American Journal of Potato Research

, Volume 89, Issue 4, pp 294–305 | Cite as

Isolation of Bacteria from the Rhizosphere and Rhizoplane of Potato (Solanum tuberosum) Grown in Two Distinct Soils Using Semi Selective Media and Characterization of Their Biological Properties

Article

Abstract

The objective of this study was to isolate and characterize genera of bacteria that had been identified as being the most common residents in the rhizosphere of potato using cpn60 pyrosequencing analysis. Using various semi-selective media targeted to specific genera of interest, 200 isolates of bacteria were collected from rhizosphere soil and the rhizoplane of potatoes grown in soils obtained from a potato farm in Prince Edward Island and Ontario. The procedures employed were successful in selecting out representative bacteria suggested by pyrosequencing to be common in the potato rhizoplane. Results of 16S rRNA sequencing showed that 44 % of the isolates represented new species. All isolates were tested for biological and biochemical activities including phosphate solubilization, phytohormone metabolism, nitrogen fixation, antibiosis, exoenzyme production, and production of acyl-homoserine lactones. Massilia spp. and Chryseobacterium spp. showed the strongest exoenzyme activities. A greater proportion of Agrobacterium tumefaciens rhizosphere strains produced acyl-homoserine lactones compared to rhizoplane strains. Pseudomonas spp. and Lysobacter capsici had the greatest antagonistic activity on laboratory media towards six potato pathogens, and also significantly decreased disease in plants grown in pathogen-infested soil. Four isolates significantly increased growth of potato nodal explants in tissue culture. By using preliminary results derived from next generation sequencing technology and a targeted cultural technique, we were able to gain a better understanding of the biological activities of the most abundant bacterial species in the rhizosphere and rhizoplane of a cultivated crop.

Keywords

Rhizosphere Biocontrol Biofertilizer Novel bacteria 

Resumen

El objetivo de este estudio fue aislar y caracterizar genero/especie de bacterias que se han identificado como los residentes mas comunes en la rizosfera de la papa, utilizando un análisis de pirosecuenciación epn60. Mediante el uso de varios medios semiselectivos enfocados a géneros específicos de interés, se colectaron 200 aislamientos de bacterias del suelo de la rizosfera y del rizoplano de papas cultivadas en suelos obtenidos de un campo de papa en la Isla del Principe Eduardo y Ontario. Los procedimientos que se usaron fueron exitosos en la selección de bacterias representativas identificadas como las más comunes en el rizoplano. Los resultados de la secuenciación del ADNr 16S mostraron que el 44 % de los aislamientos representaron nuevas especies. Se probaron todos los aislamientos para actividades biológicas y bioquímicas, incluyendo la solubilización de fosfatos, metabolismo de las fitohormonas, la fijación de nitrógeno, antibiosis, producción de exoenzimas, y la producción de lactonas acil-homoserinas. Massilia spp. y Chryseobacterium spp. mostraron las actividades enzimáticas más fuertes. Una proporción mas grande de variantes de Agrobacterium tumefasciens de la rizosfera produjeron lactonas acil-homoserinas comparadas con las del rizoplano. Pseudomonas spp. y Lysobacter capsici tuvieron la mayor actividad antagonistica en medio en laboratorio hacia seis patógenos de la papa, y también disminuyeron significativamente enfermedades en plantas cultivadas en suelos infestados de patógenos. Cuatro aislamientos aumentaron significativamente el crecimiento de plantas nodales de papa en cultivo de tejidos. Mediante el uso de resultados preliminares derivados de la tecnología de secuenciación de la siguiente generación, y una técnica de cultivo enfocada, pudimos ganar un mejor entendimiento de las actividades biológicas de las especies de bacterias más abundantes en la rizosfera y el rizoplano de un cultivo.

Supplementary material

12230_2012_9253_MOESM1_ESM.doc (34 kb)
Table S1Exoenzymes produced by bacteria isolated from roots using various laboratory media (DOC 33 kb)
12230_2012_9253_MOESM2_ESM.doc (51 kb)
Table S2Signalling molecules produced by bacteria isolated from roots using various laboratory media. (DOC 51 kb)
12230_2012_9253_MOESM3_ESM.doc (36 kb)
Table S3Nutrient usage of root-associated bacteria in petri plate-based assays (DOC 36 kb)
12230_2012_9253_MOESM4_ESM.doc (43 kb)
Table S4Antibiotic activity of bacterial isolates towards six potato pathogens (DOC 43 kb)
12230_2012_9253_MOESM5_ESM.doc (30 kb)
Table S5(DOC 00 kb)

