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Documentos de Cultura
DOI 10.1007/s11104-014-2186-6
METHODS PAPER
Abstract
Background and aim Nitrogen-fixing bacteria or
diazotrophs have been isolated for many years using
different formulations of N-free semi-solid media. However, the strategies used to isolate them, and the recipes
of these media, are scattered through the published
literature and in other sources that are more difficult to
access and which are not always retrievable. Therefore,
the aim of this work was to collate the various methods
and recipes, and to provide a comprehensive methodological guide and their use by the scientific community
working in the field of biological nitrogen fixation
(BNF), particularly with non-leguminous plants.
414
Introduction
The development of the acetylene reduction (AR) technique to evaluate nitrogenase activity in the 1960s
(Dilworth 1966; Schllhorn and Burris 1966) and its
subsequent application to investigate BNF associated
with non-legume crops (Yoshida and Ancajas 1970;
Balandreau and Dommergues 1971; Dbereiner et al.
1972) generated great interest in the concept that nonlegumes may be able to benefit from N inputs from this
process. Without doubt, one of the most important pioneers in the area of BNF associated with non-legume
plants was Dr Johanna Dbereiner, the founder of our
institute, now known as Embrapa Agrobiologia. Since
she started work at the research institute at Km 47 in
Seropdica, Rio de Janeiro, in 1951, she showed a great
interest in N2-fixing (or diazotrophic) bacteria isolated
from rhizosphere soil (Dbereiner 1953) or associated
with sugarcane (Dbereiner and Ruschel 1958) and
other grasses, such as Paspalum notatum (Dbereiner
1966). Up until her death in 2000, the team she headed
discovered and named nine new species of diazotrophs
and had developed new techniques to isolate and identify these bacteria (Baldani and Baldani 2005). Even
now, more than 10 years after her passing, all the different techniques that she and her team developed are
scattered across the literature and many of these publications are from the pre-electronic (PDF) period, some
are only in Portuguese, and many of them are difficult to
access. The interest in the bacteria which associate,
sometimes endophytically, with non-nodulating plants
continues unabated. For just the keyword
Azospirillum, the best known nitrogen-fixing and
plant growth-promoting genus, the Web of ScienceTM
lists over 3199 publications in all databases since its
discovery in 1978 until 2014 (updated March 17,
2014). The accumulated literature, especially on
Azospirillum, indicates that in addition to BNF, principally in association with sugarcane (Oliveira et al.
2006), these bacteria also benefit host plants through
other mechanisms, such as phytohormone and
415
416
Bacterial species
Carbon source
NFb
Azospirillum lipoferum
Malic acid
pH
References
6.8
Azospirillum brasilense
6.8
Azospirilum irakense
7.0 8.5
Azospirillum melinis
6.5 6.8
Azoarcus olearus
6.5 7.3
Azospirillum doebereinerae
LGI
Azospirillum amazonense
Sucrose
6.0 6.2
Baldani (1984)
FAM
Azospirillum amazonense
Sucrose
6.0
JNFb
Herbaspirillum seropedicae
Malic acid
5.8
Herbaspirillum rubrisubalbicans
Herbaspirillum frisingense
JNFb
Sphingomonas spp.
Malic acid
5.8
LGI-P
Gluconacetobacter
5.5
JMV
diazotrophicus
G. johannae
G. azotocaptans
Burkholderia kururiensis
5.0 5.4
Mannitol
Burkholderia tropica
Burkhoderia silvatlantica
Baz
Diazotrophic Burkholderia
Azelaic acid
5.7
Bac
Diazotrophic Burkholderia
Azelaic acid+L-citrulline
5.7
417
418
419
Treatments
of roots
intact
12947
crushed
13326
intact
846
crushed
8631
30
intact
758
30
crushed
1895
60
intact
60
crushed
583
420
Fig. 3 Inoculation of nitrogen-free semi-solid media and monitoring the pellicle formed in the media. a. Veil like pellicle formed
2 days after inoculation. b. Surface/subsurface pellicle formed
7 days after inoculation. The black arrows in figures indicate the
characteristic pellicle of the diazotrophic bacteria during growth in
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422
423
424
425
426
Future Perspectives
In conclusion, the introduction of the N-free semi-solid
medium opened new opportunities for those working in
the area of BNF with non-legumes, not only for the
important role played by microorganisms associated
with cereals and energy crops but also because some
of these diazotrophs, most notably Azospirillum spp.,
are being recommended as inoculants for sugarcane,
maize and wheat in Brazil and in other countries, such
as Argentina, Mexico, Colombia, Egypt and South Africa, among others. Azospirillum spp. have also been
used as co-inoculant plant growth-promoting bacteria to
be applied with soybean rhizobia in Argentina and
South Africa for many years. The recommended methodologies applied to count these bacteria in formulations
include recipes of semi-solid and solid media, thus
illustrating their importance in the general field of BNF.
