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The formulation of in vitro cultured entomopathogenic nematodes
Thesis (MScAgric)--Stellenbosch University, 2023.
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867613748516093952 |
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| access_status_str | Open Access |
| author | Schlesinger, Subrina |
| author2 | Malan, Antoinette Paula |
| author_browse | Malan, Antoinette Paula Schlesinger, Subrina |
| author_facet | Malan, Antoinette Paula Schlesinger, Subrina |
| author_sort | Schlesinger, Subrina |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MScAgric)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/129309 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:41:04.390Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/129309 The formulation of in vitro cultured entomopathogenic nematodes Schlesinger, Subrina Malan, Antoinette Paula Stellenbosch University. Faculty of AgriSciences. Dept. of Conservation Ecology & Entomology. Entomopathogenic nematodes -- Formulation Insect nematodes -- Biological control Steinernema yirgalemense Biological pest control agents -- South Africa Pesticides -- Formulation -- Technological innovations UCTD Thesis (MScAgric)--Stellenbosch University, 2023. ENGLISH ABSTRACT: Entomopathogenic nematodes (EPNs), of the genera Steinernema and Heterorhabditis, are employed as biological control agents to control insect pests, due to their insect-parasitic nature. Bacteria belonging to the genera Xenorhabdus and Photorhabdus, which coexist symbiotically with EPNs, are key to their pathogenicity and biocontrol potential. For applications on a small scale, EPNs can be mass-cultured in vivo by using live insects, and in vitro for commercial use, by means of using shake flasks and bioreactors. Key to the commercial success of EPNs is the development of a formulation protocol that possesses prolonged shelf-life, without negatively affecting their pathogenicity. The EPN stage that is formulated as a product is the free-living, infective juvenile (IJ) stage, which is a morphologically adapted life stage that is geared towards long-term survival under adverse conditions in the soil. Although different formulation techniques exist, extended shelf-life is a limiting factor and, therefore, the development of high-quality EPN formulations with increased storage periods using different formulation techniques should be investigated. To prepare the nematodes physiologically before formulation, with a focus on partial anhydrobiosis, three glycerol concentrations were screened. Different carboxymethyl cellulose (CMC) concentrations were used as a novel medium for the formulation of Steinernema yirgalemense with 15% glycerol, and storage stability and survivability were determined weekly at three different temperatures for 3 weeks. Steinernema yirgalemense showed poor survival at 8 °C, regardless of the CMC concentration used. Alternatively, for the other temperatures of 14 °C and 25°C, the survival of the IJs increased with increasing CMC concentration. The highest survival rate (79%) after 21 days was obtained at 14 °C, when using 5% CMC. Microbial contamination was found to affect the quality of the formulations. Generally, contaminants compete with nematodes for oxygen, leading to a decreased survival rate of the IJs involved. To optimise the formulation, different antimicrobial products were tested for their efficacy in suppressing microbial growth. However, only the use of trans-cinnamaldehyde (TCA) prevented microbial growth. Furthermore, secondary metabolites were also investigated as an antimicrobial and yielded positive results. Lastly, formulation methods that can prolong the shelf-life of the IJs of Heterorhabditis bacteriophora and of S. yirgalemense, without negatively affecting their pathogenicity, were investigated over the course of 3 weeks, when stored at 14 °C. Such methods included the use of CMC, diatomaceous earth, cellulose fibres and the encapsulation of IJs in alginate beads. The study illustrated that the use of CMC and cellulose fibre formulations did not negatively affect the longevity of IJs, as survival rates of >87% and >90%, respectively, were achieved, while maintaining their pathogenicity over time in soil bioassays, with >95% false codling moth mortality. Furthermore, for the first time, metabolites of X. indica were investigated in an EPN formulation, as an alternative option of reducing microbial contamination. The results obtained in such a case were not promising, as a decline in the survival rate of S. yirgalemense was reported. In contrast, as the survival rate was not affected by TCA, it was, therefore, regarded as a promising option for preventing microbial growth in EPN formulations, achieving a survival rate of 87% after 21 days, when formulated with CMC. The above findings provided two EPN formulations that can be applied in the South African agriculture industry as effective biological control agents, in terms of an integrated pest management programme. AFRIKAANSE OPSOMMING: Entomopatogeniese