References

  1. Abbasi, P.A., K.L. Conn, and G. Lazarovits. 2004. Suppression of Rhizoctonia and Pythium damping-off of radish and cucumber seedlings by addition of fish emulsion to peat mix or soil. Canadian Journal of Plant Pathology 26: 177–187.CrossRefGoogle Scholar
  2. Ashelford, K.E., S.J. Norris, J.C. Fry, M.M. Bailey, and M.J. Day. 2000. Seasonal population dynamics and interactions of competing bacteriophages and their host in the rhizosphere. Applied and Environmental Microbiology 66: 4139–4199.CrossRefGoogle Scholar
  3. Barber, L.E. 1979. Use of selective agents for recovery of Rhizobium meliloti from soil. Soil Science Society of America Journal 43: 1145–1148.CrossRefGoogle Scholar
  4. Berg, G., A. Krechel, M. Ditz, R.A. Sikora, A. Ulrich, and J. Hallmann. 2005. Endophytic and ectophytic potato-associated bacterial communities differ in strucutre and antagnoistic function against plant pathogenic fungi. FEMS Microbiology Ecology 51: 215–229.PubMedCrossRefGoogle Scholar
  5. Boehm, M.J., T. Wu, A.G. Stone, B. Kraakman, D.A. Iannotti, G.E. Wilson, L.V. Madden, and H.A.J. Hoitink. 1997. Cross-polarized magic-angle spinning 13 C nuclear magnetic resonance spectroscopic characterization of soil organic matter relative to culturable bacterial species composition and sustained biological control of Pythium root rot. Applied and Environmental Microbiology 63: 162–168.PubMedGoogle Scholar
  6. Bonanomi, G., V. Antignani, M. Capodilupo, and F. Scala. 2010. Identifying the characteristics of organic soil amendments that suppress soilborne plant diseases. Soil Biology and Biochemistry 42: 136–144.CrossRefGoogle Scholar
  7. Buyer, J.S., D.P. Roberts, and E. Russek-Cohen. 2002. Soil and plant effects on microbial community structure. Canadian Journal of Microbiology 48: 955–964.PubMedCrossRefGoogle Scholar
  8. Cavalcante, V.A., and J. Dobereiner. 1988. A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane. Plant and Soil 108: 23–31.CrossRefGoogle Scholar
  9. Chen, W.-M., L. Moulin, C. Bontemps, P. Vandamme, G. Béna, and C. Biovin-Masson. 2003. Legume symbiotic nitrogen fixation by b-proteobacteria is widespread in nature. Journal of Bacteriology 185: 7266–7272.PubMedCrossRefGoogle Scholar
  10. Compant, S., B. Duffy, J. Nowak, C. Clément, and E. Ait Barka. 2005. Use of plant growth-promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects. Applied and Environmental Microbiology 71: 4951–4959.PubMedCrossRefGoogle Scholar
  11. Conn, K.L., J. Nowak, and G. Lazarovits. 1997. A gnotobiotic bioassay for studying interactions between potatoes and plant growth-promoting rhizobacteria. Canadian Journal of Microbiology 43: 801–808.CrossRefGoogle Scholar
  12. Dandie, C.E., D.L. Burton, B.J. Zebarth, J.T. Trevors, and C. Goyer. 2007. Analysis of denitrification genes and comparison of nosZ, cnorB, and 16 S rDNA from culturable denitrifying bacteria in potato cropping systems. Systematic and Applied Microbiology 30: 128–138.PubMedCrossRefGoogle Scholar
  13. Den Herder, G., G. Van Isterdael, T. Beeckman, and I. De Smet. 2010. The roots of a new green revolution. Trends in Plant Science 15: 600–607.CrossRefGoogle Scholar
  14. Dumonceaux, T.J., J. Schellenberg, V. Goleski, J.E. Hill, W. Jaoko, J. Kimani, D. Money, T.B. Ball, F.A. Plummer, and A. Severini. 2009. Multiplex detection of bacteria associated with normal microbiota and with bacterial vaginosis in vaginal swabs by use of oligonucleotide-coupled fluorescent microspheres. Journal of Clinical Microbiology 47: 4067–4077.PubMedCrossRefGoogle Scholar
  15. Eisen, M.B., P.T. Spellman, P.O. Brown, and D. Botstein. 1998. Cluster analysis and display of genome-wide expression patterns. Proceedings of the National Academy of Sciences 95: 14863–14868.CrossRefGoogle Scholar
