Bigdye terminator reagent kit

A partial portion of TEF1α was sequenced with the BigDye Terminator reagent kit (Applied Biosystems) in a SeqStudio Genetic Analyzer (Applied Biosystems) using HS392, HS393, EF11, and EF21 primers (O’Donnell et al., 1998 (link); Scheel et al., 2013 (link); Muraosa et al., 2014 (link); Moretti et al., 2018 (link)). The portion rDNA was sequenced using the primers ITS4, ITS5, NL1, and NL4 (Table 1). DNA sequences were edited and assembled by Sequencher version 5.2.4 (Gene Codes, USA). For identification, a homology search for the sequences of TEF1α and rDNA genes was done using the BLAST tool of the NCBI database (GenBank), the database FUSARIUM-ID (https://isolate.fusariumdb.orgl/), and the Fusarium MLST (http://www.cbs.knaw.nl/fusarium). ß-tubulin and Calmodulin genes were sequenced and used for the pairwise alignment in the NCBI database (GenBank) for the identification of species of Aspergillus (Glass and Donaldson, 1995 (link); Hong et al., 2005 (link)). The sequence of primers used are described in Table 1.

DNA was extracted from 1933P^T culture using the PowerSoil DNA Isolation Kit (MoBio, USA), according to the manufacturer’s recommendations. The 16S rRNA gene of the strain 1933P^T was amplified with the 27F and 1492R primers [33 ], and purified PCR products were sequenced with an ABI Prism 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA) using the Big Dye Terminator reagent kit, version 3.1. The 16S rRNA gene sequence analysis was performed using the EzBioCloud [34 (link)]. Phylogenetic analysis of the 16S rRNA gene sequences was carried out using the maximum-likelihood, neighbour-joining, and maximum-parsimony algorithms. The sequences were first aligned by MUSCLE [35 (link)], and the maximum-likelihood tree was inferred using the GTR + F + I + G4 model recommended by ModelFinder [36 (link)] in IQ-Tree [37 (link)]. Neighbour-joining and maximum-parsimony trees were reconstructed using the MEGA7 software package [38 (link)]. Bootstrap values were calculated from 1000 alternative trees.

DNA for the 16S rRNA gene or genome sequencing was extracted from biomass of the strains HO-Ch2^T and HO-62b1 grown aerobically on the R2A medium with 2.0% (w/v) NaCl at 28 °C. Cells were harvested after 7 days of cultivation. The cetyltrimethylammonium bromide (CTAB) method [35 (link)] was used to purify DNA from cell biomass. The 1492R and 27F primers were used to amplify the 16S rRNA genes of the strains HO-Ch2T and HO-62b2 [36 ]. The PCR products were sequenced using the Big Dye Terminator reagent kit, v. 3.1, at the ABI Prism 3730 DNA analyzer (Applied Biosystems, Waltham, MA, USA). Analysis of the 16S rRNA gene sequences was carried out using EzBioCloud [37 (link)]. The sequences were analyzed using the maximum-likelihood, neighbor-joining, and maximum-parsimony algorithms. First, the sequences were aligned by MUSCLE [38 (link)], and a maximum-likelihood tree was constructed using the model GTR+F+I+G4 recommended by ModelFinder [39 (link)] in IQ-Tree [40 (link)]. The MEGA7 software package was used to reconstruct the neighbor-joining and maximum-parsimony trees [41 (link)]. Bootstrap values were calculated from 1000 alternative trees. The GenBank/EMBL/DDBJ accession numbers of the 16S rRNA gene sequence of strains HO-Ch2^T, HO-62b1, and HO-Met1 are MT225794, OK336454, and MT218393, respectively.