Three novel Erwinia billingiae phages isolated from organic waste represent three new genera.

Despite the ecological significance of viral communities, phages remain insufficiently studied. Current genomic databases lack high-quality phage genome sequences linked to specific bacteria. Bacteria of the genus Erwinia are known to colonize the phyllosphere of plants, both as commensals and as pathogens. We isolated three Erwinia billingiae phages-Zoomie, Pecta, and Snitter-from organic household waste. Based on sequence similarity to their closest relatives, we propose that they represent three new genera: "Pectavirus" within the family Zobellviridae, "Snittervirus" in the subfamily Tempevirinae, family Drexlerviridae, and "Zoomievirus" within the family Autographiviridae, which, together with the genus Limelightvirus, may constitute a new subfamily.

Four novel Curtobacterium phages isolated from environmental samples.

Despite Curtobacterium spp. often being associated with the plant phyllosphere, i.e., the areal region of different plant species, only one phage targeting a member of the genus Curtobacterium has been isolated so far. In this study, we isolated four novel plaque-forming Curtobacterium phages, Reje, Penoan, Parvaparticeps, and Pize, with two novel Curtobacterium strains as propagation hosts. Based on the low nucleotide intergenomic similarity (<32.4%) between these four phages and any phage with a genome sequence in the NCBI database, we propose the establishment of the four genera, "Rejevirus", "Pizevirus", "Penoanvirus", and "Parvaparticepsvirus", all in the class of Caudoviricetes.

A novel genus of Pectobacterium bacteriophages display broad host range by targeting several species of Danish soft rot isolates.

The bacterial diseases black leg and soft rot in potatoes cause heavy losses of potatoes worldwide. Bacteria within the genus Pectobacteriaceae are the causative agents of black leg and soft rot. The use of antibiotics in agriculture is heavily regulated and no other effective treatment currently exists, but bacteriophages (phages) have shown promise as potential biocontrol agents. In this study we isolated soft rot bacteria from potato tubers and plant tissue displaying soft rot or black leg symptoms collected in Danish fields. We then used the isolated bacterial strains as hosts for phage isolation. Using organic waste, we isolated phages targeting different species within Pectobacterium. Here we focus on seven of these phages representing a new genus primarily targeting P. brasiliense; phage Ymer, Amona, Sabo, Abuela, Koroua, Taid and Pappous. TEM image of phage Ymer showed siphovirus morphotype, and the proposed Ymer genus belongs to the class Caudoviricetes, with double-stranded DNA genomes varying from 39 kb to 43 kb. In silico host range prediction using a CRISPR-Cas spacer database suggested both P. brasiliense, P. polaris and P. versatile as natural hosts for phages within the proposed Ymer genus. A following host range experiment, using 47 bacterial isolates from Danish tubers and plants symptomatic with soft rot or black leg disease verified the in silico host range prediction, as the genus as a group were able to infect all three Pectobacterium species. Phages did, however, primarily target P. brasiliense isolates and displayed differences in host range even within the species level. Two of the phages were able to infect two or more Pectobacterium species. Despite no nucleotide similarity with any phages in the NCBI database, the proposed Ymer genus did share some similarity at the protein level, as well as gene synteny, with currently known phages. None of the phages encoded integrases or other genes typically associated with lysogeny. Similarly, no virulence factors nor antimicrobial resistance genes were found, and combined with their ability to infect several soft rot-causing Pectobacterium species from Danish fields, demonstrates their potential as biocontrol agents against soft rot and black leg diseases in potatoes.

Pheno- and Genotyping of Three Novel Bacteriophage Genera That Target a Wheat Phyllosphere Sphingomonas Genus.

