Penicillium marneffei is the most important thermal dimorphic fungus causing systemic mycosis in HIV-infected patients in Southeast Asia. However, laboratory diagnosis based on microscopic morphology and mycelial-to-yeast conversion is time-consuming and expertise-dependent. The performance of the Bruker MALDI-TOF MS system for identification of mold and yeast cultures of 59 P. marneffei strains were evaluated by using the direct transfer method. Using the Bruker databases, BDAL v4.0.0.1 and Filamentous Fungi Library 1.0, the 59 P. marneffei strains grown in mold and yeast phase were identified as P. funiculosum (score <1.7) and P. purpurogenum (<1.7) respectively. When the combined database was expanded with inclusion of spectra from 20 P. marneffei strains grown in mold, yeast or both phases, all the remaining 39 P. marneffei strains grown in both mold and yeast phase were correctly identified to the species level with score >2.0. The spectra of P. marneffei exhibited significant difference to those of the closely related species, P. brevi-compactum, P. chrysogenum, Talaromyces aurantiacus and T. stipitatus (one strain included for each species). P. brevi-compactum was identified to the genus level (as P. brevi-compactum but with score <2.0) and P. chrysogenum was unidentified (as P. chrysogenum but with score <1.7) using the combined database with or without spectra from P. marneffei. Both T. aurantiacus and T. stipitatus were unidentified (as wrong species with score <1.7). MALDI-TOF MS is useful for rapid identification of both yeast and mold cultures of P. marneffei, but this requires expansion of the database using P. marneffei strains. Since the susceptibilities of P. marneffei to the newer antifungal drugs are not well studied, their in vitro antifungal activities against the 59 isolates of P. marneffei were also investigated in accordance with CLSI M27-A3 microdilution method. MICs of itraconazole, voriconazole, posaconazole and anidulafungin for yeast form of P. marneffei were determined. The MICs of itraconazole, voriconazole, posaconazole and anidulafungin were 0.00128-0.00256 μg/ml, 0.01565-0.0625 μg/ml, 0.000978-0.001956 μg/ml, 2-8 μg/ml respectively. The results suggested that the azoles have similarly good activities against P. marneffei, whereas anidulafungin was the least active. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Thesis (doctoral)--K. Bayr. Julius-Maximilians-Universität Würzburg. / Vita. Includes bibliographical references.

Thesis (doctoral)--K. Bayr. Julius-Maximilians-Universität Würzburg. / Vita. Includes bibliographical references.

Diss. Hels.

