Microbial Ecology

, Volume 63, Issue 3, pp 628–638 | Cite as

Independent Origins of Vectored Plant Pathogenic Bacteria from Arthropod-Associated Arsenophonus Endosymbionts

  • Alberto Bressan
  • Federica Terlizzi
  • Rino Credi
Invertebrate Microbiology


The genus Arsenophonus (Gammaproteobacteria) is comprised of intracellular symbiotic bacteria that are widespread across the arthropods. These bacteria can significantly influence the ecology and life history of their hosts. For instance, Arsenophonus nasoniae causes an excess of females in the progeny of parasitoid wasps by selectively killing the male embryos. Other Arsenophonus bacteria have been suspected to protect insect hosts from parasitoid wasps or to expand the host plant range of phytophagous sap-sucking insects. In addition, a few reports have also documented some Arsenophonus bacteria as plant pathogens. The adaptation to a plant pathogenic lifestyle seems to be promoted by the infection of sap-sucking insects in the family Cixiidae, which then transmit these bacteria to plants during the feeding process. In this study, we define the specific localization of an Arsenophonus bacterium pathogenic to sugar beet and strawberry plants within the plant hosts and the insect vector, Pentastiridius leporinus (Hemiptera: Cixiidae), using fluorescence in situ hybridization assays. Phylogenetic analysis on 16S rRNA and nucleotide coding sequences, using both maximum likelihood and Bayesian criteria, revealed that this bacterium is not a sister taxon to “Candidatus Phlomobacter fragariae,” a previously characterized Arsenophonus bacterium pathogenic to strawberry plants in France and Japan. Ancestral state reconstruction analysis indicated that the adaptation to a plant pathogenic lifestyle likely evolved from an arthropod-associated lifestyle and showed that within the genus Arsenophonus, the plant pathogenic lifestyle arose independently at least twice. We also propose a novel Candidatus status, “Candidatus Arsenophonus phytopathogenicus” novel species, for the bacterium associated with sugar beet and strawberry diseases and transmitted by the planthopper P. leporinus.


Sugar Beet Strawberry Plant Plant Pathogenic Bacterium Endosymbiotic Bacterium Ancestral State Reconstruction 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We are grateful to four anonymous reviewers for suggestions on the original version of the manuscript and to Elisabeth Boudon-Padieu at INRA Dijon for providing P. leporinus planthoppers and sugar beet plants used for the FISH assays. Research was supported by the University of Hawaii Start-up and Hatch funds to Alberto Bressan.

Supplementary material

248_2011_9933_MOESM1_ESM.pdf (8.3 mb)
Figure S1 Results of fluorescence in situ hybridization assays on a thin section of Pentastiridius leporinus abdomen hybridized with both universal eubacterial probe, EUB338-Alexa Fluor488 (a) and SBR proteobacterium probe, SBR450-Texas Red (b). B 1 a bacteriome containing “Ca. Sulcia muelleri”-like cells, B 2 a bacteriome containing “Ca. Purcelliella pentastirinorum”-like cells and other large cells from an underscribed β-proteobacterium (***); Oo oocyte. Arrows indicate localization of SBR proteobacterium. Scale bars = 50 μm. (PDF 8457 kb)
248_2011_9933_MOESM2_ESM.pdf (642 kb)
Figure S2 Cladogram showing ancestral state reconstruction using parsimony analysis mapped onto 16S rRNA tree (PDF 642 kb)
248_2011_9933_MOESM3_ESM.pdf (603 kb)
Figure S3 Cladogram showing ancestral state reconstruction using parsimony analysis mapped onto spoT-spoU-recG tree. (PDF 603 kb)
248_2011_9933_MOESM4_ESM.doc (99 kb)
Table S1 (DOC 99 kb)


