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In vitro liquid culture of the entomopathogenic nematode, Steinernema jeffreyense
Thesis (MScConsEcol)--Stellenbosch University, 2019.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2019
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| _version_ | 1867614083698655232 |
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| access_status_str | Open Access |
| author | Murray David, Dunn |
| author2 | Malan, Antoinette P. |
| author_browse | Malan, Antoinette P. Murray David, Dunn |
| author_facet | Malan, Antoinette P. Murray David, Dunn |
| author_sort | Murray David, Dunn |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MScConsEcol)--Stellenbosch University, 2019. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/106971 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:46:23.902Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2019 |
| publishDateRange | 2019 |
| publishDateSort | 2019 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/106971 In vitro liquid culture of the entomopathogenic nematode, Steinernema jeffreyense Murray David, Dunn Malan, Antoinette P. Stellenbosch University. Faculty of Agrisciences. Dept. of Conservation Ecology and Entomology. Entomopathogenic nematodes -- Species -- South Africa Steinernema jeffreyense Plant nematodes -- Biological control Biological pest control agents Bioreactors Integrated pest management UCTD Thesis (MScConsEcol)--Stellenbosch University, 2019. ENGLISH ABSTRACT: Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis are efficient and safe biological control agents of many agriculturally important above-ground and soil-dwelling insect pests. The key to their success as biological control agents is their symbiotic association with the bacteria of the genera Xenorhabdus and Photorhabdus, respectively. EPNs, which have proven to be effective when used in an integrated pest management programme, can be used in conjunction, or in tandem, with chemical pesticides. Essential to the EPN commercialisation potential, is the fact that they can be mass-produced, using large-scale industrial bioreactors and in vitro liquid culture technology. The first steps in the mass production of EPNs are taken with small shake flasks of 250- ml capacity each, with a standard protocol for mass production being developed and optimised. The optimised protocol is then upscaled to desktop bioreactors, and, finally, to industrial-sized bioreactors, with each step having an optimisation phase. This report investigated the mass production potential of the endemic South African EPN species, Steinernema jeffreyense. Due to the initial success of mass-producing S. jeffreyense in 250-ml shake flasks, and the development of a standard protocol, the method of production was optimised and upscaled to a small, custom-made glass desktop bioreactor vessel. Optimisation was measured using the recovery of infective juveniles (IJs), and the yield of IJs after 14 days post-IJ inoculum. The first stage of optimisation focused on the mechanical aspects of the mass production in shake flasks, or on those aspects unrelated to the diet or complex liquid media. The aspects concerned were the volume of liquid media, the difference between the two different shaking platforms, and the initial IJ inoculum density. Yields of between 90 000 to 150 000 IJs/ml were achieved. Moreover, the pathogenicity of the in vitro-cultured IJs was compared with that of those cultured using in vivo methods. The second part of the optimisation focused on the dietary requirements of the nematodes, investigating new additions to the liquid complex medium of agar and glucose, as well as the impact of different initial bacterial inoculum densities. The final aspect of optimisation in shake flasks investigated the growth dynamics of the symbiotic bacteria, Xenorhabdus khoisanae, using optical density. A growth curve was developed to find the optimal time of bacterial inoculation. Finally, the optimised protocol developed was upscaled and adapted for a small, custom-made glass desktop bioreactor vessel. Initial attempts were unsuccessful, due to contamination issues and an ineffective design. The design of the bioreactor was then optimised, and IJs of S. jeffreyense were successfully cultured, albeit with low yields. Future prospects include further optimising of the bioreactor design and growth conditions, and additional upscaling to bioreactors of 100 L and 1000 L each. AFRIKAANSE OPSOMMING: Entomopatogenies nematodes (EPNs) van die genera Steinernema en Heterorhabditis is effektiewe en veilige biologiese