Bacterial leaf spot of cucurbits (BLS) is an emerging disease in the southeastern United States that is capable of causing widespread outbreaks under conducive conditions. Historically attributed solely to the bacterium Pseudomonas syringae pv. lachrymans, recent studies have identified additional P. syringae pathovars as causal agents of the disease. To further investigate the identity and diversity of P. syringae strains associated with BLS in the southeastern United States, 47 bacterial isolates were recovered from symptomatic cucurbits from Florida, Alabama, and Georgia. Strains were characterized using the LOPAT testing scheme, fluorescence, and pathogenicity to watermelon and squash seedlings. Thirty-eight fluorescent isolates underwent whole-genome sequencing and were further characterized with 16S rRNA, four gene multilocus sequence analysis (MLSA) phylogeny, and average nucleotide identity analysis. Thirty-four isolates were identified as members of the P. syringae species complex, including P. syringae sensu stricto (12), P. alliivorans (12), P. capsici (nine), and P. viridiflava (one). An additional four isolates were found to belong to the Pseudomonas genus outside of the syringae species complex, though they did not share 95% or greater average nucleotide identity to any validly published species and are believed to belong to three novel Pseudomonas species. These results reveal an unpredicted level of diversity of Pseudomonas strains associated with BLS in the region and show the benefits of whole-genome sequencing for strain identification. Identification of P. capsici, which is capable of causing disease at higher temperatures than P. syringae, as a causal agent of BLS may also affect management strategies in the future.

Peptidoglycan hydrolases are enzymes responsible for breaking the peptidoglycan present in the bacterial cell wall, facilitating cell growth, cell division and peptidoglycan turnover. Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus canker, encodes an Escherichia coli M23 peptidase EnvC homolog. EnvC is a LytM factor essential for cleaving the septal peptidoglycan, thereby facilitating the separation of daughter cells. In this study, the investigation focused on EnvC contribution to the virulence and cell separation of X. citri. It was observed that disruption of the X. citri envC gene (ΔenvC) led to a reduction in virulence. Upon inoculation into leaves of Rangpur lime (Citrus limonia Osbeck), the X. citri ΔenvC exhibited a delayed onset of citrus canker symptoms compared with the wild-type X. citri. Mutant complementation restored the wild-type phenotype. Sub-cellular localization confirmed that X. citri EnvC is a periplasmic protein. Moreover, the X. citri ΔenvC mutant exhibited elongated cells, indicating a defect in cell division. These findings support the role of EnvC in the regulation of cell wall organization, cell division, and they clarify the role of this peptidase in X. citri virulence.

In 2022, a fluorescent bacterial isolate, designated 22-AL-CL-001, was isolated from diseased cantaloupe (Cucumis melo) foliage collected in Alabama, USA, displaying marginal necrosis and general blighting. Whole-genome sequencing and subsequent comparisons to publicly available bacterial genomes identified Pseudomonas isolate USDA-ARS-USMARC-56711, isolated from a clinically healthy calf (Bos taurus) in Kansas, USA, as genetically similar to isolate 22-AL-CL-001. The two isolates were then characterized using phenotypic and genetic analyses, including fluorescence on King’s medium B; LOPAT reactions; pathogenicity assays on cantaloupe, watermelon and squash plants; 16S rRNA gene sequence analysis; phylogeny based on multi-locus sequence analysis (MLSA) of four housekeeping genes; average nucleotide identity based on blast (ANIb); in silico DNA–DNA hybridization (isDDH) including comparison to the Type Genome Server and biochemical profiling using the Biolog Gen III microplate system. MLSA phylogeny placed both isolates into a single clade within the genus Pseudomonas, distant from all included reference strains. ANIb and isDDH analyses definitively identified the isolates as members of the same species (ANIb, 97.25; isDDH, 78.5%), though comparison scores to reference strains were all below the accepted thresholds for species determination. Biolog biochemical profiling, as well as MALDI-TOF mass spectrometry of isolate 22-AL-CL-001, was also unable to identify the isolate as a member of any existing bacterial species. Using a combination of genetic and phenotypic data, we conclude that the two isolates belong to a new species of Pseudomonas, for which we propose the name Pseudomonas alabamensis. The specific epithet, alabamensis, was chosen to represent the location where the type strain 22-AL-CL-001^T (NCPPB 4760^T=LMG 33363^T) was isolated, Alabama, USA. Taxonomic classification of the two isolates by the Genome Taxonomy Database revealed other previously sequenced bacterial strains that, according to ANI and isDDH results, also belong to P. alabamensis.

Three fluorescent bacterial strains, K1, K13 and K18, were obtained from watermelon (Citrullus lanatus) foliage symptomatic of bacterial leaf spot of cucurbits in Florida. The strains underwent phenotypic characterization, including LOPAT (levan production, oxidase activity, pectolytic activity on potato, arginine dihydrolase production and hypersensitive response (HR) on both tobacco and tomato) and pathogenicity testing on watermelon and squash seedlings. Whole-genome sequencing of the isolates was performed, and multi-locus sequence analysis (MLSA) utilizing housekeeping genes gltA, rpoD, gapA and gyrB placed the isolates into two distinct clades within the Pseudomonas genus. Average nucleotide identity based on blast (ANIb) was used to compare the isolates to Pseudomonas reference genomes. Using ANIb, the closest relatives to the novel strains were identified as Pseudomonas wayambapalatensis (K1 : 82.58%; K13 : 83.77%) and Pseudomonas kilonensis (K18 : 87.16%), although ANIb values were below the 95% threshold for species delineation. DNA–DNA hybridization (genome–genome distance calculation method), comparison to the online Type Genome Server, Biolog biochemical profiling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were also unable to identify the isolates as any known species of Pseudomonas. Based on the combination of genetic and phenotypic data, we conclude that these isolates represent two novel Pseudomonas species, for which we propose the names Pseudomonas boreofloridensis sp. nov. (K1, K13^T, NCPPB 4759=LMG 33364) and Pseudomonas citrulli sp. nov. (K18^T, NCPPB 4761=LMG 33365). The specific epithet boreofloridensis was chosen for the geographic location of isolation (northern Florida), while citrulli designates the host of origin (C. lanatus).

