The barley chafer grub, Coptognathus curtipennis, is an important field pest of barley, wheat and linseed seedlings at Tikur Inchini district of the West Shewa zone of Ethiopia in Ormoia regional state. Two entomopathogenic nematodes (EPN; Heterorhabditis bacteriophora and Steinernema yirgalemense, strains ANEPN21, ANEPN22, respectively) and three entomopathogenic fungal isolates of Metarhizium anisopliae (MM, EE and PPRC66) were compared separately at different rates of concentration for their effect against C. curtipennis in laboratory bioassays. Heterorhabditis bacteriophora and M. anisopliae isolate MM were found to be the most pathogenic to C. curtipennis at 1000 infective juveniles (IJ)/grub and 10 8 conidia (g soil) −1 concentration, causing 88.7% and 88.6% mortality, respectively, after 3-4 weeks exposure. These two most effective entomopathogens were further tested against the same insect in simultaneous and sequential combination with each other. Insect larvae were exposed to optimal concentration of H. bacteriophora and M. anisopliae isolate MM, which caused 50% larval mortality, i.e., 380 IJ/grub and 1.7 × 10 7 conidia (g soil) −1 , respectively, as observed in the first experiments. The combined application increased larval mortality in an additive and synergistic manner. To achieve a strong synergistic effect, larva had to be exposed to M. anisopliae isolate MM 3 weeks before the addition of nematodes. The result of the whole experiment suggests that the damage caused by barley chafer grub can be controlled or managed by applying EPN and/or fungi. Furthermore, the combined use of M. anisopliae with nematodes may offer an integrated approach to increase the efficacy of EPN for C. curtipennis control and perhaps other insect pests.

Entomopathogenic nematodes (EPNs) suffer from trait deterioration, a potential problem when these antagonists are transferred into artificial environments for mass production. In order to improve beneficial traits of EPN genetic selection and hybridization has been successfully carried out. Should these selected strains deteriorate during serial culturing the efforts would be in vain. Inbreeding might offer a possibility to stabilize traits but can also result in inbreeding depression. This study attempted to increase heat tolerance of Heterorhabditis bacteriophora by selective breeding for seven cycles either with nematodes propagated in vivo in Galleria mellonella or with in vitro propagated nematodes which were exposed to heat stress in monoxenic liquid culture. After release of the selection pressure, the tolerance was monitored over 15 additional reproductive cycles to compare the stability of the trait. Virulence of the selected strains was assessed to check for negative tradeoff effects. Heat tolerance was successfully increased in vivo (from 39.03 to 40.85°C) and in vitro (from 39 to 40°C) propagated H. bacteriophora, but could only be maintained in populations which were serially reared in liquid culture. When H. bacteriophora is cultured in vivo, reproduction by cross fertilization is possible. In in vitro culture male and female cannot mate and reproduction is solely by self-fertilizing hermaphrodite resulting in homozygous inbred lines. Trait deterioration seems to be restricted to in vivo propagated H. bacteriophora, whereas monoxenic liquid cultures handling large numbers of inbred lines provided genetically stable and virulent nematode populations. Selection using liquid culture technology is thus superior over in vivo propagation to sustain beneficial traits in H. bacteriophora not only for selective breeding but also for mass production.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is used in biological plant protection to control pest insects. In the past, several attempts targeted at an enhancement of the desiccation tolerance of EPN by genetic selection in order to improve their storage stability. The subsequent loss of improved beneficial traits after release of selection pressure has often been reported. In order to stabilize progress of selective breeding, selection during liquid culturing was tested against propagation in host insects. After release of the selection pressure, the tolerance was monitored over additional reproductive cycles in vivo and in vitro to compare the stability of the trait. Furthermore, it was tested whether the virulence of the selected strains would be impaired. Exposure to desiccation stress prior to propagation, in vivo or in vitro, both resulted in increasing desiccation tolerance. When selection pressure was released, the gained tolerance was lost again during in vivo production, whereas the tolerance was maintained at a high level when EPNs were cultured in liquid culture. In Heterorhabditis sp., liquid culture conditions produce highly homozygous, genetically stable inbred lines. The investigation provides easily applicable methods to improve and stabilize beneficial traits of heterorhabditid EPNs through selective breeding in liquid culture. Compared to nematodes from in vivo propagation, production in liquid media yielded EPN of higher virulence.

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The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a destructive pest of maize, Zea mays L. in North America and Europe. Larvae and pupae can be controlled with entomopathogenic nematodes (EPN) of the genus Heterorhabditis. When maize is attacked by WCR, the roots of some maize varieties emit (E)-b-caryophyllene that attract EPN to the pest larvae. To use synthetic (E)-b-caryophyllene in bioassays for the genetic selection of EPN strains with enhanced chemotactic response to a volatile emitted from the damaged root when attacked by larvae of the WCR, different laboratory bioassays were tested. Three sand assays and one agar assay used synthetic (E)-b-caryophyllene as an attractant for H. megidis. In none of the assays, attraction of the nematodes to (E)-bcaryophyllene was recorded. Possible reasons why (E)-b-caryophyllene was not attracting EPN in the bioassays are discussed.