  16. Fierer, N., M.A. Bradford, and R.B. Jackson. 2007. Toward an ecological classification of soil bacteria. Ecology 88: 1354–1364.PubMedCrossRefGoogle Scholar
  17. Folman, L.B., J. Postma, and J.A. Van Veen. 2003. Inability to find consistent bacterial biocontrol agents of Pythium aphanidermatum in cucumber using screens based on ecophysiological traits. Microbial Ecology 45: 72–87.PubMedCrossRefGoogle Scholar
  18. Fuqua, C., and S.C. Winans. 1996. Conserved cis-acting promoter elements are required for density-dependent transcription of Agrobacterium. Journal of Bacteriology 178: 435–440.PubMedGoogle Scholar
  19. Gilbert, N. 2009. The disappearing nutrient. Nature 461: 716–718.PubMedCrossRefGoogle Scholar
  20. Gogleva, A.A., E.N. Kaparullina, N.V. Doronina, and Y.A. Trotsenko. 2010. Methylophilus flavus sp. nov., and Methylophilus luteus sp. nov., aerobic methylotrophic bacteria associated with plants. International Journal of Systematic and Evolutionary Microbiology 60: 2623–2628.PubMedCrossRefGoogle Scholar
  21. Gomes, N.C.M., D.F.R. Cleary, F.N. Pinto, C. Egas, A. Almeida, A. Cunha, L.C. Mendonça-Hagler, and K. Smalla. 2010. Taking root: enduring effect of rhizosphere bacterial colonization of mangroves. PLoS One 5: e14065.PubMedCrossRefGoogle Scholar
  22. Green, S.J., F.C. Michel Jr., Y. Hadar, and D. Minz. 2007. Contrasting patterns of seed and root colonization by bacteria from the genus Chryseobacterium and from the family Oxalobacteraceae. ISME Journal 1: 291–299.PubMedGoogle Scholar
  23. Grinwis, M.E., C.D. Sibley, M.D. Parkins, C.S. Eshaghurshan, H.R. Rabin, and M.G. Surette. 2009. Characterization of Streptococcus milleri group isolates from expectorated sputum of adult cystic fibrosis patients. Journal of Clinical Microbiology 48: 395–401.PubMedCrossRefGoogle Scholar
  24. Haas, D., and C. Keel. 2003. Regulation of antibiotic production in root-colonizing Pseudomonas spp. and relevance for biological control of plant disease. Annual Review of Phytopathology 41: 117–153.PubMedCrossRefGoogle Scholar
  25. Haluschak, P., C. McKenzie, and K. Panchuk. 2003. Guide to commercial potato production on the Canadian prairies. Canada: Western Potato Council.Google Scholar
  26. Hayward, A.C., N. Fegan, M. Fegan, and G.R. Stirling. 2010. Stenotrophomonas and Lysobacter: ubiquitous plant associated gamma-proteobacteria of developing significance in applied microbiology. Journal of Applied Microbiology 108: 756–770.PubMedCrossRefGoogle Scholar
  27. Horticulture and Special Crops Division. 2007. Potato situation and trends 2006–2007. Canada: Agriculture and Agri-Food Canada.Google Scholar
  28. Janssen, P.H., P.S. Yates, B.E. Grinton, P.M. Taylor, and M. Sait. 2002. Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. Applied and Environmental Microbiology 68: 2391–2396.PubMedCrossRefGoogle Scholar
  29. Kado, C.I., and M.G. Heskett. 1970. Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas, and Xanthomonas. Phytopathology 60: 967–976.CrossRefGoogle Scholar
  30. Kinkle, B.K., M.J. Sadowsky, K. Johnstone, and W.C. Koskinen. 1994. Tellurium and selenium resistance in rhizobia and its potential use for direct isolation of Rhizobium meliloti from soil. Applied and Environmental Microbiology 60: 1674–1677.PubMedGoogle Scholar
  31. Konopka, A. 2009. What is microbial community ecology? ISME Journal 3: 1223–1230.PubMedCrossRefGoogle Scholar
  32. Krechel, A., A. Faupel, J. Hallmann, A. Ulrich, and G. Berg. 2002. Potato-associated bacteria and their antagonistic potential towards plant-pathogenic fungi and the plant-parasitic nematode Meloidogyne incognita (Kofoid & White) Chitwood. Canadian Journal of Microbiology 48: 772–786.PubMedCrossRefGoogle Scholar
