There are many species and strains of Steinernema and Heterorhabditis and of their respective symbionts, Xenorhabdus and Photorhabdus. Selective use of the most appropriate nematode species/strain and/or selective utilization of the differing characters of these species and strains can temporarily decrease target insect populations. Genetic improvement and the exchange of bacterial symbionts may lead to improved biological control of insects. Enormous advances have occurred in the in vitro, mass production of entomopathogenic nematodes, utilizing industrial fermentation technology, which has enabled the production of the very large numbers of nematodes required for field applications. As well, improved storage, transport and application methods for the nematodes have ensured more consistent insect control. Nevertheless, understanding of the biology of Steinernema and Heterorhabditis in the soil is very incomplete. The attraction of Steinernema spp. to their target hosts is complex, and secretions of plants and the microflora, in particular, appear to change the nematode's behaviour.
The beneficial characteristics of this nematode-bacterium complex to agroforestry are not limited to that of controlling insects. Many secondary metabolites produced by the bacterial symbionts have significant bioactive properties. Some of these compounds are antimycotic (e.g., xenocoumacins, xenorhabdins, nematophin), antibiotic (e.g., xenorxides, indoles), nematicidal (e.g., stilbenes) and insecticidal. The effective field use of some of these substances or of their derivatives can now be realized, and may play a key role when integrated in pest management strategies in the agroforestry industries.

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The infective, third stage juveniles (IJs) of entomopathogenic rhabditid nematodes are relatively poor at surviving desiccation when compared, for example, with the survival/infective stages of many plant parasitic nematodes. Nevertheless, some species and strains of entomopathogenic nematodes are known to be much better at surviving desiccation than others and such observations may have important implications for the improved formulation, storage and application of such nematodes as biopesticides. The present paper reviews the potential role of the cuticle, neutral and phospholipids, and carbohydrates in the survival of IJs under anhydrobiotic conditions.

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Entomopathogenic nematodes and their symbiotic bacteria have their own guild of natural enemies, but quantitative data to document the long-term effects of these natural enemies on entomopathogenic nematode populations are scant. Because entomopathogenic nematodes occur in the soil, it is difficult to quantify the effects of these natural enemies in the soil habitat. However, even in non-soil situations, the natural enemies of the nematode/bacterium complex are not well known. Thus, the only documented pathogens of the symbiotic bacteria, Xenorhabdus and Photorhabdus, are phages that can cause lysogeny and reduce the developing nematodes' food supply or negatively affect the nematode/bacterium's efficacy. Protozoan natural enemies (i.e., three microsporidian species) infect steinernematid nematodes, and one species adversely affects the longevity of the infective juveniles. In the nematodes' more natural habitat, nematode-trapping fungi, such as Arthrobotrys oligospora, Monacrosporium eudermatum and Geniculifera paucispora, can reduce infective juvenile populations in pasteurized and raw soil. With the endoparasitic fungus, Hirsutella rhossiliensis, higher mortality of Steinernema glaseri occurred compared with Heterorhabditis bacteriophora. Differential susceptibility to the fungus may be associated with the retention of the second-stage cuticle by H. bacteriophora. Invertebrate predators including mites and collembolans feed on the infective juveniles of entomopathogenic nematodes. Scavengers, especially several ant species feeding on nematode-killed insects, can adversely affect the developing entomopathogenic nematodes. Ants readily feed on steinernematid-killed insects but tend to avoid heterorhabditid-killed insects. Photorhabdus bacteria associated with heterorhabditid nematodes appear to produce a deterrent factor that prevents the ants from feeding on the cadaver.

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Since soil ecosystems are constructed of complex biological webs, it has been generally difficult to control soil pests with a single biological agent. Combinations of two or more agents tend to compensate each other for any deficiency or may have a synergistic effect. This paper discusses the possibilities of the integrated control of soil pests by the following combinations of biological agents: 1) Entomopathogenic nematodes with different searching strategies against two or more target insects. 2) Entomopathogenic and fungivorous nematodes against insect pests and soil-borne fungal diseases. 3) Entomopathogenic nematodes and microbial agents, such as Bacillus thuringiensis (Bt) or Pasteuria penetrans (Pp), against one target insect and plant parasitic nematodes. 4) Fungivorous nematodes and microbial agents, such as Pp, against soil fungal diseases and plant parasitic nematodes. 5) Beneficial nematodes and microbial agents against insect pests, fungal diseases, and plant nematodes. For the actual practice, discussions will be made on the following issues: 1) Compatibility of the employed bioagents with other agents. 2) Sensitivity of one biological agent to the additives or activators employed for the other. 3) Optimum temperature range for combined application. 4) Persistence of the employed agents in soils.