  1. 1.
    Alison PG (2003) Epidemiology meets evolutionary ecology. Trends Ecol Evol 18:132–139CrossRefGoogle Scholar
  2. 2.
    Arneodo JD, Bressan A, Lherminier J, Michel J, Boudon-Padieu E (2008) Ultrastructural detection of an unusual intranuclear bacterium in Pentastiridius leporinus (Hemiptera: Cixiidae). J Inver Pathol 97:310–313CrossRefGoogle Scholar
  3. 3.
    Bai X, Zhang J, Ewing A, Miller SA, Jancso Radek A, Shevchenko DV, Tsukerman K, Walunas T, Lapidus A, Campbell JW, Hogenhout SA (2006) Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts. J Bacteriol 188:3682–3696PubMedCrossRefGoogle Scholar
  4. 4.
    Bové JM, Garnier M (2002) Phloem-and xylem-restricted plant pathogenic bacteria. Plant Science 163:1083–1098CrossRefGoogle Scholar
  5. 5.
    Bressan A, Sémétey O, Nusillard B, Clair D, Boudon-Padieu E (2008) Insect vectors (Hemiptera: Cixiidae) and pathogen types associated with syndrome “basses richesses” disease of sugar beet in France. Plant Disease 92:113–119CrossRefGoogle Scholar
  6. 6.
    Bressan A, Arneodo JD, Simonato M, Haines WP, Boudon-Padieu E (2009) Characterization and evolution of two bacteriome-inhabiting symbionts in cixiid planthoppers (Hemiptera: Fulgoromorpha: Pentastirini). Environ Microbiol 11:3265–3279PubMedCrossRefGoogle Scholar
  7. 7.
    Bressan A, Sémétey O, Arneodo J, Lherminier J, Boudon-Padieu E (2009) Vector transmission of a plant pathogenic bacterium in the Arsenophonus clade sharing ecological traits with facultative insect endosymbionts. Phytopathology 99:1289–1296PubMedCrossRefGoogle Scholar
  8. 8.
    Chiel E, Gottlieb Y, Zchori-Fein E, Mozes-Daube N, Katzir N, Inbar M, Ghanim M (2007) Biotype-dependent secondary symbiont communities in sympatric populations of Bemisia tabaci. Bull Entomol Res 97:407–413PubMedCrossRefGoogle Scholar
  9. 9.
    Dale C, Beeton M, Harbison C, Jones T, Pontes M (2006) Isolation, pure culture, and characterization of “Candidatus Arsenophonus arthropodicus,” an intracellular secondary endosymbiont from the hippoboscid louse fly Pseudolynchia canariensis. Appl Env Microbiol 72:2997–3004CrossRefGoogle Scholar
  10. 10.
    Danet J-L, Foissac X, Zreik L, Salar P, Verdin E, Nourrisseau JG, Garnier M (2003) “Candidatus Phlomobacter fragariae” is the prevalent agent of marginal chlorosis of strawberry in French production fields and is transmitted by the planthopper Cixius wagneri (China). Phytopathology 93:644–649PubMedCrossRefGoogle Scholar
  11. 11.
    Darby AC, Choi JH, Wilkes T, Hughes MA, Werren JH, Hurst GDD, Colbourne JK (2010) Characteristics of the genome of Arsenophonus nasoniae, son-killer bacterium of the wasp Nasonia. Insect Mol Biol 19:75–89PubMedCrossRefGoogle Scholar
  12. 12.
    Davis MJ, Ying Z, Brunner BR, Pantoja A, Ferwerda FH (1998) Rickettsial relative associated with papaya bunchy top disease. Curr Microbiol 36:80–84PubMedCrossRefGoogle Scholar
  13. 13.
    Duron O, Bouchon D, Boutin S, Bellamy L, Zhou L, Engelstadter J (2008) The diversity of reproductive parasites among arthropods: Wolbachia do not walk alone. BMC Biol 6:27PubMedCrossRefGoogle Scholar
  14. 14.
    Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797PubMedCrossRefGoogle Scholar
  15. 15.
    Erickson DL, Waterfield NR, Vadyvaloo V, Long D, Fischer ER, French-Constant R, Hinnebusch BJ (2007) Acute oral toxicity of Yersinia pseudotuberculosis to fleas: implications for the evolution of vector-borne transmission of plague. Cellular Microbiol 9:2658–2666CrossRefGoogle Scholar
  16. 16.
    Fukatsu T, Watanabe K, Sekiguchi J (1998) Specific detection of intracellular symbiotic bacteria of aphids by oligonucleotide-probed in situ hybridization. Appl Entomol Zool 33:461–472Google Scholar
  17. 17.
    Gatineau F, Jacob N, Vautrin S, Larrue J, Lherminier J, Richard-Molard M, Boudon-Padieu E (2002) Association with the syndrome “basses richesses” of sugar beet of a phytoplasma and a bacterium-like organism transmitted by a Pentastiridius sp. Phytopathology 92:384–392PubMedCrossRefGoogle Scholar
  18. 18.
