In 2020, tomato foliage samples exhibiting symptoms were collected from Cook County and Grady County in Georgia, United States. For bacterial isolation, the margins of healthy and symptomatic tomato leaf tissues were excised using a sterile scalpel and then macerated in sterile distilled water (sdH₂O). The resulting tissue macerates were then streaked onto the nutrient agar (NA) medium. The NA plates were incubated at 28°C for 2 days. The dominant colonies on the isolation plates were selected and streaked onto new NA plates to obtain pure cultures. In addition, three P. capsici strains (Pc19-1^T, Pc19-2, and Pc19-3) isolated from pepper in Georgia (Zhao et al., 2021), two Pseudomonas strains (GEV417 and GEV1127) isolated from tomato in Florida (Timilsina et al., 2017), and three Pseudomonas strains (NCPPB1511, NCPPB2479, and NCPPB3928) obtained from the National Collection of Plant Pathogenic Bacteria (NCPPB) were also included and characterized in this study. The location, host, and year of isolations for these strains are shown in Table 1.

The Pseudomonas strains were routinely cultured on NA at 28°C for 2 days. To prepare the inoculum, the strains were grown in nutrient broth in a shaking incubator (MaxQ 4,450, Thermo Scientific, Waltham, MA) at 28°C and 200 rpm for around 16 h. After centrifugation at 16,100 x g for 1 min, the supernatants were then removed, and the resulting pellets were resuspended in sdH₂O. The bacterial concentrations were then adjusted to an optical density of 0.3 at 600 nm, which corresponds to approximately 10⁸ colony-forming units (CFU/mL), using a Biophotometer (Eppendorf, Hamburg, Germany).

The host range assays were performed on pepper cv. Aristotle, tomato cv. Glacier, Chinese cabbage (Brassica rapa ssp. chinensis) cv. Rubicon, eggplant (Solanum melongena) cv. Nadia, lettuce (Lactuca sativa) cv. Dragoon, broccoli (Brassica oleracea var. italica) cv. Arcadia F1, and endive (Cichorium endivia) cv. Curlesi. Seedlings (n = 10 seedlings per host per experiment) were grown in plastic pots filled with commercial potting mix and maintained at a temperature of 28°C in a greenhouse. Two independent experiments were conducted. To inoculate the seedlings, leaves of four to six-week-old seedlings of each host were infiltrated with bacterial suspensions of Pseudomonas strains in Table 1 at a concentration of 10⁶ CFU/mL using a syringe. Seedlings inoculated with sdH₂O were used as negative controls. The symptom was evaluated qualitatively (presence/absence) in the inoculated seedlings at 7 days post-inoculation (dpi). The bacteria were re-isolated from the symptomatic tissues and the identities of the isolated bacteria were confirmed by BOX-PCR (Brusetti et al., 2008). Briefly, isolated bacterial strains were inoculated into 3 mL of nutrient broth and incubated on a rotary shaker (Innova; New Brunswick Scientific Co., Edison, NJ) at 250 rpm for 18 h. After incubation, cells were harvested by centrifugation at 6,000 × g (Allegra 25R, Beckman Coulter, Fullerton, CA) for 5 min and DNA was extracted using the UltraClean Microbial DNA Kit (MO BIO, Carlsbad, CA) according to the manufacturer’s instructions. For BOX-PCR, 2 μL of bacterial DNA were amplified using 10 μM of BOXA1R primer (5’-CTA CGG CAA GGC GAC GCT GAC G-3′) according to PCR conditions as described previously (Brusetti et al., 2008). PCR products (10 μL) were separated by electrophoresis at 125 V for 4 h on a 1.5% agarose gel in 1X Trisborate ethylenediaminetetraacetic acid (EDTA) buffer.

