Olaf Strauch

Food signal production of Photorhabdus luminescens inducing the recovery of entomopathogenic nematodes Heterorhabditis spp. in liquid culture

Photorhabdus luminescens are bacterial symbionts of entomopathogenic nematodes of the genus Heterorhabditis. The bacto-helminthic complexes are used in biocontrol of insect pests in cryptic environments. For in vitro production, liquid media are incubated with P. luminescens for 24 h prior to the inoculation of nematode dauer juveniles. The nematodes develop to self-fertilizing hermaphrodites and produce offspring. The exit from the developmentally arrested dauer stage (recovery) is a response to a yet undescribed food signal. Major process instability is caused by low and unsynchronized recovery of the dauers. In living insects, dauer recovery is approximately 95% within 1 day. In liquid cultures of P. luminescens the recovery is spread over several days and varies between 0 and 81%. In complex culture media no food signal was detected. A food signal is produced by P. luminescens and excreted into the culture medium. The maximum food signal production was recorded during the late exponential growth phase. Compared to the food signal found in insects, the efficacy of the bacterial signal is much lower. The reasons for the variable activity of the bacterial food signal and its function during the nematode life cycle are discussed.

Storage and formulation of the entomopathogenicnematodes Heterorhabditis indica and H. bacteriophora

Successful control of insect pests through theapplication of entomopathogenic nematode dauerjuveniles of H. bacteriophora and H.indica can only be achieved when the nematodematerial reaches the end user in good condition.Storage and formulation techniques must provideoptimum conditions to guarantee a maximum survival andinfectivity of the nematodes. Nematode survival wastested at temperatures ranging between 5–25 °C.A maximum survival of H. indica was achieved at15 °C and the highest mortality at 5 °C.H. bacteriophora survived best at 7.5 °Cand least at 25 °C. An increase of the saltconcentration had positive effects on dauer juvenilesurvival in aqueous suspensions. Low pH between 6 and4 reduced the bacterial growth and prolonged survivalof stored dauer juveniles. Of the organic acidsascorbic, benzoic, citric and sorbic acid, onlyascorbic acid had a positive effect on H. indicasurvival. Extracts of the dried spice plants cinnamon,cloves, rosemary and oregano were tested. Enhancementof H. indica survival was recorded for cinnamonand cloves. Survival and infectivity of nematodesstored in attapulgite and bentonite clays and spongewere recorded over several weeks at different storagetemperatures. Infectivity was not influenced by thedifferent formulation materials. When stored insponge at 25 °C nematodes survived less than 1week and the formulation in clay could only prolongthis period for another week. At 5 °C thesurvival of H. bacteriophora in sponge wassuperior to that in clay, whereas H. indicasurvived less well in sponge than in clay at15 °C. Storage in aerated water at 5 °Cfor H. bacteriophora and at 15 °C for H. indica resulted in the lowest mortality. Forstorage at controlled conditions (temperature, pH andosmolarity), aerated water is superior to all othermethods tested and the addition of preservatives willincrease survival.

Identification and ecological characterisation of three entomopathogenic nematode-bacterium complexes from Turkey

Two heterorhabditid nematode strains (TUR-H1 and TUR-H2), and their bacterial symbionts isolated from soil samples taken at the campus of the Agriculture Faculty of the University of Ankara, Turkey, were identified by molecular methods and by cross-breeding with Heterorhabditis bacteriophora . The bacterial symbionts shared >99% similarity in the 16S rDNA sequence with Photorhabdus luminescens subsp. laumondii . Results of the restriction fragment length analysis of the ITS region assigned both nematode strains to the species H. bacteriophora . Cross-breeding confirmed the species designation for strain TUR-H2. Crosses of TUR-H1 with a hybrid strain of H. bacteriophora or with TUR-H2 resulted in infertile offspring. Both strains reproduced in monoxenic cultures of the symbionts P.luminescens isolated from H. bacteriophora and H. megidis and the resulting dauer juveniles retained cells of the bacteria. Reproduction on the symbionts isolated from H. indica failed. Infectivity at variable soil moisture and heat tolerance of the two heterorhabditid strains was compared with a Turkish isolate of Steinernema feltiae . Significantly more nematodes invaded the insect Galleria mellonella in a sandy soil assay at 10% water content than at lower values. A higher water content significantly reduced the invasion rate. Steinernema feltiae was better adapted to a temperature of 32°C than the heterorhabditid strains. Hardly any nematodes of all strains survived for longer than 4 h at 36°C.

