Xanthomonas fragariae is a Gram-negative bacterium that causes angular leaf spot (ALS) in strawberry [1]. The pathogen is known to infect only Fragaria ssp. and cannot live for any extended period outside of its host [2]. Consequently, the pathogen is almost exclusively transmitted to production fields through contaminated nursery stock [3, 4]. No fully resistant strawberry varieties exist, and disease management is achieved through a combination of horticultural practices and chemical applications, each of which has its limitations. A potential alternative or addition to these traditional management strategies may be the use of bacteriophages. Research on the potential of bacteriophages as biocontrol agents for plant pathogens has increased in recent years [5]. However, no reports are available on the isolation or characterization of bacteriophages from X. fragariae.

Xanthomonas phage RiverRider was isolated through an enrichment culture using strawberry leaves collected from ‘Festival’ strawberry plants naturally infected with X. fragariae. A single plaque was selected and serially purified to produce pure lysates. RiverRider produces clear plaques, approximately 1.5 cm in diameter. Transmission electron microscopy revealed an icosahedral head and a short noncontractile tail, suggesting that this virus is a member of the family Podoviridae family (Fig. 1A). The mean capsid size for RiverRider was 66.14 nm.

Fig. 1
figure 1

(a) Transmission electron micrograph of Xanthomonas phage RiverRider stained with 2% uranyl acetate. The scale bar represents 100 nm (b) Genome map of the RiverRider genome. ORFs are color coded according to their functional classification

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DNA was extracted from lysates using a Promega Wizard DNA Clean Up Kit (Promega, Madison, WI), with modifications [6]. DNA library preparation and genome sequencing were done at the University of Miami Center for Genome Technology using an Illumina MiSeq 125-bp paired-end run. The sequenced reads were filtered, merged and assembled de novo using Geneious R8 version 8.1.4 (Biomatter, Auckland, New Zealand). Direct terminal repeats (DTRs) were characterized in Geneious by identifying genome assembly regions that had reads mapped to more than twice the average depth of coverage [7, 8]. The distinct start and end points of DTRs were confirmed computationally using PhageTerm [9]. The genome was auto-annotated using Glimmer [10] and GeneMark [11], and predicted open reading frames (ORFs) were annotated manually in DNA Master (https://cobamide2.bio.pitt.edu/computer.htm). Functional assignments of ORFs were made using BLASTp [12] and HHPred [13]. Identification of tRNAs was cone using Aragorn [14] and tRNAscan-SE [15].

The genome of RiverRider is 76,335 bp in length with a GC content of 49.4% and has 568-bp DTRs. A total of 90 ORFs were predicted, and seven tRNAs were identified. BLASTn analysis of the complete genome sequence showed the best match with the podovirus Stenotrophomonas phage Pokken, with 72.48% identity (ID) and 67% query coverage (QC). The next-closest matches were two N4-like bacteriophages, Achromobacter phages JWAlpha and JWDelta, both with 79% ID and only 24% QC. BLASTp analysis of protein sequences identified 25 RiverRider ORFs with predicted functional matches to N4-like phage with substantial similarity (Table S1). The genome of RiverRider shares the N4-like bacteriophage conserved architecture, with genes for transcription, DNA metabolism, and DNA replication transcribed in the forward direction, followed by a gene encoding a large viral RNA polymerase and lysis and structural genes transcribed in the reverse direction [16, 17] (Fig. 1B, Table S2).

The genes for transcription of N4-like phages consist of one large viral RNA polymerase (vRNAP) and two additional smaller RNA polymerases [18]. The vRNAP (ORF70) of RiverRider is 3442 aa in length, making its coding region the largest ORF in the genome. The vRNAP, which is a distinguishing characteristic of N4-like phages, is packaged in the phage capsid and is responsible for host-independent transcription of early genes [19, 20]. Two smaller RNA polymerases, RNAP1 and RNAP2, were identified at beginning of the genome suggesting that RiverRider follows an early and middle transcription scheme similar to N4-like phages.

RiverRider contains a complete N4-like DNA replication cluster, including genes for DNA helicase (ORF56), DNA polymerase (ORF58), DNA primase (ORF64), and single-strand binding proteins (ORF67). There is a conserved synteny for the genes in the replication cluster among N4-like phage genomes, which is also present in RiverRider [17]. Genes for DNA metabolism, including deoxycytidine deaminase (ORF33) and thymidylate synthase (ORF50), were also identified. Phylogenetic analysis of RiverRider DNA polymerase and terminase (ORF90) amino acid sequences was conducted using MEGA7 [21], revealing that RiverRider forms a clade with Stenotrophomonas phage Pokken that is distinct from the neighboring Achromobacter and Erwinia N4-like bacteriophages (Fig. 2A and B).

Fig. 2
figure 2

Phylogenetic analysis of the terminase large subunit (a) and DNA polymerase (b) amino acid sequences from Xanthomonas phage RiverRider. ClustalW alignments were used to generate neighbor-joining trees with 1000 bootstrap replicates (values at nodes). The corresponding sequence from Alteromonas phage vB_AmaP_AD45-P1 was included as an outgroup in each tree

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The lysis cassette in RiverRider consists of the holin (ORF82), endolysin (ORF81) and spanin genes (ORF80), which are all upstream of the portal protein gene (ORF79), as in other N4-like phage. The holin protein contains two transmembrane domains, a characteristic of class II holins [22]. The endolysin gene encodes N-acetylmuramidase, a well-described murein hydrolyase from Escherichia phage N4 that is involved in cell lysis [23]. Both tail fiber proteins in RiverRider are clearly distinct from those of other N4-like phages and share homology with tail fibers in Xanthomonas and Xylella phages (Table S2). Similar exchanges of complete tail fibers and adsorption-related genes have been identified in other N4-related phages [24, 25], and may facilitate adaptation to new hosts. Host range analysis showed that RiverRider could infect seven of eight X. fragariae strains tested and none of the 14 other Xanthomonas species tested (Table S2).

FormalPara Nucleotide sequence and accession number

The GenBank accession number for Xanthomonas phage RiverRider is MG983743.