Itamar Glazer1, Dana Ment1, Duarte Toubarro2, Nelson Simoes2, Bart Vandenbossche3, Carlos Molina3, Ralf-Udo Ehlers3, Olaf Strauch3, Christine Griffin4, Apostolos Kapranas4, Julien Chuche4, Iwona Skrzecz5, Padraig O'Tuama6

1 ARO, Volcani Centre, Israel,. 2 UAc, Fundação Gaspar Frutuoso, University of Azores, Portugal. 3 E-nema GmbH, l, Germany. 4 NUIM, Department of Biology, Maynooth University, Ireland. 5 IBL, Forest Research Institute, Poland. 6 Coillte, Ireland. *All authors and institutions have enormously contributed to the development of this international and highly interdisciplinary research project

Overview of the research focused on the application of entomopathogenic nematodes in Europe

Genetics for trait improvement in Heterorhabditis bacteriophora

Conclusions • Basic knowledge about the genetic basis of

longevity and virulence in Heterorhabditis bacteriophora has been gathered

• A set of virulence- and stress-related candidate genes has been generated for H. bacteriophora

• Molecular markers associated to phenotypes of interest is a mayor priority task.

• EPNs applications show good efficiency against pine weevil in coniferous forests

• New stress tolerant H. bacteriophora strains will be tested in field trials for crops protection aside clear-felled coniferous forests

References: Yaari, M., et al. (2015). Transcriptome analysis of stress tolerance in entomopathogenic nematodes of the genus Steinernema. Int J Parasitol.

• At the Volcani Centre (Israel) Differentially expressed genes (DEG) related to stress tolerance were detected in H. bacteriophora DJs guided by a previous S. feltiae transcriptome study conducted under stress conditions (Yaari et al. 2015)

• Conserved sequence regions were defined for each DEG by C. elegans sequence homology. Primers for qPCR were designed

• Gene expression was tested under two assays: i) evaporative desiccation (97% RH), and ii) hygroscopic desiccation (20% PEG) (Figure 1)

Results • In H. bacteriophora, candidate genes were

most highly expressed under evaporative desiccation after 16 hours, and under hygroscopic desiccation after 36 hours (Figure 2)

• A group of seven genes was selected as marker-set for further analyses assessing polymorphisms between stress-tolerant and sensitive materials.

Figure 1: Experimental setup for desiccation assays in Heteorhabditis bacteriophora DJs (A) and additional heat-tolerance assays (B)

Figure 2: Relative gene expression in Heteorhabditis bacteriophora DJs desiccated by 97% RH for 16 and 36h and 20% PEG for 16 and 36h, respectively. Error bars: SD of three technical replicates

Figure 3: Selection pressure effects in mean survival time under oxidative stress (MST; A), and mean tolerated water activity (WA; B), in Heterorhabditis bacteriophora DJs from an inbred line (IL3) and a synthetic pool of accessions.

Figure 4: Mean lethal dose (LD50) natural Heterorhabditis bacteriophora accessions assessed in meal worm (Tenebrio molitor) assays

• At e-nema GmbH (Germany) H. bacteriophora accessions were characterized for their DJ-longevity under oxidative stress

• A positive correlation has been found between DJ-longevity under control and oxidative stress conditions

• A synthetic pool of accessions has been established and subjected to selection pressure

• In parallel, the individual H. bacteriophora accessions have been characterized for virulence. Interesting accessions with high longevity and virulence have been identified (data not shown)

Results • In H. bacteriophora, positive effects after

selection are observed in oxidative stress-tolerance and desiccacion (Figure 3)

• Inbred lines with contrasting DJ longevity have been crossed for further molecular and phenotypic analyses

• More than 40 natural accessions have been characterized for virulence (LD50) in meal worm bioassays (Tenebrio molitor). Extreme LD50 ranged from <5 up to > 30 DJs/insect (Figure 4).

LD50

(IJs

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BIOCOMEs: further development of entomopathogenic nematodes as biocontrol products Within the EU-BIOCOMES project, the improved use of entomopathogenic nematodes (EPNs) in agriculture and forestry is a major interdisciplinary objective. The work package Genetics for trait improvement focuses on the understanding of the longevity and virulence traits in Heterorhabditis bacteriophora. Within this framework, predictive longevity bioassays were developed and natural isolates have been characterized and selected for longevity and virulence (E-nema, Germany). Additionally, desiccation-related gene markers were determined by RNA-seq and qRT-PCR validation (Volcani Centre, Israel). In parallel, differentially-expressed virulence genes were identified via RNA-seq (University of Azores, Portugal). Within the work package Biocontrol of forest pests, the efficacy of EPNs against the large pine weevil (Hylobius abietis) in clear-felled coniferous forests has been assessed. Steinernema and Heterorhabditis EPNs were compared for their effect on pine weevils in roots and stumps. In addition, the effect of soil type and application method was analysed. Field trials were carried out by the Maynooth University and Coillte in Ireland, and by the Forest Research Institute (Poland). Both EPN species resulted effective in field tests and showing considerable mortality of Hylobius larvae in treated stumps. In the work package Production technologies, the improvement of the downstream processing of H. bacteriophora is a main objective (E-nema, Germany). Two principles for separation of DJs from the culture broth were compared and a small-scale system was developed to monitor the shelf-life and virulence of stored EPNs after production. The support by the EC to the BIOCOMES project will mark a milestone in the further establishment of EPNs as alternative control measure to chemical pesticides in Europe.

• Pine weevil (Hylobius abietis) is a serious pest of reforestation throughout northern Europe. Weevils develop in tree-stumps and adults destroy young trees

• At the Forest Research Institute (Poland) EPNs efficacy against the large pine weevil in clear-felled coniferous forests was assessed. (Figure 5):

• Parallel small- and large-scale field trials against the large pine weevil were conducted in Irish locations by the Maynooth University in cooperation with Coillte (Ireland, Figure 6)

• In both, Polish and Irish locations, the efficacy of Steinernema and Heterorhabditis EPNs was directly compared

Results • In Poland the highest level of parasitized pine weevil larvae

was found in stumps treated with H. downesi. Data from two consecutive years is shown (Table 1)

• Small scale trials in Ireland showed that both Steinernema carpocapsae and Heterorhabditis downesi are efficient in reducing numbers of adult weevils emerging from stumps

• EPNs efficacy after application around and on the top of P. sylvestris stumps can be further improved. Weather conditions and soil type have influence on the EPNs performance.

• Stress-resistant nematode lines are expected to reduce the effects of the environmental conditions on EPNs applications

A) B)

C) D)

Figure 5: Application of EPN treatments in Pinus sylvestris stumps (A, B). Digging of roots of from experimental stumps (C). Debarking of roots and estimation of parasitized pine weevil larvae (D). Time of application: Beginning of October.

Table 1: Efficiency of the different EPN treatments applied on pine stumps against pine weevil. Data collected from two experimental seasons in Polish locations

Biocontrol of forest pests

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B)

A)

Figure 6: setup of small-scale (A) and large-scale (B) field trials for the control of pine weevil in Irish locations. For both, Polish and Irish locations, EPNs were produced by in large fermenters at e-nema (Germany).

A)

B)