The soil ecology of Serratia entomophila, A bacterial pathogen of the New Zealand grass grub, Costelytra zealandica (White)

Serratia entomophila (Enterobacteriaceae) is a newly described species of bacterium, currently being developed as a biological control agent for the New Zealand grass grub Costelytra zealandica (White) (Coleoptera: Scarabaeidae). The bacterium causes amber disease; the larvae stop feeding and develop a characteristic amber coloration before death. Insect pest control by pathogens is often not optimized because of insufficient knowledge of the pathogen's attributes. An understanding of the ecology of s. entomophila, in particular its persistence in the grass grub's environment, is essential if full advantage is to be gained from its pathogenicity towards the insect. Laboratory and field experiments were conducted to determine the occurrence and persistence of S. entomophila in soil. Studies were conducted using viable plate counting on a selective medium, caprylate thallous agar (CTA). This medium was tested for its selectivity for Serratia species. All Serratia strains tested grew well on CTA; the mean efficiency of colony formation on CTA was 93% of that on a non-selective medium. The identity of colonies growing on CTA was checked by replica plating colonies onto 3 test media: DNase toluidine blue agar, chitinase agar and adonitol agar. The identification scheme developed allowed the selective recovery of Serratia isolates from field soils containing a diverse microflora. Quantitative recovery of cells added to soil was possible so persistence of S. entomophila applied to soil could be determined. S. entomophila was recovered from 27 sites throughout New Zealand, from a range of soil types, indicating the bacterium was widely distributed. Sampling for S. entomophila in a range of habitats in the pasture ecosystem showed the species was restricted to the soil and the grass grub larva. The incidence of naturally occurring amber disease was monitored at 2 sites in Canterbury, from 1983 to 1987. Grass grub populations increased with increasing age of pasture. Amber disease incidence was low in young pastures but increased as grass grub numbers increased, until there was sufficient disease to cause a population collapse. The incidence of natural outbreaks of disease and the frequent isolation of S. entomopbila from many sites suggests the bacterium is a stable member of some soil-larvae ecosystems, and is capable of persistence in that habitat. Field trials were conducted to examine the persistence of S. entomopbila populations applied to soil. Bacterial numbers declined quite rapidly in soil, with the time taken for 90% of the population to decline ranging from 17 to 36 days. Laboratory experiments were conducted to examine biotic and environmental factors affecting persistence of S. entomopbila in soil. Competition with indigenous soil microorganisms, nutrient availability and soil moisture were the most important factors in the decline of the bacteria in soil. S. entomopbila was capable of rapid growth in soil given adequate nutrients and no competition from soil micro-organisms, but declined slowly in natural soil. Numbers of S. entomopbila declined at a significantly faster rate in drying soil than in soil held at constant soil moisture. The use of an appropriate application method is essential to the effective use of S. entomopbila for grass grub control. The method should place the bacteria beneath the surface where they can be ingested by susceptible larvae. Conventional boom spray, jet stream and turf treatment application methods were evaluated for their success in placement of S. entomopbila into pasture. Turf treatment deposited more bacteria into the soil than surface treatments, jet stream and spray. Turf-applied bacteria survived in higher numbers than jet stream and sprayed bacteria for the 30 days of the trial. The role of larvae in the maintenance of soil bacterial populations was examined by following persistence of bacteria applied to soil containing high and low densities of larvae. Numbers of S. entomopbila declined steadily where no larvae were present but were maintained for at least 8 months where larvae were found. Diseased larvae contained, on average, 1.4 x 10⁶ bacteria, which could contribute to the soil inoculum pool at death of the insect. The role of the insect in disease transmission is discussed