Report on The Experiments on Light Incubation

In the first experiment, our results supported the hypothesis that T. zuluensis reacts differently on exposure of light. However, it did not grow best in the darkness. The growth of pathogen was significantly different under different volumes of light and dark incubation. The growth under 25% light volume was significantly the higher compared to all other light intensities and the darkness in each media treatment (Table 5). Our results also showed that there is a significant difference in fungal growth upon exposure of different light intensities. Incubation in different light volumes revealed a different diameter growth of T. zuluensis. The growth was lower in the darkness as well as in 75% of light but higher in 25% light volume. It indicated that the pathogen needs a light but in a limited intensity to grow fast. Dark incubation exhibited the lowest growth compared to all light volumes.

Though, the light preferences vary among different fungi as some fungi grow best in the darkness and others prefer the light conditions. Hatakeyama et al. , 2017 found that the light exerted the negative effects on diameter growth of A. oryzae and conidial production was also repressed in Light incubation. Light pulse is also required in many fungi to initiate conidia formation, yet several other mechanisms are also involved in its regulation. Likewise, different stages of growth also regulate conidial initiation and the processes that elicit enzymatic production (Tisch and Schmoll, 2010). Biomass production is also another imperative parameter that can be compared in different lights in the future research as it is also regulated by the light and darkness (Murthy et al. , 2015). In their research, the biomass of A. oryzae was increased by 11% in darkness compared to the light. Light often persuade or suppresses the spores production in fungi and it can have varied effects on reproductive development, growth and metabolism (Atoui et al. , 2010). Besides the effects observed on growth by light and darkness, it also affects the nutritional balance in fungi (Carlile, 1965).

The availability/utilization of substrates to fungi is closely interlinked with the sunlight exposure (Friedl et al. , 2008). The regulation of light not only effects the utilization but also the uptake of substrates that ultimately affects whole metabolic process in the cell as they lack corresponding compounds (Hill, 1976). The limitation of these essential compounds inhibits the growth of fungi. (Tisch and Schmoll, 2010) gave the example of glucose uptake in A. ornatus was inhibited significantly when incubated under the light (Hill, 1976) however it induced the formation of conidia. The interrelation of glucose and conidial formation was emphasized through finding (Betina and Zajacova, 1978) where higher concentration of glucose reduce the conidia production however it does not influence on the growth. In other studies (Graafmans, 1976), light stimulated the synthesis of polysaccharides in Penicillium isariiforme. Cell wall of fungi is the first spot that is exposed to photons upon illumination (Tisch and Schmoll, 2010).

Several studies revealed where light altered the cell wall composition and doubled the amount of chitin in A. giganteus compared to dark incubation (Fiema, 1983). Temporary alteration was also observed in the cell wall structure of P. blakesleeanus in research carried by Estrella and Herrera, 1983. The examples and literature reviewed, where the light effect is detected in some species cannot be entirely granted or ruled out without further experimentation on other species. In this experiment, we only recorded the growth data over petri dishes using a simple scale. However, there are further research possibilities to test these strains to record other parameters like biomass production, conidial count, enzymatic and other cellular changes as an effect of light. In second experiment, we successfully implemented the Phenotypic Microarray (PM) approach on T. zuluensis to figure out its preference of nutritional substrates.

The experiment demonstrated that the PM approach is useful tool to explore the cellular phenotypes of fungi. It enabled us to determine the substrate utilization by the fungi over the period of time. In the Phenotypic Microarray experiment, the two assayed fungal strains T. zuluensis belong to different origins South Africa SA and Paraguay P exhibited the same range of substrate utilization but at the different utilization rates. Results showed that the ability of SA strain to catabolize the substrates was much higher compared to P strain. This indicates that both the strains are capable of utilizing the same number of nutritional sources however, their metabolic efficiency is different. The SA strain utilized all the substrates at a higher rate than the P strain.

The substrate utilization of SA strain was at peak between the time period of 120h and 144h while for P strain it was uppermost between 168h to 192h for most of the nutrient profiles, this indicated the slower rate of substrate utilization in the P strain. Our results showed that the pathogen of Eucalyptus T. zuluensis originated from South Africa utilized the nutrient profiles faster than the strain isolated from Paraguay. This fast utilization demonstrates its better metabolism that leads to rapid growth of the pathogen. The SA strain also showed the broad spectrum of substrates utilization rate that was 72% compared to 70% of P strain. It also clarified the fact that SA strain can relatively rely on a broader range of nutrients. The result was expressively different in three major groups of chemical compounds that include oligosaccharides, hexoses, and glucosides. Maximum growth was shown in these substrates classified into above-mentioned groups. Formulation of the new medium using these substrates can potentially enhance the growth of T. zuluensis. The experiment was terminated after 216h of incubation; however, the growth was still continuous at that time. For future studies, measurements can be continued until the growth has ceased.

Conclusion

In this study we focus on slow growing Eucalyptus pathogen Teratosphaeria zuluensis and tested under light volumes of 25%, 50%, and 75% and in the darkness, respectively. The results showed significant differences in pathogen growth under different light exposures. The pathogen showed the highest growth in 25% light compared to other light intensities and in the dark incubation. We also observed the lowest growth of T. zuluensis while incubated in darkness.

In our second experiment, we assayed two strains of T. zuluensis originated from South Africa and Paraguay using Phenotypic Microarray approach. The substrates classified into the groups of Oligosaccharides, Hexoses and Glucosides exhibited the highest values for AWCD and the mycelial growth. Both of the strains revealed the same growth pattern but South African strain exhibited the elevated growth compared to the strain originated from Paraguay.

In both experiments, the South African strain showed higher growth in comparison to Paraguayan strain. We could show that the pathogen has potential to grow faster in the media formulated by using the oligosaccharides, hexoses and glucosides and incubated under 25% light. These conditions are recommended to be used in further studies of this pathogen.