Plant and Soil

, Volume 435, Issue 1–2, pp 385–393 | Cite as

Localization of typical and atypical Frankia isolates from Casuarina sp. in nodules formed on Casuarina equisetifolia

  • Spandana Vemulapally
  • Trina Guerra
  • Dittmar HahnEmail author
Regular Article



Members of the nitrogen-fixing actinobacterial genus Frankia are typically isolated from root nodules and generally infective on the same plant species. Several Frankia strains originally isolated from Casuarina species, however, have been found to be non-infective on Casuarina species. The goal of this study was to investigate the potential role of infective isolates from Casuarina species on the potential establishment of these non-infective Frankia strains in root nodule formation on Casuarina equisetifolia.


Soil microcosms were established with plants of C. equisetifolia and inoculated with Frankia casuarinae strain CcI3 or cluster 3 strain R43, or combinations of both at different densities. Basic plant growth characteristics, root nodule formation and localization of both Frankia strains in nodule periderm and cortex, as well as population development in soils were monitored.


The presence of strain R43 did not affect plant growth performance nor root nodule formation, while inoculation with strain CcI3 enhanced plant growth and resulted in root nodule formation. qPCR analyses on selected nodule lobes revealed the presence of strain CcI3 in cortex samples in all treatments, while strain R43 was not detected in any cortex samples but in 40% of the periderm samples from lobes from treatments with highest inoculation values. In situ hybridization detected cells of strain R43 on the outside of the nodules, i.e. on the periderm only.


These results demonstrate that the cluster 3 Frankia strain R43 is not co-infecting root nodules formed by the F. casuarinae strain CcI3 on C. equisetifolia, but has likely been isolated as a surface contaminant from Casuarina nodules.


FISH Frankia In situ hybridization nifNitrogenase Root nodules 



The authors are indebted to the Graduate College (Doctoral Research Support Fellowship to S. Vemulapally), and the Department of Biology at Texas State University for financial support.


