In vitro, in planta, and comparative genomic analyses of Pseudomonas syringae pv. syringae strains of pepper (Capsicum annuum var. annuum)

Abstract

Pseudomonas syringae pv. syringae (Pss) is an emerging phytopathogen that causes Pseudomonas leaf spot (PLS) disease in pepper plants. Pss can cause serious economic damage to pepper production, yet very little is known about the virulence factors carried by Pss that cause disease in pepper seedlings. In this study, Pss strains isolated from pepper plants showing PLS symptoms in Ohio between 2013 and 2021 (n = 16) showed varying degrees of virulence (Pss populations and disease symptoms on leaves) on 6-week-old pepper seedlings. In vitro studies assessing growth in nutrient-limited conditions, biofilm production, and motility also showed varying degrees of virulence, but in vitro and in planta variation in virulence between Pss strains did not correlate. Comparative whole-genome sequencing studies identified notable virulence genes including 30 biofilm genes, 87 motility genes, and 106 secretion system genes. Additionally, a total of 27 antimicrobial resistance genes were found. A multivariate correlation analysis and Scoary analysis based on variation in gene content (n = 812 variable genes) and single nucleotide polymorphisms within virulence genes identified no significant correlations with disease severity, likely due to our limited sample size. In summary, our study explored the virulence and antimicrobial gene content of Pss in pepper seedlings as a first step toward understanding the virulence and pathogenicity of Pss in pepper seedlings. Further studies with additional pepper Pss strains will facilitate defining genes in Pss that correlate with its virulence in pepper seedlings, which can facilitate the development of effective measures to control Pss in pepper and other related P. syringae pathovars.

Importance: Pseudomonas leaf spot (PLS) caused by Pseudomonas syringae pv. syringae (Pss) causes significant losses to the pepper industry. Highly virulent Pss strains under optimal environmental conditions (cool-moderate temperatures, high moisture) can cause severe necrotic lesions on pepper leaves that consequently can decrease pepper yield if the disease persists. Hence, it is important to understand the virulence mechanisms of Pss to be able to effectively control PLS in peppers. In our study, in vitro, in planta, and whole-genome sequence analyses were conducted to better understand the virulence and pathogenicity characteristics of Pss strains in peppers. Our findings fill a knowledge gap regarding potential virulence and pathogenicity characteristics of Pss in peppers, including virulence and antimicrobial gene content. Our study helps pave a path to further identify the role of specific virulence genes in causing disease in peppers, which can have implications in developing strategies to effectively control PLS in peppers.

Keywords: Pseudomonas syringae pv. syringae; comparative genomic analysis; pepper plants; phytobacteria; virulence genes; whole-genome sequence analysis.

Fig 1

Disease severity and bacterial populations on “California Wonder” pepper seedlings 3 and 7 dpi with 16 Pss strains. Disease severity is represented by the number of lesions on two lower leaves of infected seedlings at 3 dpi (A) and 7 dpi (B) and populations of Pss strains in terms of log CFU/g at 3 dpi (C) and 7 dpi (D). The experiment was conducted twice with a minimum of four replicates for each strain at different time points, and data from the two experiments were combined. The black bars represent the median number of lesions or log (CFU/g). Different letters represent strains that are significantly different (P < 0.05), and the error bars represent the standard deviation.

Fig 2

In vitro (growth, biofilm, and motility) characteristics of 16 Pss strains. (A) The growth rate and doubling time of Pss strains, calculated using the growthcurver 0.3.1 package in R 4.3.1. The OD₆₀₀ measurement of Pss strains taken over 24 h in M9 media was used in growthcurver 0.3.1 in R to calculate the growth rate and doubling time. (B) Quantity of biofilm produced by Pss strains after 72 h of incubation at 28°C in M9 minimal broth. OD₅₇₀ was measured to quantify biofilm production. (C) Motility of Pss strains was measured after 24 h incubation on Luria–Bertani (LB) semi-solid agar medium (0.3%) at 28°C. Motility was determined by measuring the diameter (in millimeter) of the halo. Each of the in vitro experiments was conducted twice with three replicates each. The bars represent standard error, and the different letters represent strains that are significantly different (P < 0.05).

Fig 3

Phylogenetic, core, and pangenome analysis of 16 Pss strains. (A) Whole-genome core SNP-based phylogeny of phytopathogenic Pseudomonas strains (n = 34). K-chooser was used to find the optimal k-mer (19-mer), and the core SNPs were calculated by KSNP3. The maximum likelihood phylogeny tree was plotted in R. The branch length of the tree correlates with the SNP distance. Each pathovar as well as host/source of isolation is indicated by a different color. Pepper pathovars colored in blue are strains collected and used in this study. The branch for Pseudomonas moraviensis used as the outgroup species is truncated as indicated by the dashed line. The disease severity of the 16 Pss strains is indicated with a color spectrum, where dark blue indicates low disease severity and yellow indicates high disease severity. The bootstrap support for the branches is indicated with a color spectrum, where dark purple indicates low bootstrap support and light yellow indicates high bootstrap support. (B) Pie chart showing the pangenome composition of the 16 Pss strains. Core genes are present in 100% of the strains, shell genes in 15%–99%, and cloud genes in <15% of strains. (C) Rarefaction curves for core and pangenome of the 16 Pss strains. The lefthand plot shows how the size of the core genome (y-axis) decreases with the addition of more genome assemblies (x-axis), with individual points showing the core genome size for each possible combination of genomes for each value of x, the number of included genomes, and the line showing the fitted line following an exponential decay function. The righthand plot is similar but shows the increase of the pangenome with an increasing number of genomes, and the fitted line follows a power-law distribution.

Fig 4

Hierarchical clustering based on presence/absence of variable genes (n=812) of Pseudomonas syringae pv. syringae (Pss) strains (n=16). On the left-hand side, colored boxes around the strain names indicate the cluster for each strain, and the disease severity score at 7 dpi is shown for each strain. On the right-hand side, each column represents a single gene with blue indicating presence of a gene, and red indicating absence of a gene.

Fig 5

Principal component analysis (PCA) of variable genes (n=812) between Pseudomonas syringae pv. syringae (Pss) strains (n=16). Each dot represents the different Pss strains. PCA grouped the Pss strains into 5 clusters (A to E).

Fig 6

Virulence (secretion system, motility, and biofilm) and antimicrobial resistance genes of Pseudomonas syringae pv. syringae (Pss) strains (n=16) in the variable genome. A total of 29 secretion system genes, 2 motility genes, 2 biofilm genes and 6 antimicrobial resistance genes were found to be part of the variable genome. The darker grey boxes indicate more than one copy of the gene.