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).