Characterisation of nematode symbiotic bacteria and the in vitro liquid culture of Heterorhabditis zealandica and Steinernema yirgalemense

Ferreira, Tiarin (2013-03)

Thesis (PhD)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Entomopathogenic nematodes have the potential to be outstanding biocontrol agents against agricultural pest insects. Combined with their bacterial symbionts, these biocontrol agents have proven to be very effective against numerous pests. The nematodes belong to the families Steinernematidae and Heterorhabditidae, and are ideal to be used in, and integrated with, pest management systems. There is a dire need for new and innovative methods to control agricultural pests, as numerous pest insects have developed resistance against broad-spectrum insecticides. Together with the environmental impact of these insecticides and the safety aspect regarding humans and animals, the need to develop new technologies, including entomopathogenic nematodes for pest management, is high. In this study, the associated symbiotic bacteria of three entomopathogenic nematodes species were isolated, and the potential of two nematode species to be successfully mass cultured in liquid medium was evaluated. Regarding the symbiotic bacteria, results from the study showed that bacteria species from all three nematode species, Heterorhabditis noenieputensis, Steinernema khoisanae and Heterorhabditis zealandica, were novel. Heterorhabditis noenieputensis was isolated in the Mpumalanga province during a previous survey conducted in citrus orchards. The bacterium isolated from this nematode belongs to the genus Photorhabdus, and bear closest similarity (98.6%) to the type strain of P. luminescens subsp laumondii (TT01T). Photorhabdus luminescens subsp. noenieputensis subsp. nov., derives its name from the area where the nematode was sourced, namely the farm Springbokvlei, near the settlement Noenieput close to the Namibian border. Thus far, 85 Steinernema spp. have been described worldwide, including S. khoisanae which was isolated in the Western Cape province of South Africa. Four S. khoisanae strains, namely SF87, SF80, SF362 and 106-C, were used for characterisating the new bacteria from different localities in South Africa. Using the neighbor-joining method, all the strains were aligned with 97% homology to the 16S rRNA sequences of several Xenorhabdus- type strains, indicating that they belonged to the same genus. The multigene approach was used to distinguish between the Xenorhabdus spp. and partial recA, dnaN, gltX, gyrB and infB gene sequences of the various strains were analysed. The bacterium species was named Xenorhabdus khoisanae sp. nov. after the nematode from which it was isolated. The results showed that the third bacterium species, which was isolated from H. zealandica, was new. The sequence of the bacteria strain clustered with the type strains of P. temperata and P. asymbiotica, indicate that it belonged to the genus Photorhabdus. This is the first study to show that H. zealandica associates with a luminescent Photorhabdus species, rather than with the known non-luminescent P. temperata. The potential of H. zealandica and Steinernema yirgalemense mass culture in liquid was investigated. Results illustrated that H. zealandica and its P. luminescens symbiont can be successfully cultured in liquid. However, two generations occurred during the process time, instead of the desirable one-generation. The growth curve of the symbiotic bacteria during the process time was measured, in order to determine when the stationary phase was reached, with the results showing this to occur after 36 h. Therefore, the optimum amount of time required for inoculating the IJs and for aiding in maximum infective juvenile (IJ) recovery is 36 h for adding the nematodes post pre-culturing of the bacteria. Future research goals should be to increase the percentage recovery in liquid culture, which would increase the number of nematodes produced per ml, which would, therefore, reduce the processing time significantly. The results from mass culturing the second nematode species, S. yirgalemense, indicated an asynchronous nematode development in the first generation. Growth curves were performed with the symbiotic bacteria that showed the exponential phase of Xenorhabdus started after 15 h, and that, after 42 h, the stationary phase was reached, with an average of 51 × 107 cfu·ml-1. Bioassays were performed to compare the virulence between in vitro- and in vivo-produced nematodes, with the results showing that the in vitro-produced nematodes were significantly less virulent than were the nematodes produced in vivo. The success obtained with the production of S. yirgalemense in liquid culture can serve as the first step in the optimising and upscaling of the commercial production of nematodes in industrial fermenters. The last aim of the current study was to determine when Xenorhabdus reached the stationary phase, when it is grown in a 20-L fermenter, as this would be the optimum time at which to add the IJs of S. yirgalemense. Such characteristics as the effect of stationary phase conditions on the bacterial cell density and on the DO2 rate in the fermenter were investigated. The results showed that the stationary phase of Xenorhabdus was reached after 36 h at 30˚C, which took 6 h less than did the same procedures followed with the Xenorhabdus sp. cultured in Erlenmeyer flasks on orbital shakers. This is the first step toward the liquid mass culturing of S. yirgalemense in industrial-size fermenters. Data from this study indicated the optimum amount of time that is required for adding nematodes to the bacterial culture in the fermenter, and for ensuring the optimum recovery of IJs, as well as a subsequent high yield of nematodes within a minimum processing time. This is the first report of its kind to investigate comprehensively the successful liquid culture of two South African entomopathogenic nematode species for the sole purpose of evaluating potential commercialisation. Results emanating from this study could be used as groundwork in future, in combination with similar research such as culturing nematodes intensively in large fermenters.