nematodes (EPNs) van die genera Steinernema en Heterorhabditis is patogeniese insek-parasitiese nematodes wat as biologiese beheermiddels gebruik word om insek peste te beheer. EPNs het ʼn simbiotiese verwantskap met bakterieë van die genera Xenorhabdus en Photorhabdus en kan in vivo (in lewendige insekte) massa gekweek word vir kleinskaalse gebruik en in vitro (met skudflesse) vir kommersiële gebruik. Die sleutel tot hul kommersiële sukses is die ontwikkeling van ʼn formuleringsprotokol wat ʼn verlengde raklewe het, sonder om hul patogenisiteit negatief te beïnvloed. Die EPN-stadium wat as ʼn produk geformuleer word, is die vrylewende, infektiewe larvale (IJ) stadium wat ʼn morfologies aangepaste lewensfase is vir langtermyn-oorlewing in ongunstige toestande in die grond. Verskillende formulerings tegnieke bestaan wel, maar verlengde raklewe is steeds ʼn beperkte faktor en daarom moet dit ondersoek word hoe om hoë gehalte EPN formulerings met verlengde berginsperiodes te ontwikkel en te verbeter. Om die nematodes fisiologies voor te berei voor formulering, met die fokus op gedeeltelike anhidrobiose, is drie gliserol konsentrasies ondersoek. In hierdie geval is verskillende karboksiemetielsellulose (CMC) konsentrasies as medium gebruik vir die formulering van Steinernema yirgalemense tesame met 15% gliserol, terwyl bergings stabiliteit asook oorlewing weekliks by verskillende temperature oor 3 weke bepaal is. Die spesie het swak oorlewing by 8 °C getoon, ongeag van die CMC-konsentrasie. Alternatiewelik, vir die ander temperature het oorlewing van die IJs toegeneem met toenemende CMC-konsentrasie. Die hoogste oorlewingsyfer (79%) na 21 dae is verkry by 14°C wanneer 5% CMC gebruik is. Mikrobiese kontaminasie het die kwaliteit van die voorgenoemde formulerings beïnvloed. Kontaminante kompeteer met nematodes vir suurstof, wat lei tot ʼn verlaagde oorlewingsyfer van die EPNs in die formulasie. Om die formulering te optimaliseer, is verskillende antimikrobiese produkte getoets vir hul doeltreffendheid om mikrobiese groei te onderdruk. Slegs trans-cinnamaldehied (TCA) het egter suksesvol mikrobiese groei onderdruk. Verder is sekondêre metaboliete as ʼn antimikrobiese middel ondersoek wat ook positiewe resultate gelewer het. Laastens is formuleringsmetodes wat die raklewe van IJs van Heterorhabditis bacteriophora en S. yirgalemense kan verleng, sonder om hul patogenisiteit negatief beïnvloed, oor 3 weke ondersoek wanneer dit by 14°C geberg word. Dit sluit in CMC, diatoomaarde, sellulosevesels en die inkapsulering van IJs in alginaat korrels. Die studie het geïllustreer dat CMC en sellulosevesels formulerings verhoogde langlewendheid van die IJs verseker het aangesien oorlewingsyfers van >87% en >90% bereik is, terwyl hul patogenisiteit oor tyd gehandhaaf word en grondbiotiese toetse 95% vals kodling mot mortaliteit getoon het. Verder is metaboliete van X. indica vir die eerste keer in ʼn EPN formulering ondersoek as ʼn alternatiewe opsie om mikrobiese kontaminasie te verminder. Resultate in hierdie geval was nie belowend nie aangesien ʼn afname in die oorlewingsyfer van S. yirgalemense aangemeld is. Daarteenoor het TCA nie die oorlewingsyfer beïnvloed nie en is gevind dat dit ʼn geskikte opsie was om mikrobiese groei in EPN-formulerings te voorkom, wat ʼn oorlewingsyfer van 87% na 21 dae behaal het wanneer dit met CMC geformuleer was. Die bogenoemde bevindinge verskaf twee EPN formulasie-metodes, CMC en sellulose vesels, wat in die Suid-Afrikaanse landboubedryf toegepas kan word met die doel om as effektiewe biologiese beheermiddels te gebruik. Masters 2023-11-17T07:42:54Z 2024-02-20T07:02:45Z 2023-11-17T07:42:54Z 2024-02-20T07:02:45Z 2023-12 Thesis https://scholar.sun.ac.za/handle/10019.1/129309 en Stellenbosch University xvii, 122 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Entomopathogenic nematodes -- Formulation Insect nematodes -- Biological control Steinernema yirgalemense Biological pest control agents -- South Africa Pesticides -- Formulation -- Technological innovations UCTD Schlesinger, Subrina The formulation of in vitro cultured entomopathogenic nematodes |
| title | The formulation of in vitro cultured entomopathogenic nematodes |
| title_full | The formulation of in vitro cultured entomopathogenic nematodes |
| title_fullStr | The formulation of in vitro cultured entomopathogenic nematodes |
| title_full_unstemmed | The formulation of in vitro cultured entomopathogenic nematodes |
| title_short | The formulation of in vitro cultured entomopathogenic nematodes |
| title_sort | formulation of in vitro cultured entomopathogenic nematodes |
| topic | Entomopathogenic nematodes -- Formulation Insect nematodes -- Biological control Steinernema yirgalemense Biological pest control agents -- South Africa Pesticides -- Formulation -- Technological innovations UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/129309 |
| work_keys_str_mv | AT schlesingersubrina theformulationofinvitroculturedentomopathogenicnematodes AT schlesingersubrina formulationofinvitroculturedentomopathogenicnematodes |