Limited vegetative compatibility following intra- and interspecific protoplast fusion in Trichoderma

A variety of auxotrophic mutants were prepared from several species and strains of Trichoderma by nitrosoguanidine (NNG) mutagenesis. Effective protoplasting from hyphae was achieved with the commercial enzyme preparation Novozym 234; however, pretreatment with 2-deoxy- d-glucose was required for several strains. Aggregation of protoplasts and subsequent fusion were monitored directly by complementary fluorescent staining and were effectively induced by polyethylene glycol and calcium regardless of the Trichoderma species, strains, or auxotrophs being fused. In all cases, about 10 6 viable colony-forming units (CFUs) were formed from about 2 × 10 8 protoplasts. However, subsequent recovery of somatic hybrid colonies was dramatically lower for interstrain fusions than for intrastrain (between two auxotrophs derived from one strain) fusions. Following intrastrain fusions, 2 to 10 × 10 −2 of the viable CFUs grew under selective conditions regardless of the auxotrophs involved, indicating that induced heterofusions were frequent and nutritional complementation was functional. In interstrain fusions, however, only about 1 to 20 × 10 −5 of the viable CFUs produced colonies under selective conditions, indicating a low level of postfusion compatibility. Restricted growth of these somatic hybrid colonies, which were not heterokaryotic, appears to result from fusion of heterologous protoplasts and vegetative incompatibility. No vegetatively compatible pairs of strains were resolved; all inter- and intraspecific protoplast fusions exhibited similarly limited compatibility. Limited compatibility may reduce the likelihood of parasexual recombination but does not preclude the possibility of genetic manipulation of Trichoderma strains by protoplast fusion.