3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item The isolation, characterisation and granular formulation of native entomopathogenic nematodes in South Africa(2020) Didiza, Linda TabileEntomopathogenic nematodes (EPNs) are obligate pathogens in nature and are used in the biological control of insect pests of agricultural crops. Nematodes are simple, colourless, unsegmented, bilaterally symmetrical, pseudocoelomic, triploblastic, worm-like animals, enclosed within a tough, elastic, and flexible, chitin containing cuticle. Entomopathogenic nematodes belonging to the genera Steinernema and Heterorhabditis are symbiotically associated with insect pathogenic bacteria belonging to the genera Xenorhabdus and Photorhabdus, respectively. The focuses of this research was to isolate, identify, phylogenetically analyse, and formulate entomopathogenic nematodes indigenous to South Africa. The entomopathogenic nematodes were isolated from soil samples originally collected in Brits and Walkerville. Subsequent to their collection the soil samples had been stored for a prolonged period (> 2years) in a dehydrated state. To isolate EPNs the soil samples were rehydrated and baited with Galleria mellonella larvae. The methods used for the identification and phylogenetic analysis of the isolated nematodes involved genomic DNA extraction, PCR amplification of 18S rDNA and Sanger sequencing of the18S rDNAamplicons. The entomopathogenic nematodes that were isolated included Heterorhabditis bacteriophora isolate 56-C and a new previously uncharacterised Steinernema species. Another focus of the research was to isolate and identify the bacterial symbionts of the isolated entomopathogenic nematodes. The methods used for the isolation of the bacterial symbionts involved haemolymph extraction from the infected larvae and homogenisation of sterilized infective juveniles. The methods used for the identification and phylogenetic analysis of the isolated entomopathogenic bacterial species involved total genomic DNA extraction, PCR amplification of 16S rDNA,and Sanger sequencing of the16 rDNA amplicon. The isolated bacteria were identified as Xenorhabdus sp VP and Photorhabdus luminescens subspecies sonorensis Carbonca. The study also showed that the isolated entomopathogenic nematodes had survived in soils that had been kept in a state of complete dehydration for a prolonged period. The survival of infective juveniles in the desiccated soil could have been due to the induction of anhydrobiosis or dehydration tolerance. Thus, the aim of this study also involved an investigation into the possible induction of anhydrobiosis or dehydration toleration in formulated infective juveniles by regulating the rate of moisture loss from various formulation media used. In the study, the Heterorhabditis bacteriophora isolate 56-C was formulated in different hydroscopic or water-absorbing powders which included diatomaceous earth, crystalline cellulose and clay. Results showed that the rate of moisture loss from the formulation media had a significant impact on the viability of formulated infective juveniles. The finding was interpreted as evidence supporting the hypothesis that the induction of anhydrobiosis or dehydration tolerance depends strongly on the rate of dehydration. Both the comparative morphometric characterization and phylogenetic analysis of the previously uncharacterised Steinernema sp confirmed that it was a new species of Steinernema. The results showed that the average length of the infective juveniles was 975μm with a standard deviation of 72μm, therefore, the species fell under the glaseri-group of Steinernema. Phylogenetic analysis showed that the new species did not form a clade with any of the local Steinernema species, therefore, confirming that the species isolated from Brits was, in fact, a new speciesItem The impact of organic compost on the infectivity and virulence of entomopathogenic nematodes and their bacterial symbionts as biocontrol agents(2019) Khumalo, Nolwandle NolenA safer and greener alternative to chemical pesticides includes the use of entomopathogenic nematodes (EPNs) and their endosymbiotic bacterial pathogens as biocontrol agents. Entomopathogenic nematodes of the genera Steinernema and Heterorhabditis cause disease in insect pests, killing them within 48 hours, with the help of their highly virulent insect pathogenic bacterial symbionts belonging to the genera Xenorhabdus and Photorhabdus, respectively. The efficacy of EPNs in the field is affected by abiotic and biotic factors, thus understanding the soil ecosystem and its interaction with EPNs will aid in improving the application of EPNs in many agricultural soils post application. The research project focused on isolating and identifying locally adapted EPNs and their bacterial symbiont isolated from the North West province in the Magaliesburg region. Investigating factors affecting the survival, virulence, and persistence of EPNs in the soil, by the use of adding soil amendments such as organic compost namely mulch, organic compost, professional potting mix, organic lawn dressing mixed with loamy soil and coarse river in soils undergoing progressive dehydration. The effects of different soil composition on the behaviour and host-seeking efficiency within the soil profile and EPN response to desiccation tolerance was also investigated. EPNs were retrieved and harvested using the soil baiting technique with Galleria mellonella and White trap method respectively. PCR amplification of the 18S rDNA and 16S rDNA sequencing of the PCR amplicon was used to establish the phylogenetic affinities of EPN isolates and their bacterial symbiont using GenBank sequence database respectively. The presumptive Heterorhabditis species and Steinernema species isolated were found to show high affinity to H. bacteriophora (MG334257.1) and S. khoisanae (MH697401.1) respectively. The symbiont bacteria only isolated of H. bacteriophora was identified as Photorhabdus luminescence subsp. laumondi (KT963833.1). The use of organic compost did not have any detrimental effect on EPNs. The Mulch soil amendment resulted in the highest insect mortality rate across different application concentration (mass: mass basis) when mixed with loamy soil. It was observed that virulence and efficacy decreased over 7 days of progressive dehydration. In soil profile larvae locating behavioural studies, H. bacteriophora was found to be a cruise predator (search and forage) in search of a new host. Soil composition affected host locating ability of H. bacteriophora infective juveniles (IJs). H. bacteriophora IJs were able to survive desiccation tolerance with resuscitation by rehydration with water for a period of 8 weeks however infectivity and survival of IJs to locate the host were affected. Overall the type of compost used based on its feedstock iii composition, the concentration of nutrients and organic matter mixed with a specific type of soil texture had a significant effect on the infectivity and survival of H. bacteriophora IJs in the soil. In conclusion soil physical structures affects EPNs infectivity post application and the knowledge obtained will be used to improve soil application strategies and formulation products so that EPNs can be used as commercially viable biocontrol agents.