Molecular approaches, such as qPCR can be applied
to detect with high efficiency a target N2-fixing species
or even a diazotrophic strain colonizing soil or plant
tissues (Quecine et al. 2012). nifH cDNA clone libraries
have also been applied to detect the functional
diazotrophic bacterial community associated with many
poaceous plants (Fischer et al. 2012; Videira et al. 2013;
Demba-Diallo et al. 2008; Ando et al. 2005). To facilitate the isolation of these in silico identified diazotrophic
bacteria, traditional microbiological methods (via isolation using semi-solid media) can be coupled to a molecular probe-directed approach that may be used to predict
the culturable diazotrophic diversity present in the plant
tissues (Hartmann et al. 2006). Several molecular probes
have already been designed (Stoffels et al., 2001;
Rothballer et al. 2006) and applied to monitor N2-fixing
proteobacteria associated with non-legumes (Schloter
et al. 1995; Oliveira et al. 2009).
The majority of the diazotrophic bacteria associated
with non-legumes have been obtained using the recipes
listed earlier. However, considering the published literature on this subject the possibilities to isolate new
species and even genera are very high using novel
semi-solid media. For example, a simple modification
in the NFb medium by the addition of 3 % NaCl allowed
the isolation from the Brazilian coral species
Mussismilia hispida of nineteen Vibrio strains belonging
to species V. harveyi, V. alginolyticus, V. campbellii, and
V. parahaemolyticus. Many of these vibrios were capable of growing six consecutive times in N-free medium
and each time showed strong nitrogenase activity
427
References
Adachi K, Nakatani M, Mochida H (2002) Isolation of an endophytic diazotroph, Klebsiella oxytoca, from sweet potato
stems in Japan. Soil Sci Plant Nutr 48:889895
Adler J (1966) Chemotaxis in bacteria. Science 153:708716
Ando S, Goto M, Meunchang S, Thongra-Ar P, Fujiwara T,
Hayashi H, Yoneyama T (2005) Detection of nifH sequences
in sugarcane (Saccharum officinarum L.) and pineapple
(Ananas comosus [L.] Merr.). J Soil Sci Plant Nutr 51:303
308
Balandreau J, Dommergues Y (1971) Mesure in situ lactivite
nitrogenasique. Comptes Rendus dAcademie du Sciences
(Paris) 273:20202023
Baldani JI (1984) Ocorrncia e caracterizao de Azospirillum
amazonense em comparao com outras espcies deste
gnero, em razes de milho, sorgo e arroz. Federal Rural
University of Rio de Janeiro, Seropdica, RJ, Brazil, MSc
Thesis
Baldani VLD (1996) Efeito da inoculao de Herbaspirillum spp.
