Newly Isolated Bacteriophages from the Podoviridae, Siphoviridae, and Myoviridae Families Have Variable Effects on Putative Novel Dickeya spp.

Abstract

Soft rot pathogenic bacteria from the genus Dickeya cause severe economic losses in orchid nurseries worldwide, and there is no effective control currently available. In the last decade, the genus Dickeya has undergone multiple changes as multiple new taxa have been described, and just recently a new putative Dickeya species was reported. This study reports the isolation of three bacteriophages active against putative novel Dickeya spp. isolates from commercially produced infected orchids that show variable host-range profiles. Bacteriophages were isolated through enrichment from Dickeya-infected orchid tissue. Convective interaction media monolith chromatography was used to isolate bacteriophages from wastewaters, demonstrating its suitability for the isolation of infective bacteriophages from natural sources. Based on bacteriophage morphology, all isolated bacteriophages were classified as being in the order Caudovirales, belonging to three different families, Podoviridae, Myoviridae, and Siphoviridae. The presence of three different groups of bacteriophages was confirmed by analyzing the bacteriophage specificity of bacterial hosts, restriction fragment length polymorphism and plaque morphology. Bacteriophage BF25/12, the first reported Podoviridae bacteriophage effective against Dickeya spp., was selected for further characterization. Its genome sequence determined by next-generation sequencing showed limited similarity to other characterized Podoviridae bacteriophages. Interactions among the bacteriophages and Dickeya spp. were examined using transmission electron microscopy, which revealed degradation of electron-dense granules in response to bacteriophage infection in some Dickeya strains. The temperature stability of the chosen Podoviridae bacteriophage monitored over 1 year showed a substantial decrease in the survival of bacteriophages stored at -20°C over longer periods. It showed susceptibility to low pH and UV radiation but was stable in neutral and alkaline pH. Furthermore, the stability of the tested bacteriophage was also connected to the incubation medium and bacteriophage concentration at certain pH values. Finally, the emergence of bacteriophage-resistant bacterial colonies is highly connected to the concentration of bacteriophages in the bacterial environment. This is the first report on bacteriophages against Dickeya from the Podoviridae family to expand on potential bacteriophages to include in bacteriophage cocktails as biocontrol agents. Some of these bacteriophage isolates also showed activity against Dickeya solani, an aggressive strain that causes the soft rot of potatoes, which indicates their broad potential as biocontrol agents.

Jumbo Bacteriophages Are Represented Within an Increasing Diversity of Environmental Viruses Infecting the Emerging Phytopathogen, Dickeya solani

“…Whilst this is not the first member of this family that we have isolated (Day et al, 2017 ), this is the first isolate we have studied in further depth. The genome of JA10 was therefore sequenced to investigate the similarity between it and previously published Dickeya -infecting Podoviridae family members (Alič et al, 2017b ).…”

“…The closest published phage is the Pectobacterium parmentieri phage PP74 isolated from potato washing waste water in Russia in 2015 (Kabanova et al, 2018 ), which shares less than 14% nucleotide identity. It shares no DNA sequence identity with the other sequenced Dickeya -infecting Podoviridae family member BF25/12 (Alič et al, 2017b ). PP74 has been designated as a T7-like virus and a member of the Autographivirinae subfamily, with a conserved core genome.…”

“…There are at least four groups of D. solani phages present in waterways around Cambridge, spanning three of the four Caudovirales families. Phages that have been isolated on other species of Dickeya that are capable of forming plaques on D. solani have also been recently described (Alič et al, 2017b ), including the fourth Caudovirales family, Siphoviridae . It is therefore apparent that we are only superficially defining the level of phage diversity present in the environment, consistent with the notion that double-stranded DNA phages alone have been predicted to outnumber their bacterial hosts by a factor of 10 to 1 (Chibani-Chennoufi et al, 2004 ).…”

“…All of the Dickeya phages isolated so far, and 96% of all known phages, are members of the order Caudovirales (Fokine and Rossmann, 2014 ), which currently consists of four families. Apart from the Siphoviridae family member BF-CIM1/14 recently described by Alič et al ( 2017b ) and three Podoviridae family members reported in our recent publication (Day et al, 2017 ), the vast majority of D. solani phages characterized so far share a high degree of similarity and have been designated members of the Ackermannviridae family (formerly known as the Vi1virus genus; Adriaenssens et al, 2018 ) based on morphology and genomic comparisons. A summary of these phages is shown in Table 1 .…”

“…Bacterial viruses (bacteriophages; phages) have been suggested as potential tools for biocontrol due to their specificity, environmental persistence and biological “organic” nature (Iriarte et al, 2007 ; Czajkowski et al, 2017 ; Svircev et al, 2018 ). Several studies have isolated phages capable of infecting Dickeya species (Adriaenssens et al, 2012c ; Czajkowski et al, 2014a , b , 2015a ; Matilla et al, 2014 ; Alič et al, 2017b ; Day et al, 2017 ). Their potential use as biocontrol agents has been trialed both in the lab and in the field (Adriaenssens et al, 2012c ) and these studies showed a partially “therapeutic” outcome with reduced crop losses.…”