Academic literature on the topic 'Yeast fungi'

Candida albicans, Candida auris, Candida glabrata, and Cryptococcus neoformans are pathogenic yeasts which can cause systemic infections in immune-compromised as well as immune-competent individuals. These yeasts undergo replicative aging analogous to a process first described in the nonpathogenic yeast Saccharomyces cerevisiae. The hallmark of replicative aging is the asymmetric cell division of mother yeast cells that leads to the production of a phenotypically distinct daughter cell. Several techniques to study aging that have been pioneered in S. cerevisiae have been adapted to study aging in other pathogenic yeasts. The studies indicate that aging is relevant for virulence in pathogenic fungi. As the mother yeast cell progressively ages, every ensuing asymmetric cell division leads to striking phenotypic changes, which results in increased antifungal and antiphagocytic resistance. This review summarizes the various techniques that are used to study replicative aging in pathogenic fungi along with their limitations. Additionally, the review summarizes some key phenotypic variations that have been identified and are associated with changes in virulence or resistance and thus promote persistence of older cells.

Organic material decomposition is the reorganizing process of the organic material by microbes in the controlled circumstances. The microbes which were used in general such as fungi, bacteria or yeast. Aerobic yeasts is one of the microbes needs oxygen to work. Yeast was obtained by the exploration of the rice straw, maize straw and cane straw in Dau, Malang, East of Java. By the result of the microscopic exploration and observation through the microscope, it was obtained 9 yeast isolates and 3 fungi isolates. 2 yeast isolates and 1 fungi isolate from the rice straw, 2 yeast isolates and 1 fungi isolate from the maize straw, and 5 yeast isolates and 1 fungi isolate from the cane straw. The identification results were obtained Candida parapsilosis, Bellera oryzae, Kluyveromyces thermotolerant, Candida tropicalis, Debaryomyces hansenii, Wickerhamomyces anomalus, Pichia membranfaciens, Cryptococcus wieringae. The highest potential yeast as an organic fertilizer decomposer is Cryptococcus wieringae and the lowest is Bullera oryzae. Keywords : Candida, Pichia, Cryptococcus, Bullera, Kluyveromyces, DebaryomycesWickerhamomyces, Fusarium and Trichoderma

Yeasts and filamentous fungi associated with smooth (non-lenticel) and lenticel bark of young and scaffold branches of peach (Prunus persica (L.) Batsch) were monitored using bark washing and direct or impression plating techniques and scanning electron microscopy during potential Cystospora canker infection periods. Total populations of fungi were high in the fall but dropped in the winter and increased during the spring. Yeasts and yeast-like fungi predominated in the spring and fall samples. The principal yeasts were Basidiomycetes in the form genera Cryptococcus, Rhodotorula, and Sporobolomyces. The yeast-like fungi were Aureobasidium and Taphrina. The principal filamentous fungi were in the form genera Alternaria, Epicoccum, Cladosporium, Coniothyrium, and Libertella. The canker pathogens, Leucostoma persoonii (Nits.) Höhn. and Leucostoma cincta (Pers. & Fr.) Höhn., were observed mainly in the spring sampling. Lenticels supported greater fungal populations than smooth (non-lenticel) bark surfaces. The impact of fungal epiphytes, particularly the yeasts, on the potential biological control of peach canker is discussed.Key words: Cytospora canker, mycoflora, yeast, bark, biological control, fungal epiphyte.

Summary This paper reports on the use of yeast as biological control agents against mould and blue-stain fungi in laboratory-based Pinus sylvestris sapwood wood block incubation tests. All 5 yeast isolates tested significantly reduced discolouration of wood surfaces by moulds and most were also effective against staining fungi. The 2 most promising yeast isolates also significantly reduced radial growth of selected mould and stain fungi in vitro solely through the liberation of volatile compounds. Five bacterial isolates were also tested for antagonism toward mould and stain fungi and were effective biological control agents in wood block tests. Again, volatile production alone by the bacteria was shown to be sufficient to significantly reduce fungal growth in vitro. Most of the bacteria and yeasts significantly reduced wood discolouration by fungi when inoculated onto block surfaces 7 days before, or at the same time as the target fungi, and in some cases even when inoculated 7 days after the fungi. The possibility of using yeasts as biological control agents to prevent wood spoilage during kiln drying and short-term storage at sawmills is considered.

On water agar, hyphae of certain fungi are able to attack microcolonies and even solitary cells of yeasts belonging to the genera Candida, Cryptococcus, Pichia, Rhodotorula, and Sporidiobolus. The ability to attack yeasts is particularly common among the Basidiomycota and 100 of 195 isolates tested positive. In the Ascomycota and Deuteromycota, only 12 of 128 isolates tested positive. Two species of the Oomycota and 10 species of the Zygomycota tested negative. Two types of initial responses were recorded: (i) a chemotropic response in which directional hyphae are attracted to microcolonies or solitary cells, and (ii) a contact response in which hyphae reach yeast colonies by chance and subsequently attack them. In both cases the invading fungus produces coralloid masses of branched assimilative hyphae within the yeast colonies. Yeast cells are utilized as a nutrient source by the invading fungus in either of two ways: (i) the walls of yeast cells are lysed releasing the contents that are absorbed by the coralloid hyphae or (ii) branched hyphae from the invading fungus attach to the yeast cells, penetrate the walls, and absorb the contents. Both methods involve necrotrophic mycoparasitism. Keywords: yeasts, Basidiomycota, necrotrophic mycoparasitism, wood decay, nitrogen requirements.

An evaluation of the mycological quality of the sand beaches of the Lisboa and Vale do Tejo coastal area in Portugal was undertaken in May-October 1994. The keratinolytic fungi, yeasts, potential pathogenic and allergic and/or environmental saprophytic fungi were analysed. The results for the yeast Candida were evaluated according to the “Proposed Guidelines for the Microbiological Quality of Sand”. The data showed good/satisfactory quality of the sand beaches for the genus Candida. The results indicate that the allergic and/or environmental saprophytic fungi were the most common in sand beaches. The fungi Scopulariopsis and the yeast Candida could be used as specific indicator organisms of sand beaches quality. A new quality objective is introduced that will contribute to improve the sand beaches quality.

Antarctica is known as one of the harshest environments on Earth, with a frigid and dry climate. Soil yeasts living in such extreme environments can grow by decomposing organic compounds at sub-zero temperatures. Thus far, a list of lichen and non-lichen fungi isolated from the area near Syowa Station, the base of the Japanese Antarctic research expedition, has been compiled and a total of 76 species of fungi have been reported. Yeast, especially basidiomycete yeast, is the dominant fungus in Antarctica. This mini-review summarizes a survey of the yeast diversity in the soil of Eastern Ongul Island and the ability of these yeasts to secrete extracellular enzymes. We also describe the yeast diversity in the soil of the Skarvesnes ice-free region and how these yeasts have adapted to the sub-zero environment. Further, we describe the secondary metabolites of these yeasts, whose production is induced by cold stress.

