The biocontrol fungus Trichoderma virens is an avirulent symbiont with the ability to control plant disease by the production of antibiotic compounds, induction of plant resistance to pathogens, and mycoparasitism of other fungi. In this document, the analysis of a putative terpene biosynthesis gene cluster (vir cluster) in T. virens is described. The vir cluster contains genes coding for four putative cytochrome P450s, an oxidoreductase, MFS transporter, and a terpene cyclase. To determine the function of this cluster in secondary metabolism biosynthesis, a strain of T. virens with a deletion of the putative cyclase, vir4, was constructed. Deletion mutants were deficient in the synthesis of sesquiterpene volatiles and complementation of vir4 restored this loss in transformants, albeit at a lower level of production. An analysis of phenotypic characteristics between mutant and wild-type strains did not identify any differences when the strain interacted with other fungi, bacteria, or Arabidopsis seedlings. Paralogs of the gene encoding the elicitor SM1 were examined as genetic sources for potential elicitors to induce systemic resistance in plants. A search of the T. virens genome revealed the presence of three paralogs of sm1. One paralog, sm3, was found to be expressed when grown in association with plant roots and in still-culture. The Pichia pastoris protein expression system was used to generate sufficient quantities of SM3 to allow characterization of its function. The purified protein from the yeast system (picSM3) was shown to be glycosylated and to increase expression of a plant defense gene in maize seedlings. Mutant strains in which sm3 was either deleted or over-expressed were constructed to further explore the potential of sm3 as an elicitor of ISR. The differential production of SM1 and SM3 by these strains suggested that SM1 and SM3 may be co-regulated and native SM3 may be glycosylated. To further understand the role of a putative glycosylation site as a mechanism to prevent dimerization and subsequent elicitor activity, a point mutation was created in a sm1 deletion strain. Analysis of the behavior of the protein demonstrates that the putative glycosylation site is not involved in protein aggregation and deletion of this site does not prevent the protein from testing positive for glycosylation. We propose that SM1is not glycosylated but instead may interact with an oligosaccharide or other small molecule. However, the mechanism of dimerization in SM1 remains unknown.

The soil-borne, rhizosphere-competent, filamentous fungus Trichoderma virens is a well-known biocontrol agent able to control pathogenic fungi through the production of antibiotics, the induction of systemic resistance in host plants, or by directly parasitizing the competing fungus. Competition for iron is another means by which Trichoderma can hinder competing microorganisms, and siderophores are a means by which microorganisms obtain iron. In silico analysis of the T. virens genome suggested that two genes putatively encoding extracellular siderophore-producing nonribosomal peptide synthetases (NRPSs) were present. In this study, a disruption was created in one of the genes, TvNPS6, to create a mutant unable to produce the NRPS TvNps6 (DeltaTvnps6). Previously, a mutant (DeltaTvsidD) had been generated with a disruption in the second gene (TvSIDD) encoding an NRPS thought to be involved in siderophore biosynthesis. A double mutant (DeltaDeltaTvsidDTvnps6) was generated by transformation of a DeltaTvsidD strain with a vector targeting disruption of TvNPS6. This resulted in transformants disrupted within both the putative siderophore-producing NRPSs. Thus, three mutants were available for analysis of the role of these genes in the ecology of T. virens. Transformants were confirmed by PCR and Southern blotting analysis. Phenotypic characterization of the mutants included both HPLC analysis of siderophore production, growth on agar and in liquid media, conidiation, germination in the presence of hydrogen peroxide, biocontrol against Pythium ultimum, in vitro confrontation against Rhizoctonia solani and growth with iron chelators to determine the contribution of reductive iron assimilation (RIA) compared to that of siderophores. The HPLC analysis demonstrated that T. virens Gv 29-8 (wild-type) produced a single siderophore peak when grown in an iron-depleted medium. This peak was not present in the DeltaTvnps6 and DeltaDeltaTvsidDTvnps6 mutants but was apparent with the DeltaTvsidD mutants. From the HPLC analysis, T. virens evidently produces a coprogen-type siderophore. Few differences were observed in the other phenotypic tests, though hydrogen peroxide showed some small inhibitory effects towards the DeltaTvnps6 mutants. The addition of chelators, which inhibit RIA, exerted some negative effects on all strains growing under iron-limited media, particularly the DeltaTvnps6 and DeltaDeltaTvsidDTvnps6 strains. This study demonstrated that although T. virens has two genes putatively encoding siderophore producing NRPSs, only the TvNPS6 gene was required for extracellular siderophore production. The greater sensitivity of the mutants towards the iron chelators suggests that unlike other other fungi studied, Trichoderma virens utilizes RIA, rather than siderophore production, as the primary means by which the fungus obtains iron in an iron-limited environment.