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The use of nematodes as biological control agents of insect pests in Korea has a short history with concentrated efforts being made since 1986. The major focus of the research has been on the obligate parasitic nematodes in the families Mermithidae, Steinernematidae, and Heterorhabditidae. Research with Agamermis unka, a naturally-occurring mermithid parasite of the brown planthopper Nilaparvata lugens in rice, has shown that it is the most important biological control agent of this insect in Korea. In some years, this mermithid will suppress the brown planthopper effectively, especially when this insect population is low. In other years when the brown planthopper populations are extremely high, chemical pesticides are required for insect suppression. The mermithid is compatible with some chemical pesticides, and through proper rice management practices, it may be conserved and work in concert with pesticides to effect integrated control of the brown planthopper. Research with the steinernematid and heterorhabditid nematodes has shown that they also occur naturally in soils throughout the country. Because these nematodes have a wide host range, they have been tested against several Korean vegetable, rice, turfgrass, and forest insects. Current research emphasis has been to increase the efficacy of these nematodes against turfgrass insects. Although the steinernematids and heterorhabditids are not commercially available in Korea, the promising results against a number of insect pests may spur private industry to pursue their development in the near future.

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Entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae are effective in the control of a wide range of soil-inhabiting insects occupying cryptic habitats. Recent field tests for commercialization with nematode-based products, for instance Steinernema carpocapsae against to hunting billbug, Sphenophrus venatus vestitus (turf), lawn grass cutworm, Spodoptera depravata (do.), bluegrass webworm, Parapediasia teterrella (do.), black cutworm, Agrotis ipsilon (do.), black vine weevil, Otiorhynchus sulcatus (potted flowers), common cutworm, Spodoptera litura (strawberry), S. glaseri to white grubs (turf.) and S. kushidai towhite grubs (do.) showed favorable results. Preliminary trials of S. carpocapsae on sweet potato weevil Cylas formicarius (sweet potato), the west Indian sweet potato weevil, Euscepes postfasciatus (do.) and of S. feltiae on darkwinged fungus gnats, Lycoriella mali (mushroom) have been conducted. Hunting billbug is an exotic insect pest from the U.S. and entered Japan in the 1970's. This insect has now spread to over 400 of the 2000 Japanese golf courses. This is not a serious pest in the U.S., but it causes severe damage for Japanese lawngrass (Zoysia grass). In 1993 Biosafe^® (S. carpocapsae) was registered for turf pest control in Japan and is very effective against larvae of the hunting billbug compared with chemical insecticides.

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The potential world market for entomopathogenic nematodes (ENs) could reach several hundred million $US per annum if these organisms are optimally developed and exploited. Currently, the market for ENs represents only a tiny fraction of this potential (even though ENs are already second only to Bacillus thuringiensis (Bt) in biopesticide sales).
However, a continually expanding research effort around the world has developed a sound knowledge base from which to focus on the range of basic, strategic and tactical R & D that is now required to develop ENs much further. By 2005 we should see the current world collections of a few hundred isolates, representing some 30 or 40 species, expanded to many thousands of isolates of over 100 species. This will have helped to increase substantially the range of targets susceptible to ENs and, because fewer of the new, superior nematodes will be required, greatly reduce application rates and therefore costs. It will also doubtless have been possible to improve good strains still further by cross breeding, artificial selection and even genetic manipulation, as well as by mixing and matching various ENs and symbiotic bacteria. Because of IP considerations, molecular biology will become increasingly important for genetically characterizing strains of both nematodes and bacteria and will be used to help screen large numbers of isolates and in field trialing mixtures of strains. A large increase in numbers of patents concerning the use of particular EN species and strains for the control of specific insect pests can be expected. A new series of application technologies will have been developed to deliver ENs as close to their insect targets as possible in the most favorable environment; some will enable improved soil penetration, others will involve baiting and there may well be better means of using ENs to control some leaf eating insects.
In addition to major cost cuttings achieved using superior nematode strains and improved application technologies, further, though not so significant, reductions can be expected from improved efficiency in production methods. Once the most appropriate EN has been developed for a large market segment it will doubtless most efficiently be produced using large-scale liquid fermentation, carefully tailored for that particular species/strain. However while species/strains are being continually improved and superseded the more flexible solid culture methods will still have a part to play. By 2005, it can be expected that there will be available some EN products with a shelf life approaching one year at ambient temperatures and others with at least 6 months. These improved shelf lives will result from careful attention to methods of induction into cryptobiosis and resulting biochemical and respiratory rate modifications specific for each species, and possibly even each strain. Packaging that enables maintenance of water activity while allowing adequate oxygen exchange will have become increasingly important as shelf life is extended. For all aspects of culture maintenance, production, formulation and packaging, strict quality control procedures will have been developed and practiced as a part of ensuring consistent results. EN commercialization will go from strength to strength hand in hand with a wealth of R & D that will benefit all working on these important organisms.

