Towards enhanced shelf life and performance : genetic improvement and quality control of the entomopathogenic nematode Heterorhabditis bacteriophora

Limited shelf-life is a major constraint to successful commercialization of the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora that is symbiotically associated with bacteria from the genus Photorhabdus spp. The optimal cold storage temperature of two H. bacteriophora strains in diatomaceous earth formulation was determined to be 8°C (28 days for strain D2D6) and 9°C (25 days for strain HB4). Both strains recorded lowest virulence at 5°C, 6% and 46% for HB4 and D2D6 respectively after 9 weeks of storage. A qPCR technique to quantify the bacteria Photorhabdus spp. inside the nematode during storage was optimized by identifying ten single copy genes with no homology to other bacterial accessions in the genome of P. laumondii. A 4.5 and 1.8-fold decline in the bacteria target gene copy number was detected inside nematodes stored at 2°C and 4°C respectively after two weeks and by week 6 an 8-fold decline was recorded at both temperatures. In comparison, a slight increase in bacterial target gene copy number was detected in nematodes stored at 7.5°C after 6 weeks. The H. bacteriophora genetic diversity was exploited and better performing wild type inbred line (WT IL) XX21 identified after phenotyping for reproductive potential, stress tolerance and virulence. Fourteen single nucleotide polymorphic (SNP) markers were found to be associated with these traits. Marker assisted breeding thereafter yielded a stress tolerant and high reproducing hybrid pool X21L3 from a cross between XX21 and a commercial line (IL3). This study defines H. bacteriophora optimal cold storage conditions and provides a qPCR quality control tool. We finally avail a stress tolerant hybrid pool X21L3 with potential for commercialization.