Bacteriophages are viral agents that infect and replicate within bacterial cells. Despite the increasing importance of phage ecology, environmental phages-particularly those targeting phyllosphere-associated bacteria-remain underexplored, and current genomic databases lack high-quality phage genome sequences linked to specific environmentally important bacteria, such as the ubiquitous sphingomonads. Here, we isolated three novel phages from a Danish wastewater treatment facility. Notably, these phages are among the first discovered to target and regulate a Sphingomonas genus within the wheat phyllosphere microbiome. Two of the phages displayed a non-prolate Siphovirus morphotype and demonstrated a narrow host range when tested against additional Sphingomonas strains. Intergenomic studies revealed limited nucleotide sequence similarity within the isolated phage genomes and to publicly available metagenome data of their closest relatives. Particularly intriguing was the limited homology observed between the DNA polymerase encoding genes of the isolated phages and their closest relatives. Based on these findings, we propose three newly identified genera of viruses: Longusvirus carli, Vexovirus birtae, and Molestusvirus kimi, following the latest ICTV binomial nomenclature for virus species. These results contribute to our current understanding of phage genetic diversity in natural environments and hold promising implications for phage applications in phyllosphere microbiome manipulation strategies.

Genetic rearrangements in Pseudomonas amygdali pathovar aesculi shape coronatine plasmids.

Plant pathogenic Pseudomonas species use multiple classes of toxins and virulence factors during host infection. The genes encoding these pathogenicity factors are often located on plasmids and other mobile genetic elements, suggesting that they are acquired through horizontal gene transfer to confer an evolutionary advantage for successful adaptation to host infection. However, the genetic rearrangements that have led to mobilization of the pathogenicity genes are not fully understood. In this study, we have sequenced and analyzed the complete genome sequences of four Pseudomonas amygdali pv. aesculi (Pae), which infect European horse chestnut trees (Aesculus hippocastanum) and belong to phylogroup 3 of the P. syringae species complex. The four investigated genomes contain six groups of plasmids that all encode pathogenicity factors. Effector genes were found to be mostly associated with insertion sequence elements, suggesting that virulence genes are generally mobilized and potentially undergo horizontal gene transfer after transfer to a conjugative plasmid. We show that the biosynthetic gene cluster encoding the phytotoxin coronatine was recently transferred from a chromosomal location to a mobilizable plasmid that subsequently formed a co-integrate with a conjugative plasmid.

Two New Dickeya dadantii Phages with Odd Growth Patterns Expand the Diversity of Phages Infecting Soft Rot Pectobacteriaceae.

Background: Bacterial soft rot caused by members of the soft rot Pectobacteriaceae afflicts plant production of both vegetable and ornamental crops. Recent outbreaks highlight Dickeya sp. in the etiology of this disease in potatoes. Since there is a lack of control strategies for these diseases, alternative approaches have been suggested, including the use of biological control mediated by bacteriophages (phages). However, phages infecting many of these members are still undiscovered or poorly described. Materials and Methods: Two phages targeting Dickeya dadantii subsp. dadantii (NCPPB 4097) were isolated from household organic waste and purified. They were then further characterized using whole-genome sequencing and comparative genomics, transmission electron microscopy, latent period and burst size. Results: Dickeya phage Sucellus displayed Siphovirus morphology and had a genome of 39,826 bp with very limited similarity to any previously described phages. Dickeya phage Amaethon had a Podovirus morphology with a genome comprising 41,436 bp and limited similarity to phages in the Kafuna genus. The phages exhibited burst sizes of app. 94 and 240 virions per cell with latent periods of 91 and 86 minutes for Sucellus and Amaethon, respectively. While both phages had similar adsorption efficiencies and latent periods, the rise periods for the two phages diverged markedly, highlighting an odd growth pattern. Conclusions: Together, the two phages isolated here expand the known diversity of phages infecting the important plant pathogen D. dadantii. As they both share limited similarity to previously described groups of phages, they likely constitute novel genera within their respective groups.

Pectobacterium Phage Jarilo Displays Broad Host Range and Represents a Novel Genus of Bacteriophages Within the Family Autographiviridae.