  1. Amann RI, Krumholz L, Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172:762–770CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco JM (2006) The role of root exudates in rhizosphere interactions with plants and other organisms. Annu Rev Plant Biol 57:233–266CrossRefGoogle Scholar
  3. Baker DD (1987) Relationships among pure cultured strains of Frankia based on host specificity. Physiol Plant 70:245–248CrossRefGoogle Scholar
  4. Baker D, Newcomb W, Torrey JG (1980) Characterization of an ineffective actinorhizal microsymbiont, Frankia sp. EuI1 (Actinomycetales). Can J Microbiol 26:1072–1089CrossRefPubMedGoogle Scholar
  5. Bashan Y, Puente ME, Rodriguez-Mendoza MN, Toledo G, Holguin G, Ferrera-Cerrato R, Pedrin S (1995) Survival of Azospirillum brasilense in the bulk soil and rhizosphere of 23 soil types. Appl Environ Microbiol 61:1938–1945PubMedPubMedCentralGoogle Scholar
  6. Ben Tekaya S, Ganesan AS, Guerra T, Dawson JO, Forstner MRJ, Hahn D (2017) SybrGreen- and TaqMan-based quantitative PCR approaches allow assessment of the abundance and relative distribution of Frankia clusters in soils. Appl Environ Microb 85. UNSP e02833
  7. Benson DR, Dawson J (2007) Recent advances in the biogeography and genecology of symbiotic Frankia and its host plants. Physiol Plant 130:318–330CrossRefGoogle Scholar
  8. Berg RH, Mcdowell L (1987) Endophyte differentiation in Casuarina actinorhizae. Protoplasma 136:104–117CrossRefGoogle Scholar
  9. Bertin C, Yang X, Weston LA (2003) The role of root exudates and allelochemicals in the rhizosphere. Plant Soil 256:67–83CrossRefGoogle Scholar
  10. Diem HG, Gauthier D, Dommergues YR (1982) Isolation of Frankia from nodules of Casuarina equisetifolia. Can J Microbiol 28:526–530CrossRefGoogle Scholar
  11. Dobritsa SV (1998) Grouping of Frankia strains on the basis of susceptibility to antibiotics, pigment production and host specificity. Int J Syst Bacteriol 48:1265–1275CrossRefGoogle Scholar
  12. Gauthier D, Diem HG, Dommergues Y (1981) In vitro nitrogen fixation by two actinomycete strains isolated from Casuarina nodules. Appl Environ Microbiol 41:306–308PubMedPubMedCentralGoogle Scholar
  13. Ghodhbane-Gtari F, Nouioui I, Chair M, Boudabous A, Gtari M (2010) 16S-23S rRNA intergenic spacer region variability in the genus Frankia. Microb Ecol 60:487–495CrossRefPubMedGoogle Scholar
  14. Grayston SJ, Wang SQ, Campbell CD, Edwards AC (1998) Selective influence of plant species on microbial diversity in the rhizosphere. Soil Biol Biochem 30:369–378CrossRefGoogle Scholar
  15. Hahn D, Amann RI, Ludwig W, Akkermans ADL, Schleifer KH (1992) Detection of microorganisms in soil after in situ hybridization with ribosomal-RNA-targeted, fluorescently labeled oligonucleotides. J Gen Microbiol 138:879–887CrossRefPubMedGoogle Scholar
  16. Hahn D, Mirza B, Benagli C, Vogel G, Tonolla M (2011) Typing of nitrogen-fixing Frankia strains by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Syst Appl Microbiol 34:63–68CrossRefPubMedGoogle Scholar
  17. Hönerlage W, Hahn D, Zepp K, Zeyer J, Normand P (1994) A hypervariable 23S rRNA region provides a discriminating target for specific characterization of uncultured and cultured Frankia. Syst Appl Microbiol 17:433–443CrossRefGoogle Scholar
  18. Kuzyakov Y, Blagodatskaya E (2015) Microbial hotspots and hot moments in soil: concept & review. Soil Biol Biochem 83:184–199CrossRefGoogle Scholar
  19. La Favre JS, Focht DD (1983) Conservation in soil of H2 liberated from N2 fixation by Hup nodules. Appl Environ Microbiol 46:304–311PubMedPubMedCentralGoogle Scholar
  20. Lalonde M, Knowles R (1975) Ultrastructure, composition, and biogenesis of the encapsulation material surrounding the endophyte in Alnus crispa var. mollis Fern. Root nodules. Can J Bot 53:1951–1971CrossRefGoogle Scholar
