Investigating formulation strategies and desiccation tolerance of a South African entomopathogenic nematode (Rhabditida: heterorhabditidae)

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirement 2018Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis are excellent biological control agents of lepidopteran and coleopteran soil dwelling insects. Their successful potential as biological control agents is attributed to the bacterial symbiont contained in their guts, together the dual kill insect host in 24 hours. The aims of the dissertation were to investigate the effects of nematode infectivity against susceptible hosts in different formulations as well as its tolerance to desiccation. Soil samples were collected from Brits, North-West province, South Africa. Insect baiting technique and White trap methods were used to recover nematodes from the collected soil samples. Preliminary identification based on symptoms revealed a red/maroon colour on EPN infected cadavers which indicated that the isolated EPN specie belonged to a Heterorhabditis genus. For molecular based confirmation of taxonomic affinities, genomic DNA was extracted, followed by PCR and sequencing of the 18S rDNA. BLASTn sequence results revealed that the presumptive Heterorhabditis sp. had a 99 % sequence affinity to Heterorhabditis bacteriophora_ isolate UP2A2 (MF033536.1), the evolutionary distance of the two species was 0.022 revealing evolutionary relatedness. Heterorhabditis sp. and Heterorhabditis _ isolate UP2A2 were clustered together in the same clade confirming the presumed Heterorhabditis sp. as a Heterorhabditis bacteriophora isolate B1. Heterorhabditis bacteriophora isolate B1 infected larvae were dissected to isolate the associated endosymbiotic bacteria and a drop of hemolymph was streaked onto sterile plates of nutrient bromothymol blue-triphenyltetrazolium chloride agar (NBTA) and MacConkey which were then stored in the dark at room temperature. After 4 days of incubation, preliminary identification of the associated bacteria was performed by observing the colony morphology. Single small sized colonies of bacteria were green with red centres on NBTA and red on MacConkey agar plates confirmed that the isolated bacterial colonies displayed phenotypic features expected of bacteria belonging to Photorhabdus genus. Molecular identification revealed the presumed Photorhabdus spp. to be 99% closely related to Photorhabdus_ temperate_subsp._khani_NC19 (KF740642) in sequence. Heterorhabditis bacteriophora isolate B1 was able to tolerate desiccation up to 15 days and following exposure to desiccation treatment the IJs induced 100% larval mortality within 96 hours post exposure to desiccation following resuscitation by rehydration. Infective juveniles desiccated in host cadavers embedded in loam soil and vermiculite emerged at a faster rate on larval cadavers placed on saturated White traps at a mean rate of 16.67 IJs/day whereas emergence of injective juveniles from undesiccated host cadavers emerged at a mean rate of 10 IJs/day. The EPN IJ populations formulated in pure loam and sandy soil induced 80% and 50% larval mortality on day 2 of exposure to Galleria mellonella larvae. Cumulative mortalities reaching a 100% larval mortality were induced on days 5 and 6. EPN infected cadavers were desiccated for 31 days by incubating them at 16 ⁰C, 25 ⁰C, and 37 ⁰C. Previously desiccated EPN infected cadavers which were stored at temperatures 25 ⁰C and 37 ⁰C induced 100% larval mortality following rehydration in water over a period of 6, 8 and 24 hours. Desiccated IJs formulated in host cadavers could serve in formulation and application technology. Applying desiccation tolerant and native strains of EPNs is important for the eradication of susceptible insect pests.MT 201