The development of a formulation for the commercialization of entomopathogenic nematodes

[摘要] 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.zaivjeffreyense 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.

[发布机构] Stellenbosch University