Thomas E. Stasz

Protoplast preparation and fusion in two biocontrol strains of Trichoderma harzianum

ABSTRACTHigh yields (approx. 108/ml) of protoplasts of high purity (>99%) can be obtained from young thalli of strains T12 and T95 of Trichoderma harzianum by digestion of cell walls with NovoZym 2...

EVALUATION OF PHENETIC SPECIES AND PHYLOGENETIC RELATIONSHIPS IN THE GENUS TRICHODERMA BY CLADISTIC ANALYSIS OF ISOZYME POLYMORPHISM

Taxonomy in Trichoderma is problematic because of the lack of reliable morphological characters. This study evaluates five morphological species using methods of isozyme analysis that allow determination of allozyme polymorphism. One hundred nine alleles were resolved at 16 putative enzyme loci among 71 strains tested. Morphological species were not characterized either by specific alleles at single loci or by specific patterns of alleles at multiple loci. Rather, most alleles were distributed among most species, and each species included several different alleles at most loci. Allele patterns were analysed by phylogenetic analysis using parsimony (PAUP). A strict consensus tree was derived from 20 equally parsimonious, shortest cladograms using Gliocladium virens as an outgroup. In the resulting cladogram, T harzianum included a main group of strains adjacent to the outgroup and other strains distributed throughout the cladogram; this species appears to be paraphyletic. A core group of T. pseudokoningii strains appears to be monophyletic and derived directly from a T. harzianum-like ancestral group. Trichoderma hamatum included three isozymically distinct strains with close affinity to Gliocladium virens and a main group which appears to have been derived from a T. harzianum-like ancestral group independently of the core group of T. pseudokoningii. Trichoderma koningii and T. viride are not well distinguished from each other, but T. viride appears to be more derived and may have arisen from a more ancestral T koningii type. Phylogenetic analysis of allozyme data identified isozymically similar core groups of strains within morphological species, clusters of strains from various species, and iso? zymically distinct strains for further study. Wide distribution of numerous alleles among morphological species suggests that extensive genetic exchange occurs among these taxa.

Nonparental progeny resulting from protoplast fusion inTrichoderma in the absence of parasexuality

Abstract The purpose of this study was to characterize a number of progeny from intra- and interstrain protoplast fusion within the genus Trichoderma . We wished to determine whether parasexuality or other genetic mechanisms occur in these fungi. When two different auxotrophs of the same strain were fused, rapidly growing prototrophic progeny were obtained in high frequencies. When single spore isolates of these strains were prepared, equal numbers of strains indistinguishable from the two parental auxotrophic strains were obtained, even though 10 9 –10 10 conidia were tested per strain. Thus, progeny from intrastrain fusions all appeared to be balanced heterokaryons, and no evidence of recombination between the two parental strains was obtained. When 16 separate interstrain fusions were conducted, very different results were obtained, regardless of whether fusions were within or between species. Following interstrain fusions, presumptive somatic hybrids developed very slowly and in low numbers as compared with hybrids from intrastrain fusions. Most were weakly prototrophic. These slow-growing progeny were unstable and sectors developed from them. Such sectors themselves were unstable and gave rise to other progeny. Usually sectors were more strongly prototrophic and more rapid growing than the original progeny strain. Sectoring gave rise to a very wide range of morphotypes. Most of these morphotype variants were stable through conidiation; thus, these types did not occur as a consequence of heterokaryosis. Isozyme analysis was conducted on over 1000 progeny strains. Nearly all progeny were identical to one or the other parental isozyme phenotypes. A few progeny, when tested as soon as possible after fusion, exhibited the isozyme phenotypes of both parents, but such biparental banding patterns were rapidly lost upon subsequent reculturing. Isozyme banding patterns of multimeric enzymes never gave band patterns indicative of heterokaryosis or heterozygosis. Banding patterns indicative of heterozygous diploids or recombinants were never detected. Despite the extreme variation in morphotype and nutritional requirements among progeny, isozyme banding patterns of derived progeny from any fusion were invariably identical to one or the other parental strains. From these results, we conclude that protoplast fusion in the genus Trichoderma gives rise to great variability, but that the classical parasexual cycle is not required for variation to occur.

