School of Life Sciences

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Epigenetic priming and in Vitro mutagenesis in sugarcane (Saccharum Spp. Hybrids) for resistance to Fusarium Species and Aldana Saccharina (Lepidoptera: Pyralidae).
(2022) Govender, Eshani.; Watt, Maria Paula Mousaco Deoliveira.; Snyman, Sandy Jane.; Rutherford, Richard Stuart.
In the South African sugar industry, there have been substantial economic losses of R1 billion/annum caused by the indigenous pyralid borer, Eldana saccharina (Lepidoptera: Pyralidae). To develop control measures for E. saccharina in sugarcane, it is important to understand the interactions between the stalk borer and Fusarium spp. In previous studies, in vitro assays have shown that Fusarium strains may be antagonistic (e.g., F. sacchari PNG40) or beneficial (e.g., F. pseudonyamai SC17). F. pseudonyamai SC17 is a potential endophytic indicator of E. saccharina infestation, as the association between borer infestation and infection by the fungus causes Fusarium stalk rot in sugarcane. Studies have reported that the presence of endophytic fungi may have several benefits to the host plant, e.g., the production of phytohormones such as indole-3-acetic acid (IAA), which promotes plant development. The study aimed to: 1) choose a suitable resistance priming agent between hexanoic acid (Hx) and cis-jasmone (CJ); 2) determine an appropriate culture filtrate (CF) concentration for in vitro screening of calli and plantlets for tolerance to F. pseudonygamai; 3) develop a protocol (epigenetic priming and mutagenesis) to generate mutants: primed only (0.6 mM Hx), a combination of priming and mutagenic agents (100 μM 5-AzaC + 16 mM EMS-induced); 4) screen for indole-3-acetic acid production by F. pseudonygamai; and 5) characterise in vitro selected mutants for E. saccharina and F. pseudonygamai resistance by comparing the levels of resistance between unprimed, primed, and primed + mutagenic plantlets through ex vitro screening. When cis-jasmone (CJ) and hexanoic acid (Hx) were investigated for their effect on priming for pathogen resistance, 0.6 mM Hx was selected as the optimum priming agent concentration for both the callus and plantlet regeneration stages. At the highest CF concentration (100 ppm) at the embryo germination stage, the number of plantlets was greatly reduced to 58 and 98 plantlets/0.1 g of callus, for cultivars 88H0019 and N41 respectively, compared to more than 600 plantlets/0.1 g of callus in the no CF control. Unexpectedly, in the plantlet regeneration stage all the tested CF concentrations had a significant positive effect on the percentage of plantlets that re-rooted compared with the control. Both cultivars showed a 95 - 100 % rooting ability of the plantlets, which was significantly higher than the percentage of plantlets that rooted in the embryo germination media (EGM1) containing no CF (60 - 70 %) (p < 0.001). Likewise, all the concentrations of the CF had a positive effect on the root length of plantlets, with 1500 ppm CF resulting in the highest root length of 31.5 mm ± 4.3 for 88H0019 and 34.05 mm ± 3.9 for N41. Hence, F. pseudonygamai SC17 could not be used as an in vitro selection agent in a root re-growth assay. Due to the enhanced effect of F. pseudonygamai SC17 CF on root growth, the fungal isolate’s potential to produce indole acetic-3-acid (IAA) was assessed. F. pseudonygamai produced the highest IAA concentration (743.1 nM) in the presence of L-tryptophan than in the treatment without L-tryptophan (457.2 nM). This suggests that the observed enhanced root growth may be due in part to the production of auxin (IAA) in the F. pseudonygamai SC17 CF. Acclimatised in vitro plantlets (8-9 months old) were inoculated only with F. pseudonygamai SC17 or dual inoculated: firstly, with F. pseudonygamai SC17, then 1-2 2nd instar E. saccharina larvae that were placed into the leaf whorls 2 weeks later. To confirm tolerance of the putative mutants, fungal isolations were performed on the stem sections above the inoculation lesion from symptomatic and asymptomatic plants. The results revealed that the putative mutant plants that were primed with Hx only and treated with a combination of mutagens (EMS and 5-AzaC) and priming agent exhibited a significant decrease in lesion severity as compared with controls. For both treatments, a mild lesion severity rating was recorded for plants inoculated with only SC17 for cultivars N41 and 88H0019. For the plants that were dual inoculated there was a significant difference in the lesion severity ratings between the two treatments (p < 0.001). The lesion severity rating was moderate for cultivar 88H0019 (primed with Hx) and mild for cultivar N41 (primed with Hx). Plants from the combined treatment for both cultivars resulted in a mild lesion severity rating. This protocol could be valuable in generating commercially important cultivars that are tolerant and resistant to F. pseudonygamai SC17 and possibly other sugarcane pathogens. Planting resistant cultivars is recommended as an economical and the best method for controlling diseases and pests. This approach used in this study will have the least impact on the environment and increase yields without the need for expensive chemical applications and labour.
