Delineation of Trichoderma harzianum into two different genotypic groups by a highly robust fingerprinting method, UP-PCR, and UP-PCR product cross-hybridization

Strains of Trichoderma harzianum possess biocontrol capabilities. As background for identification of strain-specific markers for monitoring strains of interest, the relationship of strains designated as T. harzianum, including representatives of three biological forms Th1, Th2 and Th3, were analysed by Universally Primed PCR UP-PCR, using UP and random primers. Cross dot blot hybridization of UP-PCR products generated with either of two different UP primers or a random primer showed unequivocal differences among strains. Using this approach, T. harzianum strains were distributed into two different genotypic groups. One of the T. harzianum groups included forms Th1 and Th2 while the other group accounted for the Th3 form. The relatedness of strains of each group was estimated by UPGMA analysis based on markers revealed with three primers. It was found that both genotypic groups are heterogeneous, and Th2 form strains definitely cluster together with those of Th1. Division of the three biological forms of T. harzianum into two groups was also supported by rDNA-ITS1 analysis, where Sau3A digestion of the amplified ITS1 region gave a restriction fragment profile specific for each genotypic group. Two strains with known biocontrol capabilities were found to relate to the genotypic group containing Th1 and Th2 forms and, based on variation within this group, to belong to a homogeneous group of form Th1 strains. The robustness and reliability of UP-PCR fingerprinting were demonstrated by obtaining identical banding profiles using different conditions for PCR in different laboratories.