The genome of the Erwinia amylovora phage PhiEaH1 reveals greater diversity and broadens the applicability of phages for the treatment of fire blight

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Abstract

The enterobacterium Erwinia amylovora is the causal agent of fire blight. This study presents the analysis of the complete genome of phage PhiEaH1, isolated from the soil surrounding an E. amylovora-infected apple tree in Hungary. Its genome is 218 kb in size, containing 244 ORFs. PhiEaH1 is the second E. amylovora infecting phage from the Siphoviridae family whose complete genome sequence was determined. Beside PhiEaH2, PhiEaH1 is the other active component of Erwiphage, the first bacteriophage-based pesticide on the market against E. amylovora. Comparative genome analysis in this study has revealed that PhiEaH1 not only differs from the 10 formerly sequenced E. amylovora bacteriophages belonging to other phage families, but also from PhiEaH2. Sequencing of more Siphoviridae phage genomes might reveal further diversity, providing opportunities for the development of even more effective biological control agents, phage cocktails against Erwinia fire blight disease of commercial fruit crops.

Introduction

Erwinia amylovora, a member of the Enterobacteriaceae family, is a Gram-negative facultative anaerobic, rod shaped, phytopathogenic bacterium. It is the causal agent of fire blight of some Rosaceae plants, such as quince, apple and pear (Starr & Chatterjee, 1972; Van Der Zwet & Keil, 1979; Van der Zwet & Beer, 1991; Vanneste, 2000).

So far, 11 E. amylovora phage genomes have been sequenced (Lehman et al., 2009; Born et al., 2011; Muller et al., 2011; Dömötör et al., 2012). They include five phages that were isolated from samples collected in Northern America (four from USA, one from Canada), and five from European samples (four from Switzerland, one from Hungary), and one is of unknown origin. All the sequenced E. amylovora phages were members of Caudovirales. Five of them belonged to the Myoviridae family, five to the Podoviridae family and PhiEaH2 (isolated in Hungary) to the Siphoviridae.

Genome of Erwinia amylovora phage PhiEaH1

The E. amylovora Siphoviridae phage PhiEaH1 was isolated from soil in Hungary. The phage lysed E. amylovora under laboratory conditions and successfully reduced the occurrence of fire blight cases in field experiments. These results supported the use of phage PhiEaH1 as a good biocontrol agent. Erwiphage (composed of PhiEaH1 and PhiEaH2, containing UV-protectant) was marketed in 2012 and 2013 in Hungary, as the first bacteriophage-based pesticide against E. amylovora.

The genome sequencing protocol and the computer tools used are given in the Supporting Information. The genomic sequence of PhiEaH1 phage is 218 339 bp in length. The graphical genome organization is shown in Fig. S1. The G + C content is 52.3 mol%. In the genome, 241 ORFs were annotated, 181 ORFs seemed to encode hypothetical proteins and 60 ORFs were annotated as functional genes. Twenty-nine ORFs were found to encode proteins involved in the structure and assembly of virions, and the deduced products of 28 ORFs are responsible for nucleic acid metabolism and modification and DNA replication (helicases, DNA-directed RNA polymerase-beta subunit, nuclease SbcCD D subunit, terminase large subunit, phosphohydrolases, thymidylate synthase, deoxyuridine 5′-triphosphate nucleotide hydrolase, ribonuclease, thymidylate kinase, SbcC protein, UvsX protein); two ORFs encode transglycosylases, and one ORF codes for an EPS depolymerase associated with phage infections (Deveau et al., 2002; Abedon, 2011; Gutierrez et al., 2012).

Despite being isolated from the same soil sample in Hungary, the nucleotide sequences of the two Siphoviridae E. amylovora phages, PhiEaH1 and PhiEaH2, are significantly different (Fig. 1). Moreover, the genome of PhiEaH1 does not have any significant similarities when compared with the other completely sequenced E. amylovora phage genomes (Fig. 1). All except one of the previously sequenced phages had a genome size less than 100 kb. Although PhiEaH1 is similar to the exception, PhiEaH2, in having genomes larger than 200 kb and although both were isolated from the same soil in Hungary, they surprisingly appear to be very distantly related: their overall sequence similarity is around 6% at the DNA level. Application of phage cocktails instead of single phage is a generally applied approach for extending the host specificity of the phage preparations (Abedon, 2011). The implication is that sequencing of more Siphoviridae phage genomes will reveal even greater diversity, providing opportunities for the development of even more effective biological control agents, phage cocktails against Erwinia fire blight disease of commercial fruit crops.

Figure 1.

Comparison of genome sequence of 11 Erwinia amylovora bacteriophages. The comparison was carried out with MAUVE software 2.3.1 (Darling et al., 2004), progressive MAUVE algorithm, using the default parameters). MAUVE is a software that attempts to align orthologous and xenologous regions among more genome sequences that have undergone both local and large-scale changes. Genomes are organized into horizontal panels containing the name of the genome sequence, a scale showing the sequence coordinates for that genome, and a single black horizontal center line. Colored block outlines surround a region of the genome sequence that aligned to part of another genome, and is presumably homologous and internally free from genomic rearrangement. Regions outside blocks lack detectable homology among the input genomes. Inside each block a similarity profile of the genome sequence is demonstrated. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Areas that are completely white were not aligned and probably contain sequence elements specific to a particular genome.

Nucleotide sequence accession number: The complete genome sequence of E. amylovora phage PhiEaH1 has been submitted to GenBank and assigned accession number KF623294.

Acknowledgements

This work was supported by the European Union and by the Hungarian Government; projects GOP-1.1.1-07/1-2008-0038, GOP-1.3.2.-09-2010-0023, GOP-1.1.1-11-2012-00136. ERANET (project: BIOMOS) and the Hungarian National Technology Program (projects FAGCNTER and MFCDiagn) also supported this work. The financial supports of HUSRB/1203/214/250 and PTE ÁOK-KA-2013/23 grant are gratefully appreciated.

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