The development of a formulation for the commercialization of entomopathogenic nematodes

Kagimu, Nicholas (2018-12)

Thesis (PhD)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis, and their associated symbiotic bacteria Xenorhabdus and Photorhabdus, are efficient biological control agents, due to their ease of culture, high rate of fatality caused against key pests, and safety. However, the large-scale commercial utilisation of EPNs as biological control agent, in integrated pest management (IPM) programmes, is limited by their finite shelf life, both in storage and formulations. Thus, efficient formulation of EPNs is essential in IPM strategies. To achieve this, nematode survival mechanisms, in terms of heat and cold tolerance, desiccation, osmotic stress / water activity (aw), hypoxia, and energy reserves, or in formulation, and their influence on the formulation of EPNs, as well as in maintaining the quality of EPN products, should be investigated. In this case, South African EPN species, including Steinernema yirgalemense, S. jeffreyense and Heterorhabditis bacteriophora, were investigated regarding their role in formulations according to various formulation techniques. These included the encapsulation of the infective juveniles (IJs) in alginate beads, as well as the use of diatomaceous earth (DE) at 6°C, 14°C and 25°C, for 4 weeks. The beads successfully retained most of the IJs with a longer storage capacity, while the survival rate for DE was still high (80%) by the fourth week. The three EPN species researched revealed poor survival and loss of virulence at low temperatures, for both formulations. The optimisation process involved testing for the viability of S. yirgalemense at room temperature, and at a higher density in DE after 4 weeks, in addition to the direct effect of antifungal agents on its efficacy. Microbial contamination unequivocally lowers the quality and shelf life of EPNs in formulations. Peroxyacetic acid (PAA), trans-cinnamic acid (TCA) and nipagin were measured as antifungal agents in the study. A decline in the survival rate and pathogenicity of S. yirgalemense, due to PAA, was reported. In contrast, TCA and nipagin did not affect the survival rate and pathogenicity of S. yirgalemense. The shelf life of IJs stored in DE formulation at room temperature improved, when measured against the 80% mean survival rate of S. yirgalemense in week 4 at 25°C. There is lack of information on the respiratory physiology of the nematode/bacterium complex of EPNs during production, storage, and formulation. Equally important, low oxygen supply jeopardises their survival. The present study determined, by means of basal measurement, the specific oxygen consumption rate (OCR) of the IJs of S. yirgalemense, S. jeffreyense, and H. bacteriophora, using fibre-optic sensors. The results showed that nematode size inversely influences its OCR, with smaller nematodes, with a higher surface-area-to-volume ratio than larger nematodes, having a higher OCR. Steinernema Stellenbosch University https://scholar.sun.ac.za iv jeffreyense and S. yirgalemense did not significantly differ from each other in terms of the results obtained, probably due to their proximity in size, with the former being slightly larger than the latter, but they differed significantly from H. bacteriophora. Water activity (aw), as a determinant of microbial contamination, as well as desiccation, was investigated in relation to the quality and shelf life of EPNs in formulation. In the current study, the concept of determining moisture content at the corresponding aw-values, using the Guggenheim-Anderson-Boer (GAB) isotherm model, has been studied concerning DE, as well as the survival of S. yirgalemense. Scanning electron microscopy was employed to determine the effect of DE on S. jeffreyense during storage in formulation. A decline in the survival rate of S. yirgalemense at high aw-values, due to bacterial sporulation and toxin production, was reported. Scanning micrographs depicted a strong desiccative effect of DE on S. jeffreyense, exceeding rejuvenation on the addition of water. Desiccation was random and limited in terms of distribution throughout the sample. Lastly, but of equal importance, because virulence remains the key standard for the measurement of nematode quality and is often determined through using either one-on-one or sand-well bioassays, which are costly in terms of laboratory consumables and time, new alternatives have been investigated. The potential for quality control of formulated S. jeffreyense and S. yirgalemense in DE, and the characterisation of different species using attenuated total reflectance (ATR), in conjunction with Fourier-transform infrared spectroscopy (FTIR) and hyperspectral imaging (HSI) tools, have been investigated. Such tools have a proven wide application in other fields of research, due to their quick, non-destructive and effective quality control techniques. Results report, for the first time, the use of ATR-FTIR spectral analysis in detecting chemometric changes in the formulated EPN product, and changes occurring over time, during storage. Such changes are mainly for purposes of nematode survival, due to environmental stresses. HSI tools were able to differentiate between variables, in terms of differences in nematode densities in the formulated sample. For EPN characterisation, the study reports close similarities among the species, as detected by the ATR-FTIR. The above findings provide a much-required working formulation for the commercial application of EPN. However, much research still needs to be done, especially in areas such as the use of fibre-optic sensors for oxygen measurement, ATR-FTIR and HSI in quality control to draw realistic and meaningful conclusions.