no processo de colonizao e infeco de plantas de arroz e
428
ocorrncia e caracterizao parcial de uma nova bactria
diazotrfica. PhD thesis, Federal Rural University of Rio de
Janeiro, Seropdica, RJ, Brazil
Baldani JI, Baldani VLD (2005) History on the biological nitrogen
fixation research in graminaceous plants: special emphasis on
the Brazilian experience. An Acad Bras Ci 77:549579
Baldani JI, Baldani VLD, Sampaio MJAM, Dbereiner J (1984) A
fourth Azospirillum species from cereal roots. An Acad Bras
Ci 56:265
Baldani JI, Baldani VLD, Seldin L, Dbereiner J (1986a)
Characterization of Herbaspirillum seropedicae gen. nov.,
sp. nov., a root-associated nitrogen-fixing bacterium. Int J
Syst Bacteriol 36:8693
Baldani VLD, Alvarez MAB, Baldani JI, Dbereiner J (1986b)
Establishment of inoculated Azospirillum spp. in the rhizosphere and in roots of field-grown wheat and sorghum. Plant
Soil 90:3546
Baldani VLD, Baldani JI, Dbereiner J (1987) Inoculation of fieldgrown wheat (Triticumaestivum) with Azospirillum spp. in
Brazil. Biol Fert Soils 4:3740
Baldani VLD, Baldani JI, Olivares FL, Dbereiner J (1992)
Identification and ecology of Herbaspirillum seopedicae
and the closely related Pseudomonas rubrisubalbicans.
Symbiosis 19:6573
Baldani JI, Pot TB, Kirchhof G, Falsen E, Baldani VLD, Olivares
FL, Hoste B, Kersters K, Hartmann A, Gillis M, Dbereiner J
(1996) Emended description of Herbaspirillum, a mild plant
pathogen, as Herbaspirillum rubrisubalbicans comb.nov.; and
classification of a group of clinical isolates (EF group 1) as
Herbaspirillum species 3. Int J Syst Bacteriol 46:802810
Baldani VLD, Baldani JI, Dbereiner J (2000) Inoculation of rice
plants with the endophytic diazotrophs Herbapirillum
seropedicae and Burkholderia spp. Biol Fert Soils 30:485
491
Baldani JI, Krieg NR, Baldani VLD, Hartmann A, Dbereiner J
(2005a) Bergeys Manual of Systemic Bacteriology The
Protobacteria. In: Brenner DJ, Krieg NR, Staley JT, Garrity
GM (eds) Genus II. Azospirillum, 2edth edn. Springer, New
York, pp 726
Baldani JI, Baldani, VLD, Dbereiner J (2005b) Genus III.
Herbaspirillum in: Bergeys Manual of Systemic
Bacteriology The Protobacteria, 2.ed. Vol. 2, part C. In:
Brenner DJ, Krieg NR, Staley JT, Garrity GM (eds) Springer,
New York, pp 629636
Beijerinck M (1925) ber ein Spirillum, welches freien Stickstoff
binden kann? Zentralb Bakteriol Parasitenkd Infektionskr
Hyg 63:353359
Bodenhausen N, Horton MW, Bergelson J (2013) Bacterial communities associated with the leaves and the roots of
Arabidopsis thaliana. PLoS One 8:e56329. doi:10.1371/
journal.pone.0056329
Brasil MS, Baldani JI, Baldani VLD (2005) Ocorrncia e
diversidade de bactrias diazotrficas associadas a
gramneas forrageiras do Pantanal Sul Matogrossense. Rev
Bras Ci Solo 29:179190
Bulgarelli D, Schlaeppi K, Spaepen S, van Themaat EVL,
Schulze-Lefert P (2013) Structure and functions of the bacterial microbiota of plants. Annu Rev Plant Physiol Plant Mol
Biol 64:807838
Burbano CS, Liu Y, Rsner KL, Reis VM, CaballeroMellado J,
ReinholdHurek B, Hurek T (2011) Predominant nifH
429
Hitchens AP (1921) Advantages of culture medium containing
small percentages of agar. J Infec Disease 29:390407
Hugh R, Leifson E (1953) The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram negative bacteria. J Bacteriol 66:2426
James EK, Olivares FL (1998) Infection and colonization of sugar
cane and other gramineous plants by endophytic diazotrophs.
Crit Rev Plant Sci 17:77119
James EK, Olivares FL, de Oliveira AL, Reis FB Jr, da Silva LG,
Reis VM (2001) Further observations on the interaction between sugar cane and Gluconacetobacter diazotrophicus under
laboratory and greenhouse conditions. J Exp Bot 52:747760
Jha B, Gontia I, Hartmann A (2012) The roots of the halophyte
Salicornia brachiata are a source of new halotolerant
diazotrophic bacteria with plant growth-promoting potential.
