duct is high, the two single strands of the PCR product bind to each other more quickly than the probe to its target sequence or binding site, so that the amount of fluorescence emitted decreases. According to Keer \[[@bib0385]\], this decrease in fluorescence does not affect the efficiency or specificity of the amplification or detection of the target DNA and this effect can be avoided by optimizing the DNA concentration in the reaction or by reducing the number of amplification cycles. In the case of *Trichoderma* BV1CR, the detection of the target DNA was affected and a late detection of the fungus was observed (high Cqs) or there were no amplification curves (in fruits completely covered with the fungus) when qPCR reactions were run using highly concentrated DNA. Adjusting the sample dilutions to 1:1000 allowed the expected detection and quantification of *Trichoderma* BV1CR present in the samples.

4.3. Primer and probe set performance {#sec0080}
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In *Botrytis*, the ITS regions also had limited species resolution, and therefore other regions of the genome have been studied for species identification. The IGS region between the ribosomal genes has evolved faster than the ITS regions and has more sequence variation, thus the IGS region is a much more useful tool to differentiate between closely related species \[[@bib0150],[@bib0390],[@bib0395]\]. The capacity of the IGS region to discriminate among species explains the high fidelity and specificity of the qPCR probe designed by Suárez et al. \[[@bib0150]\] to quantify *B. cinerea*. That probe successfully quantified *B. cinerea* in multiplex and single probe assays using total DNA extracted from the BcLLCR isolate and from composite biological matrices in the present investigation, and has been used by other researchers to study this pathogen applying qPCR technology \[[@bib0165],[@bib0170],[@bib0395]\].

In the case of *Trichoderma*, generic probes, as well as taxon-specific probes, have been developed to monitor and qu