Development of a Method for Detection of the Biocontrol Agent Penicillium oxalicum Strain 212 by Combining PCR and a Selective Medium

Abstract

The registration of biological control agents requires the development of monitoring systems to detect and quantify the agent in the environment. Penicillium oxalicum strain 212 (PO212) is being developed for the control of tomato pathogens. In this study, we demonstrated that PO212 was more effective for controlling Fusarium oxysporum f. sp. lycopersici in tomato plants than 13 other P. oxalicum strains. A new semiselective medium was developed as a preliminary screen for P. oxalicum from soil. This semiselective medium was a modified Fusarium selective medium that contained 0.006 g of nystatin per liter. The growth of P. oxalicum strain 212 was not inhibited on this medium, but it did inhibit the growth of 11 fungal species. Specific identification of the biocontrol strain and its quantification were achieved using a polymerase chain reaction with a strain-specific pair of primers (POITS1F/POITS2R1) and dilution plating. This primer set differentiated the biocontrol strain from 13 other strains of P. oxalicum. There were differences in the nucleotide sequences of the internal transcribed spacer (ITS) regions of the ribosomal DNA of 25 strains of P. oxalicum and those of PO212. Based on the differences in the nucleotide sequences of the ITS regions in rDNA of PO212 and other P. oxalicum strains, a relationship between the nucleotide sequences in the ITS region and biocontrol efficacy is postulated.