Desiccation tolerance among different isolates of the entomopathogenic nematode Steinernema feltiae (Fillipjev).

Poor storage capacity is a major constraint limiting further expansion of the use of entomopathogenic nematodes. In order to prolong shelf life, a quiescent state of the dauer juveniles (DJs) should be induced. This can be attained by means of desiccation of DJs. In this study, 24 natural isolations of Steinernema feltiae were exposed to desiccation stress in non-ionic polyethylene glycol 600. The dehydrating conditions were measured as water activity, a(w)-value. Non-adapted and adapted DJs were tested separately under a series of dehydrating conditions. The mean tolerated a(w)-value (MW50) ranged from 0.85 for the isolate NEP1 to 0.95 for FIN1, ISR5 and ITA2 when not adapted to desiccation stress and from MW50 of 0.822 for CR1 to 0.98 for ISR6 when adapted to the stress conditions. CR1 tolerated the lowest desiccation stress at an a(w)-value for the most tolerant 10% of the population (MW10) at 0.65 when DJs had been adapted to stress. No significant differences were recorded between all isolates in non-adapted DJs populations MW10 was compared. No correlation between tolerance under non-adapted and adapted conditions were found. Most tolerant isolates will now be used for cross-breeding and subsequent genetic selection to enhance desiccation tolerance.

The influence of humidity on the effect of Steinernema feltiae against diapausing codling moth larvae (Cydia pomonella L.) (Lepidoptera: Tortricidae).

Codling moth (CM) is a serious and global pest of pome fruit. It overwinters in cryptic habitats as cocooned diapausing larvae. Field trials with the entomopathogenic nematode Steinernema feltiae (Rhabditida: Steinernematidae) report control of diapausing CM of up to 70%, but results are variable. The objective of this study was to define environmental conditions favouring the performance of the nematodes. Cocooned larvae were more susceptible than non-cocooned larvae. S. feltiae was unable to infect CM at a water activity (aw-values) < or = 0.9. Mortality of cocooned larvae was reported at lower aw-values than of non-cocooned larvae. Exposure time and impact of external relative humidity (RH) was studied. Mortality of cocooned larvae did not further increase after half an hour of exposure, whereas the mortality increased with increasing exposure time in non-cocooned larvae. LC50 and LC90 considerably decreased with increasing RH. The influence of the relative humidity was less pronounced when surpassing 80% than the effect of the volume of applied water. When S. feltiae was formulated in a surfactant-polymer-formulation (SPF), mortality significantly increased when compared to application in water.

Attraction of Heterorhabditis sp. toward synthetic (E)-beta-cariophyllene, a plant SOS signal emitted by maize on feeding by larvae of Diabrotica virgifera virgifera.

Most plants, when damaged by herbivore insects, synthesize and release various chemicals as indirect defence mechanism that attract parasitic or predatory insects that are natural enemies of the herbivores. When attacked by Western Corn Rootworms, the roots of many maize plant varieties emit (E)-beta-caryophyllene that attracts the neighbouring entomopathogenic nematodes to kill the feeding pest. Through plant genetics and biotechnology it was possible to manipulate this volatile compound in order to increase the effectiveness of entomopathogenic nematodes in reducing the damage of the pest. In order to further use this strategy to improve the effectiveness of Heterorhabditis bacteriophora by selective breeding, we invesa tigated the applicability of the strategy in different standard laboratory bioassays using three different sand and agar plate assays. The synthetic form of (E)-beta-caryophyllene and H. megidis (the strain, which in previous investigation, showed significant attraction to caryophyllene) were used in the study. In all bioassays no significant difference was observed in attraction of nematodes between the caryophyllene treatments and the controls. The results contradict results of previous investigations done by other investigators (Rasmann et al., 2005). Future investigations for the genetic improvement of the host finding ability of entomopathogenic nematodes can therefore not target attraction to caryophyllene.

Intraspecific variability of Steinernema feltiae strains from Cemoro Lawang, eastern Java, Indonesia.

Four strains of Steinernema feltiae from Eastern Java, Indonesia were characterized based on morphometric, morphological and molecular data. In addition, their virulence against last instar Tenebrio molitor and heat tolerance was tested. Infective juvenile have a mean body length ranging from 749 to 792 microm. The maximum sequence difference among the four strains was 7 bp (8.8%) in the ITS and 2 bp (0.3%) in D2D3 regions of the rDNA. All the strains are not reproductively isolated and can reproduce with European strain S. feltiae Owiplant. The lowest LC50 was observed for strain SCM (373) and the highest for S. feltiae strain Owiplant (458) IJs/40 T. molitor. All four strains showed relatively better mean heat tolerance when compared with S. feltiae Owiplant, both in adapted and non-adapted heat tolerance experiments.

Effect of temperature on the development of Steinernema carpocapsae and Steinernema feltiae (Nematoda: Rhabditida) in liquid culture.