  21. Leul M, Mohapatra A, Sellstedt A (2005) Biodiversity of hydrogenases in Frankia. Curr Microbiol 50:17–23CrossRefPubMedGoogle Scholar
  22. Meesters TM, van Genesen ST, Akkermans ADL (1985) Growth, acetylene reduction activity and localization of nitrogenase in relation to vesicle formation in Frankia strains Cc1.17 and Cp1.2. Arch Microbiol 143:137–142CrossRefGoogle Scholar
  23. Mirza BS, Welsh A, Hahn D (2007) Saprophytic growth of inoculated Frankia sp. in soil microcosms. FEMS Microbiol Ecol 62:280–289CrossRefPubMedGoogle Scholar
  24. Mirza BS, Welsh A, Rasul G, Rieder JP, Paschke MW, Hahn D (2009a) Variation in Frankia populations of the Elaeagnus host infection group in nodules of six host plant species after inoculation with soil. Microb Ecol 58:384–393CrossRefPubMedGoogle Scholar
  25. Mirza BS, Welsh AK, Rieder JP, Paschke MW, Hahn D (2009b) Diversity of frankiae in soils from five continents. Syst Appl Microbiol 32:558–570CrossRefPubMedGoogle Scholar
  26. Navarro E, Nalin R, Gauthier D, Normand P (1997) The nodular microsymbionts of Gymnostoma spp. are Elaeagnus-infective Frankia strains. Appl Environ Microbiol 63:1610–1616PubMedPubMedCentralGoogle Scholar
  27. Newcomb W, Pankhurst CE (1982) Fine structure of actinorhizal root nodules of Coriaria arborea (Coriariaceae). N Z J Bot 20:93–103CrossRefGoogle Scholar
  28. Normand P, Orso S, Cournoyer B, Jeannin P, Chapelon C, Dawson J, Evtushenko L, Misra AK (1996) Molecular phylogeny of the genus Frankia and related genera and emendation of the family Frankiaceae. Int J Syst Bacteriol 46:1–9CrossRefPubMedGoogle Scholar
  29. Normand P, Nouioui I, Pujic P, Fournier P, Dubost A, Schwob G, Klenk HP, Nguyen A, Abrouk D, Herrera-Belaroussi A, Pothier JF, Pfluger V, Fernandez MP (2018) Frankia canadensis sp nov., isolated from root nodules of Alnus incana subspecies rugosa. Int J Syst Evol Microbiol 68:3001–3011CrossRefPubMedGoogle Scholar
  30. Nouioui I, Ghodhbane-Gtari F, Beauchemin NJ, Tisa LS, Gtari M (2011) Phylogeny of members of the Frankia genus based on gyrB, nifH and glnII sequences. Anton Leeuw 100:579–587CrossRefGoogle Scholar
  31. Nouioui I, Ghodhbane-Gtari F, Montero-Calasanz MD, Goker M, Meier-Kolthoff JP, Schumann P, Rohde M, Goodfellow M, Fernandez MP, Norrnand P, Tisa LS, Klenk HP, Gtari M (2016) Proposal of a type strain for Frankia alni (Woronin 1866) Von Tubeuf 1895, emended description of Frankia alni, and recognition of Frankia casuarinae sp nov and Frankia elaeagni sp nov. Int J Syst Evol Microbiol 66:5201–5210CrossRefPubMedGoogle Scholar
  32. Nouioui I, Ghodhbane-Gtari F, Montero-Calasanz MD, Rohde M, Tisa LS, Gtari M, Klenk HP (2017a) Frankia inefficax sp nov., an actinobacterial endophyte inducing ineffective, non nitrogen-fixing, root nodules on its actinorhizal host plants. Anton Leeuw 110:313–320CrossRefGoogle Scholar
  33. Nouioui I, Ghodhbane-Gtari F, Rohde M, Klenk HP, Gtari M (2017b) Frankia coriariae sp nov., an infective and effective microsymbiont isolated from Coriaria japonica. Int J Syst Evol Microbiol 67:1266–1270CrossRefPubMedGoogle Scholar
  34. Nouioui I, Ghodhbane-Gtari F, Rhode M, Sangal V, Klenk HP, Gtari M (2018) Frankia irregularis sp nov., an actinobacterium unable to nodulate its original host, Casuarina equisetifolia, but effectively nodulates members of the actinorhizal Rhamnales. Int J Syst Evol Microbiol 68:2883–2890CrossRefPubMedGoogle Scholar
  35. Pernthaler A, Pernthaler J, Amann RI (2002) Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl Environ Microbiol 68:3094–3101CrossRefPubMedPubMedCentralGoogle Scholar
