The social ecology of Serratia: Pathogen of the New Zealand grass grub (Costelytra giveni) : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Abstract

The New Zealand grass grub (Costelytra giveni) is an endemic New Zealand pest species which feeds on the roots of common pastoral plants. Grass grub populations in the soil can be managed using biopesticides, such as those based on Serratia entomophila, a host-specific pathogen which causes a chronic disease in C. giveni larvae called amber disease. Following ingestion of this bacterium by C. giveni, the larvae cease feeding, and a clearance of the gut results in amber discolouration, with the infection leading to death after two-to-three months. The amber disease virulence determinants are encoded by the Serratia amber disease associated plasmid (pADAP), which is also found in pathogenic S. proteamaculans strains. Non-pathogenic plasmid-free strains of S. entomophila and S. proteamaculans have also been isolated from pasture soils. The interactions between pathogenic and non-pathogenic Serratia spp. within the soil are not well defined. Therefore, increasing knowledge on the conditions and outcome of intra-species competition between pathogenic and non-pathogenic Serratia spp. will provide important information regarding both the ecology of the strains in the field, and how competition can impact pathogenic efficacy. The results generated will assist in predicting the outcome of field applications and improving biopesticide efficacy. The interactions between pathogenic and non-pathogenic Serratia strains were explored through in vitro and in vivo competition assays using constructed strains containing antibiotic markers positioned in intergenic regions of the chromosome and the pADAP backbone. This allowed for the selective recovery of strains during mixed inoculation assays in liquid media, soil microcosms, and within the larvae. The assays were conducted by competing the pathogenic S. entomophila strain A1MO2 against an isogenic non-pathogenic plasmid-cured variant, 5.6. Additional experiments were conducted using 477, a naturally occurring plasmid-free S. entomophila soil isolate, and AGR96X, a hyperpathogenic strain of S. proteamaculans associated with increased virulence, causing mortality in challenged larvae within five to ten days. Due to this rapid kill time, AGR96X has also been identified as a potential biopesticide agent of C. giveni larvae. The results obtained demonstrated an increased competitive fitness for 5.6 in all mixed inoculation experiments with A1MO2, including a faster doubling time than A1MO2 during the exponential growth phase in in vitro assessments, which was validated with Lotka/Volterra-style competition models. Comparative transcriptomics between 5.6+pADAP (an A1MO2 proxy strain) and 5.6 cultured to the mid-exponential growth phase revealed an increased expression of operons relating to carbohydrate metabolism and transport in 5.6 relative to 5.6+pADAP, including operons related to maltose and trehalose transport. This finding was validated when 5.6 outcompeted A1MO2 during mixed inoculation in M9 medium with a maltose carbon source. However, during mixed inoculation in liquid media A1MO2 outcompeted the natural plasmid-free S. entomophila isolate 477. In soil microcosms and within the larvae 5.6 was also able to outcompete A1MO2, showing a higher persistence in microcosms in the presence of healthy C. giveni larvae. Using the antibiotic markers, the occurrence of pADAP horizontal transfer between A1MO2 and other non-pathogenic Serratia spp. was observed at a low rate in challenged C. giveni larvae during in vivo assessments. A key result obtained from in vivo assessments was that pre-inoculation of larvae with non-pathogenic strains, such as 5.6 or 477, led to a reduction in amber disease occurrence when challenged with A1MO2. Furthermore, mixed inoculation of larvae with both A1MO2 and 5.6, or A1MO2 and 477 led to a delay in amber disease onset compared to larvae challenged only with A1MO2. These findings were validated with a pot trial, where the pre-inoculation of soil with 5.6 or 477 led to a reduction in amber disease rates in larvae challenged with A1MO2, with amber disease reduced from 75 to 14 %. A pot trial was also conducted with AGR96X, a hyperpathogenic S. proteamaculans strain, where the pre-inoculation of soil with 5.6 and 477 reduced mortality rates in larvae challenged with AGR96X from 62 to 25 %. These results demonstrate that the presence of non-pathogenic Serratia spp. in the soil can detrimentally impact the efficacy of pathogenic Serratia strains such as A1MO2 and AGR96X, limiting their effectiveness against grass grub larvae. These results will have important implications in both the improvement of Serratia-based biopesticides and predicting the field efficacy of the applied Serratia strains.