Background. At the height of the novel coronavirus infection, the risk of fungal coinfection increases due to a decrease in the immune status of patients and massive antibacterial therapy, which leads to a complication in the course of the underlying disease and increases the risk of an unfavorable outcome. Therefore, it is necessary not only to identify all associates that cause the infectious process, but also to determine their sensitivity to antibacterial and antifungal drugs in order to select an adequate treatment regimen for a patient.The aim of the study was to investigate the species composition of fungal associates in corona positive and corona negative patients with community-acquired pneumonia (CAP), as well as to determine the spectrum of sensitivity/resistance of yeasts and yeast-like fungi to antimycotic drugs.Material and methods. The species composition of the fungal microflora in sputum samples from 723 patients with CAP, sent from a health care facility in Rostov-on-Don, was investigated. The isolated cultures were identified using bacteriological and mass spectrometric methods. The sensitivity of yeasts to antimycotic drugs was determined by disk-diffusion method.Results. It has been established that yeast and yeast-like fungi were present in sputum samples in 31–32% of patients with CAP in etiologically significant quantities. 10 different types of yeast and yeast-like fungi were identified. Candida albicans was the predominant species in terms of the number of isolated cultures (83%). A re-examination of corona positive patients after their hospital stay revealed a tendency towards an increase in the number and intensity of manifestation of candidal lesions. Moreover, a change in Candida species has been registered, which entails a change in their sensitivity to antifungal drugs. A high percentage of strains resistant to fluconazole was identified after determining the sensitivity of fungi to antimycotics.Conclusion. The data obtained demonstrate that yeast and yeast-like fungi are the dominant associates in complicated forms of CAP. In this regard, it seems appropriate to provide individual microbiological support to patients during their treatment with the identification of all pathogens.

Fungal mycotoxins are secondary metabolites that can be present in green forage, hay, or silage. Consumption of contaminated plants or agricultural products can cause various animal and human diseases, which is why problems associated with mycotoxins have received particular attention. In addition, public pressure to produce healthy food and feed is also increasing. As the results of several surveys indicate that yeasts can decrease toxic effects by binding or converting secondary metabolites or control growth of harmful fungi, this article provides an overview of the yeast species that can have great potential in detoxification. The most important antagonistic yeast species against toxigenic fungi are described and the mode of their inhibitory mechanisms is also discussed. We provide an insight into toxin binding and biotransformation capacities of yeasts and examples of their use in silo. Issues requiring further study are also mentioned.

