Fig 1.
Morphological characterization of Prototheca sp.
A. Macroscopic aspect of colonies cultured in SDA for 72 hours at 32°C, showing corrugated edges with central elevation of the colonies and their cream-white pigmentation. B. Wet preparation from SDA colonies showing microscopic characteristics of spherical sporangia (arrows) and sporangiospores (asterisk). Bar: 10 μm. C, D. Detail of morula-like sporangia (arrowhead) and sporangiospores (asterisk) in lactophenol cotton blue stain. Bar: 10 μm.
Fig 2.
Morphometric and growth evaluation of Prototheca stages.
A. Prototheca stages in SDA have a size of 8.09 + 0.93 μm for sporangia and 3.84 ± 0.57 μm for sporangiospores. Digital images analyzed by KS400 software (Carl Zeiss, Oberkochen, Germany). B. Cell cycle pattern in PYEG liquid medium after an inoculum of 100 sporangiospores/mL.
Fig 3.
Sugar assimilation assay using striation and pour plate techniques.
The assays were performed in agar peptone medium (10 g/L bacteriological peptone, 1.7% bacteriological agar, 20mg/L bromophenol blue, pH 7.0) containing 40% of each carbohydrate. Image obtained after 96h of incubation at 32°C.
Table 1.
Identification of Prototheca species by biochemical assays including the investigated isolate in this study.
Fig 4.
Unrooted phylogenetic tree of partial ITS1, 5.8S, and ITS2 DNA sequences from various Prototheca and Chlorella (Auxenochlorella) species (accessions numbers shown on the tree).
The Brazilian isolated clustered with other P. wickerhamii DNA sequences (red rectangle). Evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model [21]. The tree with the highest log likelihood (-940.82) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (6 categories [+G, parameter = 2.5023]). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.