known to be an opportunistic pathogen in immunocompromised patients. Cr. flavescens used to be considered a synonym of Cr. laurentii but nowadays it is classified as a separate species that belongs to the phylogenetic group I of the Cr. laurentii group. In the experimental part 28 strains of species Cr. laurentii, Cr. flavescens a Cr. victoriae were biotyped using MALDI-TOF MS. The yeasts were cultivated on three different media (Sabouraud, YPD and potato agar) and three methods were used for the protein extraction. The impact of growth medium composition from which the strains were inoculated on the quality of spectra was studied together with the suitability of individual methods for use on different media. Then the impact of growth medium composition on the quality of acquired spectra was evaluated. Finally, all strains were compared mutually and with the type strain of Cr. laurentii CCY 17 3-2. The composition of the medium cells were inoculated from was found to have little impact on the spectra quality. The same result was determined for the composition of the actual growth medium cells were cultivated on. Crucial for the quality of mass spectrum is the method of cells preparation. Best results were acquired when cultivating cells on YPD agar, washing the cells with ethanol and using mix of sinapinic and ferulic acid as a matrix. Potato agar was found not suitable for cultivating yeasts of the Cryptococcus genus due to significant production of extracellular polysaccharides which complicate the protein isolation process. All strains were compared to Cr. laurentii type strain CCY 17-3-2 and MSP dendrograms were created based on the spectra similarity. In the MSP dendrograms all strains were successfully divided into relevant species on all tested media. Finally sequences of D1/D2 domain of LSU gene were compared and phylogenetic tree was created. This tree was then compared to the MSP dendrograms.

A radiação solar é um dos principais fatores responsáveis pelo controle das populações de fungos no meio ambiente. A inatividade de estruturas de dispersão e de infecção pela radiação UVA e UVB é ainda mais importante no controle de fungos que se dispersam pelo ar e que podem infectar o hospedeiro, quando inalados, como é o caso de espécies do gênero Cryptococcus. C. neofarmans é capaz de produzir melanina na presença de substratos exógenos, como a L-dopa. A melanização é capaz de proteger o fungo contra diversos fatores ambientais bióticos e abióticos. Neste trabalho, foi avaliado o efeito de exposições a uma intensidade ambiental de radiação UVB (I.000mW m-2) na sobrevivência de células melanizadas e não melanizadas de quatro linhagens de Cryptococcus neoformans e de quatro linhagens de Cryptococcus laurentii. Foi determinada a sobrevivência relativa (sobrevivência das células expostas em relação à das células não expostas à radiação de células com 2, 4, 6 e 8 dias, crescidas em meio com e sem L-dopa, após exposição às doses de 1.8 e 3.6 kJ m-2. Tanto a irradiância como as doses, são observadas no meio ambiente, mesmo em regiões temperadas. Foram observadas diferenças na tolerância à radiação UVB, tnato entre as linhagens de C. neoformans, como entre as linhagens de C. laurentii. As linhagens de C. neoformans, foram observbadas diferenças na tolerância à radiação ao longo do desenvolvimento, tanto nas células melanizadas como nas não melanizadas. Na maioria das situações (linhagem, tempo de crescimento, dose de UVB), não houve diferença significativa entre a tolerância das células melanizadas e não melanizadas e, quando isso ocorreu, a diferença foi menor do que a observada anteriormente com UVC. E, C laurentii, não houve diferença na tolerância à radiação ao longo do desenvolvimento das células melanizadas e não melanizadas. Também não foram observadas diferenças significativas entre a tolerância das células melanizadas e não melanizadas em nenhuma das linhagens. / Solar radiation is one of the major factors responsible for the control of fungus populations in the environment. The inactivation of dispersal and infection structures by UVA and UVB radiation is even more important for the control of fungi that disperse through the air and that can produce melanin in the presence of oxogeneous substrates such as L-dopa. Melanization can protect the fungus against various biotic and abiotic factors. In the present study, we investigated the effect of exposure to an environmental intensity of UVB radiation of 1,000 mWm-2 on the survival of melanized and non-melanized cells of four strains of Cryptococcus neoformans and of four strains of Cryptococcus laurentii. The relative survival (survival of cells exposed to radiation in relation to cells not exposed) of 2, 4, 6 and 8 day cells grown on medium with and