longibrachiatum*CanadaSoilDAOM 166989\[[@bib0105]\]EU248618.1*T. martiale*Brazil*Theobroma cacao*CBS 123052\[[@bib0105]\]FJ463324.1*T. rifaii*Ecuador*Theobroma gileri*CBS 130746= Dis 355b\[[@bib0235]\]MK644113*T. rifaii*Costa Rica*Rubus adenotrichos*J2-2CRPresent studyDQ841726.1*T. scalesiae*Ecuador*Scalesia pedunculata*CBS 120069 T = G.J.S. 03-74\[[@bib0355]\]AY376053*T. viride*NetherlandsCellulosic tissueCBS 101526\[[@bib0105]\][^1]

2.5. Primer and probe design {#sec0035}
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Two primers and a hydrolysis probe (TaqMan®) were designed based on the tef1-α sequence of the BV1CR isolate using BioEdit v7.2.5 \[[@bib0275]\] and Primer Express® v3.0 from Applied Biosystems®, following the manufacturer's recommendations \[[@bib0360]\]. The oligo set (primer and probe) encompassed a sequence between the third intron and fourth exon of the tef1-α (92 nucleotides amplicon; [Fig. 1](#fig0005){ref-type="fig"}) of *T. atroviride* BV1CR (Accession MK644115) (Positions 866--946 of the tef1-α gene of *T. asperellum* NW_020208831.1 genomic DNA used as reference). The probe was fluorescently labeled at the 5´-end with VIC™ and a minor groove binder (MGB) conjugated with a non-fluorescent quencher (NFQ) was attached at the 3´-end. Primers and probe were synthesized and purified by standard desalting and HPLC by Invitrogen™. Both primers and labeled probe had a predicted annealing temperature of 58 °C and 68 °C, respectively.Fig. 1Multiple alignment of partial sequences of the translation elongation factor 1-alpha (tef1-α), generated by BioEdit, showing the position of the TrichoBV1 F/R primers (arrows) and the TrichoBV1 probe (line) designed to detect *Trichoderma atroviride* isolate BV1CR through qPCR.Fig. 1

Primer and probe specificity were evaluated by comparing all the aligned tef1-α sequences from the *Trichoderma* isolates ([Table 1](#tbl0005){ref-type="table"}), *B. cinerea* and *R. adenotrichos* and by *in silico* PCR tests using an online *in silico* PCR amplification to