Plant Soil 356:265277
Khammas KM, Ageron E, Grimont PAD, Kaiser P (1989)
Azospirillum irakense sp. nov., a nitrogen-fixing bacterium
associated with rice roots and rhizosphere soil. Rev
Microbiol 140:679693
Kirchhof G, Eckert B, Stoffels M, Baldani JI, Reis VM, Hartmann
A (2001) Herbaspirillum frisingense sp. nov., a new
nitrogen-fixing bacterial species that occurs in C4-fiber
plants. Int J Syst Evol Microbiol 51:157168
Kloepper JW, Schippers B, Bakker PAHM (1992) Proposed elimination of the term Endorhizosphere. Phytopathol 82:726
727
Li RP, MacRae IC (1992) Specific identification and enumeration
of Acetobacter diazotrophicus in sugarcane. Soil Biol
Biochem 24:413419
Lipman JG (1904) Soil bacteriological studies. Further contributions to the physiology and morphology of the members of
the Azotobacter group. Rep New Jersey State Agric Exp
Station 25:237289
LonhienneT G, PaungfooLonhienne C, Yeoh YK, Webb RI,
Lakshmanan P, Chan CX, Hugenholtz P (2014) A new
species of Burkholderia isolated from sugarcane roots promotes plant growth. Microbial Biotech 7:154
Magalhes FMM, Dbereiner J (1984) Occurrence of
Azospirillum amazonense in some Amazonian ecosystems.
Rev Microbiol 4:246252
Magalhes FM, Baldani JI, Souto SM, Kuykendall JR, Dbereiner
J (1983) A new acid-tolerant Azospirillum species. An Acad
Bras Ci 55:417430
Mehnaz S, Weselowski B, Lazarovits G (2007) Azospirillum
canadense sp. nov., a nitrogen-fixing bacterium isolated from
corn rhizosphere. Int J Syst Evol Microbiol 57:620624
Okon Y, Albrecht SL, Burris RH (1977) Methods for growing
Spirillum lipoferum and for counting it in pure culture
and in association with plants. Appl Environ Microbiol
33:8588
Oliveira E (1992) Estudo da associao entre bactrias
diazotrficas e arroz. Federal Rural University of Rio de
Janeiro, Seropdica, RJ, Brazil, MSc Thesis
Oliveira ALM, Canuto EL, Silva EE, Reis VM, Baldani JI (2004)
Survival of endophytic diazotrophic bacteria in soil under
different moisture levels. Braz J Microbiol 35:295299
Oliveira ALM, Canuto EL, Urquiaga S, Reis VM, Baldani JI
(2006) Yield of micropropagated sugarcane varieties in different soil types following inoculation with diazotrophic
bacteria. Plant Soil 284:2332
430
Oliveira ALM, Stoffels M, Schmid M, Reis VM, Baldani JI,
Hartmann A (2009) Colonization of sugarcane plantlets by
mixed inoculations with diazotrophic bacteria. Euro J Soil
Biol 45:106113
Patriquin DG, Dbereiner J (1978) Light microscopy observations of tetrazolium-reducing bacteria in the
endorhizosphere of maize and other grasses in Brazil.
