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 morphology, and the Ymer genus belongs to the class Caudoviricetes, with double-stranded DNA genomes varying from 39kb to 43kb. 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 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 Ymer 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 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.
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.
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.
Abstract 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”.
Genome modifications are central components of the continuous arms race between viruses and their hosts. The archaeosine base (G+), which was thought to be found only in archaeal tRNAs, was recently detected in genomic DNA of Enterobacteria phage 9g and was proposed to protect phage DNA from a wide variety of restriction enzymes. In this study, we identify three additional 2'-deoxy-7-deazaguanine modifications, which are all intermediates of the same pathway, in viruses: 2'-deoxy-7-amido-7-deazaguanine (dADG), 2'-deoxy-7-cyano-7-deazaguanine (dPreQ0) and 2'-deoxy-7- aminomethyl-7-deazaguanine (dPreQ1). We identify 180 phages or archaeal viruses that encode at least one of the enzymes of this pathway with an overrepresentation (60%) of viruses potentially infecting pathogenic microbial hosts. Genetic studies with the Escherichia phage CAjan show that DpdA is essential to insert the 7-deazaguanine base in phage genomic DNA and that 2'-deoxy-7-deazaguanine modifications protect phage DNA from host restriction enzymes.
ABSTRACT 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”.
Modern agriculture is expected to face an increasing global demand for food while also needing to comply with higher sustainability standards. Therefore, control of crop pathogens requires new, green alternatives to current methods. Potatoes are susceptible to several bacterial diseases, with infections by soft rot Enterobacteriaceae (SRE) being a significant contributor to the major annual losses. As there are currently no efficient ways of combating SRE, we sought to develop an approach that could easily be incorporated into the potato production pipeline. To this end, 46 phages infecting the emerging potato pathogen Dickeya solani were isolated and thoroughly characterized. The 46 isolated phages were grouped into three different groups based on DNA similarity, representing two distinct clusters and a singleton. One cluster showed similarity to phages previously used to successfully treat soft rot in potatoes, whereas the remaining phages were novel and showed only very limited similarity to previously isolated phages. We selected six diverse phages in order to create the hereto most complex phage cocktail against SRE. The cocktail was applied in a proof-of-principle experiment to treat soft rot in potatoes under simulated storage conditions. We show that the phage cocktail was able to significantly reduce the incidence of soft rot as well as disease severity after 5 days of storage post-infection with Dickeya solani. This confirms results from previous studies that phages represent promising biocontrol agents against SRE infection in potato.
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