Soil dilution plates were prepared from different soil samples using a solid synthetic selective medium containing (i). glucose as carbon source, (ii). thymine as nitrogen source, (iii). vitamins, (iv). minerals, and (v). chloramphenicol as antibacterial agent. Using the Diazonium Blue B colour reaction, it was found that both ascomycetous and basidiomycetous yeasts were able to grow on this medium. Subsequently, the medium was used to enumerate yeasts in soil microcosms prepared from four different soil samples, which were experimentally treated with the fungicide copper oxychloride, resulting in copper (Cu) concentrations of up to 1000 ppm. The selective medium supplemented with 32 ppm of Cu was used to enumerate Cu-resistant yeasts in the microcosms. The results showed that the addition of Cu at concentrations >or=approximately 1000 ppm did not have a significant effect on total number of yeasts in the soil. Furthermore, it was found that Cu-resistant yeasts were present in all the soil samples, regardless of the amount of Cu that the soil was challenged with. At the end of the incubation period, yeasts in the microcosms with zero and approximately 1000 ppm of additional Cu were enumerated, isolated, and identified with sequence analyses of the D1/D2 600-650 bp region of the large subunit of ribosomal DNA. Hymenomycetous species dominated in the control soil, while higher numbers of the urediniomycetous species were found in the soil that received Cu. These observations suggest that urediniomycetous yeasts may play an important role in re-establishing overall microbial activity in soils, following perturbations, such as the addition of Cu-based fungicides.
While plant genome analysis is gaining speed worldwide, few plant genomes have been sequenced and analyzed on the African continent. Yet, this information holds the potential to transform diverse industries as it unlocks medicinally and industrially relevant biosynthesis pathways for bioprospecting. Considering that South Africa is home to the highly diverse Cape Floristic Region, local establishment of methods for plant genome analysis is essential. Long-read sequencing is becoming standard procedure for plant genome research, as these reads can span repetitive regions of the DNA, substantially facilitating reassembly of a contiguous genome. With the MinION, Oxford Nanopore offers a cost-efficient sequencing method to generate long reads; however, DNA purification protocols must be adapted for each plant species to generate ultra-pure DNA, essential for these analyses. Here, we describe a cost-effective procedure for the extraction and purification of plant DNA and evaluate diverse genome assembly approaches for the reconstruction of the genome of rooibos (Aspalathus linearis), an endemic South African medicinal plant widely used for tea production. We discuss the pros and cons of nine tested assembly programs, specifically Redbean and NextDenovo, which generated the most contiguous assemblies, and Flye, which produced an assembly closest to the predicted genome size.
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