A framework to gauge the epidemic potential of plant pathogens in environmental reservoirs: the example of kiwifruit canker

Abstract

New economically important diseases on crops and forest trees emerge recurrently. An understanding of where new pathogenic lines come from and how they evolve is fundamental for the deployment of accurate surveillance methods. We used kiwifruit bacterial canker as a model to assess the importance of potential reservoirs of new pathogenic lineages. The current kiwifruit canker epidemic is at least the fourth outbreak of the disease on kiwifruit caused by Pseudomonas syringae in the mere 50 years in which this crop has been cultivated worldwide, with each outbreak being caused by different genetic lines of the bacterium. Here, we ask whether strains in natural (non-agricultural) environments could cause future epidemics of canker on kiwifruit. To answer this question, we evaluated the pathogenicity, endophytic colonization capacity and competitiveness on kiwifruit of P. syringae strains genetically similar to epidemic strains and originally isolated from aquatic and subalpine habitats. All environmental strains possessing an operon involved in the degradation of aromatic compounds via the catechol pathway grew endophytically and caused symptoms in kiwifruit vascular tissue. Environmental and epidemic strains showed a wide host range, revealing their potential as future pathogens of a variety of hosts. Environmental strains co-existed endophytically with CFBP 7286, an epidemic strain, and shared about 20 virulence genes, but were missing six virulence genes found in all epidemic strains. By identifying the specific gene content in genetic backgrounds similar to known epidemic strains, we developed criteria to assess the epidemic potential and to survey for such strains as a means of forecasting and managing disease emergence.

Keywords: co-existence; effector repertoire; emerging pathogens; host range.

Figure 1

Bayesian tree constructed on the concatenated sequences cts, gyrB, rpoD and gapA (1852 bp) of the strains tested for pathogenicity, endophytic growth and molecular markers. Bootstrap values are indicated at each node. External and internal symptoms are indicated with black boxes (severe disease), dark grey boxes (mild symptoms), light grey boxes (faint symptoms) and white boxes (no symptoms). The presence of the catechol operon involved in aromatic compound degradation, and of hopZ3 and ITS markers, is indicated by black (present) and white (absent) squares. For the catechol operon, all the genes were present for the positive strains. The origin of strains and their biovar affiliation are indicated with colour: light blue for Pseudomonas syringae pv. actinidiae (Psa) bv. 3, turquoise for Psa bv. 1, dark blue for Psa bv. 4, brown for strains isolated from woody hosts, green for strains isolated from herbaceous hosts and red for the environmental strains.

Figure 2

Characteristic external symptoms observed on kiwifruit stems at 30 days post‐inoculation. External necrosis and canker caused by the strain from Actinidia deliciosa  CFBP 7286 (A), necrosis caused by the environmental strain AF0015 (B), swelling caused by the strain NCPPB 3335 isolated from Olea europaea (C) and the absence of symptoms on stems inoculated with strain ICMP 18882 isolated from Actinidia deliciosa.

Figure 3

Growth of strains in 3‐week‐old kiwifruit plants. Population densities were determined at the point of inoculation for strains CFBP 7286 (blue) and USA007 (red) of Pseudomonas syringae (A) and for strains CFBP 7286 (blue) and CC1544 (green) (B). Broken lines indicate the growth of each strain independently and full lines represent growth for mixed inoculations. Two replicate experiments were conducted with three independent measurements of population density per time and per replicate experiment. The mean ± standard error at each time point indicated in the figure is based on all six observations pooled over the two replicates, because there were no significant differences in mean density between the two replicate experiments (P < 0.05), except for CFBP 7286 alone at 14 and 21 dpi. In addition, the strains showed the same trend during the two experiments.

Figure 4

External symptoms recorded on kiwifruit plants 3 weeks after inoculation. The same symptoms were observed on all plants (three per time point). Necrosis observed with strain CFBP 7286 of Pseudomonas syringae (A). No symptoms were recorded on kiwifruit 3 weeks after co‐inoculation with environmental strains (USA0007 and CC1544) and CFBP 7286 (B).

Figure 5

Bayesian tree of the concatenated complete sequences of phenol‐MetA, flavin adenine dinucleotide (FAD)‐dependent oxygenase, short‐chain alcohol dehydrogenase and dienelactone hydrolase. Posterior probabilities are shown at each node. Phylogroups 1 and 3, Pseudomonas syringae pv. actinidiae (Psa) biovars and the names of the strains are indicated on the branches. The tree was rooted with the sequences extracted from the genomes of strain KF707 of Pseudomonas pseudoalcaligenes and strain CEB98818 of Pseudomonas fuscovaginae. The origin of the strains and their biovar affiliation are indicated with colour: light blue for Psa bv. 3, turquoise for Psa bv. 1, dark blue for Psa bv. 4, brown for strains isolated from woody hosts, and red for the environmental strains.

Figure 6

Host range of strains CFBP 7286, ICMP 18882, USA0007 and CC1544. The severity of the disease is expressed with a grey colour scale. For endophytic growth, black boxes indicate that strains grow into the host plant and that they were re‐isolated even in the absence of symptoms. For strains pathogenic on soybean, faba bean, clover, sunflower, geranium and ranunculus, disease was observed only on stems (*). Pseudomonas syringae pv. actinidiae (Psa) bv. 3 is indicated in blue, Psa bv. 4 in dark blue and environmental strains in red.

Figure 7

Type III secretion gene repertoires in strains of Pseudomonas syringae. Results for Pseudomonas syringae pv. actinidiae (Psa) bv. 1, Psa bv. 3 and Psa bv. 4 were compressed as the results were identical for all the strains analysed (all the genomes available in GenBank). Strains USA0007 and CC1544 were isolated from river headwaters. Other strains indicated include MAFF 302280 (P. syringae pv. morsprunorum), NCPPB 2598 (P. syringae pv. theae), BPIC 631 (P. syringae pv. avellanae), DC3000 (P. syringae pv. tomato), B728a (P. syringae pv. syringae) and Pph1448a (P. syringae pv. phaseolicola). Black boxes indicate that the genes were found in full length, white boxes indicate the absence of the genes and grey boxes indicate that the genes were truncated. The origin of strains and their biovar affiliation are indicated with colour: light blue for Psa bv. 3, turquoise for Psa bv. 1, dark blue for Psa bv. 4, brown for strains isolated from woody hosts, green for strains isolated from herbaceous hosts and red for the environmental strains.