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EnvironmentalpH has been shown to be important for regulating OA production by

S. sclerotiorum ascospores. Principally pH substrates above pH 5 have been shown to

induce OA production (Maxwell and Lumsden 1970, Rollins and Dickman 2001, Culbertson et al. 2007). This was observed when SDB was buffered at pH 5 there was a reduction in the amount of OA produced and as a result pH 5 was chosen as the optimum starting pH for the medium used within the biosensor.

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pH has been shown to strongly regulate the pac homologue gene in S. sclerotiorum

which in turn regulates a range of pH dependent developmental genes in the fungus. The more oxalic acid secreted, the lower the pH which activates many developmental genes. However once a certain acidic pH is reached, oxalic acid production is inhibited (Rollins and Dickman 2001). It has been suggested that by buffering the medium, prolonged OA could be achieved. This was certainly observed for the 50mM HEPES buffer added to SDB which had prolonged high levels of OA production (Figure 31 c-d). At day 6 and 7, the OA amounts were nearly double that of the control SDB for both high and low spore dilutions. However for the biosensor, 6 days for sample incubation is too long a period and instead 4 days is a better incubation time frame to allow a suitable fungicide spray window for growers. At day 4 there was very little difference between the amounts of OA produced in the control SAB medium or the HEPES buffered medium. As a result, no buffer was added to the final medium.

3.4.3 The relationship between S. sclerotiorum ascospore number, biomass and oxalic

acid production.

In this series of experiments, no evidence was obtained that ascospore number positively correlated to increases in OA concentration or biomass. This finding supports other published studies, where the highest biomass of a mycelial plug inoculated culture did not correspond to the highest amount of OA measured (Culbertson et al. 2007). Instead it was observed in this study that a baseline biomass was required before OA production was induced. This is observed as the lower ascospore doses only reached this required biomass a day later compared with the highest ascospore doses and only then was OA secreted. It is important to note that biomass did not differ significantly between the three spore doses and this is most likely because once the spores germinate, the wells can only support so much biomass as there is a limited nutrient supply, principally the glucose supply, which was not replenished. The 2 ml of medium will only support the growth and development of a finite number of fungal cells and it was also observed that by 11 days there was very liquid remaining in the wells. In addition, it was difficult to assess out of the 2000 spores, for example, how many were actually viable.

Biomass reduced later on during the infection course probably due to a combination of pH environmental signals and reduced availability of nutrients for biomass growth. By 11 days growth, the pH had increased and cultures began to synthesise sclerotia, a process which is linked to gene regulation by the alkaline dependent pac1 gene. This gene actively promotes transcription of alkaline-expressed genes (Rollins 2003). This occurs when pac1

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recognises alkaline expressed genes which contain multiple copies of a 5’GCCARG- 3’binding site situated in a zinc finger DNA-binding domain (Espeso et al. 1997). Under alkaline conditions this gene is activated and so ensures that only pH responsive genes are activated under the right pH conditions (Espeso et al. 1997). The production of sclerotia may account for the decrease in biomass in the older colonies as the fungus potentially recycles biomass to make the sclerotia.

Obtaining accurate biomass measurements was a challenge during this experiment as it was not possible to gather 100% of the biomass from the plastic wells for freeze drying which will make the data more variable.

During the first few days of incubation, the OA concentration increased for all three ascospore doses and the pH dropped which was an expected result. The OA concentrations eventually reduced in all spore concentrations which correlated with an increase in pH. This could be a result of putative oxalate decarboxylase enzymes which are secreted into the medium, and hydrolyse OA into formate and carbon dioxide. Secretion of formate oxidases may then be involved in the breakdown of formate. Formate oxidases have been shown to be part of glucose-methanol-choline oxidoreductase family (Doubayashi et al. 2011). Two putative oxalate decarboxylase enzymes as well as ten putative secreted glucose-methanol-choline oxidoreductase family proteins were predicted in the refined S.

sclerotiorum secretome (Chapter 5). These genes could be investigated for further

extracellular function. Another possibility is that S. sclerotiorum may possess oxalate:formate transporters which are present in other fungi including Saccharomyces

cerevisiae and Aspergillus fumigatus (Cheng et al. 2007, Nierman et al. 2005). These may

assist in the efflux of oxalate and influx of formate but this has not been identified yet and so requires further information.

From the above experiments there remains little understanding about why the OA concentrations vary so much between different ascospore doses and different biomasses. For the purpose of the biosensor this is not a problem as even the smaller amounts of ascospores captured within the sensor will be able to produce detectable levels of OA. From an academic perspective it may be interesting to repeat this experiment but monitor the expression of pH-dependent genes to determine at which point specific genes are expressed and how these might further induce growth.

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