Physiological responses of a South African high-latitude coral community to global warming.
(2021) Hanekom, Tanja.; Porter, Sean Nixon.; Sshleyer, Michael Henry.
The health of the world’s coral reefs is deteriorating rapidly due to global climate change and increasing localised anthropogenic stressors. The substantial benefits resulting from coral reef ecosystems, economically and ecologically, requires that research be conducted on their responses to rising sea temperatures driven by climate change. Millions of people depend on the natural resources that coral reefs provide, whether for food or eco-tourism, trade and other indirect sources of income. Although South African coral reef communities lie within a long-established marine protected area, the iSimangaliso Wetland Park World Heritage Site, this status does not preclude them from being affected by the potential effects of global warming. Therefore, the objective of this study was to quantify several physiological parameters, including net community calcification (total alkalinity), growth (dimension and weight) and photosynthetic efficiency in the hard coral Acropora appressa, the soft coral Sinularia brassica and ‘live rock’ under local historical-average (24.4°C), future (26.9°C) and bleaching-threshold (28.8°C) temperatures indicative of climate change conditions projected at Sodwana Bay. Corals and live rock were exposed to the three different temperature treatments during a 10-week long mesocosm experiment that consisted of three phases: the initial phase during which temperatures were increased from 24.4°C over four weeks to reach setpoints of 26.9°C (future) and 28.8°C (bleaching threshold), respectively; the middle phase during which temperatures were held stable at each treatment’s setpoint for the proceeding four weeks; and the final phase during which a further 1°C increase was done over two weeks in the bleaching-threshold treatment to simulate an extreme warming scenario. An initial increase in size was evident in both coral species exposed to the historical-average control temperature and the future temperature projected for Sodwana Bay in 2100 by the representative concentration pathway (RCP) 4.5 climate change scenario. Although the growth trends of both species persisted in the control treatment, the overall linear growth of A. appressa was lower under the RCP 4.5 climate change temperature scenario and bleaching-threshold temperature relative to the control temperature. While no significant treatment effects were found, a decrease in the linear extension of A. appressa was evident at the end of the experiment at the bleaching-threshold temperature relative to the control temperature. Continuous growth trends were evident in the control and RCP 4.5 climate change scenario for S. brassica, however a reduction in diameter after 5 weeks was apparent in the bleaching-threshold treatment. A gradual increase in buoyant weight of A. appressa was evident across all treatments and experimental phases, with a slower growth rate only apparent towards the end of the experiment in the bleaching-threshold treatment. The buoyant weight of S. brassica decreased up until the start of the middle phase in the control treatment and RCP 4.5 treatment. However, an increasing trend in the weight of S. brassica was measured in the same two treatments from the end of the middle phase until the experiment concluded. Contrastingly, the weight of S. brassica in the bleaching-threshold treatment continued to decrease throughout the course of the experiment. Pulse-amplitude modulated fluorometry measurements of the photosynthetic efficiency of both A. appressa and S. brassica were lower under the temperature conditions projected by the RCP 4.5 scenario and by the bleaching-threshold temperature, relative to the historical-average control temperature. Contrastingly, live rock showed no significant differences in photosynthetic efficiency among the different temperature treatments. On average, total alkalinity levels were higher under future temperature conditions projected by the RCP 4.5 temperature scenario and by the bleaching-threshold temperature, relative to the control temperature, indicating suppressed net community calcification. Suppressed net community calcification was particularly evident during (week 6) and at the end (week 8) of the middle phase of the experiment. The experiment revealed that exposure to temperatures equivalent to those projected by the RCP 4.5 climate change scenario in 2100 and the local bleaching threshold are likely to be deleterious to high-latitude corals and coral reef communities in South Africa: buoyant weight and dimension, as well as photosynthetic efficiency were negatively affected in both species of coral and net community calcification was supressed under the two future climate scenarios of warming. Due to the location of Sodwana Bay reefs, the results indicate that calcification processes will be an essential physiological response to consider under global warming conditions. However, as high-latitude reef areas generally fared better during recent bleaching conditions, these reefs can be utilised to improve climate-change projection models. Such model improvements can guide climate policymakers in enhanced conservation efforts that will further stakeholder engagement and outreach. Accordingly, urgent action is needed to reduce greenhouse gas emissions to minimise the effects of global warming on coral reef communities as much as possible. Such efforts will further help to attain the 2°C Paris Climate Agreement and improve socioeconomic development for the management of reefs.
