Pseudomonas capsici sp. nov., a plant-pathogenic bacterium isolated from pepper leaf in Georgia, USA

Three phytopathogenic bacterial strains (Pc19-1T, Pc19-2 and Pc19-3) were isolated from seedlings displaying water-soaked, dark brown-to-black, necrotic lesions on pepper (Capsicum annuum) leaves in Georgia, USA. Upon isolation on King’s medium B, light cream-coloured colonies were observed and a diffusible fluorescent pigment was visible under ultraviolet light. Analysis of their 16S rRNA gene sequences showed that they belonged to the genus Pseudomonas , with the highest similarity to Pseudomonas cichorii ATCC 10857T (99.7 %). The fatty acid analysis revealed that the majority of the fatty acids were summed feature 3 (C16  :  1 ω7c/C16  :  1 ω6c), C16  :  0 and summed feature 8 (C18  :  1 ω7c/C18  :  1 ω6c). Phylogenomic analyses based on whole genome sequences demonstrated that the pepper strains belonged to the Pseudomonas syringae complex with P. cichorii as their closest neighbour, and formed a separate monophyletic clade from other species. Between the pepper strains and P. cichorii , the average nucleotide identity values were 91.3 %. Furthermore, the digital DNA–DNA hybridization values of the pepper strains when compared to their closest relatives, including P. cichorii , were 45.2 % or less. In addition, biochemical and physiological features were examined in this study and the results indicate that the pepper strains represent a novel Pseudomonas species. Therefore, we propose a new species Pseudomonas capsici sp. nov., with Pc19-1T (=CFBP 8884T=LMG 32209T) as the type strain. The DNA G+C content of the strain Pc19-1T is 58.4 mol%.

The members of Pseudomonas species have been isolated from various environmental sources including soil, water, plants and animals [1]. In the past, DNA-DNA hybridization (DDH) was considered the gold standard for prokaryotic species differentiation [2]. However, DDH is laborious and a difficult approach. Sequence analysis of 16S rRNA genes and conserved housekeeping genes are often conducted for characterization of Pseudomonas species [3,4], but not accepted as valid standards for bacterial species delineation. Instead, recent developments of whole-genome sequencing technologies have advanced the sequence-based taxonomy for bacteria [5].
Multiple Pseudomonas species and pathovars have been associated with causing diseases on a wide range of plant hosts [6]. Among the Pseudomonas species, Pseudomonas syringae has been widely studied and taxonomically well-characterized compared with other plant-pathogenic bacterial species. The multi-locus sequence analysis of housekeeping genes has been used to assign 13 phylogroups and nine genomospecies within the P. syringae complex [4,6]. Due to the advances in sequencing technology, whole-genome sequences (WGS) have been used for classifying Pseudomonas species taxonomically [5]. Specifically, multiple pairwise comparative approaches like average nucleotide identity (ANI) and genome-to-genome distance calculations have been used for species differentiation using WGS [7]. Recently, three fluorescent Pseudomonas strains were isolated from symptomatic pepper foliage in Georgia, USA. Using a polyphasic taxonomic approach, we provide evidence that these strains represent a novel Pseudomonas species. The pepper strains Pc19-1, Pc19-2 and Pc19-3 are being proposed as Pseudomonas capsici sp. nov.

ISOLATION AND ECOLOGY
Leaf blight symptoms were observed in pepper seedlings in greenhouses in Georgia, USA in 2019. Foliar symptoms included water-soaked, dark brown-to-black, irregularly shaped lesions (Fig. S1, available in the online version of this article). Three fluorescent Pseudomonas strains were isolated from the symptomatic tissue of pepper leaves on King's medium B. Pathogenicity of isolated bacterial strains was confirmed by leaf infiltration of pepper foliage (cv. Aristotle) using a syringe with bacterial suspensions containing approximately 1×10 6 c.f.u. ml −1 [8]. Disease symptoms similar to the original natural infections were observed 48 h after inoculation by strains Pc19-1 T , Pc19-2 and Pc19-3, while P. cichorii type strain NCPPB943 T did not cause necrosis on pepper leaf (Fig. S1). The standard lopat tests [9], which consist of determining levan production on 5 % sucrose medium, oxidase activity, potato soft rot ability, arginine dihydrolase assay and tobacco hypersensitivity, were conducted for preliminary characterization. The three pepper strains were positive for the oxidase test, caused potato rot (Fig. S2), and showed a hypersensitive reaction on tobacco. However, they were unable to produce levan and were negative for the arginine-dihydrolase assay. These characteristics are unique when compared to the lopat profiles of the fluorescent plant pathogenic Pseudomonas species reported by Lelliott et al. [9], but closely matched with P. cichorii except for the potato rot test.

