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 backgr ound for identification of strain-specific markers for monitoring strains o f interest, the relationship of strains designated as T. harzianum, includi ng representatives of three biological forms Th1, Th2 and Th3, were analyse d by Universally Primed PCR UP-PCR, using UP and random primers. Cross dot blot hybridization of UP-PCR products generated with either of two differen t UP primers or a random primer showed unequivocal differences among strain s. Using this approach, T. harzianum strains were distributed into two diff erent genotypic groups. One of the T. harzianum groups included forms Th1 a nd 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 rev ealed with three primers. It was found that both genotypic groups are heter ogeneous, and Th2 form strains definitely cluster together with those of Th 1. Division of the three biological forms of T. harzianum into two groups w as also supported by rDNA-ITS1 analysis, where Sau3A digestion of the ampli fied ITS1 region gave a restriction fragment profile specific for each geno typic group. Two strains with known biocontrol capabilities were found to r elate to the genotypic group containing Th1 and Th2 forms and, based on var iation within this group, to belong to a homogeneous group of form Th1 stra ins. The robustness and reliability of UP-PCR fingerprinting were demonstra ted by obtaining identical banding profiles using different conditions for PCR in different laboratories.