Dissertation (PhD)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: In the yeast Saccharomyces cerevtstee, two biochemical pathways ensure that activated cytoplasmic or peroxisomal acetyl-groups are made available for mitochondrial energy production when the cells utilise non-fermentable carbon sources. The first pathway is the glyoxylate cycle, where two activated acetyl-groups are incorporated into each cycle, which releases a C4 intermediate. This intermediate is then transported to the mitochondria where it can enter the tricarboxylic acid cycle. The second pathway is the carnitine shuttle. Activated acetyl-groups react with carnitine to form acetylcarnitine, which is then transported to the mitochondria where the acetyl group is transferred. In this study it was shown that the deletion of the glyoxylate cycle specific citrate synthase, encoded by CIT2, results in a strain that is dependent on carnitine for growth on non-fermentable carbon sources. Using a /::"cit2 strain, mutants affected in carnitine-dependent metabolic activities were generated. Complementation of the mutants with a genomic library resulted in the identification of four genes involved in the carnitine shuttle. These include: (i) the mitochondrial and peroxisomal carnitine acetyltransferase, encoded by CAT2; (ii) the outer-mitochondrial carnitine acetyltransferase, encoded by YA T1; (iii) the mitochondrial carnitine translocase, encoded by CRC1; and (iv) a newly identified carnitine acetyltransferase, encoded by YAT2. All three carnitine acetyltransferases are essential in a carnitine-dependent strain. The dependence on exogenous carnitine of the /::"cit2 strain when grown on nonfermentable carbon sources suggested that S. cerevisiae does not biosynthesise carnitine. Measurements using electrospray mass spectrometry confirmed this hypothesis. As a result an investigation was initiated into carnitine biosynthesis in order to genetically engineer a S. cerevisiae strain that could endogenously biosynthesise carnitine. The filamentous fungus, Neurospora crassa, was one of the first organisms used in the seventies to identify the precursor and intermediates of carnitine biosynthesis. However, it was only about twenty years later that the first genes encoding these enzymes where characterised. Carnitine biosynthesis is a four-step process, which starts with trimethyllysine as precursor. Trimethyllysine is converted to hydroxytrimethyllysine by the enzyme trimethyllysine hydroxylase (TMLH). Hydroxytrimethyllysine is cleaved to trimethylamino-butyraldehyde by the hydroxytrimethyllysine aldolase (HTMLA) releasing glycine. Trimethylaminobutyraldehyde is dehydrogenated to trimethylamino-butyrate (y-butyrobetaine) by trimethylamino-butyraldehyde dehydrogenase (TMABA-DH). In the last step, ybutyrobetaine is converted to t-carnltine by y-butyrobetaine hydroxylase (BBH). The N. crassa TMLH homologue was identified in the genome database based on the protein sequence homology of the human TMLH. Due to the high amount of introns predicted for this gene, the cDNA was cloned and subjected to sequencing, which then revealed that the gene indeed had seven introns. Functional expression of the gene in S. cerevisiae and subsequent enzymatic analysis revealed that the gene coded for a TMLH. It was therefore named cbs-1 for "carnitine biosynthesis gene no. 1JJ. Most of the kinetic parameters were similar to that of the human TMLH enzyme. Following this, a genomic copy of the N. crassa BBH homologue was cloned and functionally expressed in S. cerevisiae. Biochemical analysis revealed that the BBH enzyme could biosynthesise L-carnitine from y-butyrobetaine and the gene was named cbs-2. In addition, the gene could rescue the growth defect of the carnitinedependent Scii? strain on non-fermentable carbon sources when y-butyrobetaine was present. This is the first report of an endogenously carnitine biosynthesising strain of S. cerevisiae. The cloning of the remaining two biosynthesis genes presents particular challenges. To date, the HTMLA has not been characterised on the molecular level making the homology-based identification of this protein in N. crassa impossible. Although the TMABA-DH has been characterised molecularly, the protein sequence is conserved for its function as a dehydrogenase and not conserved for its function in carnitine biosynthesis, as in the case of TMLH and BBH. The reason for this is probably due to the fact that the enzyme is involved in other metabolic processes. The use of N. crassa carnitine biosynthesis mutants would probably be one way in which to overcome these obstacles. The !1cit2 mutant proved useful in studying carnitine related metabolism. We therefore searched for suppressors of !1cit2, which resulted in the cloning of RAS2. In S. cerevisiae, two genes encode Ras proteins, RAS1 and RAS2. GTP-bound Ras proteins activate adenylate cyclase, Cyr1 p, which results in elevated cAMP levels. The cAMP molecules bind to the regulatory subunit of the cAMP-dependent kinase (PKA), Bcy1 p, thereby releasing the catalytic subunits Tpk1 p, Tpk2p and Tpk3p. The catalytic subunits phosphorylate a variety of regulators and enzymes involved in metabolism. Overexpression of RAS2 could suppress the growth defect of the Sclt? mutant on glycerol. In general, overexpression of RAS2 enhanced the proliferation of wild-type cells grown on glycerol. However, the enhancement of proliferation was much better for the !1cit2 strain grown on glycerol. In this respect, the retrograde response may play a role. Overexpression of RAS2 resulted in elevated levels of intracellular citrate and citrate synthase activity. It therefore appears that the suppression of !1cit2 by RAS2 overexpression is a result of the general upregulation of the respiratory capacity and possible leakage of citrate and/or citrate synthase from the mitochondria. The phenotype of RAS2 overexpression contrasts with the hyperactive RAS2val19 allele, which causes a growth defect on glycerol. However, both RAS2 overexpression and RAS2val19activate the cAMP/PKA pathway, but the RAS2val19dependent activation is more severe. Finally, this study implicated the Ras/cAMP/PKA pathway in the proliferation effect on glycerol by showing that in a Mpk1 strain, the growth effect is blocked. However, the enhanced proliferation was still observed in the Mpk2 and Mpk3 strains when RAS2 was overexpressed. Therefore, it seems that Tpk1 p plays an important role in growth on non-fermentable carbon sources, a notion that is supported by the literature.
AFRIKAANSE OPSOMMING: In die gis Saccharomyces cerevtstee, is daar twee metaboliese weë waarmee geaktiveerde asetielgroepe na die mitochondrium vervoer kan word wanneer die sel op nie-fermenteerbare koolstofbronne groei. Die een weg is die glioksilaatsiklus, waar die geaktiveerde asetielgroepe geïnkorporeer word in die siklus en dan vrygestel word as Ca-intermediêre. Hierdie intermediêre word dan na die mitochondrium vervoer waar dit in die trikarboksielsuursiklus geïnkorporeer word. Die ander weg is die karnitiensiklus, waar geaktiveerde asetielgroepe met karnitien reageer om asetielkarnitien te vorm wat dan na die mitochondrium vervoer word waar dit die asetielgroep weer vrygestel. Hierdie studie het getoon dat die delesie van die glioksilaatsiklus spesifieke sitraatsintetase, gekodeer deur CIT2, die gisras afhanklik maak van karnitien vir groei op nie-fermenteerbare koolstofbronne. Deur gebruik te maak van 'n ócit2 gisras, kon mutante, wat geaffekteer is in karnitien-verwante metaboliese aktiwiteite, gegenereer word. Komplementering van die mutante met 'n genomiese biblioteek het gelei tot die identifisering van vier gene betrokke by die karnitiensiklus. Hierdie gene sluit in: (i) die mitochondriale en die peroksisomale karnitienasetieltransferase, gekodeer deur CAT2; (ii) die buite-mitochondriale karnitienasetieltransferase, gekodeer deur YAT1; (iii) die mitochondriale karnitientranslokase, gekodeer deur CRC1; en (iv) 'n nuutgeïdentifiseerde karnitienasetieltransferase, gekodeer deur YAT2. Daar benewens, is ook gewys dat al drie karnitienasetieltransferases noodsaaklik is in 'n karriltienafhanklike gisras. Die afhanklikheid van eksogene karnitien van die ócit2 gisras, wanneer dit gegroei word op nie-fermenteerbare koolstofbronne, was aanduidend dat S. cerevisiae nie karnitien kan biosintetiseer nie. Metings deur middel van elektronsproeimassaspektrometrie het hierdie veronderstelling bevestig. Gevolglik is 'n ondersoek deur ons geïnisieer in die veld van karnitienbiosintese om 'n S. cerevisiae gisras geneties te manipuleer om karnitien sodoende endogenies te biosintetiseer. Die filamentagtige fungus, Neurospora crassa, was een van die eerste organismes wat in die sewentiger jare gebruik is om die voorloper en intermediêre van karnitienbiosintese te identifiseer. Dit was egter eers sowat twintig jaar later dat die eerste gene wat vir hierdie ensieme kodeer, gekarakteriseer is. Karnitienbiosintese is 'n vierstap-proses wat met trirnetlellisten as voorloper begin. Trimetiellisien word omgeskakel na hidroksi-trimetiellisien deur die ensiem trimetiellisienhidroksilase (TMLH). Hidroksietrimetlelllsien word dan gesplits om trimetielaminobuteraldehied te vorm deur die werking van die hidroksitrimetiellisienaldolase (HTMLA) met die gevolglike vrystelling van glisien. Trimetielaminobuteraldehied word dan na trimetielaminobuteraat (y-butirobeteïen) deur trimetielaminobuteraldehied dehidrogenase (TMABA-DH) gedehidrogeneer. In die laaste stap word y-butirobeteïen deur middel van die y-butirobeteïen hidroksilase (BBH) na L-karnitien omgeskakel. Op grond van die proteïenvolgordehomologie in die genoomdatabasis tussen die menslike TMLH en N. crassa se TMLH is laasgenoemde geïdentifiseer. As gevolg van die groot getal introns wat vir hierdie geen voorspel is, is die cDNA-weergawe daarvan gekloneer en aan volgordebepaling onderwerp. Dit het getoon dat die geen inderdaad sewe introns bevat. Funksionele uitdrukking van die geen in S. cerevisiae en ensiematiese analise het getoon dat die geen vir 'n TMLH kodeer en is gevolglik cbs-1 genoem; dit staan vir "karnitien biosintese geen no. 1tt. Meeste van die kinetiese parameters was ook soortgelyk aan die van die menslike TMLH-ensiem. Hierna is 'n genomiese kopie van N. crassa se BBH-homoloog gekloneer en funksioneel in S. cerevisiae uitgedruk. Biochemiese analise het getoon dat die uitgedrukte BBH-ensiem L-karnitien vanaf y-butirobeteïen kan biosintetiseer en die geen is cbs-2 genoem. Daar benewens kon die geen die groeidefek van die karnitien-afhanklike tlcit2-gisras ophef wanneer dit op nie-fermenteerbare koolstofbronne in die teenwoordigheid van y-butirobeteïen aangekweek is. Hierdie is die eerste verslag oor 'n endogeniese karnitien-biosintetiserende ras van S. cerevisiae. Die klonering van die oorblywende twee karnitienbiosintetiserende gene het sekere uitdagings. Tot op datum, is die HTMLA nog nie tot op genetiese vlak gekarakteriseer nie, wat dan die homologie-gebaseerde identifikasie van hierdie proteïen in N. crassa onmoontlik maak. Alhoewel die TMABA-DH geneties gekarakteriseer is, is die proteïenvolgorde ten opsigte van sy funksie as 'n dehidrogenase gekonserveer, maar nie vir sy funksie in karnitienbiosintese soos in die geval van TMLH en BBH nie. Die rede hiervoor is moontlik omdat die ensiem ook in ander metaboliese prosesse betrokke is. Die gebruik van N. crassa karnitienmutante sal moontlik een manier wees om hierdie probleme te oorkom. Die tlcit2-mutant het handig te pas gekom vir die bestudering van karnitienverwante metabolisme. Dus is daar vir onderdrukkers van die tlcit2-mutant gesoek wat gelei het tot die klonering van die RAS2-geen. In S. cere visiae , kodeer twee gene vir Ras-proteïene, RAS1 en RAS2. GTP-gebonde Ras-proteïene aktiveer adenilaatsiklase, Cyr1 p, wat verhoogde intrasellulêre cAMP-vlakke tot gevolg het. Die cAMP bind aan die regulatoriese subeenheid van die cAMP-proteïenkinase (PKA), Bcy1 p, en daardeur word die katalitiese subeenhede, Tpk1 p, Tpk2p en Tpk3p, vrygestel. Die katalitiese subeenheid fosforileer 'n verskeidenheid van reguleerders en ensieme betrokke by metabolisme. Ooruitdrukking van RAS2 het die groeidefek van die tlcit2-mutant op gliserolonderdruk. Oor die algemeen, verbeter die ooruitdrukking van RAS2 die proliferasie van die wildetipe op gliserol bevattende media. Alhoewel, die verbetering van proliferasie was baie meer opmerklik in die tlcit2-gisras. In hierdie verband, speel die gedegenereerde response dalk 'n rol. Ooruitdrukking van RAS2 het verhoogde intrasellulêre vlakke van sitraat- en sitraatsintetase-aktiwiteit tot gevolg gehad. Dit wou dus voorkom asof die onderdrukking van die ócit2-groeidefek deur RAS2 se ooruitdrukking die gevolg was van algemene opreguiering van respiratoriese kapasiteit en die lekkasie van sitraat en/of sitraatsintetase uit die mitochondria. Die fenotipe van RAS2 ooruitdrukking kontrasteer die hiperaktiewe RAS2va / 19 alleel, wat 'n groeidefek op gliserol media veroorsaak. Alhoewel beide RAS2-00ruitdrukking en RAS2va / 19 die cAMP/PKA-weg aktiveer, is gevind dat die RAS2va/19-afhanklike aktivering strenger is. Ten slotte, die cAMP/PKA-weg is in die proliferasie effek op gliserol media geïmpliseer deur te wys dat in 'n Mpk1-gisras, die groeieffek geblokkeer is. Alhoewel, die verbeterde proliferasie is steeds waargeneem in die Mpk2-en Mpk3-gisrasse toe die RAS2-geen ooruitgedruk is. Dus, dit wil voorkom asof Tpk1 p 'n belangrike rol in die groei van gisselle op nie-fermenteerbare koolstofbronne speel; 'n veronderstelling wat deur die literatuur ondersteun word.

