Detection of <em>Candida albicans</em> DNA from blood samples using a novel electrochemical assay

The genus Candida contains a number of yeast species which are opportunistic pathogens and are associated with life-threatening infections in immunocompromised individuals. Provision of appropriate therapy relies on the rapid identification of the infecting species, and existing methods of identifying Candida species in clinical samples are time and resource intensive and are not always specific enough to differentiate between drug-susceptible and drug-resistant species. We have previously developed a system for the rapid detection of yeast pathogens in clinical samples using PCR followed by hybridization with a suite of five species-specific, electrochemically labelled DNA probes. The limit of detection of the assay was shown to be 37 fg (∼1 genome) per reaction using extracted genomic DNA. We carried out a study to test the limit of detection of one of the probes, CA PR3, using blood samples from a healthy donor that were spiked with genomic DNA or with C. albicans cells. Our results demonstrated a limit of detection of 37 fg (ml blood)−1 (∼1 genome ml−1) using extracted DNA or 10 c.f.u. (ml blood)−1 using C. albicans cells, indicating that the assay is capable of detecting C. albicans nucleic acid at levels that are encountered in clinical samples.</jats:p

Acquisition of flocculation phenotype by Kluyveromyces marxianus when overexpressing GAP1 gene encoding an isoform of glyceraldehyde-3-phosphate dehydrogenase

The use of flocculating yeast strains has been considered as a convenient approach to obtain high cell densities in bioreactors with increasing productivity in continuous operations. In Kluyveromyces marxianus ATTC 10022, the GAP1 gene encodes an isoform of glyceraldehyde-3-phosphate dehydrogenase–p37—that is accumulated in the cell wall and is involved in flocculation. To test the use of p37 as a tool for engineering Kluyveromyces cells to display a flocculation phenotype, K. marxianus CCT 3172 was transformed with an expression vector containing GAP1. This vector is based on the pY37 previously described, harbouring a S11 Kluyveromyces origin of replication, and the expression of GAP1 is under the control of GAL1. Kluyveromyces cells overexpressing GAP1 acquired a flocculent phenotype together with the accumulation of p37 in the cell wall. The results support the use of GAP1 gene as a molecular tool for inducing flocculation.Fundação para a Ciência e a Tecnologia (FCT) - BD/18203/98

Disease epidemiology and genetic diversity of fusarium oxysporum f. sp. elaeidis, cause of fusarium wilt of oil palm (Elaeis guineensis Jacq.)

Vascular wilt disease caused by Fusarium oxysporum f. sp. elaeidis (Foe) has devasted oil palm in west and central Africa. This study investigates the spatial distribution of Foe, whereby non-random, clustered patterns of the disease were recorded in four separate plantations in Ghana; infection from tree to tree via elongating roots therefore plays a more significant role than aerial distribution by conidiospores, with management implications. Control of Foe with disease-resistant palm lines can depend on the genetic variability of Foe isolates. Twenty-two putative Foe isolates from several African countries, including Ghana, were obtained from oil palms in infected areas for phylogenetic analysis along with 19 fungal outgroups, using the TEF-1a gene. The data showed Foe isolates have a monophyletic origin, and therefore limited diversity. Palm adapted isolates of F. oxysporum appear to have evolved independently, as ff. spp. elaeidis, albedinis and canariensis were nested into three independent groups. Slowly developing (chronic) and fast, severe (acute) Fusarium wilt are both evident in plantations and we provide preliminary evidence that Foe isolates' different aggressiveness might contribute to this variation. Sampling for Foe infection from xylem in extracted stem cores revealed the deficiency of field surveys based only on visual symptoms.</p

Disease epidemiology and genetic diversity of fusarium oxysporum f. sp. elaeidis, cause of fusarium wilt of oil palm (Elaeis guineensis Jacq.)

Vascular wilt disease caused by Fusarium oxysporum f. sp. elaeidis (Foe) has devasted oil palm in west and central Africa. This study investigates the spatial distribution of Foe, whereby non-random, clustered patterns of the disease were recorded in four separate plantations in Ghana; infection from tree to tree via elongating roots therefore plays a more significant role than aerial distribution by conidiospores, with management implications. Control of Foe with disease-resistant palm lines can depend on the genetic variability of Foe isolates. Twenty-two putative Foe isolates from several African countries, including Ghana, were obtained from oil palms in infected areas for phylogenetic analysis along with 19 fungal outgroups, using the TEF-1a gene. The data showed Foe isolates have a monophyletic origin, and therefore limited diversity. Palm adapted isolates of F. oxysporum appear to have evolved independently, as ff. spp. elaeidis, albedinis and canariensis were nested into three independent groups. Slowly developing (chronic) and fast, severe (acute) Fusarium wilt are both evident in plantations and we provide preliminary evidence that Foe isolates' different aggressiveness might contribute to this variation. Sampling for Foe infection from xylem in extracted stem cores revealed the deficiency of field surveys based only on visual symptoms.</p

Mutants affecting plasmodium formation in a homothallic strain of Physarum polycephalum.

A re-investigation of the strain Colonia of the Myxomycete Physarum polycephalum showed it to be homothallic. All amoebae can give rise to plasmodia within clones. Using genetic analysis it was shown that this ability was due to an allele of mating-type (mt), mt, which allowed (1) crossing of amoebae both carrying mt, (2) outcrossing with amoebae carrying heterothallic mating-types, Micro-spectrophotometric measurements on amoebal and plasmodial nuclei showed a change in DNA content consistent with a change in ploidy. Both results independently eliminate apogamy as an explanation for these results. Many interesting observations were made on amoebal growth during progress towards synthesising an axenic medium for amoebae. New mutagenic techniques devised to mutagenise amoebae all failed to increase mutagenic rate. An independent check on the work of Haugli using his method confirmed that (1) mutation rate could be enhanced from 10 to 100 fold by ultraviolet light with the synergistic effect of caffeine, (2) the maximum expression of mutations under these conditions occumed after 30 hours. Using this method 4 mutants affecting the amoebal-plasmodial transition were isolated. These amoebae fail to undergo the necessary developmental switch and do not produce plasmodia in clones. Genetic analysis showed them to be recessive, functionally different and, in the detailed analysis of one of these mutants, freely recombining with all other genetic markers including mt. This groundwork showed the feasibility of genetic analysis of a developmental process in great detail. A review of developmental genetics was made. The processes of cell fusion, nuclear fusion and mitosis in other organisms was discussed. Cell fusion seems to be a rare but important biological event. Nuclear fusion of interphase nuclei is restricted to gametic cells. All syncytial cells have a closed mitosis but not all cells with a closed mitosis are syncytial. The relevance of these processes to the amoebal-plasmodiel transition in other Myxomycstss was discussed

Rapid identification of Saccharomyces eubayanus and its hybrids

The Saccharomyces species Saccharomyces eubayanus was recently discovered in Patagonia. Genomic, genetic and phylogenetic data all suggest that it is one of the two parents of the hybrid yeast S. pastorianus (S. cerevisiae being the other). Saccharomyces eubayanus genomes can also be found in strains of the hybrid species S. bayanus. Here, we describe a novel pair of PCR primers targeted against the S. eubayanus FSY1 gene that will specifically detect S. eubayanus and hybrids containing this allele of the gene. The primer pair can be used to identify the species using a rapid, inexpensive colony PCR method suitable for a preliminary identification of wild isolates.Keyword