  36. Personeni E, Nguyen C, Marchal P, Pagès L (2007) Experimental evaluation of an efflux-influx model of C exudation by individual apical root segments. J Exp Bot 58:2091–2099CrossRefPubMedGoogle Scholar
  37. Poindexter JS (1981) Oligotrophy. Adv Microb Ecol 5:63–89CrossRefGoogle Scholar
  38. Pozzi AC, Bautista-Guerrero HH, Abby SS, Herrera-Belaroussi A, Abrouk D, Normand P, Menu F, Fernandez MP (2018) Robust Frankia phylogeny, species delineation and intraspeciesdiversity based on multi-locus sequence analysis (MLSA) and single-locus strain typing (SLST) adapted to a large sample size. Syst Appl Microbiol 41:311–323CrossRefPubMedGoogle Scholar
  39. Ramirez-Saad H, Janse J, Akkermans ADL (1998) Root nodules of Ceanothus caerulens contain both the N2-fixing Frankia endophyte and a phylogenetically related nod/fix actinomycete. Can J Microbiol 44:140–148CrossRefGoogle Scholar
  40. Roller C, Ludwig W, Schleifer K-H (1992) Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes. J Gen Microbiol 138:1167–1175CrossRefPubMedGoogle Scholar
  41. Samant S, Sha Q, Iyer A, Dhabekar P, Hahn D (2012) Quantification of Frankia in soils using SYBR green based qPCR. Syst Appl Microbiol 35:191–197CrossRefPubMedGoogle Scholar
  42. Samant S, Amann RI, Hahn D (2014) Evaluation of the 23S rRNA gene as target for qPCR based quantification of Frankia in soils. Syst Appl Microbiol 37:229–234CrossRefPubMedGoogle Scholar
  43. Samant S, Dawson JO, Hahn D (2016a) Growth responses of introduced Frankia strains to edaphic factors. Plant Soil 400:123–132CrossRefGoogle Scholar
  44. Samant S, Huo T, Dawson JO, Hahn D (2016b) Abundance and relative distribution of Frankia host infection groups under actinorhizal Alnus glutinosa and non-actinorhizal Betula nigra trees. Microb Ecol 71:473–481CrossRefPubMedGoogle Scholar
  45. Schönhuber W, Fuchs B, Juretschko S, Amann R (1997) Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification. Appl Environ Microbiol 63:3268–3273PubMedPubMedCentralGoogle Scholar
  46. Sellstedt A (1989) Occurrence and activity of hydrogenase in symbiotic Frankia from field-collected Alnus incana. Physiol Plant 75:304–308CrossRefGoogle Scholar
  47. Sellstedt A, Huss-Danell K, Ahlqvist AS (1986) Nitrogen fixation and biomass production in symbioses between Alnus incana and Frankia strains with different hydrogen metabolism. Physiol Plant 66:99–107CrossRefGoogle Scholar
  48. Van Elsas JD, Kijkstra AF, Govaert JM, van Veen JA (1986) Survival of Pseudomonas fluorescens and Bacillus subtilis introduced into two soils of different texture in field microplots. FEMS Microb Ecol 38:151–160CrossRefGoogle Scholar
  49. van Veen JA, van Overbeek LS, van Elsas JD (1997) Fate and activity of microorganisms introduced into soil. Microbiol Mol Biol Rev 61:121–135PubMedPubMedCentralGoogle Scholar
  50. Welsh A, Mirza BS, Rieder JP, Paschke MW, Hahn D (2009a) Diversity of frankiae in root nodules of Morella pensylvanica grown in soils from five continents. Syst Appl Microbiol 32:201–210CrossRefPubMedGoogle Scholar
  51. Welsh AK, Dawson JO, Gottfried GJ, Hahn D (2009b) Diversity of Frankia in root nodules of geographically isolated Arizona alders in central Arizona (USA). Appl Environ Microbiol 75:6913–6918CrossRefPubMedPubMedCentralGoogle Scholar
  52. Zhang Z, Lopez MF, Torrey JG (1984) A comparison of cultural characteristics and infectivity of Frankia isolates from root nodules of Casuarina species. Plant Soil 78:79–90CrossRefGoogle Scholar
  53. Zimpfer JF, Igual JM, McCarty B, Smyth C, Dawson JO (2004) Casuarina cunninghamiana tissue extracts stimulate the growth of Frankia and differentially alter the growth of other soil microorganisms. J Chem Ecol 30:439–452CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Department of BiologyTexas State UniversitySan MarcosUSA

Personalised recommendations