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A dominant selection marker is an important prerequiste for gene transformation studies in most organisms. In fungi, two types or selection marker are usually used; the introduction of an antibiotic resistant gene or by using auxotrophic mutant. Due to the biosafety issues, auxotrophic genetic markers are widely used for the genetic manipulation, especially for downstream industrial applications. The aim of this work is to develop a uracil/uridine (ΔpyrG) auxotrophic mutant of Trichoderma virens UKM1, a locally isolated fungus capable of producing interesting industrial enzymes. The T. virens ΔpyrG mutant was developed by the deletion of the gene encoding for orotidine-5-phosphate decarboxylase (pyrG), an enzyme involved in the biosynthesis of pyramidine, using a homologous recombination approach. Successful ΔpyrG mutants exhibited a uracil auxotrophy phenotype and were able to grow on media containing 5-FOA (5-fluoroorotic acid) supplemented with uracil and uridine, that inhibit the growth of the wild-type strain. Transformation of the ΔpyrG mutant with the pyrG from Aspergillus oryzae restored the pyrimidine biosynthesis pathway, transforming the ΔpyrG mutant into a uracil prototroph. These results suggest uracil/uridine auxotrophic phenotype resulted from the deletion of the pyrG gene in T. virens UKM1. Hence, in this work we have successfully developed a Trichoderma virens UKM1 ΔpyrG auxotrophic mutant that is amenable to further efforts in the genetic engineering of this interesting fungus. [ABSTRACT FROM AUTHOR]