  33. Lazarovits, G, Turnbull, A.L., Haug, B., Links, M.G., Hill, J.E. and Hemmingsen, S.M. 2011. Unravelling the rhizosphere using the cpn60 genomic marker and pyrosequencing. In Molecular Microbial Ecology of the Rhizosphere, ed. F.J. de Bruijn. Wiley-Blackwell.Google Scholar
  34. Lottmann, J., H. Heuer, J. de Vries, A. Mahn, K. Düring, W. Wackernagel, K. Smalla, and G. Berg. 2000. Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effects on the bacterial community. FEMS Microbiology Ecology 33: 41–49.PubMedCrossRefGoogle Scholar
  35. Manter, D.K., J.A. Delgado, D.G. Holm, and R.A. Strong. 2010. Pyrosequencing reveals a highly diverse and cultivar-specific bacterial endophyte community on potato roots. Microbial Ecology 60: 157–166.PubMedCrossRefGoogle Scholar
  36. Nunes da Rocha, U., F.D. Andreote, J.L. de Azevedo, J.D. van Elsas, and L.S. van Overbeek. 2010. Cultivation of hitherto-uncultured bacteria belonging to the Verrucomicrobia subdivision 1 from the potato (Solanum tuberosum L.) rhizosphere. Journal of Soils and Sediments 10: 326–339.CrossRefGoogle Scholar
  37. Park, J.H., R. Kim, Z. Aslam, C.O. Jeon, and Y.R. Chung. 2008. Lysobacter capsici sp. nov., with antimicrobial activity, isolated from the rhizosphere of pepper, emended description of the genus Lysobacter. International Journal of Systematic and Evolutionary Microbiology 58: 387–392.PubMedCrossRefGoogle Scholar
  38. Pierson, E.A., and D.M. Weller. 1994. Use of mixtures of fluorescent pseudomonads to suppress take-all and improve the growth of wheat. Phytopathology 84: 940–947.CrossRefGoogle Scholar
  39. Pliego, C., C. Ramos, A. de Vicente, and F.M. Cazorla. 2011. Screening for candidate bacterial biocontrol agents against soilborne fungal plant pathogens. Plant and Soil 340: 505–520.CrossRefGoogle Scholar
  40. Postma, J., M.T. Schider, J. Bloem, and W.K. van Leeuwen-Haagsma. 2008. Soil suppressiveness and functional diversity of the soil microflora in organic farming systems. Soil Biology and Biochemistry 40: 2394–2406.CrossRefGoogle Scholar
  41. Reynolds, H.L., A. Packer, J.D. Beaver, and K. Clay. 2003. Grassroots ecology: plant-microbe-soil interactions as drivers of plant community structure and dynamics. Ecology 84: 2281–2291.CrossRefGoogle Scholar
  42. Robertson, B.K., B. Dreyfus, and M. Alexander. 1995. Ecology of stem-nodulating Rhizobium and Azorhizobium in four vegetation zones of Senegal. Microbial Ecology 29: 71–81.CrossRefGoogle Scholar
  43. Ryu, J., M. Madhaiyan, S. Poonguzhali, W. Yim, P. Indiragandhi, K. Kim, R. Anandham, J. Yun, K.H. Kim, and T. Sa. 2006. Plant growth substances produced by Methylobacterium spp. and their effect on tomato (Lycopersicon esculentum L.) and red pepper (Capsicum annuum L.) growth. Journal of Microbiology and Biotechnology 16: 1622–1628.Google Scholar
  44. Sawar, M., and R.J. Kremer. 1995. Determination of bacterially derived auxins using a microplate. Letters in Applied Microbiology 20: 282–285.CrossRefGoogle Scholar
  45. Schnürer, J., and T. Rosswall. 1982. Fluorescein diacetate hydrolysis as a measure of total microbial activity in soil and litter. Applied and Environmental Microbiology 43: 1256–1261.PubMedGoogle Scholar
  46. Sessitsch, A., B. Reiter, U. Pfeifer, and E. Wilhelm. 2002. Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes. FEMS Microbiology Ecology 39: 23–32.PubMedCrossRefGoogle Scholar
  47. Sessitsch, A., T. Coenye, A.V. Sturz, P. Vandamme, E. Ait Barka, J.F. Salles, J.D. Van Elsas, D. Faure, B. Reiter, B.R. Glick, G. Wang-Pruski, and J. Nowak. 2005. Burkholderia phytofirmans sp. nov., a novel plant-associated bacterium with plant-beneficial properties. International Journal of Systematic and Evolutionary Microbiology 55: 1187–1192.PubMedCrossRefGoogle Scholar
