Evidence for an Opportunistic and Endophytic Lifestyle of the Bursaphelenchus xylophilus-Associated Bacteria Serratia marcescens PWN146 Isolated from Wilting Pinus pinaster

Abstract

Pine wilt disease (PWD) results from the interaction of three elements: the pathogenic nematode, Bursaphelenchus xylophilus; the insect-vector, Monochamus sp.; and the host tree, mostly Pinus species. Bacteria isolated from B. xylophilus may be a fourth element in this complex disease. However, the precise role of bacteria in this interaction is unclear as both plant-beneficial and as plant-pathogenic bacteria may be associated with PWD. Using whole genome sequencing and phenotypic characterization, we were able to investigate in more detail the genetic repertoire of Serratia marcescens PWN146, a bacterium associated with B. xylophilus. We show clear evidence that S. marcescens PWN146 is able to withstand and colonize the plant environment, without having any deleterious effects towards a susceptible host (Pinus thunbergii), B. xylophilus nor to the nematode model C. elegans. This bacterium is able to tolerate growth in presence of xenobiotic/organic compounds, and use phenylacetic acid as carbon source. Furthermore, we present a detailed list of S. marcescens PWN146 potentials to interfere with plant metabolism via hormonal pathways and/or nutritional acquisition, and to be competitive against other bacteria and/or fungi in terms of resource acquisition or production of antimicrobial compounds. Further investigation is required to understand the role of bacteria in PWD. We have now reinforced the theory that B. xylophilus-associated bacteria may have a plant origin.

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Acknowledgments

The authors would like to thank Prof. John Jones (The James Hutton Institute) for advice on an earlier draft of this manuscript; Sonia Humphris, Jenny A. Morris, and Pete Hedley (The James Hutton Institute) for all the support given in Serratia sp. PWN146 sequencing. This work was supported by the JSPS KAKENHI Grant numbers P14394 (to CSLV) and 26450204 (to KH); the European Project REPHRAME—Development of improved methods for detection, control and eradication of pine wood nematode in support of EU Plant Health policy, European Union Seventh Framework Programme FP7-KBBE-2010-4; and FEDER Funds through the Operational Programme for Competitiveness Factors - COMPETE and National Funds through FCT—Foundation for Science and Technology under the Strategic Project PEst-C/AGR/UI0115/2011.

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Correspondence to Cláudia S. L. Vicente.

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Sequence Data

The nucleotide sequence data reported is available in the EMBL database under the accession number ERS1151563.

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ESM 1

Figure S1 Genome-to-genome alignment of Serratia marcescens PWN146 with S. marcescens CAV1492, S. marcescens SmUNAM836 and S. marcescens subsp. marcescens Db11 using MAUVE. Locally collinear blocks (LCD) with the same colour indicate syntenic regions. In each LCD, the height of the similarity profile indicates the level of conservation in that genome region. White areas may indicate specific sequences of the genome. The genome rearrangements are indicated by different colour lines. (TIF 973 kb)

ESM 2

Table S1 Gene Ontology Serratia marcescens PWN146 complete genome (chromosome and plasmids). These results are based upon BLAST2GO [31] analysis of S. marcescens PWN146 proteome. Table S2 List of genomic islands predicted by at least one method (SIGI-HMM, IslandPath-DIMOB, IslandPick) in IslandViewer 3.0 [39], using the available genomes as reference: S. marcescens Db11, S. marcescens CAV1492, S. marcescens WW4, S. marcescens SM39, and S. marcescens FGI94. A, indicate the unique genes found in S. marcescens PWN146 genome using OrthoFinder [41] in all-against-all BlastP between PWN146 and all S. marcescens completely sequenced (Table 1). Table S3 Nematicidal effect of Serratia marcescens PWN146 to Bursaphelenchus xylophilus. Determination in accordance with Barbosa et al. []. Table S4 List of genes putatively involved in Serratia marcescens PWN146 plant-associated life-style. The list is complemented with KEGG annotation and the description accordingly. Table S5 Biochemical characterization of Serratia marcescens PWN146 according to the VITEK 2 Systems, with GN (Gram-negative) cards. Positive and negative results are indicated, respectively, by (+) and (−). (DOCX 119 kb)

ESM 3

Figure S2 Classification of KEGG KO terms of Serratia marcescens PWN146 complete genome (chromosome and plasmids). This classification was obtained in KEGG BLASTKoala tool [32]. (TIF 30 kb)

ESM 4

Figure S3 SEM images of Bursaphelenchus xylophilus Ka4 without bacteria association. (TIF 1202 kb)

ESM 5

Figure S4 Pathogenicity trials in 4-year-old Pinus thunbergii. The treatments established were: (1) control P. thunbergii (inoculation with sterile ddH2O); (2) P. thunbergii inoculated with B. xylophilus Ka4; (3) P. thunbergii inoculated with S. marcescens PWN146; and (4) P. thunbergii inoculated with B. xylophilus Ka4 in association with S. marcescens PWN146.WAI indicates weeks after inoculation. (TIF 180 kb)

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Vicente, C.S.L., Nascimento, F.X., Barbosa, P. et al. Evidence for an Opportunistic and Endophytic Lifestyle of the Bursaphelenchus xylophilus-Associated Bacteria Serratia marcescens PWN146 Isolated from Wilting Pinus pinaster . Microb Ecol 72, 669–681 (2016). https://doi.org/10.1007/s00248-016-0820-y

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Keywords

  • Bursaphelenchus xylophilus
  • Endophyte
  • Nematode
  • Serratia marcescens
  • Pine wilt disease