Penicillium marneffei is a thermal dimorphic fungus that causes systemic mycosis in HIV-positive patients. The fungus displays unique phenotypic properties, including the yellow and black pigments on its conidia as well as the secretion of a diffusible red pigment during growth in mycelial phase. However, all these pigments have not been characterized. Investigation into the pigment production of the fungus can provide insights into the functions of the respective pigment to the fungus as well as their roles in fungal pathogenesis. This study reports the identification and characterization of 23 polyketide synthase (PKS) and 2 polyketide synthase non-ribosomal peptide synthase hybrid (PKS-NRPS) genes in the genome of P. marneffei. Systematic knockdown of the PKS genes showed a loss of black pigment on the conidia of the pks4 (alb1) knockdown mutant, a loss of yellow pigment in the mycelial form of pks11 and pks12 knockdown mutants and a loss of red pigment production in the pks3 knockdown mutant. PKS4 in P. marneffei is responsible for melanin production. Knockdown of pks4 resulted in the loss of melanin production and reduced ornamentation on the conidial surface. Mice that were challenged with the pks4 knockdown mutant survived significantly better than those challenged with wild type conidia (P<0.005). The sterilizing doses of hydrogen peroxide giving a 50% survival reduction of the fungal conidia were 11 minutes and 6 minutes for wild type and the pks4 knockdown mutant, respectively. These together suggested that melanin in P. marneffei contributed to its pathogenesis by reducing its susceptibility to killing by hydrogen peroxide. HPLC-MS analysis revealed the identity of the yellow pigment of P. marneffei to be mitorubrinic acid and mitorubrinol. Mice that were challenged with the pks11and pks12 knockdown mutants survived significantly better than those challenged with wild type conidia (P<0.05). The survival of the pks11and pks12 knockdown mutants in J774 and THP1 macrophages were also both significantly lower than the wild type, suggesting mitorubrinic acid and mitorubrinol contribute to fungal pathogenesis by improving its survival in macrophages. The red pigment secreted by P. marneffei was found to compose of monascorubrin, rubropunctatin, ankaflavin, citrinin and different amino acid conjugated with monascorubrin/rubropunctatin. The biosynthetic pathway of the red pigment involved a polyketide synthase (pks3), a transcription activator (rp1), a fatty acid synthase subunit beta (rp2), a 3-oxoacyl-[acyl-carrier-protein] synthase (rp3) and an oxidoreductase (rp4). RP2, PR3 and RP4 are responsible for fatty acid production. PKS3 is responsible for the biosynthesis of an intermediate polyketide, and RP1 is responsible for the biosynthetic activation. Through esterification, the fatty acid attaches to the intermediate polyketide to form monascorubin, an orange pigment, which is secreted out of the cell. Amino acids in the culture medium were found to conjugate with monascorubrin to form pigments ranging from orange to red in color. Ankaflavin is synthesized by the reduction of monascorubrin. PKS3 and RP1 are also responsible for the biosynthesis of citrinin. In conclusion, the chemical composition, biosynthetic pathways and potential roles in virulence of the black, yellow and red pigments in P. marneffei were characterized. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Penicillium marneffei is the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia. While miRNAs are increasingly recognized for their roles in post-transcriptional regulation of gene expression in animals and plants, the existence of miRNAs in fungi was less well studied and their potential roles in fungal dimorphism were largely unknown. Based on available genome sequence of P. marneffei, it is hypothesized that miRNA-like small RNAs (milRNAs) may be expressed in the dimorphic fungus and dicer- or argonuate-like proteins may be involved in dimorphism or virulence in P. marneffei. I attempted to identify milRNAs in P. marneffei in both mycelial and yeast phase using high-throughput sequencing technology. Small RNAs were more abundantly expressed in mycelial than yeast phase. Sequence analysis revealed 24 potential milRNA candidates, including 17 (2502 reads) candidates in mycelial and seven (232 reads) in yeast phase. Two genes, dcl-1 and dcl-2, encoding putative Dicer-like proteins and the gene, qde-2, encoding Argonaute-like protein, were identified in P. marneffei. Phylogenetic analysis showed that dcl-2 and qde-2 of P. marneffei were more closely related to the homologues in the thermal dimorphic pathogenic fungi, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Coccidioides immitis than to Penicillium chrysogenum and Aspergillus spp., suggesting the co-evolution of dcl-2 and qde-2 among other thermal dimorphic fungi. Moreover, dcl-2 and qde-2 demonstrated higher mRNA expression levels in mycelial than yeast phase by 7 folds and 2 folds respectively (P<0.001). Northern blot analysis confirmed the expression of two milRNAs, PM-milR-M1 and PM-milR-M2, only in mycelial phase. Using dcl-1KO, dcl-2KO, dclDKO and qde-2KO deletion mutants, it was shown that the biogenesis of both milRNAs were dependent on dcl-2 but not dcl-1 or qde-2. While deletion of qde-2, but not the two dcl genes, was found to decrease the virulence level of P. marneffei in mice model, the deaths of the qde-2KO conidia challenged mice were delayed for over 10 days. The qde-2KO conidia have lower recovery rate both in human THP1 and murine J774 macrophage cell lines and also reduced resistance to hydrogen peroxide than the wild type. This study provided the first evidence for differential expression of milRNAs in different growth phases of thermal dimorphic fungi and shed light on the evolution of fungal proteins involved in milRNA biogenesis and possible role of post-transcriptional control in governing thermal dimorphism. This is also the first study to reveal the relationship between argonuate-like QDE-2 protein and virulence in P. marneffei in mice model. This study provides a foundation for the milRNAs study in pathogenic thermal dimorphic fungi. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Abstract A newapproach to the control of postharvest pathogens, while maintaining fruit quality, has been implemented by the application of essential oil amended coatings to citrus. This approach eliminates the need for synthetic fungicides, thereby complying with consumer preferences, organic requirements and reducing environmental pollution. In vitro studies indicated that the essential oils and some of the terpenoid components tested were active against Penicillium digitatum. In a series of subsequent semi-commercial and commercial trials, Mentha spicata and Lippia scaberrima essential oils, as well as pure (d)-limonene and R-(−)-carvone were incorporated into a variety of commercial citrus coatings. These amended coatingswere applied postharvest to ‘Tomango’ oranges in the absence of the standard fungicide dip. Excellent disease control was achieved with the amended coatings, while measured quality parameters indicated that overall fruit quality was maintained. Moreover, moisture loss was decreased significantly in fruit treated with essential oil enriched coatings. The efficacy of amended coatings as a viable alternative or supplement to existing fruit protection strategies was demonstrated in a commercial trial.

a b s t r a c t A newapproach to the control of postharvest pathogens, while maintaining fruit quality, has been implemented by the application of essential oil amended coatings to citrus. This approach eliminates the need for synthetic fungicides, thereby complying with consumer preferences, organic requirements and reducing environmental pollution. In vitro studies indicated that the essential oils and some of the terpenoid components tested were active against Penicillium digitatum. In a series of subsequent semi-commercial and commercial trials, Mentha spicata and Lippia scaberrima essential oils, as well as pure (d)-limonene and R-(−)-carvone were incorporated into a variety of commercial citrus coatings. These amended coatingswere applied postharvest to ‘Tomango’ oranges in the absence of the standard fungicide dip. Excellent disease control was achieved with the amended coatings, while measured quality parameters indicated that overall fruit quality was maintained. Moreover, moisture loss was decreased significantly in fruit treated with essential oil enriched coatings. The efficacy of amended coatings as a viable alternative or supplement to existing fruit protection strategies was demonstrated in a commercial trial.

Thesis (M.S.)--University of Wisconsin--Madison, 1964. / Title from title screen (viewed Nov. 15, 2007). Includes bibliographical references (leaves 43-44). Online version of the print original.

Thesis (Ph. D.)--Cornell University, 1915. / "Reprinted from the American journal of botany, 5 ... May, 1916." Bibliography: p. 259-260.