Serratia proteamaculans Strain AGR96X Encodes an Antifeeding Prophage (Tailocin) with Activity against Grass Grub (Costelytra giveni) and Manuka Beetle (Pyronota Species) Larvae.

A highly virulent Serratia proteamaculans strain, AGR96X, exhibiting specific pathogenicity against larvae of the New Zealand grass grub (Costelytra giveni; Coleoptera: Scarabaeidae) and the New Zealand manuka beetle (Pyronota festiva and P. setosa; Coleoptera: Scarabaeidae), was isolated from a diseased grass grub larva. A 12-day median lethal dose of 4.89 × 103 ± 0.92 × 103 cells per grass grub larva was defined for AGR96X, and death occurred within 5 to 12 days following the ingestion of a high bacterial dose. During the infection period, the bacterium rapidly multiplied within the insect host and invaded the hemocoel, leading to a mean bacterial load of 8.2 × 109 cells per larva at 6 days postingestion. Genome sequencing of strain AGR96X revealed the presence of a variant of the Serratia entomophila antifeeding prophage (Afp), a tailocin designated AfpX. Unlike Afp, AfpX contains two Afp16 tail-length termination protein orthologs and two putative toxin components. A 37-kb DNA fragment encoding the AfpX-associated region was cloned, transformed into Escherichia coli, and fed to C. giveni and Pyronota larvae, causing mortality. In addition, the deletion of the afpX15 putative chaperone component abolished the virulence of AGR96X. Unlike S. entomophila Afp, the AfpX tailocin could be induced by mitomycin C. Transmission electron microscopy analysis revealed the presence of Afp-like particles of various lengths, and when the purified AfpX tailocin was fed to grass grub or manuka beetle larvae, they underwent phenotypic changes similar to those of larvae fed AGR96X.IMPORTANCESerratia proteamaculans strain AGR96X shows dual activity against larvae of endemic New Zealand pasture pests, the grass grub (Costelytra giveni) and the manuka beetle (Pyronota spp.). Unlike Serratia entomophila, the causal agent of amber disease, which takes 3 to 4 months to kill grass grub larvae, AGR96X causes mortality within 5 to 12 days of ingestion and invades the insect hemocoel. AGR96X produces a unique variant of the S. entomophila antifeeding prophage (Afp), a cell-free phage-like entity that is proposed to deliver protein toxins to the grass grub target site, causing a cessation of feeding activity. Unlike other Afp variants, AGR96X Afp, named AfpX, contains two tail-length termination proteins, resulting in greater variability in the AfpX length. AfpX shows dual activity against both grass grub and manuka beetle larvae. AGR96X is a viable alternative to S. entomophila for pest control in New Zealand pasture systems.

Identification of Diverse Toxin Complex Clusters and an eCIS Variant in Serratia proteamaculans Pathovars of the New Zealand Grass Grub (Costelytra Giveni) and Manuka Beetle (Pyronota Spp.) Larvae.

The grass grub endemic to New Zealand, Costelytra giveni (Coleoptera: Scarabaeidae), and the manuka beetle, Pyronota festiva and P. setosa (Coleoptera: Scarabaeidae), are prevalent pest species. Through assessment of bacterial strains isolated from diseased cadavers of these insect species, 19 insect-active Serratia proteamaculans variants and a single Serratia entomophila strain were isolated. When independently bioassayed, these isolates differed in host range, the rate of disease progression, and 12-day mortality rates, which ranged from 60 to 100% of the challenged larvae. A Pyronota spp.-derived S. proteamaculans isolate caused a transient disease phenotype in challenged C. giveni larvae, whereby larvae appeared diseased before recovering to a healthy state. Genome sequence analysis revealed that all but two of the sequenced isolates contained a variant of the S. entomophila amber-disease-associated plasmid, pADAP. Each isolate also encoded one of seven distinct members of the toxin complex (Tc) family of insect-active toxins, five of which are newly described, or a member of the extracellular contractile injection (eCIS) machine family, with a new AfpX variant designated SpF. Targeted mutagenesis of each of the predicted Tc- or eCIS-encoding regions abolished or attenuated pathogenicity. Host-range testing showed that several of the S. proteamaculans Tc-encoding isolates affected both Pyronota and C. giveni species, with other isolates specific for either Pyronota spp. or C. giveni. The isolation of several distinct host-specific pathotypes of Serratia spp. may reflect pathogen-host speciation. IMPORTANCE New pathotypes of the insect pathogen Serratia, each with differing virulence attributes and host specificity toward larvae of the New Zealand manuka beetle and grass grub, have been identified. All of the Serratia proteamaculans isolates contained one of seven different insect-active toxin clusters or one of three eCIS variants. The diversity of these Serratia-encoded virulence clusters, resulting in differences in larval disease progression and host specificity in endemic scarab larvae, suggests speciation of these pathogens with their insect hosts. The differing virulence properties of these Serratia species may affect their potential infectivity and distribution among the insect populations. Based on their differing geographic isolation and pathotypes, several of these Serratia isolates, including the manuka beetle-active isolates, are likely to be more effective biopesticides in specific environments or could be used in combination for greater effect.