Improvement of heat and desiccation tolerance in Heterorhabditis bacteriophora through cross-breeding of tolerant strains and successive genetic selection

Genetic selection can be a powerful tool to increase beneficial traits in biological control agents. In this study the heat and desiccation tolerance of the entomopathogenic nematode Heterorhabditis bacteriophora Poinar (Rhabditidomorpha: Strongyloidea) were significantly increased by cross breeding tolerant parental strains and successive genetic selection. These strains originated from a prior screening among 60 strains for increased stress tolerance. During genetic selection, the selection pressure was constantly increased and only the most tolerant 10% of the nematode populations were propagated for further selection steps. Assessment of tolerance and selection for both traits was performed with and without prior adaptation to the stress conditions. Eleven selection steps were performed to increase heat tolerance. A final overall increase in mean heat tolerance of 5.5°C was achieved when nematodes had been adapted to heat stress. For non-adapted tolerance an increase of 3.0°C from 40.1°C to 43.1°C was recorded. For comparison, a commercial strain had a mean tolerated temperature after adaptation of 38.2°C and of 36.5°C without adaptation. For assessment of the desiccation tolerance the mean tolerated water activity (aw-value) of a population was measured. Cross-breeding most tolerant strains reduced the aw-value from 0.67 to 0.65 after adaptation and from 0.9 to 0.7 without prior adaptation. The following six selection steps could not increase the tolerance whether nematodes had been adapted to stress or not. In comparison, the commercial strain tolerated a mean aw-value of 0.985 after adaptation and 0.951 without adaptation. Further investigation will have to assess trait stability and possible trade-off effects. This study is a first important step on the road towards domestication of the entomopathogenic nematode H. bacteriophora.

Mass production potential of the bacto-helminthic biocontrol complex Heterorhabditis indica-Photorhabdus luminescens.

Heterorhabditis indica is a potential agent for the biological control of grubs in sugarcane fields in India. The type strain LN 2 was transferred to monoxenic cultures on its symbiont Photorhabdus luminescens and successfully produced on solid media. In liquid cultures, a mean dauer juvenile yield of 457 000 was obtained with a maximum of 648 000 per ml. Comparatively high yields have not been reported before. Therefore, costs related to the liquid culture production of H. indica will be lower than for other entomopathogenic nematodes currently used in biocontrol. Different bacterial clones had no significant influence on the dauer juvenile yields in liquid media. The exit from the dauer juvenile stage (recovery) after inoculation and the number of hermaphrodites significantly decreased when culture temperature was increased from 25-30 ° C; the dauer juvenile yields were not affected. The cell density of P. luminescens in batch cultures was higher at 25 and 30 ° C than at growth temperatures of 35 and 37 ° C. In continuous culture, the bacterial growth was inhibited when the growth temperature reached 38 ° C. After approximately 60 h, the bacteria adapted to higher temperature and the growth rate increased again. When the temperature was further increased to 40 ° C, the bacterial growth was inhibited.

Selective breeding for desiccation tolerance in liquid culture provides genetically stable inbred lines of the entomopathogenic nematode Heterorhabditis bacteriophora

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.

Variability in desiccation tolerance among different strains of the entomopathogenic nematode Heterorhabditis bacteriophora.

The shelf life of biological control products based on the entomopathogenic nematode Heterorhabditis bacteriophora is rather short. In order to prolong shelf life, the metabolism of nematodes during storage must be reduced. This can be achieved by means of desiccation of the infective third-stage dauer juveniles (DJ). The tolerance can be increased by an adaptation to moderate desiccation conditions. Previous investigations indicate that the heritability of the desiccation tolerance is high, justifying a genetic selection for enhanced tolerance. This investigation screened the desiccation tolerance of 43 strains of Heterorhabditis spp. and 18 hybrid/inbred strains of H. bacteriophora . Dehydrating conditions measured as water activity ( a w values) were produced by treating DJ with different concentrations of the non-ionic polymer poly(ethylene glycol) 600. Significant inter-specific variation was recorded between nematode strains and species. The mean tolerated a w value (MW 50 ) ranged from 0.90 to 0.95 for non-adapted and 0.67 to 0.99 for adapted nematode populations. For selective breeding, only the 10% most tolerant individuals would be used. The lowest a w value tolerated by 10% of a population (MW 10 ) ranged from of 0.845 to 0.932 for non-adapted nematode populations and 0.603 to 0.950 for adapted nematode populations. Adaptation significantly increased the desiccation tolerance and a weak correlation was recorded for tolerance with and without adaptations. The most tolerant nematode strains will form the basis for the foundation of a parental stock produced by cross-breeding and following genetic selection for enhanced tolerance. Thus, this investigation is another milestone on the road to domestication of H. bacteriophora for commercial use in sustainable pest management.

