Genome Organization And Reproduction

One of the major difficulties with Trichoderma biocontrol strains is their genetic instability, whose reason is only poorly understood at present. This is in part due to the fact that only little is known about the genome organization and its plasticity of Trichoderma. Not even the number of chromosomes is known with certainty: Fekete et al. (1996) separated six chromosomes in five Trichoderma biocontrol strains with sizes ranging from 3.7 to 7.7 Mb; estimated genome sizes were between 30.5 and 35.8Mb. When fractionated chromosomes of the five species were probed with a fragment of the ech42 (endochitinase-encoding) gene, strong hybridization signals developed, but their physical position varied among species indicating a polymorphic chromosomal location. Herrera-Estrella et al. (1993) compared the molecular karyotype of T. reesei with that of T. atroviride (named erroneously T. harzianum in their study), and T. viride, and detected largely similar chromosomal organization of genes in different species, although T. viride seemed to lack the smallest chromosome. Similarly, Hayes et al. (1993), when karyotyping three biocontrol strains of T. harzianum (one parent and two mutants derived from it), found that the smallest chromosome was not present in the mutants. While all these studies revealed a low degree of chromosome polymorphism at the species level, the karyotypes were relatively constant. A report to the contrary (Gomez et al. 1997) is probably flawed by the use "T. harzianum" strains which in fact consisted of several different species (CP Kubicek, unpublished data). Thus, as expected for an asexual fungus, chromosome plasticity is unlikely responsible for the genetic instability of Trichoderma biocontrol strains.

Molecular genetic work with Trichoderma spp. is still limited by the only rudimentary information about its genomic organization as is available for Aspergillus fumigatus (http://www.tigr.org/tdb/e2k1/afu1/) and Neuro-spora crassa (http://www-genome.wi.mit.edu/annotation/ fungi/neurospora). Genetic maps could so far not be constructed, because the teleomorphs of biocontrol species of Trichoderma (see Table 1) do not cross in axenic culture (CP Kubicek, unpublished data). Also, at the time of this writing, genome sequencing projects on selected species of Trichoderma have only just been initiated at a few places, and no results from these are yet available. However, a collection of 1151 ESTs of T. reesei grown on glucose and the sequence of the complete mitochondrial genome is already available in the Internet (http://trichoderma.iq.usp.br/TrEST.html), and can (because of the high similarity of nucleotide sequences of protein encoding genes within the genus (unpublished data) be used for picking genes from biocontrol strains as well).

Interestingly, Seiboth and Hofmann (2002) found a similar genomic organization of several genes of galactose metabolism in T. reesei and N. crassa. This finding is highly interesting, as N. crassa has evolved about 200 million years ago (Berbee and Taylor 1993), whereas H. jecorina evolved only about 100 million years ago (Kullnig-Gradinger et al. 2003), and thus the genomic organization of these genes has been maintained constant for about 100 million years. Hamer et al. (2001) have also recently reported that a 53-kb region of the genome of Magnaporthe grisea was also syntenic to a corresponding portion of the Neurospora genome. In a comprehensive study on hemiascomycetous yeasts, Llorente et al. (2000) demonstrated that even phylogenetic distant species such as S. cerevisiae and Yarrowia lipolytica exhibit 10.1 % of conserved synteny. If there is indeed a high degree of synteny between Neurospora and Trichoderma, this may be useful for studying the genomic organization of Trichoderma biocontrol strains.

Probably due to reproduction, largely via asexual mechanisms, many species of Trichoderma reveal a high level of genetic stability (cf. Kubicek et al. 2002; Kullnig et al. 2000). T. harzianum, however, is a noteworthy exception, showing a remarkable intraspecific genetic and phenotypic variation, and this may also be related to the instability of the respective biocontrol species. The reason for this has not been explained yet. As the respective teleomorph (H. lixii) is known, the possibility of sexual recombination still needs rigorous testing. Transposons, which have been isolated from phylogenetically close fungal genera such as Tolypocladium or Fusarium, are another possibility. We have recently observed a very high noninduced mutation rate in one biocontrol strain of T. harzianum which would be compatible with the presence of a mobile element (C Gallhaup, RL Mach and CP Kubicek, unpublished).

As far as nonchromosomal elements are concerned, plasmids have been detected in filamentous fungi almost exclusively in the mitochondrium (Bertrand 2002). They are generally stable genetic elements and vary between 1-6kb size. In accordance with this situation, Meyer (1991) detected mitochondrial plasmids in strains of T. viride and the biocontrol-relevant species T. asperellum (then named "T. viride 2"). A circular plasmid called pThr1, with a monomer size of 2.6 kb, was identified in the mitochondria of the biocontrol isolate T. harzianum T95 (Antal et al. 2002). It revealed no DNA sequence similarity with the mitochondrial genome of the isolate and contained a single 1818 bp open reading frame. The derived amino acid sequence exhibited similarity to the reverse transcriptases of the circular Mauriceville and Varkud retroplasmids of Neurospora spp. and the linear pFOXC2 and pFOXC3 retroplasmids of Fusarium oxysporum strains. In the regions of homology all of the seven conserved amino acid blocks characteristic of RTs could be found. In Fusarium oxysporum f. sp. conglutinans, these mito-chondrial plasmids have been identified as factors determining the host specificity (Kistler and Leong 1986); unfortunately, corresponding investigations are still lacking for Trichoderma.

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