A dissertation submitted to the Faculty of Science, University of the
Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Master of Science. May 2013 / The purpose of the study was to isolate and identify indigenous nematode species and use them as model organisms for studying the responses of indigenous entomopathogenic nematodes (EPNs) to soil desiccation, soil rehydration, and also to study their behaviour with regards to the infection and location of insect larvae, Galleria mellonella in an 18cm column filled with sterilised sandy loamy soil. Two unknown nematodes were isolated from soil samples collected in Walkerville, South of Johannesburg and their sequences were found to have high affinity to Steinernema australe (accession number FJ235125) and Heterorhabditis bacteriophora isolate 56-C (FJ217351) when aligned with existing sequences in the NCBI database. Furthermore, a polymerase chain reaction was used to amplify the 16S rDNA region in order to identify bacterial symbiots of these EPNs. Symbiotic bacteria isolated from the Heterorhabditis spp had high affinity to Photorhabdus sp carborca (JF12345). Desiccation tolerance studies revealed that EPN5T and EPN3T were able to withstand desiccated conditions or continuous dehydration for 20 days. EPN5T caused 80% larval mortality after day 20 when the last row of Petri dishes was rehydrated. EPN3T caused only 60% larval mortality. All of the sandy controls gave 0% larval mortality by day 20. Comparative dose-response assays involved exposing G. mellonella insect larvae to different IJs concentrations (0, 5, 25, 100, 300 and 500 IJs/ml) were carried for both EPN5T and EPN3T. Larval mortality was recorded daily over a week. Insect mortality was high for both S. australe and H. bacteriphora, at 100IJs\ml, 300 IJs\ml and 500 IJs\ml. Mortality was observed within 48 to 96 hours and insects larvae showed signs of infection after 48hours. Significant differences were observed at EPN concentrations containing 5 IJs/ml and 25 IJs/ml. At these low IJ concentrations H. bacteriphora was able to kill 20% of the larvae by day 3 while S. australe displayed mortality only after day 4 and 5 for the two respective IJ concentrations. The significance of this observation was supported by the two-way Post-hoc analysis. Further studies were conducted to investigate the effect of soil humidity on EPNs behaviour in a column of soil. Results supported that humidity was crucial for EPNs locomotion or mobility and infection efficiency, as 100% mortality was observed in all columns by day 4. The study also investigated different IJ application concentrations in order to determine suitable field application doses. Even at the lowest IJ concentration of both EPNs 33.3% mortality was observed in each column. H. bacteriophora displayed increased mobility because high mortality percentage was obtained within 48hours in almost all three arenas of the column, proving that this species is an effective cruiser foraging deeper into the soil. S. australe was capable of cruising further down to 18cm searching for host as high mortality was observed even at the deeper or the bottom arena near the bottom of the soil column. Lastly field capacity was determined where the sandy loamy soil was saturated with water and allowed to drain so as to remove excess water from the 8 vertical columns for 48hours, resulting in a soil moisture content associated with the soil’s field capacity for water. Overall results of this study gave ideas for IJ formulation IJ storage and IJ application strategies of the identified EPNs which proved to have promising potential as biological control agents.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/12925 |
| Date | 30 July 2013 |
| Creators | Lephoto, Tiisetso Elizabeth |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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