Can J Microbiol 24:734742
Peng G, Wang H, Zhang G, Hou W, Liu Y, Wang ET, Tan Z (2006)
Azospirillum melinis sp. nov., a group of diazotrophs isolated
from tropical molasses grass. Intern J Syst and Evol
Microbiol 56:12631271
Pereira TP, do Amaral FP, DallAsta P, Brod FCA, Arisi ACM
(2014) Real-Time PCR Quantification of the Plant Growth
Promoting Bacteria Herbaspirillum seropedicae Strain SmR1
in Maize Roots. Molec Biotech 111. doi:10.1007/s12033014-9742-4
Perin L, Martinez-Aguilar L, Paredes-Valdez G, Baldani JI,
Estrada-de-Los-Santos P, Reis VM, Caballero-Mellado J
(2006) Burkholderia silvatlantica sp. nov., a diazotrophic
bacterium associated with sugarcane and maize. Int J Syst
Evol Microbiol 56:19311937
Quecine MC, Arajo WL, Rossetto PB, Ferreira A, Tsui S, Lacava
PT, Mondin M, Azevedo JL, Pizzirani-Kleiner AA (2012)
Sugarcane growth promotion by the endophytic bacterium
Pantoea agglomerans. Appl Environ Microbiol 78:75117518
Reinhold B, Hurek T, Niemann EG, Fendrik I (1986) Close
association of Azospirillum and diazotrophic rods with different root zones of Kallar grass. Appl Environ Microbiol 52:
520526
Reinhold B, Hurek T, Fendrik I, Pot B, Gillis M, Kersters K,
Thielemans S, De Ley J (1987) Azospirillum halopraeferens
sp. nov., a nitrogen-fixing organism associated with roots of
Kallar grass (Leptochloa fusca (L.) Kunth). Int J Syst
Bacteriol 37:4351
Reinhold-Hurek B, Hurek T (2006) In The Prokaryotes. In:
Dworkin M, Falkow S, Rosenberg E, Schleifer KHE S
(eds) The genera Azoarcus, Azovibrio, Azospira and
Azonexus, 3rd edn. Springer, New York, pp 873891
Reinhold-Hurek B, Hurek T, Gillis M, Hoste B, Vancanneyt M,
Kersters K, De Ley J (1993) Azoarcus gen. nov., nitrogenfixing proteobacteria associated with roots of Kallar grass
(Leptochloa fusca (L.) Kunth), and description of two species, Azoarcus indigens sp. nov. and Azoarcus communis sp.
nov. Int J Syst Bacteriol 43:574584
Reis Junior FB, Silva LG, Reis VM, Dbereiner J (2000)
Ocorrncia de bactrias diazotrficas em diferentes
gentipos de cana-de-acar. Pesq Agropec Bras 35:985994
Reis VM, Olivares FL, Dbereiner J (1994) Improved methodology for isolation of Acetobacter diazotrophicus and confirmation of its endophytic habitat. World J Microbiol Biotech
10:401405
Reis VM, Estrada-de-Los-Santos P, Tenrio-Salgado S, Vogel J,
Stoffels M, Guyon S, Mavingui P, Baldani VLD, Schmid M,
Baldani JI, Balandreau J, Hartmann A, Caballero-Mellado J
(2004) Burkholderia tropica sp. nov., a novel nitrogen-fixing,
plant-associated bacterium. Int J Syst Evol Microbiol 54:
21552162
Rennie RJ (1981) A single medium for the isolation of acetylenereducing (dinitrogen-fixing) bacteria from soils. Can J
Microbiol 27:814
431
Watt M, Hugenholtz P, White R, Vinall K (2006) Numbers and
locations of native bacteria on fieldgrown wheat roots quantified by fluorescence in situ hybridization (FISH). Environ
Microbiol 8:871884
Weber OB, Baldani VLD, Teixeira KRS, Kirchhof G, Baldani JI,
Dbereiner J (1999) Isolation and characterization of
diazotrophic bacteria from banana and pineapple plants.
Plant Soil 210:103113
Whittenbury R (1963) The Use of Soft Agar in the Study of
Conditions Affecting the Utilization of Fermentable Substrates
by Lactic Acid Bacteria. J Gen Microbiol 32:375384
Xie C, Yokota A (2005) Azospirillum oryzae sp. nov., a nitrogenfixing bacterium isolated from the roots of the rice plant
Oryza sativa. Int J Syst Evol Microbiol 55:14351438
Yoshida T, Ancajas RR (1970) Application of the acetylene reduction method in nitrogen fixation studies. Soil Sci Plant
Nutr 16:234237
ZoBell CE (1932) Factors influencing the reduction of nitrates and
nitrites by bacteria in semisolid media. J Bacteriol 24:273281