Defining genomic drivers of evolution in the entomopathogenic Serratia spp. A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

There is a growing demand for new and sustainable solutions as alternatives to chemical pesticides both in Aotearoa New Zealand and worldwide. Since the early 1990s, isolates of the bacterium Serratia entomophila have been utilised as biocontrol agents, causing amber disease specific to larvae of the New Zealand endemic grass pest, Costelytra giveni (Coleoptera. Scarabaeidae). Serratia proteamaculans, another causal agent of amber disease, is as yet unutilised in commercial biocontrol but has been implicated in causing diverse disease pathotypes in grass grub larvae and larvae of other endemic scarabs (Pyronota spp.). As some isolates have a broader host range than S. entomophila, there is potential for S. proteamaculans to be a more suitable candidate for future biocontrol approaches. Virulence determinants for both chronic and hypervirulent isolates reside on variants of a megaplasmid, pADAP (amber-disease associated plasmid), which encodes the Afp (antifeeding prophage) and Sep-Toxin complex. Pathogenic isolates of both species are found coexisting with non-pathogenic conspecifics (predicted to be around 44% of the S. entomophila population) in sampled field sites around New Zealand. It is yet to be elucidated why the pADAP plasmid is only found in a proportion of the population, if this is a stable population, or what benefit can be conferred by the presence or absence of the plasmid. There is potential that disease expression and retention of pADAP in the population is in some way mediated by the chromosome. This study seeks to further investigate the potential role of the chromosome of Serratia spp. in the population dynamic and variability of disease and plasmid persistence. The presence of conspecific plasmid-free isolates compounds the need for characterisation of the production of accessory virulence determinants in entomopathogenic Serratia. Through assessment of 52 whole chromosomal sequences, it was found that while S. entomophila pADAP bearing isolates shared a relatively conserved chromosome, that of S. proteamaculans were heterogeneous. The consistent and predictable disease phenotype caused by S. entomophila isolates could therefore infer that S. entomophila may favour a chronic disease state. Pangenome wide analysis of S. entomophila isolates found differences in those from areas devoid of grass grub, suggesting the native pADAP carrying isolates have undergone speciation, coevolving with C. giveni larvae. Comparative genomics showed chromosomal inversions and larger chromosome size in isolates from areas where amber disease has not previously been documented. Evidence of horizontal gene transfer (HGT) was found in both S. entomophila and S. proteamaculans, though predictions of pangenome bounds and putative islands suggest that these events are more commonplace in S. proteamaculans. Plasmid transconjugants to non-native backgrounds were constructed to investigate the potential coevolution of the plasmid and chromosome. Differential expression of accessory virulence determinants between transconjugants and wildtype isolates demonstrated an association between down-regulation of various factors and acquisition of a non-native plasmid type. In vivo analysis supported this finding, where transconjugants with non-native plasmids displayed a more erratic disease phenotype. In silico assessment found chronic isolates to encode a similar number of accessory virulence determinants to their hypervirulent counterparts, suggesting a higher regulatory mechanism could be responsible for the decrease in time to infectivity found in disease caused by these isolates. Overall, the expression of accessory virulence determinants was found to be similar between pathotypes. Differences were exclusive to extracellular nuclease production (in non-pathogenic isolates and S. entomophila vs S. proteamaculans) and siderophore production in hypervirulent isolates. While the plasmid disease-encoding genes are well characterised, the mechanism driving retention of chromosomally located accessory virulence determinants such as phospholipases, chitinases, and extracellular proteases in S. entomophila and S. proteamaculans are yet to be described. Bioinformatics and both in vitro (plate assays) and in vivo bioassays were used to assess accessory virulence factors (associated with host tissue damage and colonisation) in Serratia spp. isolates. Results were correlated with geographic/phenotypic data to determine any evolutionary links to places of isolation or virulence. Overall, this study has shown that the pADAP plasmid of S. entomophila and S. proteamaculans have coevolved, where regulatory mechanisms of chromosomally bound accessory virulence determinants potentially mediate pathogenic adhesion and host tissue degradation. Though evidence currently indicates that the S. entomophila genome is not reducing, it is hypothesised that the indicators of reduced HGT opportunity and evidence of trophic specialisation could suggest coevolution with C. giveni.