    Gherna RL, Werren JH, Weisburg W, Cote R, Woese CR, Mandelco L, Brenner R (1991) Arsenophonus nasoniae, genus novel, species novel, causative agent of son killer trait in the parasitic wasp, Nasonia vitripennis. Int J Syst Bacteriol 41:563–565CrossRefGoogle Scholar
  19. 19.
    Gotoh T, Noda H, Ito S (2007) Cardinium symbionts cause cytoplasmic incompatibility in spider mites. Heredity 98:13–20PubMedCrossRefGoogle Scholar
  20. 20.
    Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. System Biol 52:696–704CrossRefGoogle Scholar
  21. 21.
    Hansen AK, Jeong G, Paine TD, Stouthamer R (2007) Frequency of secondary symbiont infection in an invasive psyllid relates to parasitism pressure on a geographic scale in California. Appl Environ Microbiol 73:7531–7535PubMedCrossRefGoogle Scholar
  22. 22.
    Hansen AK, Trumble JT, Stouthamer R, Paine TD (2008) A new Huanglongbing species, “Candidatus Liberibacter psyllaurous”, found to infect tomato and potato, is vectored by the psyllid Bactericera cockerelli (Sulc). Appl Environ Microbiol 74:5862–290PubMedCrossRefGoogle Scholar
  23. 23.
    Hogenhout SA, Ammar E-D, Whitfield AE, Redinbaugh MG (2008) Insect vector interactions with persistently transmitted viruses. Ann Rev Phytopathol 46:327–359CrossRefGoogle Scholar
  24. 24.
    Hoshia A, Oshimaa K, Kakizawaa S, Ishiia Y, Ozekia J, Hashimotoa M, Komatsua K, Kagiwadab S, Yamajia Y, Nambaa S (2009) A unique virulence factor for proliferation and dwarfism in plants identified from a phytopathogenic bacterium. PNAS 2:6416–6421CrossRefGoogle Scholar
  25. 25.
    Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17:754–755PubMedCrossRefGoogle Scholar
  26. 26.
    Hypsa V, Dale C (1997) In vitro culture and phylogenetic analysis of “Candidatus Arsenophonus triatominarum”, an intracellular bacterium from the triatomine bug, Triatoma infestans. Int J Syst Bacteriol 47:1140–1144PubMedCrossRefGoogle Scholar
  27. 27.
    IRPCM (2004) ‘Candidatus Phytoplasma’, a taxon for the wall-less, nonhelical prokaryotes that colonize plant phloem and insects. Int J Syst Evol Microbiol 54:1243–1255CrossRefGoogle Scholar
  28. 28.
    Lambrechts L, Scott TW (2009) Mode of transmission and the evolution of Arbovirus virulence in mosquito vectors. Proc R Soc B 276:1369–1378PubMedCrossRefGoogle Scholar
  29. 29.
    Maddison WP, Maddison DR (2010) Mesquite: a modular system for evolutionary analysis. Version 2.73 ( November 16, 2010
  30. 30.
    Montllor C, Maxmen A, Purcell AH (2002) Facultative bacterial endosymbionts benefit pea aphids Acyrthosiphon pisum under heat stress. Ecol Entomol 27:189–195CrossRefGoogle Scholar
  31. 31.
    Moran NA, McCutcheon JP, Nakabachi A (2008) Genomics and evolution of heritable bacterial symbionts. Annu Rev Genet 42:165–190PubMedCrossRefGoogle Scholar
  32. 32.
    Murray RGE, Schleifer KH (1994) Taxonomic notes: a proposal for recording the properties of putative taxa of procaryotes. Int J Syst Bacteriol 44:174–176PubMedCrossRefGoogle Scholar
  33. 33.
    Nishigawa H, Oshima K, Kakizawa S, Jung H-Y, Kuboyama T, Miyata S, Ugaki M, Namba S (2002) A plasmid from a non-insect-transmissible line of a phytoplasma lacks two open reading frames that exist in the plasmid from the wild-type line. Gene 298:195–201PubMedCrossRefGoogle Scholar
  34. 34.
    Nourrisseau JG, Lansac M, Garnier M (1993) Marginal chlorosis, a new disease of strawberries associated with a bacterium like organism. Plant Dis 77:1055–1059CrossRefGoogle Scholar
  35. 35.
    Novakova E, Hypsa V, Moran NA (2009) Arsenophonus, an emerging clade of intracellular symbionts with a broad host distribution. BMC Microbiol 9:143PubMedCrossRefGoogle Scholar
  36. 36.
    Oshima K, Kakizawa S, Nishigawa H, Jung HY, Wei W, Suzuki S, Arashida R, Nakata D, Miyata S, Ugaki M, Namba S (2004) Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma. Nature Genet 36:27–29PubMedCrossRefGoogle Scholar
  37. 37.
    Pagel M (1999) The maximum likelihood approach to reconstructing ancestral character states of discrete characters on phylogenies. Syst Biol 48:612–622CrossRefGoogle Scholar