For commercial use of the entomopathogenic nematodes Steinernema carpocapsae and Steinernema feltiae in biological control of insect pests, they are produced in liquid culture on artificial media pre-incubated with their symbiotic bacteria Xenorhabdus nematophila and Xenorhabdus bovienii, respectively. After 1 day of the bacterial culture, nematode dauer juveniles (DJs) are inoculated, which recover development. The adult nematodes produce DJ offspring, which are harvested and can be sprayed. This study determined optimal temperatures to obtain high DJ progeny within a short process time. Temperatures assessed were 23 degrees C, 25 degrees C, 27 degrees C, and 29 degrees C for S. carpocapsae and 20 degrees C, 23 degrees C, 25 degrees C, and 27 degrees C for S. feltiae. The recovery of inoculated DJs was hardly affected and was reduced only in S. carpocapsae at 29 degrees C. The fecundity (eggs in uterus) in S. carpocapsae reached a maximum at 27 degrees C; whereas, maximum yields were recorded at 25 degrees C. For both Steinernema spp., highest DJ densities were obtained after 15 days incubation at 25 degrees C. Optimal culture temperature for both nematode species is 25 degrees C. S. carpocapsae was more sensible to suboptimal temperature than S. feltiae. Results on total DJ density and DJ proportion of the total nematode population were more variable at non-optimal temperature condition for S. carpocapsae than for S. feltiae. Suboptimal culture temperature also reduced DJ infectivity.

Influence of cell density and phase variants of bacterial symbionts (Xenorhabdus spp.) on dauer juvenile recovery and development of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida).

The rhabditid nematodes Steinernema carpocapsae and Steinernema feltiae are used in biological control of insect pests. Mass production is done in liquid culture media pre-incubated with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, before nematode dauer juveniles (DJs) are inoculated. As a response to food signals produced by the bacterial symbionts, the DJs exit from the developmentally arrested dauer stage (they recover development) and grow to adults, which produce DJ offspring. Variable DJ recovery after inoculation often causes process failure due to non-synchronous population development and low numbers of adult nematodes. This contribution investigated the influence of the bacterial cell density on DJ recovery and development to adults. At higher density of 10(10) bacterial cells ml(-1), a higher percentage of DJ recovery was induced, and adults occurred earlier in both Steinernema spp. than at lower density of 10(9) and 10(8) cells ml(-1). Xenorhabdus symbionts produce phase variants. Recovery in bacteria-free supernatants was lower than in supernatants containing bacterial cells for both primary and secondary phase Xenorhabdus spp. and lower in secondary than in primary phase supernatants or cell suspensions. In general, recovery was lower for Steinernema feltiae and the time at which 50% of the population had recovered after exposure to the food signal was longer (RT(50) = 17.1 h) than for Steinernema carpocapsae (RT(50) = 6.6 h). Whereas >90% S. carpocapsae DJs recovered in hemolymph serum of the lepidopteran insect Galleria mellonella, recovery of S. feltiae only reached 31%. Penetration into a host insect prior to exposure to the insect's food signal did not enhance DJ recovery. Consequences for liquid culture mass production of the nematodes and differences between species of the genera Steinernema and Heterorhabditis are discussed.

Combining Steinernema carpocapsae and Bacillus thuringienis strains for control of diamondback moth (Plutella xylostella).

The diamondback moth (DBM), Plutella xylostella, is a major pest of crucifers and has developed resistance against all chemical insecticides, even against the biological insecticide Bacillus thuringiensis (Bt). Entomopathogenic nematodes (EPN) as novel biological insecticide were successfully used against DBM. We investigated the potential of Bt to enhance the virulence of EPN and the possibility to reduce the application density by exploiting synergistic effects. The interaction between different combinations of Bt and EPN were tested against early 3rd instar P. xylostella on 2 cm2 cabbage leaf discs. All of the combinations had additive effects. Synergistic results were exceptional. The result indicate that a joint application of Bt and EPN is not recommended. But alternating applications are a measure to manage resistance development against Bt.

Screening of entomopathogenic nematodes for virulence against the invasive western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) in Europe.

Entomopathogenic nematode species available in Europe were screened for their efficacy against both the root-feeding larvae and silk-feeding adults of the western corn rootworm, Diabrotica virgifera virgifera LeConte. Laboratory screening tests were aimed at the selection of candidate biological control agents for the management of this invasive alien pest in Europe. Steinernema glaseri, S. arenarium, S. abassi, S. bicornutum, S. feltiae, S. kraussei, S. carpocapsae and Heterorhabditis bacteriophora were studied to determine their virulence against third instar larvae and adults of D. v. virgifera in small-volume arenas (using nematode concentrations of 0.5, 0.8, 7.9 and 15.9 infective juveniles cm-2). All nematode species were able to invade and propagate in D. v. virgifera larvae, but adults were rarely infected. At concentrations of 7.9 and 15.9 cm-2, S. glaseri, S. arenarium, S. abassi and H. bacteriophora caused the highest larval mortality of up to 77%. Steinernema bicornutum, S. abassi, S. carpocapsae and H. bacteriophora appeared to have a high propagation level, producing 5970+/-779, 5595+/-811, 5341+/-1177 and 4039+/-1025 infective juveniles per larva, respectively. Steinernema glaseri, S. arenarium, S. feltiae, S. kraussei and H. bacteriophora were further screened at a concentration of 16.7 nematodes cm-2 against third instar larvae in medium-volume arenas (sand-filled trays with maize plants). Heterorhabditis bacteriophora, S. arenarium and S. feltiae caused the highest larval mortality with 77+/-16.6%, 67+/-3.5%, and 57+/-17.1%, respectively. In a next step, criteria for rating the entomopathogenic nematode species were applied based on results obtained for virulence and propagation, and for current production costs and availability in Europe. These criteria were then rated to determine the potential of the nematodes for further field testing. Results showed the highest potential in H. bacteriophora, followed by S. arenarium and S. feltiae, for further testing as candidate biological control agents.