beheer agente van verskeie grondlewende en bogrondse insek peste van groot belang in landbou. Die sleutel tot hul sukses as biologiese beheer agente, is hul simbiotiese assosiasie met die bakterieë van die genera Xenorhabdus en Photorhabdus, onderskeidelik. EPNs is effektief as deel van ʼn geïntegreerde pes beheer program en kan in samewerking of in tandem met chemiese plaagdoders gebruik word. ʼn Noodsaaklike faktor in die kommersialisering potensiaal van EPNs is die feit dat massaproduksie moontlik is, met die gebruik van grootskaalse industriële bioreaktore en in vitro vloeistof-kultuur-tegnologie. Die eerste stappe in die massaproduksie van EPNs word geneem met klein 250 ml skudflesse en die ontwikkeling en optimalisering van ʼn standaard protokol vir massaproduksie. Die geoptimaliseerde protokol word dan opgeskaal na klein bioreaktore en uiteindelik na industriële-grootte bioreaktore, met elke stap wat ʼn optimalisering fase vereis. Hierdie studie het die massaproduksie potensiaal van die endemiese Suid-Afrikaanse spesie, Steinernema jeffreyense, ondersoek. Nadat groot sukses behaal is met die massaproduksie van S. jeffreyense in 250 ml skudflesse, asook met die ontwikkeling van ʼn konstant suksesvolle standaard protokol, was die produksie metode geoptimaliseer en opgeskaal na ʼn klein, glas-bioreaktor wat spesiaal gemaak is vir die doel. Optimalisering was gemeet aan die aktivering van infektiewe larwes (ILs), asook die hoeveelheid ILs wat geproduseer is 14 dae na inokulasie met ILs. Die eerste fase van optimalisering was gefokus op die meganiese aspekte van die massaproduksie in die skudflesse, of op die aspekte wat nie verband hou met die dieet of die saamgestelde vloeistof media nie. Die aspekte waarop daar gefokus was, was die volume van die vloeistof media, die effek van die twee verskillende skudplatforms en die aanvanklike IL inokulum digtheid. Infektiewe larwe opbrengste van tussen 90 000 en 150 000 ILs/ml is bereik. Die patogenisiteit van die larwes wat in vitro geteel is, is daarna vergelyk met die wat in vivo geteel is. Die tweede fase van die optimalisering was gefokus op die voedings vereistes van die nematodes, die byvoeging van agar en glukose by die saamgestelde medium, sowel as die impak van verskillende aanvanklike bakteriële inokulum digthede. Die finale aspek van die optimalisering in skudflesse was die ondersoek van die groei dinamika van die simbiotiese bakterium, Xenorhabdus khoisanae, deur die optiese digtheid te meet. ʼn Groeikurwe was ontwikkel om die perfekte tydstip te vind vir die inokulasie van bakterieë. Laastens was die geoptimaliseerde protokol wat ontwikkel is, opgeskaal en aangepas vir ʼn klein, glas bioreaktor wat spesiaal gemaak is vir die doel en op ʼn toonbank kan staan. Die eerste probeerslae was onsuksesvol as gevolg van kontaminasie en ʼn oneffektiewe ontwerp. Die ontwerp van die bioreaktor was toe geoptimaliseer en ILs van S. jeffreyense was suksesvol geproduseer, ten spyte van lae getalle. Toekomstige vooruitsigte behels die verdere optimalisering van die bioreaktor ontwerp en die groei omstandighede, sowel as die verdere opskaal na bioreaktore van 100 L en 1000 L. Masters 2019-11-19T13:33:42Z 2019-12-11T06:41:02Z 2021-03-01T03:00:11Z 2019-12 Thesis http://hdl.handle.net/10019.1/106971 en_ZA Stellenbosch University xx, 156 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Entomopathogenic nematodes -- Species -- South Africa Steinernema jeffreyense Plant nematodes -- Biological control Biological pest control agents Bioreactors Integrated pest management UCTD Murray David, Dunn In vitro liquid culture of the entomopathogenic nematode, Steinernema jeffreyense |
| title | In vitro liquid culture of the entomopathogenic nematode, Steinernema jeffreyense |
| title_full | In vitro liquid culture of the entomopathogenic nematode, Steinernema jeffreyense |
| title_fullStr | In vitro liquid culture of the entomopathogenic nematode, Steinernema jeffreyense |
| title_full_unstemmed | In vitro liquid culture of the entomopathogenic nematode, Steinernema jeffreyense |
| title_short | In vitro liquid culture of the entomopathogenic nematode, Steinernema jeffreyense |
| title_sort | in vitro liquid culture of the entomopathogenic nematode steinernema jeffreyense |
| topic | Entomopathogenic nematodes -- Species -- South Africa Steinernema jeffreyense Plant nematodes -- Biological control Biological pest control agents Bioreactors Integrated pest management UCTD |
| url | http://hdl.handle.net/10019.1/106971 |
| work_keys_str_mv | AT murraydaviddunn invitroliquidcultureoftheentomopathogenicnematodesteinernemajeffreyense |