During the 2021-22 and 2022-23 seasons (December to February), onion plants (Allium cepa L.) showing decay, leaf blight, chlorosis and water soak lesions were collected in Central Chile. Five symptomatic plants were sampled from 20 different onion fields. Brown rot of the external scales was observed in bulbs from two fields: one planted with the cv. Campero (20 ha; O'Higgins Region), and another with cv. Marenge (2 ha; Metropolitan Region). The disease incidence in these fields ranged from 2% to 5%. Isolations were carried out from symptomatic leaves and bulbs from these fields on King's B medium, resulting in small white colonies with smooth margin. Three isolates were selected, two from first field (QCJ3A & QCJ2B), and one from second field (EPB1). A preliminary identification based on 16S rRNA sequences was conducted. BLAST analyses of strains QCJ3A, QCJ2B and EPB1 (GenBank Accession No. PP345601 to PP345603) against the NCBI Database resulted in a match with strains (GenBank Accession No. ON255770.1 and ON255825.1) isolated from infected bulbs in Texas, USA identified as Erwinia spp. (Khanal et al. 2023), with 100% coverage and 100% identity (707 bp out of 707). To evaluate the pathogenicity of these three strains, onion bulbs were inoculated (Guajardo et al. 2023). Toothpicks previously immersed in a bacterial suspension at ~ 108 colony forming units (CFU)/mL were pricked at a 4 cm depth into the shoulders of onion bulbs bought from commercial store and incubated at room temperature. Bulbs inoculated with sterile water served as negative control. A known onion bulb rotting bacterial strain of Dickeya sp. was used as a positive control. At the end of the incubation period (20 days), bulbs were opened longitudinally across their inoculation site, showing that the external scales had a brown color. Negative control remained asymptomatic. Strains were re-isolated from damaged tissue and identified as Erwinia sp. This assay was repeated three times with the same results. For further identification, genomic DNA extraction was carried out using the Blood & Cell Culture DNA Kit (Qiagen), and genome sequencing was performed in the Illumina HiSeq 2500 platform. The Whole Genome Shotgun project for strains QCJ3A, QCJ2B and EPB1 have been deposited at DDBJ/ENA/GenBank under the accession JBANEI010000000, JBANEJ010000000 and JBANEK010000000. The average nucleotide identity (ANI) values were 99.6% (EPB1), 98.2% (QCJ2B), and 99.6% (QCJ3A) and DNA-DNA hybridization (dDDH) values were 96.9% (EPB1), 83.7% (QCJ2B), and 97.1% (QCJ3A), when compared with the type strain Erwinia aphidicola JCM 21238 (GenBank accession No. GCF_014773485.1). The three strains were deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM). Erwinia aphidicola has been previously described causing diseases in common bean (Phaseolus vulgaris) and pea (Pisum sativum), in Spain (Santos et al. 2009) and in pepper (Capsicum annuum) in China (Luo et al. 2018). Its close relative E. persicina has been reported causing bulb rot in onion in Korea (Cho et al. 2019) and garlic in Europe (Galvez et al. 2015). To our knowledge, this is the first report of E. aphidicola causing a bulb rot of onion in Chile. Although the distribution and prevalence of this bacterium in Chilean agroecosystems is not known, it can be a potential cause of losses in onions and other crops such as beans, peas, and peppers. Additional studies should be conducted to determine the host range of Chilean Erwinia aphidicola strains.

Six Pseudomonas strains isolated from muskmelon and watermelon seedlings affected by stem rot and wilting in Serbia were reported as P. cichorii based on pathogenicity, LOPAT and cell wall fatty acid analyses. Recent bacterial isolates from cucurbit crops displaying P. cichorii-like symptoms in Alabama, USA, were identified as P. capsici, prompting polyphasic re-evaluation of the Serbian strains. All six strains were found to cause severe disease in watermelon and squash seedlings under greenhouse conditions. Strains KFB 138 and KFB 140 underwent whole-genome sequencing and were found to have the highest level of 16S rRNA similarity to P. lijiangensis LJ2^T (both 99.87%). Phylogenies based on housekeeping genes and core-genome analysis placed both strains into phylogroup 11 of the Pseudomonas syringae species complex, with KFB 138 forming a lineage basal to all other phylogroup 11 members. In core-genome phylogeny, KFB 140 was placed into a clade alongside P. lijiangensis LJ2^T. Average nucleotide identity based on blast (ANIb) identified KFB 140 as a member of P. lijiangensis (95.85%), though KFB 138 did not produce an ANIb value over 95% to any Pseudomonas type strain to which it was compared. Values for in silico DNA–DNA hybridization for both strains were below 70% to all reference strains tested, though KFB 140 was found to be most similar to P. lijiangensis (68.2%). KFB 138 and KFB 140 were further characterized using the online Type Genome Server, biochemical profiling with the Biolog Gen III MicroPlate system, matrix-assisted laser desorption/ionization time of flight mass spectrometry and imaged with transmission electron microscopy. From the results of the above analyses, we conclude that KFB 140 is a member of the species P. lijiangensis and that KFB 138 represents a novel Pseudomonas species, for which we propose the name Pseudomonas serbiensis (KFB 138^T, NCPPB 4762=LMG 33366), named for its location of isolation.