16S rRNA GENE PHYLOGENY
Phylogenetic analysis was conducted using 16S rRNA gene sequences. The 16S rRNA genes of the three pepper strains was amplified using primers 27F and 1492R, and the resulting products were sequenced with primers 27F and 1492R using Sanger sequencing by Eurofins Genomics (Louisville, KY). The partial 16S rRNA gene sequences of strains Pc19-1, Pc19-2 and Pc19-3 were deposited in GenBank (accession numbers: MW583591, MW583592 and MW583593). The sequences were compared with sequences of the type strains of 17 closely related Pseudomonas species downloaded from NCBI. Comparative analysis revealed that the three pepper strains had identical partial 16S rRNA gene sequences and their sequence identities with P. cichorii type strain ATCC 10857 T were all 99.7 % ( Table 1). The next species was Pseudomonas ovata F51T T with 98.8 % 16S rRNA gene sequence identities. The sequences were aligned using mafft (version Table 1. Genomic relationship between strain Pc19-1 T and the type strains of closely related Pseudomonas species as well as other Pseudomonas capsici sp. nov. strains The 16S rRNA gene sequences were retrieved from GenBank by similarity searches using the strain Pc19-1 T sequence as a query. ANIb and dDDH values were calculated using JSpeciesWS version 1.2.1 [17] and the Genome-to-Genome Distance Calculator 2.1 (formula 2) [18], respectively. 7.294b) [10] within Geneious Prime 2019. A maximumlikelihood phylogenetic tree using 16S rRNA gene sequences (1266 nucleotides) was reconstructed using the phyml package [11]. The model for phylogenetic analysis was determined using the Akaike Information Criterion (AIC) statistic within jModelTest version 2.1.10 [12]. Based on the AIC, a TIM3 substitution model with invariable sites (+I) and rate variation among sites (+G) was used [13]. The robustness of the topology was estimated using 1000 bootstrap replicates. Phylogenetically, the strains were closely related to P. cichorii ( Fig. 1).
The taxonomic position of the three pepper strains at the species level was determined by comparing the ANI values and digital DNA-DNA hybridization (dDDH) values between the pepper strains and 17 closely related Pseudomonas species.
Pairwise ANI values based on blast (ANIb) were calculated between Pc19-1 T and phylogenetically closely related Pseudomonas species using jSpeciesWS version 1.2.1 [17]. The dDDH values were calculated using formula 2 of the Genometo-Genome distance calculator 2.1 [18] using the Type Strain Genomic Server (TYGS) [19]. The calculated ANIb and dDDH values are shown in Table 1. The highest ANIb value for Pc19-1 T was observed with P. cichorii ATCC10857 T at 91.3 % (Table 1), which is lower than the threshold value of 95 % considered as the standard ANI value for species differentiation [20,21]. Moreover, the dDDH value estimated for Pc19-1 T and the P. cichorii type strain was 45.2 %, which is below the 70 % threshold for prokaryotic species delineation [20,21].
The phylogenomic analysis was carried out using the TYGS [19]. Pairwise genome comparisons were conducted using the genome blast distance phylogeny (GBDP) approach [18], and intergenomic distances were inferred under the algorithm 'trimming' and distance formula d 5 . The resultant intergenomic distances were used to generate a minimumevolution tree via FastME 2.1.6.1 [22] including subtree pruning and regrafting post processing. Branch supports were inferred from 100 pseudo-bootstrap repetitions. The three  pepper strains were positioned independently of the other Pseudomonas species and formed a monophyletic clade with a high support value of 100 % (Fig. 2). Pseudomonas cichorii was placed closest to the three pepper strains (Fig. 2).
To clarify the species assignment of 19 P. cichorii strains available in the NCBI genome database, we calculated the ANIb and dDDH values of these strains compared with the P. cichorii type strain ATCC10857 T and the proposed P. capsici type strain Pc19-1 T (

