4 Discussion
Two successive invasions of Dickeya potato pathogens occurred in Europe: first D. dianthicola in the middle of the 20th century, this pathogen being now considered as endemic, and second, D. solani at the beginning of the 21th century. Dickeya solani and D. dianthicola cause similar symptoms on potato plants, so that they are expected to compete for the same resources. Over ten years, our epidemiologic surveys revealed a successful establishment of D. solani together with a maintenance of D. dianthicola . Our experiments contributed to explain such puzzling co-existence by contrasted advantages in different parts of the plants and therefore partially different ecological niches.
By comparing ecological traits of D. solani and D. dianthicola , using multiple strains representative of the population diversity, we indeed found contrasted behaviors in these species with respect to aerial parts versus tubers of S. tuberosum . The resident D. dianthicola more efficiently exploited host stems after an inoculation of the wounded and unwounded roots than did the invader D. solani, i.e., causing higher symptom incidence, and exhibiting greater proliferation and relative fitness in competition assays. In contrast, D. solani more efficiently initiated the rotting process (especially at a low bacterial inoculum) and outcompetedD. dianthicola in tubers, with potential consequences on tubers in the fields and during storage. Our experimental assays suggested that tubers represent an entry route of the D. solani invader into the potato agrosystems.
The capacity of D. solani to cause rooting at a lower bacterial load than D. dianthicola is likely at least partly due to a higher expression of the pel enzymes, especially PelE that acts as an initiator of the plant cell wall degradation (Duprey et al., 2016). By comparing the expression of some pel genes in emerging lesions in potato stems and tubers, we observed a higher expression of the virulence genes pelD and pelE in D. solanicompared to D. dianthicola. Remarkably, by comparing pelgenes in several Dickeya species, Duprey et al. (2016) showed that D. solani evolved specific regulatory sequences that contributes to a different expression level of the pelD andpelE genes compared to D. dianthicola . The pelAgene is truncated in D. dianthicola (Duprey et al., 2016; Raoul des Essarts et al., 2019), but full-length and expressed in D. solani (as shown in this study), reinforcing their virulence arsenal. Other metabolic traits could also contribute to the fitness advantage ofD. solani for exploiting tubers: comparative transcriptomics revealed a higher expression of the glyoxylate shunt in D. solanithan in D. dianthicola, a pathway that contributes to exploit alternative carbon sources when sugar availability is low (Raoul des Essarts et al., 2019).
Beside the tripartite interaction between potato plant host, D. solani and D. dianthicola , additional environmental (soil and climate) and biological factors (microbiota includingPectobacterium species, other hosts and non-host plants) may facilitate or limit D. solani establishment (Charkowsky 2018; Shyntum et al., 2019; Toth et al., 2011). We showed thatD. solani efficiently outcompeted D. dianthicola in hyacinths, in line with the epidemiologic data supporting the bulb plants as potential intermediate hosts with an important role in the recurrent invasion of potato agrosystems (Chen, Zhang, & Chen, 2015; Slawiak et al., 2009; van der Wolf et al., 2014). Prophylactics may allow circumventing the propagation of D. solani from an agrosystem to another. Competition and facilitation processes have been well studied in pathogenic fungi (Al-Naimi, Garrett, & Bockus, 2005; Abdullah et al., 2017; Gladieux et al., 2015; Zhan & McDonald, 2013). Another factor that could reduce competition between the twoDickeya species is their natural low abundance in soils and surface waters, as well as in tuber seeds that are subjected to prophylactic diagnosis. A small population size belonging to a single species could proliferate in a plant individual without any interactions with another Dickeya or Pectobacterium pathogen species. These effects linked to size population and dispersal are expected to delay invasion. In line with this hypothesis, the observed slow increase of the percentage of Dickeya -positive fields over one decade (2004-2015) suggested that the Dickeya invasion was still ongoing. Among the emerging literature on microbial invasion, theD. solani pathogen appears as a good example illustrating how different ecological components (here, the plant host and a resident pathogen) should be considered to understand the biological invasion by a bacterial pathogen (Cadotte et al., 2018; Germain, Mayfield, & Gilbert, 2018).
The genome sequencing data indicating low diversity in D. solanisuggest a bottleneck during introduction in Europe. The dispersal modes (either horizontally via soil, surface water, insects and some agricultural practices or vertically by asymptomatically contaminated tuber seeds) could contribute to further bottlenecks in D. solani(Charkowsky 2018; Toth et al., 2011). Selection on some genes may also have further reduced genetic diversity through selective sweeps across the whole genomes as these bacteria are clonal. Allelic changes related to quorum-sensing systems were already observed in different plant and animal pathogens along the host-colonization process (Feltner et al., 2016; Guidot et al., 2014; Tannières, Lang, Barnier, Shykoff, Faure, 2017), indicating that balancing selection contributed to maintain their variability. Remarkably, we observed balanced frequencies of two alleles in the VfmB protein involved in the Vfm-type quorum-sensing inDickeya bacteria. The chemical structure of the Vfm signal is not known, but it has been involved in tuber rotting and upregulation of some virulence genes including pectate lyases (pel genes), proteases (prt genes) and cellulases (cel genes) (Nasser et al., 2013; Potrykus et al., 2018). Using tuber assays, we observed a slightly higher aggressiveness in VfmBSer strains compared to VfmBPro strains. Using transcriptomics ofD. solani pathogens recovered from tuber symptoms, we confirmed an enrichment of upregulated genes, including pel and prtgenes, in the VfmBSer strain Ds0432.1 compared to VfmBPro strain IPO2222. Because of the role of the VfmB protein in Vfm signaling is not completely elucidated, it was premature to go deeper into the mechanistic characterization of the VfmBSer and VfmBPro alleles. In a near future, the reconstruction of allelic mutants and comparison with the wild strain with otherwise the same genetic background should help characterizing the role of VfmB in Vfm quorum-sensing and ecology ofD. solani .
In this work, we strongly suggested that the VfmBSerstrains were more aggressive than the VfmBPro strains in potato tubers, but not more virulent in potato stem assays, and that the VfmBSer strains were less competitive than the VfmBPro strains in tuber and stem symptoms. Such an environment-dependent advantage of VfmBser and VfmBPro alleles in plant infection assays predicts balancing selection in natural populations. The VfmBserallele could thus provide an advantage when competition is reduced at the beginning of the establishment in potato agrosystems and under a high dispersal condition. In contrast, VfmBser could be outcompeted by VfmBPro when D. solani is already established. Our epidemiologic data revealed up and down variation of VfmBser relative abundance in field populations over the past decade, in agreement with an increase of competition betweenD. solani VfmB alleles in the more recent sampling year. There is a very low number of SNPs/InDels (<12) in the genomes beyond those in Vfm, however, functional genetics will be required to fully validate the causal role of the Vfm alleles.
In conclusion, this study using complementary approaches, multiple strains representative of the population diversity and multiple ecological conditions, brings novel insights allowing a better understanding of the pattern and causes of the D. solani invasion into potato production agrosystems, and the reasons whyD. dianthicola nevertheless persisted. More broadly, this study contributes to our understanding the ecological determinants of pathogen invasion and of the conditions for the maintenance of endemic competitors.