Prepman ultra sample preparation reagent kit

Genomic DNA of all Fusarium isolates was extracted with the PrepMan Ultra Sample Preparation Reagent kit (Applied Biosystems, Palo Alto, CA, USA) according to the manufacturer’s instructions. PCRs were performed for the amplification of the largest subunit of RNA polymerase (RPB2) and the translation elongation factor-1α (TEF1α) following the methods published by Salah et al. [15 ]. The primer pairs for TEF1α were EF1 & EF2 [16 (link)] and for RPB2 were RPB2-7cR & RPB2 -5F [17 (link)]. PCR products were sequenced with the same primers used for amplification. The ABI Prism® Big DyeTM Terminator v. 3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems) was used for sequencing PCR according to the manufacturer’s instructions. The samples were run on an ABI 3730XL automatic sequencer (Applied Biosystems). A BLAST search of TEF1 and RPB2 sequences against the database FUSARIUM-ID (http://isolate.fusariumdb.orgl/), the Fusarium database (http://www.cbs.knaw.nl/fusarium) and GenBank databases (www.ncbi.nlm.nih.gov) were used as an initial step to identify isolates to species and/or species complex. The TEF1 and RPB2 nucleotide sequences of all isolates were deposited in GenBank.

According to Abuhena et al. (2022b) (link), the PrepMan^® Ultra Sample Preparation Reagent Kit (Applied Biosystems, United States) was used to isolate genomic DNA from a single colony of a Trichoderma isolate. PCR was performed in a MyGeneTM Series Peltier Thermal Cycler using genomic DNA isolated from a Trichoderma isolate and a positive control of Saccharomyces cerevisiae with standard primers provided in the Fast MicroSEQ^® D2 LSU rDNA Fungal PCR Kit (Applied Biosystems, United States). The MicroSeq^® D2 LSU rDNA Fungal Sequencing Kit’s Forward Primer Reaction Mix and purified PCR product were used in cycle sequencing (Applied Biosystems, United States). Using the NCBI BLAST system,¹ DNA similarity was examined. To identify species, a criterion of 98% sequence similarity was applied. The isolated strains’ D2 LSU rDNA sequences were added to the NCBI GenBank database and given an accession number. The phylogenetic analyses were conducted in MEGA11.0 (Maitra et al., 2021 (link)).