PHYSIOLOGY AND CHEMOTAXONOMY
Pc19-1 T grown on Luria-Bertani (LB) agar plate was cultured in LB medium at 28 °C overnight. Cell size, morphology and flagellar insertion were observed using transmission electron microscopy jeol JEM1011 at Georgia Electron Microscopy (https:// gem. uga. edu/) at the University of Georgia. The bacterial cells of Pc19-1 T were determined to be rod-shaped with multiple polar flagella (Fig.  S3). The mean cell size (±standard error) of Pc19-1 T was 2.4±0.2×0.7±0.1 µm (n=7).
The phenotypic characteristics of the three pepper strains were characterized. Carbon source utilization and tests for sensitivity to different chemicals were determined using the Biolog GEN III MicroPlate system. Bacterial cells from nutrient agar plates were suspended in Biolog inoculation fluid at 95 % transmission (optical density=0.022), and 100 µl of the suspension were added to each well of the GENIII MicroPlate and incubated at 33 °C for 22 h according to the manufacturer's protocol. The results were recorded manually. The Biolog GENIII assay differentiated pepper strains from other closely related type strains of Pseudomonas species based on differences in various carbon source utilization ( Table 3). The Biolog assay also showed growth at pH 5 and pH 6, and growth at 1 and 4% NaCl, but not at 8 % NaCl. API 20 NE strips (bioMérieux) were performed according to the instructions of the manufacturer. In API 20 NE assays, the pepper strains assimilated glucose, arabinose, mannose, mannitol, gluconate, caprate, malate and citrate, and were positive for nitrate reduction to nitrite, urease and β-glucosidase. The pepper strains were negative for indole production, arginine dihydrolase, gelatin hydrolysis, β-galactosidase, assimilation of N-acetylglucosamine, maltose, adipate and phenyl-acetate. Bacterial growth of the three pepper strains was observed at 4, 20, 28, 30 and 37 °C in LB medium, but not at 42 °C for a 48 h incubation period.
The three pepper strains were sent to the Microbial Identification System (midi, Newark, DE, USA) laboratory to determine the cellular fatty acid composition. The cellular fatty acid content was analysed by gas chromatography (Agilent 6890 N unit) with the midi Microbial Identification System using the RTSBA6 version 6.2 library and the midi Sherlock software version 6.3 [23]. The major fatty acids were summed feature 3 (C 16 : 1 ω7c/C 16 : 1 ω6c), C 16 : 0 and summed feature 8 (C 18 : 1 ω7c/C 18 : 1 ω6c) ( Table 4). The fatty  Table 1. The tree was generated with FastME 2.1.6.1 [22] from GBDP distances calculated from genome sequences on the TYGS [19]. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers at nodes are genome blast distance phylogeny approach pseudo-bootstrap support values (>60 %) from 100 replications, with an average branch support of 94.9 %. The tree was rooted at the midpoint [29]. GenBank accession numbers are shown within parentheses, with T indicating type strains.
acid profiles of the pepper strains were closely related to those of P. cichorii.
In addition, analyses of polar lipids and respiratory quinones were carried out by the Identification Service of the DSMZ (Braunschweig, Germany) based on previously described methods [24][25][26][27]. The major polar lipids found in Pc19-1 T were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol (Fig. S4), which is consistent with species of the genus Pseudomonas [28]. Minor amounts of phosphatidylcholine, aminophospholipid and one unidentified lipid were also detected (Fig. S4). The major respiratory quinone was Q9 (96.9 %), which is consistent with other Pseudomonas species. Additional respiratory quinones detected were Q8 (1.7 %) and Q10 (1.4 %).
Considering the results of polyphasic analyses based on phenotypic characteristics, metabolic reactions, fatty acid composition, phylogenetic studies and whole-genome sequence comparisons, the three strains isolated from pepper in Georgia represent a novel species, and the name Pseudomonas capsici sp. nov. is proposed.

Funding information
This study was supported by USDA-NIFA-SCRI 2019-51181-30010 and the Georgia Agricultural Commodity Commission for Vegetables.