PubMed · 15411243
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1950. SCLERODERMA (acrosclerosis).. https://pubmed.ncbi.nlm.nih.gov/15411243/
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High-quality genomic DNA extraction from basidiomycete fungi remains challenging due to polysaccharide co-purification and nuclease-mediated DNA degradation. We systematically compared 22 experimental conditions combining five DNA extraction methods (one Dellaporta-based method, three CTAB-based variants, and one commercial kit) with five mycelium preparation techniques (scalpel scraping, fungal suspension, silica bead homogenization, liquid fermentation, and liquid nitrogen treatment) for Phanerodontia australis (BRM62389). DNA quality was assessed by 1% agarose gel electrophoresis, NanoDrop spectrophotometry, and Qubit fluorimetry. The optimal protocol combined liquid-fermentation mycelium with an adapted CTAB method, yielding Qubit-quantified concentrations of 34.2 and 62.6 ng/µL (samples S21 and S22), 260/280 ratios of 2.06 and 2.05, 260/230 ratios of 1.88 and 1.85, and no detectable DNA degradation by agarose gel electrophoresis. This protocol enabled whole-genome sequencing, yielding a 37.62 Mb assembly with 98.5% completeness as assessed by BUSCO v5 (basidiomycota_odb10 lineage dataset, n = 1,764). Liquid fermentation is associated with the production of younger, actively growing hyphae with reduced cell wall thickness, and the adapted CTAB method effectively removed residual polysaccharides. This protocol provides a reliable, cost-effective approach for obtaining sequencing-grade DNA from P. australis.
A semicontinuous perfusion culture process (repeated medium renewal with cell retention) was evaluated together with batch and repeated fed-batch processes for astaxanthin production in shake-flask cultures of Xanthophyllomyces dendrorhous. The perfusion process with 25% medium renewal every 12 h for 10 days achieved a biomass density of 65.6 g/L, a volumetric astaxanthin yield of 52.5 mg/L, and an astaxanthin productivity of 4.38 mg/L-d, which were 8.4-fold, 5.6-fold, and 2.3-fold of those in the batch process, 7.8 g/L, 9.4 mg/L, and 1.88 mg/L-d, respectively. The incorporation of hydrogen peroxide (H(2)O(2)) stimulation of astaxanthin biosynthesis into the perfusion process further increased the astaxanthin yield to 58.3 mg/L and the productivity to 4.86 mg/L-d. The repeated fed-batch process with 8 g/L glucose and 4 g/L corn steep liquor fed every 12 h achieved 42.2 g/L biomass density, 36.5 mg/L astaxanthin yield, and 3.04 mg/L-d astaxanthin productivity. The lower biomass and astaxanthin productivity in the repeated fed-batch than in the perfusion process may be mostly attributed to the accumulation of inhibitory metabolites such as ethanol and acetic acid in the culture. The study shows that perfusion process plus H(2)O(2) stimulation is an effective strategy for enhanced astaxanthin production in X. dendrorhous cultures.
Evidence for the participation of reactive oxygen species (ROS) and antioxidant systems in ectomycorrhizal (ECM) establishment is lacking. In this paper, we evaluated ROS production and the activities of superoxide dismutase (SOD) and catalase (CAT) during the early contact of the ECM fungus Pisolithus tinctorius with the roots of Castanea sativa (chestnut tree). Roots were placed in contact with P. tinctorius mycelia, and ROS production was evaluated by determining the levels of H(2)O(2) and O(2) (.-) during the early stages of fungal contact. Three peaks of H(2)O(2) production were detected, the first two coinciding with O(2) (.-) bursts. The first H(2)O(2) production peak coincided with an increase in SOD activity, whereas CAT activity seemed to be implicated in H(2)O(2) scavenging. P. tinctorius growth was evaluated in the presence of P. tinctorius-elicited C. sativa crude extracts prepared during the early stages of fungal contact. Differential hyphal growth that matched the H(2)O(2) production profile with a delay was detected. The result suggests that during the early stages of ECM establishment, H(2)O(2) results from an inhibition of ROS-scavenging enzymes and plays a role in signalling during symbiotic establishment.