The aim of the project was to determine whether a by-product of industrial fermentations, Saccharomyces cerevisiae, could be utilized to bioaccumulate heavy metal cations and to partially define the mechanism of accumulation. S. cerevisiae cells were found to be capable of accumulating Cu²⁺in a manner that was proportional to the external Cu²⁺ concentration and inversely proportional to the concentration of biomass. The accumulation process was only minimally affected by temperature variations between 5 and 40°C or high ambient concentrations of sodium chloride. The accumulation process was however considerably affected by variations in pH, bioaccumulation being most efficient at pH 5 - 9 but becoming rapidly less so at either extreme of pH. Selection for copper resistant or tolerant yeast diminished the yeast's capacity for Cu²⁺ accumulation. For this and other reasons the development of heavy metal tolerance in yeasts was deemed to be generally counterproductive to heavy metal bioaccumulation. The yeast biomass was also capable of accumulating other heavy metal cations such as c0²⁺ or Cd²⁺. The yeast biomass could be harvested after bioaccumulation by tangential filtration methods, or alternatively could be packed into hollow fibre microfilter membrane cartridges and used as a fixed-bed bioaccumulator. By immobilizing the yeast in polyacrylamide gel and packing this material into columns, cu²⁺, C0²⁺ or Cd²⁺ could be removed from influent aqueous solutions yielding effluents with no detectable heavy metal, until breakthrough point was reached. This capacity was hypothesized to be a function of numerous "theoretical plates of equilibrium" within the column. The immobilized biomass could be eluted with EDTA and recycled for further bioaccumulation processes with minor loss of bioaccumulation capacity. Yeast cells were fractionated to permit identification of the major cell fractions and molecular components responsible for metal binding. Isolation of the yeast cell walls permitted investigation of their role in heavy metal accumulation. Although the amino groups of chitosan and proteins, the carboxyl groups of proteins, and the phosphate groups of phosphomannans were found to be efficient groups for the accumulation of copper, the less effective hydroxyl groups of the carbohydrate polymers (glucans and mannans) had a similar overall capacity for copper accumulation owing to their predominance in the yeast cell wall. The outer (protein-mannan) layer of the yeast cell wall was found to be a better Cu²⁺ chelator than the inner (chitinglucan) layer. It appeared that the physical condition of the cell wall may be more important than the individual macromolecular components of the cell wall in metal accumulation. It was apparent that the cell wall was the major, if not the sole contributor to heavy metal accumulation at low ambient heavy metal concentrations. At higher ambient metal concentrations the cytosol and vacuole become involved in bioaccumulation. Copper and other metals caused rapid loss of 70% of the intracellular potassium, implying permeation of the plasma membrane. This was followed by a slower "leakage" of magnesium from the vacuole which paralleled Cu²⁺ accumulation, suggesting that it may represent some form of ion-exchange. An intracellular copper chelating agent of approximately 2 kDalton molecular mass was isolated from copper tolerant yeast. This chelator was not a metallothionein and bound relatively low molar equivalents of copper compared to those reported for metallothionein. Treatment of the biomass with hot alkali yielded two biosorbents, one soluble (which could be used as a heavy metal flocculent), and an insoluble biosorbent which could be formed into a granular product to be used in fixed-bed biosorption columns. The granular biosorbent could accumulate a wide range of heavy metal cations in a semispecific manner and could be stored in a dehydrated form indefinitely, and rehydrated when required. Bioaccumulation by live algae was investigated as an alternative to yeast based processes. Various strains of algae, of which Scenedesmus and Selenastrum were the most effective, were found to be capable of accumulating heavy metals such as Cu²⁺, Pb²⁺ and Cr³⁺.