Background: Soft rot Pectobacteriaceae includes the genera Pectobacterium and Dickeya, which are important plant pathogens being responsible for diseases in a wide range of plant species, with potatoes as the main group. Both genera cause pre- and postharvest losses of potatoes, resulting in huge economic losses linked with the soft rot diseases. Materials and Methods: Organic waste was used to isolate phages, with Pectobacterium carotovorum subsp. carotovorum DSM 30170 as host. Complete genome sequencing, comparative genomics, and electron microscopy were used to characterize the phage. An adsorption assay was used to estimate adsorption rate. Twenty-three strains from the genera Pectobacterium and Dickeya were used to examine the host range of the phage. Results: Pectobacterium phage Jarilo represents a novel genus of bacteriophages within the family Autographiviridae, order Caudovirales. Jarilo possesses a double-stranded DNA genome of 40557 bp with a G+C% content of 50.08% and 50 predicted open reading frames. Gene synteny and products seem to be partly conserved between Pectobacterium phage Jarilo and Enterobacteria phage T7, but limited nucleotide similarity is found between Jarilo and other phages within the family Autographiviridae. The adsorption rate of phage Jarilo increased continuously for 1 h upon infection. Phage Jarilo was not able to infect any strains of P. carotovorum and Dickeya tested with the exception of the P. carotovorum strain used for isolation. However, phage Jarilo infected 10 of 16 Pectobacterium atrosepticum strains tested. Conclusion: We propose Pectobacterium phage Jarilo as the first member of a new genus of bacteriophages within the family Autographiviridae, order Caudovirales, displaying a broad host range within the genera of Pectobacterium.

Isolation and characterisation of novel phages infecting Lactobacillus plantarum and proposal of a new genus, "Silenusvirus".

Bacteria of Lactobacillus sp. are very useful to humans. However, the biology and genomic diversity of their (bacterio)phage enemies remains understudied. Knowledge on Lactobacillus phage diversity should broaden to develop efficient phage control strategies. To this end, organic waste samples were screened for phages against two wine-related Lactobacillus plantarum strains. Isolates were shotgun sequenced and compared against the phage database and each other by phylogenetics and comparative genomics. The new isolates had only three distant relatives from the database, but displayed a high overall degree of genomic similarity amongst them. The latter allowed for the use of one isolate as a representative to conduct transmission electron microscopy and structural protein sequencing, and to study phage adsorption and growth kinetics. The microscopy and proteomics tests confirmed the observed diversity of the new isolates and supported their classification to the family Siphoviridae and the proposal of the new phage genus "Silenusvirus".

A novel six-phage cocktail reduces Pectobacterium atrosepticum soft rot infection in potato tubers under simulated storage conditions.

Pectobacterium atrosepticum is a species of plant pathogenic bacteria responsible for significant losses in potato production worldwide. Pectobacterium atrosepticum can cause blackleg disease on potato stems as well as the tuber disease termed potato soft rot. Methods for the effective control of these diseases are limited and are primarily based on good agricultural practices. Bacteriophages, viruses of bacteria, could be used as an alternative, environmentally friendly, control measure. Here, we describe the isolation and characterization of 29 phages virulent to P. atrosepticum. The phages belong to 12 different species based on a 95% sequence identity cut-off. Furthermore, based on sequence diversity and propagation results, we selected six of these phages to form a phage cocktail. The phages in the cocktail was tested on a number of P. atrosepticum strains in order to determine their host range. The phages was found to lyse 93% of the tested strains. The cocktail was subsequently tested for its effectiveness in combatting potato soft rot under simulated storage conditions. Use of the phage cocktail reduced both disease incidence and disease severity by 61% and 64%, respectively, strongly indicating that phage biocontrol has the potential to reduce the economic impact of soft rot in potato production.

The first characterized phage against a member of the ecologically important sphingomonads reveals high dissimilarity against all other known phages.

This study describes the first molecular characterization of a bacteriophage infecting a member of the environmentally important Sphingomonadaceae family. Both bacteriophage Lacusarx and its host Sphingobium sp. IP1 were isolated from activated sludge from a wastewater treatment plant. Genome sequencing revealed that the phage genes display little similarity to other known phages, despite a remarkable conservation of the synteny in which the functional genes occur among distantly related phages. Phylogenetic analyses confirmed that Lacusarx represents a hitherto undescribed genus of phages. A classical lysis cassette could not be identified in Lacusarx, suggesting that the genes encoding endolysin, holin, and spanin are host-specific and not found in phages infecting other bacteria. The virus harbors 24 tRNA genes corresponding to 18 different amino acids and furthermore has a significantly different codon usage than its host. Proteomic analysis of Lacusarx revealed the protein components of the phage particle. A lysogeny test indicated that Lacusarx is not a temperate phage.