Bringing Omics and new quality parameters together to improve the EPNs industrial performance

Carlos Molina

Biological control products based on the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora can vary in virulence and longevity. Along the mass production, the success of a given process is determined by the final yield (DJ/volume). Within the past 10 years, e-nema GmbH invested large resources to establish platforms enabling marker-assisted breeding and genome-based studies for the improvement of Heterorhabditis bacteriophora beneficial traits. What is the legacy of this initiative for the future? Concerning biologic materials, 48 WT inbred lines have been derived out of natural H. bacteriophora isolates collected worldwide. These lines are highly homozygous (>95%) and constitute an “eternal” resource for large comparative phenotyping studies. Up to now the lines have been phenotyped for stress-tolerance and virulence. The genome from two of these WT inbred lines has been also re-sequenced and annotated. Further on, >5.000 SNPs have been characterized in these lines, and association analyses identified markers linked to stress-tolerance and virulence. Aside, a set of >160 highly homozygous (>95%) EMS-mutant lines has been produced, and the lines were phenotyped for DJ-recovery. More than 190 SNPs have been as well identified for 96 of these lines and association analysis identified markers linked to DJ-recovery using this platform. This mutant collection is a consolidated system for future phenotype-genotype correlation studies. We have also developed novel parameters as quality predictors for EPN. Concerning virulence, the presence of the bacteria in the DJ is crucial for its biocontrol potential. We provide a qPCR-based method to quantify the bacterial load inside the DJs. We tested our qPCR system in DJ-populations carrying defined proportions of bacteria-free (axenic) vs bacteria-carrying nematodes. With an increasing proportion of axenic DJ in a population, virulence declined, and the insect mortality was proportional to the amount of bacterial DNA detected in the population by qPCR. Along liquid storage over long time, virulence also decreased, and this correlated with the reduction of bacterial DNA on the respective DJ-population. Storage temperature was shown to influence the bacterial survival. In formulated DJ, the loss of bacterial DNA on the DJ-population accelerated under storage temperatures below 7.5°C. Concerning storage potential, we aimed to predict whether living DJ have substantial changes in their reserve compounds along different storage times. For this, we analyzed H. bacteriophora commercial batches in different storage time points using luminescence enzymatic assays. We present an overview of the different assays and the correlation with the DJ-fitness. Concerning the industrial culture process, we have developed qPCR assays to monitor very large fermenters (>3000 L) for potential contamination. Results of the qPCR assays show correlation between contamination events and the decrease of the final DJ-yield. Future markets and new application scenarios can only be reached on the long term if EPN consequently meet standard quality parameters.