Elucidating the dual physiological induced effect of gliotoxin on plants
Bezuidenhout, Johannes Jacobus
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Fungi and Oomycetes represent the two most important groups of eukaryotic plant pathogens. Besides chemical and physical control of these pathogens, biological control is an approach enjoying increasingly more focus. One of the biological agents increasingly employed in biological control of plant pathogenic fungi is ironically the fungus Trichoderma, more specifically Trichoderma harzianum. Besides control of the fungal plant pathogens, another interesting aspect observed when plants are treated with Trichoderma harzianum are effects such as complete and even stand of plants, faster seed germination, increases in plant height and overall enhanced plant growth. Though there have been various studies on this effect, almost no research has yet been conducted to elucidate the mechanism by which these effects occur. In particular, effects such as faster seed germination suggest that Trichoderma harzianum produces a metabolite that may mimic the plant growth hormone gibberellic acid. Through an evaluation of the various metabolites produced by Trichoderma harzianum; gliotoxin seemed structurally most similar to gibberellic acid. To verify that gliotoxin can indeed serve as an analogue for gibberellic acid and elicit similar physiological responses in plants, a two–pronged approach was followed. Firstly, molecular similarity evaluation through common pharmacophore evaluation was conducted, followed by docking simulations into the recently discovered receptor for gibberellic acid. Common pharmacophore evaluation between gibberellic acid and gliotoxin showed successful alignment of gliotoxin into the gibberellic acid based pharmacophore space. Furthermore, docking simulations further strengthened this by the similarity in docking scores calculated and the similar poses of the ligands (gliotoxin and gibberellic acid) in the receptor space. However, similarity in pharmacophore alignment and docking simulation results only suggest that gliotoxin should be able to occupy the receptor space, but it is not a guarantee that similar physiological responses will be elicited. In the second part of the project, the ability of gliotoxin to elicit similar physiological responses in plants to gibberellic acid was investigated. For this, a–amylase induction; plant emergence and height; and chlorophyll fluorescence were compared for both gliotoxin and gibberellic acid treatments. In terms of a–amylase induction, gliotoxin was able to induce production of the enzyme as visualised by starch–containing native gel electrophoresis (zymograms). Gliotoxin induced the strongest response at a 10–6 M dilution which is typically the range expected for hormones in biological systems in de–embryonated seeds of Phaseolus vulgaris. Gibberellic acid was able to induce the strongest response at a 10–7 M dilution. In essence, similar physiological responses were observed. In terms of plant emergence and plant height, treatment with gliotoxin or gibberellic acid resulted in plant emergence a day earlier than the untreated control. However, even though there were slight differences in plant height favouring the gliotoxin or gibberellic acid treated plants, the differences were not statistically significant. Thus, in this regard similar responses were again observed for both gliotoxin and gibberellic acid treatments. In the final evaluation the effect of gliotoxin and gibberellic acid treatments on the chlorophyll fluorescence of mature plants was investigated. Overall, both gliotoxin and gibberellic acid elicited beneficial effects on plant vitality, expressed through PI(Abs) with the gliotoxin treatment performing better than the equivalent gibberellic acid treatment. Overall, the physiological tests demonstrated that gliotoxin can indeed elicit similar positive physiological responses to gibberellic acid in Phaseolus vulgaris. Furthermore the test used in this project can serve as a standard evaluation bench for screening for gibberellic acid analogues on a laboratory scale before larger scale field trials are considered.