Limited vegetative compatibility following intra- and interspecific protoplast fusion inTrichoderma

Abstract 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.

Methods of Isozyme Electrophoresis for Trichoderma and Gliocladium Species

Species of Trichoderma and the closely related genus Gliocladium are ubiquitous, active components of the soil microflora. As biocontrol agents, these fungi can protect a wide range of agronomic, horticultural, and ornamental plants against a variety of plant pathogens (6, 13), and a few strains have been patented for commercial use. Some strains enhance plant growth or rooting of cuttings even in the absence of pathogens (4, 14). Trichoderma spp. have also been studied extensively as potential sources of cellulases for utilization of lignocellulosic materials (10, 17). The taxonomy, genetics and population composition of these fungi are poorly understood. Limited knowledge of variability in these fungi and infrequent culturing of their sexual stages make delineation of narrowly defined species difficult (16). Additionally, little is known about such genetic components as ploidy levels, frequency of heterokaryosis, or the prevalence of parasexual events. Finally, methods are lacking to differentiate among strains for patent purposes, or to determine the variability and abundance of strains in natural ecosystems. Isozyme analysis by starch gel electrophoresis is a powerful method for taxonomic, genetic and population studies. Descriptions of general techniques and of numerous enzyme assay systems have been published (9, 12, 18, 20, 21). However, for studies of specific organisms, suitable enzyme assays and corresponding electrophoretic buffers must be determined empirically (8, 12, 18). For an enzyme assay to be useful, enzyme activity must be reliably detected and activity bands must be clearly resolved. In addition, polymorphism must exist among isolates of the study population. Also, the usefulness of an enzyme assay for a particular study often depends on the number of loci detected. Assays detecting products of single loci can be particularly useful because polymorphism among individuals reflects allelic differences, and electrophoretic variants can be scored as allozymes. The genetic basis of an isozyme phenotype usually can be inferred from the complexity of the activity band pattern visualized (9, 12, 18). In this study, 63 enzyme assays were tested in combination with four electrophoretic buffers to assess detection of enzyme activity, resolution of activity bands, and complexity ofphenotypes visualized. This report summarizes the most useful systems for studies of Trichoderma and Gliocladium spp. and identifies those which apparently visualize products of single loci, i.e., allo-

TRANSFORMATION OF TRICHODERMA SPP. WITH PLASMIDS CONFERRING HYGROMYCIN B RESISTANCE

Several plasmids with different promoter sequences, all containing the HygB gene that confers resistance to the antibiotic hygromycin B, were evaluated for their ability to transform Trichoderma viride strain T 105-227 Nic-. Plasmid pH IB, which contains a promoter sequence from Cochliobolus heterostrophus, was found to give the largest number of putative transformants. Transformation of T. harzianum strains T12-2 His- and T95-1 Lys- was attempted, and the time at which transformed protoplasts were exposed to hygromycin B was found to be critical. If hygromycin B was added before protoplasts had regenerated for at least 6 h, no transformants were obtained. However, if regeneration proceeded for more than 12 h, many nontransformed protoplasts formed thalli. Putative transformants were heterokaryotic. Even if thalli were grown in the presence of hygromycin B, the number of transformed nuclei was low; only 0.5 to 0.8% of conidia from putative transformants were capable of producing thalli in the presence of hygromycin B. Subcultures derived from single hygromycin B-resistant conidia were stable. Southern analysis indicated that the plasmid was integrated into the genome in either tandem repeat pattern or was single copy insertion.

Protoplast Fusion for the Production of Superior Biocontrol Fungi

Biological control (biocontrol) of plant pathogens is becoming an important component of weed and plant disease management practices. Biocontrol potentially offers answers to many persistent problems in agriculture, including problems of resource limitations, nonsustainable agricultural systems, and overreliance on pesticides (7). Much emphasis is now being placed on the use of specific biocontrol agents, as opposed to modification of the environment, to improve the level of naturally occurring biocontrol (7). Many fungi and other microorganisms have been shown to control various plant pathogens and weeds; products sold commercially include the mycoherbicides COLLEGO and DeVINE.