This thesis presents a systematic investigation ofyeasts associated with wine grapes cultivated in several Australian vineyards during the 2001-2003 vintages. Using a combination of cultural and molecular methods, yeast populations of red (Cabernet sauvignon, Merlot, Tyrian) and white (Sauvignon blanc, Semilion) grape varieties were examined throughout grape cultivation. The yeast-like fungus, Aureobasidium pullulans, was the most prevalent species found on grapes. Various species of Cryptococcus, Rhodotorula and Sporobolomyces were frequently isolated throughout grape maturation. Ripe grapes showed an increased incidence of Hanseniaspora and Metschnikowia species for the 2001-2002 seasons, but not for the drought affected, 2002-2003 seasons. Atypical, hot and dry conditions may account for this difference in yeast flora and have limited comparisons of data to determine the influences of vineyard location, grape variety and pesticide applications on the yeast ecology. More systematic and controlled studies of these variables are required. Damaged grape berries harboured higher yeast populations and species diversity than intact healthy berries. PCR-DGGE analysis was less sensitive than plate culture for describing the diversity of yeast species on grapes; it detected prevalent species, but subdominant populations below 103 CFU/g were not detected. In some cases, PCR-DGGE revealed the presence ofyeasts (Candida galli, C. zemplinina) not isolated by culture. Fermentative wine species (Kluyveromyces, Torulaspora, Saccharomyces) were rarely isolated, and only detected by enrichment cultures. Significant morphological and genetic variability were detected among A. pullulans and other black yeasts isolates from grapes. Taxonomic characterization of 61 strains by ITS-RFLP and rDNA sequencing revealed that they belonged to several distinct species within the generic groupings ofAureobasidium, Hormonema and Kabatiella. Isolates were strong producers of extracellular enzymes and polysaccharides that could have oenological significance, and, using a plate assay, some were antagonistic towards Bacillus thuringiensis, several wine yeasts, and some spoilage and mycotoxigenic fungi found on grapes. Growth of Saccharomyces cerevisiae was not inhibited by these organisms in grape juice. A species-specific probe was developed for the identification of the wine spoilage yeast, Zygosaccharomyces bailii in a microtitre plate hybridization assay. The probe detected 102 cells/ml in wine, reliably differentiating Z. bailii from other Zygosaccharomyces and other wine-related yeasts.

Studies on the capacity of growing and non-growing cells to be induced to grow in different morphological forms suggested that metabolically active cells are better able to undergo dimorphism than metabolically quiescent cells. Accelerated growth kinetics requiring de novo RNA and or protein biosynthesis but not DNA synthesis were observed in metabolically active cells undergoing a yeast to hyphal transition. The roles that divalent cation calcium plays in eukaryotic growth and morphogenesis are illustrated in this study with regard to C. albicans. Free calcium was required for germ tube emergence from stationary phase yeast cells but not for subsequent growth or hyphal extension. The galvanotropic response of C. albicans hyphae was investigated and was shown to be quantitatively dependent on the concentration of available calcium in the medium and also on the integrity of calcium dependent second messenger systems. The mechanism of galvanotropism of C. albicans may be due to the electrophoretic movement of charged proteins, possibly calcium transport channels, in the plasma membrane as shown by the dependence of cathodotropic growth on external pH. Although the yeast to hyphal transition can be easily controlled and studied in vitro, attempts to use such methods to control morphological development, with a view to comparing the relative infectivity of each morphology, are complicated in the environment in vivo. Yeast cells were introduced into the rat vagina, or systemically into mice, along with chemical effectors which had been shown to prevent the yeast to mycelial transition in vitro without affecting the growth rate of the cells per se. It was concluded tentatively from this study, that, in the rat vaginal model of candidosis, the hyphal form of growth is better adapted and more pathogenic than the yeast form.

Yeast are exposed to many physical and chemical stresses when used in large-scale industrial fermentations, particularly the initial stages in which yeast are shifted from anaerobic storage to aerated wort. This work investigated the transcriptional and physiological responses of yeast that had been shifted from anaerobic to aerobic growth conditions. Microarray technology was employed to determine the transcriptional changes that occurred in the first hour of a pilot-plant fermentation compared to the 23rd hour. It was found that over 100 genes were up-regulated initially including genes involved in the synthesis of the essential membrane sterol ergosterol and genes for the protection of cells against oxidative stress. It was also determined that cells which accumulate ergosterol precursors in the absence of ergosterol were more sensitive to exogenous oxidative stresses, indicating a role for ergosterol in oxidative stress tolerance. Aeration of anaerobically grown cells did not affect their growth kinetics or viability. However, anaerobically grown cells were hypersensitive to exogenous oxidative stress compared to their aerobic counterparts. Anaerobic cells that underwent a short period of aeration prior to treatment with hydrogen peroxide generated a tolerance to the oxidant, indicating that the period of aeration produced an adaptive-like response. Microarray analysis of the cells during the period of aeration showed that representative genes from the oxidative stress response family were up-regulated rapidly and it was determined that the response was controlled by the Yap1p and Skn7p transcription factors. Deletion of the transcription factor genes indicated that they were responsible for the creation of tolerance to oxidant. Target gene products of the two transcription factors (Gpx2p, Gsh1p and Trx2p) were shown to be induced during the shift to aeration; however, the glutathione redox balance did not seem to be affected as the cells were shifted from highly reduced to oxidising environments. Unexpectedly, it was discovered that genes involved in the synthesis of amino acids were up-regulated during anaerobic growth and stringently downregulated upon aeration of cells. The transcriptional activator of those genes (Gcn4p) was essential for growth in anaerobic media which included amino acid supplementation.

