Antimicrobial production by Pectobacterium carotovorum subspecies brasiliensis and its role in competitive fitness of the potato pathogen

Abstract

Pectobacterium carotovorum subspecies brasiliensis (P. c. subsp. brasiliensis), a member of the soft rot Erwinias (SREs), was first described as the causative agent of a stem disease in potato called blackleg. Blackleg describes the blackening, wilting and necrosis of potato stem tissue. Initially detected in Brazil, P. c. subsp. brasiliensis subsequently emerged as a pathogen in temperate regions, although the mechanisms that contributed to its emergence are unknown. A second SRE pathogen, Dickeya solani, also emerged as an aggressive potato pathogen in Europe. Dickeya solani successfully displaced the previously dominant blackleg causing pathogens, such as P. atrosepticum. Comparative genomic studies, using the genome of D. solani plus other SRE genomes such as Pectobacterium, identified some D. solani specific genes. Three of these loci were identified as novel non-ribosomal synthetase/polyketide synthatase (NRPS/PKS) genes, which all encoded previously unknown products. It was predicted that the combination of these novel gene clusters provided the adaptive advantage, which enabled D. solani to successfully emerge as a pathogen. The genome of a P. c. subsp. brasiliensis strain isolated from infected potato plants in New Zealand, P. c. subsp. brasiliensis ICMP 19477, was recently sequenced. The bacterium was found to encode many genes associated with antimicrobial production, including bacteriocin and carbapenem synthesis, as well as a putative novel NRPS locus. A number of the identified loci were not present in the genomes of other SREs. One of these antimicrobial clusters, or a combination of these clusters, may be an important mechanism in the emergence of P. c. subsp. brasiliensis. However, the ecological significance of antimicrobial molecules is not understood. It has previously been reported that, P. c. subsp. brasiliensis PBR1692, is antagonistic to P. atrosepticum SCRI1043 in vitro (Marquez-Villavicencio et al., 2011). However, in planta significance of this interaction appeared minimal during co-inoculation studies in potato stems. Pectobacterium betavasculorum, was also reported to inhibit the growth of other Pectobacterium species when co-inoculated in potato tubers. This study found that P. c. subsp. brasiliensis ICMP 19477 outcompetes P. atrosepticum SCRI1043 in both in vitro plate and in planta competition assays, when co-inoculated in potato tubers. However, this was not observed in in vitro liquid competition assays. This suggested that the antagonistic effect of P. c. subsp. brasiliensis ICMP 19477 on P. atrosepticum SCRI1043 only occurred in structured environments. Functional studies identified that P. c. subsp. brasiliensis ICMP 19477 produces a secreted antimicrobial molecule at late exponential / early stationary phase. A random transposon (Tn5) mutant library of P. c. subsp. brasiliensis ICMP 19477 identified three mutants, within the genes carR, slyA and carI, which were unable to inhibit the growth of P. atrosepticum SCRI1043 in vitro. These mutated genes are known to be involved in carbapenem regulation in P. c. subsp. carotovorum. Furthermore, these mutants also lost the competitive advantage against P. atrosepticum SCRI1043 when co-inoculated in potato tubers. This evidence suggested that a carbapenem molecule, produced by P. c. subsp. brasiliensis ICMP 19477, enhances the competitive fitness of the bacterium in planta. Overall, this study provided novel insights into the ecological significance of antimicrobial production by plant pathogens, thereby, identifying possible mechanisms for pathogen emergence.