Influence of the aeration rate on the yields of the biocontrol nematode Heterorhabditis megidis in monoxenic liquid cultures

Abstract The entomopathogenic nematode–bacterium complex Heterorhabditis megidis–Photorhabdus luminescens was cultured in 10-l internal loop bioreactors with marine impellers at aeration rates of 0.3 vvm and 0.7 vvm. Process parameters like impeller velocity and oxygen saturation were controlled at equal set points. The bacterial density was assessed at 24 h. Nematode dauer juveniles (DJ) were then inoculated and the development to adults after 8 days and final DJ yields after 16 days were recorded. The bacterial population density and the nematode inoculum development was variable and was not influenced by the aeration rate. A significant effect on the yield was recorded at the highest aeration rate. This result was confirmed by a direct comparison in two 5-l internal loop glass bioreactors at 0.3 vvm and 1.0 vvm, which were inoculated with nematode and bacterium pre-cultures from the same flask culture. Possible reasons for the positive correlation between aeration rate and DJ yield are discussed.

Influence of humidity and a surfactant-polymer-formulation on the control potential of the entomopathogenic nematode Steinernema feltiae against diapausing codling moth larvae (Cydia pomonella L.) (Lepidoptera: Tortricidae)

The codling moth (Cydia pomonella L.) is a serious pest of pome fruit. Diapausing cocooned larvae overwinter in cryptic habitats in the soil or in the bark of infested trees. The entomopathogenic nematode Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) is used to control diapausing codling moth larvae. 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. Susceptibility of pupae was low. To determine the influence of decreasing water activity (aw-value) on the activity of the nematodes, mortality of codling moth larvae and Galleria mellonella L. were tested in sand-sodium-polyacrylate mixtures of variable water activity. S. feltiae was able to infect both insects at aw-values >0.9. Cocooned larvae of both insects died at lower aw-values than non-cocooned larvae. Mortality of cocooned larvae did not further increase after half an hour of exposure to nematodes, whereas the mortality of non-cocooned larvae increased with increasing exposure time. LC50 and LC90 considerably decrease with increasing RH. The negative influence of the relative humidity (macro environment) was less important than the effect of the water activity in the bark substrate (micro environment). The micro environment can be manipulated by applying S. feltiae with higher volumes of water. A surfactant-polymer-formulation significantly increased nematode efficacy and can buffer detrimental environmental effects.

Life cycle and population development of the entomopathogenic nematodes Steinernema carpocapsae and S. feltiae (Nematoda, Rhabditida) in monoxenic liquid culture

The life cycle and population dynamics of the entomopathogenic nematodes Steinernema carpocapsae and S. feltiae were studied in monoxenic liquid culture with their symbiotic bacteria Xenorhabdus nematophila and X. bovienii . To distinguish between the different juvenile and adult stages, their size was recorded. No differences were observed between the species in the size of the juvenile stages but significant differences were recorded in the length of the F1 adults, pre-dauer (J2d) and dauer juvenile stages (DJ). On average, 90% of inoculated DJ of S. feltiae recovered and 77% of S. carpocapsae . In general, S. feltiae developed from the inoculum DJ to the adult approximately 1 day faster than S. carpocapsae . The sex ratio was female-biased (59.2 ± 2.2% in S. carpocapsae , 66.7 ± 2.6% in S. feltiae ) in the parental population but not in the F1 generations. Steinernematid adults, like heterorhaditids, respond to depleting food resources with the cessation of egg laying. Juveniles hatch inside the uterus and develop at cost of the maternal body content causing the death of the adult ( endotokia matricida ). In contrast to Heterorhabditis spp. and in vivo observations of steinernematids by other authors, who reported that readily developed DJ leave the carcass of the dead adult, in this study J2d emerged 12 h after cessation of egg laying. The density of both bacterial cultures decreased due to the feeding of the parental juveniles. However, X. nematophila continued at very low density, whereas the density of X. bovienii increased again until 15 days post-inoculation. The vast majority of F1 S. carpocapsae offspring developed to DJ, whereas in S. feltiae a significant second and third generation of adults was observed, probably due to the increasing bacterial population. However, second and third generation adults in S. feltiae cultures did not contribute significantly to the DJ yield. Mean yields of 158 × 10 3 DJ ml –1 were recorded for S. carpocapsae and 106 × 10 3 DJ ml –1 for S. feltiae . The results provide valuable information for future process improvement.