Subterranean termites encounter numerous kinds of fungi during foraging and feeding activities. Nearly nine decades of research have exposed only a small fraction of the termitefungal interactions that exist in nature. The first portion of research presented here focused on how feeding behaviors of Reticulitermes virginicus (Banks) were affected by the fungus Trichoderma virens (Miller, Giddens & Foster) von Arx. Tests were performed with 'P' (GLT+) and 'Q'(GLT-) strains of T. virens. Both strains were applied to filter paper and wood disks cut from southern yellow pine and Sentricon® monitoring devices. The first bioassay assessed the termites' feeding responses to fungal extracts removed from liquid media on days 2 through 7, and again on day 15. Only the GLT+ extracts from days 6 and 7 inhibited termite feeding significantly from the controls (16% and 54% less area loss, respectively). Response to wood covered by live T. virens mycelia was tested in the second bioassay. No significant differences in termite consumption were seen between fungal strains, but both substantially reduced the area loss due to termite feeding of treated wood by an average of 35%. A vacuum impregnation system was used to inoculate wood disks with fungal homogenate in the third bioassay. The wood treated with either GLT+ or GLT- homogenates did not have significant differences in area loss due to termite feeding. Overall, these results reiterated the plasticity that exists with termitefungal relationships. The second research topic addressed the applicability of labrum-based identification techniques to Reticulitermes Holmgren in Texas. Soldier labral morphology of four species, R. flavipes (Kollar), R. hageni (Banks), R. tibialis Banks, and R. virginicus (Banks), was evaluated as a character to separate species. Length and width measurements of five soldier labra were taken from each of the eight collection sites. These results were then judged against molecular analysis of the mtDNA 16S rRNA gene. Findings showed that labral shape was an unreliable diagnostic characteristic when comparing all species. A combination of length and length-towidth ratio successfully segregated all four Reticulitermes species. Comparison of a morphology-based dendogram to the phylogenetic analysis revealed a shared pattern between phenotypic and genotypic variations.

The diploma thesis was focused on evaluation of non-thermal (cold) plasma seed treatment of cabbage (Brassica oleracea var. capitata), on its health and production properties and on the effects against Xanthomonas campestris pv. campestris (Xcc). A soil fungus Trichoderma virens (TVI) was used as additional seed treatment. The field experiment showed that microwave, low pressure, 500 W non-thermal plasma and 4 minute exposure time did not have a significant effect on Xcc, but had a positive influence - in combination with Xcc and TVI - on plants´ vitality and yield. Further, this plasma treatment, in combination with Xcc, had a negative impact on seeds´ germination. Non-thermal plasma treatment with the above-mentioned parameters proved to be unsuitable for cabbage seed treatment.

The dissertation is focused on the findings, whether it is combination of physical and biological seed treatment for selected field crops (oilseed rape, spring barley) comparable with chemical treatment. Evaluated the effectiveness of the physical seed treatment, low temperature plasma (Gliding Arc, microwave), a biological treatment culture mycoparasitic of the fungus Trichoderma virens and culture entomopathogenic fungus Metarhizium anisopliae. In the context of experiments in laboratory conditions, was evaluated the effect of treatment on the evolution grains (the seeds), the percentage of germination grains (the seeds) and the percentage of healthy grains (the seeds). Individual plasma treatment in combination with biological treatment had a significant positive effect on the germination rate of grains (the seeds).There was no negative effect of physical and biological treatments on the grains (seeds). Involved the evaluation of emergence of the treated seed in laboratory and field conditions. In the field conditions for the selected crops in different localities and in different years, was assessed the effect of treatment on the development of the crop health and yield parameters. Plasma and plasma with T. virens treatments positively affected by the model of the plant better integration of crops, the length of the cob, (shell) and yield. The combined adjustment should in oilseed rape positive effect on the faster development of the spring, the length and strength of the root, earlier flowering and ripening. In contrast, the combined adjustment for barley spring positively influenced the emergence and tillering. According to the obtained results it seems that the combined physical and biological seed treatment is an effective alternative to the chemical treatment of seeds.

The thesis is focused on biological seed treatment of selected field crops using adhesive in combination with entomopathogenic fungi Metarhizium anisopaliae and mycoparasitic fungi Trichoderma virens. Guar gum, xanthan gum and carboxymethylcellulose were chosen as the adhesive. Within the experiments in laboratory conditions, the percentage of kernels germination of spring barley, winter oilseed rape and a cucumber was evaluated after 24 hours and energy of germination and vitality during 2 5 days. The adhesive in combination with the filamentous fungi had positive influence on growth and development of embryonic roots of spring barley and growth and development of seedlings in all tested crops. The evaluation of plant emergence took place under laboratory and field conditions and the plants were biologically treated. Furthermore, the ability to colonize environment of the entomopathogenic fungus M. anisopliae was verified in this thesis. The fungus was injected into the substrate using seed dressings. According to the results, the introduced fungus is able to infect larvae Tenebrio molitor and Galleria mellonella after specific time.

This M. Sc. thesis is based on using of mycoparasitic fungi Trichoderma virens, Clonostachys rosea f. catenulata in biological control against phytopathogenic fungi Sclerotinia sclerotiorum, Rhizoctonia solani, Botrytis cinerea and Fusarium solani. The efficacy of mycoparasitic fungi against pathogens was evaluated in dual cultural tests. The strains of T. virens and C. rosea f. catenulata isolated from soils in the Czech Republic were tested in the experiment. Reference strain was GL 21 fungus T. virens reisolated from commercially available bio-preparation SoilGard and strain C. rosea f. catenulata reisolated from Prestop Mix. All the strains were tested for biological and production properties. All strains are able to colonize the substrate and to suppress the growth and development of pathogens. Strain GL 21 of T. virens was used for seed coating of variety Scirocco in combination with products Guar gum and Carboxymethyl cellulose, which served as a carrier for stick on conidia. After 3 days, the effect of fungus T. virens was evaluated on energy of germination, development of roots of grain. The grain health was determined after 7 days. The fungus T. virens has a positive effect on the grains germination and grain health. During the vegetation the influence of seed coating by T. virens was observed on growth and development of spring wheat. The parameters such as number of plants per m2, tiller numbers, plants health, stand height, number of grains in the spike and thousand grain weight (TGW) were evaluated. During the vegetation the fungus T. virens has positive effect on the plant height.