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. 2024 Oct 3;12(10):e0132424.
doi: 10.1128/spectrum.01324-24. Epub 2024 Sep 3.

Pathogenicity, phylogenomic, and comparative genomic study of Pseudomonas syringae sensu lato affecting sweet cherry in California

Affiliations

Pathogenicity, phylogenomic, and comparative genomic study of Pseudomonas syringae sensu lato affecting sweet cherry in California

Tawanda E Maguvu et al. Microbiol Spectr. .

Abstract

To gain insights into the diversity of Pseudomonas syringae sensu lato affecting sweet cherry in California, we sequenced and analyzed the phylogenomic and genomic architecture of 86 fluorescent pseudomonads isolated from symptomatic and asymptomatic cherry tissues. Fifty-eight isolates were phylogenetically placed within the P. syringae species complex and taxonomically classified into five genomospecies: P. syringae pv. syringae, P. syringae, Pseudomonas cerasi, Pseudomonas viridiflava, and A. We annotated components of the type III secretion system and phytotoxin-encoding genes and correlated the data with pathogenicity phenotypes. Intact probable regulatory protein HrpR was annotated in the genomic sequences of all isolates of P. syringae pv. syringae, P. syringae, P. cerasi, and A. Isolates of P. viridiflava had atypical probable regulatory protein HrpR. Syringomycin and syringopeptin-encoding genes were annotated in isolates of all genomospecies except for A and P. viridiflava. All isolates of P. syringae pv. syringae caused cankers, leaf spots, and fruit lesions in the field. In contrast, all isolates of P. syringae and P. cerasi and some isolates of P. viridiflava caused only cankers. Isolates of genomospecies A could not cause any symptoms suggesting phytotoxins are essential for pathogenicity. On detached immature cherry fruit pathogenicity assays, isolates of all five genomospecies produced symptoms (black-dark brown lesions). However, symptoms of isolates of genomospecies A were significantly (P < 0.01) less severe than those of other genomospecies. We also mined for genes conferring resistance to copper and kasugamycin and correlated these data with in vitro antibiotic sensitivity tests.

Importance: Comprehensive identification of phytopathogens and an in-depth understanding of their genomic architecture, particularly virulence determinants and antibiotic-resistant genes, are critical for several practical reasons. These include disease diagnosis, improved knowledge of disease epidemiology, pathogen diversity, and determination of the best possible management strategies. In this study, we provide the first report of the presence and pathogenicity of genomospecies Pseudomonas cerasi and Pseudomonas viridiflava in California sweet cherry. More importantly, we report a relatively high level of resistance to copper among the population of Pseudomonas syringae pv. syringae (47.5%). This implies copper cannot be effectively used to control bacterial blast and bacterial canker of sweet cherries. On the other hand, no isolates were resistant to kasugamycin, an indication that kasugamycin could be effectively used for the control of bacterial blast and bacterial canker. Our findings are important to improve the management of bacterial blast and bacterial canker of sweet cherries in California.

Keywords: Prunus avium; Pseudomonas syringae; antibiotic resistance; bacterial blast; bacterial canker; blossom blast; comparative genomics; genome mining; genomics; pathogenicity; phylogenetic analysis; sweet cherry.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Symptoms of bacterial blast and bacterial canker on sweet cherry. (A) Trunk canker with amber-colored gumballs on the margins. (B) Blasted flowers; they turn brown and wither while still attached to the plant. (C) Dead buds with amber-colored gumming exuding from the base of a dead spur; the bacteria are most likely initiating branch canker. (D) Typical fruit rot caused by Pseudomonas syringae pv. syringae showing a sunken black–dark brown lesion.
Fig 2
Fig 2
Detached immature fruit pathogenicity test. Rating scale for lesions/rot on the fruits. The image shows severity score ratings (0-5) from left to right.
Fig 3
Fig 3
Balanced minimum evolutionary tree inferred from inter-genomic distances of the whole genome sequences calculated using GGDC 2.1. Using phylogenomic analyses, fluorescent pseudomonads isolated from sweet cherry clustered into two main clades designated as P. syringae species complex (indicated by the black outer border) and other fluorescent pseudomonads (indicated by the green outer border). The outgroups consisted of non-pseudomonad isolates. Isolates highlighted in red are representatives of the established P. syringae phylogroups.
Fig 4
Fig 4
A close-up of the phylogenomic analyses of the P. syringae species complex from Fig. 3. Isolates from this study are highlighted in black, and isolates highlighted in red are representatives of the established P. syringae phylogroups. Isolates from this study fall into two main phylogroups (PG) PG2 and PG7 (see key). They were taxonomically classified into at least five genomospecies based on dDDH; P. syringae pv. syringae, P. syringae, A, P. cerasi, and P. viridiflava.
Fig 5
Fig 5
Some of the virulence determinants and epiphytic fitness-related genes annotated from the genomic sequences of isolates belonging to the P. syringae species complex. The % represents the % of isolates with the corresponding gene annotated from their genomic sequences from each of the five identified genomospecies (P. syringae pv. syringae, P. syringae, A, P. cerasi, and P. viridiflava).
Fig 6
Fig 6
Canker pathogenicity tests in November 2023. (A) % of disease incidences; (B) disease severity ratings; data are the average of all replications, and error bars indicate ±standard deviation of the mean.
Fig 7
Fig 7
Canker pathogenicity tests in March 2024. (A) % of disease incidences; (B) disease severity ratings; data are the average of all replications, and error bars indicate ±standard deviation of the mean. Different letters indicate significant differences (P < 0.01) in the severity ratings among isolates of the different genomospecies.
Fig 8
Fig 8
Results of leaf pathogenicity tests. Data are the average of all replications, and error bars indicate ±standard error of the mean. Different letters indicate significant differences (P < 0.01) in the disease ratings among isolates of the different genomospecies.
Fig 9
Fig 9
Detached immature fruit pathogenicity test. (A) Fruit rot score ratings 3 days post-inoculation. (B) Fruit rot score ratings 6 days post-inoculation. Data are the average of all replications, and error bars indicate ±standard deviation of the mean. Different letters indicate significant differences (P < 0.01) in the fruit rot score ratings among isolates of the different genomospecies.

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