AFRIKAANSE OPSOMMING: Entomopatogeniese nematodes (EPNs) van die genera Steinernema en Heterorhabditis, tesame met hul geassosieerde simbiotiese bakterieë, is welbekende effektiewe biologiese beheer agente. Dit word toegeskryf aan die gemak waarmee hul aangeteel kan word, hul sukses in die beheer van belangrike pes insekte, asook hul veiligheid. Die kommersiële gebruik van EPNs as ʼn biologiese beheermiddel in geïntegreerde pes beheer (GPB) programme word egter gekniehalter deur hul beperkte rakleeftyd na formulering en opberging. Die effektiewe formulasie van EPNs is dus noodsaaklik vir GPB strategieë. Om die formulasie van EPNs te optimaliseer, moet die oorlewingsmeganismes van nematodes ondersoek word in terme van hitte en koue toleransie, uitdroging, osmotiese stres / water aktiwiteit (aw), hipoksie en energie reserwes, asook die invloed daarvan op die formulasie van EPNs en die handhaaf van die kwaliteit van EPN produkte. In hierdie studie was drie Suid-Afrikaanse EPN spesies, Steinernema yirgalemense, S. jeffreyense en Heterorhabditis bacteriophora ondersoek vir faktore wat hul formulering sal beïnvloed, volgens verskillende formulasie tegnieke. Die twee tegnieke wat ondersoek was, was die formulering van infektiewe larwes (ILs) in alginaat korrels, asook die gebruik van diatomiet, by 6°C, 14°C en 25°C, vir 4 weke. Die korrels het die meeste van die ILs suksesvol binne gehou en ook ʼn langer bergingskapasiteit gehad, terwyl die oorlewings van die nematodes in diatomiet steeds hoog was (80%) teen die vierde week. Al drie EPN spesies het lae oorlewingsgetalle en verminderde infektiwiteit getoon by laer temperature, in beide formulasies. Die optimaliseringsproses was gefokus daarop om die lewensvatbaarheid te toets van S. yirgalemense by kamertemperatuur, asook in diatomiet by ‘n hoër temperatuur na 4 weke, tesame met die direkte effek van teen-swam middels op die effektiwiteit van die nematode. Mikrobiese kontaminasie verlaag die kwaliteit en rakleeftyd van EPNs in formulasie. Peracetic suur (PAS), trans-kaneel suur (TKS) en Nipagin was die teen-swam middels wat getoets was in hierdie studie. Resultate het getoon dat PAS gelei het tot ʼn afname in die oorlewing en infektiwiteit van S. yirgalemense. TKS en Nipagin het egter nie oorlewing of infektiwiteit beïnvloed nie. Die rakleeftyd van ILs wat gestoor was in diatomiet by kamertemperatuur het verbeter, teenoor die 80% gemiddelde oorlewingskoers van S. yirgalemense in week 4 by 25°C. Daar is ʼn tekort aan informasie oor die respiratoriese fisiologie van die nematode/bakterieë kompleks van EPNs gedurende produksie, berging en formulering. Dit is wel bekend dat lae suurstof vlakke in die formulasie die oorlewing van die nematodes benadeel. Met gebruik van veseloptiese sensors en basale metings, is die spesifieke koers waarteen suurstof verbruik word deur die ILs van S. yirgalemense, S. jeffreyense en H. bacteriophora, bepaal. Resultate het getoon dat nematode grootte ʼn omgekeerde effek het op suurstofverbruik, wat beteken dat kleiner nematodes wat ʼn hoër oppervlak-tot-volume verhouding gehad het, hoër suurstofverbruik getoon het. Daar was nie