Nutrient agar supplemented with 0.8 mM copper sulphate (CuSO₄· 5H₂O) was prepared following published protocols (Walcott et al., 2004; Stice et al., 2018). Ten microliters of each 1 × 10⁸ CFU/mL bacterial suspension in sdH₂O were spotted onto the plates, and growth was visually evaluated and recorded as positive or negative after two days of incubation at 28°C. Acidovorax citrulli group I strain M6, and group II strain AAC00-1 were used as copper-tolerant and sensitive controls, respectively. Each strain was tested in triplicate, and the experiment was conducted twice.

Genome annotation was performed using Rapid Annotation using Subsystem Technology (RAST v2.0) server (Overbeek et al., 2014). The subsystems of the annotated genomes related to copper were further manually reviewed on the RAST server. Copper tolerance gene clusters unique to the copper-tolerant strains were identified and their nucleotide sequences were extracted and aligned using MAFFT in Geneious. Protein secretion systems were predicted using TXSScan (Abby et al., 2014, 2016). Operon maps of the identified copper tolerance clusters and protein secretion systems were generated using Gene Graphics (Harrison et al., 2018) and annotated in PowerPoint. The assembly files were used as input data for in silico secondary metabolite gene cluster analysis using antiSMASH v 6 with default parameters and the ‘knownclusterblast’ flag (Blin et al., 2021).

The general features of the 12 Pseudomonas sequenced genomes are summarized in Table 2. The genome lengths of the assemblies ranged from 5.82 Mbp (NCPPB2479) to 6.05 Mbp (NCPPB1511) with an average length of 5.91 Mb. The number of contigs in each genome assembly ranged from 40 to 59. The N50 of the assemblies ranged from 201,021 bp (GEV417) to 362,351 bp (NCPPB3928). The total number of genes in each genome ranges from 5,104 (Pc19-2) to 5,367 (NCPPB1511). The predicted total number of protein-coding genes varies from 4,991 (Pc19-2) to 5,248 (NCPPB1511). The GC content of the twelve genomes ranges from 58.37 to 58.58%, with an average of 58.43% (Table 2). The LINs for the twelve Pseudomonas genomes are shown in Table 3.

To determine the species identity of the 12 Pseudomonas genomes, their dDDH and ANIb values were calculated by comparing them with the P. capsici type strain Pc19-1^T. All strains displayed dDDH values ranging from 77.0 to 100.0% when compared with P. capsici Pc19-1^T, exceeding the cut-off value of 70% for species delineation based on dDDH (Table 4). Similarly, all strains exhibited ANIb values ranging from 97.1 to 100.0% when compared with P. capsici Pc19-1^T, surpassing the commonly accepted threshold of 95–96% for species delineation based on ANIb (Table 4). These observations indicate that all twelve strains belonged to the same species. Furthermore, 13 genomes listed under the P. cichorii genome list in the NCBI database had dDDH values exceeding 70%, indicating that they are in fact P. capsici. The list of strains that were wrongly speciated as P. cichorri in the NCBI database but were indeed P. capsici based on the above genome-based assays include; 473, MAFF 302698, Ku1409-10-1, NB15027, LCDW06, LCFQ22, 481, 482, 136, 474, WSXC14, s-2-2-1, and ICMP 1649.

The phylogenomic tree, which was generated using the M1CR0B1AL1Z3R web server, is based on the alignment of the core-proteome (4,216 genes) of all 25 P. capsici strains, with P. cichorii ATCC 10857^T as an outgroup. This core-proteome tree showed that the Georgia pepper strains Pc19-1^T, Pc19-2, and Pc19-3 formed a cluster that was closely related to another cluster consisting of Georgia tomato strains Pc20-3, Pc20-4, Pc20-5, and a tomato strain from Florida GEV1127, with 100% support values (Figure 1). These observations suggest that these strains have a close evolutionary relationship with each other. However, strain Pc20-2, which was isolated from a separate tomato outbreak in Cook County, Georgia, was found to be distinct from other strains, indicating a unique source of introduction and outbreak (Figure 1).