Polyhydroxyalkanoate production by Bacillus thuringiensis: an aspect of biorefining pulp and paper mill sludge.
(2021) Singh, Sarisha.; Govinden, Roshini.; Sithole, Bishop Bruce.; Lekha, Prabashni.; Permaul, Kugenthiren.
The substantial success of plastic as a material is owed to its unparalleled designs with unique properties and proved versatility in an extensive range of applications. Unfortunately, the reliance on single-use plastic commodities consequently results in the incorrect disposal and accumulation of this waste at staggering rates in our environment and landfill sites. In this regard, there is a vested interest in replacing petrochemical plastics with natural, biodegradable plastics (bioplastics). Of the many natural polymers available, microbially synthesized polyhydroxyalkanoates (PHAs) have gained popularity. Eco-friendly PHA-based bioplastics are characteristically as robust and as durable as their oil-based equivalents. Pulp and paper mill sludge (PPMS) is another solid waste stream that is predominantly disposed of via landfilling. The environmentally hazardous gases and leachate emitted from PPMS together with limited landfill space availability and the implementation of strict waste management legislation may not make landfilling practicable in the future. However, this carbohydrate-rich biomass has favorable traits that make it applicable as a feedstock for microbial biomass and PHA production. Hence, in the interest of addressing the issues mentioned above, this study aimed to beneficiate PPMS into PHAs by applying it as the sole feedstock for microbial cell proliferation and subsequent PHA production. Presently, to the best of the author’s knowledge, there are no reports on PHA production as a route for valorization of PPMS from South African pulp and paper mills. Thus, the novelty of the present study is marked by the unique ways of incorporating PPMS as a low-cost substrate as well as the various fermentative strategies navigated to enhance both microbial cell biomass and PHA productivity. In the present study, it was established that Bacillus thuringiensis had promising PHA-producing capability. The strain synthesized a copolymer and terpolymer using untreated (raw) neutral semi-sulphite chemical pulping and cardboard recycling mill (NSSC-CR) and prehydrolysis kraft and kraft pulping mill (PHKK) PPMS in a consolidated bioprocessing fermentation. A separate hydrolysis and fermentation strategy was pursued whereby a glucose-rich hydrolyzate was obtained from enzymolysis of PPMS and subsequently utilized in a cyclic fed-batch fermentation (CFBF) strategy to obtained enhanced yields of cell biomass and PHAs. Response surface methodology (RSM) was first implemented to optimize the conditions for enzymatic saccharification of de-ashed PHKK PPMS. The optimized variables were; pH 4.89; 51°C; hydrolysis time 22.9 h; 30 U/g β-glucosidase and 60 U/g cellulase; and 6.4% of dried de-ashed PPMS fiber resulting in a hydrolyzate comprising of 48.27% glucose. Thereafter, CFBF was pursued where the glucose-rich hydrolyzate was employed as the sole carbon source for cell proliferation and PHA production. The statistically optimized fermentation conditions to obtain high cell density biomass (OD600 of 2.42) were; 8.77 g L-1 yeast extract; 66.63% hydrolyzate (v/v); a fermentation pH of 7.18; and an incubation time of 27.22 h. The CFBF comprised of three cycles and after the third cyclic event, maximum cell biomass (20.99 g L-1) and PHA concentration (14.28 g L-1) were achieved. This cyclic strategy yielded an almost 3-fold increase in biomass concentration and a 4-fold increase in PHA concentration compared with batch fermentation. The properties of the synthesized PHAs were similar to commercial polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) and also displayed slightly higher thermostability and lower crystallinity compared with commercial PHB and PHBV. This is the first report detailing the proof of concept of using PPMS from South African pulp and paper making mills for cell biomass and PHA production by B. thuringiensis. In addition, this study reports on the practicality and novelty of utilizing PPMS either in its raw, untreated state or as enzymatically saccharified glucose-rich hydrolyzate as cheap substrates applicable for both cell biomass and PHA production using different fermentation strategies. Finally, to the best of our knowledge, this is also the first report that has successfully applied B. thuringiensis in a CFBF strategy coupled with glucose-rich hydrolyzate as the sole carbon source for the production of high cell density biomass and enhanced PHA production. From this study, it is intended that innovative insights and prospective solutions to valorizing pulp and paper mill sludge are provided, whilst simultaneously generating a value-added product.

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