The dimorphic yeast Geotrichum candidum (teleomorph: Galactomyces candidus) is commonly used to inoculate washed-rind and bloomy-rind cheeses. However, little is known about the phylogenetic lineage of this microorganism. We have sequenced the complete 18S, 5.8S, 26S ribosomal RNA genes and their internal transcribed spacers (ITS1) and ITS2 regions (5126 nucleotides) from 18 G. candidum strains from various environmental niches, with a focus on dairy strains. Multiple sequence alignments revealed the presence of 60 polymorphic sites, which is generally unusual for ribosomal DNA (rDNA) within a given species because of the concerted evolution mechanism. This mechanism drives genetic homogenization to prevent the divergent evolution of rDNA copies within individuals. While the polymorphisms observed were mainly substitutions, one insertion/deletion (indel) polymorphism was detected in ITS1. No polymorphic sites were detected downstream from this indel site, that is, in 5.8S and ITS2. More surprisingly, many sequence electrophoregrams generated during the sequencing of the rDNA had dual peaks, suggesting that many individuals exhibited intragenomic rDNA variability. The ITS1-5.8S-ITS2 regions of four strains were cloned. The sequence analysis of 68 clones revealed 32 different ITS1-5.8S-ITS2 variants within these four strains. Depending on the strain, from four to twelve variants were detected, indicating that multiple rDNA copies were present in the genomes of these G. candidum strains. These results contribute to the debate concerning the use of the ITS region for barcoding fungi and suggest that community profiling techniques based on rDNA should be used with caution.

The new API 20C yeast identification system together with appropriate microscopic morphology determinations achieved a 97% correlation with a rapid conventional method. Whereas a group composed of Candida, Torulopsis, Saccharomyces, and Rhodotorula was identified with ease (98% overall correlation), a second group, containing Cryptococcus, Trichosporon, and Geotrichum species, appeared to give the system the most difficulty (90% correlation). Within this group particular difficulty was encountered in identifying varieties of Cryptococcus albidus, C. terreus, C. laurentii, Trichosporon beigelli, and Geotrichum spp. as to species. The API 20C system should be incubated the full 72 h prescribed by the manufacturer. However, when used in conjunction with appropriate morphological tests, presumptive identifications of some Candida and Torulopsis species may be made at 24 to 48 h. To facilitate identifications of the more difficult group of yeasts, ancillary tests for determining nitrate reductase, urease, and phenol oxidase activities should be considered as additions to the strip. Incorporating the phenol oxidase test would be especially important for identification of Cryptococcus neoformans, a yeast which should be identified as quickly and as accurately as possible. The API 20C system with computer assistance has proved to be an easy-to-inoculate, versatile, and fairly rapid method of yeast identification, giving results comparable to those obtained by conventional methodologies.

The yeast-like genera Geotrichum and Trichosporon are heterogeneous and are related with anamorphs of both ascomycetous and basidiomycetous fungi. A rearrangement can be obtained using carbohydrate composition of intact cells, studied with the aid of gas-liquid chromatography. The genus Geotrichum is restricted to ascomycetous species with a dominance of galactomannans, whereas Trichosporon is reserved for basidiomycete-like, xylose-containing species. Consequently, some new combinations are introduced in both genera. Representatives of related genera are included for comparison: e.g. Dipodascus, Hyphophichia, Cryptococcus and Filobasidium.

Among the clinical isolates hitherto identified as Geotrichum capitatum, two groups were defined from DNA-DNA reassociation experiments. This confirms the existence of two closely related, human-pathogenic Geotrichum species, namely, G. capitatum and G. clavatum. A third group of strains from cactus rots, though morphologically identical to G. capitatum, has a lower moles percent G + C of DNA. The three groups can be recognized by a combination of morphological and physiological characters.

Opportunistic infections by yeasts have been implicated as one of the major causes of complications in the compromised patient. Rapid recognition and identification of these yeasts is essential for patient management, but conventional liquid medium methods for completing identification tests are cumbersome and time consuming. Rapid tests have been devised based on modifications of methods commonly used in bacteriology. These rapid methods included tests for carbohydrate and nitrate assimilation, fermentation, and urease production. These were compared with several current methods for accuracy of results, for time to final identification, and for economy of time and reagents. In addition, the usual tests for pseudogerm tube formation, for production of hyphae or pseudohyphae, and for growth temperatures were included. The rapid tests achieved 96% or better accuracy compared with expected results, and 46 species of yeasts were identified in 1 to 2 days compared with the 10 to 14 days required by conventional liquid culture methods.