Mycotoxins, the secondary fungal metabolites are important contaminants of food and feed. Among the other contaminants, aflatoxin B1 (AFB1) and OTA are frequently detected in the animal feed product. In the present study, the mixed dairy cow feed products were collected from the supermarkets in Qatar and analyzed for the presence of AFB1 and OTA. Yeast strains were isolated and tested for their biological control activities against aflatoxigenic and ochratoxin fungi. We demonstrated that local 15 yeasts isolates have important antifungal potential activities through the synthesis of volatile organic compounds (VOC) that are able to act against the mycotoxigenic fungi and their synthesis of the mycotoxins. Two Yeast strains (4&2) isolated from fermented food, have shown a great antifungal inhibition growth in-vitro as well as spores inhibition and mycotoxins synthesis.

The aim of this study was to estimate maize silage hygienic sanitary parameters, contamination with mycotoxins in dairy farms in Lithuania. In 2011–2012 maize silage samples were collected from 20 dairy farms: prior to ensiling and 3 and 8 months after ensiling. In maize samples, prior to ensiling, L. monocytogenes was detected 25.0 % and after 3 months of ensiling – 10 %. Average of yeast and filamentous fungi in raw material samples were respectively – 71.39 ± 32.17 and 47.67 ± 18.31 CFU/g, after 3 months – 50.06 ± 16.09 CFU/g and 28.27 ± 13.18, after 8 months – 213.81 ± 55.98 and 215.08 ± 56.16 CFU/g. In raw maize dominated Fusarium spp. and in silage – Aspergillus, Penicillium spp. Average of lactic acid bacteria – 189.01 ± 57.79 (3 months after ensilage) and 436.11 ± 93.97 CFU/g (8 months after ensilage). Compared prior to ensilage and 3 and 8 months after ensilage maize samples, higher mycotoxins concentrations of DON – 36.96 % (P < 0.05) and ZEA – 77.32 % (P < 0.05) were detected in 3 month after ensilage, T-2 toxin – 72.69 % (P < 0.05), AFL – 94.31 % (P < 0.05) were detected in 8 month after ensilage. Concentration of OTA was higher 34.99 % (P > 0.05) in samples after 3 months of ensiling. Dry matter and pH respectively: prior to ensiling – 36.96 % and 5.26, 3 months after ensiling – 33.17 % and 3.84, 8 months after ensiling – 40.52 % and 3.91. The current results indicate the presence of yeast, viable fungi spores and mycotoxins AFL (total), ZEA, DON, T-2 and OTA contamination in maize silage in Lithuania.

This research addresses the potential of microbial implications in bentonite for use as a buffer and backfill material in final disposal site for low-level radioactive waste (LLRW) in Taiwan, where has a special island-type climate. Microbe activities naturally present in this site were analyzed, and buffer materials (BM) consisted of 100%, 70% or 50% bentonite were prepared for laboratory studies. A total of 39 microbial strains were isolated, and the predominant strains included four bacterial, one yeast and four fungal strains. Growth inhibition was not detected in any tested strain cultured in a radiation field with a dose rate of 0.2 Gy/h. Most of the isolated strains grew under a dose rate of 1.4 Gy/h. The D10 values of the tested strains ranged from 0.16 to 2.05 kGy. The mycelia of tested fungal strains could spread over 5 cm during six months of inoculation in BM. The spreading activity of the tested bacteria was less than that of the fungi. Moreover, biofilms were observed on the surfaces of the BM. Since a large and diverse population of microbes is present in Taiwan, microbes may contribute to the mobilization of radionuclides in the disposal site.

Histoplasma is a thermally dimorphic fungus with endemic and opportunistic behavior, which causes a systemic disease known as histoplasmosis. The habitat for this fungus is soil laden with bird and bat droppings, in caves and henhouses, and it persists in the environment long after the contamination. This fungus is widely disseminated in the American continent. In South American countries, the disease is mainly present in Venezuela, Colombia, Peru, Brazil, Argentina, and Uruguay. Man is contaminated by inhaling conidia present in nature, and most infections are mild and subclinical. After being inhaled, conidia undergo phagocytosis by macrophages and mononuclear cells, which are unable to destroy them. They multiply inside these cells, traveling through mediastinal and hilar lymph nodes and into the bloodstream, spleen, bone marrow, liver, skin, and subcutaneous tissue. The diagnosis is based on the detection of the fungus in secretions or tissues and in serology tests. Among these tests, enzyme-linked immunosorbent assays are more sensitive and specific than complement fixation. Tissue biopsies show epithelioid granulomas, with or without necrosis, and fungi within phagocytic cells. Gomori-Groccot staining is required for the visualization of the fungus. A 22-year-old female patient, an undergraduate psychology student, from the urban area of the inner state of Sergipe, no comorbidities, vegetarian, visited a mastologist due to the recent appearance of a nodule in the right breast associated with signs of inflammation and no fever. The clinical examination showed a 2 cm palpable, retroareolar thickening, and thickening of the areolar skin with discrete hyperemia, and no palpable axillary lymph nodes. The patient was initially treated with amoxicillin and clavulanic acid for 7 days. After treatment, there was regression of the inflammation signs upon physical examination; however, the thickening remained and the areolar skin was still thickened and hard. An ultrasound of the right breast showed a well-defined heterogeneous, superficial, and elongated retroareolar nodular image, measuring 3.4×1.2 cm. A breast ultrasound-guided fine-needle aspiration (FNA) was performed, and the cytology test suggested an inflammatory process. After 1 month, the patient returned with two areolar fistulas with yellowish discharge. A new cycle of antimicrobial therapy was started with clindamycin for 14 days. The secretion was decreased over the antibiotic period; however, 14 days after the treatment, the two areolar fistulas were still present with yellowish discharge. A third cycle of antibiotic therapy with metronidazole was administered with no improvement. An excisional biopsy was performed of the area around the fistula and the underlying breast tissue. Two specimens were examined — one skin specimen with the fistulizing areas measuring 1.9×0.8×0.8 cm, and the other specimen measuring 1.7 cm, corresponding to the breast tissue beyond the fistulas, measuring 1.7×1×0.2 cm. Histopathological evaluation of the specimen showed a chronic, granulomatous inflammatory process, with exudative foci and formation of a fistulous tract, chronic inflammatory lymphoplasmacytic reaction, fibrosis, and giant cell reaction. Screening for fungi (Groccot) showed small, clustered yeast-like structures in the cytoplasm of macrophages, suggestive of histoplasmosis. The patient’s clinical tests included hemoglobin of 9 and a white blood cell count of 3,500, with a normal differential count. Screenings for HIV, hepatitis B, and hepatitis C were negative, fasting blood glucose was normal, and liver function was normal. The anemia investigation revealed only a ferroprivic component because of the vegetarian diet. The patient was subjected to general chest and abdominal examinations with no abnormalities. The patient was started on itraconazole 200 mg a day for 1 year, with no relapse until the end of the treatment.