ʼn beduidende verskil in die suurstofverbruik resultate van S. jeffreyense en S. yirgalemense nie, moontlik omdat hulle bykans dieselfde grootte is, met S. jeffreyense wat effens groter is as S. yirgalemense. Albei het wel beduidende verskille getoon in vergelyking met die suurstofverbruik van H. bacteriophora. Water aktiwiteit (aw) as ʼn bepaler van mikrobiese kontaminasie, sowel as uitdroging, was ondersoek met betrekking tot die kwaliteit en rakleeftyd van EPNs in formulasie. In die huidige studie, is die konsep van die bepaling van vog inhoud by ooreenstemmende aw waardes ondersoek in diatomiet, deur gebruik te maak van die Guggenheim-Anderson-Boer (GAB), sowel as die oorlewing van S. yirgalemense. Skandeer elektron mikroskopie is gebruik om die effek van diatomiet op S. jeffreyense te ondersoek in berging, sowel as in formulasie. Die resultate het ʼn afname getoon in die oorlewing van S. yirgalemense by hoë aw-waardes, as gevolg van die groei van bakterieë en toksien produksie. Skandeer mikrograwe het getoon dat diatomiet ʼn sterk uitdrogingseffek het op S. jeffreyense, sterker as die herstelproses van die nematode wat plaasgevind het wanner water bygevoeg was. Uitdroging was lukraak en beperk in terme van verspreiding deur die monster. Die laaste deel van die studie was gefokus op die infektiwiteit van die nematode. Infektiwiteit is een van die belangrikste faktore in die bepaling van die kwaliteit van nematodes en word gewoonlik getoets met een-tot-een of sand biotoetse. Hierdie metodes verg egter baie tyd in terme van laboratorium verbruiksgoedere, wat gelei het tot die ontwikkeling van nuwe metodes. Die potensiaal vir die kwaliteitsbeheer S. jeffreyense en S. yirgalemense, geformuleer in diatomiet, asook die karakterisering van verskillende spesies is ondersoek, met gebruik van verswakte totale refleksie (VTR) tesame met Fourier-transformasie infrarooi (FTIR) spektroskopie asook hiperspektrale beeldanalise (HSB) tegnieke. Hierdie metodes word algemeen gebruik in ander navorsingsvelde, as gevolg van hul vinnige, nie-afbrekende en effektiewe kwaliteitsbeheer tegnieke. Die resultate toon dat VTR-FTIR spektrale analise vir die eerste keer gebruik was om chemometriese veranderinge op te spoor in die geformuleerde EPN produk, sowel as veranderinge oor tyd gedurende berging. Die funksie van sulke veranderinge is meestal vir die oorlewing van nematodes, as gevolg van omgewingstres. HSB tegnieke was suksesvol gebruik om te onderskei tussen veranderlikes, in terme van verskille in nematode digthede in die geformuleerde voorbeeld. In terme van die karakterisering van EPNs soos ondersoek deur die VTR-FTIR, het die resultate nabye ooreenkomste tussen spesies getoon. Die bevindinge van die studie verskaf ʼn belangrike werkende formulasie vir die kommersiële gebruik van EPNs. Baie navorsing moet egter steeds gedoen word, veral in areas soos die gebruik van veseloptiese sensors vir die meet van suurstofvlakke, VTR-FTIR en HSB in kwaliteitsbeheer, om betekenisvolle gevolgtrekkings te kan maak.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/104858
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