A phylogenetic analysis was conducted using the concatenated sequences of gyrB and rpoD, which had a total length of 961 nucleotides. Fifteen validly described Pseudomonas type strains were included in the analysis, with P. graminis DSM 11363^T serving as an outgroup. The resulting phylogenetic tree showed that the strains claimed to be a new phylogroup of P. cichorii, as well as 12 P. capcisi strains whose genomes were sequenced in this project including P. capsici type strain Pc19-1^T, clustered together and formed a separate clade from the P. cichorii clade (Figure 2). This clade included P. capsici strains from diverse crops and diverse geographical locations. Additionally, the Georgia tomato strains Pc20-2, Pc20-3, and Pc20-4 were clustered together with the 17 Florida tomato strains isolated in 2012 (Figure 2). Pepper strains from Georgia were clustered together and close to tomato strains.

The host range of twelve P. capsici strains was evaluated on seven different plant species, including pepper cv. Aristotle, tomato cv. Glacier, Chinese cabbage cv. Rubicon, eggplant cv. Nadia, lettuce cv. Dragoon, broccoli cv. Arcadia F1, and endive cv. Curlesi. All twelve strains induced symptoms on the leaves of pepper, tomato, Chinese cabbage, eggplant, and lettuce, but did not cause any symptoms on broccoli and endive (Table 5). The symptoms observed on the tested hosts were more or less similar with initial water-soaked lesions that gradually turn necrotic. Control plants of each respective host did not exhibit any symptoms. The responses of seedlings were consistent across replicates and experiments. The isolates obtained from artificially inoculated plants were confirmed to be identical to the original inoculated strain through BOX-PCR analysis for DNA fingerprinting comparisons.

All 12 tested P. capsici strains were able to grow on NA medium that was not amended with CuSO₄· 5H₂O. Out of the 12 strains, seven strains were able to grow on NA medium amended with 0.8 mM CuSO₄· 5H₂O. However, the remaining five P. capsici strains were not able to grow on the same copper amended medium. Analysis of the copper-tolerance related subsystems in RAST revealed that copper tolerance gene clusters are present only in seven copper-tolerant strains (Pc19-1, Pc19-2, Pc19-3, GEV1127, Pc20-3, Pc20-4, and Pc20-5), but are absent in the remaining five strains (NCPPB3928, NCPPB2479, Pc20-2, GEV417, and NCPPB1511). Interestingly, two types of copper tolerance gene clusters were observed in the seven copper-tolerant strains listed above (Figure 3). Three strains (Pc19-1, Pc19-2, and Pc19-3) had identical sequences, while the other four strains (GEV1127, Pc20-3, Pc20-4, and Pc20-5) had identical sequences for their respective aligned region (Figure 3). The pairwise identity of the copper tolerance gene clusters between Pc19-1^T and GEV1127 was 77.3%. The copper tolerance gene cluster in strain GEV1127 included four genes that were absent in the Pc19-1^T cluster, including a DUF1289 domain-containing protein, a hypothetical protein, a CDF family Co (II) / Ni (II) efflux transporter DmeF, and a metal/formaldehyde-sensitive transcriptional repressor (Figure 3). These copper tolerance gene clusters were not found in other publicly available P. capsici genomes.

The P. capsici strains possess an array of secretion systems, including one set of type I secretion system, two sets of type II secretion systems, one set of type III secretion system, two sets of type V secretion systems, three sets of type VI secretion systems (Figure 4). In addition, 10 gene clusters associated with secondary metabolites were identified using antiSMASH and are listed in Table 6. These gene clusters are linked to a variety of products, including a non-ribosomal peptide synthetase (NRPS) and type I polyketide synthase product (similar to yersiniabactin), an NRPS product (similar to cichopeptin), an NRPS product (similar to pyoverdine), an NRPS beta-lactam product (similar to thanamycin), an aryl polyene product, an N-acetylglutaminylglutamine amide product, an NRPS lipopeptide product (similar to cichofactin), a type III polyketide synthase product (similar to fisherindole), a redox-cofactor type product (similar to lankacidin C), and an NRPS product (similar to fragin).