  48. Singh, B.K., P. Millard, A.S. Whiteley, and J.C. Murrell. 2004. Unravelling rhizosphere-microbial interactions: opportunities and limitations. Trends in Microbiology 12: 386–393.PubMedCrossRefGoogle Scholar
  49. Sugimoto, E.E., H.A.J. Hoitink, and O.H. Tuovinen. 1990. Oligotrophic pseudomonads in the rhizosphere: suppressiveness to Pythium damping-off of cucumber seedlings. Biology and Fertility of Soils 9: 231–234.CrossRefGoogle Scholar
  50. Sy, A., E. Giraud, P. Jourand, N. Garcia, A. Willems, P. de Lajudie, Y. Prin, M. Neyra, M. Gillis, C. Biovin-Masson, and B. Dreyfus. 2001. Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes. Journal of Bacteriology 183: 214–220.PubMedCrossRefGoogle Scholar
  51. Teixeira, L.C.R.S., R.S. Peixoto, J.C. Cury, W.J. Sul, V.H. Pellizari, J. Tiedje, and A.S. Posado. 2010. Bacterial diversity in the rhizosphere from Antarctic vascular plants of Admiralty Bay, maritime Antartica. ISME Journal 4: 989–1001.PubMedCrossRefGoogle Scholar
  52. Tong, Z., and M.J. Sadowsky. 1994. A selective medium for the isolation and quantification of Bradyrhizobium japonicum and Bradyrhizobium elkanii strains from soils and inoculants. Applied and Environmental Microbiology 60: 581–586.PubMedGoogle Scholar
  53. Trujillo, M.E., A. Willems, A. Abril, A.-M. Plancheulo, R. Rivas, D. Lundena, P.F. Mateos, E. Martinez-Molina, and E. Velázquez. 2005. Nodulation of Lupinus albus by strains of Ochrobactrum lupini sp. nov. Applied and Environmental Microbiology 71: 1318–1327.PubMedCrossRefGoogle Scholar
  54. Uroz, S., M. Buée, C. Murat, P. Frey-Klett, and F. Martin. 2010. Pyrosequencing reveals a contrasted bacterial diversity between oak rhizosphere and surrounding soil. Environmental Microbiology 2: 281–288.CrossRefGoogle Scholar
  55. van Bruggen, A.H.C., and A.M. Semenov. 2000. In search of biological indicators for soil health and disease suppression. Applied Soil Ecology 15: 13–24.CrossRefGoogle Scholar
  56. van Overbeek, L., and J.D. van Elsas. 2008. Effects of plant genotype and growth stage on the structure of bacterial communities associated with potato (Solanum tuberosum L.). FEMS Microbiology Ecology 64: 283–296.PubMedCrossRefGoogle Scholar
  57. Wanandy, S., N. Brouwer, Q. Liu, A. Mahon, S. Cork, P. Karuso, S. Vemulpad, and J. Jamie. 2005. Optimisation of the fluorescein diacetate antibacterial assay. Journal of Microbiological Methods 60: 21–30.PubMedCrossRefGoogle Scholar
  58. Weinert, N., Y. Piceno, G.-C. Ding, R. Meincke, H. Heuer, G. Berg, M. Schloter, G. Andersen, and K. Smalla. 2011. PhyloChip hybridization uncovers an enormous bacterial diversity in the rhizosphere of different potato cultivars: many common and few cultivar-dependent taxa. FEMS Microbiology Ecology 75: 497–506.PubMedCrossRefGoogle Scholar
  59. Xu, L. 2008. Root colonization of Burkholderia phytofirmans strain PsJN and its relation to the bacterial plant growth promoting ability. In Department of Biology. University of Western Ontario, London, Canada.Google Scholar
  60. Yu, F., K. Zaleta-Rivera, X. Zhu, J. Huffman, J.C. Millet, S.D. Harris, G. Yuen, X.-C. Li, and L. Du. 2007. Structure and biosynthesis of heat-stable antifungal factor (HSAF), a broad spectrum antimycotic with a novel mode of action. Applied and Environmental Microbiology 51: 64–72.Google Scholar

Copyright information

© Potato Association of America 2012

Authors and Affiliations

  • Amy L. Turnbull
    • 1
  • Yibin Liu
    • 1
  • George Lazarovits
    • 1
  1. 1.A&L BiologicalsAgroecology Research Services CentreLondonCanada

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