AFRIKAANSE OPSOMMING: Entomopatogeniese nematodes het die potensiaal om as doeltreffende biologiese beheeragente teen sleutelplaaginsekte gebruik te word. Elke nematood werk interaktief met ‘n spesifieke bakterium. Entomopatogeniese nematodes, behorende tot die families Steinernematidae en Heterorhabditidae, is ideale kandidate vir gebruik in ‘n geïntegreerde plaagbestuurprogram. Tans is daar ʼn behoefte vir nuwe metodes vir die beheer van plaaginsekte, omdat meeste insekte reeds weerstand opgebou het teen bestaande plaagdoders. As gevolg van die negatiewe impak van plaagdoders op die omgewing, asook kommer oor veiligheid vir die mens en diere, is die ontwikkeling en gebruik van alternatiewe plaagbeheermiddels noodsaaklik. In die eerste deel van die studie word drie nuwe bakterie spesies geïsoleer en beskryf. Resultate van hierdie studie het aangetoon dat die bakterië spesies vanuit die nematode spesies, Heterorhabditis noenieputensis, Steinernema khoisanae, en Heterorhabditis zealandica, tot dusver onbeskryf was. Eersgenoemde, H. noenieputensis, is afkomstig van ʼn sitrusboord in die Mpumalanga Provinsie. Die bakterie hieruit geïsoleer behoort tot die genus Photorhabdus en is biologies verwant (98.6%) aan P. luminescens subsp laumondii (TT01T). Die bakterie is benaam as Photorhabdus luminescens subsp. noenieputensis nov. en is na die nematood waaruit dit geïsoleer is vernoem. Tot dusver is wêreldwyd 82 spesies van Steinernema spp. beskryf, insluitende S. khoisanae van die Weskaap provinsie. Vier bakterie isolate is van S. khoisanae, SF87, SF80, SF362 en 106-C geïsoleer. Die buur-koppeling metode was gebruik om te bepaal dat hierdie bakterie isolate tot 97% ooreenstem met verskeie isolate van Xenorhabdus se 16S rRNA DNS volgordebepalings. Om tussen Xenorhabdus spp. te onderskei is ʼn multi-geen benadering gebruik deur gedeeltelike recA, dnaN, gltX, gyrB en infB DNS basispaar volgordebepalings van die verskeie isolate te bepaal. Hierdie bakterie isolaat is soortgelyk ook vernoem as, Xenorhabdus khoisanae sp. nov., na die nematood waaruit dit geïsoleer is. Die derde onbekende bakteriële spesie is uit H. zealandica geïsoleer. Die DNS basispaar volgordebepaling van die 16S geen van SF41 toon aan dat dit in dieselfde groep as P. temperata en P. asymbiotica val en sodoende aan die genus Photorhabdus behoort. Hierdie is die eerste studie met die bevinding dat H. zealandica ook met ʼn ander bakterie spesie geassosieer kan word buiten die normale P. temperata spesie. Die tweede deel van die studie gaan oor die teling van twee nematood spesies, H. zealandica en Steinernema yirgalemense, en hulle is geëvalueer vir hulle potensiaal om geteel te word in ʼn vloeibare medium. Die resultate het gewys dat H. zealandica met sy P. luminescens simbiont suksesvol in vloeistof aangeteel kan word, ten spyte van die feit dat daar twee generasies ontwikkel het, in plaas van die meer ideale enkel generasie. Die groeikurwe van die simbiotiese bakterie was gemonitor om te bepaal wanneer die stasionêre fase bereik word. Die resultate toon dat hierdie fase na 36 uur bereik was. Dus was die infektiewe nematode larwes eers na 36 uur tot die vloeibare medium waarin die bakterie geteel was bygevoeg. Navorsing in die toekoms moet dus gefokus wees om die persentasie herwinning van die infektiewe larwes te verhoog. Dit sal daartoe lei dat meer nematodes per ml geproduseer kan word en ook die prosesseringstyd van die nematodes verminder. ʼn Tweede nematode spesie, S. yirgalemense, was ook in vloeistof geteel. Hier het ʼn asinkroniese ontwikkeling in die eerste generasie plaasgevind wat problematies is. Groeikurwes is bepaal van die bakteriële simbiont en die resultate het gewys dat die groeifase van Xenorhabdus na 15 uur in aanvang geneem het en dat die stasionêre fase bereik was na 42 uur met ʼn gemiddelde van 51 × 107 selle·ml-1. Die virulensie van nematodes wat in vitro geteel is, is vergelyk met die virulensie van nematodes wat in vivo geteel is en die resultate het getoon dat die in vitro geteelde nematodes minder virulent was. Die teling van S. yirgalemense in vloeistof was oor die algemeen meer suksesvol as die teling van H. zealandica in dieselfde medium. Die doelwit van die laaste gedeelte van hierdie studie was om te bepaal wanneer Xenorhabdus die stasionêre fase bereik wanneer dit in ʼn 20-L fermenter gekweek word. Dit bepaal sodoende die optimale tyd wanneer die infektiewe larwes van S. yirgalemense bygevoeg behoort te word. Die uitwerking van die stasionêre fase op die bakteriële selle, asook die DO2-konsentrasie in die fermenter, was geëvalueer. Resultate het gewys dat die stasionêre fase van Xenorhabdus na 36 uur bereik was, wat 6 uur korter is as toe dit gekweek is in Erlenmeyer flesse. Hierdie studie is die eerste stap om die massa teling van S. yirgalemense in industriële fermenters suksesvol te bemeester. Die data wat verkry was, het aangedui wat die ideale tydsduur sal wees om die bakteriegetalle te vermeerder voordat die nematode bygevoeg word. Hierdie is die eerste studie wat die teling van twee Suid-Afrikaanse nematode spesies omvattend in vloeistof evalueer het. Die hoof doelwit is om die potensiaal van hierdie nematode spesies, met die oog op kommersiële gebruik, te meet. Die resultate van hierdie studie kan gekombineer word met toekomstige studies in hierdie spesifieke navorsingsveld.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/80294
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