MYCOLOGICAL METHODS AND RESULTS
Morphology and Phenotypic Assessment. Isolate UTHSCSA DI20-341was three-point inoculated onto Creatinine agar (Crea), Czapek yeast extract agar (CYA), malt extract agar (MEA), yeast extract sucrose agar (YES), and potato flakes agar (PFA) and were incubated in the dark at 25 ºC for 7 d. Growth was also assessed at 30 ºC, 37 ºC, 45 ºC and 50 ºC. Slide cultures were set up and was mounted on lactophenol cotton blue after 7 d. Morphological and microscopic characteristics typical ofAspergillus citrinoterreus showed cinnamon-colored colonies on MEA at 7d at 25, 30 and 37 °C (Figure 4 A, B, C) with intense yellow diffusing pigment, globose to subglobose yellowish conidia, and smaller obovoid accessory conidia (Figure 4 D, E). Growth at 25, 30, 37, and 45 °C were observed but not at 50 °C. Sporulation was abundant on MEA compared to the other culture media.
DNA Sequencing and Phylogenetic Analysis. Genomic DNA was extracted from harvested mycelia on PFA and sequenced using Bt2a and Bt2b primers for the partial beta-tubulin (BenA ) gene region and CF1 and CF4 primers for the partial calmodulin (CaM ) gene region as previously described.2-4 Newly generated sequences were deposited in the GenBank database under the accession numbers MW419109 (BenA ), MW419108 (CaM ). BLASTn searches were conducted for presumptive species identification. BLASTn results forBenA and caM showed 99-100% identity withAspergillus citrinoterreus sequences in GenBank. Many of theAspergillus citrinoterreus sequences in GenBank were identified as Aspergillus terreus but were re-identified later as A. citrinoterreus .5
Based on BLASTn results, the dataset was compiled using the newly generated sequences and publicly available representative sequences of authentic and type strains of species in the Aspergillus sectionTerrei . Maximum likelihood trees were generated on individual locus and combined using the IQ-Tree.6 The best fitting model for each locus and combined was determined by corrected Akaike Information Criterion as implemented in IQ-TREE. Branch supports were estimated by 1000 bootstrap replicates (BS) and Bayesian inference (BI) with MrBayes v.3.2.6 using a Markov Chain Monte Carlo (MCMC) algorithm.7,8 Aspergillus fumigatus NRRL 163T was used as the outgroup species. Phylogenetic analysis of each individual loci and combined showed the isolate consistently grouping with the type and other strains of A. citrinoterreus and confirmed the identity of the isolate with high branch support (caM = BI/BS = 1.00/88%; BenA = BI/BS 0.97/ 94%; caM +BenA = BI/BS 1.00/ 99% BS) (Figure 5 and Figure 6).