Molecular and biological characterization of ϕRs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum

RESEARCH ARTICLE Molecular and biological characterization of ϕRs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum Abdelmonim Ali Ahmad1,2, Michael J. Stulberg1¤, John Patrick Mershon1, Dimitre S. Mollov3, Qi Huang1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Floral and Nursery Plants Research Unit, United States National Arboretum, U.S. Dept. of AgricultureAgricultural Research Service, Beltsville, Maryland, United States of America, 2 Department of Plant Pathology, Faculty of Agriculture, Minia University, El-minia, Egypt, 3 National Germplasm Resources Laboratory, U.S. Dept. of Agriculture-Agricultural Research Service, Beltsville, Maryland, United States of America ¤ Current address: CPHST-Beltsville Lab, USDA/APHIS, Beltsville, Maryland, United States of America * Abstract OPEN ACCESS Citation: Ahmad AA, Stulberg MJ, Mershon JP, Mollov DS, Huang Q (2017) Molecular and biological characterization of ϕRs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum. PLoS ONE 12(9): e0185034. https://doi.org/10.1371/ journal.pone.0185034 Editor: Ulrich Melcher, Oklahoma State University, UNITED STATES Received: July 14, 2017 Accepted: September 4, 2017 Published: September 21, 2017 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are within the paper. Funding: This research was financially supported by the U. S. Department of Agriculture, Agricultural Research Service. A filamentous bacteriophage, designated ϕRs551, was isolated and purified from the quarantine and select agent phytopathogen Ralstonia solanacearum race 3 biovar 2 strain UW551 (phylotype IIB sequevar 1) grown under normal culture conditions. Electron microscopy suggested that ϕRs551 is a member of the family Inoviridae, and is about 1200 nm long and 7 nm wide. ϕRs551 has a genome of 7929 nucleotides containing 14 open reading frames, and is the first isolated virion that contains a resolvase (ORF13) and putative type-2 phage repressor (ORF14). Unlike other R. solanacearum phages isolated from soil, the genome sequence of ϕRs551 is not only 100% identical to its prophage sequence in the deposited genome of R. solanacearum strain UW551 from which the phage was isolated, but is also surprisingly found with 100% identity in the deposited genomes of 10 other phylotype II sequevar 1 strains of R. solanacearum. Furthermore, it is homologous to genome RS-09-161, resulting in the identification of a new prophage, designated RSM10, in a R. solanacearum strain from India. When ORF13 and a core attP site of ϕRs551 were either deleted individually or in combination, phage integration was not observed, suggesting that similar to other filamentous R. solanacearum ϕRSM phages, ϕRs551 relies on its resolvase and the core att sequence for site-directed integration into its susceptible R. solanacearum strain. The integration occurred four hours after phage infection. Infection of a susceptible R. solanacearum strain RUN302 by ϕRs551 resulted in less fluidal colonies and EPS production, and reduced motilities of the bacterium. Interestingly, infection of RUN302 by ϕRs551 also resulted in reduced virulence, rather than enhanced or loss of virulence caused by other ϕRSM phages. Study of bacteriophages of R. solanacearum would contribute to a better understanding of the phage-bacterium-environment interactions in order to develop integrated management strategies to combat R. solanacearum. Competing interests: The authors have declared that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0185034 September 21, 2017 1 / 19 Filamentous phage ϕRs551 isolated from a r3b2 strain of Ralstonia solanacearum Introduction Ralstonia solanacearum causes bacterial wilt, a soil-borne vascular disease that is arguably one of the most economically important bacterial diseases in the world. It attacks over 450 plant species and limits the production of such economically important crops as tomato, tobacco, potato and banana [1]. The race 3 biovar 2 (r3b2) strains (phylotype IIB, sequevars 1 and 2) of R. solanacearum causing devastating potato brown rot are quarantine pathogens in many countries and are also select agents in the United States [2]. R. solanacearum normally enters plants from soil through wounds in the roots and then multiplies in the xylem vessels and spreads through the plants’ vascular system. Control of R. solanacearum depends mainly on quarantines, use of pathogen-free propagating materials and eradication. The use of resistant cultivars, when available, and proper rotation or fallow has also been used with limited success due to the pathogen’s wide host range, broad distribution, great variability and ability to survive in soil and water [3,4]. Recently, the potential of using bacteriophages to control R. solanacearum has also been explored [5–7]. At present, a wide range of bacteriophages specifically infecting R. solanacearum have been isolated from soil of crop fields and include: filamentous phages ϕRSM1, ϕRSS1 [8], RS603 [9] and PE226 [10] of the family Inoviridae, icosahedral phages of the family Myoviridae [5,8], and lytic phages of the families Podoviridae [5,11,12], and Siphoviridae [13]. All of the R. solanacearum phages reported so far were isolated from soil in Japan, with the exceptions being the phages studied by Bhunchoth et al. [5] and Murugaiyan et al. [10] that were isolated from soil in Thailand and Korea, respectively. The filamentous Ff-type phages ϕRSS1 and ϕRSM1 have been studied in detail, including morphological and genomic characterization [8,14], integration mechanism [15,16], potential as a vector/expression vector [14,17], and effect on virulence of their R. solanacearum host strains [8,18,19]. While the phage ϕRSS1 was found to enhance virulence [18], infection by the phage ϕRSM1 resulted in loss of virulence of R. solanacearum on tomato plants [19], suggesting different effects caused by the two filamentous phages. In addition to the R. solanacearum phages isolated from the soil, Askora et al. [15,20] identified seven ϕRSM-like prophage sequences in the deposited genomes of R. solanacearum and R. pickettii with different evolutionary origins, including prophage RSM3 in MAFF7390139 (Asia), RSM4 in UW551 (Americas), RSM5 in IPO1609 (Americas), RSM6 in CMR15 (Africa), RSM7 in Y45 (Asia), RSM8 in R. syzygii R24 (Indonesia) and RSM9 (R. pickettii 12J). Askora et al. [15] converted one of the prophages, RSM3, into an infectious phage by PCR and cloning. In addition, open reading frames encoding for different types of putative phage repressors were identified in RSM phages and prophage (...truncated)