56 SA FRUIT JOURNAL JUNE/JULY 2013 HORTGRO SCIENCE Introduction Entomopathogenic nematodes (EPNs) are insect parasites with the ability to control insect pests and are considered to be an ef- fective alternative to chemical insecticides within an integrated pest management programme. EPNs from the families Steinerne- matidae and Heterorhabditidae are produced on a commercial scale as bio-insecticides and have been used for the biocontrol of numerous insect pests. Two of the most important aspects for a biological control agent to be successful is the ability to be mass- produced and formulated. Freshly harvested nematodes at high concentrations will perish if stored in water, mainly because of a lack of oxygen. Therefore, nematodes should be formulated to keep them stable during storage by limiting mortality and main- taining infectivity. Apart from preserving nematodes, formula- tions assist in the handling, transport, application and enhance- ment in field efficacy of the nematodes. Despite the successes obtained using EPNs as biocontrol agents, development of for- mulations that extend shelf-life as long as a year, while ensuring delivery of high-quality nematodes, have not yet been realised on the international commercial front. The unique challenge of maintaining biological activity in formulations containing nematodes is that these living organ- isms demand sufficient levels of oxygen and moisture. When de- veloping or selecting a formulation, the requirements specified should be taken into account. To determine the requirements of specific nematode species, the physiological chemistry, ecol- ogy and behaviour of the species need to be understood. The absence of a stable and effective formulation, capable of main- taining nematode quality for a few months, might be due to the complexity of the combination of factors that needs to be known and taken into consideration during formulation development. Two distinct approaches to formulating nematodes have been used, from which a variety of formulations have branched. First- ly, the use of such inert carriers as sponge, and secondly, the use of inert carriers containing such additional functional ingredients as adjuvants and antimicrobial agents. Small-scale nematode pro- ducers generally use low-technology and -cost formulations, such as sponge, or store nematodes in aerated water under refrigeration conditions, under which conditions it can be kept for up to several months. Although inert carriers are less expensive and assist in the transport and handling of nematodes, nematodes can move around freely and, in some cases, migrate from the formulation. The high rate of activity of nematodes in such formulations leads to the depletion of stored energy reserves, which, in turn, leads to increased sensitivity to extreme temperatures and reduced infective juvenile (IJ) viability and pathogenicity. Furthermore, nematodes that migrate from the formulation, dry out and die. To overcome such problems, nematodes are increasingly being formulated into non- or semi-liquid substrates, such as absorbent clays and water-dispersible granules (WDG), which physically trap nematodes, contain metabolic inhibitors and induce partial anhydrobiosis. Anhydrobiosis is a reversible, physiologically ar- rested state of dormancy resulting from a total loss of body water. However, EPNs in the families Heterorhabditidae and Steinerne- matidae can only enter a state of partial anhydrobiosis. The development and modification of granular formulations by Capinera and Hibbard, Connick et al. and Silver et al. contrib- uted significantly to the advancement of formulation develop- ment. Steinernema IJs survived for up to six months in an anhyd- robiotic state at 4-25°C in a WDG. The nematodes were encased within the granule, consisting mainly of various types of clays and starches. The WDG formulation was the first formulation al- lowing Steinernematids to survive for up to six months at 25°C. Unfortunately, the shelf-life of heterorhabditids is much shorter than that of steinernematids, due to higher sensitivity to desicca- tion and temperature, different foraging behaviour and a faster rate of lipid use. The few commercially produced heterorhab- ditid formulations also require constant refrigeration (4-6°C) to ensure nematode storage stability. Due to the extensive advances that had been made in at- tempting to gain a better understanding of different nematode species requirements, formulations can be developed to con- form, to an extent, to specific requirements. However, variation in nematode stability during long-term storage still occurs with- in such formulations. Accordingly, effective quality control (QC) measures should be set in place to ensure nematode stability in formulations, as well as during the entire production process. QC is defined by Grewal and Peters as “a system of maintain- ing standards in manufactured products by testing a sample of the output against the specification”. Nematode virulence is one of the most important indicators of nematode quality, and sev- eral measures exist to determine it. With the most effective and Storing requirements of entomopathogenic nematodes CAROLIEN VAN ZYL & ANTOINETTE P. MALAN & MATTHEW ADDISON Department of Conservation Ecology and Entomology, Faculty of AgriSciences, Stellenbosch University A comparative study was conducted to determine the storage stability of Heterorhabditis zealandica in modified Pesta granules and coconut fibres compared to the control, which represented the suspension of nematodes in distilled water. Nematode survival within each formulation was determined after 24 h and thereafter at 7, 14 and 21 day intervals and formulations were stored at 4°C. Carolien van Zyl