  38. 38.
    Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25:1253–1256PubMedCrossRefGoogle Scholar
  39. 39.
    Purcell AH (1982) Evolution of the insect vector relationship. In: Lacy GH, Mount MS (eds) Phytopathogenic prokaryotes. Academic Press, New York, pp 121–156Google Scholar
  40. 40.
    Regassa LB, Gasparich GE (2006) Spiroplasmas: evolutionary relationships and biodiversity. Front Biosci 11:2983–3002PubMedCrossRefGoogle Scholar
  41. 41.
    Salar P, Sémétey O, Danet JL, Boudon-Padieu E, Foissac X (2010) ‘Candidatus Phlomobacter fragariae’ and the proteobacterium associated with the low sugar content syndrome of sugar beet are related to bacteria of the Arsenophonus clade detected in hemipteran insects. Europ J Plant Pathol 126:123–127CrossRefGoogle Scholar
  42. 42.
    Scarborough CL, Ferrari J, Godfray HCJ (2005) Aphid protected from pathogen by endosymbionts. Science 310:1781PubMedCrossRefGoogle Scholar
  43. 43.
    Sémétey O, Bressan A, Gatineau F, Boudon-Padieu E (2007) Development with RISA of a specific assay for detection of the bacterial agent of syndrome “basses richesses” of sugar beet. Confirmation of Pentastiridius sp. (Fulgoromopha, Cixiidae) as the economic vector. Plant Pathol 56:797–804CrossRefGoogle Scholar
  44. 44.
    Sémétey O, Bressan A, Richard-Molard M, Boudon-Padieu E (2007) Monitoring of proteobacteria and phytoplasma in sugar beet naturally or experimentally affected by the disease syndrome ‘basses richesses’. Europ J Plant Pathol 117:187–196CrossRefGoogle Scholar
  45. 45.
    Sémétey O, Gatineau F, Bressan A, Boudon-Padieu E (2007) Characterization of a γ-3 Proteobacteria responsible for the syndrome “basses richesses” of sugar beet transmitted by Pentastiridius sp. (Hemiptera: Cixiidae). Phytopathology 97:72–78PubMedCrossRefGoogle Scholar
  46. 46.
    Stavrinides J (2009) Origin and evolution of phytopathogenic bacteria. In: Jackson RW (ed) Plant pathogenic bacteria: genomics and molecular biology. Caister Academic Press, Norfolk, p 330Google Scholar
  47. 47.
    Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  48. 48.
    Tanaka M, Nao M, Usugi T (2006) Occurrence of strawberry marginal chlorosis caused by “Candidatus Phlomobacter fragariae” in Japan. J Gen Plant Pathol 72:374–377CrossRefGoogle Scholar
  49. 49.
    Terlizzi F, Babini AR, Credi R (2006) First report of stolbur phytoplasma (16SrXII-A) on strawberry in northern Italy. Plant Dis 90:831CrossRefGoogle Scholar
  50. 50.
    Terlizzi F, Babini AR, Lanzoni C, Pisi A, Credi R, Foissac X, Salar P (2007) First report of a γ-3 proteobacterium associated with diseased strawberries in Italy. Plant Dis 91:1688CrossRefGoogle Scholar
  51. 51.
    Thao ML, Baumann P (2004) Evidence for multiple acquisitions of Arsenophonus by whitefly species (Sternorrhyncha: Aleyrodidae). Curr Microbiol 48:140–144PubMedCrossRefGoogle Scholar
  52. 52.
    Weinert LA, Werren JH, Aebi A, Stone GN, Jiggins FM (2009) Evolution and diversity of Rickettsia bacteria. BMC Biol 7:6PubMedCrossRefGoogle Scholar
  53. 53.
    Weintraub PG, Beanland L (2006) Insect vectors of phytoplasmas. Annu Rev Entomol 51:91–111PubMedCrossRefGoogle Scholar
  54. 54.
    Werren JH, Skinner SW, Huger AM (1986) Male-killing bacteria in a parasitic wasp. Science 231:990–992PubMedCrossRefGoogle Scholar
  55. 55.
    Zreik L, Bové JM, Garnier M (1998) Phylogenetic characterization of the bacterium-like organism associated with marginal chlorosis of strawberry and proposition of a Candidatus taxon for the organism, ‘Candidatus Phlomobacter fragariae’. Int J Syst Bacteriol 48:257–261PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Alberto Bressan
    • 1
  • Federica Terlizzi
    • 2
  • Rino Credi
    • 2
  1. 1.Department of Plant and Environmental Protection SciencesUniversity of Hawaii at ManoaHonoluluUSA
  2. 2.Dipartimento di Scienze e Tecnologie Agroambientali-Patologia VegetaleUniversity of BolognaBolognaItaly

Personalised recommendations