Much literature is available on fungal lipids and their capability as a renewable oil platform for alternate fuels, chemicals, and food products. Microbial oils will not displace all edible oils soon, given techno-economical hurdles in commercialization. However, continued research & development can flatten the curve of deforestation and land-use impacts associated with cultivating these crops. To better understand how oleaginous yeasts and fungi could alleviate the challenges related to the energy-environment-food nexus, it becomes critical to investigate their potential environmental impacts quantitively compared to other feedstocks. Life cycle analysis or assessment (LCA) is a standard tool used for this purpose. LCA studies are not being conducted on a broader scale for fungus-derived oils than their phototrophic algal counterparts. The different stages in the life cycle of fungal lipid production that can be analyzed for environmental implications include cultivation and fermentation, oil extraction; further downstream processing; and end-use. The LCA method for fungal lipid-derived biofuel production systems should cover the main sustainability concerns of biofuel production systems: energy efficiency, climate change, and land occupation. With the scope of microbial oil applications expanding beyond non-fuel encompassing food, supplements, and medicines, their subsequent environmental implications need to be investigated. Further work is required in this area. There are significant knowledge gaps in life cycle inventory and impact assessment information for non-fuel applications.

The vacuolar H+-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It pumps protons into the vacuolar system of eukaryotic cells and provides the energy for numerous transport systems. Through our BARD grant we discovered a novel family of membrane chaperones that modulate the amount of membrane proteins. We also elucidated the mechanism by which assembly factors guide the membrane sector of V-ATPase from the endoplasmic reticulum to the Golgi apparatus. The major goal of the research was to understand the mechanism of action and biogenesis of V-ATPase in higher plants and fungi. The fundamental question of the extent of acidification in organelles of the vacuolar system was addressed by studying the V-ATPase of lemon fruit, constructing lemon cDNAs libraries and study their expression in mutant yeast cells. The biogenesis of the enzyme and its function in the Golgi apparatus was studied in yeast utilizing a gallery of secretory mutants available in our laboratories. One of the goals of this project is to determine biochemically and genetically how V-ATPase is assembled into the different membranes of a wide variety of organelles and what is the mechanism of its action.The results of this project advanced out knowledge along these lines.

One of the emerging technologies is the use of microbial agents for the control of postharvest diseases of fruits and vegetables. A number of antagonistic microorganisms have been discovered which have the potential to effectively control postharvest diseases. Some of this technology has been patented and commercial products such as AspireTM (Ecogen Corporatin, Langhorne, PA, USA), Biosave 10TM and Biosave 11TM (Ecoscience Inc., Worchester, MA, USA) have been registered for commercial use. The principal investigator of this project was involved in developing the yeast-based biofungicide-AspireTM and testing its efficacy under commercial conditions. This research project was initiated to fill the gap between the knowledge available on development and commercial implementation of yeast biocontrol agents and basic understanding of various aspects related to introducing yeast antagonists to fruit surfaces, along with verification of population genetics. The main objectives of this study were: Study ecology, population dynamics and genetic diversity of the yeast antagonists Candida guilliermondii, C. oleophila, and Debaryomyces hansenii, and study the effect of preharvest application of the yeast antagonist C. oleophila naturally occurring epiphytic microbial population and on the development of postharvest diseases of citrus fruit during storage. Our findings, which were detailed in several publications, have shown that an epiphytic yeast population of grapefruit able to grow under high osmotic conditions and a wide range of temperatures was isolated and characterized for its biocontrol activity against green mold decay caused by Penicillium digitatum. Techniques based on random amplified polymorphic DNA (RAPD) and arbitrary primed polymerase chain reaction (ap-PCR), as well as homologies between sequences of the rDNA internal transcribed spacers (ITS) and 5.8S gene, were used to characterize the composition of the yeast population and to determine the genetic relationship among predominant yeast species. Epiphytic yeasts exhibiting the highest biocontrol activity against P. digitatum on grapefruit were identified as Candida guilliermondii, C. oleophila, C. sake, and Debaryomyces hansenii, while C. guilliermondii was the most predominant species. RAPD and ap-PCR analysis of the osmotolerant yeast population showed two different, major groups. The sequences of the ITS regions and the 5.8S gene of the yeast isolates, previously identified as belonging to different species, were found to be identical. Following the need to develop a genetically marked strain of the yeast C. oleophila, to be used in population dynamics studies, a transformation system for the yeast was developed. Histidine auxotrophy of C. oloephila produced using ethyl methanesulfonate were transformed with plasmids containing HIS3, HIS4 and HIS5 genes from Saccharomyces cerevisiae. In one mutant histidin auxotrophy was complemented by the HIS5 gene of S. cerevisiae is functionally homologous to the HIS5 gene in V. oleophila. Southern blot analysis showed that the plasmid containing the S. cerevisiae HIS5 gene was integrated at a different location every C. oleophila HIS+ transformant. There were no detectable physiological differences between C. oleophila strain I-182 and the transformants. The biological control ability of C. oleophila was not affected by the transformation. A genetically marked (with b-glucuronidase gene) transformant of C. oleophila colonized wounds on orange fruits and its population increased under field conditions. Effect of preharvest application of the yeast C. oleophila on population dynamics of epiphytic microbial population on wounded and unwounded grapefruit surface in the orchard and after harvest was also studied. In addition, the effect of preharvest application of the yeast C. oleophila on the development of postharvest decay was evaluated. Population studies conducted in the orchard showed that in control, non-treated fruit, colonization of wounded and unwounded grapefruit surface by naturally occurring filamentous fungi did not vary throughout the incubation period on the tree. On the other hand, colonization of intact and wounded fruit surface by naturally occurring yeasts was different. Yeasts colonized wounded surface rapidly and increased in numbers to about two orders of magnitude as compared to unwounded surface. On fruit treated with the yeast and kept on the tree, a different picture of fungal and yeast population had emerged. The detected fungal population on the yeast-treated intact surface was dramatically reduced and in treated wounds no fungi was detected. Yeast population on intact surface was relatively high immediately after the application of AspireTM and decreased to than 70% of that detected initially. In wounds, yeast population increased from 2.5 x 104 to about 4x106 after 72 hours of incubation at 20oC. Results of tests conducted to evaluate the effect of preharvest application of AspireTM on the development of postharvest decay indicated the validity of the approach.