The wide genotypic and phenotypic diversity of Geotrichum candidum strains does not facilitate its classification as yeast or a yeast-like fungus that is still a matter of debate. Whatever its classification, G. candidum possesses many different metabolic pathways that are of particular interest to the dairy industry. G. candidum is of importance in the maturation of cheese, and much is known about its direct contribution to cheese ripening and flavour formation. Its diverse metabolic potential means that G. candidum can play an important role in the ripening of many soft and semi-hard cheeses and make a positive contribution to the development of taste and aroma. It may also influence the growth of other microorganisms, both valuable and detrimental. The significance of the presence of G. candidum in cheese depends on the particular type of production and on the presence of biotypes featuring specific types of metabolism. However, in situ metabolic pathways involved in cheese ripening and their regulations are mainly unknown. The information available provides a good understanding of the potential of G. candidum strains that are used in cheese manufacture, and permits a better choice of strain depending on the characteristics required. The biochemical activities of G. candidum and its application in the dairy industry are presented in this review.

The diversity of French fungus-ripened cheeses is due partly to the succession of fungi that colonize the cheese during ripening. Geotrichum candidum appears in the early stages of ripening on soft cheeses such as Camembert and semihard cheeses such as St. Nectaire and Reblochon. Its lipases and proteases promote flavor development, and its aminopeptidases reduce bitterness imparted by low-molecular-weight peptides in cheese. We assessed the genetic diversity of G. candidum strains by using random amplification of polymorphic DNA (RAPD)-PCR correlated with phenotypic tests for carbon assimilation and salt tolerance. Strains were isolated from milk, curd, and cheese collected in seven major cheesemaking regions of France. Sixty-four isolates were characterized. We found high genetic diversity of G. candidum even within the same cheesemaking regions. Strains did not group according to region. All of the strains from the Haute-Savoie were able to assimilate lactate as the sole source of carbon, while lactate assimilation varied among strains from the Auvergne. Strains varied in D-mannitol assimilation, and none used citrate as the sole source of carbon. Yeast-like colony morphology predominated in Reblochon, while all of the strains isolated from St. Nectaire were filamentous. The RAPD-PCR technique readily differentiated Geotrichum fragrans isolated from milk and curd in a St. Nectaire cheesemaking facility. This study reveals an enormous diversity of G. candidum that has been empirically selected through the centuries by the cheesemakers of France.

The Abbott yeast identification system (Abbott Laboratories, Diagnostics Division, Irving, Tex.) is a 24-h, instrumental method for identifying medically important yeasts, based on matrix analysis of 19 biochemical reactions and the germ tube test. The system was evaluated in two clinical laboratories by using 179 coded isolates, which included a high percentage of the less frequently encountered species. Based upon results with these coded isolates and from previously obtained laboratory data, the system software was adjusted and accuracy of the yeast identification system was further evaluated with 378 isolates from clinical sources. Of the 378 clinical yeast isolates tested, 364 (96%) were correctly identified with the Abbott system. Isolates were deliberately selected so that germ tube-positive isolates made up less than 10% of the clinical isolates tested.

A disk-diffusion method for identification of yeasts was developed that depended on their different but distinct susceptibilities to the following chemicals: janus green, ethidium bromide, 2, 3, 5-triphenyltetrazolium chloride, brilliant green, cycloheximide and rhodamine 6G. For 594 of 623 routinely isolated yeasts, the disk-diffusion and the commercial API 20C auxanogram tests gave the same identification, an agreement of 95.3%. Only 8 of 1052 isolates from clinical specimens were not identified by the disk-diffusion method. The method is simple, inexpensive and technically straightforward and for most isolates gives an identification in 24 h.

The new genus Arxula is proposed for the classification of xerotolerant, ascomycetous, anamorphic, arthroconidial yeasts. The genus is considered to be of endomycetaceous affinity.

AIM: Decolourization of black olive mill wastewaters (OMW) by depolymerization of phenolic compounds by Geotrichum candidum. METHODS AND RESULTS: Our results show that G. candidum is able to grow on black OMW supplemented with carbon source and nitrogen. The Geotrichum growth decreased the pH and induced a 49% of colour removal when the black OMW was supplemented with glycerol and diammonium tartrate (20 mm ammonium). An improvement of 10% of colour removal was observed when the culture was supplemented with veratryl alcohol. The decolourization was inhibited with glutamate as nitrogen source. CONCLUSION: Our results suggest the potential use of G. candidum in black OMW decolourization and support the concept that lignin peroxidase (LiP) of G. candidum is involved in the depolymerization of phenolic compounds. Significance and Impact of the Study: This is the first report of LiP production by G. candidum on OMW.