without L-dopa was determinated after exposure to doses of 1.8 and 3.6kJ m-2. Both irradiance and these doses are observed in the environment even in temperate regions. Difference in tolerance to UVB radiation were observed both in the C. neoformans and C. laurentii strains. The C. neofarmans strains were more sensitive to radiation than the C. laurentii strains. In C. neoformans, diferences in tolerance to radiation were observed during development both in melanized and non-melanized cells. For most features (strains, time of growth and UVB dose) there was no difference in tolerance between melanized and non-melanized cell and when differences occurred they were smaller than those previously observed with UVC. In C. laurentii, there was no difference in tolerance to radiation during development between melanized and non-melanized cells., Also, no significant differences in tolerance were observed between melanized and non-melanized cells of any strain

A radiação solar é um dos principais fatores responsáveis pelo controle das populações de fungos no meio ambiente. A inatividade de estruturas de dispersão e de infecção pela radiação UVA e UVB é ainda mais importante no controle de fungos que se dispersam pelo ar e que podem infectar o hospedeiro, quando inalados, como é o caso de espécies do gênero Cryptococcus. C. neofarmans é capaz de produzir melanina na presença de substratos exógenos, como a L-dopa. A melanização é capaz de proteger o fungo contra diversos fatores ambientais bióticos e abióticos. Neste trabalho, foi avaliado o efeito de exposições a uma intensidade ambiental de radiação UVB (I.000mW m-2) na sobrevivência de células melanizadas e não melanizadas de quatro linhagens de Cryptococcus neoformans e de quatro linhagens de Cryptococcus laurentii. Foi determinada a sobrevivência relativa (sobrevivência das células expostas em relação à das células não expostas à radiação de células com 2, 4, 6 e 8 dias, crescidas em meio com e sem L-dopa, após exposição às doses de 1.8 e 3.6 kJ m-2. Tanto a irradiância como as doses, são observadas no meio ambiente, mesmo em regiões temperadas. Foram observadas diferenças na tolerância à radiação UVB, tnato entre as linhagens de C. neoformans, como entre as linhagens de C. laurentii. As linhagens de C. neoformans, foram observbadas diferenças na tolerância à radiação ao longo do desenvolvimento, tanto nas células melanizadas como nas não melanizadas. Na maioria das situações (linhagem, tempo de crescimento, dose de UVB), não houve diferença significativa entre a tolerância das células melanizadas e não melanizadas e, quando isso ocorreu, a diferença foi menor do que a observada anteriormente com UVC. E, C laurentii, não houve diferença na tolerância à radiação ao longo do desenvolvimento das células melanizadas e não melanizadas. Também não foram observadas diferenças significativas entre a tolerância das células melanizadas e não melanizadas em nenhuma das linhagens. / Solar radiation is one of the major factors responsible for the control of fungus populations in the environment. The inactivation of dispersal and infection structures by UVA and UVB radiation is even more important for the control of fungi that disperse through the air and that can produce melanin in the presence of oxogeneous substrates such as L-dopa. Melanization can protect the fungus against various biotic and abiotic factors. In the present study, we investigated the effect of exposure to an environmental intensity of UVB radiation of 1,000 mWm-2 on the survival of melanized and non-melanized cells of four strains of Cryptococcus neoformans and of four strains of Cryptococcus laurentii. The relative survival (survival of cells exposed to radiation in relation to cells not exposed) of 2, 4, 6 and 8 day cells grown on medium with and without L-dopa was determinated after exposure to doses of 1.8 and 3.6kJ m-2. Both irradiance and these doses are observed in the environment even in temperate regions. Difference in tolerance to UVB radiation were observed both in the C. neoformans and C. laurentii strains. The C. neofarmans strains were more sensitive to radiation than the C. laurentii strains. In C. neoformans, diferences in tolerance to radiation were observed during development both in melanized and non-melanized cells. For most features (strains, time of growth and UVB dose) there was no difference in tolerance between melanized and non-melanized cell and when differences occurred they were smaller than those previously observed with UVC. In C. laurentii, there was no difference in tolerance to radiation during development between melanized and non-melanized cells., Also, no significant differences in tolerance were observed between melanized and non-melanized cells of any strain