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Genetics is a powerful means for improving crops and livestock. In recent years we and others have utilized a variety of genetic approaches aimed at enhancing beneficial traits in entomopathogenic nematodes (EPN). We review here some of the approaches we have taken for enhancing tolerance of Heterorhabditis bacteriophora to environmental extremes, in particular heat. Selection for heat tolerance proved to be effective, but was associated in deterioration of reproductive potential. On the other hand selection for resistance to nematicides was very effective, lasted when the selection pressure was removed, and did not compromise other parameters of biocontrol efficacy. Screening for natural isolates resulted in the identification of a heterorhabditid heat tolerant strain IS-5. Using genetic markers and cross hybridization we demonstrated that the trait was dominant and transferable to the commercial strain HP88 without concomitant reduction in biocontrol efficacy. Mutagenesis is useful for generating mutants displaying either desired beneficial traits or marker mutations. We demonstrate the utility of the latter. Finally, genetic engineering is a most promising tool for enhancing beneficial traits in EPN. The success in genetic transformation of EPN opens the way for generating transgenic nematodes carrying genes conferring resistance to various environmental extremes, most notably heat shock genes.

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Entomopathogenic nematode formulation technology has made significant progress in the past 15 years. Formulations ranging from the impregnation of nematodes on artificial sponge to highly advanced granular formulations have been developed. Major challenges have included the development of room-temperature shelf stability, ease of use, and contamination control. Enhanced storage stability has been achieved through the reduction in the rate of stored energy consumption by the infective juveniles. This has been achieved through the reduction in nematode activity by either physically trapping them in gels or through reduced water activity of the substrates leading to partial anhydrobiosis. At present, a maximum room temperature shelf-life of 4-5 months has been achieved for only Steinernema carpocapsae in a water dispersible granular formulation (WDG). Other Steinernema spp.(S. feltiae and S. riobrave) can only be stored for 2-3 months at room temperature. A new wettable powder formulation has been developed that allows ambient storage of the first Heterorhabditis product for 2-3 months.

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The use of nematodes as biological control agents of insects has evolved significantly since this pioneering concept was first employed by Rudolf GLASER in the early 1930s. GLASER and his colleagues (see 4) demonstrated that the entomopathogenic nematode, Steinernema glaseri (Rhabditida: Steinernematidae) could be mass produced and used as an inoculative biological control agent of the Japanese beetle, Popillia japonica. Although other Steinernema species were described after the discovery of S. glaseri, it was the independent finding of S. carpocapsae by DUTKY & HOUGH (2) and WEISER (9) that stimulated further efforts in biological control by beneficial nematodes. Of equal significance was the finding that there is a mutualistic bacterial relationship with these steinernematid nematodes (i.e., Xenorhabdus spp.)(see 3). Since finding this nematode species, S. carpocapsae and its mutualistic bacterium, X. nematophilus, were used in a number of experiments to control various insect species (see 8). In the 1970s, other nematodes would contribute to the biological control efforts because insect resistance and environmental and public health problems from the use of chemical insecticides led to an increasing demand for alternatives to insect control. Beneficial insect-parasitic nematodes may offer one of these alternatives. These would include the entomopathogenic nematodes in the genus Heterorhabditis (Rhabditida: Heterorhabditidae) and their mutualistic bacteria, Photorhabdus spp., both of which have a similar mode of infection as Steinernema (see 3), the mermithid, Romanomermis culicivorax (Stichosomida: Mermithidae)(7) and the tylenchid, Deladenus siricidicola (Tylenchida: Phaenopsitylenchidae)(see 1).
Several international forums resulting in published reports on the topic of beneficial nematodes for biological control have been held [e.g., Asilomar, California (3) and Beijing, China (1)]. COST (Committee on Scientific Technology), a European organization, has held a number of scientific meetings to discuss and consider many biological facets of entomopathogenic nematodes. Because entomopathogenic nematodes are of commercial interest and can be used as biological insecticides, these international forums have understandably focused on them. In addition, ISHIBASHI (5, 6) has summarized much of the research efforts of Japanese scientists. In this Tsukuba Symposium sponsored by the Japanese Nematological Society, the emphasis has focused on the recent developments by leading scientists working in the field of biological control by beneficial nematodes, particularly on the entomopathogenic ones. We report herein a synopsis of the major points made by the speakers and of the discussion that followed the presentations.

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