Following preliminary observations by one of the collaborating scientists on this project and the completion of a 1-year, BARD-supported feasibility study (IS-1908-90F), this 3-year BARD project has been executed. The main objectives of the research were to elucidate biochemical and pathological aspects of UV-induced resistance in fruits and vegetables, to characterize physical and biological variables of induced resistance and delay of ripening, and to explore the application of the treatment as a control practice of postharvest diseases and shelf-life extension of fruits and vegetables. Our findings, which are detailed in numerous joint publications, have shown that the effect of UV-C light on induction of resistance and delay of ripening is a general one and of wide oddurrence. Apart from surface sterilization of the commodity, the reduction of decay of different fungi has been associated with and induced resistance phenomenon which gradually builds up within 24 to 48 hours after the UV treatment and can be reversed by visible light. In citrus, induced resistance has been associated with increased activity of the enzymes phenylalanine ammonia-lyase and peroxidase, and with the levels of endglucanase and chitinase. In tomato, resistance was correlated with the production of high levels of tomatine. Our study of some molecular aspects of the induced resistance in grapefruit has revealed the induction of a cDNA which represents a gene encoding for an isoflavone reductase-like protein that, in legumes, has been associated with phytoalexin biosynthesis. This gene was cloned and sequenced. Delay of ripening was associated in tomato with inhibition of ethylene production, carotenoid synthesis, and chlorophyll degradation and with the presence of high levels of polyamines. In peach fruit epiphytic populations of a yeast increased following the UV treatment. Pilot-size treatment and packing lines were constructed in the US and Israel to test the application of the UV treatment on a semi-commercial scale. Although effective in reduction of decay and delay of ripening, a number of problems will have to be addressed before practical application of this methodology can be realized. The main issues are associated with the temporal and variable response to the treatment, and its relationship to the maturity and date of harvest of the commodity.

Review question / Objective: Various bacteria and fungi colonize the skin surface of patients with AD. The colonized fungi mainly include Malassezia, non-Malassezia yeasts, and molds. Among them, Malassezia occupies 63%~86% of the fungal colonization community on the skin surface of AD patients. Although the relationship between the level of Malassezia on the skin surface and disease severity remains controversial, many studies have shown that the level of serum anti-Malassezia-specific immunoglobulin E (IgE) antibodies in AD patients is related to the disease severity, especially in patients with AD in the head and neck. The specific mechanism by which Malassezia causes or aggravates AD is unclear, but damage to the skin barrier in AD patients is a key component of the mechanism. The presence of Malassezia on the skin also seems to change its barrier function, resulting in more Malassezia and its antigens colonizing the skin surface area that is exposed to the immune system. This produces a large number of specific IgE antibodies and cytokines to aggravate the disease.

Plant pathogenic fungi have various life styles and different plant infection strategies. Hemibiotrophs like Magnaporthe grisea and Colletotrichum species develop specialized structures during plant infection. The goal of this study was to identify, characterize, and compare genes required for plant infection in M. grisea and C. gloeosporioides. Specific objectives are to: 1) further characterize genes identified in the preliminary studies of C. gloeosporioides and M. grisea;2) identify and characterize additional fungal genes tagged by GFP; and 3) identify in planta growth and appressorium-specific genes by subtractive hybridization and transcript profiling by the LongSAGE method. In this study, the PI and Co-PI collaborated closely on studies in M. grisea and C. gloeosporioides. In M. grisea, REMI and ATMT were used to transform the wildtype with promoter-less EGFP constructs. A total of 28 mutants defective in different plant infection processes or expressing EGFP during plant infection were identified. Genes disrupted in five selected mutants have been identified, including MG03295 that encodes a putative Rho GTPase. In transformant L1320, the transforming vector was inserted in the MIRI gene that encodes a nuclear protein. The expression of MIRI was highly induced during infection. Deletion and site-directed mutagenesis analyses were used to identify the promoter regions and elements that were essential for induced in planta expression of MIRI. This was the first detailed characterization of the promoter of an in planta gene in M. grisea and the MIRI promoter can be used to monitor infectious growth. In addition, the Agilent whole-genome array of M. grisea was used for microarray analyses with RNA samples from appressoria formed by the wild-type shain and the pmkl and mstl2 mutants. Over 200 genes were downregulated in the mst I 2 and pmkl mutants. Some of them are putative transcription factors that may regulate appressorium formation and infectious hyphal growth. In C. gloeosporioides, various REMI mutants showing different pathogenic behavior were identified and characterized. Mutants N3736 had a single insertion and was hyper-virulent. The gene disrupted in mutant3736 (named CgFMOI) encodes a FAD-dependent monooxygenase. Expression analyses linked the expression of the CgFMOI gene with the necrotrophic phase of fungal infection, and also suggest that expression of CgFMOl is unnecessary for the first stages of infection and for biotrophy establishment. All CgFMOl-silenced mutants had reduced virulence. In REMI mutant N159, the tagged gene encodes a putative copper transporter that is homologue of S. cerevisiae CTR2. In yeast, Ctr2 is a vacuolar transporter for moving copper from the vacuole to the cytoplasm. The gene was therefore termed CgCTR2. In addition to characterization of CgCTR2, we also conducted comparative analyses in M. grisea. The M. grisea CgCTR-2 homolog was isolated, knockout strains were generated and characterized and the M. grisea was used to complement the Nl 59 C. gloeosporioides mutant. Overall, we have accomplished most of proposed experiments and are in the process of organizing and publishing other data generated in this project. For objective 3, we used the microarray analysis approach. Several genes identified in this study are novel fungal virulence factors. They have the potential to be used as targets for developing more specific or effective fungicides. In the long run, comparative studies of fungal genes, such as our CgCTR2 work, may lead to better disease control strategies.

Cheese, a traditional food incorporated into many cuisines, is used as an ingredient in cooking or consumed directly as an appetizer or dessert, often with wine or other suitable beverages. Great numbers of cheese varieties are produced, reflecting in part the versatility of the microorganisms used in cheese-making that this FAQ report will describe. Cheese is one of the few foods we eat that contains extraordinarily high numbers of living, metabolizing microbes, leading some participants to say, “Cheese is alive!” The broad groups of cheese-making microbes include many varieties of bacteria, yeast, and filamentous fungi (molds). This report focuses on the microbiology of “natural” cheeses, those made directly from milk, including hard and soft varieties such as Cheddar, Mozzarella, and Camembert. Pasteurized process cheese, the other broad category of cheese, is made by blending natural cheeses with emulsifying agents, preservatives, thickeners, flavorings, and seasonings. “American cheese” is perhaps the classic example of a process cheese, notwithstanding recent examples of American artisanal cheese-making and changing tastes among consumers of those cheeses.